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Recent Articles in Endocrinology

Stanley FM
Insulin-Increased Prolactin Gene Expression Requires Actin Treadmilling: Potential Role for P21 Activated Kinase.
Endocrinology. 2007 Sep 20; .
Insulin-increased prolactin gene transcription in GH4 cells was enhanced by binding on fibronectin. This was mediated by receptor-like protein tyrosine phosphatasealpha that activated Src, Rho and PI 3-kinase (1). This suggested that insulin signaling to gene transcription was partly dependent on actin rearrangement. This was confirmed through studies using inhibitors of actin treadmilling. Cytochalasin D, jasplakinolide, latrunculin B, and swinolide A altered the actin cytoskeleton of GH4 cells as assessed by Alexa fluor phalloidin staining and inhibited insulin-increased prolactin gene transcription. These reagents did not affect the controls. Nor was it due to a gross defect of insulin signaling since activation/translocation of glycogen synthase kinase 3beta (GSK3beta) and mTOR were not affected. Expression of wild-type and mutant actin treadmilling agents, Cdc42, TC10, neuronal Wiscott-Aldrich syndrome protein (N-WASP) and Nck, indicated that they were essential to insulin-increased prolactin gene expression and suggested that activation of p21 associated kinase (PAK) might also be essential to this process. PAK expression also increased and PAK mutants decreased prolactin promoter activity in insulin treated cells. The activation of PAK in the presence of inhibitors was also consistent with a role in activation of insulin-increased prolactin gene expression. Finally, siRNA-mediated reduction of PAK decreased the effect of insulin on prolactin gene expression. Thus, it is likely that insulin activation of actin treadmilling through Cdc42/TC10 and N-Wasp activates PAK and prolactin gene transcription. [Abstract/Link to Full Text]

Damdimopoulos AE, Spyrou G, Gustafsson JA
Ligands Differentially Modify the Nuclear Mobility of Estrogen Receptors {alpha} and {beta}
Endocrinology. 2007 Sep 20;
Signaling of nuclear receptors depends on the structure of their ligands, different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on ERalpha and ERbeta mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERalpha, ligands can be classified into three distinct groups: 1. Ligands that do not affect the mobility of the receptor; 2. ligands that cause a moderate effect and 3. ligands that strongly impact mobility of ERalpha. Interestingly, we found that for ERbeta such a classification was not possible as ERbeta ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response towards the anti-estrogens ICI and raloxifene which was not attributable to differential sub-nuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERalpha, while ERbeta retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteosome machinery. This novel finding that ERbeta retains its mobility in the presence of anti-estrogens, could be important for its ability to regulate genes that do not contain classic ERE sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity. [Abstract/Link to Full Text]

Emi Y, Ikushiro SI, Kato Y
Thyroxine-metabolizing Rat UDP-glucuronosyltransferase 1A7 is Regulated by Thyroid Hormone Receptor.
Endocrinology. 2007 Sep 20;
Exposure of rats to microsomal enzyme inducers perturbs thyroid hormone (TH) homeostasis through a variety of mechanisms. Glucuronidation is an important metabolic pathway for THs and is catalyzed by UDP-glucuronosyltransferase (UGT) family proteins. Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats markedly increases the biliary clearance of glucuronidated T4 and results in reduced plasma T4 levels. Determination of the UGT1 isoforms responsible for glucuronidation of T4 has yet to be conclusively established. We here provide evidence for the involvement of TCDD-inducible UGT1A7 in the glucuronidation of T4 and TH-controlled UGT1A7 expression. Among a number of rat UGT1 isoenzymes examined in this study, UGT1A7 was the most active in catalyzing glucuronidation of T4. Expression of UGT1A7 was positively regulated by T4 through specific binding of thyroid hormone receptor-retinoid X receptor heterodimers to a DR-5 sequence located between -109 and -93 in the UGT1A7 promoter. Overproduction of UGT1A7 protein decreased T4-responsiveness of a reporter gene containing the T4-responsive UGT1A7 promoter sequence. These results raise the possibility that UGT1A7 plays a key role in the glucuronidation of T4 leading to inactivation of T4, functioning via feedback regulation to control T4 levels in an autoregulatory manner, and that T4 regulate their own metabolism and subsequent clearance from cells. Our findings also predict that accumulation of TCDD-inducible UGT1A7 proteins in TH-target cells might disrupt the TH signaling by lowering the intracellular pool of T4. [Abstract/Link to Full Text]

Wang T, Wang Y, Kontani Y, Kobayashi Y, Sato Y, Mori N, Yamashita H
Evodiamine improves diet-induced obesity in a UCP1-independent manner: Involvement of anti-adipogenic mechanism and ERK/MAPK signaling.
Endocrinology. 2007 Sep 20;
Evodiamine is an alkaloidal compound with anti-obesity effects that have been thought to be due to uncoupling protein-1 (UCP1) thermogenesis similar to the effects of capsaicin, but the underlying mechanisms are poorly understood. To clarify the mechanisms, we first examined whether the anti-obesity effect of evodiamine could be attributed to the involvement of UCP1. When UCP1-KO mice were fed a high fat diet with 0.03% evodiamine (w/w) for 2 months, the increases in body weight, adiposity, and the serum levels of leptin and insulin were reduced in a manner indistinguishable from control mice fed a high fat diet with evodiamine, suggesting that evodiamine triggered a UCP1-independent mechanism to prevent diet-induced obesity. By using preadipocyte cultures, we found that evodiamine, but not capsaicin, increased phosphorylation of extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinases (MAPK), reduced the expression of transcription factors such as peroxisome proliferator-activated receptor gamma, and strongly inhibited adipocyte differentiation. Evodiamine treatment also reduced insulin-stimulated phosphorylation of Akt, a crucial regulator of adipocyte differentiation; and the reduction of phosphorylated-Akt and augmentation of phosphorylated-ERK were reversed by blockade of the MAPK kinase (MEK)/MAPK signaling pathway, restoring adipogenesis in the cultures. The changes in ERK and Akt phosphorylation levels were also observed in white adipose tissues of UCP1-KO mice fed the evodiamine diet. These findings suggest that evodiamine has a potential to prevent the development of diet-induced obesity in part by inhibiting adipocyte differentiation through ERK activation and its negative cross-talk with the insulin signaling pathway. [Abstract/Link to Full Text]

Santos SJ, Haslam SZ, Conrad SE
Estrogen and Progesterone are Critical Regulators of Stat5a Expression in the Mouse Mammary Gland.
Endocrinology. 2007 Sep 20;
Signal transducer and activator of transcription (Stat)5a is a well-established regulator of mammary gland development. Several pathways for activating Stat5a have been identified, but little is known about the mechanisms that regulate its expression in this tissue. In this report, we used immunofluorescent staining to examine Stat5a expression in mammary epithelial cells during normal development and in response to treatment with the ovarian hormones estrogen (E) and progesterone (P). Stat5a was present at very low levels in the pre-pubertal gland, and was highly induced in a subset of luminal epithelial cells during puberty. The percentage of positive cells increased in adult virgin, pregnant, and lactating animals, dropped dramatically during involution and then increased again post-weaning. Ovariectomy ablated Stat5a expression in virgin animals, and treatment with both E and P was necessary to restore it. Double labeling experiments in animals treated with E+P for three days demonstrated that Stat5a was localized exclusively to cells containing both estrogen and progesterone receptors. Together, these results identify a novel role for E and P in inducing Stat5a expression in the virgin mammary gland, and suggest that these hormones act at the cellular level through their cognate receptors. [Abstract/Link to Full Text]

Ziegler CG, Sicard F, Lattke P, Bornstein SR, Ehrhart-Bornstein M, Krug AW
Dehydroepiandrosterone (DHEA) induces a neuroendocrine phenotype in nerve growth factor (NGF)-stimulated chromaffin pheochromocytoma PC12 cells.
Endocrinology. 2007 Sep 20;
The adrenal androgen dehydroepiandrosterone (DHEA) is produced in the inner zone of the adrenal cortex which is in direct contact to adrenal medullary cells. Due to their close anatomical proximity and tightly intermingled cell borders a direct interaction of adrenal cortex and medulla has been postulated. In humans congenital adrenal hyperplasia due to 21-hydroxylase (OH) deficiency results in androgen excess accompanied by severe adrenomedullary dysplasia and chromaffin cell dysfunction. Therefore, in order to define the mechanisms of DHEA action on chromaffin cell function, we investigated its effect on cell survival and differentiation processes on a molecular level in the chromaffin cell line PC12. DHEA lessened the positive effect of NGF on cell survival and neuronal differentiation. NGF-mediated induction of a neuronal phenotype was inhibited by DHEA as indicated by reduced neurite outgrowth and decreased expression of neuronal marker proteins such as SNAP-25 and VAMP-2. We examined whether DHEA may stimulate the cells towards a neuroendocrine phenotype. DHEA significantly elevated catecholamine release from unstimulated PC12 cells in the presence, but not in the absence of NGF. Accordingly, DHEA enhanced the expression of the neuroendocrine marker protein chromogranin A. Next, we explored the possible molecular mechanisms of DHEA and NGF interaction. We demonstrate that NGF-induced ERK1/2 phosphorylation was reduced by DHEA. In summary, our data show that DHEA influences cell survival and differentiation processes in PC12 cells, possibly by interacting with the ERK1/2 MAPK pathway. DHEA drives NGF-stimulated cells towards a neuroendocrine phenotype, suggesting that the interaction of intraadrenal steroids and growth factors is required for the maintenance of an intact adrenal medulla. [Abstract/Link to Full Text]

Muhlhausler BS, Duffield JA, McMillen IC
Increased Maternal Nutrition Increases Leptin Expression in Perirenal and Subcutaneous Adipose Tissue in the Postnatal Lamb.
Endocrinology. 2007 Sep 20;
The present study tested the hypothesis that exposure to an increased level of maternal nutrition before birth results in altered expression of adipogenic, lipogenic and adipokine genes in adipose tissue in early postnatal life. Pregnant ewes were fed either at or approximately 50% above maintenance energy requirements (MER) during late pregnancy and qRT-PCR was used to measure Peroxisome Proliferator-Activated Receptor-gamma (PPARgamma), Lipoprotein Lipase (LPL), Glycerol-3-phosphate-dehydrogenase (G3PDH), adiponectin and leptin mRNA expression in perirenal (PAT) and subcutaneous (SCAT) adipose tissue in the offspring on postnatal day30. Relative SCAT mass was higher in lambs of Well Fed ewes (40.0 +/- 4.0 vs 22.8 +/- 3.3 g/kg, P < 0.05) and was directly related to plasma insulin in the first 24h after birth and to G3PDH and LPL expression. The expression of leptin mRNA in both the SCAT and PAT depots was higher (P < 0.05) in lambs of Well Fed ewes. PPARgamma adiponectin, LPL and G3PDH mRNA expression were not, however, different between Well Fed and Control groups in either depot. Relative PPARgamma expression in SCAT was directly related to plasma insulin concentrations in the first 24h after birth (r(2) = 0.23; P < 0.05) and G3PDH and LPL expressions were also positively correlated with PPARgamma expression (r(2) = 0.27; P < 0.05). We have demonstrated that exposure to increased prenatal nutrition increases leptin expression at 1 month of age in both perirenal and subcutaneous adipose tissue. The results of this study provide evidence that the nutritional environment before and immediately after birth can influence the development of adipose tissue in early postnatal life. [Abstract/Link to Full Text]

Cecconi S, Mauro A, Capacchietti G, Berardinelli P, Bernab� N, Di Vincenzo AR, Mattioli M, Barboni B
MEIOTIC MATURATION OF INCOMPETENT PREPUBERTAL SHEEP OOCYTES IS INDUCED BY PARACRINE FACTOR(S) RELEASED BY GONADOTROPIN-STIMULATED OOCYTE-CUMULUS CELL COMPLEXES AND INVOLVES MAPK ACTIVATION.
Endocrinology. 2007 Sep 20;
In this study, sheep oocyte-cumulus cell complexes derived from medium antral follicles (M-OCC) were in vitro matured alone or in co-culture with complexes derived from small antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage (MII), and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to MII and S-CC expanded following co-culture with gonadotropin-stimulated M-OCC and M-CC, but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were co-cultured with M-denuded oocytes, or when S-denuded oocytes were co-cultured with M-CC. The capacity of these paracrine factor(s) to activate MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MEK/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, firstly in cumulus cells and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes co-cultured with M-OCC or M-CC. These results demonstrate that soluble factor(s) specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via cumulus cells. These factor(s) can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding cumulus cells to respond to gonadotropin stimulation. [Abstract/Link to Full Text]

Kuhl AJ, Ross SM, Gaido KW
C/ebp {beta}, but not SF-1, modulates the phthalate-induced dysregulation of rat fetal testicular steroidogenesis.
Endocrinology. 2007 Sep 20;
Prolonged in utero exposure of fetal male rats to di-butyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes, and therefore reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semi-quantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including StAR, SR-BI, CYP11A1, and CYP17A1A1, that are down-regulated following an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by electrophoretic mobility shift assay, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein beta (c/ebp beta) and steroidogenic factor-1 (SF-1). ChIP confirmed declines in DNA-protein interactions of c/ebp beta in DBP treated animals, while SF-1 was observed to be reduced in both Di-ethyl phthalate (DEP; non-toxic) and DBP (toxic) treatments. These results suggest inhibition of c/ebp beta, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. Additionally, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure. [Abstract/Link to Full Text]

Poncin S, G�rard AC, Boucquey M, Senou M, Calderon PB, Knoops B, Lengel� B, Many MC, Colin IM
Oxidative stress in the thyroid gland: from harmlessness to hazard depending on the iodine content.
Endocrinology. 2007 Sep 20;
In basal conditions, thyroid epithelial cells produce moderate amounts of reactive oxygen species (ROS) that are physiologically required for thyroid hormone synthesis. They are not necessarily toxic because they are continuously detoxified either in the process of hormone synthesis or by endogenous antioxidant systems. Using a rat model of goiter formation and iodine-induced involution, we found that compared to control thyroids, the oxidative stress, assessed by the detection of 4-hydroxynonenal (4-HNE), was strongly enhanced both in hyperplastic and involuting glands. The level of antioxidant defences (glutathione peroxidases and peroxiredoxins) was also up-regulated in both groups, although somewhat less in the latter. Of note, increased oxidative stress came along with an inflammatory reaction, but only in involuting glands suggesting that although antioxidant systems can adequately buffer heavy load of ROS in goiter, it is not necessarily the case in involuting glands. The effects of 15 deoxy-Delta(12,14)-prostaglandin J2 (15dPGJ2), an endogenous ligand of PPARgamma with anti-inflammatory properties, were then investigated in involuting glands. This drug strongly reduced both 4-HNE staining and the inflammatory reaction indicating that it can block iodine-induced cytotoxicity. When experiments were carried out with the PPARgamma antagonist, bisphenol A diglycidyl ether (BADGE), 15dPGJ2-induced effects remained unchanged suggesting that these effects were not mediated by PPARgamma. In conclusion, thyroid epithelial cells are well adapted to endogenously produced ROS in basal and goitrous conditions. In iodine-induced goiter involution, the increased oxidative stress is accompanied by inflammation that can be blocked by 15dPGJ2 through PPARgamma-independent protective effects. [Abstract/Link to Full Text]

Minekawa R, Sakata M, Okamoto Y, Hayashi M, Isobe A, Takeda T, Yamamoto T, Koyama M, Ohmichi M, Tasaka K, Imai K, Okamoto T, Murata Y
Involvement of RelA-associated inhibitor (RAI) in regulation of trophoblast differentiation via interaction with transcriptional factor Sp1.
Endocrinology. 2007 Sep 13;
Glucose transporter-1 (GLUT1), one of the key functional indicators of placental differentiation, has an important role in placental glucose transport. We previously showed that the protein levels of GLUT1 and nuclear transcription factor Sp1 in rat choriocarcinoma cells (Rcho-1 cells) decreased during the differentiation of these cells to giant cells. We also showed that Sp1 was involved in the regulation of GLUT1 gene expression during this process. RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor kappa B (NF-kappaB) that was identified by a yeast two-hybrid screen and is preferably expressed in human placenta and heart. RAI was also found to interact with Sp1 and to exert an inhibitory effect against the DNA-binding activity of Sp1. We first show here that RAI mRNA expression increased as gestation proceeded, and that RAI was localised mainly in the syncytiotrophoblast throughout pregnancy. The chloramphenicol acetyltransferase (CAT) activity assay in Rcho-1 cells revealed that cotransfection of RAI expression vector resulted in decreased activity of the rGLUT1 promoter, but not in that of a mutated rGLUT1 promoter lacking the Sp1 binding site. Furthermore, the protein level of RAI increased during differentiation. In addition, transfection of RAI expression vector promoted the morphological differentiation of Rcho-1 cells, and RAI knockdown using RAI-specific siRNA reveals inhibitory effects on the morphological differentiation, as assessed by photomicroscopy. Taken together, these findings suggest that RAI may be involved in the regulation of trophoblast differentiation via interaction with Sp1. [Abstract/Link to Full Text]

Kawagoe J, Ohmichi M, Tsutsumi S, Ohta T, Takahashi K, Kurachi H
Mechanism of the divergent effects of estrogen on the cell proliferation of human umbilical endothelial versus aortic smooth muscle cells.
Endocrinology. 2007 Sep 13;
Diverse estrogen actions are controlled via estrogen receptors (ER). Mechanisms of action of ER are modulated by various factors such as ER subtypes, conformation of the ER-ligand complex and recruitment of coregulator complexes to a target gene promoter. Estrogen exerts divergent actions on vascular cells, namely, it increases endothelial cell and inhibits smooth muscle cell growth, resulting in a vasoprotective action. We particularly focused on these divergent effects and examined the mechanisms. The effects of raloxifene, which shows estrogen-like vasoprotective actions, were also examined. To examine the effects of 17beta-estradiol (E2) and raloxifene on human aortic smooth muscle cells (HASMCs) and human umbilical venous endothelial cells (HUVECs), we evaluated the effect of E2 and raloxifene on transcriptional activity, recruitment of the coregulator complex to a target gene promoter, and acetylation of histone, of both the IGF-1 and COX-2 genes. Treatment with E2 or raloxifene increased both IGF-1 and COX-2 mRNA expression in HUVECs, whereas they attenuated the serum-induced increase of these genes in HASMCs. Treatment by E2 and raloxifene induced recruitment of coactivator complex and histone acetylation at both the IGF-1 and COX-2 gene promoter in HUVECs. In contrast, in HASMCs, E2 and raloxifene attenuated the serum-induced recruitment of coactivator complexes and histone acetylation at both the IGF-1 and COX-2 gene promoters. Estrogen and raloxifene exert divergent transcriptional regulation on both mRNA expression and the remodeling of IGF-1 and COX-2 gene promoters in HUVECs versus HASMCs. [Abstract/Link to Full Text]

Tonack S, Kind K, Thompson JG, Wobus AM, Fischer B, Santos AN
Dioxin affects glucose transport via the arylhydrocarbon receptor signal cascade in pluripotent embryonic carcinoma cells.
Endocrinology. 2007 Sep 13;
Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR) the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells (ECC). Undifferentiated P19 cells were exposed to 1 to 100 nM TCDD and characterized for AhR signaling. For studying cell differentiation P19 cells were exposed to 10 nM TCDD at stage of embryoid body (EB) formation and analyzed on glucose metabolism and cardiac differentiation during the next 3 weeks. TCDD treatment activated the AhR-signaling cascade within 1 hour, confirmed by AhR translocation, induction of CYP1A1 expression and activation of the xenobiotic response element. Although cell viability and transcription of the cardiac marker protein alpha-MHC were affected, TCDD did not inhibit the differentiation of P19 cells to pulsating cardiomyocytes. TCDD significantly downregulated the expression levels of the glucose transporter isoforms (GLUT) 1 and 3. After 24 hours of TCDD treatment GLUT1 was no longer localized in the plasma membrane of P19 cells. The impaired GLUT expression correlated with a lower glucose uptake in 5 day-old EBs. The TCDD effects were mediated by AhR as shown by pre-culture with the AhR antagonist alpha-naphthoflavone. Our data demonstrate that an AhR-mediated disturbance in GLUT expression and insufficient glucose uptake may be major mechanisms in TCDD embryo toxicity. [Abstract/Link to Full Text]

Sakamoto H, Matsuda KI, Hosokawa K, Nishi M, Morris JF, Prossnitz ER, Kawata M
Expression of GPR30, a G protein-Coupled Membrane Estrogen Receptor, in Oxytocin Neurons of the Rat Paraventricular and Supraoptic Nuclei.
Endocrinology. 2007 Sep 13;
The regulatory actions of estrogens on magnocellular oxytocin (OT) neurons of the paraventricular (PVN) and supraoptic (SON) nuclei are well documented. Although the expression and distribution of nuclear estrogen receptor beta (ERbeta), but not ERalpha, in the OT neuron has been described, the nuclear receptors may not explain all aspects of estrogen function in the hypothalamic OT neuron. Recently, a G protein-coupled receptor for estrogens, GPR30, has been identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we therefore investigated the expression and localization of GPR30 in magnocellular OT neurons to understand the mode of rapid estrogen actions within these neurons. Here we show that, in the PVN and SON, GPR30 is expressed in magnocellular OT neurons at both mRNA and protein levels but is not expressed in vasopressin neurons. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was localized mainly in the Golgi apparatus of the neurons but could not be detected at the cell surface. In addition, the expression of GPR30 is also detected in the neurohypophysis. These results suggest that GPR30 may serve primarily as a nongenomic transducer of estrogen actions in the hypothalamo-neurohypophyseal system. [Abstract/Link to Full Text]

Naef L, Woodside B
Prolactin / leptin interactions in the control of food intake in rats.
Endocrinology. 2007 Sep 13;
Recent evidence suggests that the peptide hormone prolactin (PRL) modulates energy balance through a number of mechanisms including acting in the brain to increase food intake. In the current studies we first demonstrated that chronic infusions of PRL into the lateral ventricles increased food intake in cycling rats without disrupting estrous cyclicity. In subsequent experiments the hypothesis that at least part of PRL's ability to increase food intake resulted from PRL-induced leptin resistance was tested. Female rats given chronic infusions of PRL (5microg/hr) into the cerebral ventricles for 10 days did not show a reduction in food intake or body weight following a central injection of 4microg murine leptin whereas the expected reduction in both of these parameters was seen in vehicle-infused rats. Leptin injections were without effect on these parameters whether they were administered to free feeding prolactin-infused rats or following 24h food deprivation. This lack of a behavioral response to leptin was accompanied by an attenuation in Fos induction and phosphorylation of STAT3 following leptin administration in PRL-infused rats in both the VMH and PVN. [Abstract/Link to Full Text]

Ogushi Y, Mochida H, Nakakura T, Suzuki M, Tanaka S
Immunocytochemical and Phylogenetic Analyses of an Arginine Vasotocin-Dependent Aquaporin, AQP-h2K, Specifically Expressed in the Kidney of the Tree Frog, Hyla japonica.
Endocrinology. 2007 Sep 13;
Water movement occurs across the plasma membrane of various cells of animals, plants, and microorganisms through specialized water-channel proteins called aquaporins (AQPs). We have identified a new member of the amphibian AQP family, AQP-h2K, from the kidneys of Hyla japonica. This protein consists of 280 amino acid residues with two NPA (Asn-Pro-Ala) sequence motifs and a mercury-sensitive cysteine residue just upstream from the second NPA motif. There are two putative N-linked glycosylation sites at Asn-120 and Asn-128 and one protein kinase A phosphorylation site at Ser-262. The AQP-h2K protein was specifically expressed in the apical membrane and/or cytoplasm of principal cells in the kidney collecting ducts. Following stimulation with arginine vasotocin (AVT), it was translocated from the cytoplasmic pool to the apical membrane. Phylogenetic analysis of AQP proteins from anurans and mammals identified six clusters of anuran AQPs: types 1, 2, 3, and 5, and two anuran-specific types, designated a1 and a2. The cluster AQPa2 contains Hyla AQP-h2 and AQP-h3, which are expressed in the anuran urinary bladder and ventral pelvic skin. AQP-h2K belongs to the type 2, together with mammalian (human and mouse) AQP2, suggesting that AQP-h2K is an anuran ortholog of the neurohypophysial hormone-regulated mammalian AQP2 and that the AQP2 molecule is already present in the anuran mesonephros. [Abstract/Link to Full Text]

Li XF, Kinsey-Jones JS, Knox AM, Wu XQ, Tahsinsoy D, Brain SD, Lightman SL, O'byrne KT
Neonatal lipopolysaccharide exposure exacerbates stress-induced suppression of LH pulse frequency in adulthood.
Endocrinology. 2007 Sep 13;
Early life exposure to immunological challenge has programming effects on the adult hypothalamo-pituitary-adrenocortical (HPA) axis stress responsivity and stress is known to suppress GnRH pulse generator activity, especially LH pulses. We investigated the effects of neonatal exposure to endotoxin on stress-induced suppression of pulsatile LH secretion and the involvement of corticotropin-releasing factor (CRF) receptor mechanisms in adult rats. Pups at 3 and 5 days of age were administered lipopolysaccharide (LPS: 50 microg/kg, ip). At 12 week of age they were ovariectomized and implanted sc 17beta-estradiol capsules and iv cannulae. Blood samples (25microl) were collected every 5 min for 5 h for LH measurement. After 2 h of sampling rats were given LPS (25 microg/kg, iv). CRF, CRF-R1 and CRF-R2 receptor mRNA was determined by RT-PCR in medial preoptic area (mPOA) micropunches collected at 3 h after LPS administration. There was no difference in basal LH pulse frequency between neonatal-LPS and neonatal-saline controls. However, neonatal endotoxin treated rats exhibited a significantly greater LPS stress-induced suppression of LH pulse frequency. Basal mPOA CRF-R1 expression was unchanged in neonatal-LPS and neonatal-saline treated rats. However, CRF-R1 expression was significantly increased in response to LPS stress in neonatal-LPS treated animals, but not in neonatal-saline controls. CRF and CRF-R2 expression was unchanged in all treatment groups. These data demonstrate that exposure to bacterial endotoxin in early neonatal life programmes long-term sensitization of the GnRH pulse generator to the inhibitory influence of stress in adulthood, an effect that might involve up-regulation of CRF-R1 expression in the mPOA. [Abstract/Link to Full Text]

Myers M, Lamont MC, van den Driesche S, Mary N, Thong KJ, Hillier SG, Duncan WC
Role of luteal glucocorticoid metabolism during maternal recognition of pregnancy in women.
Endocrinology. 2007 Sep 13;
The human corpus luteum (hCL) is an active, transient and dynamic endocrine gland. It will experience extensive tissue and vascular remodeling followed by i) demise of the whole gland without any apparent scarring or ii) maintenance of structural and functional integrity dependent on conceptus-derived hCG. As cortisol has well characterized roles in tissue remodeling and repair, we hypothesized that it may have a role in controling luteal dissolution during luteolysis, and would be locally produced towards the end of the luteal cycle. Glucocorticoid metabolizing enzymes (11betaHSD types 1 and 2) and the glucocorticoid receptor (GR) were assessed in hCL and cultures of luteinized granulosa cells (LGC) using immunofluoresence and qRT-PCR. Furthermore the effect of cortisol on steroidogenic cell survival and fibroblast-like cell activity was explored in vitro. The hCL expressed 11betaHSD isoenzymes in LGC and nuclear GR in several cell types. HCG up-regulated the expression and activity of 11betaHSD type 1 (p<0.05) and down-regulated type 2 enzyme (p<0.05) in vitro and tended to do the same in vivo. Cortisol increased the survival of LGC treated with RU486 (p<0.05) and suppressed the activity of a proteolytic enzyme associated with luteolysis in fibroblast-like cells (p<0.05). Our results suggest that, rather than during luteolysis, it is luteal rescue with hCG that is associated with increased local cortisol generation by 11betaHSD type 1. Locally generated cortisol may therefore act on the hCL through GR to have a luteotropic role in the regulation of luteal tissue remodeling during maternal recognition of pregnancy. [Abstract/Link to Full Text]

Meilleur MA, Akpovi CD, Pelletier RM, Vitale ML
TNF-{alpha}-induced anterior pituitary folliculostellate TtT/GF cell uncoupling is mediated by Cx43 dephosphorylation.
Endocrinology. 2007 Sep 13;
The anterior pituitary folliculostellate cells are key elements of the paracrine control of the pituitary function. These cells are the source and the target of growth factors and cytokines and are connected to other pituitary cells via Cx43-mediated gap junctions. Here, we show that acute treatment of the folliculostellate TtT/GF cell line with TNF-alpha caused a transient cell uncoupling that was accompanied by the dephosphorylation of Cx43 in Ser368. These TNF-alpha-evoked effects were dependent on protein phosphatase 2A (PP2A) and PKC activities. TNF-alpha did not affect total cell Cx43-PP2A catalytic subunit (PP2Ac) interaction, but it did induce PP2Ac recruitment to the Triton-X-100-insoluble subcellular fraction where Cx43-gap junction plaques are recovered. This recruitment temporally coincided with P(Ser368)-Cx43 dephosphorylation. Cx43 did not interact with the conventional PKC-alpha, but it did interact with the atypical PKC-zeta. Moreover, this interaction was weakened by TNF-alpha. Cx43 dephosphorylation in Ser368 was followed by the tyrosine phosphorylation of the protein. The temporary closure of gap junctions during acute TNF-alpha challenge may constitute a protective mechanism to limit or confine the spread of inflammatory signals amongst the FS cells. [Abstract/Link to Full Text]

Chandrashekar V, Dawson CR, Martin ER, Rocha JS, Bartke A, Kopchick JJ
Age-related Alterations in Pituitary and Testicular Functions in Long-lived Growth Hormone Receptor Gene-disrupted Mice.
Endocrinology. 2007 Sep 13;
The somatotropic axis, growth hormone (GH) and insulin-like growth factor 1 (IGF-1) interact with the hypothalamic-pituitary-gonadal (HPG) axis in health and disease. GH-resistant GH receptor-disrupted (GHRKO) male mice are fertile but exhibit delayed puberty and decreases in plasma FSH levels, testicular content of LH and prolactin (PRL) receptors, while PRL levels are elevated. Because the lifespan of GHRKO mice is much greater than the lifespan of their normal siblings, it was of interest to compare age-related changes in the HPG axis in GHRKO and normal animals. Plasma IGF-1, insulin, PRL, LH, FSH, androstenedione and testosterone levels, and acute responses to GnRH and LH were measured in young (2-4 and 5-6 months of age) and old (18-19 and 23-26 months of age) male GHRKO mice and their normal siblings. Plasma IGF-1 was not detectable in GHRKO mice. Plasma PRL levels increased with age in normal mice but declined in GHRKO males and did not differ in old GHRKO and normal animals. Plasma LH responses to acute GnRH stimulation were attenuated in GHRKO mice, but increased with age only in normal mice. Plasma FSH levels were decreased in GHRKO mice regardless of age. Plasma testosterone responses to LH stimulation were attenuated in old mice regardless of genotype while plasma androstenedione responses were reduced with age only in GHRKO mice. Testicular IGF-1 mRNA levels were normal in young and increased in old GHRKO mice, whereas testicular concentrations and total IGF-1 levels were decreased in these animals. These findings indicate that GH resistance due to targeted disruption of the Ghr gene in mice leads to suppression of testicular IGF-1 levels, and modifies the effects of aging on plasma PRL levels and on responses of the pituitary and the testes to GnRH and LH stimulation. Plasma testosterone levels declined during aging in normal but not in GHRKO mice, and the age-related increase in the LH responses to exogenous GnRH was absent in GHRKO mice, perhaps reflecting a delay of aging in these remarkably long-lived animals. [Abstract/Link to Full Text]

Carlo AS, Pyrski M, Loudes C, Faivre-Baumann A, Epelbaum J, Williams LM, Meyerhof W
Leptin sensitivity in the developing rat hypothalamus.
Endocrinology. 2007 Sep 13;
In adults, the adipocyte-derived hormone, leptin, regulates food intake and body weight principally via the hypothalamic arcuate nucleus (ARC). During early postnatal development, leptin functions to promote the outgrowth of neuronal projections from the ARC, while a selective insensitivity to the effects of leptin on food intake appears to exist. To investigate the mechanisms underlying the inability of leptin to regulate food intake during early development, leptin signalling was analysed both in vitro using primary cultures of rat embryonic ARC neurones and in vivo by challenging early postnatal rats with leptin. In neuronal cultures, despite the presence of key components of the leptin signalling pathway, no detectable activation of either signal transducer and activator of transcription 3 (STAT3) or the mitogen-activated kinase (MAPK) pathways by leptin was detected. However, leptin down-regulated mRNA levels of pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) and decreased somatostatin secretion. Leptin challenge in vivo at postnatal days 7 (P7), P14, P21 and P28 revealed that, in contrast to adult and P28 rats, mRNA levels of NPY, POMC, agouti-related peptide (AgRP) and cocaine- and amphetamine-regulated transcript (CART) were largely unaffected at P7, P14 and P21. Furthermore, leptin stimulation increased the suppressor of cytokine signalling-3 (SOCS-3) mRNA levels at P14, P21 and P28 in several hypothalamic nuclei, but not at P7, indicating that selective leptin insensitivity in the hypothalamus is coupled to developmental shifts in leptin receptor signalling. Thus, the present study defines the onset of leptin sensitivity in the regulation of energy homeostasis in the developing hypothalamus. [Abstract/Link to Full Text]

Henke A, Luetjens CM, Simoni M, Gromoll J
Chorionic gonadotropin beta subunit gene expression in the marmoset pituitary is controlled by steroidogenic factor 1 (SF1), early growth response protein 1 (Egr1) and pituitary homeobox factor 1 (Pitx1).
Endocrinology. 2007 Sep 13;
In most mammals the gonads are under the control of the pituitary gonadotropins LH and FSH. However, in the common marmoset monkey Callithrix jacchus no LH is detectable in the pituitary but chorionic gonadotropin (CG) instead, normally produced in the placenta. This study investigated the mechanism of CGbeta subunit activation in the pituitary and why humans do not express CG in the pituitary. 5'-RACE, EMSA and promoter-driven luciferase assays performed with the gonadotropic LbetaT2 cells showed that marmoset monkey CGbeta is GnRH-responsive and activated similar to human LHbeta by the transcription factors SF1, Egr1 and Pitx1, and displayed a transcriptional start site (TSS) 7 bp upstream of exon 1. In contrast, the human CGbeta promoter displayed in the binding elements for Pitx1 and Egr1 three consensus sequence mismatches, leading to very low activity that could be drastically increased by mutation to the consensus sequences. Vice versa, marmoset CGbeta promoter activity was reduced after introduction of the human CGbeta mismatches. An in vivo study in pregnant marmoset monkeys showed that during pregnancy there is no significant decrease of pituitary CG production, contrasting human LH down-regulation. In conclusion, pituitary CG production is lacking in humans due to the absence of appropriate DNA-binding elements, which are present in marmosets, thereby enabling GnRH-activation of expression. However, during pregnancy of marmosets, pituitary CG expression is not inhibited. [Abstract/Link to Full Text]

Campbell RE, Herbison AE
Definition of brainstem afferents to gonadotropin-releasing hormone (GnRH) neurons in the mouse using conditional viral tract tracing.
Endocrinology. 2007 Sep 6;
Brainstem monoamines have long been considered to play a role in regulating the activity of gonadotropin-releasing hormone (GnRH) neurons although their neuroanatomical relationship with these cells has remained unclear. Using a Cre-dependent Pseudorabies virus (Ba2001) technique that permits retrograde tracing selectively from GnRH neurons in the mouse, we have examined the organization of brainstem inputs to rostral preoptic area (rPOA) GnRH neurons. Two days following injection of Ba2001 into the rPOA of adult female GnRH-Cre transgenic mice, 5-9 GnRH neurons located immediately adjacent to the injection site were found to express GFP, the marker of virus infection, with no GFP expression anywhere else in the brain. In mice killed 24h later (3 days after injection), GFP-expressing cells were identified (in order of density) in the raphe nuclei, periaqueductal grey, locus coreuleus, nucleus tractus solitarius and area postrema. This time-course is compatible with these neurons representing primary afferent inputs to the GnRH neurons. Four and 6 days after Ba2001 injection, GFP-expressing cells were found in additional brain regions. Dual-label immunofluorescence experiments in 3 day post-injection mice demonstrated that 100% of GFP-expressing neurons in the raphe were positive for tryptophan hydroxylase, whereas 100% and approximately 50% of GFP neurons in the locus coreuleus and nucleus tractus solitarius, respectively, expressed tyrosine hydroxylase. These observations demonstrate that rPOA GnRH neurons receive direct projections from brainstem A2 and A6 NE neurons and that, surprisingly, the largest afferent input from the brainstem originates from raphe serotonin neurons in the mouse. [Abstract/Link to Full Text]

Maney DL, Goode CT, Lake JI, Lange HS, O'brien S
Rapid Neuroendocrine Responses to Auditory Courtship Signals.
Endocrinology. 2007 Sep 6;
In many species, courtship signals enhance reproductive function in the receiver. How these social signals are processed by the brain, particularly how they induce an endocrine response, is not well understood. Songbirds provide an ideal model in which to study this phenomenon because of the large existing literature on both their auditory neurobiology and the control of their reproductive physiology by environmental cues. To date, all of the relevant studies on songbirds have involved measuring the effects of male vocalizations on ovarian function over a period of weeks, a time course that precludes detailed analysis of the neuroendocrine mechanisms operating during song perception. We played recordings of conspecific male song to laboratory-housed female white-throated sparrows and quantified the resulting rapid changes in LH as well as the induction of the immediate early gene Egr-1in the GnRH system and mediobasal hypothalamus (MBH). Hearing song for 42 min induced LH release and Egr-1 expression in the MBH, but did not alter Egr-1 expression in GnRH neurons. The time course of LH release and the pattern of Egr-1 expression together suggest that song acts as a trigger to induce GnRH release in a manner resembling photostimulation. The Egr-1 response in the MBH was qualitatively distinguishable from the responses to either photostimulation or pharmacologically-induced LH release, but seemed to involve overlapping neuronal populations. Song-induced Egr-1 expression in the MBH was correlated with the expression in midbrain and forebrain auditory centers, further supporting a role for the MBH in processing social information. [Abstract/Link to Full Text]

Gallardo N, Bonz�n-Kulichenko E, Fern�ndez-Agull� T, Molt� E, G�mez-Alonso S, Blanco P, Carrascosa JM, Ros M, Andr�s A
Tissue-specific effects of central leptin on the expression of genes involved in lipid metabolism in liver and white adipose tissue.
Endocrinology. 2007 Sep 6;
Leptin reduces adiposity and exerts anti-steatotic effects on non-adipose tissues. However, the mechanisms underlying leptin effects on lipid metabolism in liver and white adipose tissue have not been fully clarified. Here, we have studied the effects of central leptin administration on key enzymes and transcription factors involved in lipid metabolism in liver and epididymal adipose tissue. Intracerebroventricular leptin infusion for 7 days did not change leptin plasma levels but decreased triacylglyceride content in liver, epididymal adipose tissue and plasma. In both tissues, this treatment markedly decreased the expression of key enzymes of the de novo fatty acid synthesis such as ACC, FAS and SCD-1, in parallel with a reduction in mRNA expression of SREBP-1c in liver and ChREBP in adipose tissue. Additionally, leptin also decreased PEPCK-C expression in adipose tissue, an enzyme involved in glyceroneogenesis in this tissue. Central leptin administration down regulates delta 6-desaturase expression in liver and adipose tissue, in parallel to the decrease of the expression of SREBP-1c in liver and PPARalpha in adipose tissue. Finally, leptin treatment, by regulating ATGL/HSL/DAGT1 expression, also established a new partitioning in the fatty acid-TAG cycling in adipose tissue, increasing lipolysis and probably the fatty acid efflux from this tissue, and favoring in parallel the fatty acid uptake and oxidation in the liver. These results suggest that leptin, acting at central level, exerts tissue-specific effects in limiting fat tissue mass and lipid accumulation in non-adipose tissues, preventing the development of obesity and type 2 diabetes. [Abstract/Link to Full Text]

Goodman RL, Lehman MN, Smith JT, Coolen LM, de Oliveira CV, Jafarzadehshirazi MR, Pereira A, Iqbal J, Caraty A, Ciofi P, Clarke IJ
KISSPEPTIN NEURONS IN THE ARCUATE NUCLEUS OF THE EWE EXPRESS BOTH DYNORPHIN A AND NEUROKININ B.
Endocrinology. 2007 Sep 6;
Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area (POA). Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are co-localized in the same cells. In this study, we tested this hypothesis using dual-immunocytochemistry and also determined if kisspeptin neurons contain melanocyte-stimulating hormone (MSH) or agouti-related peptide (AGRP). To assess co-localization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n=5). Almost all ARC, but no POA, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In Exp. 2, we examined co-localization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary-intact ewes (n=9). Over three-quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or AGRP. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone receptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function. [Abstract/Link to Full Text]

Lavery GG, Hauton D, Hewitt KN, Brice SM, Sherlock M, Walker EA, Stewart PM
Hypoglycaemia with enhanced hepatic glycogen synthesis in recombinant mice lacking hexose-6-phosphate dehydrogenase.
Endocrinology. 2007 Sep 6;
Hexose-6-phosphate dehydrogenase (H6PDH) knockout (KO) mice have reduced NADPH generation within the endoplasmic reticulum. As a consequence, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme activity switches from a reductase to a dehydrogenase leading to glucocorticoid inactivation. 11beta-HSD1 has emerged as an important factor in regulating hepatic glucose output; therefore we examined aspects of glucose homeostasis in KO mice. Compared to wild type (WT) mice, KO mice have reduced weight gain, display peripheral fasting hypoglycaemia, improved glucose tolerance and elevated plasma corticosterone concentrations. Plasma insulin levels in fed and fasted KO mice are normal, however insulin and plasma glucose levels are reduced 4 hours after fasted animals are re-fed, indicating improved insulin sensitivity. There is preserved induction and activity of the glucocorticoid responsive gluconeogenic enzymes PEPCK and G6Pase in fasted KO mice. Glycogen storage is elevated in fed KO liver, with fed glycogenesis rates increased in KO mice. There is normal flux of lactate through gluconeogenesis recovered as plasma glucose, coupled with increased glycogen derived from lactate. These data suggest partial retention of glucocorticoid sensitivity at the level of the liver. We therefore postulate that increased glycogen synthesis may reflect increased flux of glucose-6-phosphate (H6PDH substrate) through to glycogen in the absence of H6PDH mediated metabolism. [Abstract/Link to Full Text]

Iqbal J, Latchoumanin O, Clarke IJ
Rapid in vivo Effects of Estradiol-17{beta} in Ovine Pituitary Gonadotropes are Displayed by Phosphorylation of ERK, Akt and CREB.
Endocrinology. 2007 Sep 6;
We have determined the time course of phosphorylation of mitogen activated protein kinase/ extracellular regulated kinase (MAPK/ERK), cAMP responsive element binding protein (CREB) and serine/threonine kinase (Akt) in ovine pituitary gonadotropes after in vivo injection (iv) of either 25microg estradiol-17beta (E17beta) or vehicle. In ovariectomized (OVX) ewes, E17beta increased the number of gonadotropes expressing pERK-1/2 and pCREB immunoreactivity (-IR) within 90 min, as assessed by immunohistochemistry. By Western blot, we also showed that pERK-1/2, pCREB and pAkt (ser 473) protein were upregulated by E17beta. In OVX-hypothalamo-pituitary disconnected (HPD) animals, gonadotrope function was restored with hourly GnRH pulses (iv) and E17beta injection (iv) reduced LH response within 1h. Immunohistochemistry showed that E17beta increased pERK-1/2-IR in gonadotropes within 15 min; peak response at 60 min. The number of cells immunoreactive for pCREB was greater in E17beta-treated animals than in vehicle-injected controls at 60 and 90 min. Western blot revealed a pERK-1/2 response within 15 min and pCREB response at 30 min. Upregulation of pAkt occurred within 60 min of E17beta treatment. Thus, rapid effects of E17beta on gonadotropes involve phosphorylation of second messenger proteins with a time-course that may relate to the rapid negative feedback effect to reduce responsiveness to GnRH. [Abstract/Link to Full Text]

McLachlan SM, Nagayama Y, Pichurin PN, Mizutori Y, Chen CR, Misharin A, Aliesky HA, Rapoport B
THE LINK BETWEEN GRAVES' DISEASE AND HASHIMOTO'S THYROIDITIS: A ROLE FOR REGULATORY T CELLS.
Endocrinology. 2007 Sep 6;
Hyperthyroidism in Graves' disease is caused by thyroid-stimulating autoantibodies to the thyrotropin receptor (TSHR) whereas hypothyroidism in Hashimoto's thyroiditis is associated with thyroid peroxidase and thyroglobulin autoantibodies. In some Graves' patients, thyroiditis becomes sufficiently extensive to "cure" the hyperthyroidism with resultant hypothyroidism. Factors determining the balance between these two diseases, the commonest organ-specific autoimmune diseases affecting humans, are unknown. Serendipitous findings in transgenic BALB/c mice, with the human TSHR A-subunit targeted to the thyroid, shed light on this relationship. Of three transgenic lines, two expressed high levels and one expressed low intrathyroidal A-subunit levels (Hi- and Lo-transgenics, respectively). Transgenics and wild-type littermates were depleted of T regulatory cells (Treg) using antibodies to CD25 (CD4+ T-cells) or CD122 (CD8+ T-cells) prior to TSHR-adenovirus immunization. Regardless of Treg depletion, Hi-expressor transgenics remained tolerant to A-sub-Ad immunization (no TSHR antibodies and no hyperthyroidism). Tolerance was broken in Lo-transgenics, although TSHR antibody levels were lower than in wild-type littermates and no mice became hyperthyroid. Treg depletion prior to immunization did not significantly alter the TSHR antibody response. However, Treg depletion (particularly CD25) induced thyroid lymphocytic infiltrates in Lo-transgenics with transient or permanent hypothyroidism (low T4, elevated TSH). Neither thyroid lymphocytic infiltration nor hypothyroidism developed in similarly treated wild-type littermates. Remarkably, lymphocytic infiltration was associated with inter-molecular spreading of the TSHR antibody response to other 'self' thyroid antigens, murine thyroid peroxidase and thyroglobulin. These data suggest a role for Treg in the natural progression of hyperthyroid Graves' disease to Hashimoto's thyroiditis and hypothyroidism in humans. [Abstract/Link to Full Text]

Fang Y, Demarco VG, Sharp GC, Braley-Mullen H
Expression of transgenic FLICE inhibitory protein (FLIP) on thyroid epithelial cells inhibits induction and promotes resolution of granulomatous experimental autoimmune thyroiditis in CBA/J mice.
Endocrinology. 2007 Sep 6;
Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of thyroglobulin-primed in vitro activated splenocytes. Thyroid lesions reach maximal severity 20 days later, and inflammation resolves or progresses to fibrosis by day 60 depending on the extent of thyroid damage at day 20. Depletion of CD8+ T cells inhibits G-EAT resolution. We showed that expression of FLICE (Fas-associated death domain-like IL-1beta-converting enzyme) inhibitory protein (FLIP) transgene (Tg) on thyroid epithelial cells (TEC) of DBA/1 mice had no effect on G-EAT induction, but promoted earlier resolution of G-EAT. However, when CBA/J WT donor cells were transferred to transgenic CBA/J mice expressing FLIP on TEC, they developed less severe G-EAT than FLIP Tg- littermates. Both strains expressed similar levels of the FLIP transgene, but endogenous FLIP was upregulated to a greater extent on infiltrating T cells during G-EAT development in DBA/1 compared to CBA/J mice. Following transient depletion of CD8+ T cells, FLIP Tg+ and Tg- CBA/J recipients both developed severe G-EAT at day 20. Thyroid lesions in CD8-depleted Tg+ recipients were resolving by day 60, whereas lesions in Tg- littermates did not resolve and most were fibrotic. FLIP Tg+ recipients had increased apoptosis of CD3+ T cells compared to Tg- recipients. The results indicate that transgenic FLIP expressed on TEC in CBA/J mice promotes G-EAT resolution, but induction of G-EAT is inhibited unless CD8+ T cells are transiently depleted. [Abstract/Link to Full Text]

Recent Articles in Molecular Endocrinology

Katzenellenbogen BS
In memoriam: Jack Gorski (1931-2006).
Mol Endocrinol. 2006 Nov;20(11):2611-2. [Abstract/Link to Full Text]

Endocrine-related resources from the National Institutes of Health.
Mol Endocrinol. 2006 Oct;20(10):2603-6. [Abstract/Link to Full Text]

Andric N, Ascoli M
A delayed gonadotropin-dependent and growth factor-mediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates aromatase expression in granulosa cells.
Mol Endocrinol. 2006 Dec;20(12):3308-20.
Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression. [Abstract/Link to Full Text]

Endocrine-related resources from the National Institutes of Health.
Mol Endocrinol. 2006 Sep;20(9):2256-9. [Abstract/Link to Full Text]

Yang CK, Kim JH, Stallcup MR
Role of the N-terminal activation domain of the coiled-coil coactivator in mediating transcriptional activation by beta-catenin.
Mol Endocrinol. 2006 Dec;20(12):3251-62.
The coiled-coil coactivator (CoCoA) is involved in transcriptional activation of target genes by nuclear receptors and the xenobiotic aryl hydrocarbon receptor, as well as target genes of the Wnt signaling pathway, which is mediated by the lymphocyte enhancer factor (LEF)/T cell factor transcription factors and the coactivator beta-catenin. The recruitment of CoCoA by nuclear receptors is accomplished by the interaction of the central coiled-coiled domain of CoCoA with p160 coactivators; the C-terminal activation domain (AD) of CoCoA is used for downstream signaling, whereas the function of the N-terminal region is undefined. Here we report that the N terminus of CoCoA contains another AD, which is necessary and sufficient for synergistic activation of LEF1-mediated transcription by CoCoA and beta-catenin. The N-terminal AD contains a p300 binding motif, which is important for synergistic cooperation of CoCoA and p300 as coactivators for LEF1 and beta-catenin. p300 contributes to the function of the CoCoA N-terminal AD primarily through its histone acetyltransferase activity. Moreover, in cultured cells, endogenous p300 is recruited to the promoter of an integrated reporter gene by the N terminus of CoCoA. Thus, the coactivator function of CoCoA for nuclear receptors and LEF1/beta-catenin involves differential utilization of two different CoCoA ADs. [Abstract/Link to Full Text]

Kara E, Cr�pieux 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]

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]

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, S�nchez 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 Kr�ppel-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]

Barr�s R, Gr�meaux 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-Mu�oz T, Romero-Avila MT, Garc�a-S�inz 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]

Recent Articles in BMC Endocrine Disorders

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Recent Articles in Endocrine Journal

Yamashita H, Yamazaki Y, Hasegawa H, Yamashita T, Fukumoto S, Shigematsu T, Kazama JJ, Fukagawa M, Noguchi S
Fibroblast growth factor-23 (FGF23) in patients with transient hypoparathyroidism: its important role in serum phosphate regulation.
Endocr J. 2007 Jun;54(3):465-70.
Hypoparathyroidism is a complication of thyroidectomy that causes hyperphosphatemia primarily due to enhanced reabsorption of phosphate in the kidney resulting from decreased parathyroid hormone (PTH) secretion. Fibroblast growth factor-23 (FGF23) is a hormone-like factor that is thought to play an important role in phosphate homeostasis. However, the changes and role of FGF23 in transient hypoparathyroidism after thyroidectomy are not clear. We examined changes in serum levels of calcium, phosphate, intact PTH, 1,25-dihydroxyvitamin D, and FGF23 in 12 patients (10 women, 2 men; mean age, 51 yr) who developed transient hypoparathyroidism after thyroidectomy. Serum phosphate reached its peak level (5.9 +/- 0.5 mg/dl) approximately 4 days after development of hypoparathyroidism, and this was followed by a peak in the serum FGF23 level (71 +/- 28 ng/l). Serum levels of calcium, phosphate, and FGF23 normalized after recovery of parathyroid function. There was a significant positive correlation between serum phosphate and FGF23 levels (P<0.05). Serum FGF23 was elevated in patients with hypoparathyroidism and hyperphosphatemia and normalized along with normalized phosphate levels after recovery of parathyroid function. The peak level of phosphate always preceded that of FGF23 by several days, suggesting that elevated phosphate is a primary stimulus for release of FGF23. This homeostatic regulation of phosphate differs considerably from that of serum calcium whose change is rapidly corrected within minutes. [Abstract/Link to Full Text]

Orzechowska-Pawilojc A, Sworczak K, Lewczuk A, Babinska A
Homocysteine, folate and cobalamin levels in hypothyroid women before and after treatment.
Endocr J. 2007 Jun;54(3):471-6.
Hypothyroidism may result in accelerated atherosclerosis. Hyperhomocysteinaemia is an independent risk factor for premature atherosclerotic vascular disease. The aim of the present study was to assess plasma total homocysteine (tHcy), folate and cobalamin concentrations in hypothyroid patients before and after treatment. Thirty-one hypothyroid and thirty health young women were studied. The hypothyroid patients were investigated in the untreated state and again after restoration of euthyroidism. The levels of homocysteine, folate, cobalamin and thyroid stimulating hormone (TSH), free thyroxine (fT(4)), free triiodothyronine (fT(3)) and renal function were measured before and after treatment. In hypothyroidism tHcy was higher but not statistically significant than in control group. Serum level of folate was higher and serum cobalamin was lower in the hypothyroid state. Following L-thyroxine therapy tHcy significantly decreased as well as the concentration of cobalamin. Level of folate remained unchanged. Univariate analysis in hypothyroid group indicated that tHcy negative correlated with creatinine clearance, fT(3), fT(4), cobalamin and positive with TSH. In multivariate analysis tHcy correlated with creatinine clearance, cobalamin and fT(4). Thyroid status influences the plasma tHcy. Free triiodothyronine and next free thyroxine have the greatest negative influence. This would account for hyperhomocysteinemia in the hypothyroid state and premature atherogenesis. [Abstract/Link to Full Text]

Oki K, Koide J, Nakanishi S, Nakashima R, Yamane K
Fenofibrate increases high molecular weight adiponectin in subjects with hypertriglyceridemia.
Endocr J. 2007 Jun;54(3):431-5.
Beneficial effects of peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists have been reported in improving insulin sensitivity and raising serum total adiponectin. High molecular weight (HMW) adiponectin, which is secreted from adipocytes, and visfatin, which is also expressed in adipose tissue, is related to glucose metabolism. In view of the additive effects of PPAR alpha agonists on these adipocytokines and glucose metabolism, we investigated male hypertriglyceridemic subjects who were treated with fenofibrate. Eleven male subjects with hypertriglyceridemia were treated with fenofibrate and serum total cholesterol (T-cho), triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), fasting glucose, fasting insulin, total and HMW adiponectin, and serum visfatin levels were determined before and 3 months after treatment. Fenofibrate treatment significantly lowered T-cho, triglyceride, and LDL-C levels. There was a statistically significant increase of HDL-C. No differences in insulin sensitivity indices (G/I ratio and HOMA-IR) were observed between before and after treatment with fenofibrate. The treatment did not alter the levels of serum total adiponectin and visfatin in the hypertriglyceridemic patients, while serum HMW adiponectin increased significantly. This study demonstrates that fenofibrate increases serum HMW adiponectin levels, whereas visfatin is not regulated by fenofibrate in hypertriglyceridemic subjects. Further investigations are warranted to determine whether the elevation of HMW adiponectin caused by fenofibrate might improve insulin sensitivity. [Abstract/Link to Full Text]

Shimizu I, Furuya K, Osawa H, Fujii Y, Makino H
A case of insulin-induced localized lobular panniculitis with evidence for the phagocytosis of insulin by histiocytes.
Endocr J. 2007 Jun;54(3):477-80.
Insulin-induced localized lipoatrophy is a well-known localized side effect. Although an immune process has been suggested as its etiology, no definitive evidence has been reported to show that insulin is involved. Here, we report the first evidence for the phagocytosis of insulin by histiocytes as a very early stage of lipoatrophy, which was reproducible in two different lobular panniculitis tissues from a 71 year-old male patient. He had taken a subcutaneous insulin injection in his arms because of a sight disturbance. Since these subcutaneous tumors were likely due to inflammation by insulin, a biopsy sample was taken from the subcutaneous tumor of his right arm with his consent. The primary antibodies for insulin (1 : 200) and CD68 (1 : 50) were obtained from DAKO (guinea pig anti-insulin and mouse anti-human CD68). HE staining revealed the infiltration of mononuclear cells and histiocytes into the subcutaneous fat tissue, and some parts of this tissue had fibrosis with rich collagen fibers. These findings are consistent with a lobular panniculitis. Some histiocytes contained intracellular substances with a positive immunoreactivity to insulin. This activity was reduced when the anti-insulin antibody was preincubated with an excess amount of insulin antigen. The same substances were also positive to CD68. Thus, the phagocytosis of insulin by histiocytes appears to occur in this region. Therefore, the activation of subcutaneous macrophages by the complex of insulin and insulin antibodies may account for the initial autoimmune process. [Abstract/Link to Full Text]

Moriyama T, Kageyama K, Nigawara T, Koyanagi M, Fukuda I, Yashiro H, Suda T
Diagnosis of a case of ectopic parathyroid adenoma on the early image of 99mTc-MIBI scintigram.
Endocr J. 2007 Jun;54(3):437-40.
We report the case of a 64-year-old woman who had a severe hypercalcemia. Serum calcium, intact parathyroid hormone (PTH), 1alpha, 25 (OH)(2 )vitamin D(3) levels were all elevated, and serum phosphorus level was decreased, which were all consistent with primary hyperparathyroidism (PHPT). (201)Tl/(99m)Tc subtraction scintigraphy failed to detect any abnormal accumulation in the neck and chest, while (99m)Tc-MIBI scintigraphy demonstrated the focal accumulation of increased radiotracer uptake in the mediastinum only on the early image, but not on the delayed image. Neck and chest computerized tomography scanning showed a small nodule at the retrosternal region, and a selective venous sampling study of the intact PTH suggested PTH production from the nodule. Together with the observation of the early image of (99m)Tc-MIBI scintigraphy, it was diagnosed that the patient had an ectopic parathyroid adenoma. Video-assisted thoracic surgery was performed. A 15-mm diameter mass, visualized by an intravenous infusion of methylene blue, was excited. The histopathology was consistent with the parathyroid adenoma. The adenoma was composed of mainly chief cells and rarely oxyphil cells. The absence of oxyphil cells would explain the lack of (99m)Tc-MIBI retention on late-phase imaging in our case. Even without uptake on the delayed image of (99m)Tc-MIBI scintigram, the early image was available for the localization of an ectopic parathyroid adenoma. [Abstract/Link to Full Text]

Mita T, Watada H, Nakayama S, Abe M, Ogihara T, Shimizu T, Uchino H, Hirose T, Kawamori R
Preferable effect of pravastatin compared to atorvastatin on beta cell function in Japanese early-state type 2 diabetes with hypercholesterolemia.
Endocr J. 2007 Jun;54(3):441-7.
While a large numbers of clinical trials using various kinds of statins has been reported, a possible preventive effect on new onset of type 2 diabetes mellitus was shown only by the subanalysis of The West of Scotland Coronary Prevention Study (WOSCOPS) using pravastatin. The aim of this study was to investigate whether pravastatin has a preferable effect on glucose tolerance among statins. An open-label prospective cross-over trial was performed to compare the effect of pravastatin (10 mg/day) or atorvastatin (10 mg/day) in Japanese early-state type 2 diabetes mellitus with hypercholesterolemia. The analyzed study subjects were treated with pravastatin (10 mg/day, n = 12) or atorvastatin (10 mg/day, n = 12) for 12 weeks. After a 4-week-washout period, the drugs were switched and treatment was continued for another 12 weeks. Oral glucose tolerance test (OGTT) was performed to evaluate several parameters including the appropriateness of beta cell function for the individual insulin sensitivity (disposition index: product of a validated secretion parameter and sensitivity) at the end of each therapy. HbA(1c) and 2 h-glucose levels during OGTT in the pravastatin treatment were significantly lower than atorvastatin treatment. Disposition index after pravastatin treatment was significantly higher than after atorvastatin treatment. In conclusion, our study suggests that pravastatin has a favorable effect on pancreatic beta cell function compared with atorvastatin. [Abstract/Link to Full Text]

Ito N, Fukumoto S, Taguchi M, Takeshita A, Takeuchi Y, Yamada S, Fujita T
Fibroblast growth factor (FGF)23 in patients with acromegaly.
Endocr J. 2007 Jun;54(3):481-4.
Fibroblast growth factor (FGF)23 is a hormone that regulates serum phosphate and 1,25-dihydroxyvitamin D levels. Hyperphosphatemia is sometimes observed in patients with acromegaly while the detailed mechanism of this abnormal phosphate metabolism remains to be elucidated. We have measured FGF23 levels in 18 patients before and after the surgery for acromegaly. Serum GH, IGF-I and phosphate significantly decreased after the surgery. In addition, FGF23 also reduced by the surgery. These results indicate that deficient action of FGF23 is not the cause of deranged phosphate metabolism in patients with acromegaly. [Abstract/Link to Full Text]

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]

Recent Articles in Minerva Endocrinologica

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Recent Articles in Reproductive Biology and Endocrinology

Julio-Pieper M, Lara HE, Bravo JA, Romero C
Effects of nerve growth factor (NGF) on blood vessels area and expression of the angiogenic factors VEGF and TGFbeta1 in the rat ovary.
Reprod Biol Endocrinol. 2006;457.
BACKGROUND: Angiogenesis is a crucial process in follicular development and luteogenesis. The nerve growth factor (NGF) promotes angiogenesis in various tissues. An impaired production of this neurotrophin has been associated with delayed wound healing. A variety of ovarian functions are regulated by NGF, but its effects on ovarian angiogenesis remain unknown. The aim of this study was to elucidate if NGF modulates 1) the amount of follicular blood vessels and 2) ovarian expression of two angiogenic factors: vascular endothelial growth factor (VEGF) and transforming growth factor beta 1 (TGFbeta1), in the rat ovary. RESULTS: In cultured neonatal rat ovaries, NGF increased VEGF mRNA and protein levels, whereas TGFbeta1 expression did not change. Sectioning of the superior ovarian nerve, which increases ovarian NGF protein content, augmented VEGF immunoreactivity and the area of capillary vessels in ovaries of prepubertal rats compared to control ovaries. CONCLUSION: Results indicate that NGF may be important in the maintenance of the follicular and luteal vasculature in adult rodents, either indirectly, by increasing the expression of VEGF in the ovary, or directly via promoting the proliferation of vascular cells. This data suggests that a disruption on NGF regulation could be a component in ovarian disorders related with impaired angiogenesis. [Abstract/Link to Full Text]

Alc�zar JL
Three-dimensional ultrasound assessment of endometrial receptivity: a review.
Reprod Biol Endocrinol. 2006;456.
Three-dimensional ultrasound (3D US) is a new imaging modality, which is being introduced into clinical practice. Although this technique will not probably replace two-dimensional ultrasound, it is being increasingly used. It has been reported that 3D US is a very high reproducible technique. The endometrium has been paid special attention when using this technique. The aim of this paper is to address some technical aspects of 3D US and to review critically its current status in evaluating endometrial function with special focus in its role in predicting pregnancy in assisted reproductive techniques. In spontaneous cycles endometrial volume grows during follicular phase remaining constant through the luteal phase. Endometrial vascularization increases during follicular phase peaking 2-3 days before ovulation, decreasing thereafter and increasing again during mid and late luteal phase. Data from studies analysing the role of 3D US for predicting IVF outcome are controversial. An explanation for these controversial findings might be different design of reported studies, specially the timing of ultrasound evaluation. [Abstract/Link to Full Text]

Ram�rez MA, Pericuesta E, Fernandez-Gonzalez R, Moreira P, Pintado B, Gutierrez-Adan A
Transcriptional and post-transcriptional regulation of retrotransposons IAP and MuERV-L affect pluripotency of mice ES cells.
Reprod Biol Endocrinol. 2006;455.
BACKGROUND: In the mouse, culture of embryonic stem (ES) cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs). In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it. METHODS: In order to investigate whether this phenotype was associated with chromosome alteration, inadvertent differentiation, or epigenetic modification, we characterized and compared this R1 ES cell line at passage 27 with an early passage and with a second ES cell line C57/CBAF1 generated in our laboratory. We assessed: i) karyotype; ii) expression of pluripotent and differentiation markers, iii) mRNA transcription by qRT-PCR of two REs, intracisternal-A particle (IAP) and murine endogenous-retrovirus-L (MuERV-L), and iv) methylation of IAP and MuERV-L. RESULTS: The R1 ES cell at passage 27, presented normal morphology, karyotype, and expression of genetic markers characteristic of pluripotent; however, it was detected an altered mRNA transcription of sense and antisense RNA strands of both REs, concomitantly with an altered methylation pattern for the IAP element but not for MuERV-L. These results indicate that besides methylation, other post-transcriptional processes are involved in gene silencing of some REs; and that culture of ES cells may decrease their pluripotency by producing inadvertent alterations in the expression of REs without significantly affecting the morphology, chromosome structure, and expression of pluripotent or differentiation markers. CONCLUSION: Inadvertent REs instability may have important consequences for the use of ES cells in transgenesis (chimera formation) or in cell therapy. [Abstract/Link to Full Text]

Karja NW, Kikuchi K, Fahrudin M, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H, Nagai T
Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions.
Reprod Biol Endocrinol. 2006;454.
BACKGROUND: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined. METHODS: In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6. RESULTS: Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA fragmentation rate than those of Pyr-Lac blastocysts. CONCLUSION: These results show that a decrease in developmental ability of embryos cultured by use of glucose instead of pyruvate and lactate after the ferilization may be due to the rise in ROS generation in Day 1 embryos. Moreover, results from this study suggest that the concentration of glucose in the medium that can be used by the Day 1-2 embryos is limited to 3.5 mM and exposure to higher glucose concentrations does not improve embryo development. [Abstract/Link to Full Text]

Telleria CM
Can luteal regression be reversed?
Reprod Biol Endocrinol. 2006;453.
The corpus luteum is an endocrine gland whose limited lifespan is hormonally programmed. This debate article summarizes findings of our research group that challenge the principle that the end of function of the corpus luteum or luteal regression, once triggered, cannot be reversed. Overturning luteal regression by pharmacological manipulations may be of critical significance in designing strategies to improve fertility efficacy. [Abstract/Link to Full Text]

Bernard DJ, Lee KB, Santos MM
Activin B can signal through both ALK4 and ALK7 in gonadotrope cells.
Reprod Biol Endocrinol. 2006;452.
BACKGROUND: Activins stimulate pituitary FSH synthesis via transcriptional regulation of the FSHbeta subunit gene (Fshb). Like other members of the TGFbeta superfamily, these ligands signal through complexes of type I and type II receptor serine/threonine kinases. The type I receptors, or activin receptor-like kinases (ALKs), propagate intracellular signals upon ligand binding and phosphorylation by associated type II receptors. ALK4 is generally regarded as the type I receptor for activins; however, recent data suggested that activin B and AB might also signal through ALK7. Here, we examined a role for ALK7 in activin B-regulated Fshb transcription. METHODS: We analyzed ALK7 mRNA expression in immortalized gonadotrope cells, LbetaT2, and adult murine pituitary by RT-PCR. We next transfected LbetaT2 cells with wild-type and kinase-deficient (Lys to Arg, KR) forms of ALK4 and ALK7 and examined the effects of these receptors on activin A and B stimulated Fshb promoter-reporter activity. Cells were also transfected with constitutively active (Thr to Asp, TD) forms of the receptors and their effects on endogenous Fshb mRNA levels and phosphorylation of transfected Smad2/3 were measured by RT-PCR and Western blot, respectively. Finally, we measured ALK4(TD) and ALK7(TD) stimulation of Fshb transcription when endogenous Smad3 levels were depleted using short hairpin RNAs. RESULTS: ALK7 mRNA was expressed in LbetaT2 cells and pituitary gland. Transfection of ALK4 cDNA potentiated the effects of both activin A and activin B on Fshb promoter-reporter activity in LbetaT2 cells. In contrast, ALK7 transfection selectively potentiated activin B's effects. Transfection of ALK4(KR) and ALK7(KR) partly inhibited basal and activin B-stimulated reporter activity, whereas ALK4(TD) and ALK7(TD) potently stimulated the Fshb promoter and endogenous mRNA levels. Transfection of both ALK4(TD) and ALK7(TD) stimulated Smad2/3 phosphorylation, and the effects of both receptors on Fshb promoter activity were inhibited by depletion of endogenous Smad3 protein levels. CONCLUSION: These data suggest that immortalized gonadotropes express ALK7 and that activin B can signal through this receptor to stimulate Fshb transcription. The relative roles of endogenous ALK4 and ALK7 receptors in mediating activin B's effects in these cells have yet to be determined. [Abstract/Link to Full Text]

Picciarelli-Lima P, Oliveira AG, Reis AM, Kalapothakis E, Mahecha GA, Hess RA, Oliveira CA
Effects of 3-beta-diol, an androgen metabolite with intrinsic estrogen-like effects, in modulating the aquaporin-9 expression in the rat efferent ductules.
Reprod Biol Endocrinol. 2006;451.
BACKGROUND: Fluid homeostasis is critical for normal function of the male reproductive tract and aquaporins (AQP) play an important role in maintenance of this water and ion balance. Several AQPs have been identified in the male, but their regulation is not fully comprehended. Hormonal regulation of AQPs appears to be dependent on the steroid in the reproductive tract region. AQP9 displays unique hormonal regulation in the efferent ductules and epididymis, as it is regulated by both estrogen and dihydrotestosterone (DHT) in the efferent ductules, but only by DHT in the initial segment epididymis. Recent data have shown that a metabolite of DHT, 5-alpha-androstane-3-beta-17-beta-diol (3-beta-diol), once considered inactive, is also present in high concentrations in the male and indeed has biological activity. 3-beta-diol does not bind to the androgen receptor, but rather to estrogen receptors ER-alpha and ER-beta, with higher affinity for ER-beta. The existence of this estrogenic DHT metabolite has raised the possibility that estradiol may not be the only estrogen to play a major role in the male reproductive system. Considering that both ER-alpha and ER-beta are highly expressed in efferent ductules, we hypothesized that the DHT regulation of AQP9 could be due to the 3-beta-diol metabolite. METHODS: To test this hypothesis, adult male rats were submitted to surgical castration followed by estradiol, DHT or 3-beta-diol replacement. Changes in AQP9 expression in the efferent ductules were investigated by using immunohistochemistry and Western blotting assay. RESULTS: Data show that, after castration, AQP9 expression was significantly reduced in the efferent ductules. 3-beta-diol injections restored AQP9 expression, similar to DHT and estradiol. The results were confirmed by Western blotting assay. CONCLUSION: This is the first evidence that 3-beta-diol has biological activity in the male reproductive tract and that this androgen metabolite has estrogen-like activity in the efferent ductules, whose major function is the reabsorption of luminal fluid. [Abstract/Link to Full Text]

Yoshino M, Mizutani T, Yamada K, Yazawa T, Ogata-Kawata H, Sekiguchi T, Kajitani T, Miyamoto K
Co-activator p120 is increased by gonadotropins in the rat ovary and enhances progesterone receptor activity.
Reprod Biol Endocrinol. 2006;450.
BACKGROUND: Ovarian follicular development is primarily dependent on pituitary gonadotropins. Identification of gonadotropin-inducible genes in the ovary is one of the effective approaches for the study of follicular development. In this study we identify rat homologue of p120, a nuclear transcription co-activator, as one of the FSH inducible genes in the rat granulosa cells. METHODS: A full-length cDNA encoding rat p120 was cloned, and expression of the gene in the ovary was examined by Northern blotting. Tissue localization of p120 was examined by in situ hybridization. Cellular functions of p120 were studied by co-transfection of rat p120 gene together with estrogen receptor (ER)-alpha, ER-beta, androgen receptor (AR), or progesterone receptor (PR) genes. RESULTS: A full-length cDNA encoding rat p120 was characterized as a protein with 957 amino acid residues. Rat p120 was expressed ubiquitously, but strongly in the ovary and the testis. Expression of p120 mRNA was also induced in vivo by PMSG or PMSG/hCG treatment. Strong expression of p120 mRNA was observed in the granulosa cells of pre-ovulatory large antral follicles. Progesterone receptor was co-localized with p120 in the large antral follicles. Co-transfection experiments revealed that rat p120 activated AR, ER-alpha, ER-beta, and PR in the presence of their respective ligands. CONCLUSION: These observations suggest that rat p120 is strongly induced in the ovarian granulosa cells, and may work together with PR in the granulosa cells of ovulatory follicles to promote the ovulation process. [Abstract/Link to Full Text]

Hong EJ, Park SH, Choi KC, Leung PC, Jeung EB
Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals.
Reprod Biol Endocrinol. 2006;449.
Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats. [Abstract/Link to Full Text]

Tatone C, Carbone MC
Possible involvement of integrin-mediated signalling in oocyte activation: evidence that a cyclic RGD-containing peptide can stimulate protein kinase C and cortical granule exocytosis in mouse oocytes.
Reprod Biol Endocrinol. 2006;448.
BACKGROUND: Mammalian sperm-oocyte interaction at fertilization involves several combined interactions between integrins on the oocyte and integrin ligands (disintegrins) on the sperm. Recent research has indicated the ability of peptides containing the RGD sequence that characterized several sperm disintegrins, to induce intracellular Ca2+ transients and to initiate parthenogenetic development in amphibian and bovine oocytes. In the present study, we investigate the hypothesis that an integrin-associated signalling may participate in oocyte activation signalling by determining the ability of a cyclic RGD-containing peptide to stimulate the activation of protein kinase C (PKC) and the exocytosis of cortical granules in mouse oocytes. METHODS: An In-Vitro-Fertilization assay (IVF) was carried in order to test the condition under which a peptide containing the RGD sequence, cyclo(Arg-Gly-Asp-D-Phe-Val), was able to inhibit sperm fusion with zona-free mouse oocytes at metaphase II stage. PKC activity was determined by means of an assay based on the ability of cell lysates to phosphorylate MARKS peptide, a specific PKC substrate. Loss of cortical granules was evaluated by measuring density in the oocyte cortex of cortical granules stained with LCA-biotin/Texas red-streptavidin. In all the experiments, effects of a control peptide containing a non RGD sequence, cyclo(Arg-Ala-Asp-D-Phe-Val), were evaluated. RESULTS: The IVF assay revealed that the fusion rate declined significantly when insemination was carried out in the presence of cyclic RGD peptide at concentrations > or = 250 microM (P < 0.05, Student-Newman-Keuls Method). When the peptide was applied to the oocytes at these concentrations, a dose-dependent increase of PKC activity was observed, in association with a loss of cortical granules ranging from 38+/-2.5 % to 52+/-5.4 %. Evaluation of meiotic status revealed that cyclic RGD peptide was ineffective in inducing meiosis resumption under conditions used in the present study. CONCLUSION: The presents results provide evidence that a cyclic RGD peptide highly effective in inhibiting sperm-oocyte interaction stimulates in mouse oocytes the activation of PKC and the exocytosis of cortical granules. These data support the view that RGD-binding receptors may function as signalling receptors giving rise integrated signalling not sufficient for a full oocyte activation response. This study may contribute to the understanding of possible negative effects of skipping gamete interaction in IVF techniques. [Abstract/Link to Full Text]

Sahlin L, Masironi B, Akerberg S, Eriksson H
Tissue- and hormone-dependent progesterone receptor distribution in the rat uterus.
Reprod Biol Endocrinol. 2006;447.
BACKGROUND: Estradiol (E2) and progesterone (P) are well known regulators of progesterone receptor (PR) expression in the rat uterus. However, it is not known which receptor subtypes are involved. Little knowledge exist about possible differences in PR regulation through ERalpha or ERbeta, and whether the PR subtypes are differently regulated depending on ER type bound. Thus, in the present study PR immunostaining has been examined in uteri of ovariectomized (ovx) rats after different treatments of estrogen and P, in comparison with that in immature, cycling, and pregnant animals. METHODS: The uteri were collected from 1) ovx rats treated with E2 and/or P; 2) immature rats, intact cycling rats and animals pregnant day 8 and 18; 3) ovx rats treated with E2 or an estrogen receptor (ER)alpha agonist or an ERbeta agonist. Two antibodies were used, one detecting PRA+B and another one specific for PRB. Real-time PCR was used to determine mRNA levels for PRAB and PRB in experiment 3. RESULTS: In stroma and myometrium faint staining was detected in ovx controls (OvxC), whereas E2 treatment resulted in strong staining. In contrast to this, in luminal epithelium (LE) the staining was strong in the OvxC group, whereas E2 treatment during the last 24 hrs before sacrifice caused a decrease. Similar to OvxC the LE of the immature animals was strongly stained. In the pregnant rats LE was negative, well in agreement with the results seen after E2 treatment. In the pregnant animals the stroma and decidua was strongly stained for PRAB, but only faint for PRB, indicating that PRA is the most expressed isoform in this state. The increase in stromal and myometrial immunostaining after E2 treatment was also found after treatment with the ERalpha agonist PPT. The ERbeta agonist DPN caused a decrease of the PR mRNA levels, which was also found for PRAB and PRB immunostaining in the GE. CONCLUSION: Stromal and myometrial PRAB levels are increased via ERalpha, as shown by treatment with E2 and the ERalpha agonist PPT, while the levels in LE are decreased. The uterine stroma of pregnant rats strongly expressed PRAB, but very little PRB, which is different to E2 treated ovx animals where both PRAB and PRB are strongly expressed. The ERbeta agonist DPN decreased the mRNA levels of PRAB and PRB, as well as the PRAB protein level in GE. These results suggest that ERbeta signals mainly down-regulate PR levels in the epithelial cells. ERalpha, on the other hand, up-regulates PR levels in the stroma and myometrium while it decreased them in LE. Thus, the effects from E2 and PPT on the mRNA levels, as determined by PCR, could be annihilated since they are increased and decreased depending on cell type. The distribution and amount of PR isoforms strongly depend on the hormonal milieu and cell type within the rat uterus. [Abstract/Link to Full Text]

MacKenzie S, Montserrat N, Mas M, Acerete L, Tort L, Krasnov A, Goetz FW, Planas JV
Bacterial lipopolysaccharide induces apoptosis in the trout ovary.
Reprod Biol Endocrinol. 2006;446.
BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines. [Abstract/Link to Full Text]

Tingaker BK, Ekman-Ordeberg G, Forsgren S
Presence of sensory nerve corpuscles in the human corpus and cervix uteri during pregnancy and labor as revealed by immunohistochemistry.
Reprod Biol Endocrinol. 2006;445.
BACKGROUND: The uterus is exposed to changes such as enlargement and distension during pregnancy and labor. In these processes and in the process of cervical ripening, proprioceptive information is likely to be of great importance. Therefore, we wanted to study the possible existence of sensory nerve corpuscles in uterine corpus and cervix during pregnancy and labor. Studies on this aspect have not previously been perfomed. METHODS: Biopsies were taken from the upper edge of the hysterotomy during caesarean section at term (n = 8), in labor (n = 5) and from the corresponding area in the non-pregnant uterus after hysterectomy (n = 7). Cervical biopsies were obtained transvaginally from the anterior cervical lip. Serial cryostat sections were prepared for immunohistochemistry using polyclonal antibodies against nerve growth factor receptor p75, protein gene product 9.5 and S-100. RESULTS: Structures with the characteristics of sensory nerve corpuscles were observed in several specimens after staining for p75, PGP 9.5 and S-100. They were observed in specimens of the non-pregnant corpus and cervix and also in specimens of the pregnant cervix before onset of labor. However, they were absent in all specimens during labor. CONCLUSION: Sensory corpuscles have here for the first time been detected in the human corpus and cervix uteri. Studies on the importance of the corpuscles in relation to the protective reflex actions that occur in the uterus during pregnancy should be performed in the future. [Abstract/Link to Full Text]

Rout UK
Valproate, thalidomide and ethyl alcohol alter the migration of HTR-8/SVneo cells.
Reprod Biol Endocrinol. 2006;444.
BACKGROUND: Valproate, thalidomide and alcohol (ethanol) exposure during the first trimester of pregnancy is known to cause several developmental disorders. All these teratogens are known to pass the placental barrier and interfere directly with the normal development of the fetus. However, these teratogens also alter the formation and function of the placenta itself which may in turn affect the proper nourishment and development of the fetus. Optimum development of the placenta requires adequate invasion of trophoblast into the maternal uterine tissues. Changes in the migratory behavior of trophoblast by maternal exposure to these teratogens during placentogenesis may therefore alter the structure and function of the placenta. METHODS: In the present study, the effects of sodium valproate, thalidomide and alcohol on the migration of human first trimester trophoblast cell line (HTR-8/SVneo) were examined in vitro. Cells were cultured in the wells of 48-well culture plates as mono or multilayers. Circular patches of cells were removed from the center of the wells by suction, and the migration of cells into the wound was studied using microscopy. Effects of low and high concentrations of valproate, thalidomide and alcohol were examined on the healing of wounds and on the migration rate of cells by determining the wound areas at 0, 3, 6, 12, 24 and 48 h. Effects of drugs and alcohol on the proliferation and the expression levels of integrin subunits beta1 and alpha5 in cells were examined. RESULTS: The migration rates of trophoblast differed between wounds created in mono and multilayers of cells. Exposure to teratogens altered the migration of trophoblast into mono and multilayer wounds. The effects of valproate, thalidomide and alcohol on the proliferation of cells during the rapid migratory phase were mild. Drug exposure caused significant changes in the expression levels of beta1 and alpha5 integrin subunits. CONCLUSION: Results suggest that exposure to valproate, thalidomide or alcohol during the first trimester of pregnancy may change the ultrastructure of the placenta by altering the migration of trophoblast cells and this effect may be mediated by drug- or alcohol-induced changes in the expression levels of beta1 and alpha5 integrin subunits. [Abstract/Link to Full Text]

Cruz ME, Flores A, Palafox MT, Mel�ndez G, Rodr�guez JO, Chavira R, Dom�nguez R
The role of the muscarinic system in regulating estradiol secretion varies during the estrous cycle: the hemiovariectomized rat model.
Reprod Biol Endocrinol. 2006;443.
There is evidence that one gonad has functional predominance. The present study analyzed the acute effects of unilateral ovariectomy (ULO) and blocking the cholinergic system, by injecting atropine sulfate (ATR), on estradiol (E2) serum concentrations during the estrous cycle. The results indicate that ULO effects on E2 concentrations are asymmetric, vary during the estrous cycle, and partially depend on the cholinergic innervation.Perforation of the left peritoneum resulted in lower E2 serum concentrations in the three stages of the estrous cycle. At proestrus, unilateral or bilateral perforation of the peritoneum resulted in lower E2 serum concentrations.ULO of the right ovary (left ovary in situ) resulted in significantly higher E2 concentrations than animals with ULO of the left ovary (right ovary in situ). ATR treatment to ULO rats on D1 resulted in a significant drop of E2 serum concentrations. ULO rats treated with ATR on D2 or P, resulted in an asymmetrical E2 secretion response; when the right ovary remained in situ an increase in E2 was observed, and a decrease when the left ovary remained in situ.The results obtained in the present study suggest that each ovary's ability to compensate the secretion of E2 from the missing ovary is different and varies during the estrous cycle. The results also suggest that the cholinergic system participates in regulating ovarian E2 secretion. Such participation varies according to the ovary remaining in situ and the stage of the estrous cycle of the animal.The results agree with previously stated hypothesis of a neural pathway arising from the peritoneum that participates in regulating E2 secretion, and also supports the idea of cross-talk between the ovaries, via a neural communication, that modulates E2 secretion. [Abstract/Link to Full Text]

Nishida T, Nishida N
Reinstatement of "germinal epithelium" of the ovary.
Reprod Biol Endocrinol. 2006;442.
BACKGROUND: The existing dogma that the former term ovarian "germinal epithelium" resulted from a mistaken belief that it could give rise to new germ cells is now strongly challenged. DISCUSSION: Two years ago, a research group of the University of Tennessee led by Antonin Bukovsky successfully demonstrated the oogenic process from the human ovarian covering epithelium now commonly called the ovarian surface epithelium. They showed the new oocyte with zona pellucida and granulosa cells, both originated from the surface epithelium arising from mesenchymal cells in the tunica albuginea, and stressed that the human ovary could form primary follicles throughout the reproductive period. This gives a big impact not only to the field of reproductive medicine, but also to the oncologic area. The surface epithelium is regarded as the major source of ovarian cancers, and most of the neoplasms exhibit the histology resembling m�llerian epithelia. Since the differentiating capability of the surface epithelium has now expanded, the histologic range of the neoplasms in this category may extend to include both germ cell tumors and sex cord-stromal cell tumors. SUMMARY: Since the oogenic capability of ovarian surface cells has been proven, it is now believed that the oocytes can originate from them. The term "germinal epithelium", hence, might reasonably be reinstated. [Abstract/Link to Full Text]

Wicherek L, Dutsch-Wicherek M, Galazka K, Banas T, Popiela T, Lazar A, Kleinrok-Podsiadlo B
Comparison of RCAS1 and metallothionein expression and the presence and activity of immune cells in human ovarian and abdominal wall endometriomas.
Reprod Biol Endocrinol. 2006;441.
BACKGROUND: The coexistence of endometrial and immune cells during decidualization is preserved by the ability of endometrial cells to regulate the cytotoxic immune activity and their capability to be resistant to immune-mediated apoptosis. These phenomena enable the survival of endometrial ectopic cells. RCAS1 is responsible for regulation of cytotoxic activity. Metallothionein expression seems to protect endometrial cells against apoptosis. The aim of the present study was to evaluate RCAS1 and metallothionein expression in human ovarian and scar endometriomas in relation to the presence of immune cells and their activity. METHODS: Metallothionein, RCAS1, CD25, CD69, CD56, CD16, CD68 antigen expression was assessed by immunohistochemistry in ovarian and scar endometriomas tissue samples which were obtained from 33 patients. The secretory endometrium was used as a control group (15 patients). RESULTS: The lowest metallothionein expression was revealed in ovarian endometriomas in comparison to scar endometriomas and to the control group. RCAS1 expression was at the highest level in the secretory endometrium and it was at comparable levels in ovarian and scar endometriomas. Similarly, the number of CD56-positive cells was lower in scar and ovarian endometriomas than in the secretory endometrium. The highest number of macrophages was found in ovarian endometriomas. RCAS1-positive macrophages were observed only in ovarian endometriomas. CD25 and CD69 antigen expression was higher in scar and ovarian endometriomas than in the control group. CONCLUSION: The expression of RCAS1 and metallothionein by endometrial cells may favor the persistence of these cells in ectopic localization both in scar following cesarean section and in ovarian endometriosis. [Abstract/Link to Full Text]

Tung JY, Rosen MP, Nelson LM, Turek PJ, Witte JS, Cramer DW, Cedars MI, Reijo-Pera RA
Novel missense mutations of the Deleted-in-AZoospermia-Like (DAZL) gene in infertile women and men.
Reprod Biol Endocrinol. 2006;440.
BACKGROUND: The Deleted-in-AZoospermia-Like (DAZL) gene has homologs required for germ cell development in many organisms. Recently, we showed that there are several common polymorphisms within the DAZL gene that are associated with age at ovarian failure/menopause and sperm count. METHODS: Here we sought to identify rare mutations in DAZL and examine their phenotypes in men and women. We sequenced the DAZL gene in 519 individuals; sequences spanned the entire coding region of the gene. RESULTS: We report the identification of four putative missense mutations in DAZL. Three individuals that were heterozygous for a DAZL mutation reported having children, while two individuals that were homozygous reported no children. These mutations were found only in infertile men and women. CONCLUSION: Given the strong data associating DAZL polymorphisms and deletions with fertility in humans and model organisms, we suggest that these mutations may be associated with age at menopause and/or sperm count and warrant further biochemical and genetic investigation. [Abstract/Link to Full Text]

Bobe J, Montfort J, Nguyen T, Fostier A
Identification of new participants in the rainbow trout (Oncorhynchus mykiss) oocyte maturation and ovulation processes using cDNA microarrays.
Reprod Biol Endocrinol. 2006;439.
BACKGROUND: The hormonal control of oocyte maturation and ovulation as well as the molecular mechanisms of nuclear maturation have been thoroughly studied in fish. In contrast, the other molecular events occurring in the ovary during post-vitellogenesis have received far less attention. METHODS: Nylon microarrays displaying 9152 rainbow trout cDNAs were hybridized using RNA samples originating from ovarian tissue collected during late vitellogenesis, post-vitellogenesis and oocyte maturation. Differentially expressed genes were identified using a statistical analysis. A supervised clustering analysis was performed using only differentially expressed genes in order to identify gene clusters exhibiting similar expression profiles. In addition, specific genes were selected and their preovulatory ovarian expression was analyzed using real-time PCR. RESULTS: From the statistical analysis, 310 differentially expressed genes were identified. Among those genes, 90 were up-regulated at the time of oocyte maturation while 220 exhibited an opposite pattern. After clustering analysis, 90 clones belonging to 3 gene clusters exhibiting the most remarkable expression patterns were kept for further analysis. Using real-time PCR analysis, we observed a strong up-regulation of ion and water transport genes such as aquaporin 4 (aqp4) and pendrin (slc26). In addition, a dramatic up-regulation of vasotocin (avt) gene was observed. Furthermore, angiotensin-converting-enzyme 2 (ace2), coagulation factor V (cf5), adam 22, and the chemokine cxcl14 genes exhibited a sharp up-regulation at the time of oocyte maturation. Finally, ovarian aromatase (cyp19a1) exhibited a dramatic down-regulation over the post-vitellogenic period while a down-regulation of Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (cmah) was observed at the time of oocyte maturation. CONCLUSION: We showed the over or under expression of more that 300 genes, most of them being previously unstudied or unknown in the fish preovulatory ovary. Our data confirmed the down-regulation of estrogen synthesis genes during the preovulatory period. In addition, the strong up-regulation of aqp4 and slc26 genes prior to ovulation suggests their participation in the oocyte hydration process occurring at that time. Furthermore, among the most up-regulated clones, several genes such as cxcl14, ace2, adam22, cf5 have pro-inflammatory, vasodilatory, proteolytics and coagulatory functions. The identity and expression patterns of those genes support the theory comparing ovulation to an inflammatory-like reaction. [Abstract/Link to Full Text]

Revelli A, Poso F, Gennarelli G, Moffa F, Grassi G, Massobrio M
Recombinant versus highly-purified, urinary follicle-stimulating hormone (r-FSH vs. HP-uFSH) in ovulation induction: a prospective, randomized study with cost-minimization analysis.
Reprod Biol Endocrinol. 2006;438.
BACKGROUND: Both recombinant FSH (r-FSH) and highly-purified, urinary FSH (HP-uFSH) are frequently used in ovulation induction associated with timed sexual intercourse. Their effectiveness is reported to be similar, and therefore the costs of treatment represent a major issue to be considered. Although several studies about costs in IVF have been published, data obtained in low-technology infertility treatments are still scarce. METHODS: Two hundred and sixty infertile women (184 with unexplained infertility, 76 with CC-resistant polycystic ovary syndrome) at their first treatment cycle were randomized and included in the study. Ovulation induction was accomplished by daily administration of rFSH or HP-uFSH according to a low-dose, step-up regimen aimed to obtain a monofollicular ovulation. A bi- or tri-follicular ovulation was anyway accepted, whereas hCG was withdrawn and the cycle cancelled when more than three follicles greater than or equal to 18 mm diameter were seen at ultrasound. The primary outcome measure was the cost of therapy per delivered baby, estimated according to a cost-minimization analysis. Secondary outcomes were the following: monofollicular ovulation rate, total FSH dose, cycle cancellation rate, length of the follicular phase, number of developing follicles (>12 mm diameter), endometrial thickness at hCG, incidence of twinning and ovarian hyperstimulation syndrome, delivery rate. RESULTS: The overall FSH dose needed to achieve ovulation was significantly lower with r-FSH, whereas all the other studied variables did not significantly differ with either treatments. However, a trend toward a higher delivery rate with r-FSH was observed in the whole group and also when results were considered subgrouping patients according to the indication to treatment. CONCLUSION: Considering the significantly lower number of vials/patient and the slight (although non-significant) increase in the delivery rate with r-FSH, the cost-minimization analysis showed a 9.4% reduction in the overall therapy cost per born baby in favor of r-FSH. [Abstract/Link to Full Text]

Mayerhofer A, Kunz L, Krieger A, Proskocil B, Spindel E, Amsterdam A, Dissen GA, Ojeda SR, Wessler I
FSH regulates acetycholine production by ovarian granulosa cells.
Reprod Biol Endocrinol. 2006;437.
BACKGROUND: It has been previously shown that cultured granulosa cells (GCs) derived from human ovarian preovulatory follicles contain choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. They also produce ACh and express functional muscarinic ACh receptors. ACh can act on GCs to increase proliferation, disrupt gap junctional communication, alter intracellular calcium levels, as well as expression of transcription factors, suggesting an unrecognized role of ACh in GC function. To gain further insights into the possible role of ACh in the ovary, we examined ChAT expression in the gland before and after birth, as well as in adults, and studied the regulation of ACh production by FSH. METHODS: ChAT immunohistochemistry was performed using ovarian samples of different species and ages (embryonic, postnatal and adult rats and mice, including embryonic ovaries from mice null for ChAT, neonatal and adult rhesus monkeys and adult humans). ACh was measured by HPLC and/or a fluorescence based method in rat ovaries and in a FSH receptor-expressing cell line (rat GFSHR-17) cultured with or without FSH. RESULTS: In adult rat, as well as in all other species, ovarian ChAT immunoreactivity is associated with GCs of antral follicles, but not with other structures, indicating that GCs are the only ovarian source of ACh. Indeed ACh was clearly detected in adult rat ovaries by two methods. ChAT immunoreactivity is absent from embryonic and/or neonatal ovaries (mouse/rat and monkey) and ovarian development in embryonic mice null for ChAT appears normal, suggesting that ACh is not involved in ovarian or follicular formation. Since ChAT immunoreactivity is present in GCs of large follicles and since the degree of the ChAT immunoreactivity increases as antral follicles grow, we tested whether ACh production is stimulated by FSH. Rat GFSHR-17 cells that stably express the FSH receptor, respond to FSH with an increase in ACh production. CONCLUSION: ACh and ChAT are present in GCs of growing follicles and FSH, the major driving force of follicular growth, stimulates ACh production. Since ACh stimulates proliferation and differentiation processes in cultured GCs, we suggest that ACh may act in the growing ovarian follicle as a local mediator of some of the actions ascribed to FSH. [Abstract/Link to Full Text]

Rago V, Siciliano L, Aquila S, Carpino A
Detection of estrogen receptors ER-alpha and ER-beta in human ejaculated immature spermatozoa with excess residual cytoplasm.
Reprod Biol Endocrinol. 2006;436.
BACKGROUND: A key role of estrogens in human sperm biology has been recently suggested by aromatase and estrogen receptor detection in human testicular germ cells and ejaculated spermatozoa. However, the involvement of these hormones in the sperm maturation process is still not defined. The aim of this work was to investigate the expression of estrogen receptors, ER-alpha and ER-beta, in human ejaculated immature spermatozoa with excess residual cytoplasm. METHODS: Immunofluorescence labelling has been used to localize ER-alpha and ER-beta proteins in immature spermatozoa isolated by Percoll gradient, while Western blot analysis was carried out on sperm protein extracts. RESULTS: Both estrogen receptors were localized in excess residual cytoplasm of immature sperm, while sperm tails showed only ER-beta. Furthermore, in the same cells, immunoblots detected the presence of the full-length (approximately 67 kDa) ER-alpha and (approximately 59 kDa) ER-beta proteins, together with a approximately 50 kDa ER-beta species, lacking in mature sperm. CONCLUSION: The present investigation demonstrated ER-alpha and ER-beta presence in excess residual cytoplasm of human abnormal sperm cells, suggesting the hypothesis that both the 'classical' ERs could be able to mediate estrogen action in spermatogenetic cells. Furthermore, the presence of the short ER-beta form in abnormal germ cells and its disappearance in mature sperm, support estrogen modulation via different ER forms during sperm maturation. [Abstract/Link to Full Text]

Bonnet A, Frappart PO, Dehais P, Tosser-Klopp G, Hatey F
Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization.
Reprod Biol Endocrinol. 2006;435.
FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized.In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts.A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin. [Abstract/Link to Full Text]

Browne RK, Seratt J, Vance C, Kouba A
Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri).
Reprod Biol Endocrinol. 2006;434.
The endangered Wyoming toad (Bufo baxteri) is the subject of an extensive captive breeding and reintroduction program. Wyoming toads in captivity rarely ovulate spontaneously and hormonal induction is used to ovulate females or to stimulate spermiation in males. With hormonal induction, ovulation is unreliable and egg numbers are low. The sequential administration of anovulatory doses of hormones (priming) has increased egg numbers and quality in both anurans and fish. Consequently, we tested the efficacy of a combination of human Chorionic Gonadotrophin (hCG) and Luteinizing Hormone Releasing Hormone analogue (LHRHa) administered as one dose, or two or three sequential doses to Bufo baxteri on egg numbers, fertilization and early embryo development. Spawning toads deposited eggs into Simplified Amphibian Ringers (SAR) solution to enable controlled in-vitro fertilization (IVF) with sperm from hormonally induced male toads. Unprimed females receiving a single mixed normally ovulatory dose of 500 IU hCG plus 4 micrograms of LHRHa produced no eggs. Whereas females primed with this dose and an anovulatory dose (100 IU hCG and 0.8 micrograms of LHRHa) of the same hormones, or primed only with an anovulatory dose, spawned after then receiving an ovulatory dose. Higher total egg numbers were produced with two primings than with one priming. Moreover, two primings produced significantly more eggs from each individual female than one priming. The cleavage rate of eggs was not found to differ between one or two primings. Nevertheless, embryo development with eggs from two primings gave a significantly greater percentage neurulation and swim-up than those from one priming. Of the male toads receiving a single dose of 300 IU hCG, 80% produced spermic urine with the greatest sperm concentration 7 hours post-administration (PA). However, peak sperm motility (95%) was achieved at 5 hours PA and remained relatively constant until declining 20 hours PA. In conclusion, Bufo baxteri egg numbers and quality benefited from sequential priming with LHRHa and hCG whereas spermic urine for IVF was produced from males with a single dose of hCG. The power of assisted reproduction technology in the conservation of endangered amphibians is shown by the release of nearly 2000 tadpoles produced by IVF during this study. [Abstract/Link to Full Text]

Piketty V, Kara E, Guillou F, Reiter E, Crepieux P
Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization.
Reprod Biol Endocrinol. 2006;433.
BACKGROUND: The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. METHODS: Human embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319-418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. RESULTS: In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319-418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. CONCLUSION: From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH. [Abstract/Link to Full Text]

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-Tornbl�m S, Stjernholm-Vladic Y, Bystr�m 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]