| Non-synchronous nuclear and cytoplasmic maturation is the main problem in the process of oocyte in vitro maturation.C-type natriuretic peptide（NPPC）has attracted much attention as an endogenous meiotic inhibitor.Hormone and transforming growth factor-9（GDF9）are the main maturation-promoting factors in the process of oocyte in vitro maturation.However,the effects of hormones and GDF9 on NPPC/NPR2 in sheep follicles are not clear.In this experiment,we investigated the expression patterns of NPPC and natriuretic peptide receptor 2（NPR2）in different diameter follicles,the effects of follicle stimulating hormone（FSH）and GDF9 on the expression pattern of NPPC/NPR2 in granulosa cells,and further investigated the effects of different hormones on the inhibition of meiosis of sheep oocytes induced by NPPC.1.The expression patterns of NPPC and NPR2 in follicles with different diameters.The expressions of NPPC and NPR2 genes in mural granulosa cells（MGCs）and cumulus cells（CCs）in follice of different diameters（small antrum≤2 mm,2 mm<middle antrum≤6 mm,large antrum>6 mm）were detected by RT-PCR,and the expression of NPR2 protein in cells was detected by Western blot.The results showed that among different diameter follicles,the expression of NPPC mRNA in MGCs and CCs of middle antrum follicles were significantly higher than those of small antrum follicles and large antral follicles（P<0.05）,but NPPC mRNA expression was not detected in CCs of large antrum follicles,and the expression of NPR2 mRNA and protein in MGCs and CCs of middle antrum follicles were significantly higher than those of small antral and large antrum follicles（P<0.05）.2.The effect of FSH and GDF9 on the expression of NPPC and NPR2 in MGCs and CCs cultured in vitro.Compare with uncultured MGCs（control group）,the levels of NPPC mRNA and protein in MGCs cultured in vitro were significantly decreased（P<0.01）;FSH could significantly increase the level of NPPC mRNA and protein in MGCs cultured in vitro（P<0.05）,but still significantly lower than those in control group（P<0.05）.GDF9 could significantly increase the level of NPPC mRNA and protein in MGCs induced by FSH（P<0.01）.The levels of NPR2 mRNA and protein in MGCs cultured in vitro were significantly decreased（P<0.05）;FSH could significantly increase the expression of NPR2 mRNA and protein in MGCs to some extent（P<0.05）,but GDF9 had no effect on the expression of NPR2 mRNA and protein in MGCs induced by FSH.Compared with uncultured CCs（control group）,the levels of NPPC mRNA and protein in CCs cultured in vitro were significantly decreased（P<0.0l）.);FSH could significantly increase the level of NPPC mRNA and protein in CCs cultured in vitro,while GDF9 significantly inhibited the promoting effect of FSH.The expression of NPR2 mRNA and protein in CCs in vitro were significantly increased（P<0.05）.FSH could significantly increase the expression of NPR2 mRNA and protein in CCs to some extent（P<0.05）,while GDF9 significantly decreased the expression of NPR2 mRNA and protein in CCs（P<0.05）.3.Effects of hormones on meiotic arrest induced by NPPC in sheep oocytes.Sheep oocytes were preincubated with conventional maturation medium（control group）,NPPC200 n M medium（positive control group）,NPPC（200 n M）+FSH（10,20,30 ug/m L）,NPPC（200 n M）+LH（10,50 ug/m L）and NPPC（200 n M）+E2（0.5,1 ug/m L）for 4h,6h and 8h,and the nuclear phase analysis was performed by immunofluorescence staining.The results showed that the meiotic arrest in 200 n M NPPC group can lasted for 6 h（P<0.05）,0.5 and 1 ug/m L E2 could significantly promote the meiotic arrest of sheep oocytes induced by NPPC（P<0.05）,while FSH and LH had no obvious interaction effect on NPPC-induced meiotic arrest of sheep oocytes.In conclusion,the antrum results showed that NPPC and NPR2 began to express in sheep small antrum follicles,and the expression of NPPC and NPR2 in small,middle and large antrum follicles increased at first and then decreased.In the granulosa cell culture in vitro,FSH could promote the expression of NPPC,NPR2 in MGCs and CCs,and GDF9could promote the expression of NPPC in MGCs induced by FSH,but inhibited the expression of NPPC,NPR2 in CCs induced by FSH.E2significantly enhanced meiosis arrest of sheep oocytes induced by NPPC.The results of this study provide a theoretical basis for further elucidating the mechanism of NPPC in in vitro maturation. |
