Interration Between RTP4 And IZUMO1 And Its Effect On Fertilization

The most important part of sexual reproduction in mammalian reproduction is fertilization.The whole process of fertilization includes sperm capacitation,zona pellucida recognition,acrosome reaction and sperm-egg membrane fusion.It is known that IZUMO1 on the sperm and JUNO on the egg are the key molecules for sperm-egg membrane fusion.However,that the somatic cells with IZUMO1 and with JUNO could bind with each other,but could not fuse,indicated that mammalian sperm-egg membrane fusion should be a process of multi-molecular cooperation.There must be other molecules implicated following IZUMO1-JUNO interaction.In this study,the interaction of RTP4 with IZUMO1 and JUNO and the effects of RTP4 on fertilization were studied by various experimental techniques,to provide new data for understanding the molecular mechanism of mammalian sperm-egg membrane fusion.Firstly,the rat rtp4 cDNA was cloned,and the yeast expression vector pGADrtp4 and eukaryotic expression vector pCMV-C-Flag-rtp4 were constructed.The yeast two hybridized experiments were performed between pGAD-rtp4 and pGBK-Izumo1,pGAD-rtp4 and pGBK-juno,respectively.The results showed that RTP4 interacted with IZUMO1,but not with JUNO.The interaction between RTP4 and IZUMO1 was confirmed then by,the coimmunoprecipitation carried out by co-transfecting pCMVC-Flag-rtp4 with pCMV-C-HA-Izumo1 into 293 T cells.Then,the prokaryotic expression plasmid pET-28a-rtp4 was constructed,and the HIS-RTP4 fusion protein was obtained and purified after the plasmid pET-28a-rtp4 was transformed into E.coli BL21(DE3).Then the mice were immunized with purified HIS-RTP4 fusion protein and anti-RTP4 ascitic polyclonal antibody were harvested.The paraffin section of mouse testis and ovary tissues were prepared and indirect immunofluorescence staining was performed with anti-RTP4 ascitic polyclonal antibody.The results showed that: RTP4 was expressed in the cytoplasm of spermatogonia,spermatocytes,sertoli cells,and mature sperm;and in the cytoplasm of cumulus cells and on the membrane of MII oocytes.In addition,indirect immunofluorescence staining was also performed to investigate the locolization of RTP4 and colocolization whith IZUMO1 in mouse and rat gametes.The results showed,that RTP4 was located in the acrosome and tail of rat sperm before acrosome reaction,and located in the front of the head,and in the front half of the tail after acrosome reaction;that RTP4 was located in the acrosome and front half of mouse sperm before acrosome reaction,and located in the whole head and front half of the tail after acrosome reaction;that RTP4 was located on the cell membrane of mouse MII oocytes;that RTP4 and IZUMO1 were co-located in the acrosome of rat or mouse sperm before acrosome reaction;but there was no co-localization of RTP4 and IZUMO1 in rat or mouse sperm after acrosome reaction.Finally,effect of treatments of mouse sperms and eggs with anti-RTP4 ascitic polyclonal antibody on fertilization was tested by IVF assay.The results showed that treatments of RTP4 slightly reduced fertilization rate,but nonsignificanly.The finds that RTP4 is expressed in the reproductive tissues and germ cells of rats and mice and interacts with IZUMO1 give speculation that RTP4 should be involved in sperm-egg fusion.