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.
ABSTRACT
Aim To study the effect of forskolin on cor-ticosterone secretion in mouse adrenocortical tumor cells. Methods Y1 cells were treated with 1μmol· L-1 or 10μmol·L-1 forskolin for 15 min to 24 h. Y1 cells growth morphology was observed, cell culture su-pernatants were collected and corticosterone was tested by ELISA kit. The cells total RNA was extracted using TRIzol kit and was reversely transcribed to obtain the cDNA, then was amplificated by real time quantitative PCR. The cells were lysed with RIPA lysis buffer and protein expressions were carried out by Western blot. Results Y1 cells morphology changed from flat adher-ent to shrink and spherical growth after 1μmol · L-1 forskolin treatment for 3 h. Compared with the control group, corticosterone levels were increased significantly by 1μmol · L-1 forskolin treatment for 24 h ( P <0. 01), then, forskolin significantly enhanced steroid ogenic acute regulatory protein ( Star) mRNA and pro-tein expression ( P <0. 01 ) , moreover, the steroido-genic enzymes such as Cyp11 a1 and Cyp11 b1 mRNA expressions were also up-regulated significantly by fors-kolin ( P <0. 01 ) . Additionally, Star mRNA expres-sion was increased significantly in a time-dependent re-sponse after 10μmol·L-1 forskolin treatment from 1 h to 12 h (P<0. 01). Furthermore, Nr4a1 and Nr4a2 mRNA expressions were up-regulated significantly after 10μmol · L-1 forskolin treatment from 15 min and reached the top at 1 h ( P<0. 01 ) . However, forsko-lin showed no effect on Mc2 r and Nr5 a1 mRNA expres-sions. Conclusion Y1 mouse adrenocortical tumor cells have a strong response to adenylate cyclase ago-nists, then, forskolin can be used to glucocorticoid se-cretion inducer reagent.