Effect Of C-type Natriuretic On Maturation And Parthenogenetic Embryo Development Of Goat Oocytes In Vitro

The decrease of cAMP concentration in vitro maturation mammalian oocytes after beingseparated from follicle environment results in spontaneous resumption of meiosis precociously,causing asynchrony between cytoplasm and nuclear maturation and decreasing its developmentalcompetence. Several substances have been used to induce synchronization between cytoplasmand nuclear maturation, but most of them can not synchronize cytoplasm and nuclear maturationand improve the quality of matured oocytes at the same time. CNP is a highly conservative smallmolecular peptide widely expressed in mammalian animals. It has been reported that CNP caninhibit meiotic resumption of mice and porcine oocytes. Research indicated CNP expressed ingranulose cells of goat follicle, while there were still no reports about the expression of NPR2ingoat ovaries and the effect of CNP on goat oocyte meiosis. In this study, we investigated the effectof CNP on goat oocyte meiosis and the effect on in vitro maturation of goat oocytes and thesubsequent development of parthenogenetic embryos by in vitro maturation culture system ofoocytes, basing on the expression of NPR2in goat follicle. The results are shown as follows:(1) Immunohistochemistry and Real-time PCR technologies were used to detect theexpression of NPR2. The result shown, both NPR2protein and mRNA expressed in goat COCs.Npr2mRNA expression increased significantly with the development of follicle (P<0.05). Inantral follicles, the expression of Npr2mRNA in COCs was significantly higher than that in GCs(P<0.05), and the expression in large antral follicles lower than that in small ones.(2) Oocytes meiosis were assessed after COCs cultured in medium containing50,100,150ng/mL CNP and without CNP for4h,6h and8h respectively. Results shown, there were69.13±1.64%GV stage oocytes freshly isolated from ovaries; after cultured for4h, the rate of GVoocytes in100ng/mL and150ng/mL CNP groups were significantly higher than50ng/mL groupand control group (P<0.05), while there were no significant difference with the oocytes freshlyisolated; after cultured for6h, the rate of GV oocytes in all groups declined significantly(P<0.05),the groups with100ng/mL and150ng/mL CNP were significant higher than50ng/mL group andcontrol group(P<0.05); after cultured for8h, most oocytes in all groups have breakdowngerminal vesicle. These results indicated that the oocytes culture medium containing more than100ng/mL CNP can effectively inhibit the resumption of goat oocytes, but the duration ofthis inhibitory effect was about4h.(3) Oocytes meiotic were assessed after COCs cultured in medium containing5nmol/L，10nmol/L，50nmol/L,1μg/mL (concentration in OM,3761nmol/L) and2μg/mL (7522nmol/L)17β-E2and with100ng/mL CNP for6h and8h respectively. Results shown, after cultured for6h,the rate of GV oocytes of groups with10nmol/L and50nmol/L17β-E2were significantly higherthan5nmol/L,1μg/mL,2μg/mL17β-E2group and control groups, there were no significancebetween the two; the GV oocytes rate of10nmol/L17β-E2group was no significant difference withfreshly isolated oocytes (P>0.05); the GV oocytes rate of groups with high concentration of17β-E2were significantly lower than that cultured in medium with low concentration of17β-E2. Aftercultured for8h, the GV oocytes rate of groups declined significantly than6h(P<0.05), there wereno significance between groups with10nmol/L and50nmol/L(P>0.05), but they both significantlyhigher than other groups(P<0.05). The results demonstrated that oocytes the medium containing100ng/mL CNP and10nmol/L17β-E2can maintain goat oocytes meiotic arrest for6h, highconcentration of17β-E2promote meiotic resumption.(4) Detected the Npr2mRNA level of COCs in vitro cultured. The results shown, The Npr2mRNA level declined rapidly in vitro culture,17β-E2could increase the Npr2mRNA levelsignificantly (P<0.05) but it could not reach to the level of freshly isolated.(5) COCs cultured cultured in medium with100ng/mL CNP and10nmol/L estradiol(OMi)for24h (OMi24h), cultured in oocyte maturation medium(OM) for24h (OM24h) and culturedin OMi for6h and then in OM for18h (OMi6h+OM18h). Oocytes with polar body I wereselected for parthenogenetic activation and culture. The mature rate, cleavage rate and blastocystdevelopmental rate of OMi6h+OM18h group were76.60%,84.05%and44.50%respectively,they all significantly higher than that in control groups(P<0.05). The total cells of blastocyst ofOMi6h+OM18h group (149.86±9.26) was significantly higher than control groups (P<0.05),and it had no significance with blastocysts developed in vivo (P>0.05).In conclusion, NPR2mainly expressed in COCs of goat follicle, Npr2mRNA level increasedwith the development of follicles and decreased before ovulation. CNP has inhibitory effect ongoat oocytes meiosis.17β-E2can enhance this effect at a certain concentration and promote thematuration of oocytes directly at high concentrations. Goat COCs cultured in medium containing100ng/mL CNP and10nmol/L17β-E2for6h and then cultured in OM for18h can significantlyimprove the maturation rate and developmental competence of mature oocyte.