Transforming growth factor-β1 mediates the SMAD4/BMF pathway to regulate ovarian granulosa cell apoptosis in small tail Han sheep

Abstract

Transforming growth factor (TGF)-β1 is an important multifunctional cytokine in the TGF-β signaling pathway, which is involved in the molecular regulation of multiple activities, including follicle development and ovulation in female reproductive physiology. However, the biological function of TGF-β1 in follicular development and in regulating the proliferation or apoptosis of granulosa cells in small tail Han sheep remain unclear. In this study, we analyzed the expression levels of TGF-β1 in the ovary at the follicular stage in small tail Han sheep. We further examined the effects of TGF-β1 on the viability, proliferation, and apoptosis of granulosa cells. Differential expression of TGF-β1 at the mRNA and protein levels was detected in the ovaries between the beginning of estrus and at preovulation. Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine (EdU) labeling, and flow cytometry assays showed that adding 5 and 10 ng/mL TGF-β1 could improve the viability and growth rate, reduce the apoptosis rate, and reduce the expression level of the pro-apoptotic factor Bcl-2-modifying factor (BMF) in granulosa cells. Treatment of 10 ng/mL TGF-β1 at all time points (except 72 h) significantly increased the positive rate of EdU labeling compared to that of the control group. RNA interference of SMAD4 reversed the decreased apoptosis rate caused by stimulation with 10 ng/mL TGF-β1, accompanied by a corresponding increase in the BMF expression level. Collectively, these results indicate that TGF-β1 plays a role in the ovarian follicular-phase activity of small tail Han sheep by inhibiting the apoptosis of sheep granulosa cells through the SMAD4/BMF pathway to promote proliferation and vitality. This study provides new insight into the molecular mechanism underlying TGF-β1 function regulation in granulosa cells, suggests a new target for the regulation of follicle development, and expands the new field of animal reproduction regulation technology.