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.

No abstract

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

The activins were discovered and named based on their abilities to stimulate FSH secretion and FSHβ (Fshb) subunit expression by pituitary gonadotrope cells. According to subsequent in vitro observations, activins also stimulate the transcription of the GnRH receptor (Gnrhr) and the activin antagonist, follistatin (Fst). Thus, not only do activins stimulate FSH directly, they have the potential to regulate both FSH and LH indirectly by modulating gonadotrope sensitivity to hypothalamic GnRH. Moreover, activins may negatively regulate their own actions by stimulating the production of one of their principal antagonists. Here, we describe our current understanding of the mechanisms through which activins regulate Fshb, Gnrhr, and Fst transcription in vitro. The activin signaling molecules SMAD3 and SMAD4 appear to partner with the winged-helix/forkhead transcription factor, forkhead box L2 (FOXL2), to regulate expression of all 3 genes. However, in vivo data paint a different picture. Although conditional deletion of Foxl2 and/or Smad4 in murine gonadotropes produces impairments in FSH synthesis and secretion as well as in pituitary Fst expression, Gnrhr mRNA levels are either unperturbed or increased in these animals. Surprisingly, gonadotrope-specific deletion of Smad3 alone or with Smad2 does not impair FSH production or fertility; however, mice harboring these mutations may express a DNA binding-deficient, but otherwise functional, SMAD3 protein. Collectively, the available data firmly establish roles for FOXL2 and SMAD4 in Fshb and Fst expression in gonadotrope cells, whereas SMAD3's role requires further investigation. Gnrhr expression, in contrast, appears to be FOXL2, SMAD4, and, perhaps, activin independent in vivo.

Activin ligands are formed from two disulfide-linked inhibin β (Inhβ) 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 functions 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 murine 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.

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.

Discovery of a novel pituitary protein suggests a new means to enhance fertility.

A sex steroid-dependent modulation of the immune function in mammals is accepted, and evidence suggests that while estrogens enhance, androgens inhibit the immune response. The aim of this study was to explore in the adult male rat the effect of either neonatal flutamide (FTM) treatment or prepubertal orchidectomy (ODX) on endocrine markers in the basal condition and peripheral tumor necrosis factor alpha (TNFα) levels during inflammatory stress. For these purposes, (1) 5-day-old male rats were subcutaneously injected with either sterile vehicle alone or containing 1.75 mg FTM, and (2) 25-day-old male rats were sham operated or had ODX. Rats were sacrificed (at 100 days of age) in the basal condition for determination of peripheral metabolite levels. Additional rats were intravenously injected with bacterial lipopolysaccharide (LPS; 25 µg/kg body weight, i.v.) and bled for up to 4 h. Data indicate that (1) ODX increased peripheral glucocorticoid levels and reduced those of testosterone, whereas FTM-treated rats displayed low circulating leptin concentrations, and (2) LPS-induced TNFα secretion in plasma was significantly enhanced in the FTM and ODX groups. Our study supports that neonatal FTM treatment affected adiposity function, and adds data maintaining that androgens have a suppressive role in proinflammatory cytokine release in plasma during inflammation.