| This study investigated the C-type natriuretic peptide(CNP)pretreatment affecting sheep oocyte meiosis block and subsequent development ability,the expression of maternal genes and cumulus cell expansion related gene during maturation.The results were as follows:1.Sheep oocyte cultred in TCM 199 with 200 nM CNP for 0 h,2 h,4 h,6 h or 8 h,then proportion of GV stage after culture were detected by DAPI staining.The results showed that CNP could effectively maintain the meiotic block of oocyte within 4 h.In vitro maturation(IVM)24 h was used as the control group,the development ability of oocytes after parthenogenetic activation(PA)in CNP pretreatment group(pretreated with 200 nM CNP 4h following in vitro maturation 24h)was compared.the results showed that blastocyst rate of parthenogenetic embryos(43.44%)in CNP pretreatment group was significantly higher than that in control group(31.72%,P<0.05),cleavage percentage and cell number in blastocyst were similar between two groups.2.IVM 24 h was as the control group,tretreatment with 200 nM CNP 4h following in vitro maturation 24 h was as CNP pretreatment group,the maternal gene Dnmt1,Zar1,and Mater expression of oocyte during maturation were detected by RT-qPCR.The results showed that the maternal gene Dnmt1,Zar1 and Mater were expressed in sheep oocytes.Regadless of CNP pretreatment,the expression levels of the maternal gene Zar1 and Mater in oocyte were significantly reduced after maturation culture(P<0.05),while the expression level of Dnmt1 was significantly increased after maturation culture(P<0.05).Before maturation culture of sheep oocytes,There was no significant difference in maternal gene(Dnmt1,Zar1 and Mater)expression between CNP pretreatment group and control group(P>0.05).After maturation culture of sheep oocytes,The expression of maternal gene Dnmt1,Zar1 and Mater in CNP pretreatment group were significantly higher than those in control group(P<0.05).3.IVM 24 h was as the control group,tretreatment with 200 nM CNP 4h following in vitro maturation 24 h was as CNP pretreatment group,the expression of PTGS2,HAS2 and PTX3 in the cumulus cell of sheep oocyte were detected by RT-qPCR.The results showed that the gene HAS2,PTX3 and PTGS2 were expressed in the cumulus cells.Regadless of CNP pretreatment,the expression levels of HAS2,PTX3 and PTGS2 in oocyte were extreme significantly increased after maturation culture(P<0.01).Before maturation culture of sheep oocytes,the expression of cumulus cell expansion related gene(HAS2,PTX3 and PTGS2)in CNP pretreatment group was significantly higher than that of control group(P<0.01);After maturation culture of sheep oocytes,the expression of PTX3 in CNP pretreatment group was extreme significantly higher than that in control group(P<0.01),while the expression level of HAS2 in CNP pretreatment group was extreme significantly lower than that in control group(P<0.01);In addition,after maturation culture of sheep oocytes,the expression of PTGS2 in CNP pretreatment group was no significantly than that in control group(P >0.05).In summary,CNP pretreatment can affect expression of cumulus cell expansion related gene HAS2,PTX3,PTGS2 and maternal gene Dnmt1,Zar1 and Mater.CNP pretreatment can temporarily hinder the recovery of sheep oocyte meiosis,and improve sheep oocyte maturation quality and early embryonic development ability.These results provides an basis for further study of the mechanism regulating sheep oocyte maturation. |
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