| Mammalian oocyte meiotic process began in the period of fetal development of primary oocyte, then arrested at the first meiotic prophase, the arrest period could last for several years or even decades. For the first time, the oocyte blocked in first meiosis diplotene forms pramordial follicle together with the surrounding pregranulosa cells. In mammals, after sexual maturation, the pramordial follicles in the ovaries gradually began to develop to maturation or atresia degradation, amoning them, the primary oocytes in mature follicles develop from ungrowth state to full growth state. Under the action of gonadotropin peak before ovulation, fully grown primary oocyte resume meiosis process, completed the first meiotic division and formed secondary oocytes. Followed by secondary oocytes going into the second meiotic division process, the oocytes blocked again in the middle of the second meiosis, until the fertilization.Research of oocytes in vitro maturation demonstrated, once out of the follicle meiosis block physical environment, the oocytes could restore spontaneously meiotic division. Now the primary oocytes may not yet have reached extranuclear fully mature, early recovery and completion of meiosis, it is unfavorable to support the subsequent development of oocytes. In the process of in vitro culture, protein synthesis inhibitors, phosphorylation inhibitors and cyclic adenosine analogues can inhibit the recovery of oocyte meiosis, avoid early nuclear maturation of oocytes, synchronize the nuclear and cytoplasmic maturation. However, the nonspecific inhibitor can affect the structure and developmental competence of oocytes, in the end, the blastocyst formation rate is similar with non-arrested oocytes.C-type natriuretic peptide(CNP) is a member of natriuretic peptide family. Recent studies have shown that CNP is naturally occurring in the follicle of mouse, and it is the key material to maintain meiotic arrest in mouse oocytes. Another research also showed that CNP can maintain the meiotic arrest in porcine oocytes, synchronized the nuclear and cytoplasmic maturation, thus improve the developmental conpetence of oocytes. The previous results in our lab showed that CNP could promote bovine oocytes maturation in vitro and the blastocyst formation rate of parthenogenetic embryos. However, the related molecular mechanism is unclear. In this study, on the basis of the detection of Npr2 mRNA in bovine follicles, combined with the effect of estrogen on the expression of Npr2 mRNA, we investigate the effect and mechanism of CNP on oocytes meiosis progress and deveiopmental competence in bovine.1. Mechanical separation method was used to separate the cow preantral follicles. The results showed that the amount of follicles reduced with the increase of follicle diameter,the number of follicles whose diameter arrange in 50-100 μm is largest, followed by the 100-200 μm follicles in diametre, the minimum number is the follicles more than 200 μm in diametre.2. Gel electrophoresis and real-time quantitative-PCR was used to detect the expression of Npr2 mRNA in different stages of follicles. The result showed Npr2 mRNA expressed in primary,secondary and antral follicles, but not in primordial follicles. The result of RT-PCR indicated expression level of Npr2 mRNA increased with the development of follicles.3. PCR and gel electrophoresis were used to detect the expression of Npr2 mRNA in mural granulosa cell, cumulus cell and oocyte of antral follicles. The result indicated Npr2 mRNA expressed in the three types of cells.4. RT-PCR was used to investigate the effect of 17β-E2 on the expression of Npr2 mRNA incumulus cells. The result showed expression of Npr2 mRNA of control group declined during in vitro culture, and mature medium added respectively with 10 ng/mL,100 ng/mL and 1000 ng/mL 17β-E2 could delay the drop of Npr2 mRNA expression levels, of which 100 ng/mL 17β-E2 can significantly maintain Npr2 mRNA expression of COCs.5. Bovine COCs were cultured in 20 nmol/L, 40 nmol/L, 80 nmol/L, 160 nmol/L CNP-OM medium for 4 h and 6 h, then the nuclear process was detected. We didn’t find the effect of CNP on the occur of GVBD in bovine oocyte.6. Bovine COCs were cultures in 20 nmol/L CNP-OM for 12 h, then cultured in OM to 24 h, The cultyre system could significantly improve the maturation rate of oocytes, as so as the cleavage rate and blastocyst formation rate parthenogenetic embryos.7. RT-PCR was used to investigate the developmental related genes of parthenogenetic blastocyst. The result indicated that expression of Bmp15 and H19 are significantly higher than untreated group(P<0.05), and significant difference didn’t exist within the two groups(P>0.05).8. ELISA was used to detect the concentration of cGMP in bovine COCs. The result showed that cGMP level continuously decreased during in vitro culture, and CNP could temporarily maintain the concentration of cGMP in bovine COCs.The research results showed that Npr2 mRNA expression level in follicles of different development stages in bovine ovarian exists significant differences; Npr2 mRNA was detected in mural granulosa cells, cumulus cells and oocytes; 17β-E2 ccould relatively slow the decrease of Npr2 mRNA expression of COCs in vitro culture; we didn’t find obvious effect of CNP on oocytes meiosis process in vitro culture, but CNP could maintain temporarily the concentration of cGMP in COCs temporary, then provide suitable concentration of cyclic adenosine monophosphate(cAMP) for oocytes development, so significantly increase the rate of oocytes maturation and the growth rate of parthenogenetic embryos in vitro. |
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