Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel.

Leptin, secreted by adipocytes, directly influences the onset of puberty in mammals. Our previous study showed that leptin stimulation could promote the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from pituitary cells in primary culture and ovarian development in chub mackerel. This study aimed to elucidate the detailed mechanism of leptin-induced effects on gonadotropin hormone-producing cells. We produced recombinant leptin using silkworm pupae and investigated the effects of leptin on FSH and LH secretion and gene expression in the primary culture of pituitary cells from chub mackerel. The presence or absence of co-expression of lepr mRNA, FSH and LH b-subunit mRNA in gonadotropic cells was examined by double-labeled in situ hybridization. The addition of leptin significantly increased the secretion and gene expression of FSH and LH from male and female pituitary cells in primary culture. In contrast, gonadotropin-releasing hormone 1 affected neither FSH secretion in cells from females nor fshb and lhb expression in cells from males and females. The expression of lepr was observed in FSH- and LH-producing cells of both males and females. The results indicate that leptin directly regulates gonadotropin synthesis and secretion and plays an important role in the induction of puberty in teleost fish.

Androgen receptor positively regulates gonadotropin-releasing hormone receptor in pituitary gonadotropes.

Within pituitary gonadotropes, the gonadotropin-releasing hormone receptor (GnRHR) receives hypothalamic input from GnRH neurons that is critical for reproduction. Previous studies have suggested that androgens may regulate GnRHR, although the mechanisms remain unknown. In this study, we demonstrated that androgens positively regulate Gnrhr mRNA in mice. We then investigated the effects of androgens and androgen receptor (AR) on Gnrhr promoter activity in immortalized mouse LßT2 cells, which represent mature gonadotropes. We found that AR positively regulates the Gnrhr proximal promoter, and that this effect requires a hormone response element (HRE) half site at -159/-153 relative to the transcription start site. We also identified nonconsensus, full-length HREs at -499/-484 and -159/-144, which are both positively regulated by androgens on a heterologous promoter. Furthermore, AR associates with the Gnrhr promoter in ChIP. Altogether, we report that GnRHR is positively regulated by androgens through recruitment of AR to the Gnrhr proximal promoter.

Long extensions with varicosity-like structures in gonadotrope Lh cells facilitate clustering in medaka pituitary culture.

Accumulating evidence indicates that some pituitary cell types are organized in complex networks in both mammals and fish. In this study, we have further investigated the previously described cellular extensions formed by the medaka (Oryzias latipes) luteinizing hormone gonadotropes (Lh cells). Extensions, several cell diameters long, with varicosity-like swellings, were common both in vitro and in vivo. Some extensions approached other Lh cells, while others were in close contact with blood vessels in vivo. Gnrh further stimulated extension development in vitro. Two types of extensions with different characteristics could be distinguished, and were classified as major or minor according to size, origin and cytoskeleton protein dependance. The varicosity-like swellings appeared on the major extensions and were dependent on both microtubules and actin filaments. Immunofluorescence revealed that Lhß protein was mainly located in these swellings and at the extremity of the extensions. We then investigated whether these extensions contribute to network formation and clustering, by following their development in primary cultures. During the first two days in culture, the Lh cells grew long extensions that with time physically attached to other cells. Successively, tight cell clusters formed as cell somas that were connected via extensions migrated towards each other, while shortening their extensions. Laser photolysis of caged Ca2+ showed that Ca2+ signals originating in the soma propagated from the soma along the major extensions, being particularly visible in each swelling. Moreover, the Ca2+ signal could be transferred between densely clustered cells (sharing soma-soma border), but was not transferred via extensions to the connected cell. In summary, Lh gonadotropes in medaka display a complex cellular structure of hormone-containing extensions that are sensitive to Gnrh, and may be used for clustering and possibly hormone release, but do not seem to contribute to communication between cells themselves.

Single-cell transcriptomics identifies divergent developmental lineage trajectories during human pituitary development.

The anterior pituitary gland plays a central role in regulating various physiological processes, including body growth, reproduction, metabolism and stress response. Here, we perform single-cell RNA-sequencing (scRNA-seq) of 4113 individual cells from human fetal pituitaries. We characterize divergent developmental trajectories with distinct transitional intermediate states in five hormone-producing cell lineages. Corticotropes exhibit an early intermediate state prior to full differentiation. Three cell types of the PIT-1 lineage (somatotropes, lactotropes and thyrotropes) segregate from a common progenitor coexpressing lineage-specific transcription factors of different sublineages. Gonadotropes experience two multistep developmental trajectories. Furthermore, we identify a fetal gonadotrope cell subtype expressing the primate-specific hormone chorionic gonadotropin. We also characterize the cellular heterogeneity of pituitary stem cells and identify a hybrid epithelial/mesenchymal state and an early-to-late state transition. Here, our results provide insights into the transcriptional landscape of human pituitary development, defining distinct cell substates and subtypes and illustrating transcription factor dynamics during cell fate commitment.

Immunohistomorphometric and Hormonal Analysis of the Pituitary Gonadotropic Cells After Application of the Nandrolone Decanoate and Swimming Training in Adult Male Rats.

The aim of the study was to investigate the effects of chronic nandrolone decanoate treatment and/or swimming training on immunohistomorphometric parameters on rat pituitary gonadotropic cells. Male Wistar albino rats, 10 weeks old, were classified into four groups: control (T−N−), nandrolone (T−N+), swimming training (T+N−), and swimming training with nandrolone (T+N+). The T+ groups swam for 4 weeks, 1 h/day, 5 days/week. The N+ groups received nandrolone decanoate (20 mg/kg) once per week for 4 weeks. Pituitary tissue sections were processed and stained for immunohistochemical analysis and immunofluorescence. The volume density of luteinizing hormone (LH) cells was decreased by 48% in T−N+ and for 35% in the T+N+ group. The volume density of follicle-stimulating hormone (FSH) cells was decreased by 39% in T−N+ and for 30% in T+N+ compared to the control. Nandrolone alone, or combined with swimming training, decreased the number of LH/FSH cells compared to the control. The levels of the immunofluorescent signal of LH/FSH cells were increased in all experimental groups. Nandrolone alone decreased the serum level of LH by 17%, whereas swimming training alone increased FSH levels by 11% compared to the control. Serum levels of testosterone were increased in all experimental groups. Nandrolone alone, or combined with swimming training, decreased immunohistomorphometric parameters of gonadotropic cells, whereas the levels of immunofluorescent signal were increased.

FSH Levels Are Related to E-cadherin Expression and Subcellular Location in Nonfunctioning Pituitary Tumors.

CONTEXT: Gonadotroph pituitary neuroendocrine tumors (PitNETs) can express follicle-stimulating hormone (FSH) and luteinizing hormone (LH) or be hormone negative, but they rarely secrete hormones. During tumor development, epithelial cells develop a mesenchymal phenotype. This process is characterized by decreased membranous E-cadherin and translocation of E-cadherin to the nucleus. Estrogen receptors (ERs) regulate both E-cadherin and FSH expression and secretion. Whether the hormone status of patients with gonadotroph PitNETs is regulated by epithelial-to-mesenchymal transition (EMT) and ERs is unknown. OBJECTIVES: To study the effect of EMT on hormone expression in gonadotroph nonfunctioning (NF)-PitNETs. DESIGN: Molecular and clinical analyses of 105 gonadotroph PitNETs. Immunohistochemical studies and real-time quantitative polymerase chain reaction were performed for FSH, LH, E-cadherin, and ERα. Further analyses included blood samples, clinical data, and radiological images. SETTING: All patients were operated on in the same tertiary referral center. RESULTS: NF-PitNET with high FSH expression had decreased immunohistochemical staining for membranous E-cadherin (P < .0001) and increased staining for nuclear E-cadherin (P < .0001). Furthermore, high FSH expression was associated with increased ERα staining (P = .0002) and ERα mRNA (P = .0039). Circulating levels of plasma-FSH (P-FSH) correlated with FSH staining in gonadotroph NF-PitNET (P = .0025). Tumor size and invasiveness was not related to FSH staining, E-cadherin, or ERα. LH expression was not associated with E-cadherin or ERα. CONCLUSION: In gonadotroph PitNETs, FSH staining is related to E-cadherin, ERα expression, and circulating levels of P-FSH. There was no association between FSH staining and invasiveness. The clinical significance of these findings will be investigated in ongoing prospective studies.

Plasticity in medaka gonadotropes via cell proliferation and phenotypic conversion.

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of the fshb and lhb promotors respectively. We found that Fsh cells appear in the pituitary at 8 dpf, while Lh cells were previously shown to appear at 14 dpf. Similar to Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juveniles and adults but not in larvae where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh cells start to produce Lhß, a phenomenon enhanced by gonadotropin-releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol-sensitive proliferation and Gnrh promoted phenotypic conversion, and moreover, show that gonadotropes lose part of their identity when kept in cell culture.

Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.

BACKGROUND: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood. RESULTS: Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription. CONCLUSION: This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process.

Nonclassical actions of estradiol-17beta are not detectable in the alphaT3-1 and LbetaT2 immortalized gonadotrope cell lines†.

The immortalized mouse gonadotrope cell lines alphaT3-1 and LbetaT2 cells have been a substitute model for primary gonadotropes. These cell lines have provided a homogeneous cell population, as compared to the dissociated anterior pituitaries, which contain a heterogeneous population of cells potentially responsive to estradiol-17beta (E2). Nonclassical actions of E2 assumed to occur through the plasma membrane estrogen receptor 1 (ESR1, also known as ERalpha). These actions have included inhibition of gonadotropin-releasing hormone (GnRH)-induced increases in intracellular calcium concentrations and phosphorylation of p44/42 mitogen-activated protein kinase (ERK-1/2) in ovine pituitaries including primary gonadotropes in vitro. The objective of the present experiment was to determine if alphaT3-1 and LbetaT2 are cell models with limitations to examine the nonclassical actions of E2 occurring in gonadotropes. Experiments were conducted to determine if the cells have ESR1 at the plasma membrane using biotinylation cell and isolation of surface protein and staining with a fluorescently labeled E2 conjugate. The alphaT3-1 cells contain ESR1 associated with but not enriched within lipid rafts of the plasma membrane and do not translocate to lipid rafts upon binding of E2. In contrast, LbetaT2 cells lack ESR1 associated with the plasma membrane. Pretreatment with E2 did not cause inhibition of GnRH-stimulated increases in intracellular concentrations of calcium for either cell type. Phosphorylation of ERK-1/2 was not stimulated by E2 in either cell type. Although these cells lines have been used extensively to study GnRH signaling, in vitro or in vivo effects of nonclassical actions of E2 cannot be replicated in either cell line.

PACAP induces FSHß gene expression via EPAC.

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are heterodimers of a common α subunit and unique ß subunits. Regulation of their levels, primarily by GnRH, is critical for reproductive function. Several other hormones modulate gonadotropin expression, either independently or by modifying the responsiveness to GnRH. Pituitary adenylate cyclase activating peptide (PACAP) is one such hormone. Four-hour treatment of female mouse primary pituitary cells by either GnRH or PACAP induced FSHß expression, while 24-h treatment repressed FSHß. Both PACAP and GnRH caused FSH secretion into the medium. In the gonadotropes, PACAP activates primarily Gαs and increases concentration of cAMP, while GnRH primarily functions via Gαq and increases calcium concentration. Herein, we compared PACAP and GnRH signaling pathways that lead to the induction of FSHß expression. Interestingly, constitutively active Gαs represses LHß and induces FSHß expression, while Gαq induces both ß-subunits. We determined that FSHß induction by PACAP requires functional EPAC, a cAMP sensor protein that serves as a guanine exchange factors for small G proteins that then bridges cAMP signaling to MAPK pathway. We further demonstrate that in addition to the prototypical small G protein Ras, two members of the Rho subfamily, Rac and CDC42 are also necessary for PACAP induction of FSHß, likely via activation of p38 MAPK that leads to induction of cFOS, a critical transcription factor that is necessary and sufficient for FSHß induction. Therefore, PACAP-induced cAMP pathway leads to MAPK activation that stimulates cFOS induction, to induce the expression of FSHß subunit and increase FSH concentration.

Histone deacetylase inhibitors suppress transdifferentiation of gonadotrophs to prolactin cells and proliferation of prolactin cells induced by diethylstilbestrol in male mouse pituitary.

Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.

Single-cell stabilization method identifies gonadotrope transcriptional dynamics and pituitary cell type heterogeneity.

Immediate-early response genes (IEGs) are rapidly and transiently induced following an extracellular signal. Elucidating the IEG response patterns in single cells (SCs) requires assaying large numbers of timed samples at high accuracy while minimizing handling effects. To achieve this, we developed and validated RNA stabilization Buffer for Examination of Single-cell Transcriptomes (RNA-Best), a versatile single-step cell and tissue preservation protocol that stabilizes RNA in intact SCs without perturbing transcription patterns. We characterize for the first time SC heterogeneity in IEG responses to pulsatile gonadotropin-releasing hormone (GnRH) stimuli in pituitary gonadotrope cells. Our study identifies a gene-specific hierarchical pattern of all-or-none transcript induction elicited by increasing concentrations of GnRH. This quantal pattern of gene activation raises the possibility that IEG activation, when accurately resolved at the SC level, may be mediated by gene bits that behave as pure binary switches.

The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding.

The erlin1/2 complex is a ∼2-MDa endoplasmic reticulum membrane-located ensemble of the ∼40-kDa type II membrane proteins erlin1 and erlin2. The best defined function of this complex is to mediate the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IP3Rs) and their subsequent degradation. However, it remains unclear how mutations of the erlin1/2 complex affect its cellular function and cause cellular dysfunction and diseases such as hereditary spastic paraplegia. Here, we used gene editing to ablate erlin1 or erlin2 expression to better define their individual roles in the cell and examined the functional effects of a spastic paraplegia-linked mutation to erlin2 (threonine to isoleucine at position 65; T65I). Our results revealed that erlin2 is the dominant player in mediating the interaction between the erlin1/2 complex and IP3Rs and that the T65I mutation dramatically inhibits this interaction and the ability of the erlin1/2 complex to promote IP3R ubiquitination and degradation. Remarkably, we also discovered that the erlin1/2 complex specifically binds to phosphatidylinositol 3-phosphate, that erlin2 binds this phospholipid much more strongly than does erlin1, that the binding is inhibited by T65I mutation of erlin2, and that multiple determinants within the erlin2 polypeptide comprise the phosphatidylinositol 3-phosphate-binding site. Overall, these results indicate that erlin2 is the primary mediator of the cellular roles of the erlin1/2 complex and that disease-linked mutations of erlin2 can affect both IP3R processing and lipid binding.

Hypothalamic-pituitary-ovarian axis perturbation in the basis of bisphenol A (BPA) reproductive toxicity in female zebrafish (Danio rerio).

Thousands of safety-related studies have been published on bisphenol A (BPA), an ubiquitous environmental pollutant with estrogenic activity and many other potential biological effects. In recent years, BPA exposure has been shown to cause anovulation and infertility through irreversible alteration of the hypothalamic-pituitary-gonadal axis in several organisms, including fish and mammals. Recently, the European Chemical Agency classified BPA as a "substance of very high concern" because of its endocrine-disrupting properties, which have serious effects on human health. Given the risk of exposure to BPA as a pollutant in the environment, food, and drinking water, the objective of our study was to assess the effects of this compound on the adeno-hypophysis by means of a histopathological and morphometric study of the gonadotroph cells. In addition, using quantitative real-time PCR (qRT-PCR) assays, we analyzed the changes in the expression of Cyp19b (an aromatase gene). Zebrafish were randomly distributed into five groups: a control group and 4 treated groups which were exposed to different BPA concentrations (1, 10, 100 and 1000 µg/L). The effects of the different doses on Cyp19b mRNA molecules followed a non-monotonic curve, with the 1 and 1000 µg/L doses causing dramatic decreases in the number of Cyp19b transcripts while the doses of 10 and 100 µg/L caused important increases. The consequences might be deregulation of gonadotropic hormones causing degeneration of gonadotropic cells, as observed in BPA treated animals. This is the first study in which the gonadotroph cells have been evaluated using histomorphological endpoints after BPA exposure in zebrafish.

Heifers express G-protein coupled receptor 153 in anterior pituitary gonadotrophs in stage-dependent manner.

We recently found that orphan G-protein-coupled receptor (GPR)153 is expressed in the anterior pituitary (AP) of heifers, leading us to speculate that GPR153 colocalizes with gonadotropin-releasing hormone receptor (GnRHR) in the plasma membrane of gonadotrophs and is expressed at specific times of the reproductive cycle. To test this hypothesis, we examined the coexpression of GnRHR, GPR153, and either luteinizing hormone or follicle-stimulating hormone in AP tissue and cultured AP cells by immunofluorescence microscopy. GPR153 was detected in the gonadotrophs, and was colocalized with GnRHR in the plasma membrane. GPR153 was also detected in the cytoplasm of cultured gonadotrophs. Real-time PCR and western blot analyses found that expression was lower (P < 0.05) in AP tissues during early luteal phase as compared to pre-ovulation or late luteal phases. The 5'-flanking region of the GPR153 gene contained a consensus response element sequence for estrogen, but not for progesterone. These data suggest that some, but not all GPR153 colocalizes with GnRHR in the plasma membrane of gonadotrophs, and its expression changes stage-dependently in the bovine AP.

Differential roles of PKC isoforms (PKCs) in GnRH stimulation of MAPK phosphorylation in gonadotrope derived cells.

The role of protein kinase C (PKC) isoforms (PKCs) in GnRH-stimulated MAPK [ERK1/2, JNK1/2 and p38) phosphorylation was examined in gonadotrope derived cells. GnRH induced a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2 and p38MAPK. Gonadotropes express conventional PKCα and PKCßII, novel PKCδ, PKCε and PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein (GFP)-PKCs constructs revealed that GnRH induced rapid translocation of PKCα and PKCßII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs) has revealed differential role for PKCα, PKCßII, PKCδ and PKCε in ERK1/2, JNK1/2 and p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in MAPKs phosphorylation may be explained by persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane. Thus, we have identified the PKCs involved in GnRH stimulated MAPKs phosphorylation in gonadotrope derived cells. Once activated, the MAPKs will mediate the transcription of the gonadotropin subunits and GnRH receptor genes.

Androgens Mediate Sex-Dependent Gonadotropin Expression During Late Prenatal Development in the Mouse.

Central organization of the hypothalamic-pituitary-gonadal axis is initiated during fetal life. At this critical time, gonadal hormones mediate sex-specific development of the hypothalamic-pituitary axis, which then dictates reproductive physiology and behavior in adulthood. Although studies have investigated the effects of prenatal androgens on central factors influencing gonadotropin-releasing hormone (GnRH) release, the impact of fetal androgens on gonadotrope function has been overlooked. In the current study, we demonstrated that gonadotropin gene expression and protein production were robustly elevated in female mice compared with males during late fetal development and that this sex difference was dependent on fetal androgens. Treatment of dams from embryonic day (E)15.5 to E17.5 with testosterone, dihydrotestosterone (DHT), or the androgen antagonist flutamide eliminated the sex difference at E18.5. Specifically, flutamide relieved the suppression in male gene expression, elevating the level to that of females, whereas testosterone or DHT attenuated female gene expression to male levels. The gonadotrope population is equivalent in males and females, and gonadotropic cells in both sexes express androgen receptors, suggesting that androgen-dependent transcriptional regulation can occur in these cells in either sex. Studies using mouse models lacking GnRH signaling show that GnRH is necessary for enhanced gonadotropin expression in females and is therefore required to observe the sex difference. Collectively, these data suggest that circuits controlling GnRH input to the fetal pituitary are unrestrained in females yet robustly inhibited in males via circulating androgens and demonstrate plasticity in gonadotropin synthesis and secretion in both sexes depending on the androgen milieu during late prenatal development.

An epigenetic switch repressing Tet1 in gonadotropes activates the reproductive axis.

The TET enzymes catalyze conversion of 5-methyl cytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) and play important roles during development. TET1 has been particularly well-studied in pluripotent stem cells, but Tet1-KO mice are viable, and the most marked defect is abnormal ovarian follicle development, resulting in impaired fertility. We hypothesized that TET1 might play a role in the central control of reproduction by regulating expression of the gonadotropin hormones, which are responsible for follicle development and maturation and ovarian function. We find that all three TET enzymes are expressed in gonadotrope-precursor cells, but Tet1 mRNA levels decrease markedly with completion of cell differentiation, corresponding with an increase in expression of the luteinizing hormone gene, Lhb We demonstrate that poorly differentiated gonadotropes express a TET1 isoform lacking the N-terminal CXXC-domain, which represses Lhb gene expression directly and does not catalyze 5hmC at the gene promoter. We show that this isoform is also expressed in other differentiated tissues, and that it is regulated by an alternative promoter whose activity is repressed by the liganded estrogen and androgen receptors, and by the hypothalamic gonadotropin-releasing hormone through activation of PKA. Its expression is also regulated by DNA methylation, including at an upstream enhancer that is protected by TET2, to allow Tet1 expression. The down-regulation of TET1 relieves its repression of the methylated Lhb gene promoter, which is then hydroxymethylated and activated by TET2 for full reproductive competence.

Transient receptor potential (TRP) channel function in the reproductive axis.

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic-pituitary-gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca2+ concentration via its own Ca2+ permeability and via the activation of voltage-gated Ca2+ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.

ErbB4 cleavage by gonadotropin-releasing hormone receptor stimulation in cultured gonadotroph cells.

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G-protein-coupled receptors, and its stimulation activates extracellular signal-regulated protein kinase (ERK). In the present study, we first examined the actions of GnRH on the ErbB family using two types of cultured gonadotroph cells. As reported previously, AG1478, an inhibitor of the ErbB family tyrosine kinase, inhibited GnRH-induced ERK activation in undifferentiated gonadotroph αT3-1 cells. However, AG1478 did not inhibit ERK activation in differentiated gonadotroph LßT2 cells, suggesting that transactivation of the ErbB family was not necessary for ERK activation in LßT2 cells. We found that ErbB4 was expressed in αT3-1 cells but not in LßT2 cells. GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment in αT3-1 cells. Pharmacological experiments suggested that Gq/11 and tumor necrosis factor-α-converting enzyme (TACE) were essential for GnRH-induced ErbB4 cleavage. GnRH increased the phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), indicating that GnRH activated protein kinase C (PKC). Down-regulation of PKC and bisindolylmaleimide I, a PKC inhibitor, strongly inhibited the GnRH-induced cleavage of ErbB4. It was surprising that GnRH treatment of LßT2 cells after overexpression of ErbB4 induced ErbB4 cleavage in a TACE-dependent manner. ErbB4 cleavage was induced also by treatment of αT3-1 cells, ErbB4-overexpressing LßT2 cells, and immortalized GnRH neurons (GT1-7 cells) with leuprorelin acetate. These results may suggest that the pharmacological effects of leuprorelin acetate are conducted through TACE-mediated proteolysis of membrane proteins, including ErbB4, in gonadotroph cells and GnRH neurons.