Effect Of Cyclic Adenosine Monophosphate On Protein Expression Of Gap Junction Protein 43 During In Vitro Maturation Of Ovine Cumulus-oocyte Complexes

Connexin 43(Cx43)is a key protein specifically expressed in mammalian granulosa cells,and plays an indispensable role in follicular development and oocyte maturation.High levels of cyclic adenosine monophosphate(cAMP)in oocytes are essential for maintaining meiotic arrest.Although oocytes can independently synthesize cAMP,studies have shown that the closure of gap junction channel caused by phosphorylation of Cx43 protein is an important reason for maintaining meiotic in oocytes by reducing cAMP transport from cumulus cells to oocytes.Another study reported that artificially raising cAMP levels in bovine cumulus-oocyte complexes(COCs)can significantly enhance the gap junction function of the complex in turn,but the relevance of the cAMP levels in ovine follicles and the expression of gap junction protein Cx43 protein or functional changes has not yet been reported.In this study,the expression of Cx43 protein in ovine immature COCs by immunohistochemical,and then changes in cAMP levels and Cx43 phosphorylation and total protein during in vitro maturation were detected respectively by competitive enzyme-linked immunosorbent assay and by immunoblotting.In the absence of gonadotropins,the time effect of cAMP non-specific regulators on cAMP in ovine COCs and the expression of cAMP on Cx43 protein were explored.The results showed that:? Cx43 protein was only expressed on ovine cumulus cells,while oocytes does not express it.? The cAMP in oocytes reached the peak at 30 minutes and showed the lowest level at 6 hours of mature culture.Compared with 0h,the two time points were significantly different(P<0.05);The two peaks of cAMP level in cumulus cells at 10 min and 1h respectively,and compared with 0h,the different was significant(P ?0.05).? The expression of Cx43 phosphorylation increased transiently at 10 minutes of mature culture(P ?0.01);the expression of Cx43 total protein appeared two peaks at 10 minutes and 1 hour of mature culture(P ?0.05).?Forskolin and IBMX were added to the culture system without hormones after 10 minutes of culture,the cAMP content of the Forskolin and IBMX treatment group increased by about 6 times compared with the non-added group,the difference was significant(P ?0.05).In summary,this study found that Cx43 protein only exists in ovine cumulus cells.During in vitro maturation,the cAMP content in ovine cumulus cells showed two peaks.The phosphorylation effect of Cx43 caused by the first increase in cAMP may be a protective mechanism of gonadotropins to prevent the decrease of cAMP level in oocytes.The expression of Cx43 total protein in ovine COCs is positively regulated by cAMP,which may play an important role in maintaining the gap junction function before oocyte meiosis recovery.