Impairments in the reproductive axis of female mice lacking estrogen receptor ß in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERß. Lack of functional ERα and ERß genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERß KO (CERßKO) and a GnRH neuron-specific ERßKO (GERßKO) mouse models. Both ERßKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERßKO mice were similar to control mice; however female CERßKO and GERßKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERßKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERß in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERßKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERßKO mouse models. In male ERßKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERß in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

Genetic mechanisms mediating kisspeptin regulation of GnRH gene expression.

Kisspeptins (Kiss) have been shown to be key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion. In vitro studies have demonstrated an increase in GnRH gene expression by Kiss suggesting regulation of GnRH at both the secretory and pretranslational levels. Here, we define genetic mechanisms that mediate Kiss action on target gene expression. In vitro, sequential deletions of the mouse GnRH (mGnRH) gene promoter fused to the luciferase (LUC) reporter gene localized at kisspeptin-response element (KsRE) between -3446 and -2806 bp of the mGnRH gene. In vivo, transgenic mice bearing sequential deletions of the mGnRH gene promoter linked to the LUC reporter localized an identical KsRE. To define the mechanism of regulation, Kiss was first shown to induce nucleosome-depleted DNA within the KsRE, and a potential binding site for the transcription factor, Otx-2, was revealed. Furthermore, increased Otx-2 mRNA, protein, and binding to the KsRE after Kiss treatment were demonstrated. In conclusion, this work identified elements in GnRH-neuronal cell lines and in transgenic mice that mediate positive regulation of GnRH by Kiss. In addition, we show for the first time that Otx-2 is regulated by Kiss, and plays a role in mediating the transcriptional response of mGnRH gene.

The gonadotropin-releasing hormone cell-specific element is required for normal puberty and estrous cyclicity.

Appropriate tissue-specific gene expression of gonadotropin-releasing hormone (GnRH) is critical for pubertal development and maintenance of reproductive competence. In these studies, a common element in the mouse GnRH (mGnRH) promoter, between -2806 and -2078 bp, is shown to mediate differential regulation of hypothalamic and ovarian mGnRH expression. To further characterize this region, we generated a knock-out mouse (GREKO(-/-)) with a deletion of the mGnRH promoter fragment between -2806 and -2078 bp. GnRH mRNA expression in the brain of GREKO(-/-) was less than the expression in wild-type mice; however, immunohistochemical analysis revealed no difference between the numbers of GnRH neurons among groups. GnRH mRNA expression in the ovary was fivefold higher in GREKO(-/-). The immunohistochemical staining for GnRH in the ovary increased in surface epithelial and granulosa cells and also in the corpora lutea of GREKO(-/-) mice. The reproductive phenotype revealed that the mean day of vaginal opening was delayed, and additionally, there was a significant decrease in the length of proestrus and diestrus-metestrus phases of the estrous cycle, resulting in a shortened estrous cycle in GREKO(-/-) mice. This work supports the hypothesis that the region of the GnRH promoter contained between -2806 and -2078 bp acts as a cell-specific enhancer in the GnRH neuron and as a repressor in the ovary. Deletion of this region in vivo implicates the GnRH promoter in mediating pubertal development and periodic reproductive cycling, and forms the foundation to define the nuclear proteins important for puberty and estrous cycling in mammals.

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

Development and Characterization of Novel Rat Anti-mERß Sera.

Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERß. Understanding the function of ERs necessitates knowing their cellular location and protein partners, which, in turn, requires reliable and specific antibodies. Several antibodies are available for ERα; however, discrepancies in immunoreactivity have been reported for ERß. Here, we have developed antisera for mouse ERß (mERß) using a specific C-terminal 18-amino acid peptide conjugated to mariculture keyhole limpet hemocyanin. Sprague Dawley rats were immunized, and the resulting antisera were characterized by Western blot analysis of nuclear extracts from tissues of wild-type (WT) mice, and mice genetically modified to lack either ERα (CERαKO) or ERß (CERßKO). An approximately 56-kDa protein was detected in the hypothalamus, uterus, ovary, mammary gland, testes, and epididymis of WT mice, consistent with the predicted molecular size of ERß. In addition, the same protein band was identified in in vitro synthesized mERß protein and in the mammary glands of CERαKO mice. The approximately 56-kDa protein was not observed in in vitro synthesized mERα protein or in any tissue examined in the CERßKO mice. Immunohistochemistry using the antisera revealed ERß staining in the granulosa cells of WT ovaries and in the mediobasal hypothalamus, paraventricular nucleus, and cerebral cortex in the WT adult mouse brain. These data suggest that the novel rat anti-mERß sera are specific to ERß to allow investigators to explore to cellular and physiological role of ERß in the brain and other mouse tissues.

Modulation of renal CNG-A3 sodium channel in rats subjected to low- and high-sodium diets.

In this work, we studied the mRNA distribution of CNG-A3, an amiloride-sensitive sodium channel that belongs to the cyclic nucleotide-gated (CNG) family of channels, along the rat nephron. The possible involvement of aldosterone in this process was also studied. We also evaluated its expression in rats subjected to diets with different concentrations of sodium or to alterations in aldosterone plasma levels. Total RNA isolated from whole kidney and/or dissected nephron segments of Wistar rats subjected to low- and high-sodium diets, furosemide treatment, adrenalectomy, and adrenalectomy with replacement by aldosterone were analyzed by the use of Western blot, ribonuclease protection assay (RPA) and/or reverse transcription followed by semi-quantitative polymerase chain reaction (RT-PCR). CNG-A3 sodium channel mRNA and protein expression, in whole kidneys of rats subjected to high-Na+ diet, were lower than those in animals given a low-salt diet. Renal CNG-A3 mRNA expression was also decreased in adrenalectomized rats, and was normalized by aldosterone replacement. Moreover, a CNG-A3 mRNA expression study in different nephron segments revealed that aldosterone modulation is present in the cortical thick ascending loop (cTAL) and cortical collecting duct (CCD). This result suggests that CNG-A3 is responsive to the same hormone signaling as the amiloride sensitive sodium channel ENaC and suggests the CNG-A3 may have a physiological role in sodium reabsorption.

Disrupted kisspeptin signaling in GnRH neurons leads to hypogonadotrophic hypogonadism.

Landmark studies have shown that mutations in kisspeptin and the kisspeptin receptor (Kiss1r) result in reproductive dysfunction in humans and genetically altered mouse models. However, because kisspeptin and its receptor are present in target cells of the central and peripheral reproductive axis, the precise location(s) for the pathogenic signal is unknown. The study described herein shows that the kisspeptin-Kiss1r signaling pathway in the GnRH neuron is singularly critical for both the onset of puberty as well as the attainment of normal reproductive function. In this study, we directly test the hypothesis that kisspeptin neurons regulate GnRH secretion through the activation of Kiss1r on the plasma membrane of GnRH neurons. A GnRH neuron-specific Kiss1r knockout mouse model (GKirKO) was generated, and reproductive development and phenotype were assessed. Both female and male GKirKO mice were infertile, having low serum LH and FSH levels. External abnormalities such as microphallus and decreased anogenital distance associated with failure of preputial gland separation were present in GKirKO males. A delay in pubertal onset and abnormal estrous cyclicity were observed in female GKirKO mice. Taken together, these data provide in vivo evidence that Kiss1r in GnRH neurons is critical for reproductive development and fertility.

Both estrogen receptor α and ß stimulate pituitary GH gene expression.

Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α- and ERß-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERß mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERß in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

Glucagon regulates hepatic kisspeptin to impair insulin secretion.

Early in the pathogenesis of type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic α cells occurs prior to impaired glucose-stimulated insulin secretion (GSIS) from ß cells. However, whether hyperglucagonemia is causally linked to ß cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on ß cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose-intolerant, high-fat-diet fed, and Lepr(db/db) mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.

Estrogen regulation of gene expression in GnRH neurons.

Estrogen plays an essential role in the regulation of the female reproductive hormone axis, and specifically is a major regulator of GnRH neuronal function in the female brain. GnRH neuronal cell lines were used to explore the direct effects of estradiol on gene expression in GnRH neurons. The presence of estrogen receptor (ER) binding sites was established by a receptor-binding assay, and estrogen receptor alpha and beta mRNA were identified in GN11 cells and ERbeta in GT1-7 cells using RT-PCR analysis of mRNA. ERalpha was more abundantly expressed in GN11 cells than ERbeta as assessed by real-time PCR. Additionally, GN11 cells expressed significantly more of both ERalpha and beta than GT1-7 cells. Functional studies in GN11 and GT1-7 demonstrated estrogen down regulation of endogenous mouse GnRH mRNA levels using quantitative real-time PCR (qRT-PCR). Correspondingly, estradiol also reduced secretion of GnRH from both the GN11 and GT1-7 cell lines. Since estradiol has been shown to regulate progesterone receptor (PR) expression; similar studies were performed demonstrating an estradiol mediated increase in PR in both cell lines. Estradiol regulation of ER expression was also explored and these studies indicated that estradiol decreased ERalpha and ERbeta mRNA levels in a dose-dependent manner in GN11 and GT1-7 cells. These effects were blocked by the addition of the estrogen receptor antagonist ICI 182,780. Both PPT, a specific ERalpha agonist, and DPN, a specific ERbeta agonist, inhibited GnRH gene expression in GN11 cells, but only DPN inhibited GnRH gene expression in GT1-7 cells, consistent with their undetectable levels of ERalpha expression. These studies characterize a direct inhibitory effect of estradiol on GnRH in GnRH neurons, and a direct stimulatory effect of estradiol on PR gene expression. In addition, the agonist studies indicate that there is a functional overlap of ERalpha and ERbeta regulation in GnRH neurons. These studies may give insight into the molecular regulation of estrogen negative feedback in the central reproductive axis.

Kisspeptin increases GnRH mRNA expression and secretion in GnRH secreting neuronal cell lines.

Kisspeptins, and their G-protein coupled receptor 54 (GPR54), are key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion in humans and other mammals. Several studies demonstrate that the central or systemic administration of kisspeptin increases GnRH and gonadotropin secretion in both prepubertal and adult animals; however, the cellular targets and intracellular mechanisms of action in the central reproductive axis are unclear. In this study, we documented the presence of GPR54 in two GnRH secreting neuronal cell lines (GT1-7 and GN11). Kisspeptin treatment increases GnRH secretion and GnRH mRNA levels in a dose and time dependent manner. 10(-9)M kisspeptin maximally stimulated GnRH secretion by 2-fold and GnRH mRNA levels up to 4-fold after 4h of treatment in both cell lines. Negative regulation by 17beta-estradiol of GnRH secretion and GnRH mRNA was antagonized by kisspeptin. Co-treatment with kisspeptin and 17beta-estradiol increased GnRH secretion by 2-fold and GnRH mRNA by 4-fold over estradiol alone in both cell lines. Intracellular signaling pathway studies showed that an ERK1/2 MAPK inhibitor (PD98059) and a PI3K inhibitor, LY29402, attenuated the effects of kisspeptin on GnRH mRNA modulation. Furthermore, Western blot analysis showed that phosphorylation of both MAPK and Akt substrates increased with kisspeptin treatment. This work demonstrates that the kisspeptin-GPR54 system plays a significant role stimulating GnRH secretion and positive regulation of GnRH mRNA levels in GnRH neurons in culture, and also, demonstrates the activation of MAPK and Akt signaling pathways by kisspeptin in GT1-7 and GN11 cell lines.

Thyroid hormones stimulate renal expression of CFTR.

CFTR is a multifunctional protein of the ATP binding cassette family that may contribute to overall electrolyte homeostasis by acting as a chloride channel in the kidney. In renal tissues CFTR does not exists only in its full-length form, but also as a kidney-specific, truncated splice variant, TNR-CFTR. In this study we show that both forms of CFTR are regulated by thyroid hormones in rat renal tissue. Four groups of male rats were used: control, hypothyroid, hypothyroid with T(4) treatment and hyperthyroid rats. The hypothyroid rats showed a decrease of both CFTR and TNR-CFTR mRNAs (44%, and 49%, respectively, n=5; p<0.05) and proteins (30% and 37%, respectively, n=5, p<0.05) expressions, compared to control group. In hyperthyroid rats, a significant increase in both CFTR and TRN-CFTR mRNAs expressions (43% and 95%, n=5; p<0.05) and proteins (250% and 38%, respectively, n=5, p<0.05) was observed when compared to control group. Treatment of immortalized rat proximal tubule cells (IRPTC) with T(3) (10(-7)M) produced also an increase of CFTR mRNA expression (95%, n=5, p<0.05). Analysis of the promoter region of CFTR transfected to IRPTC showed that T(3) (10(-7) M) stimulates the CFTR promoter (38%, n=4, p<0.05).