| C-type natriuretic peptide (CNP) is one of the main members of the natriuretic peptides, which widely express in many tissues of mammal. It has been reported that CNP plays an important role in meiotic resumption. The aim of this study was to investigate the effect of CNP on meiotic resumption of buffalo oocytes and its regulation meschanism, so as to provide a foundation for further optimizing the IVM culture system and improving the efficiency of in vitro embryos production.Firstly, the mRNA expression of the mainly natriuretic peptides and their receptors in buffalo ovary were detected, the result of qRT-PCR showed that the mRNA expression of CNP was significantly higher than ANP and BNP (P<0.05), and the mRNA expression of NPR2 was significantly higher than NPR1 (P<0.05). Then the expression of CNP and its receptor NPR2 in buffalo follicles and COCs were further investigated by using the qRT-PCR, immunochemistry and immunofluorescence technology. The result indicated that the protein of CNP and NPR2 were detected in all stages of follicles, CNP mainly expressed in mural granulosa cells and NPR2 primarily presented in cumulus cells. Moreover, the mRNA expression of CNP in granulosa cells was significantly higher than in cumulus cells, the mRNA expression of NPR2 in cumulus cells was significantly higher than in granulosa cells. However, the both of CNP and NPR2 had no expression in oocyte.Secondly, the effect of CNP on meiotic resumption of buffalo oocytes and its regulation meschanism were preliminary studied. Buffalo COCs were respectively cultured in the maturation medium supplemented with different concentrations of CNP (0 nM,100 nM,200 nM,400 nM) for 6h, then moved into the maturation medium without CNP to culture for 18h, which meaned COCs totally culturing for 24h. The result showed that 200 nM CNP significantly improved (P<0.05) the maturity rate of oocytes (68.36% vs 61.21%), the cleavage rate (75.20% vs 64.86%) and the blastocysts rate(23.64% vs 16.29%) of the subsequent IVF embryos. Then, the suitable treatment time of CNP was groped after selecting 200 nM as the appropriate treatment concentration. The result indicated when COCs were cultured in the culture medium with 200 nM CNP for 6h or 9h, the maturity rate of oocytes (68.72% and 66.85), the cleavage rate (75.03% and 73.96%) and the blastocysts rate (24.38% and 23.19) of the subsequent IVF embryos were significantly higher than that of the control group (62.03% ,66.83 %and 18.35%, P<0.05). Otherwise, when denuded oocytes were cultured in the maturation medium supplemented with 200 nM CNP for 6 h, there were no significant effect (P>0.05) on the maturity rate of the denuded oocytes (42.36% vs 41.97%), the cleavage rate (49.73% vs 50.36%) and the blastocysts rate (2.35% vs 2.46%) of the subsequent IVF embryos. Result of cudbear dye stainning revealed that the time of GVBD in the oocytes could be significantly delayed (P<0.05) after treating COCs with 200 nM CNP for 6 h. QRT-PCR results showed that treating COCs with 200 nM CNP for 6 h could significantly up-regulate the expression level of Cx37, Cx43, NPR2 on cumulus cells and down-regulate the expression of PDE3A on oocytes. The result of Elisa revealed that the concentration of cGMP in cumulu cells and oocytes, as well as the concentration of cAMP in oocytes were significantly enhanced after buffalo COCs treated with 200 nM CNP for 6 h. Futhermore, the result also found that Go 6976 as the inhibitor of receptor of CNP, could reduced the extent of CNP increasing the concentration of cGMP in cumulu cells and cAMP in oocytes.In conclusion:(1) CNP and NPR2 strongly express in buffalo ovary, CNP mainly expresses in mural granulosa cells and NPR2 primarily presents in cumulus cells, but both of them have no expression in oocyte. (2) Treating buffalo COCs with 200nM CNP for 6h can delay the meiotic resumption of buffalo oocyte. The regulation meschanism of CNP is that CNP maybe need to combine with the receptor NPR2 in cumulus cells, and generate a large amount of cGMP, then cGMP diffuses into the oocyte via the gap junctions, inhibits the PDE3A activity and enhances the leves of cAMP in oocyte, so that oocyte maintains meiotic arrest. |
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