Regulation Effect Of AMH On In-vitro Maturation Of Oocytes And Cumulus Expansion In Mouse

Anti-Mullerian hormone(AMH)is a member of the transforming growth factor β(TGF-β)superfamily,secrets from the ovaries of female animals and exerts its biological effects through the type II receptor(AMHR2).AMH is involved in the regulation of follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH.AMH can also be used as an effective marker to evaluate ovarian reserve and predict superovulation.However,the research on the biological function of AMH is mainly focused on regulating steroid hormone in granulosa cells,little research on the in vitro maturation of oocytes,especially the expansion of cumulus.Therefore,the regulation action of AMH on in vitro maturation of DOs and COC as well as cumulus expansion were performed using methods of IVM,RT-PCR,Western Blotand ELISA.The results can lay a foundation for improving the utilization rate of female oocytes.(1)Expression of AMH and its receptor in mouse ovary.Expression of AMH and AMHR2 was detected in different aged mice,results showed that AMH and AMHR2 had the highest expression at 14 days of age compared with other mice.AMH is only expressed in cumulus cells,while AMHR2 is expressed in both cumulus cells and oocytes.In addition,expression of AMHR2 decreased gradually with the development of oocyte,AMHR2 expression in GV oocytes was significantly higher than that in MI oocytes(P<0.05).(2)Effect of AMH on the in vitro maturation of Denuded oocyte(DOs).Different concentrations of rh-AMH(1 ng/mL,10 ng/mL,and 100 ng/mL)were added to the culture medium of DOs,after 14 h in vitro culture,the maturation rate of DOs was found to be dose-dependent with the concentration of AMH.The high-dose(100 ng/mL)group got the highest maturation rate,and it was significantly higher than that of the control group and the low-dose group(P<0.05).With the increase of AMH concentration,the MPF level gradually increased,and the expression of BMP15 gradually increased.Furthermore,the AMH-added group could significantly reduce the content of cAMP in the cytoplasm(P<0.01).It can be seen that AMH may promotes the in vitro maturation ofDOs by promoting the expression of BMP15,increasing the content of MPF and decreasing the expression of cAMP.(3)Effect of AMH on in vitro maturation of Cumulus oocyte complexes(COC).The effect of AMH on COC in vitro maturation was analyzed by adding 100 ng/mL AMH and 100 ng/mL FSH alone or in combination into the COC in vitro culture system.The results showed that compared with the control group,100 ng/mL AMH treated group had no significant effect on the maturation of COC(P >0.05);Compared with FSH alone treated group,the maturation rate of COC in the combination of AMH and FSH group was significantly decreased(P<0.05),suggesting that AMH could inhibit the promoting effect of FSH on COC maturation in vitro.In addition,it was found that AMH could significantly inhibit the promoting effect of FSH on MPF in COC.(4)The role of AMH in regulation of cumulus expansion.The regulation of AMH on cumulus expansion was analyzed by adding 100 ng/mL AMH and 100 ng/mL FSH alone or in combination with the COC in vitro culture system.The results showed that the AMH treated group had no effect on the expansion of the cumulus,while the FSH treated group significantly increased the expansion of the cumulus(P<0.05).Compared with the FSH alone treated group,the combination of AMH and FSH group significantly decreased the cumulus expansion index(P<0.05),suggesting that AMH can inhibit the promotion of FSH on cumulus expansion.RT-PCR was used to detect the expression of genes associated with cumulus expansion in each treatment.It was found that AMH could significantly increase the expression of Ptgs2 mRNA in cumulus cells,whereas the expressions of Has2,PTX3 and Tnfaip6 genes were no significant difference(P >0.05).But FSH can significant upregulate the expression of of Has2,PTX3 and Tnfaip6(P<0.05),and AMH could inhibit this action.In conclusion,high concentration of AMH could increase the maturation rate of DOs,and AMH had no significant effect on COC maturation and cumulus expansion.However,AMH can inhibit the promotion of FSH on oocyte maturation and cumulus expansion in COC.