Selective Smad4 knockout in ovarian preovulatory follicles results in multiple defects in ovulation

The TGF-β signaling pathway is involved with multiple processes in the mammalian ovary, including primordial follicle formation, granulosa cell (GC) proliferation, follicle atresia, ovulation, and feedback regulation between the pituitary and ovary. The transcriptional factor SMAD4 (Sma- and Mad-related protein 4) is the central component of the canonical TGF-β signaling pathway. Smad4 knockout (KO) using Amhr2-Cre, which is expressed in GCs of immature developing follicles, causes premature luteinization. In this study, we specifically depleted Smad4 in GCs of preovulatory follicles using Cyp19-Cre mice. As different from results with Smad4(fl/fl);Amhr2-Cre mice, Smad4 depletion in preovulatory follicles did not cause premature luteinization or suppress GC proliferation; rather, it increased follicle atresia. In addition, Nppc and Npr2 expressions were reduced by Smad4 depletion; thus, their effect of maintaining oocyte meiotic arrest was weakened in Smad4 conditional KO mice. Smad4(fl/fl);Cyp19-Cre female mice were subfertile and had irregular estrous cycles and ovulation defects. Smad4 KO also blocked LH-induced cumulus expansion and follicle rupture, but not oocyte meiotic resumption. Our results also indicated that SMAD4 was required for LH-stimulated activation of ERK1/2 and the expressions of ovulation-related genes. The defects arising from SMAD4 depletion could not be rescued by intraovarian mediators of LH actions, such as epidermal growth factor-like factors and prostaglandin E2. Furthermore, corpus lutea did not form in Smad4(fl/fl);Cyp19-Cre female mice, indicating that SMAD4 was crucial for GCs terminal differentiation. Thus, by characterizing the ovarian phenotypes of preovulatory follicle-specific Smad4 KO mice, we identified the developmental stage-specific functions of the canonical TGF-β signaling pathway in ovulation and luteinization.