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Recent Articles in Endocrine Reviews

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The Endocrine Society 2004 annual awards.
Endocr Rev. 2004 Aug;25(4):680-92. [Abstract/Link to Full Text]

Doherty TM, Fitzpatrick LA, Inoue D, Qiao JH, Fishbein MC, Detrano RC, Shah PK, Rajavashisth TB
Molecular, endocrine, and genetic mechanisms of arterial calcification.
Endocr Rev. 2004 Aug;25(4):629-72.
Pathologists have recognized arterial calcification for over a century. Recent years have witnessed a strong resurgence of interest in atherosclerotic plaque calcification because it: 1) can be easily detected noninvasively; 2) closely correlates with the amount of atherosclerotic plaque; 3) serves as a surrogate measure for atherosclerosis, allowing preclinical detection of the disease; and 4) is associated with heightened risk of adverse cardiovascular events. There are two major types of calcification in arteries: calcification of the media tunica layer (sometimes called Mönckeberg's sclerosis), and calcification within subdomains of atherosclerotic plaque within the intimal layer of the artery. There are important similarities and differences between these two entities. Of particular interest are increasing parallels between cellular and molecular features of arterial calcification and bone biology, and this has led to accelerating interest in understanding how and why bone-like mineral deposits may form in arteries. Here, we review the two major pathological types of arterial calcification, the proposed models of calcification, and endocrine and genetic determinants that affect arterial calcification. In addition, we highlight areas requiring further investigation. [Abstract/Link to Full Text]

Vincent AM, Russell JW, Low P, Feldman EL
Oxidative stress in the pathogenesis of diabetic neuropathy.
Endocr Rev. 2004 Aug;25(4):612-28.
Oxidative stress results from a cell or tissue failing to detoxify the free radicals that are produced during metabolic activity. Diabetes is characterized by chronic hyperglycemia that produces dysregulation of cellular metabolism. This review explores the concept that diabetes overloads glucose metabolic pathways, resulting in excess free radical production and oxidative stress. Evidence is presented to support the idea that both chronic and acute hyperglycemia cause oxidative stress in the peripheral nervous system that can promote the development of diabetic neuropathy. Proteins that are damaged by oxidative stress have decreased biological activity leading to loss of energy metabolism, cell signaling, transport, and, ultimately, to cell death. Examination of the data from animal and cell culture models of diabetes, as well as clinical trials of antioxidants, strongly implicates hyperglycemia-induced oxidative stress in diabetic neuropathy. We conclude that striving for superior antioxidative therapies remains essential for the prevention of neuropathy in diabetic patients. [Abstract/Link to Full Text]

Ferrara N
Vascular endothelial growth factor: basic science and clinical progress.
Endocr Rev. 2004 Aug;25(4):581-611.
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. Hypoxia has been shown to be a major inducer of VEGF gene transcription. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high-affinity VEGF receptors. The role of VEGF in developmental angiogenesis is emphasized by the finding that loss of a single VEGF allele results in defective vascularization and early embryonic lethality. VEGF is critical also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with various VEGF inhibitors in a variety of malignancies are ongoing. Very recently, an anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration. [Abstract/Link to Full Text]

Pacak K, Eisenhofer G, Goldstein DS
Functional imaging of endocrine tumors: role of positron emission tomography.
Endocr Rev. 2004 Aug;25(4):568-80.
This article provides an update on functional imaging approaches for diagnostic localization of endocrine tumors, with emphasis on positron emission tomography (PET). [18F]Fluorodeoxyglucose PET scanning is now a widely accepted imaging approach in clinical oncology. Benefits include improved patient outcome facilitated by staging and monitoring of disease and better treatment planning. [18F]Fluorodeoxyglucose PET is also useful in some endocrine tumors, particularly in recurrent or metastatic thyroid cancer where the degree of accumulation of the radionuclide has prognostic value. However, this imaging approach does not take full advantage of the unique characteristics of endocrine tumors. Endocrine tumor cells take up hormone precursors, express receptors and transporters, and synthesize, store, and release hormones. These characteristics offer highly specific targets for PET. Radiopharmaceuticals developed for such approaches include 6-[18F]fluorodopamine, and [11C]hydroxyephedrine for localization of pheochromocytomas, [11C]5-hydroxytryptophan and [11C]L-dihydroxyphenylalanine for carcinoid tumors, and [11C]metomidate for adrenocortical tumors. These functional imaging approaches are not meant to supplant conventional imaging modalities but should be used conjointly to better identify specific characteristics of endocrine tumors. This represents a relatively new and evolving approach to imaging that promises to answer specific questions about the behavior and growth of endocrine tumors, their malignant potential, and responsiveness to different treatment modalities. [Abstract/Link to Full Text]

Fang ZY, Prins JB, Marwick TH
Diabetic cardiomyopathy: evidence, mechanisms, and therapeutic implications.
Endocr Rev. 2004 Aug;25(4):543-67.
The presence of a diabetic cardiomyopathy, independent of hypertension and coronary artery disease, is still controversial. This systematic review seeks to evaluate the evidence for the existence of this condition, to clarify the possible mechanisms responsible, and to consider possible therapeutic implications. The existence of a diabetic cardiomyopathy is supported by epidemiological findings showing the association of diabetes with heart failure; clinical studies confirming the association of diabetes with left ventricular dysfunction independent of hypertension, coronary artery disease, and other heart disease; and experimental evidence of myocardial structural and functional changes. The most important mechanisms of diabetic cardiomyopathy are metabolic disturbances (depletion of glucose transporter 4, increased free fatty acids, carnitine deficiency, changes in calcium homeostasis), myocardial fibrosis (association with increases in angiotensin II, IGF-I, and inflammatory cytokines), small vessel disease (microangiopathy, impaired coronary flow reserve, and endothelial dysfunction), cardiac autonomic neuropathy (denervation and alterations in myocardial catecholamine levels), and insulin resistance (hyperinsulinemia and reduced insulin sensitivity). This review presents evidence that diabetes is associated with a cardiomyopathy, independent of comorbid conditions, and that metabolic disturbances, myocardial fibrosis, small vessel disease, cardiac autonomic neuropathy, and insulin resistance may all contribute to the development of diabetic heart disease. [Abstract/Link to Full Text]

Jorgensen JS, Quirk CC, Nilson JH
Multiple and overlapping combinatorial codes orchestrate hormonal responsiveness and dictate cell-specific expression of the genes encoding luteinizing hormone.
Endocr Rev. 2004 Aug;25(4):521-42.
Normal reproductive function in mammals requires precise control of LH synthesis and secretion by gonadotropes of the anterior pituitary. Synthesis of LH requires expression of two genes [alpha-glycoprotein subunit (alphaGSU) and LHbeta] located on different chromosomes. Hormones from the hypothalamus and gonads modulate transcription of both genes as well as secretion of the biologically active LH heterodimer. In males and females, the transcriptional tone of the genes encoding alphaGSU and LHbeta reflects dynamic integration of a positive signal provided by GnRH from hypothalamic neurons and negative signals emanating from gonadal steroids. Although alphaGSU and LHbeta genes respond transcriptionally in the same manner to changes in hormonal input, different combinations of regulatory elements orchestrate their response. These hormone-responsive regulatory elements are also integral members of much larger combinatorial codes responsible for targeting expression of alphaGSU and LHbeta genes to gonadotropes. In this review, we will profile the genomic landscape of the promoter-regulatory region of both genes, depicting elements and factors that contribute to gonadotrope-specific expression and hormonal regulation. Within this context, we will highlight the different combinatorial codes that control transcriptional responses, particularly those that mediate the opposing effects of GnRH and one of the sex steroids, androgens. We will use this framework to suggest that GnRH and androgens attain the same transcriptional endpoint through combinatorial codes unique to alphaGSU and LHbeta. This parallelism permits the dynamic and coordinate regulation of two genes that encode a single hormone. [Abstract/Link to Full Text]

Kaltsas GA, Besser GM, Grossman AB
The diagnosis and medical management of advanced neuroendocrine tumors.
Endocr Rev. 2004 Jun;25(3):458-511.
Neuroendocrine tumors (NETs) constitute a heterogeneous group of neoplasms that originate from endocrine glands such as the pituitary, the parathyroids, and the (neuroendocrine) adrenal, as well as endocrine islets within glandular tissue (thyroid or pancreatic) and cells dispersed between exocrine cells, such as endocrine cells of the digestive (gastroenteropancreatic) and respiratory tracts. Conventionally, NETs may present with a wide variety of functional or nonfunctional endocrine syndromes and may be familial and have other associated tumors. Assessment of specific or general tumor markers offers high sensitivity in establishing the diagnosis and can also have prognostic significance. Imaging modalities include endoscopic ultrasonography, computed tomography and magnetic resonance imaging, and particularly, scintigraphy with somatostatin analogs and metaiodobenzylguanidine. Successful treatment of disseminated NETs requires a multimodal approach; radical tumor surgery may be curative but is rarely possible. Well-differentiated and slow-growing gastroenteropancreatic tumors should be treated with somatostatin analogs or alpha-interferon, with chemotherapy being reserved for poorly differentiated and progressive tumors. Therapy with radionuclides may be used for tumors exhibiting uptake to a diagnostic scan, either after surgery to eradicate microscopic residual disease or later if conventional treatment or biotherapy fails. Maintenance of the quality of life should be a priority, particularly because patients with disseminated disease may experience prolonged survival. [Abstract/Link to Full Text]

van der Lely AJ, Tschöp M, Heiman ML, Ghigo E
Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin.
Endocr Rev. 2004 Jun;25(3):426-57.
Ghrelin is a peptide predominantly produced by the stomach. Ghrelin displays strong GH-releasing activity. This activity is mediated by the activation of the so-called GH secretagogue receptor type 1a. This receptor had been shown to be specific for a family of synthetic, peptidyl and nonpeptidyl GH secretagogues. Apart from a potent GH-releasing action, ghrelin has other activities including stimulation of lactotroph and corticotroph function, influence on the pituitary gonadal axis, stimulation of appetite, control of energy balance, influence on sleep and behavior, control of gastric motility and acid secretion, and influence on pancreatic exocrine and endocrine function as well as on glucose metabolism. Cardiovascular actions and modulation of proliferation of neoplastic cells, as well as of the immune system, are other actions of ghrelin. Therefore, we consider ghrelin a gastrointestinal peptide contributing to the regulation of diverse functions of the gut-brain axis. So, there is indeed a possibility that ghrelin analogs, acting as either agonists or antagonists, might have clinical impact. [Abstract/Link to Full Text]

Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, Ohlsson C
Androgens and bone.
Endocr Rev. 2004 Jun;25(3):389-425.
Loss of estrogens or androgens increases the rate of bone remodeling by removing restraining effects on osteoblastogenesis and osteoclastogenesis, and also causes a focal imbalance between resorption and formation by prolonging the lifespan of osteoclasts and shortening the lifespan of osteoblasts. Conversely, androgens, as well as estrogens, maintain cancellous bone mass and integrity, regardless of age or sex. Although androgens, via the androgen receptor (AR), and estrogens, via the estrogen receptors (ERs), can exert these effects, their relative contribution remains uncertain. Recent studies suggest that androgen action on cancellous bone depends on (local) aromatization of androgens into estrogens. However, at least in rodents, androgen action on cancellous bone can be directly mediated via AR activation, even in the absence of ERs.Androgens also increase cortical bone size via stimulation of both longitudinal and radial growth. First, androgens, like estrogens, have a biphasic effect on endochondral bone formation: at the start of puberty, sex steroids stimulate endochondral bone formation, whereas they induce epiphyseal closure at the end of puberty. Androgen action on the growth plate is, however, clearly mediated via aromatization in estrogens and interaction with ERalpha. Androgens increase radial growth, whereas estrogens decrease periosteal bone formation. This effect of androgens may be important because bone strength in males seems to be determined by relatively higher periosteal bone formation and, therefore, greater bone dimensions, relative to muscle mass at older age. Experiments in mice again suggest that both the AR and ERalpha pathways are involved in androgen action on radial bone growth. ERbeta may mediate growth-limiting effects of estrogens in the female but does not seem to be involved in the regulation of bone size in males.In conclusion, androgens may protect men against osteoporosis via maintenance of cancellous bone mass and expansion of cortical bone. Such androgen action on bone is mediated by the AR and ERalpha. [Abstract/Link to Full Text]

Somboonporn W, Davis SR
Testosterone effects on the breast: implications for testosterone therapy for women.
Endocr Rev. 2004 Jun;25(3):374-88.
Androgens have important physiological effects in women. Postmenopausal androgen replacement, most commonly as testosterone therapy, is becoming increasingly widespread. This is despite the lack of clear guidelines regarding the diagnosis of androgen insufficiency, optimal therapeutic doses, and long-term safety data. With respect to the breast specifically, there is the potential for exogenous testosterone to exert either androgenic or indirect estrogenic actions, with the latter potentially increasing breast cancer risk. In experimental studies, androgens exhibit growth-inhibitory and apoptotic effects in some, but not all, breast cancer cell lines. Differing effects between cell lines appear to be due primarily to variations in concentrations of specific coregulatory proteins at the receptor level. In rodent breast cancer models, androgen action is antiproliferative and proapoptotic, and is mediated via the androgen receptor, despite the potential for testosterone and dehydroepiandrosterone to be aromatized to estrogen. The results from studies in rhesus monkeys suggest that testosterone may serve as a natural endogenous protector of the breast and limit mitogenic and cancer-promoting effects of estrogen on mammary epithelium. Epidemiological studies have significant methodological limitations and provide inconclusive results. The strongest data for exogenous testosterone therapy comes from primate studies. Based on such simulations, inclusion of testosterone in postmenopausal estrogen-progestin regimens has the potential to ameliorate the stimulating effects of combined estrogen-progestin on the breast. Research addressing this is warranted; however, the number of women that would be required for an adequately powered randomized controlled trial renders such a study unlikely. [Abstract/Link to Full Text]

Dey SK, Lim H, Das SK, Reese J, Paria BC, Daikoku T, Wang H
Molecular cues to implantation.
Endocr Rev. 2004 Jun;25(3):341-73.
Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and receptive uterus. Although various cellular aspects and molecular pathways of this dialogue have been identified, a comprehensive understanding of the implantation process is still missing. The receptive state of the uterus, which lasts for a limited period, is defined as the time when the uterine environment is conducive to blastocyst acceptance and implantation. A better understanding of the molecular signals that regulate uterine receptivity and implantation competency of the blastocyst is of clinical relevance because unraveling the nature of these signals may lead to strategies to correct implantation failure and improve pregnancy rates. Gene expression studies and genetically engineered mouse models have provided valuable clues to the implantation process with respect to specific growth factors, cytokines, lipid mediators, adhesion molecules, and transcription factors. However, a staggering amount of information from microarray experiments is also being generated at a rapid pace. If properly annotated and explored, this information will expand our knowledge regarding yet-to-be-identified unique, complementary, and/or redundant molecular pathways in implantation. It is hoped that the forthcoming information will generate new ideas and concepts for a process that is essential for maintaining procreation and solving major reproductive health issues in women. [Abstract/Link to Full Text]

Mansmann G, Lau J, Balk E, Rothberg M, Miyachi Y, Bornstein SR
The clinically inapparent adrenal mass: update in diagnosis and management.
Endocr Rev. 2004 Apr;25(2):309-40.
Clinically inapparent adrenal masses are incidentally detected after imaging studies conducted for reasons other than the evaluation of the adrenal glands. They have frequently been referred to as adrenal incidentalomas. In preparation for a National Institutes of Health State-of-the-Science Conference on this topic, extensive literature research, including Medline, BIOSIS, and Embase between 1966 and July 2002, as well as references of published metaanalyses and selected review articles identified more than 5400 citations. Based on 699 articles that were retrieved for further examination, we provide a comprehensive update of the diagnostic and therapeutic approaches focusing on endocrine and radiological features as well as surgical options. In addition, we present recent developments in the discovery of tumor markers, endocrine testing for subclinical disease including autonomous glucocorticoid hypersecretion and silent pheochromocytoma, novel imaging techniques, and minimally invasive surgery. Based on the statements of the conference, the available literature, and ongoing studies, our aim is to provide practical recommendations for the management of this common entity and to highlight areas for future studies and research. [Abstract/Link to Full Text]

Heinlein CA, Chang C
Androgen receptor in prostate cancer.
Endocr Rev. 2004 Apr;25(2):276-308.
The normal development and maintenance of the prostate is dependent on androgen acting through the androgen receptor (AR). AR remains important in the development and progression of prostate cancer. AR expression is maintained throughout prostate cancer progression, and the majority of androgen-independent or hormone refractory prostate cancers express AR. Mutation of AR, especially mutations that result in a relaxation of AR ligand specificity, may contribute to the progression of prostate cancer and the failure of endocrine therapy by allowing AR transcriptional activation in response to antiandrogens or other endogenous hormones. Similarly, alterations in the relative expression of AR coregulators have been found to occur with prostate cancer progression and may contribute to differences in AR ligand specificity or transcriptional activity. Prostate cancer progression is also associated with increased growth factor production and an altered response to growth factors by prostate cancer cells. The kinase signal transduction cascades initiated by mitogenic growth factors modulate the transcriptional activity of AR and the interaction between AR and AR coactivators. The inhibition of AR activity through mechanisms in addition to androgen ablation, such as modulation of signal transduction pathways, may delay prostate cancer progression. [Abstract/Link to Full Text]

Millar RP, Lu ZL, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR
Gonadotropin-releasing hormone receptors.
Endocr Rev. 2004 Apr;25(2):235-75.
GnRH and its analogs are used extensively for the treatment of hormone-dependent diseases and assisted reproductive techniques. They also have potential as novel contraceptives in men and women. A thorough delineation of the molecular mechanisms involved in ligand binding, receptor activation, and intracellular signal transduction is kernel to understanding disease processes and the development of specific interventions. Twenty-three structural variants of GnRH have been identified in protochordates and vertebrates. In many vertebrates, three GnRHs and three cognate receptors have been identified with distinct distributions and functions. In man, the hypothalamic GnRH regulates gonadotropin secretion through the pituitary GnRH type I receptor via activation of G(q). In-depth studies have identified amino acid residues in both the ligand and receptor involved in binding, receptor activation, and translation into intracellular signal transduction. Although the predominant coupling of the type I GnRH receptor in the gonadotrope is through productive G(q) stimulation, signal transduction can occur via other G proteins and potentially by G protein-independent means. The eventual selection of intracellular signaling may be specifically directed by variations in ligand structure. A second form of GnRH, GnRH II, conserved in all higher vertebrates, including man, is present in extrahypothalamic brain and many reproductive tissues. Its cognate receptor has been cloned from various vertebrate species, including New and Old World primates. The human gene homolog of this receptor, however, has a frame-shift and stop codon, and it appears that GnRH II signaling occurs through the type I GnRH receptor. There has been considerable plasticity in the use of different GnRHs, receptors, and signaling pathways for diverse functions. Delineation of the structural elements in GnRH and the receptor, which facilitate differential signaling, will contribute to the development of novel interventive GnRH analogs. [Abstract/Link to Full Text]

Sherwood OD
Relaxin's physiological roles and other diverse actions.
Endocr Rev. 2004 Apr;25(2):205-34.
Relaxin has vital physiological roles in pregnant rats, mice, and pigs. Relaxin promotes growth and softening of the cervix, thus facilitating rapid delivery of live young. Relaxin also promotes development of the mammary apparatus, thus enabling normal lactational performance. The actions of relaxin on the mammary apparatus vary among species. Whereas relaxin is required for development of the mammary nipples in rats and mice, it is essential for prepartum development of glandular parenchyma in pregnant pigs. During pregnancy relaxin also inhibits uterine contractility and promotes the osmoregulatory changes of pregnancy in rats. Recent studies with male and nonpregnant female rodents revealed diverse therapeutic actions of relaxin on nonreproductive tissues that have clinical implications. Relaxin has been reported to reduce fibrosis in the kidney, heart, lung, and liver and to promote wound healing. Also, probably through its vasodilatory actions, relaxin protects the heart from ischemia-induced injury. Finally, relaxin counteracts allergic reactions. Knowledge of the diverse physiological and therapeutic actions of relaxin, coupled with the recent identification of relaxin receptors, opens numerous avenues of investigation that will likely sustain a high level of research interest in relaxin for the foreseeable future. [Abstract/Link to Full Text]

Watson RT, Kanzaki M, Pessin JE
Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes.
Endocr Rev. 2004 Apr;25(2):177-204.
Since the discovery of insulin roughly 80 yr ago, much has been learned about how target cells receive, interpret, and respond to this peptide hormone. For example, we now know that insulin activates the tyrosine kinase activity of its cell surface receptor, thereby triggering intracellular signaling cascades that regulate many cellular processes. With respect to glucose homeostasis, these include the function of insulin to suppress hepatic glucose production and to increase glucose uptake in muscle and adipose tissues, the latter resulting from the translocation of the glucose transporter 4 (GLUT4) to the cell surface membrane. Although simple in broad outline, elucidating the molecular intricacies of these receptor-signaling pathways and membrane-trafficking processes continues to challenge the creative ingenuity of scientists, and many questions remain unresolved, or even perhaps unasked. The identification and functional characterization of specific molecules required for both insulin signaling and GLUT4 vesicle trafficking remain key issues in our pursuit of developing specific therapeutic agents to treat and/or prevent this debilitating disease process. To this end, the combined efforts of numerous research groups employing a range of experimental approaches has led to a clearer molecular picture of how insulin regulates the membrane trafficking of GLUT4. [Abstract/Link to Full Text]

Fonseca V, Desouza C, Asnani S, Jialal I
Nontraditional risk factors for cardiovascular disease in diabetes.
Endocr Rev. 2004 Feb;25(1):153-75.
People with type 2 diabetes are disproportionately affected by cardiovascular disease (CVD), compared with those without diabetes. Traditional risk factors do not fully explain this excess risk, and other "nontraditional" risk factors may be important. This review will highlight the importance of nontraditional risk factors for CVD in the setting of type 2 diabetes and discuss their role in the pathogenesis of the excess CVD morbidity and mortality in these patients. We will also discuss the impact of various therapies used in patients with diabetes on nontraditional risk factors. [Abstract/Link to Full Text]

Colao A, Ferone D, Marzullo P, Lombardi G
Systemic complications of acromegaly: epidemiology, pathogenesis, and management.
Endocr Rev. 2004 Feb;25(1):102-52.
This review focuses on the systemic complications of acromegaly. Mortality in this disease is increased mostly because of cardiovascular and respiratory diseases, although currently neoplastic complications have been questioned as a relevant cause of increased risk of death. Biventricular hypertrophy, occurring independently of hypertension and metabolic complications, is the most frequent cardiac complication. Diastolic and systolic dysfunction develops along with disease duration; and other cardiac disorders, such as arrhythmias, valve disease, hypertension, atherosclerosis, and endothelial dysfunction, are also common in acromegaly. Control of acromegaly by surgery or pharmacotherapy, especially somatostatin analogs, improves cardiovascular morbidity. Respiratory disorders, sleep apnea, and ventilatory dysfunction are also important contributors in increasing mortality and are advantageously benefitted by controlling GH and IGF-I hypersecretion. An increased risk of colonic polyps, which more frequently recur in patients not controlled after treatment, has been reported by several independent investigations, although malignancies in other organs have also been described, but less convincingly than at the gastrointestinal level. Finally, the most important cause of morbidity and functional disability of the disease is arthropathy, which can be reversed at an initial stage, but not if the disease is left untreated for several years. [Abstract/Link to Full Text]

Shimasaki S, Moore RK, Otsuka F, Erickson GF
The bone morphogenetic protein system in mammalian reproduction.
Endocr Rev. 2004 Feb;25(1):72-101.
Using molecular, cellular, and genetic approaches, recent studies examining the role of the bone morphogenetic protein (BMP) family of growth factors in the reproductive system have led to significant breakthroughs in our understanding of mammalian reproduction and fertility. Gene expression studies have revealed that key components of the BMP system (ligands, receptors, signaling molecules, and binding proteins) exhibit coordinated spatial and temporal expression patterns in fundamental cell types throughout the reproductive system. Availability of recombinant BMPs has enabled functional studies that have demonstrated important biological activities of BMPs in controlling cellular proliferation, differentiation, and apoptosis in reproductive tissues. The physiological importance of the BMP system for mammalian reproduction has been further highlighted by the elucidation of the aberrant reproductive phenotypes of animals with naturally occurring mutations or targeted deletions of certain BMP family genes. Collectively, these studies have established the concept that the BMP system plays a crucial role in fertility in female and male mammals. The purpose of this article is to review the evidence underpinning the importance of the BMP system in mammalian reproduction. [Abstract/Link to Full Text]

Smith CL, O'Malley BW
Coregulator function: a key to understanding tissue specificity of selective receptor modulators.
Endocr Rev. 2004 Feb;25(1):45-71.
Ligands for the nuclear receptor superfamily control many aspects of biology, including development, reproduction, and homeostasis, through regulation of the transcriptional activity of their cognate receptors. Selective receptor modulators (SRMs) are receptor ligands that exhibit agonistic or antagonistic biocharacter in a cell- and tissue context-dependent manner. The prototypical SRM is tamoxifen, which as a selective estrogen receptor modulator, can activate or inhibit estrogen receptor action. SRM-induced alterations in the conformation of the ligand-binding domains of nuclear receptors influence their abilities to interact with other proteins, such as coactivators and corepressors. It has been postulated, therefore, that the relative balance of coactivator and corepressor expression within a given target cell determines the relative agonist vs. antagonist activity of SRMs. However, recent evidence reveals that the cellular environment also plays a critical role in determining SRM biocharacter. Cellular signaling influences the activity and subcellular localization of coactivators and corepressors as well as nuclear receptors, and this contributes to gene-, cell-, and tissue-specific responses to SRM ligands. Increased understanding of the effect of cellular environment on nuclear receptors and their coregulators has the potential to open the field of SRM discovery and research to many members of the nuclear receptor superfamily. [Abstract/Link to Full Text]

Barzon L, Boscaro M, Palù G
Endocrine aspects of cancer gene therapy.
Endocr Rev. 2004 Feb;25(1):1-44.
The field of cancer gene therapy is in continuous expansion, and technology is quickly moving ahead as far as gene targeting and regulation of gene expression are concerned. This review focuses on the endocrine aspects of gene therapy, including the possibility to exploit hormone and hormone receptor functions for regulating therapeutic gene expression, the use of endocrine-specific genes as new therapeutic tools, the effects of viral vector delivery and transgene expression on the endocrine system, and the endocrine response to viral vector delivery. Present ethical concerns of gene therapy and the risk of germ cell transduction are also discussed, along with potential lines of innovation to improve cell and gene targeting. [Abstract/Link to Full Text]

Medh RD
Genetically modified animals in endocrinology. Knockout mouse models for bone studies.
Endocr Rev. 2003 Dec;24(6):836-9. [Abstract/Link to Full Text]

Prabhakar BS, Bahn RS, Smith TJ
Current perspective on the pathogenesis of Graves' disease and ophthalmopathy.
Endocr Rev. 2003 Dec;24(6):802-35.
Graves' disease (GD) is a very common autoimmune disorder of the thyroid in which stimulatory antibodies bind to the thyrotropin receptor and activate glandular function, resulting in hyperthyroidism. In addition, some patients with GD develop localized manifestations including ophthalmopathy (GO) and dermopathy. Since the cloning of the receptor cDNA, significant progress has been made in understanding the structure-function relationship of the receptor, which has been discussed in a number of earlier reviews. In this paper, we have focused our discussion on studies related to the molecular mechanisms of the disease pathogenesis and the development of animal models for GD. It has become apparent that multiple factors contribute to the etiology of GD, including host genetic as well as environmental factors. Studies in experimental animals indicate that GD is a slowly progressing disease that involves activation and recruitment of thyrotropin receptor-specific T and B cells. This activation eventually results in the production of stimulatory antibodies that can cause hyperthyroidism. Similarly, significant new insights have been gained in our understanding of GO that occurs in a subset of patients with GD. As in GD, both environmental and genetic factors play important roles in the development of GO. Although a number of putative ocular autoantigens have been identified, their role in the pathogenesis of GO awaits confirmation. Extensive analyses of orbital tissues obtained from patients with GO have provided a clearer understanding of the roles of T and B cells, cytokines and chemokines, and various ocular tissues including ocular muscles and fibroblasts. Equally impressive is the progress made in understanding why connective tissues of the orbit and the skin in GO are singled out for activation and undergo extensive remodeling. Results to date indicate that fibroblasts can act as sentinel cells and initiate lymphocyte recruitment and tissue remodeling. Moreover, these fibroblasts can be readily activated by Ig in the sera of patients with GD, suggesting a central role for them in the pathogenesis. Collectively, recent studies have led to a better understanding of the pathogenesis of GD and GO and have opened up potential new avenues for developing novel treatments for GD and GO. [Abstract/Link to Full Text]

van der Eerden BC, Karperien M, Wit JM
Systemic and local regulation of the growth plate.
Endocr Rev. 2003 Dec;24(6):782-801.
The growth plate is the final target organ for longitudinal growth and results from chondrocyte proliferation and differentiation. During the first year of life, longitudinal growth rates are high, followed by a decade of modest longitudinal growth. The age at onset of puberty and the growth rate during the pubertal growth spurt (which occurs under the influence of estrogens and GH) contribute to sex difference in final height between boys and girls. At the end of puberty, growth plates fuse, thereby ceasing longitudinal growth. It has been recognized that receptors for many hormones such as estrogen, GH, and glucocorticoids are present in or on growth plate chondrocytes, suggesting that these hormones may influence processes in the growth plate directly. Moreover, many growth factors, i.e., IGF-I, Indian hedgehog, PTHrP, fibroblast growth factors, bone morphogenetic proteins, and vascular endothelial growth factor, are now considered as crucial regulators of chondrocyte proliferation and differentiation. In this review, we present an update on the present perception of growth plate function and the regulation of chondrocyte proliferation and differentiation by systemic and local regulators of which most are now related to human growth disorders. [Abstract/Link to Full Text]

Cabrera-Vera TM, Vanhauwe J, Thomas TO, Medkova M, Preininger A, Mazzoni MR, Hamm HE
Insights into G protein structure, function, and regulation.
Endocr Rev. 2003 Dec;24(6):765-81.
In multicellular organisms from Caenorhabditis elegans to Homo sapiens, the maintenance of homeostasis is dependent on the continual flow and processing of information through a complex network of cells. Moreover, in order for the organism to respond to an ever-changing environment, intercellular signals must be transduced, amplified, and ultimately converted to the appropriate physiological response. The resolution of the molecular events underlying signal response and integration forms the basis of the signal transduction field of research. An evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide-binding proteins (G proteins) are key determinants of the specificity and temporal characteristics of many signaling processes and are the topic of this review. Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to heptahelical membrane receptors coupled to heterotrimeric G proteins. These highly specialized transducers can modulate the activity of multiple signaling pathways leading to diverse biological responses. In vivo, specific combinations of G alpha- and G beta gamma-subunits are likely required for connecting individual receptors to signaling pathways. The structural determinants of receptor-G protein-effector specificity are not completely understood and, in addition to involving interaction domains of these primary acting proteins, also require the participation of scaffolding and regulatory proteins. [Abstract/Link to Full Text]

Edmondson SR, Thumiger SP, Werther GA, Wraight CJ
Epidermal homeostasis: the role of the growth hormone and insulin-like growth factor systems.
Endocr Rev. 2003 Dec;24(6):737-64.
GH and IGF-I and -II were first identified by their endocrine activity. Specifically, IGF-I was found to mediate the linear growth-promoting actions of GH. It is now evident that these two growth factor systems also exert widespread activity throughout the body and that their actions are not always interconnected. The literature highlights the importance of the GH and IGF systems in normal skin homeostasis, including dermal/epidermal cross-talk. GH activity, sometimes mediated via IGF-I, is primarily evident in the dermis, particularly affecting collagen synthesis. In contrast, IGF action is an important feature of the dermal and epidermal compartments, predominantly enhancing cell proliferation, survival, and migration. The locally expressed IGF binding proteins play significant and complex roles, primarily via modulation of IGF actions. Disturbances in GH and IGF signaling pathways are implicated in the pathophysiology of several skin perturbations, particularly those exhibiting epidermal hyperplasia (e.g., psoriasis, carcinomas). Additionally, many studies emphasize the potential use of both growth factors in the treatment of skin wounds; for example, burn patients. This overview concerns the role and mechanisms of action of the GH and IGF systems in skin and maintenance of epidermal integrity in both health and disease. [Abstract/Link to Full Text]

Kahl CR, Means AR
Regulation of cell cycle progression by calcium/calmodulin-dependent pathways.
Endocr Rev. 2003 Dec;24(6):719-36.
Many hormones, growth factors, and cytokines regulate proliferation of their target cells. Perhaps the most universal signaling cascades required for proliferative responses are those initiated by transient rises in intracellular calcium (Ca(2+)). The major intracellular receptor for Ca(2+) is calmodulin (CaM). CaM is a small protein that contains four EF-hand Ca(2+) binding sites and is highly conserved among eukaryotes. In all organisms in which the CaM gene has been deleted, it is essential. Although Ca(2+)/CaM is required for proliferation in both unicellular and multicellular eukaryotes, the essential targets of Ca(2+)/CaM-dependent pathways required for cell proliferation remain elusive. Potential Ca(2+)/CaM-dependent targets include the serine/threonine phosphatase calcineurin and the family of multifunctional Ca(2+)/CaM-dependent protein kinases. Whereas these enzymes are essential in Aspergillus nidulans, they are not required under normal growth conditions in yeast. However, in mammalian cells, studies demonstrate that both types of enzymes contribute to the regulation of cell cycle progression. Unfortunately, the mechanism by which Ca(2+)/CaM and its downstream targets, particularly calcineurin and the Ca(2+)/CaM-dependent protein kinases, regulate key cell cycle-regulatory proteins, remains enigmatic. By understanding how Ca(2+)/CaM regulates cell cycle progression in normal mammalian cells, we may gain insight into how hormones control cell division and how cancer cells subvert the need for Ca(2+) and its downstream targets to proliferate. [Abstract/Link to Full Text]


The Endocrine Society and Pfizer, Inc. International Award for Excellence in Published Clinical Research in the Journal of Clinical Endocrinology & Metabolism.
Endocr Rev. 2003 Oct;24(5):718. [Abstract/Link to Full Text]

Tomer Y, Davies TF
Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function.
Endocr Rev. 2003 Oct;24(5):694-717.
The autoimmune thyroid diseases (AITD) are complex diseases that are caused by an interaction between susceptibility genes and environmental triggers. Genetic susceptibility, in combination with external factors (e.g., dietary iodine), is believed to initiate the autoimmune response to thyroid antigens. Abundant epidemiological data, including family and twin studies, point to a strong genetic influence on the development of AITD. Various techniques have been used to identify the genes contributing to the etiology of AITD, including candidate gene analysis and whole genome screening. These studies have enabled the identification of several loci (genetic regions) that are linked with AITD, and in some of these loci putative AITD susceptibility genes have been identified. Some of these genes/loci are unique to Graves' disease (GD) and Hashimoto's thyroiditis (HT), and some are common to both diseases, indicating that there is a shared genetic susceptibility to GD and HT. The putative GD and HT susceptibility genes include both immune modifying genes (e.g., human leukocyte antigen, cytotoxic T lymphocyte antigen-4) and thyroid-specific genes (e.g., TSH receptor, thyroglobulin). Most likely these loci interact, and their interactions may influence disease phenotype and severity. It is hoped that in the near future additional AITD susceptibility genes will be identified and the mechanisms by which they induce AITD will be unraveled. [Abstract/Link to Full Text]

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Recent Articles in Endocrinology

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Wang JM, Liu L, Brinton RD
Estradiol-17{beta}-induced Human Neural Progenitor Cell Proliferation Is Mediated by an Estrogen Receptor {beta}-pERK Pathway.
Endocrinology. 2007 Oct 25; .
Estradiol-17beta (E2) induces rodent hippocamal neural progenitor cell (rNPC) proliferation in vitro (1, 2), in vivo (3, 4) and following brain injury (5). The purpose of the present investigation was to determine whether E2-induced proliferation observed in rodent model systems generalized to cells of human neural origin and the signaling pathway by which E2 promotes mitosis of human neural progenitor cells (hNPC). Results of these analyses indicate that E2 induced a significant increase in hNPC proliferation in a time- and dose-dependent manner. E2-induced hNPC DNA replication was paralleled by elevated cell cycle protein expression and centrosome amplification, which was associated with augmentation of total cell number. To determine whether estrogen receptor (ER) and which ER subtype were required for E2-induced hNPC proliferation, ER expression was first determined by real time RT-PCR followed by Western blot analysis and subsequently verified pharmacologically using ERalpha or beta-selective ligands. Results of these analyses indicated that ERbeta expression was predominant relative to ERalpha, which was barely detectable in hNPC. Activation of ERbeta by the ERbeta-selective ligand, diarylpropionitrile, led to an increase in phosphorylated extracellular signal-regulated kinase, and subsequent centrosome amplification and hNPC proliferation, which were blocked by the MEKK antagonist, UO126, but not its inactive analogue, UO124. These findings, for the first time, demonstrate the molecular cascade and related cell biology events involved in E2-induced hNPC proliferation in vitro. Therapeutic implications of these findings relevant to hormone therapy and prevention of neurodegenerative disease are discussed. [Abstract/Link to Full Text]

Styer AK, Sullivan BT, Puder M, Arsenault D, Petrozza JC, Serikawa T, Chang S, Hasan T, Gonzalez RR, Rueda BR
Ablation of leptin signaling disrupts the establishment, development and maintenance of endometriosis-like lesions in a murine model.
Endocrinology. 2007 Oct 25;
Leptin, a 16kDa cytokine, has been implicated in several reproductive processes and disorders. Notably, elevated leptin levels in the peritoneal fluid of women with mild endometriosis has been demonstrated, suggesting a role for this cytokine in the early stages of disease establishment. To gain insight into the functional significance of leptin during the initial requisite proliferative and neovascularization events involved in endometriosis, we investigated the effect of disruption of in vivo leptin signaling on the establishment and/or maintenance of an endometriosis-like lesion in a syngeneic immunocompetent mouse model of endometriosis. Findings of this study show that the disruption of leptin signaling by intraperitoneal injection of the pegylated leptin peptide receptor antagonist (LPrA) impairs the establishment of endometriosis-like lesions (derived from uteri of C57BL/6 female siblings) and results in a reduction of viable organized glandular epithelium, VEGF-A expression, and mitotic activity. LPrA treatment resulted in a significant reduction of microvascular density in endometriosis-like lesions following continuous and acute courses. Endometriosis-like lesions (derived from tissue with functional leptin receptor) of Lep(db) hosts (nonfunctional leptin receptor) were phenotypically similar to those of LPrA treated mice. Our results confirm that leptin signaling is a necessary component in lesion proliferation, early vascular recruitment, and maintenance of neoangiogenesis in a murine model of endometriosis. [Abstract/Link to Full Text]

Yan J, Brown TR
Cell Proliferation and Expression of Cell Cycle Regulatory Proteins that Control the G1/S Transition are Age-Dependent and Lobe-Specific in the Brown Norway Rat Model of Prostatic Hyperplasia.
Endocrinology. 2007 Oct 25;
Age-dependent epithelial cell hyperplasia in the dorsal and lateral lobes of Brown Norway rats is analogous to benign prostatic hyperplasia in aging men. A major question is whether differential lobe-specific and age-dependent proliferation of cells, rather than cell survival, contributes to the hyperplasia. Although serum testosterone levels decline in aged rats, active cell proliferation was detected as Ki67-positive cells in the dorsal and lateral lobes. We determined whether androgens differentially affect cell proliferation and cell-cycle regulatory proteins in the prostate lobes of young and aged rats. Castrated rats were treated with different doses of testosterone to restore serum levels to those of intact young or aged rats. Rates of cell proliferation, measured by BrdU-labeling, peaked after 3 days of testosterone treatment in all lobes. BrdU-labeling indices were higher in the dorsal and lateral lobes of aged than of young rats with equivalent serum testosterone levels. No age-dependent difference was seen in the ventral lobe. Cell proliferation was marked by increased levels of cyclins D1 and E and cyclin-dependent kinases 4 and 6, decreased p27 and increased phosphorylation of Rb. Levels of cyclins D1 and E were higher in the dorsal and lateral lobes of intact and testosterone-treated aged than young rats. Confocal immunofluorescent microscopy documented changes in cdk4 and cyclin D1 subcellular localization. Cyclin D1 nuclear localization correlated with the timeframe for cell proliferation. In conclusion, rates of cell proliferation and levels of cell-cycle regulatory proteins that control the G1/S transition exhibit lobe-specific and age-dependent differences in response to androgens. [Abstract/Link to Full Text]

Santos AN, Ramin N, Tonack S, Fischer B
Cell lineage-specific signalling of insulin and insulin like growth factor (IGF) 1 in rabbit blastocysts.
Endocrinology. 2007 Oct 25;
The insulin/insulin growth factor (IGF) system plays a critical role in embryo growth and development. We have investigated the expression of insulin and IGF1 receptor (IR, IGF1R) and the activation of their downstream pathways in rabbit 6 day old blastocysts. IR was expressed in embryoblast (Em, ICM) and trophoblast (Tr) cells, whereas IGF1R was localized mainly in Em. Isoform A (IR-A) represents the main insulin isoform in blastocysts and was found in Em and Tr cells. IR-B was detectable only in Tr. IR/IGF1R signalling pathways were analysed after stimulation with insulin (17 nM) or IGF1 (1.3 nM) in cultured blastocysts. Insulin stimulated Erk1/2 in Em and Tr, and Akt in Tr but not in Em. IGF1 activated both kinases exclusively in Em. The target genes c-fos (for MKK1/Erk signalling) and phosphoenolpyruvate carboxykinase (PEPCK; for PI3K/Akt signalling) were also specifically regulated. Insulin downregulated PEPCK RNA amounts in Tr by activation of the PI3K/Akt pathway. Expression of c-fos by insulin and IGF1 was different in respect to time and fortitude of expression, mirroring again the specific IR and IGF1R expression patterns in Em and Tr. Taken together we show that IGF1 acts primarily mitogenic, an effect which is cell lineage-specifically restricted to the Em. By contrast, insulin is the growth factor of the Tr stimulating mitogenesis and downregulating metabolic responses. As soon as blastocyst differentiation in embryoblast and trophoblast has been accomplished, insulin and IGF1 signalling is different in both cell lineages, implying a different developmental impact of both growth factors. [Abstract/Link to Full Text]

Freitas HS, Anhê GF, Melo KF, Okamoto MM, Oliveira-Souza M, Bordin S, Machado UF
SGLT2 mRNA expression in kidney of diabetic rats correlates with glycemic levels: involvement of HNF-1{alpha} expression and activity.
Endocrinology. 2007 Oct 25;
Mutations in SGLT2 and HNF-1alpha genes have been related to renal glycosuria and Maturity-Onset Diabetes of the Young 3 (MODY3), respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here, in kidney of diabetic rats, we tested the hypotheses that: SGLT2 mRNA expression is altered; HNF-1alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1alpha into the SGLT2 promoter region in renal cortex was confirmed by Chromatin Immunoprecipitation assay. SGLT2 and HNF-1alpha mRNA expression (by Northern and RT-PCR analysis), and HNF-1 binding activity of nuclear proteins (by Electrophoretic Mobility Shift Assay - EMSA) were investigated in diabetic rats, treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1alpha mRNA expression ( approximately 50%), and binding of nuclear proteins to a HNF-1 consensus motif ( approximately 100%). Six days of insulin or phlorizin treatment restores these parameters to non-diabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF1-alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease. [Abstract/Link to Full Text]

Gieske MC, Kim HJ, Legan SJ, Koo Y, Krust A, Chambon P, Ko C
Pituitary gonadotroph estrogen receptor alpha (ER{alpha}) is necessary for fertility in females.
Endocrinology. 2007 Oct 18;
Estrogens play a central role in regulating female reproduction throughout the reproductive axis, and the pituitary is one of the major targets of estrogen action. We hypothesized that estrogen receptor alpha (ERalpha) mediates estrogen action in the pituitary gonadotroph. To test this hypothesis, we generated a mouse line with a selective ERalpha deletion in the gonadotropin alpha subunit (alphaGSU)-expressing pituitary cells (pituitary-specific ERalpha knockout; ERalpha(flox/flox) alphaGSU(cre)). While the ERalpha(flox/flox) alphaGSU(cre) female mice maintain a basal level of serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) and their ovulatory capacity is comparable to that in controls, they do not display regular estrous cycles and are infertile, indicating a potential disorder in regulating LH and/or FSH secretion. The ERalpha(flox/flox) alphaGSU(cre) female mice express equivalent levels of LHbeta and alphaGSU mRNA compared to wild type mice as determined by microarray analysis. Taken together, these findings indicate that pituitary gonadotroph ERalpha carries out the effects of estrogens with regard to estrous cyclicity and ultimately fertility. [Abstract/Link to Full Text]

Solini A, Cuccato S, Ferrari D, Santini E, Gulinelli S, Callegari MG, Dardano A, Faviana P, Madec S, Di Virgilio F, Monzani F
Increased P2X7 receptor expression and function in thyroid papillary cancer: a new potential marker of the disease?
Endocrinology. 2007 Oct 18;
Nucleotides are increasingly recognized as non-redundant extracellular signals for chemotaxis, cell growth and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X7 subtype is attracting increasing attention for its involvement in apoptosis, cell growth and cytokine release. Recent studies showed that P2X7 is overexpressed in chronic lymphocytic leukemia, breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X7 receptor expression in normal and cancer human thyroid tissues. P2X7 receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic and papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, express the P2X7 receptor (P2X7R) to a much higher level than normal thyroid tissue. The P2X7R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X7R inhibitors. Finally, the thyroid carcinoma cell lines had an at least three fold higher intracellular ATP concentration, and maintained an at least three fold higher extracellular ATP level compared to control cells. These data suggest that an enhanced P2X7R function might be a feature of human thyroid cancer. [Abstract/Link to Full Text]

Lalmansingh AS, Uht RM
Estradiol (E2) Regulates Corticotropin Releasing Hormone Gene (crh) Expression in a Rapid and Phasic Manner that Parallels Estrogen Receptor -alpha (ER{alpha}) and -beta ({beta}) Recruitment to a cAMP regulatory element (CRE) in the proximal crh promoter.
Endocrinology. 2007 Oct 18;
In the central nervous system, corticotropin-releasing hormone (CRH) regulates several affective states. Dysregulation of neuronal crh expression in the paraventricular nucleus of the hypothalamus (PVH) correlates with some forms of depression, and amygdalar crh expression may modulate levels of anxiety. Because estrogens modulate these states, we sought to determine E2effects on crh expression. CRH mRNA levels were measured in the AR-5 amygdaloid cell line by RT-PCR analysis. They increased by one minute of E2 treatment, suggesting that crh behaves as an immediate-early gene (IEG). After peaking at 3 minutes, CRH mRNA returned to basal levels, then increased by 60 minutes. To dissect some of the molecular mechanisms underlying these events, we measured occupancy of the crh promoter by ERs and co-activators, using chromatin immunoprecipitation. Because this promoter does not contain palindromic estrogen response elements (EREs), we targeted the region of a cAMP regulatory element (CRE), implicated in crh regulation. The temporal pattern of the mRNA response was mimicked by recruitment of ERalpha, beta, phospho-CREB, co-activators SRC-1 and CBP, and an increase in histone 3 and 4 acetylation. Lastly, ERalpha and beta loading were temporally dissociated, peaking at 1 and 3 minutes, respectively. The ER peaks were associated with coactivators and acetylation patterns. ERalpha associated with pCREB, CBP, SRC-1, and increased acetylated histone 3 (Ac-H3). ERbeta associated with CBP, and increased Ac-H4. The tight temporal correlation between E2-induced CRH mRNA levels and promoter occupancy by ERs strongly suggest that E2 regulates crh expression through an ERalpha- and/or ERbeta- CRE alternate pathway. [Abstract/Link to Full Text]

Renlund N, Pieretti-Vanmarcke R, O'Neill FH, Zhang L, Donahoe PK, Teixeira J
JNK inhibitor II (SP600125) activates Mullerian Inhibiting Substance type II receptor-mediated signal transduction.
Endocrinology. 2007 Oct 18;
Müllerian Inhibiting Substance (MIS), the hormone required for Müllerian duct regression in fetal males, is also expressed in both adult males and females but its physiological role in these settings is not clear. The expression of the MIS type II receptor (MISRII) in ovarian cancer cells and the ability of MIS to inhibit proliferation of these cells suggests that MIS might be a promising therapeutic for recurrent ovarian cancer. Using an MISRII-dependent activity assay in a small molecule screen for MIS mimetic compounds, we have identified the c-Jun N-terminal Kinase (JNK) inhibitor, SP600125, as an activator of the MIS signal transduction pathway. SP600125 increased the activity of a BMP-responsive reporter gene in a dose-dependent manner and exerted a synergistic effect when used in combination with MIS. This effect was specific for the MISRII and was not seen with other receptors of the transforming growth factor-b (TGFbeta) family. Moreover, treatment of mouse ovarian cancer cells with a combination of SP600125 and paclitaxel, an established chemotherapeutic agent used in the treatment of ovarian cancer, or with MIS enabled inhibition of cell proliferation at a lower dose than with each treatment alone. These results offer a strong rationale for testing the therapeutic potential of SP600125, alone or in combination with already established drugs, in the treatment of recurrent ovarian cancer with a much-needed decrease in the toxic side effects of currently employed therapeutic agents. [Abstract/Link to Full Text]

Devost D, Carrier ME, Zingg HH
Oxytocin-induced activation of eukaryotic elongation factor eEF2 in myometrial cells is mediated by protein kinase C.
Endocrinology. 2007 Oct 18;
The nonapeptide oxytocin mediates a wide spectrum of biological action many of them related to reproduction. Recently, we have shown that oxytocin exerts a trophic effect on uterine smooth muscle cells and induces dephosphorylation, and thus activation, of the translation elongation factor eEF2 (eukaryotic elongation factor 2). The present study was designed to elucidate the mechanisms underlying this novel action of oxytocin in the well characterized human myometrial cell line hTERT-C3. Pathways known to induce eEF2 dephosphorylation are mTOR and the MAP kinases ERK1/2 and p38. Using a panel of chemical inhibitors of specific signaling pathways, we determined that none of these pathways played a role in oxytocin-mediated eEF2 dephosphorylation. Because the oxytocin receptor is a G protein-coupled receptor linked to Galphaq, we tested the possibility that this oxytocin action was mediated via protein kinase C (PKC). PKC activity was blocked by application of the general PKC chemical inhibitor Go6983 or by incubation with the cell-permeable PKC inhibitor peptide myr-psi PKC. With either approach, the effect of oxytocin on eEF2 dephosphorylation was suppressed, indicating that the PKC pathway is essential for this oxytocin action. Consistent with this idea, we also found that direct stimulation of PKC with the phorbol ester PMA induced eEF2 dephosphorylation. Moreover, we observed that the stimulatory effect of oxytocin on [(35)S]methionine incorporation into nascent proteins was blocked by PKC inhibition. Overall, these results define a novel hormonal signaling pathway that leads to eEF2 dephosphorylation and activation of protein synthesis. [Abstract/Link to Full Text]

Patisaul HB, Fortino AE, Polston EK
Sex differences in serotonergic (5-HT) but not GABAergic projections to the rat ventromedial nucleus of the hypothalamus (VMN).
Endocrinology. 2007 Oct 18;
Hormonal conditions that elicit lordosis in female rats are ineffective in males, suggesting that this behavior is actively suppressed in males. Previous studies theorize that serotonergic and GABAergic inputs to the ventrolateral division of the ventromedial nucleus of the hypothalamus (VMNvl) may contribute to lordosis inhibition in males. Using triple-label immunofluorescent techniques, the present studies explored potential sex differences in the density of these projections within three hypothalamic sites: the VMNvl, the arcuate nucleus (ARC), and the dorsomedial nucleus of the hypothalamus (DMN). Antibodies directed against HuC/D, estrogen receptor alpha (ERalpha) and either serotonin (5-HT) or the GABA synthetic enzyme GAD65 were used to compare the densities of GAD65- and 5-HT- containing fibers in each brain area, the percentage of VMNvl HuC/D immunoreactive (ir) neurons that contained ERalpha, and the percentage of HuC/D and ERalpha double labeled cells receiving apparent contacts from 5-HT fibers between adult, gonadectomized male and female rats. The densities of VMNvl and ARC 5-HT immunolabeled fibers were significantly higher in the males, and the percentage of VMNvl HuC/D-ir neurons containing ERalpha was significantly higher in the females. The percentage of HuC/D-ir cells contacted by 5-HT fibers was significantly higher in the males compared to the females, but there was no sex difference in the proportion of those cells receiving contacts that were ERalpha-ir. Neonatal administration of estradiol (E2) but not genistein (GEN) masculinized 5-HT content in the adult female VMNvl, but the percentage of HuC/D-ir cells co-labeled with ERalpha was not significantly affected by treatment. A similar, but not statistically significant, pattern was observed in the ARC. These findings suggest that the development of serotonergic inputs to the male VMNvl is orchestrated by neonatal E2 exposure. The hormone-dependent organization of these 5-HT projection patterns may be an important developmental mechanism accounting for sex-specific behaviors in adulthood. [Abstract/Link to Full Text]

Reyes BA, Valentino RJ, Van Bockstaele EJ
Stress-induced intracellular trafficking of corticotropin-releasing factor receptors in rat locus coeruleus neurons.
Endocrinology. 2007 Oct 18;
Corticotropin-releasing factor (CRF) activates locus coeruleus (LC)-norepinephrine neurons during stress. Previous stress or CRF administration attenuates the magnitude of this response by decreasing postsynaptic sensitivity to CRF. Here we describe the fate of CRF receptors in LC neurons following stress. Rats were exposed to swim stress or handling and perfused one or twenty-four hours later. Sections through the LC were processed for immunogold-silver labeling of CRF receptor (CRFr). CRFr in LC dendrites was present on the plasma membrane and within the cytoplasm. In control rats, the ratio of cytoplasmic to total dendritic labeling was 0.55 +/- 0.01. Swim stress increased this ratio to 0.77 +/- 0.01 and 0.80 +/- 0.02 at one and twenty-four hours post-stress, respectively. Internalized CRFr was associated with different organelles at different times after stress. At one hour post-stress, CRFr was often associated with early endosomes in dendrites and perikarya. By twenty-four hours, more CRFr was associated with multivesicular bodies, suggesting that some of the internalized receptor is targeted for degradation. In perikarya, more internalized CRFr was associated with Golgi apparati twenty-four hours vs. one hour post-stress. This is suggestive of changes in CRFr synthesis. Alternatively this may indicate communication between multivesicular bodies and Golgi apparati in the process of recycling. Administration of the selective CRF1 antagonist, antalarmin, prior to swim stress attenuated CRFr internalization. The present demonstration of stress-induced internalization of CRFr in LC neurons provides evidence that CRF is released in the LC during swim stress to activate this system and initiate cellular trafficking of the receptor that determines subsequent sensitivity of LC neurons to CRF. [Abstract/Link to Full Text]

Douard V, Cui XL, Soteropoulos P, Ferraris RP
Dexamethasone sensitizes the neonatal intestine to fructose-induction of GLUT5 transport function.
Endocrinology. 2007 Oct 18;
The recent dramatic increase in fructose consumption is tightly correlated with an equally dramatic surge in incidence of type II diabetes and obesity in children, but little is known about dietary fructose metabolism and absorption in neonates. The expression of the rat intestinal fructose transporter GLUT5 can be specifically induced by its substrate fructose but only after weaning begins at 14 d of age. In suckling rats < 14 d old, dietary fructose cannot enhance GLUT5 expression. The aim of this study was identify the mechanisms allowing fructose to stimulate GLUT5 during weaning. After intestines were perfused with fructose or glucose (control), we showed using microarray hybridization that of 5K genes analyzed in 10 d old pups, only 13 were fructose-responsive. Previous work found approximately 50 fructose-responsive genes in 20 d old pups. To identify fructose-responsive genes whose expression also changed with age, intestines of 10 and 20 d old littermate pups perfused with fructose were compared by microarray. Intestines of 10 and 20 d olds perfused with glucose were used to segregate age- but not fructose-responsive genes. About 28 genes were up- and 22 downregulated in 20 relative to 10 d old pups, under conditions of fructose perfusion, and many were found, by cluster analysis, to be regulated by corticosterone. When dexamethasone (dex) was injected into suckling pups prior to fructose perfusion, GLUT5 but not SGLT1 nor GLUT2 expression, and fructose but not glucose uptake increased dramatically. Thus, dex which allows dietary fructose to precociously stimulate intestinal fructose absorption can mimic the effect of age and modify developmental timing mechanisms regulating GLUT5. [Abstract/Link to Full Text]

Wersinger SR, Temple JL, Caldwell HK, Young WS
Inactivation of the oxytocin and the vasopressin 1b receptor genes, but not the vasopressin 1a receptor gene, differentially impair the Bruce effect in laboratory mice (Mus musculus).
Endocrinology. 2007 Oct 18;
The Bruce effect is a pheromonally mediated process whereby exposure to chemosensory cues from an unfamiliar male terminates pregnancy in a recently mated female. Pharmacological and genetic evidence implicates both oxytocin (Oxt) and vasopressin (Avp) in the regulation of social memory in males, but less work has been done in females. We tested the extent to which the Avp 1a (Avpr1a) and 1b (Avpr1b) receptors and Oxt are essential for the Bruce effect, a phenomenon that relies on olfactory memory. Adult female mice were paired with stimulus males and monitored for the presence of sperm plugs. Wildtype, heterozygous and homozygous knockout (KO) females for either the Avpr1a, Avpr1b, or Oxt genes were randomly assigned to one of the following treatment groups: 1. Alone (mate removed, no second exposure to another animal); 2. Paired continuously (mate kept with female for 10-14 days); 3. Familiar Male (mate removed, reintroduced 24 h later); 4. Unfamiliar Male (mate removed, BalbC male introduced 24 h later). Regardless of genotype, 90-100% of females in the alone or paired continuously groups became pregnant. The Oxt KO females terminated their pregnancies regardless of whether their original mate or an unfamiliar male was reintroduced. The Avpr1b KO mice failed to terminate pregnancy in the presence of an unfamiliar male. The Avpr1a KO mice exhibited a normal Bruce effect. These data demonstrate that both Oxt and the Avpr1b are critical for the normal expression of the Bruce effect but have different effects on the interpretation of social cues. [Abstract/Link to Full Text]

Ricu M, Paredes A, Greiner M, Ojeda SR, Lara HE
Functional Development of the Ovarian Noradrenergic Innervation.
Endocrinology. 2007 Oct 18;
A substantial fraction of the noradrenergic innervation targeting the mammalian ovary is provided by neurons of the celiac ganglion. Although studies in the rat have shown that noradrenergic nerves reach the ovary near the time of birth, it is unknown how the functional capacity of this innervation unfolds during postnatal ovarian development. To address this issue we assessed the ability of the developing ovary to incorporate and release (3)H-norepinephrine ((3)H-NE). Incorporation of (3)H-NE was low during the first three weeks of postnatal life, but pharmacological inhibition of NE neuronal uptake with cocaine showed that an intact transport mechanism for NE into nerve terminals is already in place by the first week after birth. Consistent with this functional assessment, the mRNA encoding the NE transporter (NET) was also expressed in the celiac ganglion at this time. During neonatal-infantile development (PN day 5 to 20), the spontaneous, vesicle-independent outflow of recently taken-up NE was high, but the NE output in response to K(+)-induced depolarization was low. After PN20, spontaneous outflow decreased and the response to K(+) increased markedly, reaching maximal values by the time of puberty. Tyramine-mediated displacement of NE stored in vesicles, which displace vesicular NE, showed that vesicle-dependent NE storage becomes functional by PN day 12, and that vesicular release increases during the juvenile-peripubertal phases of sexual development. These results indicate that vesicular release of NE from ovarian noradrenergic nerves begins to operate by the third week of postnatal life, becoming fully functional near the time of puberty. [Abstract/Link to Full Text]

Danaher RN, Loomes KM, Leonard BL, Whiting L, Hay DL, Xu LY, Kraegen EW, Phillips AR, Cooper GJ
Evidence that alpha calcitonin gene-related peptide is a neurohormone that controls systemic lipid availability and utilization.
Endocrinology. 2007 Oct 11;
Alpha calcitonin gene-related peptide (alphaCGRP) is released mainly from sensory and motor nerves in response to physiological stimuli. Despite well-documented pharmacological effects its primary physiological role has thus far remained obscure. Increased lipid content, particularly in skeletal muscle and liver, is strongly implicated in the pathogenesis of insulin resistance, but the physiological regulation of organ lipid is imperfectly understood. Here, we report our systematic investigations of the effects of alphaCGRP on in vitro and in vivo indices of lipid metabolism. In rodents, levels of alphaCGRP similar to those in the blood markedly stimulated fatty acid beta-oxidation and evoked concomitant mobilization of muscle lipid via receptor-mediated activation of muscle lipolysis. alphaCGRP exerted potent in vivo effects on lipid metabolism in muscle, liver and the blood, via receptor-mediated pathways. Studies with receptor antagonists were consistent with tonic regulation of lipid metabolism by an endogenous CGRP agonist. These data reveal that alphaCGRP is a newly recognized regulator of lipid availability and utilization in key tissues and that it may elevate the availability of intramyocellular FFA to meet muscle energy requirements generated by contraction by evoking their release from endogenous TG. [Abstract/Link to Full Text]

Makeeva N, Roomans GM, Myers JW, Welsh N
TAB1{alpha}, but not TAB1{beta}, mediates cytokine-induced p38 MAPK phosphorylation and cell death in insulin producing cells.
Endocrinology. 2007 Oct 11;
Previous studies have indicated that the p38 MAPK participates in signaling events that lead to the death of the insulin-producing beta-cell. The aim of the present study was to elucidate the role of the transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1) in the cytokine-induced activation of p38. Levels of TAB1 mRNA and protein were analyzed by real time PCR and immunoblotting, and TAB1 expression in mouse and human islet cells was down-regulated using lipofection of diced-siRNA. TAB1 over-expression in beta-TC6 cells was achieved by transient transfections followed by fluorescence activated cell sorting. Phosphorylation of p38, JNK and ERK was assessed by immunoblotting and viability was determined using vital staining with bisbenzimide and propidium iodide. We observed that TAB1 is expressed in insulin producing cells. Cytokine (IL-1beta + IFN-gamma)-stimulated p38 phosphorylation was significantly increased by TAB1alpha overexpression, but not by TAB1beta overexpression, in beta-TC6 cells. The TAB1alpha-augmented p38 phosphorylation was paralleled by an increased cell death rate. Treatment of islet cells with diced-siRNA specific for TAB1, but not for TAK1, resulted in lowered cytokine-induced p38 phosphorylation and protection against cell death. The cytokine-induced phosphorylation of JNK and ERK was not affected by changes in TAB1 levels. Finally, TAB1 phosphorylation was decreased by the p38 inhibitor SB203580. We conclude that TAB1alpha, but not TAB1beta, plays an important role in the activation of p38 in insulin-producing cells and therefore also in cytokine-induced beta-cell death. [Abstract/Link to Full Text]

Galli C, Zella LA, Fretz JA, Fu Q, Pike JW, Weinstein RS, Manolagas SC, O'brien CA
Targeted deletion of a distant transcriptional enhancer of the RANKL gene reduces bone remodeling and increases bone mass.
Endocrinology. 2007 Oct 11;
Receptor activator of NFkappaB ligand (RANKL) is essential for osteoclast differentiation, and hormones and cytokines that stimulate bone resorption increase RANKL expression in stromal/osteoblastic cells. We have previously shown that parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) control murine RANKL gene expression in vitro, in part, via an evolutionarily-conserved transcriptional enhancer, designated the distal control region (DCR), located 76 kb upstream from the transcription start-site. Herein we describe the phenotype of mice lacking this enhancer. Deletion of the DCR reduced PTH- and 1,25(OH)2D3-stimulation of RANKL mRNA and osteoclast formation in primary bone marrow cultures, as well as stimulation of RANKL mRNA in bone. DCR deletion also reduced basal RANKL mRNA levels in bone, thymus, and spleen. Moreover, mice lacking the DCR exhibited increased bone mass and strength. The increase in bone mass was due to reduced osteoclast and osteoblast formation leading to a low rate of bone remodeling similar to that observed in humans and mice with hypoparathyroidism. These findings demonstrate that hormonal control of RANKL expression via the DCR is a critical determinant of the rate of bone remodeling. [Abstract/Link to Full Text]

Trevaskis JL, Meyer EA, Galgani JE, Butler AA
Counter-intuitive effects of double heterozygous null Mc4r and Lep genes on diet-induced obesity and insulin resistance in C57BL/6J mice.
Endocrinology. 2007 Oct 11;
Circulating levels of leptin correlate with food intake and adiposity. A decline in serum leptin associated with calorie restriction instigates behavioral and metabolic adaptation, increasing appetite and conserving energy. Brain melanocortin-4 receptors (Mc4r) are important mediators of leptin's effects on appetite and energy expenditure. Since subtle changes in function associated with heterozygous null mutations for either the Leptin (Lep-HET) or Mc4r genes (Mc4r-HET) increase adiposity, we tested the hypothesis that combined heterozygous mutations (Dbl-HET) would severely exacerbate diet-induced obesity (DIO) and insulin resistance in C57BL/6J mice. Serum leptin levels were lower as a function of adiposity in heterozygous Leptin mutants (Lep-HET, Dbl-HET) matched with mice homozygous for the wild type Lep gene (WT, Mc4r-HET). Evidence for an additive interaction on adiposity in Dbl-HET mice maintained on low fat diet was observed at 10 weeks of age. Male but not female mice developed DIO and insulin resistance on high fat diet. Compared to WT, DIO was more severe in Mc4r-HET but not Lep-HET mice, irrespective of sex. However, the response of male and female Dbl-HET mice was different, with males being less and females more responsive relative to Mc4r-HET. Glucose tolerance of Dbl-HET mice was not significantly different from WT in either sex. These results show a complex interaction between the Leptin and Mc4r genes that is influenced by age, gender and diet. Remarkably, while heterozygous Lep mutations initially exacerbate obesity, in situations of severe obesity reduced leptin levels may act oppositely and have beneficial effects on energy homeostasis. [Abstract/Link to Full Text]

Haisenleder DJ, Burger LL, Walsh HE, Stevens J, Aylor KW, Shupnik MA, Marshall JC
PULSATILE GnRH STIMULATION OF GONADOTROPIN SUBUNIT TRANSCRIPTION IN RAT PITUITARIES: EVIDENCE FOR THE INVOLVEMENT OF JUN N-TERMINAL KINASE (JNK), BUT NOT p38.
Endocrinology. 2007 Oct 11;
We investigated whether JNK and p38, mediate gonadotropin subunit transcriptional responses to pulsatile GnRH in normal rat pituitaries. A single pulse of GnRH or vehicle was given to female rats in vivo, pituitaries collected, and phosphorylated JNK and p38 measured. GnRH stimulated an increase in JNK phosphorylation within 5 min, which peaked 15 min post-GnRH (3 fold). GnRH also increased p38 phosphorylation 2.3 fold, 15 min post-stimulus. Rat pituitary cells were given 60 min pulses of GnRH or media plus the JNK inhibitor SP600125 (SP, 20uM), p38 inhibitor SB203580 (SB, 20uM) or vehicle. In vehicle-treated groups, GnRH pulses increased LHbeta and FSHbeta primary transcript (PT) levels 3 fold. SP suppressed both basal and GnRH-induced increases in FSHbeta PT by half, but the magnitude of responses to GnRH was unchanged. In contrast, SP had no effect on basal LHbeta PT, but suppressed the stimulatory response to GnRH. SB had no effect on the actions of GnRH on either LH or FSHbeta PTs. Lbeta-T2 cells were transfected with dominant/negative (DN) expression vectors for MKK-4 and/or MKK-7 plus a rat LHbeta promoter-luciferase construct. GnRH stimulated a 50-fold increase in LHbeta promoter activity, and the combination of MKK-4 and 7 DNs suppressed the response by 80%. Thus, JNK (but not p38) regulates both LHbeta and FSHbeta transcription in a differential manner. For LHbeta, JNK is essential in mediating responses to pulsatile GnRH. JNK also regulates FSHbeta transcription (i.e. maintaining basal expression), but does not play a role in responses to GnRH. [Abstract/Link to Full Text]

Mazella J, Liang S, Tseng L
Expression of Delta-like protein 4 in the Human Endometrium.
Endocrinology. 2007 Oct 4;
Activation of Delta-Notch signaling pathway promotes the development of the vascular system in embryo, normal adult tissues and cancerous lesions. Delta and Notch genes are known to be expressed in endothelial cells and little is known of their expression beyond the vascular system. The purpose of this study was to investigate whether Delta gene would be expressed in cells of the uterine endometrium. In this study, we found that the human endometrial cells expressed one of the Delta ligands, Delta-like 4 protein (Dll4). Dll4 was expressed in human endometrium in a spatiotemporal fashion. Immunohistochemistry studies showed the cytoplasm as well as membrane staining with apical localization both in the luminal and glandular epithelium and moderate diffuse staining in the cytoplasm of the stromal cells. Western blot analysis showed that the size of the endometrial Dll4 was identical to that in the human umbilical endothelial cells. The expression Dll4 mRNA in human endometrial cells was quantitatively determined by Real-time PCR. Dll4 mRNA expressed in the glandular epithelium showed large variations and it was significantly elevated in the mid and late proliferative and early secretory endometrium. Endometrial stromal cells contained less Dll4 mRNA and had no clear correlation with the menstrual cycle. The effect of hormones was studied in the primary culture of isolated glandular epithelial and stromal cells. In glandular cells, estradiol (E2) had little effect and medroxyprogesterone acetate (MPA) significantly reduced the mRNAs compared to that of control. Relaxin induced the Dll4 mRNA. In stromal cells, both E2 and MPA reduced the Dll4 mRNA. To our knowledge, this is the first report of the expression of Dll4 in the endometrium. We propose that endometrial Dll4 may enhance the development of the endometrial microvascular system and facilitate the implantation of blastocyst in a fertile cycle. [Abstract/Link to Full Text]

Huang Z, Sjöholm A
Ethanol acutely stimulates islet blood flow, amplifies insulin secretion, and induces hypoglycemia via NO and vagally mediated mechanisms.
Endocrinology. 2007 Oct 4;
Hypoglycemia induced by alcohol ingestion is a well known problem in diabetic patients. However, the mechanisms underlying this phenomenon have largely remained elusive. Since insulin secretion in vivo can be rapidly tuned by changes in pancreatic microcirculation, we evaluated the influence of acute alcohol administration on pancreatic islet blood flow and dynamic changes in insulin secretion and glycemia in the rat. Ethanol (10%) or saline was intravenously injected as a bolus into Wistar rats, yielding serum ethanol concentrations of approximately 8 mmol/l. Measurements of pancreatic blood flow were performed by a microsphere technique in combination with a freeze-thawing technique after 10 minutes of injection. Ethanol preferentially and significantly increased pancreatic islet blood flow approx four-fold, while not influencing whole pancreatic blood flow. The alcohol also augmented late phase insulin secretion and induced late hypoglycemia upon intraperitoneal glucose tolerance tests. The nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl ester and atropine prevented the increased pancreatic islet blood flow, enhanced insulin secretion, and hypoglycemia evoked by ethanol. Our findings thus demonstrate that ethanol acutely exerts substantial influences on pancreatic microcirculation by evoking a massive redistribution of pancreatic blood flow from the exocrine into the endocrine part via mechanisms mediated by nitric oxide and vagal stimuli, augmenting late phase insulin secretion, and thereby evoking hypoglycemia. This effect may in part underlie the well known hypoglycemic properties of alcohol in diabetic patients or in alcoholics with hepatic failure. [Abstract/Link to Full Text]

Schwenke DO, Tokudome T, Shirai M, Hosoda H, Horio T, Kishimoto I, Kangawa K
Exogenous ghrelin attenuates the progression of chronic hypoxia-induced pulmonary hypertension in conscious rats.
Endocrinology. 2007 Oct 4;
Chronic exposure to hypoxia (CH), a common adverse consequence of most pulmonary disorders, can lead to a sustained increase in pulmonary arterial pressure (PAP), right ventricular hypertrophy and is, therefore, closely associated with heart failure and increased mortality. Ghrelin, originally identified as an endogenous growth hormone secretagogue, has recently been shown to possess potent vasodilator properties - likely involving modulation of the vascular endothelium and its associated vasoactive peptides. In this study we hypothesized that ghrelin would impede the pathogenesis of pulmonary arterial hypertension (PAH) during CH. PAP was continuously measured using radio-telemetry, in conscious male Sprague Dawley rats, in normoxia and during two weeks of CH (10% O2). During this hypoxic period, rats received a daily subcutaneous injection of either saline or ghrelin (150 microg/kg). Subsequently, heart and lung samples were collected for morphological, histological and molecular analyses. CH significantly elevated PAP in saline-treated rats, increased wall thickness of peripheral pulmonary arteries and, consequently, induced right ventricular hypertrophy. In these rats, CH also lead to the over-expression of endothelial nitric oxide synthase (eNOS) mRNA and protein as well as endothelin-1 (ET-1) mRNA within the lung. Exogenous ghrelin administration attenuated the CH-induced over-expression of eNOS mRNA and protein as well as ET-1 mRNA. Consequently, ghrelin significantly attenuated the development of PAH, pulmonary vascular remodeling, and right ventricular hypertrophy. These results demonstrate the therapeutic benefits of ghrelin for impeding the pathogenesis of pulmonary hypertension and right ventricular hypertrophy, particularly in subjects prone to CH (e.g. pulmonary disorders). [Abstract/Link to Full Text]

Umahara M, Okada S, Yamada E, Saito T, Ohshima K, Hashimoto K, Yamada M, Shimizu H, Pessin JE, Mori M
Tyrosine phosphorylation of Munc18c regulates PDGF-stimulated Glut4 translocation in 3T3L1 adipocytes.
Endocrinology. 2007 Oct 4;
PDGF stimulation of skeletal muscle, cultured myotubes, and 3T3L1 adipocytes result in Glut4 translocation, albeit to a reduced level compared to insulin. To address the mechanism of PDGF action, we have determined that the Syntaxin 4 negative regulatory protein, Munc18c undergoes PDGF stimulated phosphorylation on tyrosine residue 521. The tyrosine phosphorylation of Munc18c on Y521 occurred concomitant with the dissociation of the Munc18c protein from Syntaxin 4 in a time frame consistent with Glut4 translocation. Moreover, expression of the wild type Munc18c protein did not inhibit PDGF-induced Glut4 translocation whereas expression of Y521A-Munc18c mutant was inhibitory and failed to dissociate from Syntaxin 4. In contrast, expression of either wild type Munc18c or the Y521A-Munc18c mutant both resulted in a marked inhibition of insulin-stimulated Glut4 translocation. Taken together these data demonstrate that one mechanism accounting for the PDGF induction of Glut4 translocation is the suppression of the Munc18c negative regulation of Syntaxin 4 function. [Abstract/Link to Full Text]

Macintyre DA, Tyson EK, Read M, Smith R, Yeo G, Kwek K, Chan EC
Contraction in Human Myometrium is Associated with Changes in Small Heat Shock Proteins.
Endocrinology. 2007 Oct 4;
The myometrium undergoes substantial remodelling at the time of labour including rearrangement of the cellular contractile machinery. The regulation of this process in human myometrium at the time of labour is poorly defined but evidence in other muscles types suggests modulation by small heat shock proteins (sHSP). The aim of this study was to investigate if similar changes in sHSP occur in the myometrium at labour. Using a quantitative proteomic approach (2D-DIGE) we found a 69% decrease in the small HSP, alphaB-crystallin, in the myometrium at labour plus multiple isoforms of HSP27. Immunoblotting using phospho-specific HSP27 antibodies (HSP27-serine15, 78 and 82) detected marked changes in HSP27 phosphorylation at labour. While total HSP27 levels were unchanged, HSP27-ser15 was 3-fold higher at labour. Co-immunoprecipitation studies showed that HSP27 co-precipitates with alphaB-crystallin and also smooth muscle alpha-actin. Co-immunoflorescence studies demonstrated a relocation of HSP27 from the perinuclear region to the actin cytoskeleton at labour. The functional significance of these changes was demonstrated in vitro where myometrial strips stimulated to contract with oxytocin exhibited increased HSP27-ser15 phosphorylation. Our findings provide data consistent with a novel pathway regulating human myometrial contraction at labour and identify HSP27 and alphaB-crystallin as potential targets for future tocolytic design. [Abstract/Link to Full Text]

Takahashi N, Itoh MT, Ishizuka B
Human Chorionic Gonadotropin Induces Nestin Expression in Endothelial Cells of the Ovary via Vascular Endothelial Growth Factor Signaling.
Endocrinology. 2007 Oct 4;
The intermediate filament protein nestin was originally found to be expressed in neuronal progenitor cells, but recent studies have shown that other cell types, including endocrine and vascular endothelial cells, express nestin. In the present study, we examined the expression and localization of nestin in the ovaries of developing, peripubertal and adult rats. RT-PCR and Western blot analyses revealed that nestin mRNA and proteins were expressed in adult rat ovaries. Immunohistochemical analyses using adult rat ovaries showed that nestin was mainly localized to capillary endothelial cells of theca interna in follicles with more than two layers of granulosa cells and that its expression increased with follicle growth. Ontogenetically, ovarian nestin expression started at the peripubertal period when the first gonadotropin surge occurs. To test the possibility that gonadotropins induce nestin expression, prepubertal (postnatal day 21) rats were subcutaneously injected with equine chorionic gonadotropin (eCG) and/or human chorionic gonadotropin (hCG). A single injection of hCG, but not eCG, was sufficient to induce nestin expression in follicles, mainly in capillary endothelial cells of theca interna. Furthermore, pretreatment with an inhibitor of vascular endothelial growth factor (VEGF) receptor prevented the induction of the nestin expression by hCG. These findings demonstrate that endogenous LH surge induces nestin expression in capillary endothelial cells of theca interna via the VEGF signaling pathway. Nestin may be involved in angiogenesis in growing follicles, which is followed by follicle maturation and subsequent ovulation. [Abstract/Link to Full Text]

Ramocki NM, Wilkins HR, Magness ST, Simmons JG, Scull BP, Lee GH, McNaughton KK, Lund PK
IRS-1 deficiency promotes apoptosis in the putative intestinal crypt stem cell region, limits Apcmin/+ tumors, and regulates Sox9.
Endocrinology. 2007 Oct 4;
Reduced apoptosis of crypt stem/progenitor cells and elevated insulin and insulin-like growth factors (IGFs) are linked to colon cancer risk. Insulin receptor substrate-1 (IRS-1) mediates the actions of insulin, IGF-I, and IGF-II, but the role of endogenous IRS-1 in crypt apoptosis and cancer is undefined. Using IRS-1(-/-), IRS-1(+/-), and IRS-1(+/+) mice, we tested the hypothesis that reduced IRS-1 expression increases apoptosis of intestinal crypt cells and protects against Apc(min/+) (Min)/beta-catenin-driven intestinal tumors. Expression of Sox9, a transcriptional target of Tcf/beta-catenin and putative biomarker of crypt stem cells, was assessed in intestine of different IRS-1 genotypes and cell lines. Irradiation-induced apoptosis was significantly increased in the crypts and crypt stem cell region of IRS-1 deficient mice. Tumor load was significantly reduced by 31.2 +/- 14.6% in IRS-1(+/-)/Min and by 64.1 +/- 7.6% in IRS-1(-/-)/Min mice, with more prominent reductions in tumor number than size. Compared with IRS-1(+/+)/Min, IRS-1(-/-)/Min mice had fewer Sox9 positive cells in intestinal crypts and reduced Sox9 mRNA in intestine. IRS-1 overexpression increased Sox9 expression in an intestinal epithelial cell line. We conclude that even small reductions in endogenous IRS-1 increase apoptosis of crypt stem or progenitor cells, protect against beta-catenin-driven intestinal tumors, and reduce Sox9, a Tcf/beta-catenin target and putative stem/progenitor cell biomarker of putative crypt stem/progenitor cells. [Abstract/Link to Full Text]

Katsu Y, Ichikawa R, Ikeuchi T, Kohno S, Guillette LJ, Iguchi T
Molecular cloning and characterization of estrogen, androgen and progesterone nuclear receptors from a freshwater turtle (Pseudemys nelsoni).
Endocrinology. 2007 Oct 4;
Steroid hormones are essential for the normal function of many organ systems in vertebrates. Reproductive activity in females and males, such as the differentiation, growth and maintenance of the reproductive system, requires signaling by the sex steroids. Although extensively studied in mammals and a few fish, amphibians and bird species, the molecular mechanisms of sex steroid hormone (estrogens, androgens and progestins) action are poorly understood in reptiles. Here we evaluate hormone receptor ligand interactions in a freshwater turtle, the Red belly slider (Pseudemys nelsoni), following the isolation of cDNAs encoding an estrogen receptor alpha (ERalpha), an androgen receptor (AR) and a progesterone receptor (PR). The full-length Red belly slider ERalpha (tERalpha), tAR and tPR cDNAs were obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequences showed high identity to the chicken orthologs (tERalpha: 90%; tAR: 71%; tPR: 71%). Using transient transfection assays of mammalian cells, tERalpha protein displayed estrogen-dependent activation of transcription from an estrogen-responsive-element containing promoter. The other receptor proteins, tAR and tPR, also displayed androgen- or progestin-dependent activation of transcription from androgen- and progestin-responsive murine mammary tumor virus promoters. We further examined the transactivation of tERalpha, tAR and tPR by ligands using a modified GAL4-transactivation system. We found that the GAL4-transactivation system was not suitable for the measurement of tAR and tPR transactivations. This is the first report of the full-coding regions of a reptilian AR and PR, and the examination of their transactivation by steroid hormones. [Abstract/Link to Full Text]

Constantin S, Wray S
GnRH-1 neuronal activity is independent of Cyclic Nucleotide Gated channels.
Endocrinology. 2007 Oct 4;
Pulsatile release of gonadotropin-releasing hormone-1 (GnRH-1) is essential for secretion of gonadotropin hormones. The frequency of GnRH-1 pulses is regulated during the reproductive cycle by numerous neurotransmitters. Cyclic nucleotide gated (CNG) channels have been proposed as a mechanism to integrate the cAMP signal evoked by many neurotransmitters. This study reports the expression of the CNGA2 subunit in GnRH-1 neurons obtained from mouse nasal explants and shows the ability of GnRH-1 neurons to increase their activity in response to forskolin (FSK, activator of adenylyl cyclases), or 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterases), even after removal of GABA(A)ergic input. Next, the endogenous activity of adenylyl cyclases (AC) was evaluated as a component of the oscillatory mechanism of GnRH-1 neurons. Inhibition of endogenous activity of AC did not alter GnRH-1 activity. The potential involvement of CNGA2 subunit in basal or induced activity was tested on GnRH-1 neurons obtained from CNGA2 deficient mice. Without upregulation of CNGA1 or CNGA3, the absence of functional CNGA2 did not alter either the endogenous GnRH-1 neuronal activity or the response to FSK, negating CNG channels from cAMP-sensitive mechanisms leading to changes in GnRH-1 neuronal activity. In addition, the potential role of CNGA2 subunit in the synchronization of calcium oscillations previously described was evaluated in GnRH-1 neurons from CNGA2 deficient explants. Synchronized calcium oscillations persisted in CNGA2 deficient GnRH-1 neurons. Taken together, these results indicate that CNGA2 channels are not necessary for either the response of GnRH-1 neurons to cAMP increases or the basal rhythmic activity of GnRH-1 neurons. [Abstract/Link to Full Text]

Goldman-Johnson DR, de Kretser DM, Morrison JR
Evidence that androgens regulate early developmental events, prior to sexual differentiation.
Endocrinology. 2007 Oct 4;
Androgen signaling is critical for normal fetal development, but is not thought to regulate events in early embryogenesis. Given the interest in factors controlling the differentiation of embryonic stem cells, we have explored the possibility that androgens may play a role. This study demonstrates expression of androgen receptor (AR) RNA and protein in four independent mouse embryonic stem (mES) cell lines and demonstrates that the AR is functional and can interact with transfected androgen response elements (ARE) to promote green fluorescent protein (GFP) expression. AR mRNA was detected throughout 10 days of differentiation in embryoid bodies (EBs). Exposure of EBs to testosterone (T) or dihydrotestosterone (DHT), at doses of 1microM and 0.1microM respectively, promoted formation of beating cardiomyocytes. Flow cytometric analyses demonstrated a significant increase in the number of alpha-actinin and tropomyosin (cardiac markers) positive cells following these treatments. Addition of flutamide (1microM) to T-treated EBs inhibited the T-induced proliferation of cardiomyocytes, confirming that, in this instance, androgens act via the classical AR-mediated genomic pathway. We also report that ES cells express key steroidogenic enzymes, as detected by RT-PCR, and during 24 hour incubations secrete T at concentrations of 1.38 +/- 0.22nM, levels comparable to those secreted by cultured Leydig cells. These novel data demonstrate the capacity of androgens to stimulate increased differentiation of mES cells to cardiomyocytes, and is in keeping with recent observations that AR-deficient mice exhibit cardiac impairment in adulthood. [Abstract/Link to Full Text]

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Recent Articles in Molecular Endocrinology

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Sundar SN, Kerekatte V, Equinozio CN, Doan VB, Bjeldanes LF, Firestone GL
Indole-3-carbinol selectively uncouples expression and activity of estrogen receptor subtypes in human breast cancer cells.
Mol Endocrinol. 2006 Dec;20(12):3070-82.
Estrogen-responsive breast cancer cells, such as MCF7 and T47D cells, express both estrogen receptor (ER)-alpha (ERalpha) and ERbeta. Indole-3-carbinol (I3C) strongly down-regulated ERalpha protein and transcript levels, without altering the level of ERbeta protein, in both cell lines. In cells transfected with the ERalpha promoter linked to a luciferase gene reporter, I3C ablated ERalpha promoter activity. Propyl pyrazole triol (PPT) is a highly selective ERalpha agonist, whereas, 17beta-estradiol activates both ERalpha and ERbeta. I3C treatment inhibited the PPT- and 17beta-estradiol-induced proliferation of breast cancer cells, disrupted the PPT and 17beta-estradiol stimulation of estrogen response element (ERE)-driven reporter plasmid activity as well as of endogenous progesterone receptor transcripts. Using an in vitro ERE binding assay, I3C was shown to inhibit the level of functional ERalpha and stimulated the level of ERE binding ERbeta even though the protein levels of this receptor remained constant. In ERalpha-/ERbeta+ MDA-MB-231 breast cancer cells, I3C treatment stimulated a 6-fold increase in binding of ERbeta to the ERE. I3C also induced ERE- and activator protein 1-driven reporter plasmid activities in the absence of an ER agonist, suggesting that ERbeta is activated in indole-treated cells. Taken together, our results demonstrate that the expression and function of ERalpha and ERbeta can be uncoupled by I3C with a key cellular consequence being a significantly higher ERbeta:ERalpha ratio that is generally highly associated with antiproliferative status of human breast cancer cells. [Abstract/Link to Full Text]

Bonomi M, Busnelli M, Persani L, Vassart G, Costagliola S
Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues.
Mol Endocrinol. 2006 Dec;20(12):3351-63.
Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition. [Abstract/Link to Full Text]

Francis J, Babu DA, Deering TG, Chakrabarti SK, Garmey JC, Evans-Molina C, Taylor DG, Mirmira RG
Role of chromatin accessibility in the occupancy and transcription of the insulin gene by the pancreatic and duodenal homeobox factor 1.
Mol Endocrinol. 2006 Dec;20(12):3133-45.
The pancreatic and duodenal homeobox factor 1 (Pdx-1) is a Hox-like transcription factor that is responsible for the activation of the insulin gene. Previous studies have demonstrated the interaction in vitro of Pdx-1 with short (20-40 nucleotide) DNA fragments corresponding to A boxes of the insulin promoter. Precisely how Pdx-1 binds to DNA in the complex milieu of chromatin, however, has never been studied. In this study, we explored how Pdx-1-DNA interactions might be influenced by chromatin accessibility at the insulin gene in beta-cells (betaTC3) vs. pancreatic ductal cells (mPAC). We demonstrate that Pdx-1 occupies the endogenous insulin promoter in betaTC3 cells but not in mPAC cells, a finding that is independent of the intracellular Pdx-1 protein concentration. Based on micrococcal nuclease protection assays, the difference in promoter binding between the two cell types appears to be secondary to chromatin accessibility at predicted Pdx-1 binding sites between bp -126 to -296 (relative to the transcriptional start site) of the insulin promoter. Binding studies using purified Pdx-1 and reconstituted chromatin in vitro suggest that the positioning of a nucleosome(s) within this crucial region of the promoter might account for differences in chromatin accessibility. Consistent with these observations, fluorescence colocalization studies show that Pdx-1 does not occupy regions of compacted, nucleosome-rich chromatin within the nucleus. Our findings suggest a model whereby insulin transcription in the beta-cell is at least partially facilitated by enhanced chromatin accessibility within a crucial regulatory region between bp -126 to -296, thereby permitting occupancy by transactivators such as Pdx-1. [Abstract/Link to Full Text]

Kara E, Crépieux P, Gauthier C, Martinat N, Piketty V, Guillou F, Reiter E
A phosphorylation cluster of five serine and threonine residues in the C-terminus of the follicle-stimulating hormone receptor is important for desensitization but not for beta-arrestin-mediated ERK activation.
Mol Endocrinol. 2006 Nov;20(11):3014-26.
Classically, the FSH receptor (FSH-R) mediates its effects through coupling to guanine nucleotide-binding protein alpha S subunit (Galpha(s)) and activation of the cAMP/protein kinase A (PKA) signaling pathway. beta-Arrestins are rapidly recruited to the FSH-activated receptor and play key roles in its desensitization and internalization. Here, we show that the FSH-R expressed in HEK 293 cells activated ERK by two temporally distinct pathways dependent, respectively, on Galpha(s)/PKA and beta-arrestins. Galpha(s)/PKA-dependent ERK activation was rapid, transient, and blocked by H89 (a PKA inhibitor), but it was insensitive to small interfering RNA-mediated depletion of beta-arrestins. beta-Arrestin-dependent ERK activation was slower but more sustained and was insensitive to H89. We identified five Ser/Thr residues in the C terminus of the receptor (638-644) as a major phosphorylation site. Mutation of these residues into Ala (5A FSH-R) significantly reduced the stability of FSH-induced beta-arrestin 1 and 2 interaction when compared with the wild-type receptor. As expected, the 5A FSH-R-mediated cAMP accumulation was enhanced, and its internalization was reduced. In striking contrast, the ability of the 5A FSH-R to activate ERK via the beta-arrestin-dependent pathway was increased. G protein-coupled receptor kinase 5 (GRK5) and GRK6 were required for beta-arrestin-dependent ERK activation by both the wild-type and 5A FSH-R. By contrast, GRK2 depletion enhanced ERK activation by the wild-type FSH-R but not by the 5A FSH-R. In conclusion, we demonstrate the existence of a beta-arrestin-dependent, GRK-regulated mechanism for ERK activation by the FSH-R. A phosphorylation cluster in the C terminus of the FSH-R, identified as a site of beta-arrestin recruitment, positively regulated both desensitization and internalization but negatively regulated beta-arrestin-dependent ERK activation. [Abstract/Link to Full Text]

Calebiro D, de Filippis T, Lucchi S, Martinez F, Porazzi P, Trivellato R, Locati M, Beck-Peccoz P, Persani L
Selective modulation of protein kinase A I and II reveals distinct roles in thyroid cell gene expression and growth.
Mol Endocrinol. 2006 Dec;20(12):3196-211.
A global gene expression profiling of TSH stimulation on differentiated (FRTL5) and partially dedifferentiated [FRT/TSHR (TSH receptor)] rat thyroid cells was performed. A total of 123 TSH-regulated genes (95 newly described) were identified in FRTL5, whereas no significant transcriptional modifications were seen in FRT/TSHR cells. Because regulatory subunit IIbeta (RIIbeta) of protein kinase A (PKA), a key element downstream of cAMP, was expressed in FRTL5 but not in cAMP-refractory FRT/TSHR cells, we hypothesized that this gene may play an important role in TSH signaling. We therefore performed a series of experiments to investigate the involvement of RIIbeta and the different PKA isoforms. A positive effect of PKA II- but not of PKA I-selective activation on gene transcription and proliferation in FRTL5 cells, as well as an impairment of TSH nuclear effects after RIIbeta silencing were observed, suggesting that PKA II plays an essential role in TSH signaling. This view was supported by the restoration of TSH nuclear effects after reexpression of RIIbeta in FRT/TSHR cells. Because PKA I stimulation could increase iodide uptake in FRTL5 cells without affecting gene transcription, PKA I may mediate TSH actions at posttranscriptional levels. Analyses on three human cancer cell lines confirmed the possible loss of RIIbeta expression and antiproliferative activity of PKA I-selective cAMP analogs ( approximately 60% at 200 microm in BRAF-mutated cells). The inhibitory effect of PKA I apparently required constitutive MAPK activation and was associated with an inhibition of ERK phosphorylation. These findings may open new therapeutic perspectives in patients with thyroid cancer. [Abstract/Link to Full Text]

Palanisamy GS, Cheon YP, Kim J, Kannan A, Li Q, Sato M, Mantena SR, Sitruk-Ware RL, Bagchi MK, Bagchi IC
A novel pathway involving progesterone receptor, endothelin-2, and endothelin receptor B controls ovulation in mice.
Mol Endocrinol. 2006 Nov;20(11):2784-95.
The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture. [Abstract/Link to Full Text]

de Groot DM, Coenen AJ, Verhofstad A, van Herp F, Martens GJ
In vivo induction of glial cell proliferation and axonal outgrowth and myelination by brain-derived neurotrophic factor.
Mol Endocrinol. 2006 Nov;20(11):2987-98.
Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of neuronal cell survival and differentiation factors but is thought to be involved in neuronal cell proliferation and myelination as well. To explore the role of BDNF in vivo, we employed the intermediate pituitary melanotrope cells of the amphibian Xenopus laevis as a model system. These cells mediate background adaptation of the animal by producing high levels of the prohormone proopiomelanocortin (POMC) when the animal is black adapted. We used stable X. transgenesis in combination with the POMC gene promoter to generate transgenic frogs overexpressing BDNF specifically and physiologically inducible in the melanotrope cells. Intriguingly, an approximately 25-fold overexpression of BDNF resulted in hyperplastic glial cells and myelinated axons infiltrating the pituitary, whereby the transgenic melanotrope cells became located dispersed among the induced tissue. The infiltrating glial cells and axons originated from both peripheral and central nervous system sources. The formation of the phenotype started around tadpole stage 50 and was induced by placing white-adapted transgenics on a black background, i.e. after activation of transgene expression. The severity of the phenotype depended on the level of transgene expression, because the intermediate pituitaries from transgenic animals raised on a white background or from transgenics with only an approximately 5-fold BDNF overexpression were essentially not affected. In conclusion, we show in a physiological context that, besides its classical role as neuronal cell survival and differentiation factor, in vivo BDNF can also induce glial cell proliferation as well as axonal outgrowth and myelination. [Abstract/Link to Full Text]

Bonofiglio D, Aquila S, Catalano S, Gabriele S, Belmonte M, Middea E, Qi H, Morelli C, Gentile M, Maggiolini M, Andò S
Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells.
Mol Endocrinol. 2006 Dec;20(12):3083-92.
The aim of the present study was to provide new mechanistic insight into the growth arrest and apoptosis elicited by peroxisome proliferator-activated receptor (PPAR)gamma in breast cancer cells. We ascertained that PPARgamma mediates the inhibition of cycle progression in MCF7 cells exerted by the specific PPARgamma agonist rosiglitazone [BRL4653 (BRL)], because this response was no longer notable in the presence of the receptor antagonist GW9662. We also provided evidence that BRL is able to up-regulate mRNA and protein levels of the tumor suppressor gene p53 and its effector p21(WAF1/Cip1) in a time- and dose-dependent manner. Moreover, in transfection experiments with deletion mutants of the p53 gene promoter, we documented that the nuclear factor-kappaB sequence is required for the transcriptional response to BRL. Interestingly, EMSA showed that PPARgamma binds directly to the nuclear factor-kappaB site located in the promoter region of p53, and chromatin immunoprecipitation experiments demonstrated that BRL increases the recruitment of PPARgamma on the p53 promoter sequence. Next, both PPARgamma and p53 were involved in the cleavage of caspases-9 and DNA fragmentation induced by BRL, given that GW9662 and an expression vector for p53 antisense blunted these effects. Our findings provide evidence that the PPARgamma agonist BRL promotes the growth arrest and apoptosis in MCF7 cells, at least in part, through a cross talk between p53 and PPARgamma, which may be considered an additional target for novel therapeutic interventions in breast cancer patients. [Abstract/Link to Full Text]

Friesema EC, Kuiper GG, Jansen J, Visser TJ, Kester MH
Thyroid hormone transport by the human monocarboxylate transporter 8 and its rate-limiting role in intracellular metabolism.
Mol Endocrinol. 2006 Nov;20(11):2761-72.
Cellular entry of thyroid hormone is mediated by plasma membrane transporters. We have identified rat monocarboxylate transporter 8 (MCT8) as an active and specific thyroid hormone transporter. The MCT8 gene is located on the X-chromosome. The physiological relevance of MCT8 has been demonstrated by the identification of hemizygous mutations in this gene in males with severe psychomotor retardation and elevated serum T(3) levels. We have characterized human (h) MCT8 by analysis of iodothyronine uptake and metabolism in cell lines transiently transfected with hMCT8 cDNA alone or together with cDNA coding for iodothyronine deiodinase D1, D2, or D3. MCT8 mRNA was detected by RT-PCR in a number of human cell lines as well as in COS1 cells but was low to undetectable in other cell lines, including JEG3 cells. MCT8 protein was not detected in nontransfected cell lines tested by immunoblotting using a polyclonal C-terminal hMCT8 antibody but was detectable in transfected cells at the expected size (61 kDa). Transfection of COS1 and JEG3 cells with hMCT8 cDNA resulted in 2- to 3-fold increases in uptake of T(3) and T(4) but little or no increase in rT(3) or 3,3'-diiodothyronine (3,3'-T(2)) uptake. MCT8 expression produced large increases in T(4) metabolism by cotransfected D2 or D3, T(3) metabolism by D3, rT(3) metabolism by D1 or D2, and 3,3'-T(2) metabolism by D3. Affinity labeling of hMCT8 protein was observed after incubation of intact transfected cells with N-bromoacetyl-[(125)I]T(3). hMCT8 also facilitated affinity labeling of cotransfected D1 by bromoacetyl-T(3). Our findings indicate that hMCT8 mediates plasma membrane transport of iodothyronines, thus increasing their intracellular availability. [Abstract/Link to Full Text]

Yang Z, Wolf IM, Chen H, Periyasamy S, Chen Z, Yong W, Shi S, Zhao W, Xu J, Srivastava A, Sánchez ER, Shou W
FK506-binding protein 52 is essential to uterine reproductive physiology controlled by the progesterone receptor A isoform.
Mol Endocrinol. 2006 Nov;20(11):2682-94.
FK506-binding protein 52 (FKBP52) is a tetratricopeptide repeat protein that associates with steroid receptors in complexes containing heat shock protein 90. To investigate the role of FKBP52 in steroid-regulated physiology, we generated FKBP52-deficient mice. FKBP52 (-/-) females are sterile due to a complete failure of implantation, a process that requires estrogen (ER) and progesterone receptors (PR). Because the uterus expresses two forms of PR, PR-A and PR-B, we investigated all three receptors as potential targets of FKBP52 action. FKBP52 (-/-) uteri showed a normal growth response to estradiol, and unaltered expression of genes controlled by ER and PR-B. In contrast, FKBP52 (-/-) uteri were neither able to express two PR-A-regulated genes, nor undergo decidualization in response to progesterone, suggesting that FKBP52 specifically regulates PR-A at this organ. Analysis of uterine PR heterocomplexes showed preferential association of FKBP52 with PR-A compared with PR-B. Loss of FKBP52 neither disrupted the PR-A/heat shock protein 90 interaction, nor impaired uterine PR-A hormone-binding function, demonstrating the essential role of FKBP52 in PR-A action to be downstream of the hormone-binding event. Transcription studies in +/+ and -/- mouse embryonic fibroblast cells showed a near-complete loss of PR-A activity at mouse mammary tumor virus and synthetic progesterone response element promoters, although partial reductions of ER and PR-B were also observed. Partial disruptions of ovulation and mammary development were also found in FKBP52 (-/-) females. Taken as a whole, our results show FKBP52 to be an essential regulator of PR-A action in the uterus, while being a nonessential but contributory regulator of steroid receptors in the mammary and ovary. These data may now provide the basis for selective targeting of steroid-regulated physiology through tetratricopeptide repeat proteins. [Abstract/Link to Full Text]

Voss TC, John S, Hager GL
Single-cell analysis of glucocorticoid receptor action reveals that stochastic post-chromatin association mechanisms regulate ligand-specific transcription.
Mol Endocrinol. 2006 Nov;20(11):2641-55.
The glucocorticoid receptor (GR) dynamically interacts with response elements in the mouse mammary tumor virus (MMTV) promoter to regulate steroid-dependent transcription. In a clonal mammary carcinoma cell line containing a tandem array of MMTV promoter-reporter gene cassettes integrated at a single genomic locus, direct binding of a green fluorescent protein (GFP)-GR fusion protein to the MMTV regulatory elements can be observed in living cells. After ligand treatment, MMTV-dependent transcription in individual cells was detected by RNA fluorescence in situ hybridization (FISH). High-resolution fluorescence images were acquired from large numbers of randomly selected cells. Images were analyzed with a novel automated computer algorithm, measuring the RNA FISH signal and the relative GFP-GR fluorescence intensity at the MMTV array for each cell. Although dexamethasone increased the mean RNA FISH signal approximately 10-fold, RU486 produced only about a 2-fold induction, as expected for this mixed antagonist. For all treatment conditions, the relative GFP-GR fluorescence at the array for the averaged cells paralleled the RNA FISH measurements, suggesting that image analysis accurately detected an increase in steady-state GR association with the MMTV array that was responsible for the increase in transcriptional activity. The antagonist-dependent decreases in GR association with the MMTV promoter were confirmed by chromatin immunoprecipitation experiments, supporting the image analysis results. A pronounced cell-to-cell variability was observed in RNA FISH signal and GR-MMTV association within treatment groups. We observed a nonlinear relationship between GR-MMTV association and RNA FISH in individual cells, indicating that differences in GR-MMTV interaction account for some, but not all, of the transcriptional heterogeneity between individual cells. In selected cell subpopulations with equal levels of GR-MMTV association, there was a decrease in RNA FISH signal with RU486 treatment compared with dexamethasone treatment. These results indicate that stochastic events occurring after GR-promoter association, such as the actions of chromatin remodeling complexes or other cofactors, change in a ligand-dependent manner and regulate heterogeneous transcription in individual cells. [Abstract/Link to Full Text]

Ezzat S, Zhu X, Loeper S, Fischer S, Asa SL
Tumor-derived Ikaros 6 acetylates the Bcl-XL promoter to up-regulate a survival signal in pituitary cells.
Mol Endocrinol. 2006 Nov;20(11):2976-86.
We reported the expression of the lymphoid zinc finger transcription factor Ikaros (Ik) in the endocrine pituitary gland, where the usual isoforms, Ik1 and Ik2, are thought to play multiple physiological roles. The gene is alternatively spliced to yield a dominant negative isoform, Ik6, in nearly half of human pituitary tumors. We show here that the tumor-specific truncated Ik6 isoform promotes pituitary tumor AtT20 corticotroph and GH4 mammosomatotroph cell growth, evidenced by increased S-phase entry, colony formation in soft agar, and growth of xenografts in vivo. Ik6-mediated cell growth was associated with enhanced protection against apoptosis and up-regulation of the antiapoptotic factor Bcl-XL but not the related Bcl-2 family member. The effect of Ik6 on Bcl-XL induction was not reproduced by small interfering RNA-mediated Ik-down-regulation, indicating that this effect is not mediated entirely by disruption of Ik1 action. In cotransfection studies, Ik1 attenuated and Ik6 enhanced Bcl-XL promoter activity. The effect of Ik6 was mimicked by histone deacetylation inhibition but not by methylation inhibition. Furthermore, chromatin immunoprecipitation confirmed the ability of Ik6 to selectively acetylate histone 3 sites but not influence methylation of the Bcl-XL promoter. Thus, the contribution of Ik6 to tumor pathogenesis involves up-regulation of an antiapoptotic signal generated through selective acetylation of the Bcl-XL promoter. [Abstract/Link to Full Text]

Looyenga BD, Hammer GD
Origin and identity of adrenocortical tumors in inhibin knockout mice: implications for cellular plasticity in the adrenal cortex.
Mol Endocrinol. 2006 Nov;20(11):2848-63.
Inhibin knockout (Inha-/-) mice develop gonadal sex-cord tumors and--when gonadectomized--adrenocortical tumors. Previous reports demonstrated that adrenocortical tumors from Inha-/- mice produce estrogen and depend on gonadotropin signaling for initiation. Here we show that, in addition to producing estrogen, the adrenocortical tumors display a global change in cellular identity, composed of two unique cell types expressing differing arrays of genes normally restricted to theca and granulosa cells of the ovary. Many of these genes are also induced in wild-type adrenals after gonadectomy or upon chronic gonadotropin stimulation, suggesting that the adrenal cortex normally contains a population of pluripotent cells that can be driven toward an adrenal or gonadal identity given the appropriate pituitary stimuli. A central feature of this altered cellular identity is the switch from predominant expression of Gata6 (endogenous to the adrenal cortex) to Gata4, which defines cellular identity in the ovary. We show that stable transfection of Gata4 in cultured adrenocortical cells is sufficient to activate ovarian-specific genes of both theca and granulose lineages. Spatial analysis of Gata4 expression reveals a distinct pattern of localization to the supcapsular region of the adrenal, which contains undifferentiated progenitor cells that continuously populate the adrenocortical zones. Although both wild-type and Inha-/- mice display this pattern, only Inha-/- mice produce tumors composed of these Gata4-positive cells. These data suggest that Inha-/- adrenocortical tumors cells are derived from pluripotent adrenocortical progenitor cells that adopt a gonadal fate due to the convergent loss of inhibin and chronic exposure to elevated gonadotropins. [Abstract/Link to Full Text]


Endocrine-related resources from the National Institutes of Health.
Mol Endocrinol. 2006 Aug;20(8):1963-6. [Abstract/Link to Full Text]

Gummow BM, Scheys JO, Cancelli VR, Hammer GD
Reciprocal regulation of a glucocorticoid receptor-steroidogenic factor-1 transcription complex on the Dax-1 promoter by glucocorticoids and adrenocorticotropic hormone in the adrenal cortex.
Mol Endocrinol. 2006 Nov;20(11):2711-23.
Numerous genes required for adrenocortical steroidogenesis are activated by the nuclear hormone receptor steroidogenic factor 1 (SF-1) (NR5A1). Dax-1 (NR0B1), another nuclear hormone receptor, represses SF-1-dependent activation. Glucocorticoid products of the adrenal cortex provide negative feedback to the production of hypothalamic CRH and pituitary ACTH. We hypothesized that glucocorticoids stimulate an intraadrenal negative feedback loop via activation of Dax-1 expression. Reporter constructs show glucocorticoid-dependent synergy between SF-1 and glucocorticoid receptor (GR) in the activation of Dax-1, which is antagonized by ACTH signaling. We map the functional glucocorticoid response element between -718 and -704 bp, required for activation by GR and synergy with SF-1. Of three SF-1 response elements, only the -128-bp SF-1 response element is required for synergy with GR. Chromatin immunoprecipitation (ChIP) assays demonstrate that dexamethasone treatment increases GR and SF-1 binding to the endogenous murine Dax-1 promoter 10- and 3.5-fold over baseline. Serial ChIP assays reveal that that GR and SF-1 are part of the same complex on the Dax-1 promoter, whereas coimmunoprecipitation assay confirms the presence of a protein complex that contains both GR and SF-1. ACTH stimulation disrupts the formation of this complex by abrogating SF-1 binding to the Dax-1 promoter, while promoting SF-1 binding to the melanocortin-2 receptor (Mc2r) and steroidogenic acute regulatory protein (StAR) promoters. Finally, dexamethasone treatment increases endogenous Dax-1 expression and concordantly decreases StAR expression. ACTH signaling antagonizes the increase in Dax-1 yet strongly activates StAR transcription. These data indicate that GR provides feedback regulation of adrenocortical steroid production through synergistic activation of Dax-1 with SF-1, which is antagonized by ACTH activation of the adrenal cortex. [Abstract/Link to Full Text]

Ezzat S, Zheng L, Winer D, Asa SL
Targeting N-cadherin through fibroblast growth factor receptor-4: distinct pathogenetic and therapeutic implications.
Mol Endocrinol. 2006 Nov;20(11):2965-75.
Several molecular aberrations have been implicated in the pathogenesis of pituitary tumors, but few have proven thus far to be of therapeutic value. Pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4) is an alternatively transcribed cytoplasmic isoform lacking most of the extracellular domain. This oncogene recapitulates the morphological features of human pituitary tumors in transgenic mice. To investigate the therapeutic potential of targeting ptd-FGFR4, we examined the impact of FGFR4 tyrosine kinase inhibition in xenografted mice. GH4 pituitary cells expressing ptd-FGFR4 develop into invasive tumors. Systemic treatment of mice bearing ptd-FGFR4 tumors with the FGFR-selective inhibitor PD173074 resulted in recovery of membranous N-cadherin staining and a significant reduction in tumor volume with less invasive growth behavior. Mutation of tyrosine Y754F in ptd-FGFR4 abrogated the effect of PD173074-mediated inhibition. The pivotal role of N-cadherin as a mediator of this pituitary cell growth was demonstrated by small interfering RNA mediated down-regulation, which promoted invasive growth in xenografted mice. To validate this model in primary human pituitary tumors, we examined the expression of ptd-FGFR4, N-cadherin, and clinical behavior. Loss of membranous N-cadherin correlated with cytoplasmic FGFR4 expression and with tumor invasiveness in surgically resected human pituitary tumors. Primary human pituitary tumor cells treated with PD173074 showed restoration of N-cadherin to the membrane with dephosphorylation of retinoblastoma protein. These data highlight the pathogenetic significance of N-cadherin misexpression and emphasize the importance of FGFR partnership in mediating its functions. [Abstract/Link to Full Text]

Ellsworth BS, Egashira N, Haller JL, Butts DL, Cocquet J, Clay CM, Osamura RY, Camper SA
FOXL2 in the pituitary: molecular, genetic, and developmental analysis.
Mol Endocrinol. 2006 Nov;20(11):2796-805.
FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone alpha-subunit (alphaGSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and alphaGSU suggested that the alphaGSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived (alphaT3-1, LbetaT2), thyrotrope-derived (alphaTSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression. [Abstract/Link to Full Text]

Jaber BM, Gao T, Huang L, Karmakar S, Smith CL
The pure estrogen receptor antagonist ICI 182,780 promotes a novel interaction of estrogen receptor-alpha with the 3',5'-cyclic adenosine monophosphate response element-binding protein-binding protein/p300 coactivators.
Mol Endocrinol. 2006 Nov;20(11):2695-710.
Estrogen receptor-alpha (ERalpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ERalpha agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ERalpha promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ERalpha ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ERalpha. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ERalpha targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ERalpha is insufficient to promote transcription of ERalpha target genes. [Abstract/Link to Full Text]

Facchiano F, D'Arcangelo D, Russo K, Fogliano V, Mennella C, Ragone R, Zambruno G, Carbone V, Ribatti D, Peschle C, Capogrossi MC, Facchiano A
Glycated fibroblast growth factor-2 is quickly produced in vitro upon low-millimolar glucose treatment and detected in vivo in diabetic mice.
Mol Endocrinol. 2006 Nov;20(11):2806-18.
Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM. [Abstract/Link to Full Text]

Zhao Y, Yang Z, Phelan JK, Wheeler DA, Lin S, McCabe ER
Zebrafish dax1 is required for development of the interrenal organ, the adrenal cortex equivalent.
Mol Endocrinol. 2006 Nov;20(11):2630-40.
Mutations in the human nuclear receptor, DAX1, cause X-linked adrenal hypoplasia congenita (AHC). We report the isolation and characterization of a DAX1 homolog, dax1, in zebrafish. The dax1 cDNA encodes a protein of 264 amino acids, including the conserved carboxy-terminal ligand binding-like motif; but the amino-terminal region lacks the unusual repeats of the DNA binding-like domain in mammals. Genomic sequence analysis indicates that the dax1 gene structure is conserved also. Whole-mount in situ hybridization revealed the onset of dax1 expression in the developing hypothalamus at approximately 26 h post fertilization (hpf). Later, at about 28 hpf, a novel expression domain for dax1 appeared in the trunk. This bilateral dax1-expressing structure was located immediately above the yolk sac, between the otic vesicle and the pronephros. Interestingly, weak and transient expression of dax1 was observed in the interrenal glands (adrenal cortical equivalents) at approximately 31 hpf. This gene was also expressed in the liver after 3 dpf in the zebrafish larvae. Disruption of dax1 function by morpholino oligonucleotides (MO) down-regulated expression of steroidogenic genes, cyp11a and star, and led to severe phenotypes similar to ff1b (SF1) MO-injected embryos. Injection of dax1 MO did not affect ff1b expression, whereas ff1b MO abolished dax1 expression in the interrenal organ. Based on these results, we propose that dax1 is the mammalian DAX1 ortholog, functions downstream of ff1b in the regulatory cascades, and is required for normal development and function of the zebrafish interrenal organ. [Abstract/Link to Full Text]

Onuma H, Vander Kooi BT, Boustead JN, Oeser JK, O'Brien RM
Correlation between FOXO1a (FKHR) and FOXO3a (FKHRL1) binding and the inhibition of basal glucose-6-phosphatase catalytic subunit gene transcription by insulin.
Mol Endocrinol. 2006 Nov;20(11):2831-47.
Insulin inhibits transcription of the genes encoding the glucose-6-phosphatase catalytic subunit (G6Pase), phosphoenolpyruvate carboxykinase, and IGF binding protein-1 through insulin response sequences (IRSs) that share the same core sequence, T(G/A)TTTT(G/T). The transcription factors FOXO1a and FOXO3a have been shown to bind these elements, but there are conflicting reports as to whether this binding correlates with the action of insulin on gene transcription. Some researchers concluded, from overexpression experiments using FOXO1a, that binding correlated with the insulin response, whereas others concluded, mainly from gel retardation competition experiments using FOXO3a, that it did not. We show here that, although these factors can differentially activate gene transcription in a context-dependent manner, these conflicting data are not explained by a difference in FOXO1a and FOXO3a binding specificity. Instead, we find that gel retardation competition and binding experiments give different results; the latter reveal a correlation between FOXO1a/3a binding and the inhibition of basal G6Pase gene transcription by insulin. In addition, these data show that the binding of FOXO1a/3a to two adjacent IRSs in the G6Pase promoter is cooperative and that promoter context alters the specific IRS base requirements for FOXO1a-stimulated fusion gene expression. Surprisingly, an analysis of insulin action mediated through the G6Pase and IGF binding protein-1 IRSs in the context of a heterologous thymidine kinase promoter reveals that signaling through the latter does not support the accepted model for insulin-stimulated FOXO nuclear exclusion. [Abstract/Link to Full Text]

Gadd SL, Clevenger CV
Ligand-independent dimerization of the human prolactin receptor isoforms: functional implications.
Mol Endocrinol. 2006 Nov;20(11):2734-46.
Prolactin (PRL) contributes to the growth of normal and malignant breast tissues. PRL initiates signaling by engaging the PRL receptor (PRLr), a transmembrane (TM) receptor belonging to the cytokine receptor family. The accepted view has been that PRL activates the PRLr by inducing dimerization of the receptor, but recent reports show ligand-independent dimerization of other cytokine receptors. Using coimmunoprecipitation assays, we have confirmed ligand-independent dimerization of the PRLr in T47D breast cancer and HepG2 liver carcinoma cells. In addition, mammalian cells transfected with differentially epitope-tagged isoforms of the PRLr indicated that long, intermediate, and DeltaS1 PRLrs dimerized in a ligand-independent manner. To determine the domain(s) involved in PRLr ligand-independent dimerization, we generated PRLr constructs as follows: (1) the TM-ICD, which consisted of the TM domain and the intracellular domain (ICD) but lacked the extracellular domain (ECD), and (2) the ECD-TM, which consisted of the TM domain and the ECD but lacked the ICD. These constructs dimerized in a ligand-independent manner in mammalian cells, implicating a significant role for the TM domain in this process. These truncated PRLrs were functionally inert alone or in combination in cells lacking the PRLr. However, when introduced into cells containing endogenous PRLr, the ECD-TM inhibited human PRLr signaling, whereas the TM-ICD potentiated human PRLr signaling. These studies indicate that the ECD-TM and the TM-ICD are capable of modulating PRLr function. We also demonstrated an endogenous TM-ICD in T47D cells, suggesting that these findings are relevant to PRL-signaling pathways in breast cancer. [Abstract/Link to Full Text]

Raetzman LT, Wheeler BS, Ross SA, Thomas PQ, Camper SA
Persistent expression of Notch2 delays gonadotrope differentiation.
Mol Endocrinol. 2006 Nov;20(11):2898-908.
Normal pituitary gland development requires coordination between maintenance of progenitor cell pools and selection of progenitors for differentiation. The spatial and temporal expression of Notch2 during pituitary development suggested that it could control progenitor cell differentiation in the pituitary. Consistent with this idea, Notch2 is not expressed in Prop1 mutants, and anterior pituitary progenitors in Prop1 mutants appear to be unable to transition from proliferation to differentiation properly, resulting in anterior lobe failed cell specification and evolving hypoplasia. To test the function of Notch2 directly, we used the alphaGSU subunit promoter to express activated NOTCH2 persistently in pre-gonadotropes and pre-thyrotropes of transgenic mice. At birth, there is a small reduction in the population of fully differentiated thyrotropes and almost no fully differentiated gonadotropes. The temporal and spatial expression of Hey1 suggests that it could be a mediator of this effect. Gonadotropes complete their differentiation program eventually, although expression of LH and FSH is mutually exclusive with NOTCH2 transgene expression. This demonstrates that activated Notch2 is sufficient to delay gonadotrope differentiation, and it supports the hypothesis that Notch2 regulates progenitor cell differentiation in the pituitary gland. [Abstract/Link to Full Text]

Son DS, Roby KF
Interleukin-1alpha-induced chemokines in mouse granulosa cells: impact on keratinocyte chemoattractant chemokine, a CXC subfamily.
Mol Endocrinol. 2006 Nov;20(11):2999-3013.
IL-1 is well known to be involved in the immune system and have a role in ovarian inflammation as well as exhibiting inhibitory effects on steroidogenesis and folliculogenesis. Because multiple aspects of ovarian function have also been shown to involve cytokine/chemokine networks, IL-1alpha-induced chemokine gene expression in mouse granulosa cells was investigated. Granulosa cells from immature mice at 28 d of age were cultured with IL-1alpha (10 ng/ml). IL-1alpha induced abundantly and specifically keratinocyte chemoattractant (KC) chemokine, a CXC subfamily. KC chemokine mRNA and protein were increased 1-2 h after IL-1alpha and then gradually decreased. The KC promoter (-701/+30) containing three nuclear factor (NF)-kappaB sites was fully responsive to IL-1alpha, whereas deletions and mutants of the NF-kappaB sites lowered the responsiveness to IL-1alpha. The proximal NF-kappaB site (-69/-59) played a critical role in regulating IL-1alpha-induced KC chemokine promoter activity. Overexpression of the inhibitor of NF-kappaB (IkappaB) blocked KC promoter activity induced by IL-1alpha, whereas overexpression of p65, a component of NF-kappaB, increased promoter activity and mRNA of KC chemokine. In addition, FSH did not affect NF-kappaB signaling or IL-1alpha-induced KC chemokine promoter activity. Within 1-3 h after ip injection of lipopolysaccharide (100 mug/mouse), a product known to stimulate release of IL-1, KC chemokine was localized in the ovary to granulosa cells as well as the thecal-interstitial layer. The results of this study indicate that KC gene is a chemokine induced acutely by IL-1alpha via NF-kappaB signaling in mouse granulosa cells. [Abstract/Link to Full Text]

Mirasierra M, Vallejo M
The homeoprotein Alx3 expressed in pancreatic beta-cells regulates insulin gene transcription by interacting with the basic helix-loop-helix protein E47.
Mol Endocrinol. 2006 Nov;20(11):2876-89.
The regulation of insulin gene expression in pancreatic beta-cells is the result of the coordinate activity of specific combinations of transcription factors assembled on different promoter elements. We investigated the involvement of the aristaless-related homeoprotein Alx3 in this process. We found that Alx3 is coexpressed with insulin in pancreatic islets, as well as in the beta-cell line MIN6, and it is also present in glucagon- and somatostatin-expressing cells. Chromatin immunoprecipitation assays indicated that Alx3 present in MIN6 cells and in mouse pancreatic islets occupies the promoter of the mouse insulin genes. EMSAs indicated that Alx3 present in MIN6 cells binds to the A3/4 regulatory element of the insulin I promoter. We found that Alx3 transactivates the insulin promoter by acting on the E2A3/4 enhancer in conjunction with the basic helix-loop-helix transcription factors E47/Pan1 and Beta2/NeuroD, and that Alx3 physically interacts via the homeodomain with E47/Pan1 but not with Beta2/NeuroD. Alx3 binds to the A3/4 element as a dimer, and the homeodomain is sufficient to recruit E47/Pan1 to the insulin promoter. Deletion studies in transfected HeLa cells indicated that proline-rich regions located at either side of the Alx3 homeodomain work together with E47/Pan1, and that this requires the integrity of the amino-terminal activation domain to transactivate. Thus, these studies support the notion that Alx3 participates in the regulation of insulin gene expression in pancreatic beta-cells. [Abstract/Link to Full Text]

Thimmarayappa J, Sun J, Schultz LE, Dejkhamron P, Lu C, Giallongo A, Merchant JL, Menon RK
Inhibition of growth hormone receptor gene expression by saturated fatty acids: role of Kruppel-like zinc finger factor, ZBP-89.
Mol Endocrinol. 2006 Nov;20(11):2747-60.
The expression and function of the GH receptor is critical for the actions of pituitary GH in the intact animal. The role of systemic factors in the reduced expression of the GH receptor and consequent GH insensitivity in pathological states such as sepsis, malnutrition, and poorly controlled diabetes mellitus is unclear. In the current study, we demonstrate that saturated (palmitic and myristic; 50 microM) fatty acids (FA) inhibit activity of the promoter of the major (L2) transcript of the GH receptor gene; unsaturated (oleic and linoleic) FA (200 microM) do not alter activity of the promoter. Comparable effects with palmitic acid and the nonmetabolizable analog bromo-palmitic acid, and failure of triacsin C to abrogate palmitic acids effects on GH receptor expression indicate that this effect is due to direct action(s) of FA. Palmitic acid, but not the unsaturated FA linoleic acid, decreased steady-state levels of endogenous L2 mRNA and GHR protein in 3T3-L1 preadipocytes. The effect of FA was localized to two cis elements located approximately 600 bp apart on the L2 promoter. EMSA and chromatin immunoprecipitation assays established that both these cis elements bind the Krüppel-type zinc finger transcription factor, ZBP-89. Ectopic expression of ZBP-89 amplified the inhibitory effect of FA on L2 promoter activity and on steady-state levels of endogenous L2 mRNA in 3T3-L1 preadipocytes. Mutational analyses of the two ZBP-89 binding sites revealed that both the sites are essential for palmitic acid's inhibitory effect on the L2 promoter and for the enhancing effect of ZBP-89 on palmitic acid-induced inhibition of the L2 promoter. Our results establish a molecular basis for FA-induced inhibition of GH receptor gene expression in the pathogenesis of acquired GH insensitivity in pathological states such as poorly controlled diabetes mellitus and small for gestational age. [Abstract/Link to Full Text]

Barrès R, Grémeaux T, Gual P, Gonzalez T, Gugenheim J, Tran A, Le Marchand-Brustel Y, Tanti JF
Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.
Mol Endocrinol. 2006 Nov;20(11):2864-75.
APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue. [Abstract/Link to Full Text]

Frank SJ, Wang X, He K, Yang N, Fang P, Rosenfeld RG, Hwa V, Chaudhuri TR, Deng L, Zinn KR
In vivo imaging of hepatic growth hormone signaling.
Mol Endocrinol. 2006 Nov;20(11):2819-30.
We developed a system to noninvasively and repeatedly image in vivo hepatic GH signaling. GH regulates postnatal growth and metabolism. It affects numerous tissues, but has major effects in liver. We used nude mice for adenoviral-mediated delivery of a signal transducer and activator of transcription 5-dependent GH response element, a luciferase reporter to detect GH signaling pathway activation. We detected by noninvasive bioluminescence imaging GH-induced hepatic GH signaling serially within intact mice. Statistically significant effects of GH dose and time dependence were detected in the liver luciferase signal that peaked 3 h after GH injection. Codelivery of GH receptor significantly enhanced GH response, an effect that was further augmented by fasting. Our imaging system allows detailed in vivo analysis of GH signaling and action and may be a paradigm for studies of additional signaling pathways in liver and other tissues. [Abstract/Link to Full Text]

Molina-Muñoz T, Romero-Avila MT, García-Sáinz JA
Insulin-like growth factor-I induces alpha(1B)-adrenergic receptor phosphorylation through G beta gamma and epidermal growth factor receptor transactivation.
Mol Endocrinol. 2006 Nov;20(11):2773-83.
IGF-I induces alpha(1B)-adrenoceptor (alpha(1B)-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC(50) approximately 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased alpha(1B)-adrenergic effect on intracellular calcium and production of inositol phosphates. The effect of IGF-I was markedly decreased in cells treated with pertussis toxin suggesting involvement of pertussis toxin-sensitive G proteins. Transfection of the carboxyl terminus of the beta-adrenergic receptor kinase or the Deltap85 mutant of phosphoinositide 3-kinase (PI3K) markedly decreased the alpha(1B)-AR phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline. Inhibitors of PI3K and protein kinase C blocked IGF-I-induced alpha(1B)-AR phosphorylation. In addition, it was observed that AG1478, an inhibitor of the epidermal growth factor (EGF) receptor kinase, and BB-94, a metalloproteinase inhibitor, also diminished IGF-I-induced adrenoceptor phosphorylation. The data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of a serpentine G protein-coupled receptor, suggesting the following hypothetical model: 1) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; 2) the growth factor action activates metalloproteinases, which catalyze heparin binding-EGF shedding, and transactivation of EGF receptors, and 3) dissociated Gbetagamma subunits and phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of protein kinase C, resulting in alpha(1B)-AR phosphorylation and desensitization. [Abstract/Link to Full Text]

Galet C, Ascoli M
A constitutively active mutant of the human lutropin receptor (hLHR-L457R) escapes lysosomal targeting and degradation.
Mol Endocrinol. 2006 Nov;20(11):2931-45.
Using biochemical and imaging approaches, we examined the postendocytotic fate of the complex formed by human choriogonadotropin (hCG) and a constitutively active mutant of the human lutropin receptor (hLHR-L457R) found in a boy with precocious puberty and Leydig cell hyperplasia. After internalization, some of the complex formed by the hLHR-wild type (hLHR-wt) and hCG recycles to the cell surface, and some is found in lysosomes where the hormone is degraded. In contrast, the complex formed by the hLHR-L457R and hCG is not routed to the lysosomes, most of it is recycled to the cell surface and hormone degradation is barely detectable. For both, hLHR-wt and -L457R, there is an hCG-induced loss of cell surface receptors that accompanies internalization but this loss cannot be prevented by leupeptin. The removal of recycling motifs of the hLHR by truncation of the C-terminal tail at residue 682 greatly enhances the lysosomal accumulation of the hormone-receptor complexes formed by the hLHR-wt or the L457R mutant, the degradation of the internalized hormone, and the loss of cell surface receptors. The degradation of the hormone internalized by these mutants as well as the loss of cell surface receptors is largely prevented by leupeptin. These results highlight a previously unrecognized complexity in the postendocytotic trafficking of the hLHR and document a clear difference between the properties of the constitutively active mutant and the agonist-activated hLHR-wt. This lack of lysosomal degradation of the L457R mutant could contribute to its constitutive activity by prolonging the duration of signaling. [Abstract/Link to Full Text]

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Recent Articles in BMC Endocrine Disorders

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Recent Articles in Endocrine Journal

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Ohmori N, Nomura K, Ohmori K, Takano K
Preclinical Cushing's disease characterized by massive adrenal hyperplasia and hormonal changes after three years of metyrapone therapy.
Endocr J. 2007 Jun;54(3):391-7.
A 66-year-old woman had massive bilateral adrenal macronodular hyperplasia, found incidentally on an abdominal ultrasonogram. Her plasma ACTH and serum cortisol levels were normal, but they were not suppressed by low-dose dexamethasone. The patient did not exhibit any typical signs or symptoms of Cushing's disease. MRI showed no evidence of a tumor in the pituitary gland. A diagnosis of preclinical Cushing's disease was made, and she was treated with 11-hydroxylase inhibitor metyrapone. As the dose of metyrapone was increased, plasma ACTH levels gradually increased. After three years of treatment, she developed moon-face. Her plasma ACTH and serum cortisol concentrations were at their highest levels. A pituitary microadenoma was detected by MRI, whose source of ACTH was demonstrated by the definite step-up of central/peripheral ratio of ACTH obtained by cavernous sinus sampling. Overt Cushing's disease was diagnosed, and a pituitary tumor was removed by transsphenoidal surgery. In conclusion, the clinically and endocrinologically overt Cushing's disease characterized by macronodular adrenal hyperplasia was converted from a preclinical form. This case offers some insight into the clinical and biological features of preclinical Cushing's disease. [Abstract/Link to Full Text]

Asano S, Ooka H, Okazaki R, Ishikawa T, Ochiai H, Nakashima M, Ide F, Hasegawa I, Miyawaki S, Nakaguchi H, Murakami M, Ogino Y, Takano K, Matsuno A
Long-term remission of cyclic Cushing's disease that was diagnosed and treated surgically in non-active phase.
Endocr J. 2007 Jun;54(3):407-12.
Cyclic Cushing's disease is a rare clinical entity that is defined as a periodic excessive production of adrenocorticotropic hormone (ACTH) and cortisol. Only 42 cases with cyclic Cushing's disease have been reported in the literature. The diagnosis is very difficult because of the fluctuating secretion of ACTH and cortisol. We report a 78-year-old woman with a pituitary adenoma presenting with cyclic Cushing's disease. In the present case, several interesting issues are pointed out: 1) MRI study detected the presence of an adenoma and selective venous sampling in the cavernous sinus disclosed the hypersecretion of ACTH from a pituitary adenoma. These neuroimaging and endocrinological studies were helpful for the diagnosis, even in the remission phase. 2) The disease was in the long-term remission phase after transsphenoidal surgery despite the high recurrence rate in this clinical entity, although it recurred four years later. Even in the remission phase of cyclic Cushing's disease, meticulous endocrinological and neuroimaging examinations can reveal the presence of a pituitary adenoma, which should be treated surgically. [Abstract/Link to Full Text]

Shimoda S, Ohnaka K, Sakai Y, Nawata H, Takayanagi R
Identification and synergism of cis-acting elements essential for basal promoter activity of the human type 1 angiotensin II receptor gene in PLC-PRF-5 cells.
Endocr J. 2007 Jun;54(3):413-24.
The basal promoter activity of the human AT(1) receptor gene was characterized using a human hepatoma cell line with a considerably high expression of AT(1), PLC-PRF-5. Four cis-acting, positively regulating elements termed AT(1)PRE1 (-113 to -102 bp), AT(1)PRE2 (-49 to -43 bp), AT(1)PRE3 (-5 to -2 bp) and AT(1)PRE4 (+44 to +50 bp) were identified. AT(1)PRE2 contained a GC-box-like sequence and bound to Sp1. AT(1)PRE1 contained two tandem GC-boxes and was bound to several nuclear proteins in addition to Sp1. Nuclear proteins that were bound sequence-specifically to AT(1)PRE1, AT(1)PRE2 and AT(1)PRE4 were found in both PLC-PRF-5 cells and 8505C cells, while those bound to AT(1)PRE3 were not found in 8505C cells, which showed no expression of AT(1) and almost no promoter activity for the AT(1) gene. Significant promoter activity was still observed even when AT(1)PRE1, AT(1)PRE2 and AT(1)PRE4 were all mutated. Mutagenesis of AT(1)PRE3, however, substantially inactivated promoter activity. AT(1)PRE1, AT(1)PRE2 and AT(1)PRE4 synergistically enhanced AT(1) gene transcription promoted by AT(1)PRE3. These results suggested that AT(1)PRE3 is responsible for the tissue-specific expression of the human AT(1) gene, and that AT(1)PRE1, AT(1)PRE2 and AT(1)PRE4 function as a general enhancer in liver-derived cells. [Abstract/Link to Full Text]

Fukata S, Hishinuma A, Kuma K, Miyauchi A, Sugawara M
Endemic goiter due to thyroglobulin gene abnormality and social ostracism.
Endocr J. 2007 Jun;54(3):485-6. [Abstract/Link to Full Text]

Miyoshi T, Otsuka F, Takeda M, Inagaki K, Otani H, Ogura T, Ichiki K, Amano T, Makino H
An elderly patient with sarcoidosis manifesting panhypopituitarism with central diabetes insipidus.
Endocr J. 2007 Jun;54(3):425-30.
We here report a 77-year-old Japanese male who suffered general fatigue with progressive thirst and polyuria. Central diabetes insipidus was diagnosed by depletion of vasopressin secretion in response to increases in serum osmolality. Secretory responses of anterior pituitary hormones including adrenocorticotropin, thyrotropin, gonadotropins and growth hormone were severely impaired. Diffuse swelling of the infundibulum as well as lack of T1-hyperintense signal in the posterior lobe was noted by pituitary magnetic resonance imaging. The presence of bilateral hilar lymphadenopathy and increased CD4/CD8 ratio in bronchoalveolar lavage fluid was diagnostic for lung sarcoidosis. Physiological doses of corticosteroid and thyroid hormone were administered in addition to desmopressin supplementation. Complete regression of the neurohypophysial swelling was notable two years after corticosteroid replacement. Diffuse damage of anterior pituitary combined with hypothalamic involvement leading to central diabetes insipidus is a rare manifestation in such elderly patients with neurosarcoidosis. [Abstract/Link to Full Text]

Makay O, Icoz G, Gurcu B, Ertan Y, Tuncyurek M, Akyildiz M, Yetkin E
The ongoing debate in thyroid surgery: should frozen section analysis be omitted?
Endocr J. 2007 Jun;54(3):385-90.
Controversies concerning the role of frozen section (FS) have been a matter of debate. The aim of this study was to identify the role of FS analysis in intraoperative decision making and analyze the effect of the cost in detecting thyroid malignancies in Turkey. Out of 214 consecutive patients who had been operated on for thyroid cancer between January 1996 and August 2004, 178 patients were evaluated retrospectively. All 178 patients were subjected to FS. Intraoperative FS correctly identified the pathology as malignant in 58.4% of patients. A true-positive FS result changed the surgical strategy in 30 (27.6%) cases False negative FS lesions were defined histologically as papillary microcarcinoma in 54%, follicular variant of papillary cancer in 18% and follicular cancer in 8% of cases. The sensitivities of FNAB and intraoperative FS in thyroid cancer patients were 22.5% and 58.4%, respectively. False negative FS results increased the cost for each informative FS from euro25 to euro42.7. Despite limitations, results of this study reject the idea that the role of FS is becoming limited. We recommend routine frozen section in the operative assessment of thyroid nodules. Omitting FS may be suggested only in cases with a FNAB revealing malignancy. [Abstract/Link to Full Text]

Kumagai A, Namba H, Akanov Z, Saenko VA, Meirmanov S, Ohtsuru A, Yano H, Maeda S, Anami M, Hayashi T, Ito M, Sagandikova S, Eleubaeva Z, Mussinov D, Espenbetova M, Yamashita S
Clinical implications of pre-operative rapid BRAF analysis for papillary thyroid cancer.
Endocr J. 2007 Jun;54(3):399-405.
The activating point mutation of the BRAF gene, BRAF(T1799A), is the most common and specific genetic alteration in adult papillary thyroid carcinoma (PTC) and a possible marker of malignant potential of PTC. We have applied the PCR-RFLP method using fine-needle aspiration biopsy samples not only to our clinical practice but also to the international medical assistance effort around the Semipalatinsk Nuclear Testing Site in Kazakhstan. Seventy-seven cases (100 nodules) from Japan and 131 cases (137 nodules) from Kazakhstan were examined. There were 14 Japanese and 76 Kazakhstani cases of cytological malignant tumors from the examined samples. We detected 12 (85.7% of PTC) and 19 (25% of PTC) cases with BRAF(T1799A) among the Japanese and Kazakhstani cases, respectively. Of these cases, we found mutations in one cytologically "suspicious" case and even in two pathologically "benign" cases (after surgery in Kazakhstan). All of the BRAF mutation-positive cases, including those three, were confirmed as PTC by careful pathological examination, including immunohistochemical analysis. In summary, our PCR-RFLP method for BRAF(T1799A) detection using FNAB samples is useful not only for preoperative diagnosis of PTC but also as a complementary diagnostic tool for accurate pathological diagnosis, even after surgery. [Abstract/Link to Full Text]

Horiguchi K, Yamada M, Umezawa R, Satoh T, Hashimoto K, Tosaka M, Yamada S, Mori M
Somatostatin receptor subtypes mRNA in TSH-secreting pituitary adenomas: a case showing a dramatic reduction in tumor size during short octreotide treatment.
Endocr J. 2007 Jun;54(3):371-8.
TSH-secreting adenoma is a rare pituitary adenoma, and the expression levels of the specific subtypes of somatostatin receptors (sstr) mRNAs have remained obscure. To determine the quantitative expression of the sstr1-5 mRNAs in TSH-secreting adenomas that may be related to the efficacy of treatment with a somatostatin analogue, expression of the sstr1-5 mRNAs was examined and compared in TSH-secreting adenomas and other pituitary adenomas. The pituitary adenomas were obtained at transsphenoidal surgery from 4 cases of TSH-secreting adenoma, including 1 patient showing a significant shrinkage of the tumor size after only 10 days of octreotide treatment, 2 patients without tumor size reduction and 1 patient without treatment, and 5 GH-secreting adenomas, 6 prolactinomas, 5 nonfunctioning adenomas, 4 ACTH-secreting adenomas and normal pituitaries at autopsy from 4 normal subjects. In comparison to the normal pituitary, sstr2A>sstr1>sstr5>sstr3 mRNAs were expressed in the TSH-secreting adenomas examined. No expression of sstr2B or sstr4 mRNA was observed. The expression level of sstr2 mRNA was significantly higher than those in normal pituitary, prolactinomas, ACTH-secreting and nonfunctioning pituitary adenomas. The patient with marked shrinkage of the tumor showed the highest expression of both sstr2 and sstr5 mRNAs among all the cases of pituitary adenoma. A TSH-secreting tumor without shrinkage showed a similar expression level of sstr2 mRNA. These findings demonstrated that TSH-secreting adenomas express sstr1, 2A, 3 and 5 mRNAs, predominantly sstr2A, and in addition to the expression of sstr2 mRNA, the expression level of sstr5 mRNA may be a factor affecting the tumor shrinkage by somatostatin analogues against TSH-secreting adenomas. [Abstract/Link to Full Text]

Chiba Y, Satoh K, Ueda S, Kanazawa N, Tamura Y, Horiuchi T
Marked improvement of psychiatric symptoms after parathyroidectomy in elderly primary hyperparathyroidism.
Endocr J. 2007 Jun;54(3):379-83.
Psychosomatic symptoms in primary hyperparathyroidism (PHPT) are various and include such conditions as obsessive-compulsive disorder, depression, anxiety, and paranoia. In the elderly the clinical features of the disease are often non-specific and difficult to diagnose. To quantify subjective symptoms of patients with hyperparathyroidism in the elderly, we determined whether these clinical manifestations resolved after surgical parathyroidectomy (PTX) in three PHPT patients over eighty years old. They were diagnosed with hypercalcemia, hypophosphatemia, high PTH concentrations, and osteoporosis. A single parathyroid adenoma was confirmed in each patient by Tc-MIBI scintigram, neck ultrasonography and computed tomographic scanning. PTX was performed in these three patients. Assessments of psychologic symptoms, using the Hamilton Rating Scale for Depression (HAM-D), serum calcium, and intact PTH were obtained before and after PTX. Mean weight of the resected adenomas was 438 +/- 138 mg (mean +/- SD). After PTX, serum calcium decreased from 11.1 +/- 0.5 to 9.2 +/- 0.5 mg/dl and intact PTH from 160.0 +/- 25.2 to 45.3 +/- 22.2 pg/ml. Total HAM-D scores in each patient decreased from 45 to 9, 17 to 1 and 15 to 5, respectively. Especially, there were marked improvements in depressive mood, psychomotor inhibition, anxiety and somatic symptoms after PTX. The quality of life in those patients was also improved by PTX. We propose here that PTX in elderly PHPT patients with psychiatric symptoms should be considered instead of oral administration, such as anti-depressants or bisphosphonates. [Abstract/Link to Full Text]

Takahashi N, Kasai H
Exocytic process analyzed with two-photon excitation imaging in endocrine pancreas.
Endocr J. 2007 Jun;54(3):337-46.
To elucidate the spatiotemporal profiles of final secretory stage, we have established two-photon extracellular polar tracer (TEP) imaging, with which we can quantify all exocytic events in the plane of focus within the intact tissues. With such technique, we can estimate the precise diameters of vesicles independently of the spatial resolution of optical microscope, and measure the fusion pore dynamics at nanometer resolution. At insulin exocytosis in the pancreatic islets, it took two seconds for the fusion pore to dilate from 1.4 nm in diameter to 6 nm in diameter, and such unusual stability of the pore may be due to the crystallization of the intragranular contents. Opening of the pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pores, supporting the idea that this structure was mainly composed of membrane lipids. TEP imaging has been also applied to other representative secretory glands, and has revealed hitherto unexpected diversity in spatial organizations of exocytosis and endocytosis, which are relevant for physiology and pathology of secretory tissues. In the pancreatic islet, compound exocytosis was characteristically inhibited (<5%), partly due to the rarity of SNAP25 redistribution into the exocytosed vesicle membrane. Such mechanisms necessitate transport of insulin granules to the cell surface for fusion, and possibly rendering exocytosis more sensitive to metabolic state. Two-photon imaging will be powerful tools to elucidate molecular and cellular mechanisms of exocytosis and related disease, and to develop new therapeutic agencies as well as diagnostic tools. [Abstract/Link to Full Text]

Takahashi S, Tanaka T, Sakai J
New therapeutic target for metabolic syndrome: PPARdelta.
Endocr J. 2007 Jun;54(3):347-57. [Abstract/Link to Full Text]

Yoshimoto T, Hirata Y
Aldosterone as a cardiovascular risk hormone.
Endocr J. 2007 Jun;54(3):359-70.
The pathophysiological role of aldosterone in the development of cardiovascular disease has long been considered to be due its potent volume expansion/hypertensive effect mainly via mineralocorticoid receptor (MR) expressed in renal tubular epithelial cells. However, recent accumulating lines of evidence from clinical and experimental studies have suggested that direct cardiovascular effect of aldosterone contributes to the development of cardiovascular injury via MRs in non-epithelial tissue. A series of recent clinical studies have revealed that patients with primary aldosteronism have higher incidence of cardiovascular and renal complications than those with essential hypertension, and that aldosterone antagonism has cardiovascular protective effect in patients with heart failure independent from blood pressure. Numerous experimental studies have shown that both inflammation and oxidative stress play an initial and key role in the development of aldosterone-induced cardiovascular injury via non-epithelial MR activation. In this review, we discuss recent research progress in aldosterone and MR effects, with special emphasis on the pathophysiological role of aldosterone in cardiovascular diseases and the possible molecular mechanism(s) of cardiovascular injury by non-epithelial MR activation. [Abstract/Link to Full Text]

Hayashi Y, Maeshima K, Goto F, Kojima I
Activin A as a critical mediator of capillary formation: interaction with the fibroblast growth factor action.
Endocr J. 2007 Apr;54(2):311-8.
The present study was conducted to elucidate the role of activin A in capillary formation. When bovine aortic endothelial cells (BAEC) were cultured in a collagen gel, basic fibroblast growth factor (FGF-2) induced tube formation. Activin A also induced tube formation and the addition of two factors together was more effective. BAEC produced both FGF-2 and activin A as autocrine factors. Exogenous FGF-2 did not affect the production of activin A but instead upregulated the type II activin receptor. On the other hand, activin A increased the expression of FGF-2 as well as the FGF receptor. Most importantly, when the action of endogenous activin A was blocked by adding follistatin, the tubulogenic action of FGF-2 was nearly completely inhibited. Activin-induced tubulogenesis was markedly inhibited by overexpression of Smad7, an inhibitory Smad. Similarly, an inhibitor of p44/42 mitogen-activated protein (MAP) kinase attenuated the activin-mediated tubulogenesis, whereas an inhibitor of p38 MAP kinase had no effect. These results indicate that FGF-2 and activin A enhance their signals each other in BAEC, and endogenous activin A is critical for FGF-2-induced capillary formation. [Abstract/Link to Full Text]

Jung TS, Kim TY, Kim KW, Oh YL, Park do J, Cho BY, Shong YK, Kim WB, Park YJ, Jung JH, Chung JH
Clinical features and prognostic factors for survival in patients with poorly differentiated thyroid carcinoma and comparison to the patients with the aggressive variants of papillary thyroid carcinoma.
Endocr J. 2007 Apr;54(2):265-74.
We performed this study to compare the clinicopathologic features and outcomes between the patients with poorly differentiated thyroid carcinoma (PDTC) and the patients with the aggressive variants of papillary thyroid carcinoma (PTC). To evaluate the prognostic factors for survival of the patients with PDTC, we selected 49 patients with PDTC and 23 patients with the aggressive variants of PTC from three hospitals during the recent 15 years. The five-year survival rate and clinicopathologic features of the patients with PDTC were not different from those of the patients with the aggressive variants of PTC. Univariate analysis revealed the significant poor prognostic factors for survival of the patients with PDTC and the aggressive variants of PTC as follows: 1) an age more than 45 years, 2) a tumor size larger than 4 cm, 3) the presence of tumor invasion to extrathyroidal tissue or the trachea, 4) the presence of cervical lymph node invasion, 5) the presence of distant metastasis, 6) the absence of high-dose radioactive iodine (RAI) therapy, and 7) TNM stage II, III and IV. Distant metastasis and high-dose RAI therapy were independent significant predictors for survival of the patients with PDTC and the aggressive variants of PTC on multivariate analysis. However, distant metastasis was the only independent significant predictors for survival of the patients with PDTC excluding patients with the aggressive variants of PTC. [Abstract/Link to Full Text]

Jung HS, Kim KS, Chung YJ, Chung HK, Min YK, Lee MS, Lee MK, Kim KW, Chung JH
USF inhibits cell proliferation through delay in G2/M phase in FRTL-5 cells.
Endocr J. 2007 Apr;54(2):275-85.
Upstream stimulatory factor (USF) has a negative effect on the cell proliferation in some cell types. However, its effect on thyrocytes is not clear. Therefore, we investigated the effects of USF on the proliferation and function of thyroid follicular cells. Complementary DNAs of the USF-1 and USF-2 were synthesized using RT-PCR from FRTL-5 cells, and each was transfected to FRTL-5 cells and papillary thyroid carcinoma cell lines. Cyclic AMP (cAMP) production and [methyl-3H] thymidine uptake after thyroid stimulating hormone (TSH) treatment were measured in FRTL-5 cells. In the carcinoma cell lines, 5-bromo-2'-deoxyuridine (BrdU) uptake was assayed to evaluate cell proliferation. Apoptosis was tested by Hoechst staining and cell cycle analysis was done using a fluorescence activated cell sorting. Expression of cell cycle regulating genes was evaluated by Northern and Western blotting. Overexpression of USF-1 and USF-2 significantly suppressed TSH-stimulated [methyl-3H] thymidine uptake (p<0.05), while it maintained TSH-stimulated cAMP production in FRTL-5 cells. Overexpression of USF significantly suppressed BrdU uptake in each carcinoma cell line, NPA and TPC-1 cells (p<0.05). It induced delay of cell cycle at the G2/M phase, but did not increase apoptosis in FRTL-5 cells. It was accompanied by a decrease of cyclin B1 and cyclin-dependent kinase (CDK)-1, and an increase of p27 expression. USF-1 and USF-2 suppressed cell proliferation of normal thyrocytes and thyroid carcinoma cells. However, they retained the ability to produce cAMP after TSH stimulation. Their inhibitory effect on cell proliferation might be caused partly by the delay in G2/M phase. [Abstract/Link to Full Text]

Saito T, Ikoma A, Saito T, Tamemoto H, Suminaga Y, Yamada S, Kawakami M, Suzuki T, Sasano H, Ishikawa SE
Possibly simultaneous primary aldosteronism and preclinical Cushing's syndrome in a patient with double adenomas of right adrenal gland.
Endocr J. 2007 Apr;54(2):287-93.
We reported a rare case of simultaneous primary aldosteronism and preclinical Cushing's syndrome due to unilateral double adrenocortical adenomas in a 57 year-old woman who had had hypertension for the last 10 years. Abdominal computed tomography showed double tumors in her right adrenal gland. Physical findings revealed simple obesity and hypertension, but no other abnormal findings were detected. Laboratory findings demonstrated that serum potassium was 3.8 mmol/l; plasma renin activity, 0.3 ng/ml/h; plasma aldosterone, 100 pg/ml, and aldosterone renin ratio (ARR), 33. Serum cortisol was 15.7 microg/dl. There was no circadian rhythm of serum cortisol, and no suppression of serum cortisol in response to exogenous dexamethasone administration. Right adrenalectomy was performed under laparoscopy. Two well-circumscribed tumors, whose sizes were 21 and 19 mm in greatest diameter, were detected. They were macroscopically composed of a golden-yellow portion admixed with a brown portion, which corresponded to clear cells and compact cells, respectively. Immunohistochemical staining for steroidogenic enzymes demonstrated the presence of all the enzymes involved in corticosteroidogenesis in these two adenomas, indicating that the two adenomas produced both cortisol and mineralocorticoid. Specifically, one adenoma mainly caused excessive production of cortisol as compared to the other one. These findings indicate that overproduction of both cortisol and mineralocorticoid was evident in the two adenomas of the right adrenal gland in immunohistochemical study for steroidogenic enzymes, whereas there was less clinical manifestation of primary aldosteronism and Cushing's syndrome in the present patient. [Abstract/Link to Full Text]

Sakurai A, Katai M, Yamashita K, Mori J, Fukushima Y, Hashizume K
Long-term follow-up of patients with multiple endocrine neoplasia type 1.
Endocr J. 2007 Apr;54(2):295-302.
Whether early surgical treatment of non-functioning pancreas islet cell tumor (NFPT) provides a favorable quality of life and life expectancy in patients with multiple endocrine neoplasia type 1 (MEN1) remains controversial. We analyzed the long-term clinical courses and surgical outcomes of 14 Japanese patients with MEN1-associated NFPTs. NFPTs smaller than 20 mm in diameter did not show any apparent growth over a long monitoring period. Furthermore, these small NFPTs did not metastasize to regional lymph nodes or the liver. On the other hand, the development of additional NFPTs or metastasis was found in five of six patients with large (35 mm or larger) NFPTs. Among the seven patients who underwent a partial pancreatectomy, six patients developed impaired glucose tolerance or diabetes. The accumulation of more prospective data is needed to clarify the optimal surgical indications for patients with NFPTs, especially among the Japanese population, which has a relatively low insulin secretion potency compared with non-Hispanic white and African-American populations. [Abstract/Link to Full Text]

Sugai M, Ohta A, Ogata Y, Nakanishi M, Ueno S, Kawata T, Saito N, Tanaka Y
Asymmetric dimethylarginine (ADMA) in the aqueous humor of diabetic patients.
Endocr J. 2007 Apr;54(2):303-9.
Asymmetric dimethylarginine (ADMA) is an endogenous NO synthase (NOS) inhibitor whose production is enhanced by oxidative stress. Recent studies have shown that ADMA may also directly stimulate the production of reactive oxygen species (ROS) by up-regulation of the renin-angiotensin system independently of NOS inhibition. In this study, to investigate the clinical association of ADMA with diabetic retinopathy, we evaluated the levels of ADMA and NO oxides (NO2- and NO3-) in serum and aqueous humor obtained during cataract surgery from non-diabetic subjects (n = 21) and diabetic patients (n = 17). We found that the ADMA existed in aqueous humor and its level was similar to that in serum. The ADMA levels in both serum and aqueous humor were higher in diabetic patients, especially those with severe retinopathy, than in the non-diabetic group (serum ADMA: 0.67 +/- 0.26 vs. 0.53 +/- 0.08 micromol/l, p<0.05; aqueous humor ADMA: 0.55 +/- 0.20 vs. 0.32 +/- 0.16 micromol/l, p<0.05). Also, the aqueous humor level of ADMA, but not the serum level, was correlated with HbA1c on analysis of all the patients (R = 0.33, p<0.05 by simple regression analysis). However, a correlation between the ADMA levels in serum and aqueous humor was not observed in either the non-diabetic group or the diabetic group. Furthermore, serum and aqueous humor levels of NOx did not differ between the two groups, and no correlation with ADMA levels was observed in either group. These results suggest that ROS production may be enhanced in the eyes of diabetics. Since ADMA may act to potentiate ROS production independently of its inhibition of NOS, further investigation is required to clarify the possible contribution of ADMA to the development or progression of retinopathy. [Abstract/Link to Full Text]

Hashizume K, Suzuki S, Komatsu A, Hiramatsu K, Mori J, Yamazaki M, Takeda T, Kakizawa T, Miyamoto T, Koizumi Y, Ichikawa K
Administration of recombinant human growth hormone normalizes GH-IGF1 axis and improves malnutrition-related disorders in patients with anorexia nervosa.
Endocr J. 2007 Apr;54(2):319-27.
High serum level of GH in the presence of low plasma level of insulin-like growth factor-I (IGF-I) is one of the endocrinological features of anorexia nervosa (AN). Whether the amount of endogenous GH is not enough to increase IGF-I is not certain. We studied the effect of recombinant human growth hormone (rhGH) on the GH-IGF-I axis and on malnutrition-related disorders in this syndrome. Twenty patients with AN were divided into two groups; one (N = 13) was given rhGH (0.33 mg/day), and the other (N = 7) was given placebo for 6 or 12 months, respectively. During each treatment, levels of serum GH, plasma IGF-I, serum thyroid hormones, serum cholesterol, fasting plasma glucose and cardiac function were monitored. Changes in body mass index (BMI) and calorie taken were also evaluated. Plasma IGF-I level increased from 74.4 +/- 41.9 to 269.0 +/- 31.2 microg/L (P<0.001) during administration of rhGH, which associated with a decrease in serum GH level from 17.0 +/- 15.0 to 1.6 +/- 0.8 microg/L (P<0.001). Administration of rhGH increased BMI, body temperature, fasting plasma glucose level, and food intake. Serum level of triiodothyronine, but not thyroxine, increased during treatment with rhGH. The treatment decreased serum levels of both total and HDL-cholesterol. Studies with echocardiography showed an increase in cardiac output during the treatment with rhGH. These improvements were not observed in patients treated with placebo. Administration of rhGH is recommended as one of the methods of managing the patients with AN. [Abstract/Link to Full Text]

Kahara T, Ueno K, Torita M, Usuda R, Abe T
Deterioration of glycemic control during octreotide LAR treatment in an acromegalic Japanese patient with type 2 diabetes mellitus.
Endocr J. 2007 Apr;54(2):329-33.
We report a case showing deterioration of glycemic control during octreotide long-acting release (LAR) treatment in an acromegalic Japanese patient with type 2 diabetes mellitus. The patient did not show much improvement of insulin sensitivity (QUICKI; 0.33 before treatment, 0.35 during octreotide LAR treatment), and showed a significant reduction in early insulin secretion (insulinogenic index; 0.28 before treatment, 0.08 during octreotide LAR treatment) on 75 g oral glucose tolerance test (75gOGTT), despite decreases in GH and IGF-I levels during the course of octreotide LAR treatment. Postoperatively, both insulin sensitivity and early insulin secretion on 75gOGTT were improved (QUICKI 0.59, insulinogenic index 0.35). There are some reports that insulinogenic index is lower in most Japanese patients with type 2 diabetes mellitus and that early insulin secretions are significantly suppressed after administration of octreotide LAR. Although the influence of octreotide LAR on glucose metabolism varies among individuals, it is necessary to manage the deterioration of glucose tolerance during octreotide LAR treatment in acromegalic Japanese patients with decreased insulinogenic index. [Abstract/Link to Full Text]

Takamura N, Bebeshko V, Aoyagi K, Yamashita S, Saito H
Ukraine urinary iodine levels; 20 years after the Chernobyl accident.
Endocr J. 2007 Apr;54(2):335. [Abstract/Link to Full Text]

Tomioka S, Ogata H, Tamura Y, Shimizu T, Watada H, Fujitani Y, Kawamori R, Hirose T
Clinical characteristics influencing the effectiveness of metformin on Japanese type 2 diabetes receiving sulfonylureas.
Endocr J. 2007 Apr;54(2):247-53.
In this study, we described the effectiveness of metformin on Japanese type 2 diabetes patients receiving sulfonylureas and the clinical characteristics of the patients whose glycemic control were significantly improved with metformin administration. Our results showed that the reduction of glycohemoglobin (HbA1C), serum concentration of total cholesterol, and diastolic blood pressure was statistically significant through the administration of metformin. The clinical characteristics of the patients who responded to metformin therapy exhibited lower systolic blood pressure in addition to higher HbA1C value just before administration of metformin when compared with DeltaHbA1C (HbA1C 6 months after administration of metformin--HbA1C before administration of metformin). Moreover, effectiveness of metformin was weakened, in comparison with non-hypertensive patients, even though the blood pressure of hypertensive patients was reduced to normal range by medication with antihypertensive drugs. But average reduction of HbA1C level of hypertensive patients without antihypertensive medications was smaller than those of patients with high blood pressure with such medication. These results suggested that high blood pressure and hypertension phenotype itself were suppressive factors of metformin but antihypertensive therapy itself enhanced the effectiveness of metformin regardless of the improvement of blood pressure. [Abstract/Link to Full Text]

Kitamura R, Ogata T, Tanaka Y, Motoyoshi K, Seno M, Takei I, Umezawa K, Kojima I
Conophylline and betacellulin-delta4: an effective combination of differentiation factors for pancreatic beta cells.
Endocr J. 2007 Apr;54(2):255-64.
Conophylline and betacellulin-delta4 reproduce differentiation-inducing activity of activin A and betacellulin, respectively. We examined the effect of conophylline and betacellulin-delta4 on beta cell differentiation. In AR42J cells, conophylline and betacellulin-delta4 converted them into insulin-producing cells. Cells treated with conophylline and betacellulin-delta4 continued to grow after differentiation. Thus, cell number and insulin content were much greater compared to cells treated with activin A and betacellulin. Furthermore, cells treated with conophylline and betacellulin-delta4 secreted insulin in response to glucose. Likewise, conophylline and betacellulin-delta4 converted pancreatic ductal cells into insulin-producing cells. Insulin content, cell number and glucose-evoked insulin secretion were significantly greater than those in cells treated with activin A and betacellulin. Transplantation of pseudoislets prepared using ductal cells treated with conophylline and betacellulin-delta4 was able to reduce effectively the plasma glucose concentration in streptozotocin-treated nude mice. Conophylline and betacellulin-delta4 are effective in inducing differentiation of beta cells from progenitors. [Abstract/Link to Full Text]

Miyai K
Congenital thyrotropin deficiency--from discovery to molecular biology, postgenome and preventive medicine.
Endocr J. 2007 Apr;54(2):191-203. [Abstract/Link to Full Text]

Howles CM, Tanaka T, Matsuda T
Management of male hypogonadotrophic hypogonadism.
Endocr J. 2007 Apr;54(2):177-90. [Abstract/Link to Full Text]

Iwasa T, Matsuzaki T, Minakuchi M, Tanaka N, Shimizu F, Hirata Y, Kuwahara A, Yasui T, Maegawa M, Irahara M
Diagnostic performance of serum total testosterone for Japanese patients with polycystic ovary syndrome.
Endocr J. 2007 Apr;54(2):233-8.
It is reported that the incidence of clinical and biochemical hyperandrogenism may be lower in Japanese patients with PCOS. Hyperandrogenism is included as a referential but not as an essential factor in the diagnostic criteria of the Japanese Society of Obstetrics and Gynecology (JSOG 1993). However, some patients with the typical clinical features of PCOS are not diagnosed with PCOS using JSOG 1993 criteria because they do not have a high LH level, which is defined as essential for diagnosis. In this study, we compared total testosterone (T) levels between Japanese patients with PCOS diagnosed using the JSOG 1993 criteria and normal menstrual women (controls). Fifty controls and 46 patients with PCOS were enrolled in this study. Furthermore, we evaluated the sensitivity and specificity of each cut-off value of T. The mean T level of patients with PCOS was significantly higher than that of the control (86 +/- 48 vs 68 +/- 46, P<0.01), and the prevalence rates of hyperandrogenism (T >114 ng/dL; defined as the mean +2SD of the control) were 10.2% in patients with PCOS and 4% in controls. The area under the ROC curve of T was 0.72, and there was no decision threshold to diagnose PCOS by T alone with both high sensitivity and high specificity. If the threshold is set as 110 ng/dL in order to gain high specificity, 94% of women whose serum level passed the threshold will be patients with PCOS. Although T should not be used as an independent essential factor of Japanese PCOS, it might be useful as a complementary factor in order to diagnose patients who have typical clinical features of PCOS but does not fulfill the JSOG 1993 criteria for PCOS. [Abstract/Link to Full Text]

Kaji H, Iida K, Takahashi Y, Okimura Y, Chihara K
Hormone replacement therapy and vascular risk disorders in adult hypopituitarism.
Endocr J. 2007 Apr;54(2):239-45.
Adult patients with hypopituitarism are treated by the replacement of deficient hormones, although GH has not been substituted until March 2006 in Japan except for clinical trial. This study examines which hormonal status influences the prevalence of vascular risk disorders in hypopituitary adults. A sample of 263 adult patients with hypopituitarism was studied, among whom there were various hormonal status such as no deficiency, treated or untreated deficiency of each pituitary hormone. Analysis of adult patients with hypopituitarism showed that hypertension was more prevalent in the older than in younger patients and in male than in female patients. Hypercholesterolemia and hypertriglyceridemia were more prevalent in patients with TSH deficiency even with thyroxine substitution than those without TSH deficiency. Both obesity and hypertension were less prevalent in patients with treated ACTH deficiency than those without ACTH deficiency. Obesity was more prevalent in patients with treated vasopressin deficiency than those without vasopressin deficiency. These results provide evidence that glucocorticoid substitution in ACTH deficient adults was favorable to prevent obesity and hypertension but that the thyroxine substitution in TSH deficient adults appeared rather insufficient to prevent hyperlipidemia. [Abstract/Link to Full Text]

Altinova AE, Toruner F, Bukan N, Yasar DG, Akturk M, Cakir N, Arslan M
Decreased plasma adiponectin is associated with insulin resistance and HDL cholesterol in overweight subjects.
Endocr J. 2007 Apr;54(2):221-6.
The purpose of this study was to investigate plasma adiponectin concentration and its relation with metabolic parameters in overweight and normal weight subjects. The study was carried out in 46 overweight subjects (20 male, 26 female; mean age 39.4 +/- 10.2 years) and 48 (19 male, 29 female; mean age 36.1 +/- 10.6 years) sex- and age-matched normal weight subjects. Anthropometric measurements were recorded and adiponectin, glucose, insulin, lipid profile, total homocysteine (tHcy) and fibrinogen levels were measured. The insulin resistance index was assessed by homeostasis model assessment for insulin resistance (HOMA-IR). Plasma mean adiponectin concentrations of the overweight subjects were significantly lower than those of normal weight subjects (15.0 +/- 4.2 vs 17.3 +/- 5.6 ng/ml) (P<0.05). In overweight subjects, adiponectin levels negatively correlated with body weight (r = -0.35, P<0.001), body mass index (BMI) (r = -0.28, P<0.006), systolic blood pressure (r = -0.21, P<0.04), fasting insulin (r = -0.19, P<0.01) and HOMA-IR (r = -0.20, P<0.01) and positively with high-density lipoprotein cholesterol (HDL-C) (r = 0.27, P<0.009). Overweight subjects with low HDL-C levels had significantly decreased plasma adiponectin levels compared to those with high HDL-C levels (P<0.05). Multiple regression analysis revealed that BMI, HOMA-IR and HDL-C explained 12%, 20% and 15% variance of the adiponectin concentrations. These findings may suggest that circulating adiponectin is associated with insulin resistance and HDL-C levels independent from BMI in overweight subjects. [Abstract/Link to Full Text]

Yang JH, Bae SJ, Park S, Park HK, Jung HS, Chung JH, Min YK, Lee MS, Kim KW, Lee MK
Bilateral pheochromocytoma associated with paraganglioma and papillary thyroid carcinoma: report of an unusual case.
Endocr J. 2007 Apr;54(2):227-31.
A 42-year old woman presented with headache, palpitation and facial flushing. Ultrasonograms and computed tomograms revealed tumors in both of the adrenal glands, anterior aspect of the inferior vena cava, and the right lobe of the thyroid gland. Fine needle aspiration biopsy of the thyroid nodule revealed papillary thyroid carcinoma. Serum calcitonin, CEA, intact PTH and calcium levels were within normal limits. Markedly elevated levels of urinary normetanephrine and vanillylmandelic acid, and the result of 131I-metaiodobenzylguanidine (131I-MIBG) scintigraphy indicated that both adrenal masses were pheochromocytoma. Bilateral adrenalectomy, paracaval mass removal and total thyroidectomy together with central lymph node dissection were performed. The final pathological diagnosis was bilateral adrenal pheochromocytoma, paraganglioma, papillary thyroid carcinoma and either parathyroid adenoma or hyperplasia. Analysis of the RET proto-oncogene mutation, von Hippel Lindau mutation, succinate dehydrogenase subunit B mutation, and succinate dehydrogenase subunit D mutation yielded negative results. The relationship of these lesions could not be determined. This is the first report of a combination of bilateral pheochromocytoma, abdominal paraganglioma, papillary thyroid carcinoma and either parathyroid adenoma or hyperplasia without hyperparathyroidism. [Abstract/Link to Full Text]

Miyakoshi M, Kamoi K, Takano T, Nishihara M, Kawashima T, Sudo N, Togashi K, Emura I, Williams D
Multiple brown tumors in primary hyperparathyroidism caused by an adenoma mimicking metastatic bone disease with false positive results on computed tomography and Tc-99m sestamibi imaging: MR findings.
Endocr J. 2007 Apr;54(2):205-10.
We encountered an unusual case of hyperparathyroidism with both hemosiderin deposits on the ribs and low intensity on T2-weighted magnetic resonance imaging (MRI) caused by a parathyroid adenoma with multiple brown tumors that mimicked metastatic bone tumor due to false positive results on computed tomography (CT) and Tc-99m sestamibi (MIBI) imaging. The patient, a middle-aged woman, had very high serum levels of calcium (14.1 mg/dl), alkaline phosphatase (9,369 IU/l) and intact-PTH (12,400 pg/ml), and a large tumor (2.5 cm in diameter) in the lower portion of the left lobe of the thyroid. Plain X-ray revealed a soft tumor in the left chest wall. On CT scan, there were multiple destructive masses in the ribs, including large intramedullary masses on both 3rd ribs. On MIBI scintigraphy, there was strong late uptake in the lower portion of the left cervical region, both 3rd ribs, and the left 7th, 8th, and 10th ribs. T2-weighted image MRI scans showed that both 3rd ribs had a low intensity with hemosiderin deposits. These findings suggested that the patient had hyperparathyroidism with multiple bone metastases due to carcinoma of the parathyroid gland. However, on pathology, the resected tumor of lower portion of the left lobe of thyroid was diagnosed as a parathyroid adenoma, and the tumors of the left 3rd and 7th ribs, as well as the right 2nd rib, were shown to be brown tumors. After resection, the patient's serum levels of calcium, alkaline phosphatase, and intact-PTH normalized. At 1.5-years follow-up, CT, MIBI, and MRI scans showed no abnormal findings. It is necessary to determine whether MRI can be used to distinguish between brown tumors and metastases caused by carcinoma of the parathyroid gland. [Abstract/Link to Full Text]

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Recent Articles in Minerva Endocrinologica

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Recent Articles in Reproductive Biology and Endocrinology

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Manase K, Endo T, Chida M, Nagasawa K, Honnma H, Yamazaki K, Kitajima Y, Goto T, Kanaya M, Hayashi T, Mitaka T, Saito T
Coordinated elevation of membrane type 1-matrix metalloproteinase and matrix metalloproteinase-2 expression in rat uterus during postpartum involution.
Reprod Biol Endocrinol. 2006;432.
BACKGROUND: The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution. METHODS: We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20). RESULTS: We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2. CONCLUSION: These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes. [Abstract/Link to Full Text]

Rodrigues RF, Carter AM, Ambrosio CE, dos Santos TC, Miglino MA
The subplacenta of the red-rumped agouti (Dasyprocta leporina L).
Reprod Biol Endocrinol. 2006;431.
BACKGROUND: Hystricognath rodents have a lobed placenta, comprising labyrinthine exchange areas and interlobular trophoblast. These correspond to the labyrinthine and spongy zones of other rodent placentae. Beneath them, however, is a structure unique to hystricognath rodents called the subplacenta. We here describe the subplacenta of the red-rumped agouti and examine the possible functional correlates of this structure. METHODS: Placentae were collected from early in midgestation to near term of pregnancy and examined by standard histological techniques, immunohistochemistry and transmission electron microscopy. In addition, to study the microvasculature of the subplacenta, vessel casts were inspected by scanning electron microscopy. RESULTS: In the subplacenta, lamellae of connective tissue support a layer of mononuclear cytotrophoblast cells. Beneath this is found syncytiotrophoblast. Clusters of multinuclear giant cells occur in the transition zone between the subplacenta and decidua. There are prominent intercellular spaces between the cytotrophoblast cells. The basal membrane of these cells is often close to fetal blood vessels. The syncytiotrophoblast surrounds an extensive system of lacunae. Microvilli project into these lacunae from the plasma membrane of the syncytiotrophoblast. The syncytial cytoplasm contains electron-dense granules. This is probably the amylase-resistant PAS-positive material identified by histochemistry. The subplacenta is supplied entirely from the fetal circulation. Within it the vessels pursue a tortuous course with sinusoidal dilatations and constrictions. CONCLUSION: The functions that have been attributed to the subplacenta include hormone production. Our findings are consistent with this interpretation, but suggest that hormone secretion is directed towards the fetal circulation rather than the maternal tissues. [Abstract/Link to Full Text]

Bretveld RW, Thomas CM, Scheepers PT, Zielhuis GA, Roeleveld N
Pesticide exposure: the hormonal function of the female reproductive system disrupted?
Reprod Biol Endocrinol. 2006;430.
Some pesticides may interfere with the female hormonal function, which may lead to negative effects on the reproductive system through disruption of the hormonal balance necessary for proper functioning. Previous studies primarily focused on interference with the estrogen and/or androgen receptor, but the hormonal function may be disrupted in many more ways through pesticide exposure. The aim of this review is to give an overview of the various ways in which pesticides may disrupt the hormonal function of the female reproductive system and in particular the ovarian cycle. Disruption can occur in all stages of hormonal regulation: 1. hormone synthesis; 2. hormone release and storage; 3. hormone transport and clearance; 4. hormone receptor recognition and binding; 5. hormone postreceptor activation; 6. the thyroid function; and 7. the central nervous system. These mechanisms are described for effects of pesticide exposure in vitro and on experimental animals in vivo. For the latter, potential effects of endocrine disrupting pesticides on the female reproductive system, i.e. modulation of hormone concentrations, ovarian cycle irregularities, and impaired fertility, are also reviewed. In epidemiological studies, exposure to pesticides has been associated with menstrual cycle disturbances, reduced fertility, prolonged time-to-pregnancy, spontaneous abortion, stillbirths, and developmental defects, which may or may not be due to disruption of the female hormonal function. Because pesticides comprise a large number of distinct substances with dissimilar structures and diverse toxicity, it is most likely that several of the above-mentioned mechanisms are involved in the pathophysiological pathways explaining the role of pesticide exposure in ovarian cycle disturbances, ultimately leading to fertility problems and other reproductive effects. In future research, information on the ways in which pesticides may disrupt the hormonal function as described in this review, can be used to generate specific hypotheses for studies on the effects of pesticides on the ovarian cycle, both in toxicological and epidemiological settings. [Abstract/Link to Full Text]

Klimaviciute A, Calciolari J, Bertucci E, Abelin-Tornblöm S, Stjernholm-Vladic Y, Byström B, Petraglia F, Ekman-Ordeberg G
Corticotropin-releasing hormone, its binding protein and receptors in human cervical tissue at preterm and term labor in comparison to non-pregnant state.
Reprod Biol Endocrinol. 2006;429.
BACKGROUND: Preterm birth is still the leading cause of neonatal morbidity and mortality. The level of corticotropin-releasing hormone (CRH) is known to be significantly elevated in the maternal plasma at preterm birth. Although, CRH, CRH-binding protein (CRH-BP), CRH-receptor 1 (CRH-R1) and CRH-R2 have been identified both at mRNA and protein level in human placenta, deciduas, fetal membranes, endometrium and myometrium, no corresponding information is yet available on cervix. Thus, the aim of this study was to compare the levels of the mRNA species coding for CRH, CRH-BP, CRH-R1 and CRH-R2 in human cervical tissue and myometrium at preterm and term labor and not in labor as well as in the non-pregnant state, and to localize the corresponding proteins employing immunohistochemical analysis. METHODS: Cervical, isthmic and fundal (from non-pregnant subjects only) biopsies were taken from 67 women. Subjects were divided in 5 groups: preterm labor (14), preterm not in labor (7), term labor (18), term not in labor (21) and non-pregnant (7). Real-time RT-PCR was employed for quantification of mRNA levels and the corresponding proteins were localized by immunohistochemical analysis. RESULTS: The levels of CRH-BP, CRH-R1 and CRH-R2 mRNA in the pregnant tissues were lower than those in non-pregnant subjects. No significant differences were observed between preterm and term groups. CRH-BP and CRH-R2 mRNA and the corresponding proteins were present at lower levels in the laboring cervix than in the non-laboring cervix, irrespective of gestational age. In most of the samples, with the exception of four myometrial biopsies the level of CRH mRNA was below the limit of detection. All of these proteins could be detected and localized in the cervix and the myometrium by immunohistochemical analysis. CONCLUSION: Expression of CRH-BP, CRH-R1 and CRH-R2 in uterine tissues is down-regulated during pregnancy. The most pronounced down-regulation of CRH-BP and CRH-R2 occurred in laboring cervix, irrespective the length of gestation. The detection of substantial expression of the CRH and its receptor proteins, as well as receptor mRNA in the cervix suggests that the cervix may be a target for CRH action. Further studies are required to elucidate the role of CRH in cervical ripening. [Abstract/Link to Full Text]

Hernandez ME, Soto-Cid A, Rojas F, Pascual LI, Aranda-Abreu GE, Toledo R, Garcia LI, Quintanar-Stephano A, Manzo J
Prostate response to prolactin in sexually active male rats.
Reprod Biol Endocrinol. 2006;428.
BACKGROUND: The prostate is a key gland in the sexual physiology of male mammals. Its sensitivity to steroid hormones is widely known, but its response to prolactin is still poorly known. Previous studies have shown a correlation between sexual behaviour, prolactin release and prostate physiology. Thus, here we used the sexual behaviour of male rats as a model for studying this correlation. Hence, we developed experimental paradigms to determine the influence of prolactin on sexual behaviour and prostate organization of male rats. METHODS: In addition to sexual behaviour recordings, we developed the ELISA procedure to quantify the serum level of prolactin, and the hematoxilin-eosin technique for analysis of the histological organization of the prostate. Also, different experimental manipulations were carried out; they included pituitary grafts, and haloperidol and ovine prolactin treatments. Data were analyzed with a One way ANOVA followed by post hoc Dunnet test if required. RESULTS: Data showed that male prolactin has a basal level with two peaks at the light-dark-light transitions. Consecutive ejaculations increased serum prolactin after the first ejaculation, which reached the highest level after the second, and started to decrease after the third ejaculation. These normal levels of prolactin did not induce any change at the prostate tissue. However, treatments for constant elevations of serum prolactin decreased sexual potency and increased the weight of the gland, the alveoli area and the epithelial cell height. Treatments for transient elevation of serum prolactin did not affect the sexual behaviour of males, but triggered these significant effects mainly at the ventral prostate. CONCLUSION: The prostate is a sexual gland that responds to prolactin. Mating-induced prolactin release is required during sexual encounters to activate the epithelial cells in the gland. Here we saw a precise mechanism controlling the release of prolactin during ejaculations that avoid the detrimental effects produced by constant levels. However, we showed that minor elevations of prolactin which do not affect the sexual behaviour of males, produced significant changes at the prostate epithelium that could account for triggering the development of hyperplasia or cancer. Thus, it is suggested that minute elevations of serum prolactin in healthy subjects are at the etiology of prostate abnormal growth. [Abstract/Link to Full Text]

Abe Y, Sinozaki H, Takagi T, Minegishi T, Kokame K, Kangawa K, Uesaka M, Miyamoto K
Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes in human amniotic epithelial cells.
Reprod Biol Endocrinol. 2006;427.
BACKGROUND: Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated primary cultures of human amniotic epithelial cells. METHODS: Human amniotic epithelial cells were dispersed by trypsin from amniotic membranes and cultured in DME/Ham's F12 medium supplemented with 10% FBS. Two weeks after plating, cells were treated with 50 nM TCDD or DMSO (control), further incubated for 48 hrs, and the gene expression was analyzed by DNA microarray technology and quantitative real-time PCR. RESULTS: Thirty eight TCDD-inducible genes, including cytochromeP4501A1 and cytochromeP4501B1, were identified. One of the remarkable profiles of the gene expression was the prominent up-regulation of interferon-inducible genes. The genes involved in the interferon gene expression and interferon signaling pathways were also up-regulated. Furthermore, the expression of genes related to collagen synthesis or degradation was enhanced by TCDD. CONCLUSION: Using DNA microarray and quantitative real-time PCR analyses, we identified TCDD-inducible genes, including interferon-inducible genes and genes related to collagen synthesis or degradation, in human amniotic epithelial cells. [Abstract/Link to Full Text]

Tosti E
Calcium ion currents mediating oocyte maturation events.
Reprod Biol Endocrinol. 2006;426.
During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. [Abstract/Link to Full Text]

Simard M, Provost PR, Tremblay Y
Sexually dimorphic gene expression that overlaps maturation of type II pneumonocytes in fetal mouse lungs.
Reprod Biol Endocrinol. 2006;425.
BACKGROUND: In human, respiratory distress of the neonates, which occurs in prematurity, is prevalent in male. Late in gestation, maturation of type II pneumonocytes, and consequently the surge of surfactant synthesis are delayed in male fetuses compared with female fetuses. Although the presence of higher levels of androgens in male fetuses is thought to explain this sex difference, the identity of genes involved in lung maturation that are differentially modulated according to fetal sex is unknown. We have studied the sex difference in developing mouse lung by gene profiling during a three-day gestational window preceding and including the emergence of mature PTII cells (the surge of surfactant synthesis in the mouse occurs on GD 17.5). METHODS: Total RNA was extracted from lungs of male and female fetal mice (gestation days 15.5, 16.5, and 17.5), converted to cRNA, labeled with biotin, and hybridized to oligonucleotide microarrays (Affymetrix MOE430A). Analysis of data was performed using MAS5.0, LFCM and Genesis softwares. RESULTS: Many genes involved in lung maturation were expressed with no sex difference. Of the approximative 14,000 transcripts covered by the arrays, only 83 genes presented a sex difference at one or more time points between GDs 15.5 and 17.5. They include genes involved in hormone metabolism and regulation (i.e. steroidogenesis pathways), apoptosis, signal transduction, transcriptional regulation, and lipid metabolism with four apolipoprotein genes. Genes involved in immune functions and other metabolisms also displayed a sex difference. CONCLUSION: Among these sexually dimorphic genes, some may be candidates for a role in lung maturation. Indeed, on GD 17.5, the sex difference in surfactant lipids correlates with the sex difference in pulmonary expression of apolipoprotein genes, which are involved in lipid transport. This suggests a role for these genes in the surge of surfactant synthesis. Our results would help to identify novel genes involved in the physiopathology of the respiratory distress of the neonates. [Abstract/Link to Full Text]

Cluff AH, Byström B, Klimaviciute A, Dahlqvist C, Cebers G, Malmström A, Ekman-Ordeberg G
Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue.
Reprod Biol Endocrinol. 2006;424.
BACKGROUND: Prolonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour. METHODS: Uterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy. RESULTS: In women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to non-pregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy. CONCLUSION: The high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility. [Abstract/Link to Full Text]

Yenugu S, Hamil KG, Grossman G, Petrusz P, French FS, Hall SH
Identification, cloning and functional characterization of novel sperm associated antigen 11 (SPAG11) isoforms in the rat.
Reprod Biol Endocrinol. 2006;423.
BACKGROUND: Sperm binding proteins and their C-terminal peptides of the Sperm Associated Antigen 11 (SPAG11) family were found to play an important role in epididymal innate immunity in addition to their role in sperm maturation. However, the expression of Spag11 transcripts in rodents is not well documented. METHODS: Computational analysis was employed to identify novel Spag11 isoforms in the rat. RT-PCR analyses were carried out on RNAs isolated from the male reproductive tract tissues of rat using gene specific primers for Spag11c and Spag11t. The identities of PCR products were confirmed by sequencing. Tissue distribution, developmental expression and androgen regulation of Spag11t and Spag11c were studied using RT-PCR. The antimicrobial activities of recombinant Spag11t and Spag11c were tested against E coli in a colony forming unit assay. RESULTS: In this study, we identified two novel Spag11 transcripts, namely, Spag11t and Spag11c derived from the long arm of chromosome 16 in the rat (Rattus norvegicus), using both in silico and molecular biology approaches. Spag11c is expressed in all three regions of the epididymis, in testis and in ovary but is absent from the seminal vesicle. Spag11t expression is confined to the caput and it is not expressed in the testis, seminal vesicle or ovary. Age dependent expression of Spag11t and Spag11c was observed in the epididymides of rats (10-60 day old). Their expression was found to be most abundant in the adult rat (60 day) suggesting roles in mature reproductive function. Further, both Spag11t and Spag11c expression was down regulated in castrated rat epididymides and the expression was maintained in the testosterone replaced castrated rats. SPAG11C is a potent antibacterial agent. SPAG11T also displayed bactericidal capacity although weaker than SPAG11C and SPAG11E. CONCLUSION: The abundant expression of Spag11t and Spag11c in the male reproductive tract suggests an important role in male reproductive tract immunity. Their expression is developmentally regulated and androgen dependent. Characterization of novel SPAG11 isoforms will contribute to our understanding of the role of epididymal proteins in sperm maturation and innate immunity. [Abstract/Link to Full Text]

Kobayashi Y, Jimenez-Krassel F, Ireland JJ, Smith GW
Evidence of a local negative role for cocaine and amphetamine regulated transcript (CART), inhibins and low molecular weight insulin like growth factor binding proteins in regulation of granulosa cell estradiol production during follicular waves in cattle.
Reprod Biol Endocrinol. 2006;422.
The ability of ovarian follicles to produce large amounts of estradiol is a hallmark of follicle health status. Estradiol producing capacity is lost in ovarian follicles before morphological signs of atresia. A prominent wave like pattern of growth of antral follicles is characteristic of monotocous species such as cattle, horses and humans. While our knowledge of the role of pituitary gonadotropins in support of antral follicle growth and development is well established, the intrinsic factors that suppress estradiol production and may help promote atresia during follicular waves are not well understood. Numerous growth factors and cytokines have been reported to suppress granulosa cell estradiol production in vitro, but the association of expression of many such factors in vivo with follicle health status and their physiological significance are not clear. The purpose of this review is to discuss the in vivo and in vitro evidence supporting a local physiological role for cocaine and amphetamine regulated transcript, inhibins and low molecular weight insulin like growth factor binding proteins in negative regulation of granulosa cell estradiol production, with emphasis on evidence from the bovine model system. [Abstract/Link to Full Text]

Kaivo-oja N, Jeffery LA, Ritvos O, Mottershead DG
Smad signalling in the ovary.
Reprod Biol Endocrinol. 2006;421.
It has now been a decade since the first discovery of the intracellular Smad proteins, the downstream signalling molecules of one of the most important growth factor families in the animal kingdom, the transforming growth factor beta (TGF-beta) superfamily. In the ovary, several TGF-beta superfamily members are expressed by the oocyte, granulosa and thecal cells at different stages of folliculogenesis, and they signal mainly through two different Smad pathways in an autocrine/paracrine manner. Defects in the upstream signalling cascade molecules, the ligands and receptors, are known to have adverse effects on ovarian organogenesis and folliculogenesis, but the role of the individual Smad proteins in the proper function of the ovary is just beginning to be understood for example through the use of Smad knockout models. Although most of the different Smad knockouts are embryonic lethal, it is known, however, that in Smad1 and Smad5 knockout mice primordial germ cell development is impaired and that Smad3 deficient mice harbouring a deletion in exon 8 exhibit impaired folliculogenesis and reduced fertility. In this minireview we discuss the role of Smad structure and function in the ovarian context. [Abstract/Link to Full Text]

Fabre S, Pierre A, Mulsant P, Bodin L, Di Pasquale E, Persani L, Monget P, Monniaux D
Regulation of ovulation rate in mammals: contribution of sheep genetic models.
Reprod Biol Endocrinol. 2006;420.
Ovarian folliculogenesis in mammals from the constitution of primordial follicles up to ovulation is a reasonably well understood mechanism. Nevertheless, underlying mechanisms that determine the number of ovulating follicles were enigmatic until the identification of the fecundity genes affecting ovulation rate in sheep, bone morphogenetic protein-15 (BMP-15), growth and differentiation factor-9 (GDF-9) and BMP receptor-1B (BMPR-1B). In this review, we focus on the use of these sheep genetic models for understanding the role of the BMP system as an intra-ovarian regulator of follicular growth and maturation, and finally, ovulation rate. [Abstract/Link to Full Text]

Thomas FH, Vanderhyden BC
Oocyte-granulosa cell interactions during mouse follicular development: regulation of kit ligand expression and its role in oocyte growth.
Reprod Biol Endocrinol. 2006;419.
Ovarian folliculogenesis is regulated by both endocrine and intraovarian mechanisms that coordinate the processes of oocyte growth and somatic cell proliferation and differentiation. Within the follicle, paracrine interactions between the oocyte and surrounding granulosa cells are critical for normal cell development and function. This review focuses on the role of paracrine interactions during early oocyte and follicular development that ensure proper coordination of oocyte and somatic cell function. Particular emphasis is given to granulosa cell-derived Kit Ligand (KitL), whose functional importance for oocyte growth has been demonstrated by a wide range of in vivo and in vitro studies. Reported interactions between KitL and oocyte-derived growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) suggest the molecular basis of oocyte-granulosa cell interactions, but also hint at the complexity of these communications. These paracrine interactions and the structure of the oocyte-granulosa cell interface are follicle stage-specific and regulated by FSH. Elucidation of the molecular mechanisms that promote the development of healthy oocytes with good developmental competence has potential applications for improving fertility and for in vitro growth systems for oocytes from domestic animals and humans. [Abstract/Link to Full Text]

Fraser HM
Regulation of the ovarian follicular vasculature.
Reprod Biol Endocrinol. 2006;418.
Angiogenesis is associated with follicular development and is regulated independently within each follicle potentially making the functioning of its vasculature critically important in determining its fate. This review examines the various ways in which follicular angiogenesis may be monitored, describes the follicular localisation and changes in pro- and anti-angiogenic factors that may regulate the process and how antagonists may be used to elucidate their physiological role in vivo. Thus, inhibition of vascular endothelial growth factor (VEGF), VEGF receptor-2, vascular endothelial cell cadherin or interference with the angiopoietin system can inhibit follicular development or prevent ovulation. [Abstract/Link to Full Text]

Abbott DH, Padmanabhan V, Dumesic DA
Contributions of androgen and estrogen to fetal programming of ovarian dysfunction.
Reprod Biol Endocrinol. 2006;417.
In female mammals, including humans, deviations from normal androgenic or estrogenic exposure during fetal development are detrimental to subsequent adult ovarian function. Androgen deficiency, without accompanying estrogen deficit, has little apparent impact on ovarian development. Fetal estrogen deficiency, on the other hand, results in impaired oocyte and follicle development, immature and abnormal adult ovaries, and excessive ovarian stimulation from endogenous gonadotropins ultimately generating hemorrhagic follicles. Complete estrogen deficiency lasting into adulthood results in partial ovarian masculinization. Fetal androgen excess, on the other hand, mediated either by direct androgen action or following androgen aromatization to estrogen, reprograms ovarian development and reproductive neuroendocrinology to mimic that found in women with polycystic ovary syndrome: enlarged, polyfollicular, hyperandrogenic, anovulatory ovaries with accompanying LH hypersecretion. Oocyte developmental competence is also compromised. Insulin is implicated in the mechanism of both anovulation and deficient oocyte development. Fetal estrogen excess induces somewhat similar disruption of adult ovarian function to fetal androgen excess. Understanding the quality of the fetal female sex steroid hormone environment is thus becoming increasingly important in improving our knowledge of mechanisms underlying a variety of female reproductive pathologies. [Abstract/Link to Full Text]

Drummond AE
The role of steroids in follicular growth.
Reprod Biol Endocrinol. 2006;416.
The steroidogenic pathway within the ovary gives rise to progestins, androgens and oestrogens, all of which act via specific nuclear receptors to regulate reproductive function and maintain fertility. The role of progestins in follicular growth and development is limited, its action confined largely to ovulation, although direct effects on granulosa cell function have been reported. Consistent with these findings, progesterone receptor knockout mice are infertile because they cannot ovulate. Androgens have been shown to promote early follicular growth, but also to impede follicular development by stimulating atresia and apoptosis. The inability of androgens to transduce a signal in mice lacking androgen receptors culminates in reduced fertility. Oestrogens are known to exert effects on granulosa cell growth and differentiation in association with gonadotrophins. Studies with oestrogen receptor knockouts and oestrogen depleted mice have shown us that oestrogen is essential for folliculogenesis beyond the antral stage and is necessary to maintain the female phenotype of ovarian somatic cells. In summary, the action of steroids within the ovary is based on the developmental status of the follicle. In the absence of any single sex steroid, ovarian function and subsequently fertility, are compromised. [Abstract/Link to Full Text]

Yin H, Duffy DM, Gosden RG
Comparative maturation of cynomolgus monkey oocytes in vivo and in vitro.
Reprod Biol Endocrinol. 2006;414.
BACKGROUND: In vitro maturation (IVM) of oocytes followed by fertilization in vitro (IVF) and embryo transfer offers an alternative to conventional IVF treatment that minimises drug administration and avoids ovarian hyperstimulation. However, the technique is less efficient than maturation in vivo. In the present study, a non-human primate model was used to address the hypothesis that the number of oocytes is increased and their nuclear and cytoplasmic maturity after IVM are improved when maturation is initiated in vivo by priming with hCG. METHODS: Young, adult cynomolgus monkeys were given recombinant human (rh) gonadotropins to stimulate the development of multiple follicles, and oocytes were aspirated 0, 12, 24, or 36 h after injection of an ovulatory dose of rhCG. The nuclear status of oocytes was determined at the time of recovery and after culture for a total elapsed time of 40-44 hours after hCG. RESULTS: Priming with hCG significantly increased the number of oocytes harvested, especially after delaying aspiration for 24 h or longer. Nuclear maturation after the full period in culture was also enhanced by priming: 71.5, 83.6, and 94.6% of oocytes collected at 0, 12, and 24 h hCG had progressed to MII by the end of the culture period, compared to 87.8% of oocytes that were retrieved at 36 h. A large proportion of oocytes reaching the MII stage had either or both abnormal spindles (>40%) and misaligned chromosomes (>60%), judging by immunofluorescence microscopy, but these abnormalities were independent of culture time. The mitochondria were evenly distributed throughout the cytoplasm at all stages of maturation. Importantly, there was no microscopic evidence that the duration of culture had any injurious effects on the cells. CONCLUSION: In conclusion, the evidence supports this non-human primate as a model for human IVM and the practice of priming with hCG to promote developmental potential. [Abstract/Link to Full Text]

Al-Bader MD
Estrogen receptors alpha and beta in rat placenta: detection by RT-PCR, real time PCR and Western blotting.
Reprod Biol Endocrinol. 2006;413.
BACKGROUND: High levels of estrogens during pregnancy not only retard placental and fetal growth but can lead to reproductive tract abnormalities in male progeny. Estrogens act through estrogen receptors (ER) to modulate the transcription of target genes. These ER exist in two isoforms, ER alpha and ER beta and recently several variants of these isoforms have been identified. METHODS: The expressions of ER isoforms and variants have been studied in rat placenta at 16, 19 and 21 days gestation (dg). Gene expression was assessed using RT-PCR and real time PCR while protein expression was studied using Western blotting followed by immunodetection. Placental homogenates were probed with: a monoclonal antibody raised against the steroid binding domain of the ER alpha (ER alpha -S), a monoclonal antibody raised against the hinge region of ER alpha (ER alpha -H) and a polyclonal antibody raised against the amino terminus of ER beta. RESULTS: ER alpha and ER beta mRNA and protein were detected from as early as 16 dg. Two PCR products were detected for ER alpha, one for the wild type ER alpha, and a smaller variant. Real time PCR results suggested the presence of a single product for ER beta. The antibodies used for detection of ER alpha protein both identified a single 67 kDa isoform; however a second 54 kDa band, which may be an ER alpha variant, was identified when using the ER alpha -H antibody. The abundance of both ER alpha bands decreased significantly between 16 and 19 dg. As for ER beta, four bands (76, 59, 54 and 41 kDa) were detected. The abundance of the 59 and 54 kDa bands decreased significantly between 16 and 19 dg. CONCLUSION: This study shows that both ER protein isoforms and their variants are present in rat placenta. The decrease in their expression near parturition suggests that the placenta may be relatively unresponsive to estrogens at this stage. [Abstract/Link to Full Text]

Uzbekova S, Roy-Sabau M, Dalbiès-Tran R, Perreau C, Papillier P, Mompart F, Thelie A, Pennetier S, Cognie J, Cadoret V, Royere D, Monget P, Mermillod P
Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells.
Reprod Biol Endocrinol. 2006;412.
BACKGROUND: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. METHODS: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. RESULTS: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. CONCLUSION: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes. [Abstract/Link to Full Text]

Flores A, Rodríguez JO, Palafox MT, Meléndez G, Barco AI, Chavira R, Cruz ME, Domínguez R
The acute asymmetric effects of hemiovariectomy on testosterone secretion vary along the estrous cycle. The participation of the cholinergic system.
Reprod Biol Endocrinol. 2006;411.
The presence of asymmetry in the capacity of the left and right ovaries to secrete testosterone was analyzed by studying the effects of hemiovariectomy along the estrus cycle one hour after surgery. The effects of ether anesthesia on hormone serum levels were also analyzed. Bilateral ovariectomy and the extirpation of the left ovary performed on the day of proestrus resulted in significantly lower testosterone levels. Compared to the anesthetized group, the effects of perforating the peritoneum unilaterally varied according to the day of the estrous cycle and the side of the peritoneum surgery was performed on. Injecting atropine sulfate (ATR) to control or anesthetized rats on D1 resulted in a significant increase of testosterone serum levels. The effects of perforating the peritoneum on testosterone levels depended on the cholinergic innervation and varied along the estrous cycle. Blocking the cholinergic system before performing unilateral or bilateral ovariectomy had different effects depending on the day of the estrous cycle. Testosterone plasma levels increased significantly when surgery was performed on the day of diestrus and dropped when surgery was performed on proestrus. Similar effects were observed when the left adrenal was extirpated from animals with the cholinergic system blocked. The results presented herein support the hypothesis of asymmetry in the ovaries' abilities to secrete steroid hormones, and that the capacity to secrete testosterone varies along the estrous cycle. [Abstract/Link to Full Text]

McIntyre MH
The use of digit ratios as markers for perinatal androgen action.
Reprod Biol Endocrinol. 2006;410.
Since the ratio of the second-to-fourth finger length was first proposed as a marker for prenatal androgen action in 1998, over 100 studies have been published that have either further tested the association between the digit ratio and prenatal androgens, or employed digit ratios as a marker to investigate the association between prenatal androgens and a variety of outcomes, including behavior, fertility, and disease risks. Despite the clear demand for an adult marker of prenatal androgen action and increased use of digit ratios as such a marker, its validity remains controversial. This review (1) evaluates current evidence for the relationship between digit ratios and prenatal androgens (using experimentation with animal models, amniotic testosterone, and congenital adrenal hyperplasia case-control studies), (2) describes opportunities for future validation tests, and (3) compares the potential advantages and disadvantages of digit ratio measures with more established methods for studying the effects of prenatal androgens. [Abstract/Link to Full Text]

Jana K, Jana S, Samanta PK
Effects of chronic exposure to sodium arsenite on hypothalamo-pituitary-testicular activities in adult rats: possible an estrogenic mode of action.
Reprod Biol Endocrinol. 2006;49.
BACKGROUND: Inorganic arsenic is a major water pollutant and a known human carcinogen that has a suppressive influence on spermatogenesis and androgenesis in male reproductive system. However, the actual molecular events resulting in male reproductive dysfunctions from exposure to arsenic remain unclear. In this context, we evaluated the mode of action of chronic oral exposure of sodium arsenite on hypothalamo-pituitary- testicular activities in mature male albino rats. METHODS: The effect of chronic oral exposure to sodium arsenite (5 mg/kg body weight/day) via drinking water without or with hCG (5 I.U./kg body weight/day) and oestradiol (25 micrograms oestradiol 3-benzoate suspended in 0.25 ml olive oil/rat/day) co-treatments for 6 days a week for 4 weeks (about the duration of two spermatogenic cycle) was evaluated in adult male rats. Changes in paired testicular weights, quantitative study of different varieties of germ cells at stage VII of spermatogenic cycle, epididymal sperm count, circulatory concentrations of hormones (LH, FSH, testosterone and corticosterone), testicular activities of delta 5, 3beta-hydroxysteroid dehydrogenase (delta 5, 3beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), sorbitol dehydrogenase (SDH), acid phosphatase (ACP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH), as well as the levels of biogenic amines (dopamine, noradrenaline and 5-hydroxytryptamine (5-HT)) in the hypothalamus and pituitary were monitored in this study. Hormones were assayed by radioimmuno- assay or enzyme- linked immunosorbent assay and the enzymes were estimated after spectrophotometry as well as the biogenic amines by HPLC electrochemistry. RESULTS: Sodium arsenite treatment resulted in: decreased paired testicular weights; epididymal sperm count; plasma LH, FSH, testosterone and testicular testosterone concentrations; and increased plasma concentration of corticosterone. Testicular enzymes such as delta 5, 3 beta-HSD, 17 beta-HSD, and sorbitol dehydrogenase (SDH) were significantly decreased, but those of acid phosphatase (ACP), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were significantly increased. A decrease in dopamine or an increase in noradrenaline and 5-HT in hypothalamus and pituitary were also noted after arsenic exposure. Histological evaluation revealed extensive degeneration of different varieties of germ cells at stage VII of spermatogenic cycle in arsenic exposed rats. Administration of human chorionic gonadotrophin (hCG) along with sodium arsenite partially prevented the degeneration of germ cells and enhanced paired testicular weights, epididymal sperm count, plasma and intratesticular testosterone concentrations, activities of delta 5, 3beta-HSD, 17 beta-HSD and sorbitol dehydrogenase along with diminution in the activities of ACP, ALP and LDH. Since many of the observed arsenic effects could be enhanced by oestradiol, it is suggested that arsenic might somehow acts through an estrogenic mode of action. CONCLUSION: The results indicate that arsenic causes testicular toxicity by germ cell degeneration and inhibits androgen production in adult male rats probably by affecting pituitary gonadotrophins. Estradiol treatment has been associated with similar effects on pituitary testicular axis supporting the hypothesis that arsenite might somehow act through an estrogenic mode of action. [Abstract/Link to Full Text]

Carlin R, Davis D, Weiss M, Schultz B, Troyer D
Expression of early transcription factors Oct4, Sox2 and Nanog by porcine umbilical cord (PUC) matrix cells.
Reprod Biol Endocrinol. 2006 Feb 6;4(1):8.
ABSTRACT: BACKGROUND: Three transcription factors that are expressed at high levels in embryonic stem cells (ESCs) are Nanog, Oct-4 and Sox-2. These transcription factors regulate the expression of other genes during development and are found at high levels in the pluripotent cells of the inner cell mass. The downregulation of these three transcription factors correlates with the loss of pluripotency and self-renewal, and the beginning of subsequent differentiation steps. The roles of Nanog, Oct-4 and Sox-2 have not been fully elucidated. They are important in embryonic development and maintenance of pluripotency in ESCs. We studied the expression of these transcription factors in porcine umbilical cord (PUC) matrix cells. METHODS: Cells were isolated from Wharton's jelly of porcine umbilical cords (PUC) and histochemically assayed for the presence of alkaline phosphatase and the presence of Nanog, Oct-4 and Sox-2 mRNA and protein. PCR amplicons were sequenced and compared with known sequences. The synthesis of Oct-4 and Nanog protein was analyzed using immunocytochemistry. FACS analysis was utilized to evaluate Hoechst 33342 dye-stained cells. RESULTS: PUC isolates were maintained in culture and formed colonies that express alkaline phosphatase. FACS analysis revealed a side population of Hoechst dye-excluding cells, the Hoechst exclusion was verapamil sensitive. Quantitative and non-quantitative RT-PCR reactions revealed expression of Nanog, Oct-4 and Sox-2 in day 15 embryonic discs, PUC cell isolates and porcine fibroblasts. Immunocytochemical analysis detected Nanog immunoreactivity in PUC cell nuclei, and faint labeling in fibroblasts. Oct-4 immunoreactivity was detected in the nuclei of some PUC cells, but not in fibroblasts. CONCLUSIONS: Cells isolated from PUC matrix express three transcription factors found in pluripotent stem cell markers both at the mRNA and protein level. The presence of these transcription factors, along with the other characteristics of PUC cells such as their colony-forming ability, Hoechst dye-excluding side population and alkaline phosphatase expression, suggests that PUC cells have properties of primitive pluripotent stem cells. Furthermore, PUC cells are an easily and inexpensively obtained source of stem cells that are not hampered by the ethical or legal issues associated with ESCs. In addition, these cells can be cryogenically stored and expanded. [Abstract/Link to Full Text]

Yenugu S, Chintalgattu V, Wingard CJ, Radhakrishnan Y, French FS, Hall SH
Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus).
Reprod Biol Endocrinol. 2006;47.
BACKGROUND: Beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation. METHODS: In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118-123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities. RESULTS: Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10-60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity. CONCLUSION: Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis. [Abstract/Link to Full Text]

Pericuesta E, Ramírez MA, Villa-Diaz A, Relaño-Gines A, Torres JM, Nieto M, Pintado B, Gutiérrez-Adán A
The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals.
Reprod Biol Endocrinol. 2006;45.
BACKGROUND: The reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity. RESULTS: Three constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells. CONCLUSION: The 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues. [Abstract/Link to Full Text]

Wang HF, Isobe N, Kumamoto K, Yamashiro H, Yamashita Y, Terada T
Studies of the role of steroid hormone in the regulation of oocyte maturation in cattle.
Reprod Biol Endocrinol. 2006;44.
BACKGROUND: The objective of this study was to investigate whether the steroid hormone(s) secreted from cumulus-oocyte complexes (COCs) is a prerequisite for bovine oocyte maturation and cumulus expansion using aminoglutethimide (AGT), a P450 cholesterol side-chain cleavage inhibitor. METHODS: In experiment 1, COCs were cultured in maturation medium with various concentrations of AGT for 22 h to determine the effective concentration of AGT to inhibit steroid hormone secretion, meiotic maturation and cumulus expansion. In experiment 2, COCs were cultured in conditioned medium (CM) and TCM-199 medium with or without 10 mM AGT to check whether steroid hormones secreted from COCs were responsible for oocyte maturation and cumulus expansion. Experiments 3 and 4 were carried out to determine whether exogenous progesterone or estradiol-17beta was able to overcome the inhibitory effects of AGT on oocytes maturation and cumulus expansion. COCs cultured in 10 mM AGT-containing medium supplemented with various concentrations of progesterone or estradiol-17beta for 22 h were examined for oocyte maturation and cumulus expansion. RESULTS: Experiment 1 showed that a concentration of 10 mM AGT in medium was sufficient to block steroid hormone secretion, oocyte maturation and cumulus expansion, and that these inhibitory effects were fully reversible. In experiment 2, the addition of 10 mM AGT to CM did not significantly prevent oocyte maturation and cumulus expansion, implying that CM contains the steroid hormone(s) secreted from COCs, which are closely associated with oocyte maturation and cumulus expansion. The results in experiments 3 and 4 demonstrated that the addition of any concentration of progesterone or estradiol-17beta in the medium did not reduce the inhibitory effects of AGT on oocyte maturation and cumulus expansion. CONCLUSION: Our results indicate that bovine oocytes surrounded by cumulus cells are prevented from maturation and cumulus expansion through the inhibition of steroid secretion due to AGT, and that these inhibitory effects of AGT on oocyte maturation and cumulus expansions can not be overcome by the addition of either progesterone or estradiol-17beta in the medium. These observations suggest that some steroid hormone(s) other than P4 and E2 secreted from bovine COCs is essential for their meiotic maturation and cumulus expansion. [Abstract/Link to Full Text]

Mourot B, Nguyen T, Fostier A, Bobe J
Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns.
Reprod Biol Endocrinol. 2006;46.
BACKGROUND: In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. METHODS: In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. RESULTS: Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. CONCLUSION: Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels. [Abstract/Link to Full Text]

Browne RK, Li H, Seratt J, Kouba A
Progesterone improves the number and quality of hormone induced Fowler toad (Bufo fowleri) oocytes.
Reprod Biol Endocrinol. 2006 Feb 1;4(1):3.
ABSTRACT: Combinations of progesterone, lutenizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotrophin (hCG), and the dopamine-2 (DA2) receptor antagonist 1-[1-[4,4-bis(4-Fluorophenyl)butyl]-4-piperidinyl]-1,3-dihydro-2H-benzimidazol-2-one (Pimozide; Orap) were tested for improvement of spawning rates, oocyte numbers, fertilization and neurulation rates of the Fowler toad (Bufo fowleri). Only treatments combined with progesterone produced large numbers of oocytes. The best treatment on oocyte numbers, neurulation rates, and the number of neurulas was with 5 mg progesterone, 20 mic.g LHRHa, and 0.25 mg Pimozide. Progesterone (5 mg) with 60 mic.g LHRHa gave high spawning rates, oocyte numbers, and fertilization rates but neurulation rates were low. Progesterone alone in high repeated doses did not result in ovulation. High doses of LHRHa (60 mic.g) with hCG, progesterone, and Pimozide gave the greatest number of toads spawning, however, they resulted in low oocyte numbers, fertilization and neurulation rates. A low dose of LHRHa (4 mic.g) with hCG, or hCG alone as a second administration, and progesterone with Pimozide produced few good quality oocytes. Toads were given normal ovulatory doses of hormones 24 or 48 hrs after their initial dose, but these resulted in low oocyte numbers followed by poor fertilization. Overall, these results suggest that progesterone with a dose between 20 mic.g and 60 mic.g of LHRHa may be optimal for the induction of ovulation in these toads. Moreover, Pimozide can supplement low doses of LHRHa but not replace it. [Abstract/Link to Full Text]

Schatz F, Krikun G, Baergen RN, Critchley HO, Kuczynski E, Lockwood CJ
Intercellular adhesion molecule-1 expression in human endometrium: implications for long term progestin only contraception.
Reprod Biol Endocrinol. 2006;42.
BACKGROUND: Neutrophils infiltrate the endometrium pre-menstrually and after long-term progestin only-contraceptive (LTPOC) treatment. Trafficking of neutrophils involves endothelial cell-expressed intercellular adhesion molecule (ICAM-1). Previous studies observed that ICAM-1 was immunolocalized to the endothelium of endometrial specimens across the menstrual cycle, but disagreed as to whether extra-endothelial cell types express ICAM-1 and whether ICAM-1 expression varies across the menstrual cycle. METHODS: Endometrial biopsies were obtained from women across the menstrual cycle and from those on LTPOC treatment (either Mirena or Norplant). The biopsies were formalin-fixed and paraffin-embedded with subsequent immunohistochemical staining for ICAM-1. RESULTS: The current study found prominent ICAM-1 staining in the endometrial endothelium that was of equivalent intensity in different blood vessel types irrespective of the steroidal or inflammatory endometrial milieu across the menstrual cycle and during LTPOC therapy. Unlike the endothelial cells, the glands were negative and the stromal cells were weakly positive for ICAM immunostaining. CONCLUSION: The results of the current study suggest that altered expression of ICAM-1 by endothelial cells does not account for the influx of neutrophils into the premenstrual and LTPOC-derived endometrium. Such neutrophil infiltration may depend on altered expression of neutrophil chemoattractants. [Abstract/Link to Full Text]