Interaction Of CDON With IZUMO1 And JUNO And Its Effect On Fertilization In Rats

The process of mammalian reproduction includes germ cells(sperm and oocyte)formation,fertilization,implantation,embryo development and so on,in which fertilization is the most important step.Fertilization is the process of sperm and oocyte combining to form the zygote,and the membrane fusion of the sperm and egg is the key step of fertilization.However,the molecular mechanism of sperm-egg fusion is still obscure.It had been known that the interaction between the sperm membrane protein IZUMO1 and its counterpart,oocyte membrane protein JUNO,is needed for the sperm-oocyte fusion.The somatic cells with IZUMO1 and with JUNO could bind with each other,but could not fuse,indicating that fusion of mammalian sperm-egg membrane should be a process of multimolecular cooperation.There must be other molecules implicated in this process following IZUMO1-JUNO interaction.In this study,CDON,a coreceptor of hedgehog signaling,was identified for its potential interaction with JUNO and IZUMO1,and the its effect on sperm-oocyte fusion in rat.Firstly,the cDNA of rat cdon was cloned.The pET-cdon prokaryotic expression vector,pGAD-cdon yeast expression vector,and p CMV-flag-cdon eukaryotic expression vector were constructed by inserting the cDNA of cdon into pET-28 a,pGAD-T7,and p CMV-C-flag plasmids,respectively.The pET-cdon prokaryotic expression vector was transformed into E.coli BL21(DE3)to induce the expression of recombinant HIS-CDON fusion protein.Then anti-CDON ascitic polyclonal antibodies were generated by immunizing mice with purified HIS-CDON fusion proteins.Immunofluorescence on rat ovaries were performed using anti-CDON ascitic polyclonal antibodies and the results showed that CDON was widespreaded in rat ovarian,mainly exists in eggs and surrounding granular cells.The immunofluorescence of rat oocyte and sperm showed that CDON located on the MII egg cell membrane,and co-located with JUNO,but do not on the sperm.In vitro fertilization rate was counted after the rat oocyte and sperm were incubated with anti-CDON ascitic polyclonal antibodies.The results showed that the fertilization rate slightly,but not statistically significantly decreased after antibody treatment.Y2H experiments were carried out with pGAD-cdon,pGBK-Juno and pGBKIzumo1.The results suggested that CDON can interact both with JUNO and IZUMO1.Then co-immunoprecipitation was performed with p CMV-Flag-cdon,p CMV-HA-Juno and p CMV-HA-Izumo1.The results further proved that CDON can interact with JUNO and IZUMO1.Taken together,that CDON interacted with JUNO and IZUMO1,and was expressed on the oocyte cell membranes,suggested the CDON play a role in spermoocyte fusion.