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

In vitro maturation (IVM) of oocytes is the basic technique of embryo production in vitro,which can guarantee more quality oocytes for IVF and somatic cell nuclear transfer. Also,IVM is used as a model to study follicular development and oocytes maturation. Although thetechnology of IVM has maturated and perfect, and the number of oocytes has advantages, theability of subsequent development is much lower than the oocytes matured in vivo.C-type natriuretic peptide (CNP) is a naturally occurring in the follicular fluid paracrinefactors. Research had shown that CNP played an important role in inhibiting meioticresumption of porcine and mouse. But there is no study about whether CNP has the samefunction of bovine oocytes IVM, and whether CNP is beneficial to improve the quality ofIVM oocytes and if CNP has the ability to support subsequent embryonic development. Themain point of this study is to discuss the effect of CNP on bovine oocytes IVM and thesubsequent development of parthenogenetic embryo. The results are shown as follows:1. This study was carried out to observe the meiosis process of bovine oocytes camefrom slaughterhouse cultured for different times in vitro, the results show that bovine oocyteshave developed into the stage of GVBD when cultured in vitro for8h, MI when cultured for8～16h, and the highest proportion of MII oocytes is showed after cultured for24h.2. After cultured in OM medium plus20~160nmol/L CNP (CNP-OM medium) for8h,all oocytes have been completed the GVBD and some oocytes entered into developmentprocess after GVBD, no obvious inhibitory effect of CNP on germinal vesiclebreaddown(GVBD) has been observed.3. Compared with the control group that bovine oocytes cultured in OM medium for24h,bovine oocytes were cultured in20nmol/L,40nmol/L,80nmol/L and160nmol/L CNP-OMmediums for8h,12h and16h, then cultured to24h in OM medium, detecting the nuclearprocess. The percentage of MII oocytes are increased in all experimental groups, especiallythe bovine oocytes cultured in20nmol/L CNP-OM medium for12h then cultured to24h inOM medium has the highest percentage at MII. So this group is selected for the right condition ofsubsequent experiments. 4. Bovine oocytes cultured in20nmol/L CNP-OM medium for12h, then cultured to24h inOM medium. The results show it did not affect the normal distribution of microfilaments andmicrotubules in bovine oocytes.5. Bovine oocytes cultured in20nmol/L CNP-OM medium for12h then cultured to24h inOM medium, the maturated oocytes are treated with parthenogenetic activation and culturedfurther, the rate of cleavage is73.58%, and the rate of blastocyst is46.14%, which are bothhigher than the control significantly(P<0.05).6. Real-time PCR is used to detect the development and apoptosis relative genes ofpartheno-blastocyst, it showed that the expressions of Bax, Oct-4and Xist are significantlyhigher(P<0.05) than control group, especially the up-regulation of the X-chromosomeinactivation specific transcript(Xist), while there is no significant difference in H19betweentwo groups (P>0.05).In conclusion, cultured in20nmol/L CNP-OM medium for12h then cultured to24h in OMmedium is an appropriate solution for bovine oocytes matured in vitro, and this solution canimprove the quality of bovine oocytes IVM.