| As a member of the natriuretic family,C-type Natriuretic Peptide(CNP)is the physiologic peptide that can maintain the anesthesia of oocyte meiosis and it has low toxicity to oocytes.Oocyte discharged from mature ovarian follicles of canine need to be matured in the tubal for 2 to 5 days before the first polar body is discharged.This physiological difference from bovine and sheep has led to the unsatisfactory in vitro maturation effect of canine oocytes developed from the in vitro maturation culture systems of bovine and sheep and other species,and has become a bottleneck restricting of canine assisted reproductive technology.In this paper,real-time fluorescence quantitative technology was used to detect the expression levels of the main members of the natriuretic family and their receptors in canine ovaries,parietal granulocytes(PCC)and cumulus cells(CC)from the different stage ovaries.The appropriate concentration and treatment time of CNP added to the in vitro maturation media and the effect of CNP on the in vitro maturation were further screened aiming to explore a method to improve the in vitro maturation effect of canine oocytes.The results showed that the mRNA expression of CNP in the ovarian follicular phase,luteal phase,and estrus phase were significantly higher than that of Brain Natriuretic Peptide(BNP)(P<0.01)and Atrial Natriuretic Peptide(ANP)(P<0.05);mRNA expression levels of Natriuretic Peptide Receptor 1(NPR1)and Natriuretic Peptide Receptor 2(NPR2)in the ovarian follicular phase were significantly higher than that in the ovarian anestrus(P<0.01),and the expression of mRNA of NPR2 in ovarian follicular phase,estrus and luteal phase were higher than that of NPR1(P<0.01).The expression of mRNA of CNP in PGC was significantly higher than that of CC(P<0.05),however,the mRNA expression of NPR2 in CC was significantly higher than that of PGC(P<0.05).Canine oocytes were treated with different concentrations of CNP(50,100,500,1000nmol/L)for 6,12,18,24h respectively(the first step of in vitro maturation),and then transferred to conventional maturation media to make up for 72h in vitro maturation(the second step of in vitro maturation).The appropriate concentration and treatment time for adding CNP were screened.The results showed that the treatment of in vitro maturation for 12h in the conventional media with 100nmol/L CNP and then transferring to the conventional media without CNP to make up 72h was the best in vitro maturation method.Parthenogenetic Activation(PA)and In Vitro Fertilization(IVF)were performed separately for the in vitro maturation(IVM)oocytes from the best method(CNP group)and conventional method(control group)and in vivo maturation canine oocytes(in vivo group),to compare their in vitro developmental capabilities.The results showed that the cleavage rate and 8-16 cell ratio of the CNP group after PA were significantly higher than that of control group(P<0.05),but significantly lower than that of in vivo group(P<0.05).The cleavage rate and 8-16 cell ratio of CNP group after IVF were not significantly different from those of control group(P>0.05),but significantly lower than that of in vivo group(P<0.05).The 4 cells ratio of in vivo group was significantly higher than that of the CNP group(P<0.05)and the control group(P<0.01).In short,the mRNA expression of CNP in dog ovary was higher than that of BNP and ANP,CNP in PGC,the mRNA expression of NPR2 in CC;ovary was higher than that of NPR1,NPR2 in CC,and the mRNA expression of NPR1,NPR2 in CC was higher than that of PGC;dog oocytes treated with 100nmol/L CNP for 12 hours,and then transferred to conventional culture medium for 72 hours,which was the best IVM method. |
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