Study On The Effect Of C-Type Natriuretic Peptide On Growth And Development Of Bovine Preantral Follicles In Vitro

The development of embryo engineering related technologies increase the utilization efficiency of oocyte, and improve reproductive capacity of the female animals. However, currently oocyte in vitro maturation and fertilization and other embryo engineering related technologies acquire the very small quantities of oocytes, being limited to obtain oocytes from antral follicles on the ovary surface. As we all know, bovine ovaries contain a large number of preantral follicles, and if we can successfully culture bovine preantral follicles development to antral follicle stage, we can collect oocytes from these antral follicles for the in vitro fertilization with other embryo engineering researchs and applications. Moreover, it can promote the development of embryo engineering techniques, and broaden their scope in application.C-type natriuretic peptide(CNP)is a naturally occurring in mammalian follicle, and natriuretic peptide receptor 2(NPR2)is specific receptor. Most studies focus on the role of CNP about mammalian oocytes meiotic arrest and in vitro maturation(IVM). A great number of studies showed that CNP could maintain the meiotic arrest in bovine oocytes, synchronize the nuclear and cytoplasmic maturation, thus improve the maturation rate of oocytes and the developmental conpetence of subsequent embryonic. Recent studies have shown that CNP can promote the development of preantral follicles in mice. In this study, combing and scraping bovine ovaries as experimental materials isolate bovine preantral follicles. Basic culture medium adding 100 ng/mL CNP or adding in combination with 100 ng/mL FSH and 100 ng/mL CNP cultured bovine preantral follicles(150~300 μm)for 12 days in vitro. Combined with the effect of FSH on bovine preantral follicles in vitro culture, we preliminarily investigated the effect of CNP on bovine preantral follicles in vitro growth, survival and follicle antrum formation as well as follcile development- and apoptosis-related genes expression, as to provide a basis for in vitro culture of preantral follicles in mammals. Experimental research contents and results are as follows:1. In this study, bovine ovaries were collected from slaughterhouse, and the isolated preantral follicles were cultured in vitro. The results showed bovine preantral follicles were cultured in basic culture medium α-MEM+ for 12 days in vitro, and the survival rate of preantral follicle was approximately 58.33±4.04%. The results may provide possibility for following study on the effect of CNP on bovine preantral follicles in vitro growth and development.2. Basic culture medium were respectively added FSH, CNP and FSH+CNP, and bovine preantral follicles were cultured for 12 days in vitro, then measured follicles’ diameter at 0, 4, 8, 12 days in vitro culture and statisticed the diameter changes of follicles growth respectively. Results showed that the follicles’ diameter in FSH group and α-MEM+ group increases with the prolongation of days of culture. CNP group and FSH+CNP group follicles’ diameter show a decreasing trend with the prolongation of days of culture. But in CNP group and FSH+CNP group, bovine preantral follicles cultured in vitro for 12 days, and follicular antrum formation rates were significantly higher than FSH group and α-MEM+ group(P< 0.05). From the above results, we believe CNP may promote not fully grown and development follicles prematurely formation into follicle antrum in vitro.3. Basic culture medium were respectively added FSH, CNP and FSH+CNP, and bovine preantral follicles were cultured for 12 days in vitro, then statisticed follicle survival rates. The results showed that FSH+CNP group preantral follicles survival rate was significantly lower than the other three groups(α-MEM+, CNP and FSH+CNP)(P< 0.05), while CNP group follicle survival rate was significantly lower than FSH group(P< 0.05), and slightly lower than the α-MEM+ group but the difference was not significant(P> 0.05). It suggest that CNP may reduce the survival rate of bovine preantral follicles in vitro.4. Reverse transcription PCR(RT-PCR)and quantitative real-time PCR(qRT-PCR)techniques were used to detect the expression of Npr2 mRNA in different culture condition and time of bovine preantral follicles as well as the expression of follicle development- and apoptosis-related genes. The results showed that Npr2 mRNA relative expression levels in CNP group bovine preantral follicles which cultured for 12 days in vitro higher than the other three groups(α-MEM+, FSH, and FSH+CNP group), but the difference were not significant(P> 0.05). Development-related gene FSHR, CYP19A1 mRNA relative expression level with the prolongation of day of culture first increased, then decreased, and the relative expression amount of PCNA mRNA increased gradually. CNP group and FSH+CNP group bovine preantral follicle cultured for 12 days in vitro, FSHR, CYP19A1 and PCNA mRNA relative expression levels were lower than the other two groups(α-MEM+ and FSH group). Apoptosis-related gene Bax and Apaf-1 mRNA relative expression levels were gradually increasing with the prolongation of day of culture. FSH+CNP group and CNP group the relative expression levels of three apoptosis-related genes were higher than α-MEM+ group and FSH group. Based on the above findings, bovine preantral follicles cultured in basic culture medium supplement with CNP for 12 days in vitro, follicle development-related gene expression decreased, while the expression of apoptosis-related genes elevated, and we speculated that CNP may not be conducive to the growth and survival of bovine preantral follicles from those results.In summary, CNP can significantly promote bovine preantral follicles into the cavity in vitro. However, CNP significantly reduces the follicle survival and is not helpful to the growth of bovine preantral follicles. Therefore, more research on the mechanism of CNP on the growth and development of bovine preantral follicles in vitro is needed.