Functional Analysis Of CGMP Signaling Pathway In Oocyte Maturation Of Zebrafish

The roles of cyclic guanosine monophosphate(c GMP)signaling in oocyte maturation attracts much attention in mammals,but its roles in fish are still largely unknown.Using zebrafish as a model,we demonstrated for the first time in fish that c GMP is involved in oocyte maturation,and its functional model in oocyte maturation is different from that of mammals.Intriguingly,we found that either the activation or inhibition of the NO/s GC/c GMP pathway in fully grown follicles could lead to oocyte maturation.c GMP could exert its effects through the PDE3 and PKG pathways,both of which are required for spontaneous and c GMP-regulated oocyte maturation.At the same time we detected the level of NO and c GMP received LH(Luteinizing hormone,LH)regulation during oocyte maturation.The main specific results are as follows:(1)c GMP could be regulated by both the natriuretic peptide(Np)system and the nitric oxide(NO)systems.Both NO system and Np system are expressed in zebrafish ovary.In the NP system,six Np ligands(nppa,nppb,nppc1,nppc2,nppc3,and nppc4)and four receptors(npr1(npr1b),npr1 a,npr2,npr3)have been identified in zebrafish.In the NO system,like mammals,there are three types of NOS(nos1,nos2 a and nos2b)and four types of soluble guanylate cyclase(s GCs,including gucy1a2,gucy1a3,gucy1b2 and gucy1b3)identified in zebrafish.(2)Np system might not be involved in the oocyte maturation of zebrafish.We synthesized the mature peptides of six types of zebrafish Np ligands(nppc1,nppc2,nppc3,nppc4,nppa,nppb).After treatment of FG stage follicles with these Np peptides,the rate of spontaneous maturation was not largely affected by these peptides.(3)Activation of NO/s GC/c GMP signaling could stimulate oocyte maturation in zebrafish.After treatment of FG stage follicles with two NO donors SNAP and NOC-5,two s GC activators protoporphyrin IX and YC-1,and two membrane-permeable c GMP analogs 8-Br-c GMP and db-c GMP,the oocyte maturation is significantly stimulated.(4)Blockade of NO/s GC/c GMP signaling could stimulate oocyte maturation in zebrafish.Three inhibitors of NOS,AG,L-NAME and Vinyl-L-NIO,one NO scavenger PTIO,and one inhibitor of s GC ODQ were used to investigate this study.Surprisingly,we found that the oocytematuration could be dramatically induced but not blocked by these inhibitors.(5)Spontaneous oocyte maturation could be inhibited by blockade of PKG and PDE3 activity in zebrafish.After treatment of both PKG specific inhibitor kt5823 and PDE3 specific inhibitor cilostamide,the spontaneous oocyte maturation could be significantly blocked in a timeand dose-dependent manner.(6)c GMP could regulate oocyte maturation through PKG and PDE3 in zebrafish.The stimulatory effects on oocyte maturation by activation or inhibition of NO/s GC/c GMP signaling could be significantly blocked by a PKG inhibitor,KT5823 and a PDE3 inhibitor,cilostamide.These results suggest that both PKG and PDE3 is required for the c GMP-regulated oocyte maturation in zebrafish.(7)Both NO and c GMP in preovulatory ovarian follicles,follicular cells and denuded oocyte is dynamically regulated by LH during oocyte maturation in zebrafish.The c GMP level is increased in the follicular cell layer but decreased in intact ovarian follicles and denuded oocytes,while NO levels increased in follicular cells and ovarian follicles but remained constant in oocytes after injection human chorionic gonadotropin(h CG).These findings prompted us to propose a hypothesis on the dual role of c GMP in regulating oocyte maturation of zebrafish: Before LH surge,the c GMP produced in follicular layer cells entered the oocyte via gap junction,which inhibited the hydrolysis of c AMP by PDE,and then blocked the meiosis of the oocyte maturation.In follicular cells the LH signal could increase the level of NO and c GMP which induces oocyte maturation through the PKG pathway,while in the oocyte the concentration of c GMP is decreased which induces PDE3 to hydrolyze c AMP to stimulate oocyte maturation.At present,we only find that the nitric oxide signaling pathway is important in the process of regulating zebrafish oocyte maturation by c GMP.The role played by the natriuretic peptide family in this group is worth further study.These findings help us to understand the molecular mechanism of fish oocyte maturation.