| Successful antral formation in vitro from bovine preantral follicles (145-170μm) has been described previously, but antrum formation from the primary follicle (50-70μm) has not yet been achieved in vitro. In order to establish the optimal bovine primary follicle culture system in vitro and investigate the related mechanisms. Primary follicles (50-70μm) were dissected from bovine ovaries, and cultured in a three-dimensional culture system for13or21days in alpha-minimum essential medium (a-MEM). Various treatments including follicle stimulating hormone (FSH), luteinizing hormone (LH),17β-estradiol (E2), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were tested. The follicular diameter and antrum formation rate were recorded, and follicular maturation markers (P450aromatase, CYP19A1; anti-Mullerian hormone, AMH; growth differentiation factor-9, GDF-9; bone morphogenetic protein-15, BMP-15; and type III transforming growth factor P receptor, TGFβR3) were analyzed by Real-Time RT-PCR. After21days of culture under each treatment condition, the follicular diameter was significantly enlarged in the presence of FSH+LH+E2+bFGF or FSH+LH+E2+bFGF+EGF (p<0.05). An addition of50ng/mL bFGF or bFGF+25ng/mL EGF initiated antrum formation by day19and day17of culture, and the antral cavity formation rate was16.7%and33.3%by21days of culture, respectively. The expression of follicular maturation markers (CYP19A1, AMH, GDF-9, BMP-15and TGFPR3) was significantly altered. We conclude that addition of50ng/mL bFGF+25ng/mL EGF to media containing FSH+LH+E2turned out to be the most effective optimized culture conditions to support the growth and maturation of bovine primary follicles in vitro.MicroRNAs (miRNAs) are a class of small (20-25nt) non-coding RNAs which function in gene post-transcriptional regulation with important roles in cell proliferation, differentiation and apoptosis. Based on our previous bovine primary follicle culture study, we observed that expression of miR-27a was abundant in bovine ovary. We investigated the molecular function of miR-27a in mouse granulosa cell. Our results showed that expression of miR-27a in mouse preantral follicles granulose cell was higher than the expression in antral follicles granulose cell. After transfected miR-27a mimics and its inhibitor into mouse granulosa cells, we found that miR-27a played a important role in suppressing granulosa cells proliferation, promoting granulosa cell apoptosis and inhibiting estrogen secretion, and these effects were mainly by targeting transcriptional factor CREB1. |
