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Follicle-stimulating hormone (FSH), a dimeric glycoprotein produced by pituitary gonadotrope cells, regulates spermatogenesis in males and ovarian follicle growth in females. Hypothalamic gonadotropin-releasing hormone (GnRH) stimulates FSHβ subunit gene (Fshb) transcription, though the underlying mechanisms are poorly understood. To address this gap in knowledge, we examined changes in pituitary gene expression in GnRH-deficient mice (hpg) treated with a regimen of exogenous GnRH that increases pituitary Fshb but not luteinizing hormone β (Lhb) messenger RNA levels. Activating transcription factor 3 (Atf3) was among the most upregulated genes. Activating transcription factor 3 (ATF3) can heterodimerize with members of the activator protein 1 family to regulate gene transcription. Co-expression of ATF3 with JunB stimulated murine Fshb, but not Lhb, promoter-reporter activity in homologous LβT2b cells. ATF3 also synergized with a constitutively active activin type I receptor to increase endogenous Fshb expression in these cells. Nevertheless, FSH production was intact in gonadotrope-specific Atf3 knockout [conditional knockout (cKO)] mice. Ovarian follicle development, ovulation, and litter sizes were equivalent between cKOs and controls. Testis weights and sperm counts did not differ between genotypes. Following gonadectomy, increases in LH secretion were enhanced in cKO animals. Though FSH levels did not differ between genotypes, post-gonadectomy increases in pituitary Fshb and gonadotropin α subunit expression were more pronounced in cKO than control mice. These data indicate that ATF3 can selectively stimulate Fshb expression in vitro but is not required for FSH production in vivo.

Inhibins are transforming growth factor-β family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common β chain with activin ligands. Follistatin is another activin antagonist, known to bind the common β chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin β chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lower affinity than follistatin 288 alone or in complex with activin A. Characterizing the inhibin A:follistatin 288 complex in an activin-responsive luciferase assay and by surface plasmon resonance indicated that the inhibitor complex readily dissociated upon binding type II receptor activin receptor type IIb, allowing both antagonists to inhibit activin signaling. Additionally, injection of the complex in ovariectomized female mice did not alter inhibin A suppression of FSH. Taken together, this study shows that while follistatin binds to inhibin A with a substochiometric ratio relative to the activin homodimer, the complex can dissociate readily, allowing both proteins to effectively antagonize activin signaling.

The inhibins control reproduction by suppressing follicle-stimulating hormone synthesis in pituitary gonadotrope cells. The newly discovered inhibin B coreceptor, TGFBR3L, is selectively and highly expressed in gonadotropes in both mice and humans. Here, we describe our initial characterization of mechanisms controlling cell-specific Tgfbr3l/TGFBR3L transcription. We identified two steroidogenic factor 1 (SF-1 or NR5A1) cis-elements in the proximal Tgfbr3l promoter in mice. SF-1 induction of murine Tgfbr3l promoter–reporter activity was inhibited by mutations in one or both sites in heterologous cells. In homologous cells, mutation of these cis-elements or depletion of endogenous SF-1 similarly decreased reporter activity. We observed nearly identical results when using a human TGFBR3L promoter–reporter. The Tgfbr3l gene was tightly compacted and Tgfbr3l mRNA expression was essentially absent in gonadotropes of SF-1 (Nr5a1) conditional knockout mice. During murine embryonic development, Tgfbr3l precedes Nr5a1 expression, though the two transcripts are fully colocalized by embryonic day 18.5 and thereafter. Collectively, these data indicate that SF-1 directly regulates Tgfbr3l/TGFBR3L transcription and is required for postnatal expression of the gene in gonadotropes.

Loss of function mutations in IGSF1/Igsf1 cause central hypothyroidism. Igsf1 knockout mice have reduced pituitary thyrotropin-releasing hormone receptor, Trhr, expression, perhaps contributing to the phenotype. Because thyroid hormones negatively regulate Trhr, we hypothesized that IGSF1 might affect thyroid hormone availability in pituitary thyrotropes. Consistent with this idea, IGSF1 coimmunoprecipitated with the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) in transfected cells. This association was impaired with IGSF1 bearing patient-derived mutations. Wild-type IGSF1 did not, however, alter MCT8-mediated thyroid hormone import into heterologous cells. IGSF1 and MCT8 are both expressed in the apical membrane of the choroid plexus. However, MCT8 protein levels and localization in the choroid plexus were unaltered in Igsf1 knockout mice, ruling out a necessary chaperone function for IGSF1. MCT8 expression was low in the pituitary and was similarly unaffected in Igsf1 knockouts. We next assessed whether IGSF1 affects thyroid hormone transport or action, by MCT8 or otherwise, in vivo. To this end, we treated hypothyroid wild-type and Igsf1 knockout mice with exogenous thyroid hormones. T4 and T3 inhibited TSH release and regulated pituitary and forebrain gene expression similarly in both genotypes. Interestingly, pituitary TSH beta subunit (Tshb) expression was consistently reduced in Igsf1 knockouts relative to wild-type regardless of experimental condition, whereas Trhr was more variably affected. Although IGSF1 and MCT8 can interact in heterologous cells, the physiological relevance of their association is not clear. Nevertheless, the results suggest that IGSF1 loss can impair TSH production independently of alterations in TRHR levels or thyroid hormone action.

Activin ligands are formed from two disulfide-linked inhibin β subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well-characterized, little is known about the biological function of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.

The orphan nuclear receptor steroidogenic factor-1 (SF-1 or NR5A1) is an indispensable regulator of adrenal and gonadal formation, playing roles in sex determination, hypothalamic development, and pituitary function. This study aimed to identify the roles of SF-1 in postnatal female reproductive function. Using a progesterone receptor–driven Cre recombinase, we developed a novel murine model, characterized by conditional depletion of SF-1 [PR-Cre;Nr5a1f/f; conditional knockout (cKO)] in the hypothalamic-pituitary-gonadal axis. Mature female cKO were infertile due to the absence of ovulation. Reduced gonadotropin concentrations in the pituitary gland that were nevertheless sufficient to maintain regular estrous cycles were observed in mature cKO females. The cKO ovaries showed abnormal lipid accumulation in the stroma, associated with an irregular expression of cholesterol homeostatic genes such as Star, Scp2, and Acat1. The depletion of SF-1 in granulosa cells prevented appropriate cumulus oöphorus expansion, characterized by reduced expression of Areg, Ereg, and Ptgs2. Exogenous delivery of gonadotropins to cKO females to induce ovulation did not restore fertility and was associated with impaired formation and function of corpora lutea accompanied by reduced expression of the steroidogenic genes Cyp11a1 and Cyp19a1 and attenuated progesterone production. Surgical transplantation of cKO ovaries to ovariectomized control animals (Nr5a1f/f) resulted in 2 separate phenotypes, either sterility or apparently normal fertility. The deletion of SF-1 in the pituitary and in granulosa cells near the moment of ovulation demonstrated that this nuclear receptor functions across the pituitary-gonadal axis and plays essential roles in gonadotropin synthesis, cumulus expansion, and luteinization.

The progesterone receptor (PR, encoded by Pgr) plays essential roles in reproduction. Female mice lacking the PR are infertile, due to the loss of the protein’s functions in the brain, ovary, and uterus. PR is also expressed in pituitary gonadotrope cells, but its specific role therein has not been assessed in vivo. We therefore generated gonadotrope-specific Pgr conditional knockout mice (cKO) using the Cre-LoxP system. Overall, both female and male cKO mice appeared phenotypically normal. cKO females displayed regular estrous cycles (vaginal cytology) and normal fertility (litter size and frequency). Reproductive organ weights were comparable between wild-type and cKO mice of both sexes, as were production and secretion of the gonadotropins, LH and FSH, with one exception. On the afternoon of proestrus, the amplitude of the LH surge was blunted in cKO females relative to controls. Contrary to predictions of earlier models, this did not appear to derive from impaired GnRH self-priming. Collectively, these data indicate that PR function in gonadotropes may be limited to regulation of LH surge amplitude in female mice via a currently unknown mechanism.

Mammalian reproduction depends on the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone, which are secreted by pituitary gonadotrope cells. The zinc-finger transcription factor GATA2 was previously implicated in FSH production in male mice; however, its mechanisms of action and role in females were not determined. To directly address GATA2 function in gonadotropes, we generated and analyzed gonadotrope-specific Gata2 KO mice using the Cre-lox system. We found that while conditional KO (cKO) males exhibited ∼50% reductions in serum FSH levels and pituitary FSHβ subunit (Fshb) expression relative to controls, FSH production was apparently normal in cKO females. In addition, RNA-seq analysis of purified gonadotropes from control and cKO males revealed a profound decrease in expression of gremlin (Grem1), a bone morphogenetic protein (BMP) antagonist. We show Grem1 was expressed in gonadotropes, but not other cell lineages, in the adult male mouse pituitary. Furthermore, Gata2, Grem1, and Fshb mRNA levels were significantly higher in the pituitaries of WT males relative to females but decreased in males treated with estradiol and increased following ovariectomy in control but not cKO females. Finally, we found that recombinant gremlin stimulated Fshb expression in pituitary cultures from WT mice. Collectively, the data suggest that GATA2 promotes Grem1 expression in gonadotropes and that the gremlin protein potentiates FSH production. The mechanisms of gremlin action have not yet been established but may involve attenuation of BMP binding to activin type II receptors in gonadotropes, facilitating induction of Fshb transcription by activins or related ligands.

Description, Discovery of a novel pituitary protein suggests a new means to enhance fertility., Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFβ type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFβ family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L’s function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.

Inhibins are members of the transforming growth factor-β family, composed of a common α-subunit disulfide-linked to 1 of 2 β-subunits (βA in inhibin A or βB in inhibin B). Gonadal-derived inhibin A and B act in an endocrine manner to suppress the synthesis of follicle-stimulating hormone (FSH) by pituitary gonadotrope cells. Roles for inhibins beyond the pituitary, however, have proven difficult to delineate because deletion of the inhibin α-subunit gene (Inha) results in unconstrained expression of activin A and activin B (homodimers of inhibin β-subunits), which contribute to gonadal tumorigenesis and lethal cachectic wasting. Here, we generated mice with a single point mutation (Arg233Ala) in Inha that prevents proteolytic processing and the formation of bioactive inhibin. In vitro, this mutation blocked inhibin maturation and bioactivity, without perturbing activin production. Serum FSH levels were elevated 2- to 3-fold in InhaR233A/R233A mice due to the loss of negative feedback from inhibins, but no pathological increase in circulating activins was observed. While inactivation of inhibin A and B had no discernible effect on male reproduction, female InhaR233A/R233A mice had increased FSH-dependent follicle development and enhanced natural ovulation rates. Nevertheless, inhibin inactivation resulted in significant embryo-fetal resorptions and severe subfertility and was associated with disrupted maternal ovarian function. Intriguingly, heterozygous Inha+/R233A females had significantly enhanced fecundity, relative to wild-type littermates. These studies have revealed novel effects of inhibins in the establishment and maintenance of pregnancy and demonstrated that partial inactivation of inhibin A/B is an attractive approach for enhancing female fertility.

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.

Gonadotropin-releasing hormone (GnRH) regulates gonadal function via its stimulatory effects on gonadotropin production by pituitary gonadotrope cells. GnRH is released from the hypothalamus in pulses and GnRH pulse frequency differentially regulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis and secretion. The GnRH receptor (GnRHR) is a G protein–coupled receptor that canonically activates Gα q/11-dependent signaling on ligand binding. However, the receptor can also couple to Gα s and in vitro data suggest that toggling between different G proteins may contribute to GnRH pulse frequency decoding. For example, as we show here, knockdown of Gα s impairs GnRH-stimulated FSH synthesis at low- but not high-pulse frequency in a model gonadotrope-derived cell line. We next used a Cre-lox conditional knockout approach to interrogate the relative roles of Gα q/11 and Gα s proteins in gonadotrope function in mice. Gonadotrope-specific Gα q/11 knockouts exhibit hypogonadotropic hypogonadism and infertility, akin to the phenotypes seen in GnRH- or GnRHR-deficient mice. In contrast, under standard conditions, gonadotrope-specific Gα s knockouts produce gonadotropins at normal levels and are fertile. However, the LH surge amplitude is blunted in Gα s knockout females and postgonadectomy increases in FSH and LH are reduced both in males and females. These data suggest that GnRH may signal principally via Gα q/11 to stimulate gonadotropin production, but that Gα s plays important roles in gonadotrope function in vivo when GnRH secretion is enhanced.

The Hippo transcriptional coactivators YAP and TAZ exert critical roles in morphogenesis, organ size determination and tumorigenesis in many tissues. Although Hippo kinase cascade activity was recently reported in the anterior pituitary gland in mice, the role of the Hippo effectors in regulating gonadotropin production remains unknown. The objective of this study was therefore to characterize the roles of YAP and TAZ in gonadotropin synthesis and secretion. Using a conditional gene targeting approach (cKO), we found that gonadotrope-specific inactivation of Yap and Taz resulted in increased circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in adult male mice, along with increased testosterone levels and testis weight. Female cKO mice had increased circulating LH (but not FSH) levels, which were associated with a hyperfertility phenotype characterized by higher ovulation rates and larger litter sizes. Unexpectedly, the loss of YAP/TAZ did not appear to affect the expression of gonadotropin subunit genes, yet both basal and GnRH-induced LH secretion were increased in cultured pituitary cells from cKO mice. Likewise, pharmacologic inhibition of YAP binding to the TEAD family of transcription factors increased both basal and GnRH-induced LH secretion in LβT2 gonadotrope-like cells in vitro without affecting Lhb expression. Conversely, mRNA levels of ChgA and SgII, which encode key secretory granule cargo proteins, were decreased following pharmacologic inhibition of YAP/TAZ, suggesting a mechanism whereby YAP/TAZ regulate the LH secretion machinery in gonadotrope cells. Together, these findings represent the first evidence that Hippo signaling may play a role in regulating pituitary LH secretion.

Despite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility forGATA3, but with TF expression forPOMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.

Activins selectively stimulate follicle-stimulating hormone (FSH) secretion by pituitary gonadotrope cells. More recently, other members of the TGFbeta superfamily, the bone morphogenetic proteins (BMPs), were reported to regulate FSH synthesis. Activins and BMPs independently and synergistically stimulate transcription of the FSHbeta subunit (Fshb) gene in immortalized gonadotrope-like cells. Both ligands can signal via the activin receptor type IIA (ACVR2A) to regulate FSH synthesis in vitro. In vivo, global Acvr2a knockout mice exhibit a 60% reduction in circulating FSH relative to wild-type animals, suggesting that activins, BMPs, or related ligands might signal through additional type II receptors to regulate FSH in vivo. Although the leading candidates are ACVR2B and the BMP type II receptor (BMPR2), only the latter mediates activin or BMP2 induction of Fshb transcription in vitro. Here, we generated mice carrying a loss of function mutation in Bmpr2 specifically in gonadotropes. Puberty onset, estrous cyclicity, and reproductive organ weights were similar between control and conditional knockout females. Serum FSH and luteinizing hormone (LH) and pituitary expression of Fshb and the LHbeta subunit (Lhb) were similarly unaffected by the gene deletion in both sexes. These results suggest that BMPR2 might not play a necessary role in FSH synthesis or secretion in vivo or that another type II receptor, such as ACVR2A, can fully compensate for its absence. These data also further contribute to the emerging concept that BMPs may not be physiological regulators of FSH in vivo.

LβT2 and αT3-1 are important, widely studied cell line models for the pituitary gonadotropes that were generated by targeted tumorigenesis in transgenic mice. LβT2 cells are more mature gonadotrope precursors than αT3-1 cells. Microsatellite authentication patterns, chromosomal characteristics, and their intercellular variation have not been reported. We performed microsatellite and cytogenetic analysis of both cell types at early passage numbers. Short tandem repeat (STR) profiling was consistent with a mixed C57BL/6J × BALB/cJ genetic background, with distinct patterns for each cell type. Spectral karyotyping in αT3-1 cells revealed cell-to-cell variation in chromosome composition and pseudodiploidy. In LβT2 cells, chromosome counting and karyotyping demonstrated pseudotriploidy and high chromosomal variation among cells. Chromosome copy number variation was confirmed by single-cell DNA sequencing. Chromosomal compositions were consistent with a male sex for αT3-1 and a female sex for LβT2 cells. Among LβT2 stocks used in multiple laboratories, we detected two genetically similar but distinguishable lines via STR authentication, LβT2a and LβT2b. The two lines differed in morphological appearance, with LβT2a having significantly smaller cell and nucleus areas. Analysis of immediate early gene and gonadotropin subunit gene expression revealed variations in basal expression and responses to continuous and pulsatile GnRH stimulation. LβT2a showed higher basal levels of Egr1, Fos, and Lhb but lower Fos induction. Fshb induction reached significance only in LβT2b cells. Our study highlights the heterogeneity in gonadotrope cell line genomes and provides reference STR authentication patterns that can be monitored to improve experimental reproducibility and facilitate comparisons of results within and across laboratories.

Bone morphogenetic proteins (BMPs) are pleiotropic ligands in the TGF-β superfamily. In the early to mid-2000s, several BMPs, including BMP2, were shown to regulate FSH synthesis alone and in synergy with activins in immortalized gonadotrope-like cell lines and primary pituitary cultures. Activins are also TGF-β family members, which were identified and named based on their abilities to stimulate FSH production selectively. Mechanistic analyses suggested that BMP2 promoted expression of the FSHβ subunit gene (Fshb) via at least two nonmutually exclusive mechanisms. First, BMP2 stimulated the production of the inhibitor of DNA-binding proteins 1, 2, and 3 (Id1, Id2, and Id3), which potentiated the stimulatory actions of homolog of Drosophila mothers against decapentaplegic 3 (SMAD3) on the Fshb promoter. SMAD3 is an intracellular signaling protein that canonically mediates the actions of activins and is an essential regulator of Fshb production in vitro and in vivo. Second, BMP2 was shown to activate SMAD3-dependent signaling via its canonical type IA receptor, BMPR1A (also known as ALK3). This was a surprising result, as ALK3 conventionally activates distinct SMAD proteins. Although these initial results were compelling, they were challenged by contemporaneous and subsequent observations. For example, inhibitors of BMP signaling did not specifically impair FSH production in cultured pituitary cells. Of perhaps greater significance, mice lacking ALK3 in gonadotrope cells produced FSH normally. Therefore, the physiological role of BMPs in FSH synthesis in vivo is presently uncertain.

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 regulatesFshbtranscription, as results vary across species and betweenin vivoandin vitroapproaches. One of the more fully developed models suggests that the murineFshbpromoter 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-inducedFshbmRNA 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 onFshbexpression. Collectively, these data suggest that class I HDACs are positive, not negative, regulators ofFshbexpressionin vitroand that, contrary to earlier reports, GnRH may not stimulateFshbby inhibiting HDAC-mediated repression of the gene.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene result in central hypothyroidism, often associated with macroorchidism. Testicular enlargement in these patients might be caused by increases in follicle-stimulating hormone (FSH) levels, as IGSF1 has been proposed to function as an inhibin B receptor or as an inhibitor of activin type I receptor (ALK4) activity in pituitary gonadotrope cells. If true, loss of IGSF1 should lead to reduced inhibin B action or disinhibition of activin signaling, thereby increasing FSH synthesis. Here, we show that FSH levels and sperm counts are normal in male Igsf1 knockout mice, although testis size is mildly increased. Sperm parameters are also normal in men with IGSF1 deficiency, although their FSH levels may trend higher and their testes are enlarged. Inhibin B retains the ability to suppress FSH synthesis in pituitaries of Igsf1-knockout mice and IGSF1 does not interact with ALK4 or alter activin A/ALK4 stimulation of FSHβ (Fshb/FSHB) subunit transcription or expression. In light of these results, it is unlikely that macroorchidism in IGSF1 deficiency derives from alterations in spermatogenesis or inhibin/activin regulation of FSH.

Intraperitoneal injection of kisspeptin-54 induces a surge-like release of luteinizing hormone that stimulates ovulation in mice.

Follicle-stimulating hormone (FSH) regulates gonadal function and fertility. Measurement of FSH in bodily fluids and tissues is possible with radioimmunoassays and enzyme-linked immunosorbent assays (ELISAs). Recently, several novel assays were developed to measure pituitary hormones including growth hormone, prolactin, and luteinizing hormone in mice from small sample volumes. Here, we describe a novel and sensitive ELISA that enables the accurate measurement of FSH in serum, plasma, and whole blood from female and male mice. The assay can also be used to measure FSH in murine pituitary lysates and cell culture media. In summary, the new methodology described here will enable investigators to measure FSH from a variety of biological samples in mice accurately, at low cost, and in their own laboratories.

Background Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor β (TGFβ) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties. Results Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8. Conclusions These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0350-1) contains supplementary material, which is available to authorized users.

Ovarian follicle development is regulated by locally produced TGFβ superfamily members. The TGFβ type III receptor (TGFBR3, or betaglycan), which regulates the actions of diverse TGFβ ligands, including inhibins, is expressed in different ovarian cell types. However, its functional roles in the ovary have not been investigated in vivo. Here, we ablated Tgfbr3 in murine oocytes using the Cre-loxP system. Oocyte-specific Tgfbr3 knockout (cKO) females were fertile, producing litters of similar size and frequency as controls. Their ovarian weights and histology were also normal. Though we confirmed efficient recombination of the floxed alleles, we did not detect Tgfbr3 mRNA in purified oocytes from superovulated cKO or control mice. These results challenge earlier observations of betaglycan protein expression in this cell type. Regardless, Tgfbr3 in the murine oocyte is clearly dispensable for female fertility.

The pituitary regulates growth, reproduction and other endocrine systems. To investigate transcriptional network epigenetic mechanisms, we generated paired single nucleus (sn) transcriptome and chromatin accessibility profiles in single mouse pituitaries and genome-wide sn methylation datasets. Our analysis provided insight into cell type epigenetics, regulatory circuit and gene control mechanisms. Latent variable pathway analysis detected corresponding transcriptome and chromatin accessibility programs showing both inter-sexual and inter-individual variation. Multi-omics analysis of gene regulatory networks identified cell type-specific regulons whose composition and function were shaped by the promoter accessibility state of target genes. Co-accessibility analysis comprehensively identified putative cis-regulatory regions, including a domain 17kb upstream of Fshb that overlapped the fertility-linked rs11031006 human polymorphism. In vitro CRISPR-deletion at this locus increased Fshb levels, supporting this domain’s inferred regulatory role. The sn pituitary multi-omics atlas (snpituitaryatlas.princeton.edu) is a public resource for elucidating cell type-specific gene regulatory mechanisms and principles of transcription circuit control.

To provide a multi-omics resource and investigate transcriptional regulatory mechanisms, we profile the transcriptome, chromatin accessibility, and methylation status of over 70,000 single nuclei (sn) from adult mouse pituitaries. Paired snRNAseq and snATACseq datasets from individual animals highlight a continuum between developmental epigenetically-encoded cell types and transcriptionally-determined transient cell states. Co-accessibility analysis-based identification of a putative Fshb cis-regulatory domain that overlaps the fertility-linked rs11031006 human polymorphism, followed by experimental validation illustrate the use of this resource for hypothesis generation. We also identify transcriptional and chromatin accessibility programs distinguishing each major cell type. Regulons, which are co-regulated gene sets sharing binding sites for a common transcription factor driver, recapitulate cell type clustering. We identify both cell type-specific and sex-specific regulons that are highly correlated with promoter accessibility, but not with methylation state, supporting the centrality of chromatin accessibility in shaping cell-defining transcriptional programs. The sn multi-omics atlas is accessible at snpituitaryatlas.princeton.edu.

It was recently reported that mutations in the insulin receptor substrate 4 (IRS4) gene cause a novel form of X-linked congenital central hypothyroidism (OMIM 300904). To date, four different mutations, three frameshift and one nonsense, have been reported, with two affected male patients showing decreased basal, pulsatile, and total thyroid-stimulation hormone (TSH) secretion (PMID 30061370). Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and their downstream effectors. IRS4/Irs4 expression is enriched in the pituitary; however, its role in the hypothalamic-pituitary-thyroid (HPT) axis has not been studied in detail. We generated novel whole-body Irs4-knockout mouse lines using CRISPR-Cas9. A specific guide RNA was used to target the Cas9 enzyme to the 5’ end of the single exon Irs4 gene. A two-nucleotide deletion was introduced into Irs4, resulting in a frameshift and premature stop codon. We hypothesized that like IRS4 deficient patients, these mice would exhibit central hypothyroidism. Given that Irs4 is X-linked, we focused our initial characterization on males. Under normal laboratory conditions, Irs4 knockout mice do not exhibit differences in pituitary expression of Tshb, which encodes one of the subunits of the TSH heterodimer. Expression of the gene encoding the thyrotropin-releasing hormone (TRH) receptor, Trhr1, is also unperturbed in these knockout mice. Additionally, there are no differences in their serum thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine). When Irs4 knockout males were placed on a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks and rendered hypothyroid, their serum TSH increased similarly to wild-type males. Overall, Irs4 knockout males do not exhibit central hypothyroidism or phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these mice.

The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity. Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

Activins are selective regulators of FSH production by pituitary gonadotrope cells. In a gonadotrope-like cell line, LβT2, activins stimulate FSH via the activin type IIA receptor (ACVR2A) and/or bone morphogenetic protein type II receptor (BMPR2). Consistent with these observations, FSH is greatly reduced, though still present, in global Acvr2a knockout mice. In contrast, FSH production is unaltered in gonadotrope-specific Bmpr2 knockout mice. In light of these results, we questioned whether an additional type II receptor might mediate the actions of activins or related TGF-β ligands in gonadotropes. We focused on the activin type IIB receptor (ACVR2B), even though it does not mediate activin actions in LβT2 cells. Using a Cre-lox strategy, we ablated Acvr2a and/or Acvr2b in murine gonadotropes. The resulting conditional knockout (cKO) animals were compared with littermate controls. Acvr2a cKO (cKO-A) females were subfertile (~70% reduced litter size), cKO-A males were hypogonadal, and both sexes showed marked decreases in serum FSH levels compared with controls. Acvr2b cKO (cKO-B) females were subfertile (~20% reduced litter size), cKO-B males had a moderate decrease in testicular weight, but only males showed a significant decrease in serum FSH levels relative to controls. Simultaneous deletion of both Acvr2a and Acvr2b in gonadotropes led to profound hypogonadism and FSH deficiency in both sexes; females were acyclic and sterile. Collectively, these data demonstrate that ACVR2A and ACVR2B are the critical type II receptors through which activins or related TGF-β ligands induce FSH production in mice in vivo.

Follicle-stimulating hormone (FSH), an essential regulator of mammalian fertility, is synthesized by pituitary gonadotrope cells in response to activins. In mice, activins signal via SMAD3, SMAD4, and FOXL2 to regulate transcription of the FSHβ subunit (Fshb) gene. Gonadotrope-specific deletion of Foxl2, alone or in combination with Smad4, renders mice FSH-deficient. Whether human FSHB expression is similarly regulated is not known. Here, we used a combination of transgenic and conditional knockout mouse strains to assess the roles of activins, FOXL2, and SMAD4 in regulation of the human FSHB gene. First, we cultured pituitaries from mice harboring a human FSHB transgene (hFSHB mice) and measured both murine Fshb and human FSHB messenger ribonucleic acid (mRNA) expression in response to exogenous activins or two antagonists of endogenous activin-like signaling (follistatin-288 and SB431542). Both murine Fshb and human FSHB expression were stimulated by activins and reduced by the inhibitors. Next, we analyzed human FSHB expression in hFSHB mice carrying floxed Foxl2 and Smad4 alleles. Cre-mediated ablation of FOXL2 and SMAD4 strongly reduced basal and activin-stimulated murine Fshb and human FSHB expression in cultured pituitaries. Finally, the hFSHB transgene was previously shown to rescue FSH production and fertility in Fshb knockout mice. However, gonadotrope-specific Foxl2/Smad4 knockout females carrying the hFSHB transgene have significantly reduced murine Fshb and human FSHB pituitary mRNA levels and are hypogonadal. Collectively, these data suggest that similar to Fshb regulation in mice, FOXL2 and SMAD4 play essential roles in human FSHB expression.

Context The X-linked immunoglobulin superfamily, member 1 (IGSF1), gene is highly expressed in the hypothalamus and in pituitary cells of the POU1F1 lineage. Human loss-of-function mutations in IGSF1 cause central hypothyroidism, hypoprolactinemia, and macroorchidism. Additionally, most affected adults exhibit higher than average IGF-1 levels and anecdotal reports describe acromegaloid features in older subjects. However, somatotrope function has not yet been formally evaluated in this condition. Objective We aimed to evaluate the role of IGSF1 in human and murine somatotrope function. Patients, Design, and Setting We evaluated 21 adult males harboring hemizygous IGSF1 loss-of-function mutations for features of GH excess, in an academic clinical setting. Main Outcome Measures We compared biochemical and tissue markers of GH excess in patients and controls, including 24-hour GH profile studies in 7 patients. Parallel studies were undertaken in male Igsf1-deficient mice and wild-type littermates. Results IGSF1-deficient adult male patients demonstrated acromegaloid facial features with increased head circumference as well as increased finger soft-tissue thickness. Median serum IGF-1 concentrations were elevated, and 24-hour GH profile studies confirmed 2- to 3-fold increased median basal, pulsatile, and total GH secretion. Male Igsf1-deficient mice also demonstrated features of GH excess with increased lean mass, organ size, and skeletal dimensions and elevated mean circulating IGF-1 and pituitary GH levels. Conclusions We demonstrate somatotrope neurosecretory hyperfunction in IGSF1-deficient humans and mice. These observations define a hitherto uncharacterized role for IGSF1 in somatotropes and indicate that patients with IGSF1 mutations should be evaluated for long-term consequences of increased GH exposure.

The glycoprotein hormone family has four well-known members in mammals. All four are dimers of a shared α subunit noncovalently bound to hormone-specific β subunits. Three of these proteins are synthesized by two cell types in the anterior pituitary gland. Gonadotropes produce the gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH). Thyrotropes synthesize thyrotropin or thyroid-stimulating hormone (TSH). The production and secretion of all three hormones depends on the activity of peptides from the brain. In turn, FSH, LH, and TSH have effects on distal target organs that produce hormones in response. FSH and LH stimulate sex steroid production by the gonads. TSH stimulates thyroid hormone synthesis in the thyroid gland. Sex steroids and thyroid hormones have actions throughout the body, regulating reproductive physiology and metabolism. They also feedback to the brain and pituitary to regulate their own synthesis. In this chapter, we review the evolution, structure, and mechanisms of action of the pituitary glycoprotein hormones. We also describe mechanisms regulating their synthesis. Finally, we highlight some gaps in knowledge that require future investigation.

Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin β-subunits. Homodimers or heterodimers of the inhibin β-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/threonine kinases to increase transcription of the FSHβ subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFβ type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation.

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.

Extracellular signal-regulated kinase (ERK) signaling regulates hormone action in the reproductive axis, but specific mechanisms have yet to be completely elucidated. In the current study, ERK1 null and ERK2 floxed mice were combined with a gonadotropin-releasing hormone receptor (GnRHR)–internal ribosomal entry site-Cre (GRIC) driver. Female ERK double-knockout (ERKdko) animals were hypogonadotropic, resulting in anovulation and complete infertility. Transcript levels of four gonadotrope-specific genes (GnRHR and the three gonadotropin subunits) were reduced in pituitaries at estrus in ERKdko females, and the postcastration response to endogenous GnRH hyperstimulation was blunted. As females aged, they exhibited abnormal ovarian histology, as well as increased body weight. ERKdko males were initially less affected, showing moderate subfertility, up to 6 months of age. Male ERKdko mice also displayed a blunted response to endogenous GnRH following castration. By 12 months of age, ERKdko males had reduced testicular weights and sperm production. By 18 months of age, the ERKdko males displayed reduced testis and seminal vesicle weights, marked seminiferous tubule degeneration, and a 77% reduction in sperm production relative to controls. As the GRIC is also active in the male germ line, we examined the specific role of ERK loss in the testes using the stimulated by retinoic acid 8 (Stra8)-Cre driver. Whereas ERK loss in GRIC and Stra8 males resulted in comparable losses in sperm production, seminiferous tubule histological degeneration was only observed in the GRIC-ERKdko animals. Our data suggest that loss of ERK signaling and hypogonadotropism within the reproductive axis impacts fertility and gonadal aging.

Postmenopausal osteoporosis has been attributed to decreased estradiol levels. In the hypothalamic-pituitary-gonadal axis, estradiol synthesis is stimulated by follicle-stimulating hormone (FSH). FSH is secreted from the anterior pituitary gland and estradiol feeds back to the hypothalamus and pituitary to suppress FSH production. In postmenopausal women, the loss of estradiol (and inhibin) negative feedback leads to elevated serum FSH levels. It was recently proposed that this increase in FSH also contributes to postmenopausal osteoporosis by stimulating differentiation and activation of bone-resorbing osteoclast cells. Our objectives were to determine whether FSH has direct actions on osteoclast differentiation in vitro and, if so, its mechanism(s) of action. First, a murine leukemic monocyte/macrophage cell line, RAW264.7, was differentiated into osteoclasts by treatment with receptor activator of nuclear factor kappa-B ligand (RANKL, 50 ng/mL) for seven days. As expected, we observed the appearance of osteoclasts, characterized as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. RANKL also induced expression of established osteoclast differentiation markers, including Trap, cathepsin K (Ctsk), and matrix metalloproteinase-9 (Mmp9). The mRNA expression of FSH receptor (Fshr), however, was low to undetectable both before and after osteoclast differentiation. Co-treatment of RAW264.7 cells with FSH (35, 70 and 140 IU/L) and RANKL did not further impact the expression of Rank, Trap, Ctsk, Mmp-9, or Fshr. Second, primary murine monocytes were differentiated into osteoclasts by treatment with RANKL (50 ng/mL) and macrophage colony-stimulating factor (M-CSF, 25 ng/mL) for five days. FSH co-treatment (70 and 140 IU/L) had no impact on the expression of osteoclast markers, and Fshr expression was low to undetectable both before and after osteoclast differentiation, consistent with the results from RAW264.7 cells. In conclusion, in our hands, FSH does not impact RANKL-induced osteoclast differentiation in immortalized or primary murine monocytes.

The pituitary gland regulates key physiological functions, including growth, sexual maturation, reproduction, and lactation. Here, we present a paired single-nuclei (sn) transcriptome and chromatin accessibility characterization of six post-mortem human pituitaries. These samples were from juvenile, adult, and elderly male and female subjects. Well-correlated snRNAseq and snATACseq datasets facilitated robust identification of the major pituitary cell types in each sample. Using latent variable pathway analysis, we uncovered previously unreported coordinated gene expression modules and chromatin accessibility programs for each major cell type as well as an age-specific program across all the endocrine cell types. These largely appear to be congruent between human and mouse datasets. Given the importance of murine models in the study of human pituitary disorders and pituitary physiology, we next sought to compare expression profiles of pituitary cell types in mouse vs. human. Murine and human cell types were well correlated, exemplified by coordinated gene expression programs, especially for undifferentiated stem cells (SCs). In both species, we identified clusters corresponding to naive and committing SCs. All human SC clusters expressed the established SC markers SOX2 and SOX9, as well as genes involved in SC regulatory pathways (WWTR1, YAP1 andPITX2). Additional markers previously reported in murine pituitary SCs were also found in human SC, including WIF1, LGR5, FOS, CDH1, EGFR, LGR4, and WLS. Remarkably, in human, the main naive SC cluster was roughly divided into a high-JUN and a low-JUN expressing subgroup, whereas Jun expression was less pronounced in the murine SC cluster. In both species, committing SC clusters expressed the endocrine markers for POU1F1, TSHB, or POMC, while SCs committing to an intermediate lobe/melanotrope cell identity were distinguishable based on PAX7 expression. In addition, in the human datasets we identify a population of cells as originating from the pars tuberalis. We offer a range of markers that can be utilized for in vivo validation of these cells. Overall, the characterization of the murine and human pituitary SCs strongly suggests the co-existence of subpopulations with different lineage commitments in addition to a single uncommitted SC population. This sn atlas of the human pituitary is a valuable resource that will be made web-accessible.