Ovarian Follicle Development Requires Smad3

Abstract

Smad3 is an important mediator of the TGF beta signaling pathway. Interestingly, Smad3-deficient (Smad3-/-) mice have reduced fertility compared with wild-type (WT) mice. To better understand the molecular mechanisms underlying the reduced fertility in Smad3-/- animals, this work tested the hypothesis that Smad3 deficiency interferes with three critical aspects of folliculogenesis: growth, atresia, and differentiation. Growth was assessed by comparing the size of follicles, expression of proliferating cell nuclear antigen, and expression of cell cycle genes in Smad3-/- and WT mice. Atresia was assessed by comparing the incidence of atresia and expression of bcl-2 genes involved in cell death and cell survival in Smad3-/- and WT mice. Differentiation was assessed by comparing the expression of FSH receptor (FSHR), estrogen receptor (ER) alpha, ER beta, and inhibin alpha-, beta(A)-, and beta(B)-subunits in Smad3-/- and WT mice. Because growth, atresia, and differentiation are regulated by hormones, estradiol, FSH, and LH levels were compared in Smad3-/- and WT mice. Moreover, because alterations in folliculogenesis can affect the ability of mice to ovulate, the number of corpora lutea and ovulated eggs in response to gonadotropin treatments were compared in Smad3-/- and WT animals. The results indicate that Smad3 deficiency slows follicle growth, which is characterized by small follicle diameters, low levels of proliferating cell nuclear antigen, and low expression of cell cycle genes (cyclin-dependent kinase 4 and cyclin D2). Smad3 deficiency also causes atretic follicles, degenerated oocytes, and low expression of bcl-2. Furthermore, Smad3 deficiency affects follicular differentiation as evidenced by decreased expression of ER beta, increased expression of ER alpha, and decreased expression of inhibin alpha-subunits. Smad3 deficiency causes low estradiol and high FSH levels. Finally, Smad3-/- ovaries have no corpora lutea, and they do not ovulate after ovulatory induction with exogenous gonadotropins. Collectively, these data provide the first evidence that reduced fertility in Smad3-/- mice is due to impaired folliculogenesis, associated with altered expression of genes that control cell cycle progression, cell survival, and cell differentiation. The findings that Smad3-/- follicles have impaired growth, increased atresia, and altered differentiation in the presence of high FSH levels, normal expression of FSHR, and lower expression of cyclin D2, suggest a possible interaction between Smad3 and FSH signaling downstream of FSHR in the mouse ovary.

TGF-β regulates the mechanical properties and composition of bone matrix

“…Smad3Ϫ͞Ϫ mice, which are defective in the expression of Smad3 in all tissues, also have a bone phenotype with an osteopenic reduction in bone volume (14,17). In addition to the direct role of TGF-␤͞Smad3 signaling in bone metabolism, some aspects of the Smad3Ϫ͞Ϫ bone phenotype may be due to immune, gastrointestinal, or endocrine defects associated with systemic loss of Smad3 expression (24)(25)(26). No gross abnormalities have been reported in Smad3ϩ͞Ϫ mice.…”

“…The similarly increased bone mass, mineral concentration, and mechanical properties in DNT␤RII and Smad3ϩ͞Ϫ bones stand in contrast to the phenotypes of D4 and D5 bones with increased TGF-␤ signaling ( Table 1). The Smad3Ϫ͞Ϫ bone phenotype likely combines bone-specific, i.e., increased osteoblast apoptosis (14), and progressive secondary effects of systemic Smad3 absence on the bone metabolism and health of the mice as mentioned above (24)(25)(26).…”

“…We hypothesized that this discrepancy was a progressive secondary effect on bone resulting from systemic loss of Smad3. Smad3Ϫ͞Ϫ mice exhibit immune, endocrine, and gastrointestinal defects (24)(25)(26), any of which might affect bone metabolism. To test this hypothesis, XTM was used to evaluate the mineral concentration of 1-day-old bones when the systemic effects of Smad3 deficiency should as yet be minimal.…”

Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality

“…As the SMAD signal transduction proteins are known to mediate signaling by members of the TGF-β superfamily, which is involved in the control of normal folliculogenesis and ovulation [7,8], and they associate with members of the Forkhead family of transcription factors, the FOXO class [34], thought to play important roles in oocyte maturation, ovulation, and possibly luteinization [34][35][36][37][38][39], with expression in human granulosa cells [40], it is possible that SMAD expression levels may also affect ovarian folliculogenesis and fertility. Female Smad3 −/− mice exhibit impaired folliculogenesis and reduced fertility [28,30] and Smad4 ovarian-specific knockout mice exhibit multiple defects in folliculogenesis and decreased fertility over time [27]. Female mice in which dominant negative Smad2 is expressed in granulosa cells exhibit impaired folliculogenesis and the females are subfertile [26].…”

“…In addition, ovarian-and granulosaspecific Smad knockout and transgenic mouse models exhibit a variety of defects in folliculogenesis, coupled with reduced fertility [26][27][28][29][30]. In humans, transcripts for SMAD2, 3 and 4 have been identified in granulosa cells collected at oocyte retrieval [31], which suggested that these and other SMADs might also be involved in human ovarian function and fertility.…”