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

Yining Li,Gauthier Schang,Ulrich Boehm,Chu-Xia Deng,Jonathan Graff,Daniel J Bernard

doi:10.1074/jbc.m116.759167

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.

Highlights

Pituitary follicle-stimulating hormone (FSH)2 is an essential regulator of gonadal function [1, 2]
Truncated SMAD3 Stimulates FSH␤ subunit gene (Fshb) Transcription in the Presence but Not the Absence of SMAD4 in Vitro—Mice lacking Smad3 exons 2 and 3 express a truncated form of SMAD3, which lacks the DNA binding MH1 domain [36]. We proposed that this protein functions equivalently to full-length SMAD3, but only in the presence of SMAD4, enabling transactivation of the Fshb gene
We reported that Smad3 cKO mice are fertile with normal FSH synthesis [36]

Summary

Results

Truncated SMAD3 Stimulates Fshb Transcription in the Presence but Not the Absence of SMAD4 in Vitro—Mice lacking Smad exons 2 and 3 express a truncated form of SMAD3, which lacks the DNA binding MH1 domain (hereafter SMAD3MH2) [36]. Consistent with the sex difference in LH secretion, pituitary Lhb and Cga mRNA levels were modestly decreased in cKO males but markedly increased in cKO females (Fig. 6, A and B). These RNA differences were not reflected at the level of intrapituitary LH protein content Basal and Activin A-stimulated Fshb Expression Are Reduced in cKO Pituitaries—To more directly assess the effects of the targeted mutations on activin signaling in gonadotropes, we cultured pituitary cells from male and female cKO and control mice. Activin A modestly stimulated Fshb in cultures from male but not female cKOs (Fig. 10, A and B)

Discussion

ND ϭ ϭ

Experimental Procedures

Facility for Flow Cytometry and Single Cell Analysis of the Life