The Expression Of C-type Natriuretic Peptide Receptor In Ovaries Of Estrous Cycle Mouse And Superovulation Mouse

C-type natriuretic peptide（CNP）is a nature paracrine factor in follicular fluid. As aoocyte maturation inhibiting factor, CNP can improves the level of cyclic guanosine3’,5’-monophosphate（cGMP）in cumulus and oocytes, and also improves the level of cyclicadenosine3’,5’-monophosphate（cAMP） in oocytes. C-type natriuretic peptide receptor2（NPR2）, with the activity of guanylic acid cyclase,is stimulated to producing high levels ofcGMP when combined with CNP,then inhibiting the resumption of meiosis in oocytes.In the present study, we use immunohistochemical method and RT-PCR method toresearch the expression of NPR2in mouse ovary during the estrous cycle and differentdevelopment periods of follicles.Then we use a superovulation for dioestrous mice to researchthe NPR2expression in mice injected with pregnant mare serum gonadotropin （PMSG） andhuman chorionic gonadotrophin （hCG）. Finally, we study the NPR2expression insuperovulation mouse injected with EGF and the effect of injection with EGF forsuperovulation and early embryo development.The results are as follows:1. The expression of NPR2in mice ovary during the estrous cycle. In mice ovary duringthe estrous cycle, NPR2was located in the granular cells, cumulus cells and the luteal cells atmetaestrus. The relative expression levels of NPR2changed regularly during the estrous cycle,its expressional level was highest in the estrus stage, but there was no significant differencebetween proestrus and estrus （P>0.05）; the relative expression of NPR2was low atmetestrous compared with proestrus and estrus（P<0.05）; its expressional level wassignificantly lower in the diestrus stage than that in other stages （P<0.05）; the changingregularity of NPR2mRNA was consistent with NPR2during the estrous cycle.2. The expression of NPR2during follicle development. Increased expression of NPR2levels were detected during follicle development. In preantral follicles, weak signal wasdetected in primordial follicle and primary follicle compared with the secondary follicle.However, In contrast to above, the expression of NPR2between early secondary follicle, latesecondary follicle,antral follicle and preovulatory follicle have a negligible variation, reaching highest levels in preovulatory follicle, but there was no significant differencebetween antral follicle and preovulatory follicle （P>0.05）. We further isolated follicles ofdifferent sizes in diameter from mouse ovaries, we found that the NPR2mRNA expressionvariation in follicles were coincident with above in a follicle development-dependent manner,but followed by a decline in mature follicles.3. Gonadotrophin regulation of NPR2expression in mouse ovaries. We performedImmunohistochemistry to elucidate the change in NPR2expression in mouse ovaries treatedwith gonadotropin. it is clear that increasing in NPR2expression in granulosa cells duringfollicles growth in mice and a slower decline induced by the preovulatory LH/hCGstimulation confirming that the slower decline was first detected at8h. We used real-timeRT–PCR to monitor changes in ovarian NPR2mRNA expression, the expression of NPR2mRNA was increased following treatment with PMSG, showing a peak at48h. No significantdifference in NPR2mRNA expression was observed between hCG-untreated and treatedovaries at2hr; however, NPR2mRNA expression was significantly decreased at4hrfollowed by a rapid decline to basal levels.4. The expression of NPR2in superovulation mouse injected with EGF.After injectionwith EGF, the expression of NPPC mRNA and NPR2protein and mRNA were significantlyimproved, but the expression of NPPC and NPR2mRNA injected with PMSG were nothigher than the mouse injected with EGF twenty-four hours. When the superovulation micewere injected with EGF, we found that it can significantly improve the amount of embryo, butnot the2-cell embryo collection rate and the blastocyst rate,it suggested that EGF canimprove the effect of superovulation and not affect the quality of embryo.In summary, the research results suggest that the expression of NPR2varies with estrouscycle in mouse ovary, and increased with the development of follicle. Furthermore, thevariation of NPR2expression in superovulation-treated mouse ovary illustrated that LHreduces the activity of the NPR2guanylyl cyclase in mouse ovarian follicles, leading to theresumption of meiosis in oocytes, this occurs by a process that does not reduce the amount ofNPR2protein. After injection with EGF, the expression of NPPC mRNA and NPR2proteinand mRNA were significantly improved, it suggest that EGF and the signal pathy ofNPPC/NPR2may play a role in Maintaining the oocyte meiosis arrest together.