miR-21, miR-125b, and let-7b contribute to the involution of atretic follicles and corpus lutea in Tibetan sheep ovaries.

Follicular granulosa cells (FGCs) are crucial for ovarian follicle functions, and miRNAs are differentially expressed at various stages of follicular developments. In this study, we confirmed that miR-21, miR-125b, and let-7b were located in FGCs/luteal cells by in situ hybridization experiments. Moreover, miR-21 and miR-125b expressions were upregulated in late corpus lutea (CL) and atretic follicles (AF); let-7b expression was increased in early AF. After transfected with inhibitor or mimic of miRNAs in FGCs, we found that FGCs apoptosis was decreased in the miR-21-mi group but increased in the miR-125b-mi group using flow cytometry. mRNA and protein expression levels were determined for apoptosis-related factors (e.g., Bcl-2 and Bax), the potential target genes of miRNAs (e.g., SMAD7, SP1, and STAT3), hormone receptors (e.g., FSHR and LHR), and genes related to hormone secretion (e.g., CYP19, CYP11, and 3βHSD). The protein levels of SMAD7 were decreased in the miR-21-mi group but opposite to SP1 and FSHR. In the let-7b-mi group, Bcl-2, SMAD7, and FSHR were suppressed but not Bax, CYP11, and 3βHSD. However, hormone secretion was not changed in the supernatant of transfected FGCs. This study provides information about ovarian miRNAs to improve the fertility in Tibetan sheep.