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The somatostatin (SST) receptor family controls pituitary hormone secretion, but the distribution and specific roles of these receptors on the excitability and voltage-gated calcium signaling of hormone producing pituitary cells have not been fully characterized. Here we show that the rat pituitary gland expressed Sstr1, Sstr2, Sstr3, and Sstr5 receptor genes in a cell type-specific manner: Sstr1 and Sstr2 in thyrotrophs, Sstr3 in gonadotrophs and lactotrophs, Sstr2, Sstr3, and Sstr5 in somatotrophs, and none in corticotrophs and melanotrophs. Most gonadotrophs and thyrotrophs spontaneously fired high-amplitude single action potentials, which were silenced by SST without affecting intracellular calcium concentrations. In contrast, lactotrophs and somatotrophs spontaneously fired low-amplitude plateau-bursting action potentials in conjunction with calcium transients, both of which were silenced by SST. Moreover, SST inhibited GPCR-induced voltage-gated calcium signaling and hormone secretion in all cell types expressing SST receptors, but the inhibition was more pronounced in somatotrophs. The pattern of inhibition of electrical activity and calcium signaling was consistent with both direct and indirect inhibition of voltage-gated calcium channels, the latter being driven by cell type-specific hyperpolarization. These results indicate that the action of SST in somatotrophs is enhanced by the expression of several types of SST receptors and their slow desensitization, that SST may play a role in the electrical resynchronization of gonadotrophs, thyrotrophs, and lactotrophs, and that the lack of SST receptors in corticotrophs and melanotrophs keeps them excitable and ready to responses to stress., Competing Interests: Declaration of competing interest This is to state that there is no any financial and personal relationships with other people or organizations that could be viewed as inappropriately influencing or bias our work., (Copyright © 2024. Published by Elsevier Ltd.)

Irisin is a hormone secreted by muscle in response to exercise. The irisin receptor (IrisinR) is a heterodimer of integrin alpha V (ITGAV) and integrin beta 5 (ITGB5) subunits. Since irisin may mediate some beneficial effects of exercise on animal reproduction, we tested the hypothesis that bovine gonadotrophs express IrisinR and irisin stimulates luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion by gonadotrophs. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of both ITGAV and ITGB5 in the anterior pituitary glands (APs) of post pubertal heifers and mouse gonadotroph cell line "LβT2." Western blotting was used to detect protein expression in bovine APs. Immunofluorescence microscopy, utilizing the same antibody, visualized IrisinR on the plasma membrane of majority of gonadotrophs. We prepared AP cells from healthy postpubertal heifers, cultured them for 3.5 d, and treated them with increasing concentrations (0, 0.01, 0.1, 1, or 10 nM) of irisin for 5 min before either no treatment or gonadotropin-releasing hormone (GnRH) stimulation. After 2 h, media were harvested for LH and FSH assays. Irisin (0.1-10 nM) stimulated basal LH and FSH secretion, and these stimulatory effects were inhibited by the extracellular signal-regulated kinase or SMAD pathway inhibitors. In the presence of GnRH, irisin at 0.01-1 nM stimulated LH and FSH secretion. A higher dose of irisin (10 nM), however, suppressed the GnRH-induced LH and FSH levels. In conclusion, bovine gonadotrophs expressed IrisinR, and irisin controlled LH and FSH secretion from bovine gonadotrophs., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anti-Müllerian hormone (AMH) is primarily produced by ovarian granulosa cells and contributes to follicle development. AMH is also produced in other tissues, including the brain and pituitary; however, its roles in these tissues are not well understood. In this study, we examined the effect of AMH on pituitary gonadotrophs. We detected AMH and AMH receptor type 2 expression in LβT2 cells. In these cells, the expression of FSHβ- but not α- and LHβ-subunits increased significantly as the concentration of AMH increased. LβT2 cells expressed Kiss-1 and Kiss-1R. AMH stimulation resulted in decreases in both Kiss-1 and Kiss-1R. The siRNA-mediated knockdown of Kiss-1 in LβT2 cells did not alter the basal expression levels of α-, LHβ-, and FSHβ-subunits. In LβT2 cells overexpressing Kiss-1R, exogenous kisspeptin stimulation significantly increased the expression of all three gonadotropin subunits. However, kisspeptin-induced increases in these subunits were almost completely eliminated in the presence of AMH. In contrast, GnRH-induced increases in the three gonadotropin subunits were not modulated by AMH. Our observations suggested that AMH acts on pituitary gonadotrophs and induces FSHβ-subunit expression with concomitant decreases in Kiss-1 and Kiss-1R gene expression. Kisspeptin, but not GnRH-induced gonadotropin subunit expression, was inhibited by AMH, suggesting that it functions in association with the kisspeptin/Kiss-1R system in gonadotrophs.

P62 is a protein adaptor for various metabolic processes. Mice that lack p62 develop adult-onset obesity. However, investigations on p62 in reproductive dysfunction are rare. In the present study, we explored the effect of p62 on the reproductive system. P62 deficiency-induced reproductive dysfunction occurred at a young age (8 week old). Young systemic p62 knockout (p62 -/- ) and pituitary-specific p62 knockout (p62 flox/flox αGSU cre ) mice both presented a normal metabolic state, whereas they displayed infertility phenotypes (attenuated breeding success rates, impaired folliculogenesis and ovulation, etc.) with decreased luteinizing hormone (LH) expression and production. Consistently, in an infertility model of polycystic ovary syndrome (PCOS), pituitary p62 mRNA was positively correlated with LH levels. Mechanistically, p62 -/- pituitary RNA sequencing showed a significant downregulation of the mitochondrial oxidative phosphorylation (OXPHOS) pathway. In vitro experiments using the pituitary gonadotroph cell line LβT2 and siRNA/shRNA/plasmid confirmed that p62 modulated LH synthesis and secretion via mitochondrial OXPHOS function, especially Ndufa2, a component molecule of mitochondrial complex I, as verified by Seahorse and rescue tests. After screening OXPHOS markers, Ndufa2 was found to positively regulate LH production in LβT2 cells. Furthermore, the gonadotropin-releasing hormone (GnRH)-stimulating test in p62 flox/flox αGSU cre mice and LβT2 cells illustrated that p62 is a modulator of the GnRH-LH axis, which is dependent on intracellular calcium and ATP. These findings demonstrated that p62 deficiency in the pituitary impaired LH production via mitochondrial OXPHOS signaling and led to female infertility, thus providing the GnRH-p62-OXPHOS(Ndufa2)-Ca 2+ /ATP-LH pathway in gonadotropic cells as a new theoretical basis for investigating female reproductive dysfunction., (© 2021. The Author(s).)

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 LβT2 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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethanolamine plasmalogens (EPls), unique alkenylacyl-glycerophospholipids, are the only known ligands of G-protein-coupled receptor 61-a novel receptor co-localised with gonadotropin-releasing hormone receptors on anterior pituitary gonadotrophs. Brain EPl decreases with age. Commercial EPl-extracted from the cattle brain (unidentified age)-can independently stimulate FSH secretion from gonadotrophs. We hypothesised that there exists an age-related difference in the quality, quantity, and ability of bovine brain EPls to stimulate bovine gonadotrophs. We compared the brains of young (about 26 month old heifers) and old (about 90 month old cows) Japanese Black bovines, including EPls obtained from both groups. Additionally, mRNA expressions of the EPl biosynthesis enzymes, glyceronephosphate O-acyltransferase, alkylglycerone phosphate synthase, and fatty acyl-CoA reductase 1 (FAR1) were evaluated in young and old hypothalami. The old-brain EPl did not stimulate FSH secretion from gonadotrophs, unlike the young-brain EPl. Molecular species of EPl were compared using two-dimensional liquid chromatography-mass spectrometry. We identified 20 EPl molecular species of which three and three exhibited lower (P < 0.05) and higher (P < 0.05) ratios, respectively, in old compared to young brains. In addition, quantitative reverse transcription-polymerase chain reaction detected higher FAR1 levels in the POA, but not in the ARC&ME tissues, of old cows than that of fertile young heifers. Therefore, old-brain EPl may be associated with age-related infertility.

The pituitary gland controls many important physiological processes in vertebrates, including growth, homeostasis, and reproduction. As in mammals, the teleost pituitary exhibits a high degree of plasticity. This plasticity permits changes in hormone production and secretion necessary to meet the fluctuating demands over the life of an animal. Pituitary plasticity is achieved at both cellular and population levels. At the cellular level, hormone synthesis and release can be regulated via changes in cell composition to modulate both sensitivity and response to different signals. At the cell population level, the number of cells producing a given hormone can change due to proliferation, differentiation of progenitor cells, or transdifferentiation of specific cell types. Gonadotropes, which play an important role in the control of reproduction, have been intensively investigated during the last decades and found to display plasticity. To ensure appropriate endocrine function, gonadotropes rely on external and internal signals integrated at the brain level or by the gonadotropes themselves. One important group of internal signals is the sex steroids, produced mainly by the gonadal steroidogenic cells. Sex steroids have been shown to exert complex effects on the teleost pituitary, with differential effects depending on the species investigated, physiological status or sex of the animal, and dose or method of administration. This review summarizes current knowledge of the effects of sex steroids (androgens and estrogens) on gonadotrope cell plasticity in teleost anterior pituitary, discriminating direct from indirect effects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Fontaine, Royan, von Krogh, Weltzien and Baker.)

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.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to play a role in the development and regulation of gonadotrophs. PACAP levels are very high in the rodent fetal pituitary, and decline substantially and rapidly at birth, followed by a significant rise in FSHβ and GnRH-R expression. Because there is evidence that PACAP stimulates its own transcription, we propose that this self-regulation is interrupted around the time of birth. To begin to examine the mechanisms for PACAP self-regulation, we used two well-established gonadotroph cell lines, αT3-1 cells and the more mature LβT2 cells which were transfected with a PACAP promoter-reporter construct As in vivo, the basal PACAP transcription level is significantly lower in the more mature LβT2 cells in which basal cAMP signaling is also much reduced. The PACAP promoter was stimulated by PACAP in both cell lines. Treatment with inhibitors of second messenger pathways implicated PKA, PKC and MAPK in PACAP transcription. Three regions of the PACAP promoter were found to confer inhibition or stimulation of PACAP transcription. By inhibiting cAMP response element binding (CREB) activity and mutating a proximal CREB binding site, we found that CREB is essential for promoter activation. Finally, overexpression of PACAP receptor HOP1 isoform, to increase the level in LβT2 cells to that of αT3-1 cells and simulate the E19 pituitary, increased PACAP- stimulated sensitivity and significantly altered downstream gene transcription. These results provide novel insight into the feed-forward regulation of PACAP expression that may help initiate gonadotroph function at birth., (Copyright © 2020 Elsevier B.V. All rights reserved.)

GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LβT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LβT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LβT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes.

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 Lhβ, 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.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In goldfish, two native isoforms of gonadotropin-releasing hormone (GnRH2 and GnRH3) stimulate luteinizing hormone (LH) and growth hormone (GH) release from pituitary cells through activation of cell-surface GnRH-receptors (GnRHRs) on gonadotrophs and somatotrophs. Interestingly, GnRH2 and GnRH3 induce LH and GH release via non-identical post-receptor signal transduction pathways in a ligand- and cell-type-selective manner. In this study, we examined the involvement of β-arrestins in the control of GnRH-induced LH and GH secretion from dispersed goldfish pituitary cells. Treatment with Barbadin, which interferes with β-arrestin and β2-adaptin subunit interaction, reduced LH responses to GnRH2 and GnRH3, as well as GH responses to GnRH2; but enhanced GnRH3-induced GH secretion. Barbadin also had positive influences on basal hormone release, and basal GH release in particular, as well as basal activity of extracellular signal-regulated kinase (ERK) and GnRH-induced ERK activation. These findings indicate that β-arrestins play permissive roles in the control of GnRH-stimulated LH release. However, in somatotrophs, β-arrestins, perhaps by mediating agonist-selective endosomal trafficking of engaged GnRHRs, participate in GnRH-isoform-specific GH release responses (stimulatory and inhibitory for GnRH2-GnRHR and GnRH3-GnRHR activation, respectively). The correlative stimulatory influences of Barbadin on basal hormone release and ERK activation suggest that β-arrestins may negatively regulate basal secretion through modulation of basal ERK activity. These results provide the first direct evidence of a role for β-arrestins in hormone secretion from an untransformed primary pituitary cell model, and establish these proteins as important receptor-proximal players in mediating functional selectivity downstream of goldfish GnRHRs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LβT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc , Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LβT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun , Egr1 , Nr5a1 and Nr0b1 , within 8 h in LβT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function., Competing Interests: The authors declare no conflict of interest.

Cyclic adenosine monophosphate (cAMP) plays a pivotal role in gonadotrope responses in the pituitary. Gonadotropin-releasing hormone (GnRH) mediated synthesis and secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are regulated by both the G s /cAMP and G q /Ca 2+ signaling pathways. Pituitary adenylate cyclase-activating polypeptide (PACAP) also regulates GnRH responsiveness in gonadotropes through the PACAP receptor, which activates the G s /cAMP signaling pathway. Therefore, measuring intracellular cAMP levels is important for elucidating the molecular mechanisms of FSH and LH synthesis and secretion in gonadotropes. The GloSensor cAMP assay is useful for detecting cAMP levels in intact, living cells. In this study, we found that increased GloSensor luminescence intensity did not correlate with cAMP accumulation in LβT2 cells under low pH conditions. This result indicates that cell type and condition must be considered when using GloSensor cAMP.

Gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are heterodimers of a common α subunit and unique β 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 FSHβ expression, while 24-h treatment repressed FSHβ. 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 FSHβ expression. Interestingly, constitutively active Gαs represses LHβ and induces FSHβ expression, while Gαq induces both β-subunits. We determined that FSHβ 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 FSHβ, likely via activation of p38 MAPK that leads to induction of cFOS, a critical transcription factor that is necessary and sufficient for FSHβ induction. Therefore, PACAP-induced cAMP pathway leads to MAPK activation that stimulates cFOS induction, to induce the expression of FSHβ subunit and increase FSH concentration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Luteinizing hormone (Lh) and follicle-stimulating hormone (Fsh) control reproduction in vertebrates. Using a transgenic line of medaka, in which green fluorescent protein expression is controlled by the endogenous lhb promotor, we studied development and plasticity of Lh cells, comparing juveniles and adults of both genders. Confocal imaging and 3D reconstruction revealed hypertrophy and hyperplasia of Lh cells in both genders from juvenile to adult stages. We show that Lh cell hyperplasia may be caused by recruitment of existing pituitary cells that start to produce lhb, as evidenced by time lapse recordings of primary pituitary cell cultures, and/or through Lh cell proliferation, demonstrated through a combination of 5-bromo-2'-deoxyuridine incubation experiments and proliferating cell nuclear antigen staining. Proliferating Lh cells do not belong to the classical type of multipotent stem cells, as they do not stain with anti-sox2. Estradiol exposure in vivo increased pituitary cell proliferation, particularly Lh cells, whereas pituitary lhb and gpa expression levels decreased. RNA-seq and in situ hybridization showed that Lh cells express two estrogen receptors, esr1 and esr2b, and the aromatase gene cyp19a1b, suggesting a direct effect of estradiol, and possibly androgens, on Lh cell proliferation. In conclusion, our study reveals a high degree of plasticity in the medaka Lh cell population, resulting from a combination of recruitment and cell proliferation.

Fertility is dependent on follicle-stimulating hormone (FSH), a product of gonadotrope cells of the anterior pituitary gland. Hypothalamic gonadotropin-releasing hormone (GnRH) and intra-pituitary activins are regarded as the primary drivers of FSH synthesis and secretion. Both stimulate expression of the FSH beta subunit gene (Fshb), although the underlying mechanisms of GnRH action are poorly described relative to those of the activins. There is currently no consensus on how GnRH regulates Fshb transcription, as results vary across species and between in vivo and in vitro approaches. One of the more fully developed models suggests that the murine Fshb promoter is tonically repressed by histone deacetylases (HDACs) and that GnRH relieves this repression, at least in immortalized murine gonadotrope-like cells (LβT2 and αT3-1). In contrast, we observed that the class I/II HDAC inhibitor trichostatin A (TSA) robustly inhibited basal, activin A-, and GnRH-induced Fshb mRNA expression in LβT2 cells and in primary murine pituitary cultures. Similar results were obtained with the class I specific HDAC inhibitor, entinostat, whereas two class II-specific inhibitors, MC1568 and TMP269, had no effects on Fshb expression. Collectively, these data suggest that class I HDACs are positive, not negative, regulators of Fshb expression in vitro and that, contrary to earlier reports, GnRH may not stimulate Fshb by inhibiting HDAC-mediated repression of the gene.

Background/objectives: Anti-Müllerian hormone (AMH) signaling is critical for sexual differentiation and gonadal function. AMH receptor type 2 (AMHR2) is expressed in extragonadal sites such as brain, and pituitary and emerging evidence indicates that AMH biological action is much broader than initially thought. We recently reported that AMH signaling enhances follicle-stimulating hormone synthesis in pituitary gonadotrope cells. However, mechanisms regulating AMHR2 expression in these extragonadal sites remain to be explored., Method/results: Here, we demonstrated in perifused murine LβT2 gonadotrope cells that Amhr2 expression is differentially regulated by GnRH pulse frequency with an induction under high GnRH pulsatility. Furthermore, we showed that GnRH transactivates the human AMHR2 promoter in LβT2 cells. Successive deletions of the promoter revealed the importance of a short proximal region (-53/-37 bp) containing an Egr1 binding site. Using site-directed mutagenesis of Egr1 motif and siRNA mediated-knockdown of Egr1, we demonstrated that Egr1 mediates basal and GnRH-dependent activity of the promoter, identifying Egr1 as a new transcription factor controlling hAMHR2 expression. We also showed that SF1 and β-catenin are required for basal promoter activity and demonstrated that both factors contribute to the GnRH stimulatory effect, independently of their respective binding sites. Furthermore, using a constitutively active mutant of FOXO1, we identified FOXO1 as a negative regulator of basal and GnRH-dependent AMHR2 expression in gonadotrope cells., Conclusions: This study identifies GnRH as a regulator of human AMHR2 expression, further highlighting the importance of AMH signaling in the regulation of gonadotrope function., (© 2018 S. Karger AG, Basel.)

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.

Preantral and small antral follicles may secret anti-Müllerian hormone (AMH) to control gonadotrophin secretion from ruminant gonadotrophs. The present study investigated whether the main receptor for AMH, AMH receptor type 2 (AMHR2), is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion. Expression of AMHR2 mRNA was detected in anterior pituitaries (APs) of postpubertal heifers using reverse transcription-polymerase chain reaction. An anti-AMHR2 chicken antibody was developed against the extracellular region near the N-terminus of bovine AMHR2. Western blotting using this antibody detected the expression of AMHR2 protein in APs. Immunofluorescence microscopy using the same antibody visualised colocalisation of AMHR2 with gonadotrophin-releasing hormone (GnRH) receptor on the plasma membrane of gonadotrophs. AP cells were cultured for 3.5 days and then treated with increasing concentrations (0, 1, 10, 100, or 1000pgmL-1) of AMH. AMH (10-1000pgmL-1) stimulated (P<0.05) basal FSH secretion. In addition, AMH (100-1000pgmL-1) weakly stimulated (P<0.05) basal LH secretion. AMH (100-1000pgmL-1) inhibited GnRH-induced FSH secretion, but not GnRH-induced LH secretion, in AP cells. In conclusion, AMHR2 is expressed in gonadotrophs of postpubertal heifers to control gonadotrophin secretion.

The aim of the study was to examine the potential of the principal soy isoflavones, genistein and daidzein, or isoflavone rich soy extract to recover pituitary castration cells in orchidectomized adult male rats in comparison with the effects of estradiol. Two weeks post orchidectomy (Orx), animals received estradiol-dipropionate, genistein, daidzein or soy extract subcutaneously for 3 weeks. Control sham-operated (So) and Orx rats received just the vehicle. Changes in the volumes of pars distalis, of individual follicle-stimulating hormone (FSH) and luteinizing hormone (LH) containing cells, their volume, numerical density and number were determined by unbiased design-based stereology. The intracellular content of βFSH and βLH was estimated by relative intensity of fluorescence (RIF). Orchidectomy increased all examined stereological parameters and RIF. Compared to Orx, estradiol increased the volume of pars distalis, but reversed RIF and all morphometric parameters of gonadotropes to the level of So rats, except their number. Treatments with purified isoflavones and soy extract decreased RIF to the control So level, expressing an estradiol-like effect. However, the histological appearance and morphometrical features of gonadotropes did not follow this pattern. Genistein increased the volume of pars distalis, decreased the volume density of LH-labeled cells and raised the number of gonadotropes. Daidzein decreased the cell volume of gonadotropic cells but increased their number and numerical density. Soy extract induced an increase in number and numerical density of FSH-containing cells. Therefore, it can be concluded that soy phytoestrogens do not fully reverse the Orx-induced changes in pituitary castration cells. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LβT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved., Competing Interests: The authors have declared that no competing interests exist.

An emerging body of evidence supports the concept that the pituitary is a site for integration of multiple physiological and metabolic signals that inform and modulate endocrine pathways. Multiple endocrine mediators of energy balance and adiposity are known to impinge on the neuroendocrine axis regulating reproduction. Observations in humans show that obesity is correlated with decreased gonadotropin secretion, and studies have also suggested that pituitary sensitivity to stimulation by gonadotropin-releasing hormone (GnRH) is decreased in obese individuals. Free fatty acids are a potential mediator of adiposity and energy balance, but their impact as an endocrine modulator of pituitary function has not been closely examined. We evaluated the impact of free fatty acids on a pituitary gonadotrope cell line and in primary pituitary cultures of female mice. We show that increasing physiologically relevant doses of the monounsaturated ω-9 fatty acid oleate induces cellular stress and increases production of reactive oxygen species in a mouse gonadotrope cell line. In contrast, the unsaturated ω-3 α-linolenic and ω-6 linoleic fatty acids do not have this effect. Additionally, oleate can activate immediate-early gene expression independent of GnRH stimulation but has a negative impact on GnRH induction and expression of the gonadotropin subunit gene Lhb. Further, oleate suppresses gonadotropin secretion in response to pulsatile stimulation by GnRH. These results indicate that free fatty acids can directly alter gonadotropin gene expression and secretion in response to GnRH and may provide a link between energy sensing and reproduction., (Copyright © 2018 Endocrine Society.)

The cyclic expression of pituitary gonadotropin-releasing hormone receptors (GnRHRs) may be an important checkpoint for leptin regulatory signals. Gonadotrope Lepr-null mice have reduced GnRHR levels, suggesting these receptors may be leptin targets. To determine if leptin stimulated GnRHR directly, primary pituitary cultures or pieces were exposed to 1 to 100 nM leptin. Leptin increased GnRHR protein levels and the percentages of gonadotropes that bound biotinylated analogs of gonadotropin-releasing hormone (bio-GnRH) but had no effect on Gnrhr messenger RNA (mRNA). An in silico analysis revealed three consensus Musashi (MSI) binding elements (MBEs) for this translational control protein in the 3' untranslated region (UTR) of Gnrhr mRNA. Several experiments determined that these Gnrhr mRNA MBE were active: (1) RNA electrophoretic mobility shift assay analyses showed that MSI1 specifically bound Gnrhr mRNA 3'-UTR; (2) RNA immunoprecipitation of pituitary fractions with MSI1 antibody pulled down a complex enriched in endogenous MSI protein and endogenous Gnrhr mRNA; and (3) fluorescence reporter assays showed that MSI1 repressed translation of the reporter coupled to the Gnrhr 3'-UTR. In vitro, leptin stimulation of pituitary pieces reduced Msi1 mRNA in female pituitaries, and leptin stimulation of pituitary cultures reduced MSI1 proteins selectively in gonadotropes identified by binding to bio-GnRH. These findings show that leptin's direct stimulatory actions on gonadotrope GnRHR correlate with a direct inhibition of expression of the posttranscriptional regulator MSI1. We also show MSI1 interaction with the 3'-UTR of Gnrhr mRNA. These findings now open the door to future studies of leptin-modulated posttranscriptional pathways., (Copyright © 2018 Endocrine Society.)

Gonadotropin-releasing hormone (GnRH) regulates gonadotropin secretion. We previously demonstrated that the expression of annexin A5 (ANXA5) is stimulated by GnRH in gonadotropes and has a significant role in gonadotropin secretion. It is therefore of interest to know whether other members of the ANXA family, which consists of twelve structurally related members, are also regulated by GnRH. Therefore, the expression of all annexins was examined in LβT2 gonadotrope cells. ANXA4, A5, A6, A7 and A11 were detected in LβT2 cells. The expression of ANXA5 and A1 mRNA was stimulated by a GnRH agonist. An increase in ANXA1 protein by this agonist was demonstrated by western blotting. Immunohistochemistry showed that ANXA1 was present in the nucleus and to a lesser extent in the cytoplasm of some rat pituitary cells. The GnRH agonist induced translocation of ANXA1 to the periphery of LβT2 cells. The presence of ANXA1 in gonadotropes and its increase upon GnRH agonist treatment were confirmed in a primary pituitary cell culture. ANXA1 expression was also demonstrated in the ovary, the testis, the thyroid gland and the pancreas in a different manner to that of ANXA5. These data suggest that ANXA1 is a novel GnRH target gene in gonadotropes. ANXA1 also may be a target of local GnRH in peripheral tissues and may have a different role than that of ANXA5.

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit ( Cga ) and the hormone-specific β-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

GPR120 is a G-protein-coupled receptor that is activated by long-chain fatty acids. In our previous study, GPR120 expression was detected in gonadotrophs of the mouse anterior pituitary gland. It is well known that the function of anterior pituitary cells is largely under the influence of circulating sex steroids. Thus, in the present study, we investigated the modulatory roles of the ovarian hormones, estrogen (E2) and progesterone (P), on the expression levels of GPR120 mRNA in mouse pituitary glands. GPR120 mRNA expression levels in the pituitary gland were increased after ovariectomy or P treatment, and were decreased after the administration of E2. Simultaneous injection of E2 and P interfered with the action of E2 on GPR120 mRNA expression. The GnRH antagonist, Cetrotide, did not inhibit the increase in GPR120 expression in ovariectomized (OVX) animals. In addition, immunohistochemistry revealed that more than 95.4% of GPR120 immunoreactive cells colocalized with the luteinizing hormone β (LHβ) in the anterior pituitary gland of intact, ovariectomized (OVX), estradiol-primed OVX (OVX+E2), or progesterone-primed OVX (OVX+P) animals. Furthermore, GPR120 mRNA expression levels were not significantly different in the pituitary gland of females throughout the ovarian cycle. It is suggested that low levels of P may mask the inhibitory effect of estradiol on the synthesis of GPR120 in the estrous stage in intact animals. These results demonstrate that ovarian hormones may directly regulate GPR120 expression in the reproductive cycle at the pituitary level.

Reproduction is regulated by gonadotropins secreted from gonadotrophs. The production and secretion of gonadotropins are mainly regulated by gonadotropin-releasing hormone (GnRH). Agonists or antagonists that influence GnRH action on gonadotrophs are important to regulate reproduction; however, these factors have not been fully characterized due to the lack of simple and easy-to-use techniques to detect gonadotropin secretion from gonadotropin-producing cells. In the present study, we found that Gaussia luciferase (Gluc), which was expressed in LβT2 cells, can be secreted like a luteinizing-hormone (LH) upon stimulation with GnRH. The Gluc secreted into the medium was easily monitored as luminescence signals. The detection range of the GnRH-induced Gluc activity was comparable to that of the enzyme-linked immunosorbent assay for LH. In addition, when the Gluc was expressed in AtT20 cells, which produce adrenocorticotropic hormone (ACTH), the Gluc activity in the medium increased in parallel with the ACTH secretion upon stimulation with corticotropin-releasing hormone. Thus, the Gluc assay in the present study can be easily used for high-throughput screening of factors that influence LH or ACTH secretion from LβT2 or AtT20 cells, respectively.

Gonadotrope cells in the anterior pituitary gland secrete gonadotropins regulating gonadal function in mammals. Recent results have implicated transient receptor potential (TRP) cation channels in pituitary physiology; however, if and how TRP channels contribute to gonadotrope function is not known. Here, we report that 14 out of 28 TRP channels encoded in the mouse genome are expressed in murine gonadotropes with highest expression levels found for canonical TRP (TRPC) channel 5 in juvenile females. We show that TRP channel expression in these cells exhibits considerable plasticity and that it depends on the sex and the developmental and hormonal status of the animal. We then combine different genetic strategies including genetic confocal Ca2+ imaging in whole-mount pituitary gland preparations to characterize TRPC5 channel function in gonadotropes from juvenile females. We show that the TRPC5 agonist Englerin A activates a cytosolic Ca2+ signal and a whole-cell current in these cells, which is absent in TRPC5-deficient mice, and demonstrate that TRPC5 forms functional heteromultimers with TRPC1 in gonadotropes. We further show that the Englerin A-activated TRPC5-dependent Ca2+ signal is mediated by Ca2+ influx both via TRPC5 and via l-type voltage-gated Ca2+ channels, activated by the depolarization through TRPC5-mediated cation influx. Finally, we demonstrate that the gonadotropin-releasing hormone (GnRH)-mediated net depolarization is significantly reduced in gonadotropes isolated from TRPC5-deficient mice. In conclusion, our data suggest that TRPC5 contributes to depolarization of the plasma membrane in gonadotropes upon GnRH stimulation and increases the intracellular Ca2+ concentration via its own Ca2+ permeability and via the activation of voltage-gated Ca2+ channels., (Copyright © 2017 Endocrine Society.)

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 LβT2 cells, suggesting that transactivation of the ErbB family was not necessary for ERK activation in LβT2 cells. We found that ErbB4 was expressed in αT3-1 cells but not in LβT2 cells. GnRH induced the cleavage of ErbB4 and accumulation of an 80-kDa fragment in αT3-1 cells. Pharmacological experiments suggested that G q/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 LβT2 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 LβT2 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The mammalian target of rapamycin (mTOR) assembles into two different multi-protein complexes, mTORC1 and mTORC2. The mTORC2 complex is distinct due to the unique expression of the specific core regulatory protein Rictor (rapamycin-insensitive companion of mTOR). mTORC2 has been implicated in regulating actin cytoskeletal reorganization but its role in gonadotrope function is unknown. Using the gonadotrope-derived LβT2 cell line, we find that the GnRH agonist buserelin (GnRHa) phosphorylates both mTOR and Rictor. Interestingly, inhibition of mTORC2 blunts GnRHa-induced cyto-architectural rearrangements. Coincident with blunting of actin reorganization, inhibition of mTORC2 also attenuates GnRHa-mediated activation of both protein kinase C (PKC) and extracellular signal regulated kinase (ERK). Collectively, our data suggests that GnRHa-mediated mTORC2 activation is important in facilitating actin reorganization events critical for initiating PKC activity and subsequent ERK phosphorylation in the gonadotrope-derived LβT2 cell line., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We examined the role of PKCs and Ca 2+ in GnRH-stimulated p38MAPK phosphorylation in the gonadotrope derived αT3-1 and LβT2 cell lines. GnRH induced a slow and rapid increase in p38MAPK phosphorylation in αT3-1 and LβT2 cells respectively, while PMA gave a slow response. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs), has revealed differential role for PKCα, PKCβII, PKCδ and PKCε in 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 p38MAPK phosphorylation may be explained by differential localization of the PKCs. Basal, GnRH- and PMA- stimulation of p38MAPK phosphorylation in αT3-1 cells is mediated by Ca 2+ influx via voltage-gated Ca 2+ channels and Ca 2+ mobilization, while in the differentiated LβT2 gonadotrope cells it is mediated only by Ca 2+ mobilization. p38MAPK resides in the cell membrane and is relocated to the nucleus by GnRH (∼5 min). Thus, we have identified the PKCs and the Ca 2+ pools involved in GnRH stimulated p38MAPK phosphorylation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Hypothalamic GnRH together with gonadal steroids and activins/inhibin regulate its receptor gene (Gnrhr) expression in vivo, which leads to crucial changes in GnRHR numbers on the plasma membrane. This is accompanied by alterations in the gonadotroph sensitivity and responsiveness during physiologically relevant situations. Here we investigated basal and GnRH-regulated Gnrhr expression in rodent pituitary gonadotrophs in vitro. In pituitary cells from adult animals cultured in the absence of GnRH and steroid hormones, the Gnrhr expression was progressively reduced but not completely abolished. The basal Gnrhr expression was also operative in LβT2 immortalized gonadotrophs never exposed to GnRH. In both cell types, basal transcription was sufficient for the expression of functional GnRHRs. Continuous application of GnRH transiently elevated the Gnrhr expression in cultured pituitary cells followed by a sustained fall without affecting basal transcription. Both basal and regulated Gnrhr transcriptions were dependent on the protein kinase C signaling pathway. The GnRH-regulated Gnrhr expression was not operative in embryonal pituitary and LβT2 cells and was established neonatally, the sex-specific response patterns were formed at the juvenile-peripubertal stage and there was a strong correlation between basal and regulated gene expression during development. Thus, the age-dependent basal and regulated Gnrhr transcription could account for the initial blockade and subsequent activation of the reproductive system during development., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Corticotrophin-releasing factor (CRF) regulates the hypothalamic-pituitary-adrenal axis response to stress through its type 1 receptor (CRF 1 ) in the corticotrophs of the anterior pituitary. Although CRF 1 mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF 1 protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177-188 sequence of the first extracellular loop of the rat CRF 1 . Using the antiserum, CRF 1 -like immunoreactivity (CRF 1 -LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF 1 -LI also appeared in the intermediate lobe cells and on the fibre-like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF 1 (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF 1 -LI-positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF 1 -LI-positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF 1 in the intermediate lobe (98.9 ± 0.2%). CRF 1 -LI and the percentage of CRF 1 -LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 μg/kg bw). These results suggest that CRF 1 is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF 1 are regulated by glucocorticoids in the rat anterior pituitary., (© 2016 British Society for Neuroendocrinology.)

Pituitary follicle-stimulating hormone (FSH) synthesis is regulated by transforming growth factorβsuperfamily ligands, most notably the activins and inhibins. Bone morphogenetic proteins (BMPs) also regulate FSHβ subunit (Fshb) expression in immortalized murine gonadotrope-like LβT2 cells and in primary murine or ovine primary pituitary cultures. BMP2 signals preferentially via the BMP type I receptor, BMPR1A, to stimulate murine Fshb transcription in vitro Here, we used a Cre-lox approach to assess BMPR1A's role in FSH synthesis in mice in vivo Gonadotrope-specific Bmpr1a knockout animals developed normally and had reproductive organ weights comparable with those of controls. Knockouts were fertile, with normal serum gonadotropins and pituitary gonadotropin subunit mRNA expression. Cre-mediated recombination of the floxed Bmpr1a allele was efficient and specific, as indicated by PCR analysis of diverse tissues and isolated gonadotrope cells. Furthermore, BMP2 stimulation of inhibitor of DNA binding 3 expression was impaired in gonadotropes isolated from Bmpr1a knockout mice, confirming the loss of functional receptor protein in these cells. Treatment of purified gonadotropes with small-molecule inhibitors of BMPR1A (and the related receptors BMPR1B and ACVR1) suppressed Fshb mRNA expression, suggesting that an autocrine BMP-like molecule might regulate FSH synthesis. However, deletion of Bmpr1a and Acvr1 in cultured pituitary cells did not alter Fshb expression, indicating that the inhibitors had off-target effects. In sum, BMPs or related ligands acting via BMPR1A or ACVR1 are unlikely to play direct physiological roles in FSH synthesis by murine gonadotrope cells., Competing Interests: Declaration of interest The authors have no conflicts of interest to disclose., (© 2016 Society for Endocrinology.)

Mammalian pituitaries exhibit a high degree of intercellular coordination; this enables them to mount large-scale coordinated responses to various physiological stimuli. This type of communication has not been adequately demonstrated in teleost pituitaries, which exhibit direct hypothalamic innervation and expression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in distinct cell types. We found that in two fish species, namely tilapia and zebrafish, LH cells exhibit close cell-cell contacts and form a continuous network throughout the gland. FSH cells were more loosely distributed but maintained some degree of cell-cell contact by virtue of cytoplasmic processes. These anatomical differences also manifest themselves at the functional level as evidenced by the effect of gap-junction uncouplers on gonadotropin release. These substances abolished the LH response to gonadotropin-releasing hormone stimulation but did not affect the FSH response to the same stimuli. Dye transfer between neighboring LH cells provides further evidence for functional coupling. The two gonadotropins were also found to be differently packaged within their corresponding cell types. Our findings highlight the evolutionary origin of pituitary cell networks and demonstrate how the different levels of cell-cell coordination within the LH and FSH cell populations are reflected in their distinct secretion patterns.

The role of PI4K and PI3K-AKT in ERK1/2 activation by GnRH was examined. A relatively long preincubation (60 min) with wortmannin (10 nM and 10 μM), and LY294002 (10 μM and 100 μM) (doses known to inhibit PI3K and PI4K, respectively), were required to inhibit GnRH-and PMA-stimulated ERK1/2 activity in αT3-1 and LβT2 gonadotrope cells. A similar preincubation protocol was required to demonstrate inhibition of IGF-1-stimulated AKT activation lending support for the need of prolonged incubation (60 min) with wortmannin in contrast to other cellular systems. To rule out that the inhibitors acted upon PI(4,5)P2 levels, we followed the [Ca(2+)]i response to GnRH and found that wortmannin has no significant effect on GnRH-induced [Ca(2+)]i responses. Surprisingly, GnRH and PMA reduced, while IGF-1 increased AKT phosphorylation. We suggest that PI3K inhibits GnRH-stimulated αGSU activity, has no effect upon GnRH-stimulated LHβ activity and enhanced the GnRH-stimulated FSHβ transcription. Hence, PI4K and PI3K-AKT play a role in GnRH to ERK1/2 signaling, while PI3K may regulate also GnRH-induced gonadotropin gene expression., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Origanum vulgare is a plant of the mint family that contains phytoestrogens. This study compared the effects of O. vulgare, LHRH-A2, and 17β-estradiol on the ultrastructure of gonadotroph cells and ovarian oogenesis in immature Trichogaster trichopterus. Fish (5.1±0.032cm and 2.1±0.043g, n=150) were randomly divided into four treatment groups (three hormonal treatments and control) and treated intramuscularly at four levels with 17β-estradiol or O. vulgare at 10, 20, 30 and 50mg/kg body weight and with LHRH-A2 at 0.001, 0.002, 0.003, and 0.005mg/kg body weight. There were three control treatments: saline, ethanol and placebo. Fish were kept in 15 tanks, with 10 fish per tank, injected a total of seven doses over 13 days. Gonadosomatic index (GSI) and oocyte diameter were lower (P≤0.05) in the control than in the three hormonal treatments. The highest GSI and oocyte diameter responses were observed in fish treated with 17β-estradiol (2.76±0.23%, 149.8±15.43mm) followed by O. vulgare (1.86±0.18%, 104.3±11.5mm) and LHRH-A2 (1.52±0.12%, 91.75±9.02mm) (P≤0.05). Moreover, there was a significant effect of dose level within all the hormonal treatments (P≤0.05). The effect of treatment on the length and weight was likely GSI. Ovarian tissue results showed faster oogenesis of oocytes in fish treated with O. vulgare, after 17β-estradiol. Ultrastructure of gonadotroph cells demonstrated less stimulation by O. vulgare than by 17β-estradiol and LHRH-A2. This study suggests that compared with the two hormonal treatments, O. vulgare dose-dependently affects ovarian oogenesis and gonadotroph cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific β-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.

4-Chloro-N-[6,8-dibromo-2-(2-thienyl)imidazo[1,2-alpyridine-3-yl] (DS1) is a GABA(A) receptor agonist that selectively binds to delta subunit-containing GABA(A) alpha4beta3delta receptors. In the present study, we examined the effect of DS1 on pituitary gonadotropin subunit gene expression using the mouse pituitary gonadotroph cell line LbetaT2. DS1 increased the promoter activity of the gonadotropin subunits luteinizing hormone beta (LHbeta), follicle-stimulating hormone beta (FSHbeta), and alpha. Gonadotropin-releasing hormone (GnRH) receptor promoters were also activated by DS1. The effects of DS1 on gonadotropin subunit promoters were obvious, but they were less than those induced by stimulation with GnRH. GnRH-stimulated gonadotropin subunit promoters were enhanced in the presence of DS1. A prototypic specific agonist for GABAA receptors, muscimol, failed to increase LHbeta and FSHbeta subunit promoter activity and had no effect on GnRH-increased LHbeta and FSHbeta promoter activity. In addition, SKF97541, a specific agonist for GABAB receptors, did not modulate basal or GnRH-induced LHbeta and FSHbeta promoter activity. A natural GABA compound failed to increase gonadotropin promoter activity and potentiated the effect of GnRH on the FSHbeta promoter. DS1 increased the activity of serum response element (SRE) and cAMP response element (CRE) promoters, which reflect the activity of the extracellular signal-regulated kinase and cAMP/protein kinase A (PKA) pathways, and GnRH-increased SRE and CRE promoter activity was enhanced in the presence of DS1. A specific inhibitor of the ERK signaling pathway, U0126, prevented DS1-induced LHbeta and FSHbeta promoter activity almost completely; however, H89, a PKA inhibitor, did not modulate the effect of DS1. Our current observations demonstrate that the GABAA alpha4beta3delta receptor agonist DS1 can stimulate gonadotropin subunit gene expression in association with the ERK signaling pathway., (© 2015 by the Society for the Study of Reproduction, Inc.)

Objectives: Polymeric PEG-b-PLA nanoparticles (NPs) were developed for delivery of poorly water-soluble drugs via blood brain barrier into brain parenchyma. We analyzed neuroendocrine disrupting effects of neonatal exposure of female rats to PEG-b-PLA NPs and diethylstilbestrol (DES) on the function of adenohypophyseal gonadotrophs of infantile or adult rats by examining in vitro luteinizing hormone releasing hormone (LHRH)-induced luteinizing hormone (LH) release., Methods: Neonatal female Wistar rats were injected intraperitoneally, daily, from postnatal day (PND) 4 to PND7 with PEG-b-PLA NPs (20 mg.kg b.w.(-1)), DES (4 µg.kg b.w.(-1)) or vehicle. At the necropsy day (PND15 in infantile and the first estrus day after PND176 in adult rats), adenohypophyseal cells were isolated by enzymatic digestion, plated in 96-well plates (5×10(4) cells.well(-1)) in serum-supplemented medium and left to recover for 96 h. LHRH (10-7 mol.L(-1)) treatment was performed in serum-free medium for 60 min and LH levels in culture media were determined by radioimmunoassay., Results: In all experimental groups, in vitro LHRH treatment significantly stimulated LH release from pituitary cells of infantile but not adult female rats. Neonatal DES treatment increased basal LH secretion from cultured pituitary cells of adult but not infantile rats. In both, infantile and adult rats, neonatal treatment with PEG-b-PLA significantly increased basal and LHRH-induced LH release from pituitary cells compared to corresponding controls and DES-treated group., Conclusions: Data indicate that neonatal exposure to PEG-b-PLA NPs may alter pituitary LH release, and thereby modify reproductive system development in infantile female rats leading to reproductive dysfunctions in adult age.

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.

Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LβT2 cells in which Activin A and BMP2 synergistically induce FSHβ. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LβT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LβT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LβT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHβ. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHβ induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHβ induction in LβT2 cells apparently due to its ability to block Activin A signaling., (© 2014 Society for Endocrinology.)

The adipokine, leptin (LEP), is a hormonal gateway, signaling energy stores to appetite-regulatory neurons, permitting reproduction when stores are sufficient. Dual-labeling for LEP receptors (LEPRs) and gonadotropins or GH revealed a 2-fold increase in LEPR during proestrus, some of which was seen in LH gonadotropes. We therefore investigated LEPR functions in gonadotropes with Cre-LoxP technology, deleting the signaling domain of the LEPR (Lepr-exon 17) with Cre-recombinase driven by the rat LH-β promoter (Lhβ-cre). Selectivity of the deletion was validated by organ genotyping and lack of LEPR and responses to LEP by mutant gonadotropes. The mutation had no impact on growth, body weight, the timing of puberty, or pregnancy. Mutant females took 36% longer to produce their first litter and had 50% fewer pups/litter. When the broad impact of the loss of gonadotrope LEPR on all pituitary hormones was studied, mutant diestrous females had reduced serum levels of LH (40%), FSH (70%), and GH (54%) and mRNA levels of Fshβ (59%) and inhibin/activin β A and β B (25%). Mutant males had reduced serum levels of GH (74%), TSH (31%), and prolactin (69%) and mRNA levels of Gh (31%), Ghrhr (30%), Fshβ (22%), and glycoprotein α-subunit (Cga) (22%). Serum levels of LEP and ACTH and mRNA levels of Gnrhr were unchanged. However, binding to GnRH receptors was reduced in LEPR-null LH or FSH gonadotropes by 82% or 89%, respectively, in females (P < .0001) and 27% or 53%, respectively, in males (P < .03). This correlated with reductions in GnRH receptor protein immunolabeling, suggesting that LEP's actions may be posttranscriptional. Collectively, these studies highlight the importance of LEP to gonadotropes with GnRH-binding sites and activin as potential targets. LEP may modulate population growth, adjusting the number of offspring to the availability of food supplies.