Study The Effect And Mechanism Of TNFα On Mouse Oocyte Maturation In Vitro

OBJECTIVE:Study the effect and the mechanism of TNFαon the mouse oocyte maturation in vitro.METHODS:Culture mouse denuded oocytes in vitro and observe the rate of GVBD and PB1 with inverted microscope, measure the contents of p-ERK₁,ERK₂ and cyclinB₁ ,c-erbB₂ mRNA and c-myb mRNA by western blot and RT-PCR in-synchronism.RESULTS:1. The GVBD rate of mice DO when culture in medium containing 1ng/ml TNFαfor 6h, 9h,12h and 24h decreased by 14.2%(P<0.05), 15.4% (P<0.05), 14.9%(P<0.05) and 18%(P<0.01) respectively and in medium containing 10ng/ml TNFαdecreased by 16%(P<0.05),16.6%(P<0.05),18.3%(P<0.01) and 21.8%(P<0.01) respectively when compared with control group.After cultured for 12h and 24h in medium containing 1ng/ml TNFα, PB1 rate of DO decreased by 10.8%(P<0.05) and 11.6% (P<0.05),and 10ng/mlTNFαdecreased by 14.3% (P<0.05) and 17.2%(P<0.01) respectively when compared with control group.These means that TNFαcan inhibit the rate of GVBD and PB1 in a dose-dependent and time-dependent manner. P can promote the GVBD and PB1 of DO and can reverse the inhibitory effect of TNFαon DO maturation; E2 alone has no obviously effect on DO,but can also reverses the inhibitory effect of TNFαon DO maturation.2.RT-PCR showed that c-erbB2mRNA and c-mybmRNA were expressed in mouse oocytes and TNFαcan decrease their expression, P and E₂ can reverse the inhibitory effect of TNFαon the two oncogenes.3.Western blot showed that the phosphorylation of ERK₁,ERK₂ and the content of cyclinB₁ protein in oocytes are parallel with the status of oocyte maturation , TNFαcan decrease the the contents of p-ERK₁,ERK₂ and cyclinB1 protein, P and E₂ can reverse the inhibitory effect of TNFαon the phosphorylation of ERK1,ERK2 and the content of cyclinB1 protein in oocytes.CONCLUSIONS:1.TNFαcan inhibit denuded oocyte maturation in vitro in a dose-dependent and time-dependent manner, P and E₂ can reverse inhibitory effect of TNFαon oocyte.2.The mechanism of TNFαregulating oocyte maturion in vitro maybe relate with regulating the expression of proto-oncogene c-erbB₂,c-myb and contents of p-ERK₁, p-ERK₂ and cyclinB1 protein.