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.

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.

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.

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.

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.

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.

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.

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs.

Cell Type-Specific Sexual Dimorphism in Rat Pituitary Gene Expression During Maturation.

The most obvious functional differences between mammalian males and females are related to the control of reproductive physiology and include patterns of GnRH and gonadotropin release, the timing of puberty, sexual and social behavior, and the regulation of food intake and body weight. Using the rat as the best-studied mammalian model for maturation, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable Pomc profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in Gh1 and Prl profiles during embryonic to juvenile age but show the amplification of Prl expression in females and Gh1 expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized Lhb, Fshb, Cga, and Gnrhr expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental Tshb profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes.

Expression of the long-chain fatty acid receptor GPR120 in the gonadotropes of the mouse anterior pituitary gland.

G-protein-coupled receptor 120 (GPR120) has been known to be a receptor of long-chain fatty acids. Here, we investigated GPR120 expression in the mouse pituitary gland via real-time PCR, in situ hybridization, and immunohistochemistry. GPR120 mRNA was abundantly expressed in the pituitary gland of ad-lib fed animals. In situ hybridization and immunohistochemistry revealed GPR120 expression in the gonadotropes of the anterior pituitary gland, but not in thyrotropes, somatotropes, lactotropes, corticotropes, melanotropes, and the posterior pituitary gland. Furthermore, 24 h of fasting induced an increase in GPR120 mRNA expression in the pituitary gland. These results demonstrate that GPR120 in mouse pituitary gonadotropes is upregulated by fasting and that it may play a role in controlling gonadotropin secretion.

Genetic variants in the upstream region of activin receptor IIA are associated with female fertility in Japanese Black cattle.

BACKGROUND: Female fertility, a fundamental trait required for animal reproduction, has gradually declined in the last 2 decades in Japanese Black cattle. To identify associated genetic variants in Japanese Black cattle, we evaluated female fertility as a metric to describe the average inverse of the number of artificial inseminations required for conception from the first through the fourth parity (ANAI4) and conducted a genome-wide association study (GWAS) using 430 animals with extreme ANAI4 values from 10,399 animals. RESULTS: We found that 2 variants, namely a single-nucleotide polymorphisms (SNP; g.48476925C > T) and a 3-bp indel (g.48476943_48476946insGGC), in the upstream region of the activin receptor IIA gene (ACVR2A) were associated with ANAI4. ACVR2A transcripts from Japanese Black cattle of the Q haplotype, defined by the SNP and the 3-bp indel, with increased ANAI4 were 1.29-1.32-fold more abundant than q-derived transcripts. In agreement, reporter assay results revealed that the activity of the ACVR2A promoter was higher in reporter constructs with the Q haplotype than in those with the q haplotype by approximately 1.2 fold. Expression of exogenous ACVR2A induced dose-dependent increases of reporter activity from the follicle-stimulating hormone, beta polypeptide (FSHB) promoter in response to activin A in a pituitary gonadotrophic cell line. The findings suggested that sequence variations in the upstream region of ACVR2A with the Q haplotype increased ACVR2A transcription, which in turn induced FSHB expression. This association was replicated using a sample population size of 1,433 animals; the frequency of the Q haplotype was 0.39, and Q-to-q haplotype substitution resulted in an increase of 0.02 in terms of ANAI4. CONCLUSIONS: This GWAS identified variants in the upstream region of ACVR2A, which were associated with female fertility in Japanese Black cattle. The variants affected the level of ACVR2A mRNA expression, which could lead to an allelic imbalance. This association was replicated with a sample population of 1,433 animals. Thus, the results suggest that the Q haplotype could serve as a useful marker to select Japanese Black cattle with superior female fertility.

Trichostatin A specifically stimulates gonadotropin FSHß gene expression in gonadotroph LßT2 cells.

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase. In the present study, TSA was found to selectively increase gene expression of the pituitary gonadotropin ß-subunit of follicle-stimulating hormone (FSH). Stimulation of mouse pituitary gonadotroph cell lines, LßT2, with TSA for 24 h resulted in no change in mRNA expression of the α- and LHß-subunit. On the other hand, FSHß-subunit mRNA expression was significantly increased in a dose-dependent fashion. Similarly, specific induction of the FSHß-subunit gene with TSA stimulation was observed in primary cultures of rat pituitary cells. Histone acetylation in whole cell lysates of LßT2 cells was significantly increased after TSA treatment, but not gonadotropin-releasing hormone (GnRH) treatment. The effect of TSA on FSHß mRNA expression was prominent compared to that of GnRH; however, TSA-stimulated FSHß mRNA expression was significantly reduced with combined TSA and GnRH treatment. TSA caused a slight increase in extracellular signal-regulated kinase (ERK) phosphorylation, while GnRH-increased ERK phosphorylation was potentiated in the presence of TSA. In addition, TSA, but not GnRH, significantly stimulated gene expression of retinaldehyde dehydrogenase 1 (RALDH1), a retinoic acid (RA) synthesizing enzyme involved in cell differentiation. These findings demonstrate that TSA specifically increases FSHß subunit gene expression with a concomitant increase in whole cell histone acetylation. Moreover, although GnRH is a stimulator of FSHß gene expression, it interfered with the stimulatory effect of TSA on FSHß mRNA expression, without modification of TSA-increased whole cell histone acetylation. This suggests that the mechanisms of TSA and GnRH-induced gonadotropin subunit gene expression are entirely distinct.

Castration-induced modifications of GnRH-elicited [Ca2+](i) signaling patterns in male mouse pituitary gonadotrophs in situ: studies in the acute pituitary slice preparation.

Gonadotropin-releasing hormone (GnRH) binds to pituitary gonadotroph receptors and initiates [Ca(2+)](i) signals and gonadotropin secretion. Here, we recorded GnRH-induced Ca(2+) signals in acute pituitary slices from both intact and castrated male mice 15 and 45 days after orchiectomy (GnX). Cells responding with "noncanonical" sequences of Ca(2+) signaling to increasing GnRH concentrations ([GnRH]; oscillatory responses at a given [GnRH] and transient responses at both lower and higher concentrations) were augmented significantly in the castrated mice. Also, 15 days after GnX the number and size of gonadotrophs were augmented, confirming earlier anatomical studies. Hypertrophied gonadotrophs after 15 days after GnX tended to display GnRH-induced Ca(2+) responses of greater amplitude. Furthermore, median effective dose (ED50) for GnRH decreased from 0.17 nM (control) to ~0.07 nM after GnX, suggesting increased GnRH responsiveness of the gonadotroph population. The progression of Ca(2+) response patterns reported in control male rat gonadotrophs (oscillations declining and spike-plateau responses dominating at increasing [GnRH]) was less conspicuous in mouse gonadotrophs in situ. Also, GnX-induced alterations in rat gonadotrophs (persistence of Ca(2+) oscillations even at [GnRH] >100 nM) were not mirrored by mouse gonadotrophs in situ. Contrary to observations in intact and 15-day castrated mice, after 45 days of GnX the hump component diminished and oscillations were augmented with increasing [GnRH], but Ca(2+) response patterns of gonadotrophs in situ remained virtually unchanged in response to [GnRH]s >1 nM, suggesting dose discrimination failure at high [GnRH]s. This study underscores the notion that GnRH responsiveness and the effects of testosterone deficiency may not be equal in pituitary gonadotrophs across species.

Possible role of PACAP and its PAC1 receptor in the differential regulation of pituitary LHbeta- and FSHbeta-subunit gene expression by pulsatile GnRH stimulation.

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are mainly under the control of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates male and female gonadal function. GnRH is released in a pulsatile manner from the hypothalamus, and the frequency of GnRH pulses determines the dominance of output of LH and FSH from pituitary gonadotrophs. That is, more rapid pulses of GnRH preferentially increase synthesis and secretion of LH, whereas FSH is preferentially stimulated by slower GnRH pulses. The detailed mechanisms underlying this phenomenon remain unknown. Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cAMP production in pituitary cells. PACAP is produced within the pituitary gonadotroph as well as in the central nervous system. PACAP stimulates gonadotropin alpha-, LHbeta-, and FSHbeta-subunits as well as receptors for GnRH in the pituitary gonadotropin-secreting cells. In addition, its own receptor, PACAP type I receptor (PAC1R), is also regulated by PACAP in gonadotrophs. GnRH stimulates expression of PACAP as well as PAC1R, and lower frequencies of GnRH pulses preferentially increase PACAP and PAC1R expression in gonadotrophs. Increasing concentrations of PACAP further increase the levels of gonadotropin subunit and that increasing amounts of PAC1R in gonadotrophs potentiates the effects of PACAP or GnRH on gonadotropin subunit expression. In addition, we have observed that GnRH-increased FSHbeta-subunit expression was prevented in the presence of PAC1R antagonist. These observations suggest the involvement of locally produced PACAP and its PAC1R in the differential regulation of specific gonadotropin subunit expression by pulsatile GnRH stimulation. Here, we review the possible involvement of PACAP and its PAC1R in gonadotropin control on the basis of our observations with gonadotroph cell lines.

Prenatal exposure to low doses of bisphenol A increases pituitary proliferation and gonadotroph number in female mice offspring at birth.

The pituitary gland is composed of hormone-producing cells essential for homeostasis and reproduction. Pituitary cells are sensitive to endocrine feedback in the adult and can have altered hormonal secretion from exposure to the endocrine disruptor bisphenol A (BPA). BPA is a prevalent plasticizer used in food and beverage containers, leading to widespread human exposure. Although prenatal exposure to BPA can impact reproductive function in the adult, the effects of BPA on the developing pituitary are unknown. We hypothesized that prenatal exposure to low doses of BPA impacts gonadotroph cell number or parameters of hormone synthesis. To test this, pregnant mice were administered 0.5 µg/kg/day of BPA, 50 µg/kg/day of BPA, or vehicle beginning on Embryonic Day 10.5. At parturition, pituitaries from female offspring exposed in utero to either dose of BPA had increased proliferation, as assessed by mKi67 mRNA levels and immunohistochemistry. Coincidently, gonadotroph number also increased in treated females. However, we observed a dichotomy between mRNA levels of Lhb and Fshb. Female mice exposed to 0.5 µg/kg/day BPA had increased mRNA levels of gonadotropins and the gonadotropin-receptor hormone (GNRH) receptor (Gnrhr), which mediates GNRH regulation of gonadotropin production and release. In contrast, mice treated with 50 µg/kg/day of BPA had decreased gonadotropin mRNA levels, Gnrhr and Nr5a1, a transcription factor required for gonadotroph differentiation. No other pituitary hormones were altered on the day of birth in response to in utero BPA exposure, and male pituitaries showed no change in the parameters tested. Collectively, these results show that prenatal exposure to BPA affects pituitary gonadotroph development in females.

Signaling responses to pulsatile gonadotropin-releasing hormone in LbetaT2 gonadotrope cells.

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is secreted in a pulsatile fashion by hypothalamic neurons, and alterations in pulse frequency and amplitude differentially regulate gonadotropin synthesis and release. In this study, we investigated the kinetics of G(s) and G(q) signaling in response to continuous or pulsatile GnRH using fluorescence resonance energy transfer reporters in live mouse LbetaT2 gonadotrope cells. cAMP and protein kinase A-dependent reporters showed a rapid but transient increase in fluorescence resonance energy transfer signal with increasing doses of constant GnRH, and in contrast diacylglycerol (DAG) and calcium reporters showed a rapid and sustained signal. Multiple pulses of GnRH caused multiple pulses of cAMP and protein kinase A activation without desensitization, but the DAG and calcium reporters were rapidly desensitized resulting in inhibition of calcium and DAG responses. At the transcriptional level, both a cAMP-dependent cAMP-response element reporter and a DAG/calcium-dependent AP-1 reporter showed a pulse frequency-dependent increase in luciferase activity. However, constant GnRH stimulation gave very little cAMP-response element activation but very strong AP-1 activation. Based on these data, we propose that both the GnRH-R-G(s) and G(q) pathways are responsive to pulses of GnRH, but only the G(q) pathway is responsive to constant GnRH. Furthermore, the G(q) pathway is subject to desensitization with multiple GnRH pulses, but the G(s) pathway is not.

Effect of the organochlorine pesticide endosulfan on GnRH and gonadotrope cell populations in fish larvae.

Endocrine-disrupting chemicals can influence the hypothalamus-pituitary-gonad axis and possibly affect reproduction in vertebrates. We analyzed the effect of 30-day endosulfan (ES) exposure in sexually undifferentiated larvae of the cichlid fish Cichlasoma dimerus. The number, area, mean cytoplasmic and nuclear diameter, and mean cytoplasmic optical density of gonadotropin-releasing hormone (GnRH) I, II, and III immunoreactive (ir-) neurons and ß follicle-stimulating hormone (ßFSH) ir-cells were measured. Animals exposed to the highest ES concentration (0.1 µg/l) showed a decrease in GnRH I nucleus/cytoplasm area ratio upon exposure. Nuclear area and mean nuclear diameter of ßFSH ir-cells was higher in ES treated fish. ßFSH nucleus/cytoplasm area ratio was high in exposed animals, and animals exposed to 0.1 µg/l ES showed smaller mean cytoplasmic optical density. These findings suggest that ES affects GnRH I and ßFSH protein synthesis/release. However, these responses seem to be insufficient to affect gonadal differentiation at this stage of development.

Expression patterns of hormones, signaling molecules, and transcription factors during adenohypophysis development in the chick embryo.

The chick embryo is an ideal model to study pituitary cell-type differentiation. Previous studies describing the temporal appearance of differentiated pituitary cell types in the chick embryo are contradictory. To resolve these controversies, we used RT-PCR to define the temporal onset and in situ hybridization and immunohistochemistry to define the spatial localization of hormone expression within the pituitary. RT-PCR detected low levels of Fshbeta (gonadotropes) and Pomc (corticotropes, melanotropes) mRNA at E4 and Gh (somatotropes), Prl (lactotropes), and Tshbeta (thyrotropes) mRNA at E8. For all hormones, sufficient accumulation of mRNA and/or protein to permit detection by in situ hybridization or immunohistochemistry was observed approximately 3 days later and in all cases corresponded to a notable increase in RT-PCR product. We also describe the expression patterns of signaling (Bmp2, Bmp4, Fgf8, Fgf10, Shh) and transcription factors (Pitx1, Pitx2, cLim3) known to be important for pituitary organogenesis in other model organisms.

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.

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.