Steroidogenic Factor 1 Regulates Transcription of the Inhibin B Coreceptor in Pituitary Gonadotrope Cells

Abstract

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.

Keywords: cell line; inhibin; knockout mouse; pituitary; receptor; transcription.

Figure 1.

SF-1 activates murine Tgfbr3l transcription via two cis-elements in the proximal promoter. (A) Alignment of the murine and human Tgfbr3l/TGFBR3L promoters. In both cases, +1 refers to the transcription start site. The most common murine 5′ untranslated region (5′ UTR) is boxed in blue and the human 5′ UTR is boxed in green. The conserved SF-1 binding sites are boxed in red and labeled as site 1 and site 2. Mutated base pairs (in B, C, and D) are indicated above in green. (B) DNAP using probes corresponding to the wild-type and mutant murine SF-1 cis-elements. Whole cell protein lysates from LβT2 cells (input) or proteins interacting with the probes were analyzed via immunoblot (IB) using an SF-1 antibody. (C) Chromatin immunoprecipitation for SF-1 of the indicated genomic regions in LβT2 cells (n = 4). (D) HEK293T cells were transfected with 225 ng/well of the indicated murine −999/+1 Tgfbr3l-luc reporters as well as 3.125 ng/well of either pcDNA3.0 (empty expression vector) or SF-1 expression vector. WT, wild-type; site 1 mut, mutated SF-1 site 1; site 2 mut, mutated SF-1 site 2; double mut, both SF-1 sites mutated. (E) LβT2 cells were transfected with 225 ng/well of the indicated promoter–reporters. (F) LβT2 cells were transfected with 225 ng/well of the −999/+1 Tgfbr3l-luc reporter and 10 nM of control or Nr5a1 siRNA. In D-F, lysates were collected and reporter activity measured by luciferase assay. Data represent the mean of three or more independent experiments performed in triplicate. Data were analyzed by two-way ANOVA followed by Holm–Sidak multiple comparisons test in (D), one-way ANOVA followed by Dunnett’s multiple comparisons test in (E), and two-tailed unpaired t test with Welch’s correction in (C and F). ns, not significant; *P < .05; ***P < .001; ****P < .0001.

Figure 2.

Mechanisms of human TGFBR3L transcriptional regulation by SF-1 are conserved. (A) DNAP was performed as in Fig. 1B, but with probes corresponding to the wild-type and mutant human SF-1 cis-elements. (B) HEK293T cells were transfected with 225 ng/well of the indicated human −996/+1 TGFBR3L-luc reporters as well as 3.125 ng/well of either pcDNA3.0 (empty expression vector) or SF-1 expression vector. WT, wild-type; site 1 mut, mutated SF-1 site 1; site 2 mut, mutated SF-1 site 2; double mut, both sites mutated. (C) LβT2 cells were transfected with 225 ng/well of the indicated promoter–reporters. (D) LβT2 cells were transfected with 225 ng/well of the −996/+1 TGFBR3L-luc reporter and 10 nM of control or Nr5a1 siRNA. In B-D, luciferase assays were performed as in Fig. 1. Data represent the mean of three independent experiments performed in triplicate and were analyzed by two-way analysis of variance followed by Holm–Sidak multiple comparisons test (B), one-way ANOVA followed by Dunnett’s multiple comparisons test (C), or by a two-tailed unpaired t test with Welch’s correction (D). ns, not significant; **P < .01; ***P < .001; ****P < .0001.

Figure 3.

Gonadotrope-specific SF-1 knockout mice exhibit hypogonadotropic hypogonadism. (A) Genomic DNA was extracted from the indicated tissues of Nr5a1^fl/fl; Gnrhr^GRIC/+ (cKO; left panels) and Nr5a1^fl/fl (control; right panels) mice and analyzed by PCR for the presence of the floxed or recombined (rec) Nr5a1 alleles. (B) Pituitary sections from 11-week-old control and cKO mice were analyzed for SF-1 by immunofluorescence (red); DAPI (blue) was used to stain nuclei. Scale bars: 50 µm. (C) cDNA was prepared from total RNA isolated from individual pituitary glands of 8- to 10-week-old control and cKO female (top) and male (bottom) mice and analyzed by RT-qPCR for expression of Lhb, Fshb, Cga, Gnrhr, and Nr5a1. (D) Serum LH and FSH levels in female (top) and male (bottom) control and cKO mice. (E) Ovarian, uterine, testicular, and seminal vesicle weights and (F) representative images of gonads and accessory sex organs from control and cKO females (top) and males (bottom). Scale bars: 5 mm. Female data in all panels represent randomly cycling females. Data were analyzed by two-tailed unpaired t tests with Welch’s correlation. ns, not significant; *P < .05; **P < .01; ***P < .001; ****P < .0001.

Figure 4.

SF-1 is required for Tgfbr3l expression in adult murine pituitary glands. (A) Pituitary cDNA from control and cKO female and male mice (described in Fig. 3) were analyzed for Tgfbr3l expression by RT-qPCR. Data were analyzed by two-tailed unpaired t tests with Welch’s correlation. ***P < .001. Chromatin accessibility, as measured with single-nucleus ATAC-seq, over the (B) Tgfbr3l and (C) Cga genes in gonadotropes of 10- to 12-week-old control (orange) and cKO (grey) males. Exon 1 and the promoter of Tgfbr3l are boxed in red in B. Shown are representative tracks from a control and a cKO animal.

Figure 5.

Nr5a1 and Tgfbr3l expression in fetal and adult murine pituitaries. (A) mRNA in situ hybridization (RNAscope) for Tgfbr3l (blue) and Nr5a1 (red) on heads of wild-type CD1 embryos at the indicated ages. Boxed regions in the top panels are magnified in the bottom panels. Scale bars: 50 μm. Duplex RNAscope for Tgfbr3l (blue) and (B) Lhb or (C) Fshb (red) on heads of CD1 embryos at E18.5. Scale bars: 200 μm (top) and 50 μm (bottom). Duplex RNAscope for Tgfbr3l (blue) and (D) Nr5a1, (E) Fshb, or (F) Lhb (red) on pituitaries of 8-week-old CD1 males. Scale bars: 50 μm.

Figure 6.

TGFBR3L and NR5A1 expression is enriched in gonadotropes of human fetal pituitaries. (A) UMAP plots of scRNAseq of 21 human embryonic pituitaries collected 7-25 weeks post-fertilization. Different colored clusters represent different pituitary cell types. (B) Feature plots of TGFBR3L and NR5A1 expression. (C) Violin plots of TGFBR3L and NR5A1 expression in the defined cell lineages.