Effect Of Protooncogene C-myb On Progesterone-Induced Mouse Germinal Vesicle Stage Oocyte Maturation In Vitro

Objectives: The experiment was carried out to identify the effect of various concentrations of progesterone and antisense c-myb ODN on maturation of germinal vesicle stage oocyte in vitro. We used mouse germinal vesicle stage oocyte cultured with special concentration progesterone, or/and antisense c-myb ODN, or/and dbcAMP, or/and Verapamil, or/and heparin for 24h, and observed oocyte maturation and analysed the relationship between them. It may provide some new knowledge about ovarian function and regulation mechanisms, and provide new methods to promote fertility and antifertility. Methods: We used exogenous PMSG to induce Kunming mouse superovulate. After 48h, we killed the mouse by decapitation, took out of hibateral ovaries, and lacerate egg by needle under the anatomical microscope. Then we used mouth haustorial tube to blow COC and collected DO. We cultured DO under the Medium 199 with progesterone, or/and antisense c-myb ODN, or/and dbcAMP, or/and Verapamil, or/and heparin for 24h, and observed oocyte maturation in vitro. Results: (1) We cultured DO under the Medium 199 with 0,5,10,20μmol/L progesterone respectively, and found that 5,10,20μmol/L progesterone all significantly promote mouse germinal vesicle stage oocyte maturation in vitro compared with group 0, 10μmol/L progesterone has more significant effect than 5μmol/L progesterone, but has not more significant effect than 20μmol/L progesterone. We analysed time-effect relationship of progesterone-induced oocyte maturation and found: 5,10,20μmol/L progesterone all significantly promote germinal vesicle breakdown after 2h, and the promotion effect was going alone until 24h; 5,10,20μmol/L progesterone all significantly promote first polar body formation after 2h, and the promotion effect was going alone until 24h. (2) We cultured DO under the Medium 199 with 0,4,8,16μmol/L antisense c-myb ODN respectively and found antisense c-myb ODN inhibited mouse germinal vesicle stage oocyte maturation in vitro in a dose-dependent manner. 8,16μmol/L antisense c-myb ODN significantly inhibited mouse germinal vesicle stage oocyte maturation in vitro compared with group 0. We analysed the time-effect relationship of antisense c-myb ODN -inhibited oocyte maturation and found:4,8,16μmol/L antisense c-myb ODN all significantly inhibited germinal vesicle breakdown after 2h, and the effect was going alone until 24h; 8,16μmol/L antisense c-myb ODN significantly inhibited first polar body formation after 16h,8h respectively, and the effect was going alone until 24h (3) We cultured DO under the Medium 199 with 16μmol/L antisense c-myb ODN or/and 10μmol/L progesterone for 24h, and found that 16μmol/L antisense c-myb ODN significantly inhibited progesterone-induced mouse germinal vesicle stage oocyte maturation in vitro. (4) 1×10-5μmol/L Verapamil also significantly inhibited progesterone-induced mouse germinal vesicle stage oocyte maturation in vitro, and enhanced the inhibition of 16μmol/L c-myb ASODN.(5) 1×10-4μmol/L dbcAMP significantly inhibited progesterone-induced mouse germinal vesicle stage oocyte maturation in vitro, and could enhanced the inhibition of 16μmol/L antisense c-myb ODN. (6) 100μg/ml heparin significantly inhibited progesterone-induced mouse germinal vesicle stage oocyte maturation in vitro, and could enhanced the inhibition of 16μmol/L antisense c-myb ODN. Conclusions: Progesterone,protooncogene c-myb,cAMP and calcium all pay important role in regulating oocyte maturation and the mechanism of Progesterone,cAMP and calcium in regulating oocyte maturation may be through the expression of protooncogene c-myb.