The Study On AREG Affecting Glucose Metabolism Of Cumulus Cells And In Vitro Maturation Of Oocytes From Sheep

The purpose of this study is to explore the differences in energy metabolism of follicular cumulus cells and oocytes from different diameters in sheep,as well as the effects of amphiregulin(AREG)and oocyte-secreted factors(OSFs)on it,and then improve quality and embryonic development ability of in vitro maturation(IVM)of sheep oocytes.The findings are as follows:1.During cultured in vitro,the mRNA expression of G6PDH,PFKL and PFKM and the cell proliferation ability of cumulus cells derived from small antral follicles were significantly lower than those of cumulus cells from medium antral follicles(P<0.05).Compared with the group without AREG,50 ng/ml and 100 ng/ml AREG significantly increased the proliferation ability of cumulus cells from small antral follicles(P<0.05),10 ng/ml AREG significantly increased the proliferation ability of cumulus cells from medium antral follicles(P<0.05),but had no significant effect on cumulus cells from small antral follicles(P>0.05);with the synergistic effect of GDF9 and BMP 15,10 ng/mlAREG can significantly increase the proliferation ability and ATP content of cumulus cells from different diameter antral follicles(P<0.05),and significantly increase glucose consumption,lactate production and NADPH production in cumulus cell culture medium(P<0.05).2.The results of in vitro culture of cumulus oocyte complexes(COCs)showed that,before IVM,the mitochondrial membrane potential and FAD++ level of oocytes from small antral follicles were significantly lower than those from medium antral follicles(P<0.05);after IVM,the CEI and MII%of oocytes from small antral follicles were significantly lower than those from medium antral follicles(P<0.05);compared with the control group,AREG+GDF9+BMP15 significantly increased the CEI,MII%,and MMP of oocytes from small antral follicles(P<0.05),and there was no significant difference compared with the oocytes from medium antral follicles(P>0.05);AREG+GDF9+BMP15 significantly increased the NAD(P)H and FAD++ level,NADPH production and ATP content of oocytes from small antral follicles(P<0.05),and there was no significant difference compared with the oocytes from medium antral follicles(P>0.05),significantly increased their glucose consumption and lactate production in the culture medium and ATP content of cumulus cells(P<0.05),and significantly higher than those from medium antral follicles(P<0.05);compared with the control group,supplemented AREG+GDF9+BMP15 in IVM medium could significantly increase the cleavage rate and blastocyst rate of oocytes from small antral follicles after in vitro fertilization((43.79±3.69)%,(28.54±4.31)%and(78.99±1.12)%,(47.46±2.50)%,respectively,P<0.05),and there was no significant difference compared with the oocytes from medium antral follicles(P>0.05).In conclusion,cumulus cells and oocytes in sheep small antral follicles had low energy metabolism levels,and oocytes in sheep small antral follicles had poor IVM quality;under the synergistic effect of GDF9 and BMP 15,AREG can enhance energy metabolism of cumulus cells and oocytes from small antral follicles,and improve oocyte IVM quality and embryo development ability after in vitro fertilization.These results provide a theoretical basis for further elucidating the mechanism of oocyte maturation and improving the IVM system of oocytes.