SMAD3 Regulates Follicle-stimulating Hormone Synthesis by Pituitary Gonadotrope Cells in Vivo

Abstract

Pituitary follicle-stimulating hormone (FSH) is an essential regulator of fertility in females and of quantitatively normal spermatogenesis in males. Pituitary-derived activins are thought to act as major stimulators of FSH synthesis by gonadotrope cells. In vitro, activins signal via SMAD3, SMAD4, and forkhead box L2 (FOXL2) to regulate transcription of the FSHβ subunit gene (Fshb). Consistent with this model, gonadotrope-specific Smad4 or Foxl2 knock-out mice have greatly reduced FSH and are subfertile. The role of SMAD3 in vivo is unresolved; however, residual FSH production in Smad4 conditional knock-out mice may derive from partial compensation by SMAD3 and its ability to bind DNA in the absence of SMAD4. To test this hypothesis and determine the role of SMAD3 in FSH biosynthesis, we generated mice lacking both the SMAD3 DNA binding domain and SMAD4 specifically in gonadotropes. Conditional knock-out females were hypogonadal, acyclic, and sterile and had thread-like uteri; their ovaries lacked antral follicles and corpora lutea. Knock-out males were fertile but had reduced testis weights and epididymal sperm counts. These phenotypes were consistent with those of Fshb knock-out mice. Indeed, pituitary Fshb mRNA levels were nearly undetectable in both male and female knock-outs. In contrast, gonadotropin-releasing hormone receptor mRNA levels were significantly elevated in knock-outs in both sexes. Interestingly, luteinizing hormone production was altered in a sex-specific fashion. Overall, our analyses demonstrate that SMAD3 is required for FSH synthesis in vivo.

Keywords: SMAD transcription factor; activin; follicle-stimulating hormone (FSH); gene knock-out; pituitary gland.

FIGURE 1.

Models of SMAD signaling to the Fshb promoter in murine gonadotrope cells of wild-type and Smad4 knock-out mice. Top panel, in wild-type mice, activins stimulate the formation of complexes of SMAD proteins and FOXL2, which act via at least three cis-elements in the proximal Fshb promoter. SMADs can bind DNA via SBEs, and FOXL2 binds via FBEs. FOXL2 binds the distal FBE1 site and recruits SMAD3 via protein-protein interaction. Complexes of SMAD3 and SMAD4 can bind SBE1, which contains binding sites for both proteins. More proximally, SMAD4 binds SBE2, FOXL2 binds FBE2, and the two proteins are linked through their mutual association with SMAD3. Bottom panel, in mice lacking SMAD4 in their gonadotropes (cKO), SMAD3/SMAD4 binding to SBE1 is lost. However, SMAD3/FOXL2 binding to FBE1 is intact. In the absence of SMAD4, SMAD3 can bind SBE2, enabling activins to stimulate Fshb production, although at reduced levels relative to wild type. MH1, DNA binding domain; MH2, protein-protein interaction domain.

FIGURE 2.

SW480.7 cells were transfected with the −1990/+1 murine Fshb-luc reporter and different combinations of FOXL2, SMAD3, SMAD3-MH2, and/or SMAD4. Cells were co-transfected with a constitutively active activin type I receptor (ALK4-TD, red bars) or empty expression plasmid (pcDNA3.0, black bars). Bars represent the means (±S.E.) of three independent experiments. Reporter activity was normalized to that of cells transfected with empty expression plasmids (first set of bars at left). Two-way analyses of variance were conducted to compare SMAD3/FOXL2 versus SMAD3-MH2/FOXL2 and SMAD3/SMAD4/FOXL2 versus SMAD3-MH2/SMAD4/FOXL2, followed by Bonferroni-corrected multiple comparison tests. Bars with different letters are significantly different from one another, whereas those without letters were not subjected to statistical analysis.

FIGURE 3.

Recombination of Smad3 and Smad4 alleles in mouse gonadotropes. A, schematic representation of the floxed Smad3 and Smad4 alleles pre- and postrecombination with Cre. Exons 2 and 3 of Smad3 and exon 8 of Smad4 were flanked with loxP sites (light gray triangles). Exons are shown as dark gray boxes. B, PCR detection of floxed and recombined Smad3 and Smad4 alleles from the indicated tissues of control and cKO mice. C–E, RT-qPCR analysis of mRNA levels of Smad3 and Smad4 in purified gonadotropes (YFP+) and non-gonadotropes (YFP−) from adult control (YFP/+;GRIC) and cKO (Smad3^fx/fx;Smad4^fx/fx;YFP/+;GRIC) mice. In C and E, primers were directed against the deleted region of the genes/transcripts. In D, primers were directed against non-targeted exons in Smad3.

FIGURE 4.

cKO males and females are FSH deficient. A, B, E, and F, FSH (A and B) and LH (E and F) levels in mouse serum were measured by multiplex ELISA. Individual data points are shown (n = 12 or 14 for control and cKO, respectively) as are the group means (horizontal lines). C and D, pituitary FSH content was assessed by RIA (n = 8 or 5 for control and cKO males; n = 11 or 7 for control and cKO females). All samples from cKO females were below the detection limit of the assay. G and H, pituitary LH content was measured by ELISA. The data were analyzed by Student's t test in each panel.

FIGURE 5.

FSH protein expression is greatly diminished in cKO pituitaries. Immunofluorescence staining for LHβ (green) and FSHβ (red) in pituitaries of adult control and cKO males (left panels) and female (right panels) mice. The nuclei are labeled with DAPI (blue). Scale bars, 100 μm.

FIGURE 6.

Fshb expression is abolished in pituitaries of cKO mice. Pituitaries were collected from 10-week-old control and cKO mice (n = 11 or 15 for control and cKO males. A, (n = 12) genotype for females; B, mRNA levels of gonadotropin subunits (Fshb, Lhb, and Cga), GnRH receptor (Gnrhr), Smad3, and Smad4 were measured by RT-qPCR. mRNA levels were normalized to the housekeeping gene Rpl19. Bars reflect group means (±S.E.). The data were analyzed by Student's t test. ***, p < 0.0001; **, p = 0.0004. n.s., not statistically significant.

FIGURE 7.

Fertility is impaired in cKO mice. Control and cKO males (n = 6 or 4) and females (n = 4) were paired with wild-type C57BL6 mice for 6 or 4 months, respectively. A and B, average number of litters (A) and litter size (B) in control and cKO males. Different colors are used to indicate data from individual animals. C, average litter size in control and cKO females.

FIGURE 8.

cKO males are oligospermic. A, testes and seminal vesicles from adult control and cKO males. B, testicular weights of 10-week-old control and cKO males (n = 13/genotype). C, sperm counts from snap frozen epididymides of 10-week-old control and cKO males (n = 9/genotype). D, sperm motility assessment from fresh cauda epididymides in adult control and cKO males (n = 8 or 6 for control and cKO, respectively). E, seminal vesicle weights of 10-week-old control and cKO males (n = 13/genotype). F, H&E staining of testicular sections from representative control and cKO males. Scale bars, 100 μm.

FIGURE 9.

cKO females do not ovulate. A, morphology of ovaries and uteri from adult control and cKO females. B and C, ovary and uterus weights from 10-week-old control and cKO females (n = 12/genotype). D, day of vaginal opening in control and cKO females (n = 6/genotype). E, H&E-stained ovaries from representative 10-week-old control and cKO females. Corpora lutea (CL) are labeled. Scale bars, 100 μm. F, COCs collected after exogenous gonadotropin stimulation in juvenile control and cKO females (n = 18 or 7 for control and cKO, respectively).

FIGURE 10.

Basal and activin A-stimulated Fshb expression were abolished in cKO pituitary cells. Primary pituitary cells were prepared from adult male (A) or female (B) control or cKO mice and treated with 1 nm activin A (red) or with vehicle (black). Fshb mRNA levels were measured by RT-qPCR. Bars represent the means (+ S.E.) of three independent experiments. Bars with different symbols (a, b, c, and d) differ significantly.