Research On Functions And Mechanisms Of CGMP-specific PDEs Regulate Maturation Of Zebrafish Oocytes

Like other vertebrates,fish oocytes enter the growth stage through mitotic proliferation in the early stage,and their development will be arrested in the prophase I of meiosis until the fully grown stage.After luteinizing hormone(LH)surge,oocytes will resume meiosis,germinal vesicle will break,the first polar body will dischange,and the development will be arrested in the middle stage II of meiosis.This process is called oocyte maturation.The importance of cyclic guanosine monophosphate(cGMP)signaling pathway in oocyte maturation has recently attracted much attention in vertebrates.In the past two years,using zebrafish as a model,we have revealed the role of cGMP and the action of cGMP protein kinase(PKG)in oocyte maturation.In the present study,the functions of cGMP specific phosphodiesterase(PDE5a,PDE6 and PDE9)are further analyzed in oocyte maturation in zebrafish.The main findings are as follows:(1)PDE5a(pde5aa and pde5ab),PDE6(pde6a,pde6 b,pde6c and pde6d),PDE9(pde9a,pde9 al and pde9as)were dentified in zebrafish.(2)For PDE5 a genes,we first examined the expression of PDE5 a coding genes in zebrafish follicles and found that pde5 aa and pde5 ab were mainly expressed in oocytes.The expression of PDE5 a and phosphorylated PDE5a(pPDE5a)of zebrafish were then analyzed in follicles using commercial antibodies targeting mammalian PDE5 a and phosphorylated PDE5 a.Both PDE5 a and phosphorylated PDE5 a were found to be expressed only in oocytes,but not in follicular cells,and phosphorylated PDE5 a can be activated in oocytes during oocyte maturation induced by human chorionic gonadotropin.Interestingly,in vitro incubation of zebrafish follicles with two different PDE5 a inhibitors sildenafil or tadalafil revealed that both inhibitors induced oocyte maturation and exhibited significant concentration,stage and time dependence,and such effects could be completely blocked by a PKG inhibitor kt5823 and two gap junction blockers(lindane and glycyrrhetinic),respectively.These experimental results show that PDE5 a is involved in zebrafish oocyte maturation and its activity plays an important role in maintaining the spontaneous maturation of oocytes.(3)For PDE6 genes,we detected the expression of PDE6 coding genes in zebrafish follicles and found that pde6 a,pde6b,pde6 c and pde6 d were expressed mainly in oocytes,in which the expression of pde6 a,pde6b and pde6 d ware regulated by LH during oocyte maturation.Thus speculate that PDE6 genes are involved in zebrafish oocyte maturation.(4)For PDE9 genes,we first detected the expression of PDE9 coding genes in zebrafish follicles and found that pde9 a,pde9al,and pde9 as were expressed mainly in oocytes,in which the expression of pde9 as was regulated by LH during oocyte maturation.Then we incubated the zebrafish follicles with a PDE9 specific inhibitor BAY736691 in vitro.We found that BAY736691 could induced oocyte maturation with obvious concentration and time dependence,and that such effects could be completely blocked by a blocker of gap junctions lindane.There experimental results show that PDE9 is involved in zebrafish oocyte maturation and its activity plays an important role in maintaining the spontaneous maturation of oocytes.In conclusion,using zebrafish as a model,are revealed the expression of cGMP specific PDEs in ovarian follicles and its role in oocyte maturation.