Studies on optimization of two-dimensional culture of preantral follicles in mice in vitro / 解放军医学杂志

ABSTRACT

Objective To optimize the two-dimensional culture system of mouse preantral follicles, and discuss the effect of follicle stimulating hormone (FSH) on the in vitro development of mouse preantral follicles. Methods The primary follicles (PM follicles, 80-100 μm) and early second follicles (ES follicles, 110-130 μm) harvested from mouse ovaries at day 14 were in vitro cultured with different concentrations of r-FSH culture medium (10 mU/ml and 100 mU/ml). The follicles growth in vitro and the oocytes maturation were observed and recorded. The growth model of antral follicles at day 10 and the levels of estradiol (E2) in the different concentrations of culture medium were examined. The expression levels of FSH receptor (FSHR), steroid hormone synthesis rate-limiting enzyme (3β-hydroxyl steroid dehydrogenase, 3β-HSD), 17α-hydroxylase (CYP17) and aromatase (CYP19) in follicles were detected by Western blotting. Results The cavity rates of PM follicles in 10 mU/ml and 100 mU/ml r-FSH media (0.00%±0.00%, 36.14%±4.02%) and oocyte maturation rates (0.00%±0.00%, 23.54%±7.62%) were obviously lower than the cavity rates of ES follicles (78.63%±4.13%, 92.74%±2.54%) and oocyte maturation rates (48.55%±3.73%, 80.88%±4.02%) with statistical significance (P<0.05). The cavity rates of ES follicles in 100 mU/ml r-FSH media (92.74%±2.54%) and oocyte maturation rates (80.88%±4.02%) were obviously higher than the ES follicles in 10 mU/ml r-FSH media (78.63%±4.13%) and oocyte maturation rates (48.55%±3.73%) with statistical significance (P<0.05). ES follicles in 10 mU/ml r-FSH presented a pattern of tiled growth, while in 100 mU/ml r-FSH presented a stereoscopic spatial growth pattern, which was closer to that of the follicles in vivo. The relative expression level of FSHR in ES follicles was obviously higher than that in PM follicles (1.86±0.32 vs. 1.19±0.28, t=4.94, P<0.05). The expressions of 3β-HSD, CYP17 and CYP19, as well as the secretion of E2 in ES follicles cultured with 100 mU/ml r-FSH were significantly higher than in ES follicles cultured with 10 mU/ml r-FSH. Conclusions FSH can not only change the in vitro development rate, but also change the development pattern of preantral follicles in vitro. The ideal follicle development rate and development mode could be obtained by selecting ES follicles cultured in medium containing 100 mU/ml r-FSH.