| In all mammals,the mature quality of oocytes is the basis of the reproductive ability of male and female animals.Studies have shown that estradiol(E2)and its receptors(Estrogen nuclear receptors,ERs)play an important role in the process of meiosis in mouse and human oocytes.Another study reported that low concentrations of estrogen promote the maintenance of C-type natriuretic peptide(CNP)on meiotic arrest in goat oocytes through ERs.Cyclic adenosine monophosphate(c AMP)is a key molecule that regulates mammalian oocyte meiosis.There is no report about the correlation between c AMP level and estrogen nuclear receptor expression during mammalian oocyte maturation.In this study,the immunohistochemical staining technique was used to detect the expression and localization of ERα and ERβ proteins in sheep antral follicles,and enzyme-linked immunosorbent assays were used to detect the changes in c AMP levels in sheep cumulus oocyte complex(COCs)after different concentrations of E2 were treated for different times(0、1、2h),Then combined with real-time quantitative RT-PCR and western bloting techniques,the effect of adding different concentrations of E2 on the expression of ERα and ERβ gene and protein expression levels in sheep COCs cultured in vitro(1h)was discussed;Finally,in an in vitro culture system the absence of other hormones,low concentrations of E2 and c AMP regulators [Forskolin(Fsk),3-isobutyl-1-methylxanthine(IBMX)and Rp-adenosine 3’5’-cyclic monathionate(Rp-c AMPs)] were added alone or in combination,to explore its possible regulation of c AMP and ERβ protein expression in sheep COCs.The results showed that:(1)Both ERα and ERβ Proteins were expressed in sheep cumulus cells and oocytes;(2)Compared with the control group,low concentration of E2(0.5 ng/m L)can significantly increase the level of c AMP in sheep COCs(P<0.05),while high concentration of E2(1 μg/m L)has no significant effect on it(P>0.05).(3)The low concentration of E2(0.5 ng/m L)can significantly up-regulate the gene expression and protein expression of ERβ,and compared with the control group,there are significant differences(P<0.05),while there was no significant difference between high concerntions E2 group and control group(P>0.05).(4)Adding Forskolin and IBMX to the culture system without hormones can significantly increase the level of c AMP in sheep COCs(P<0.05).(5)In the hormone-free culture system alone or in combination with Forskolin and IBMX and Rp-c AMPs for 1 hour,the Forskolin and IBMX treatment groups can significantly up-regulate the expression of ERβ protein,and the promotion can be offset by Rp-c AMPs(P<0.05).(6)In the culture system without hormones,adding low concentration of E2(0.5ng/m L)significantly promoted the expression of ERβ protein,and this effect was also significantly offset by Rp-c AMPs(P<0.05).In summary,the results show that both ERα and ERβ proteins exist in ovine cumulus cells and oocytes.In the process of in vitro culture,low concentration of E2(0.5 ng/m L)promotes c AMP level in sheep COCs,and subsequently up-regulates ERβ gene and protein expression.The results of this study will be helpful to further clarify the role of E2 in the maturation of oocytes,which is of great significance for improving the quality of oocytes and subsequent embryonic development. |
