The Interaction Of LGALS1 And LGALS3 With JUNO,IZUMO1 And CD9 In Rats

Sexual reproduction is the basic pathway for the reproduction of higher eukaryotes.The fusion of sperm and egg is a key step in sexual reproduction,including the process of gamete binding and membrane fusion.Mammalian sperm-egg fusion is a multi-molecular process,but the detailed underlying cellular and molecular mechanisms are still unclear.Interaction between IZUMO1 on sperm and JUNO,CD9 on egg is known to be indispensable for sperm-egg membrane fusion.Among them,IZUMO1 and JUNO identify each other and mediate the adhesion between sperm and egg.However,that somatic cells expressing IZUMO1 bind to those expressing JUNO,but they do not fuse,remind that other gametic proteins might assist IZUMO1 and JUNO during the sperm-egg fusion process.In this paper,two members of galectins family,LGALS1 and LGALS3,were investigated for their interaction with IZUMO1 and JUNO by yeast-two hybrid and indirect immunofluorescence assays,as a part of mission of searching the proteins that bind to IZUMO1 or/and JUNO.Firstly,the cDNA of rat lgals1 and lgals3 were cloned,and yeast expression vector pGAD-lgals1 and pGAD-lgals3 were constructed.Then yeast two-hybrid were performed for the pairs of pGAD-lgals1 with pGBK-Juno,pGAD-lgals1 with pGBK-Izumo1 and pGAD-lgals1 with pGBK-Cd9 respectively.The results showed that LGALS1 could bind to JUNO,but could not bind to IZUMO1 and CD9,whereas LGALS3 did not bind to JUNO and IZUMO1.The prokaryotic expression vector pGEX-lgals1 was constructed and transformed into E.coli BL21(DE3).GST-LGALS1 fusion protein was isolated and purified from the bacteria by inducing the pGEX-lgals1 expression with IPTG.Anti-GST-LGALS1 ascites polyclonal antibodies were generated by immunizing mice with the GST-LGALS1 fusion protein and were purified by glutaraldehyde cross-linking method to remove the antibodies against GST.Immunohistochemical stains were performed on serial sections of rat testis and ovary using the purified anti-LGALS1 ascites polyclonal antibodies.The results showed that in rat testis,LGALS1 was located in the cytoplasm of the sertoli cells and spermatozoa among various developmental stages of sperm cells;while in rat ovary,LGALS1 was located in the cytoplasm and on the plasma membrane of cumulus cells and the oocyte.Indirect immunofluorescence cytochemistry was carried out on sperm and egg with purified anti-LGALS1 ascites polyclonal antibodies and anti-JUNO antibody to reveal the subtle location and co-localization of LGALS1 and JUNO.The results showed that LGALS1 was located on the whole head and the first half of the tail of rat sperm,on the mouse egg of MII phase,LGALS1 was located on the cell membrane and the zona pellucida around.Co-localization of LGALS1 with JUNO was observed on the plasma membrane of MII oocyte.This experiment found that LGALS1 interacts with JUNO,and LGALS1 is expressed in both reproductive tissues and germline cells.It is speculated that LGALS1 may be involved in the process of sperm-egg membrane fusion,providing new clues for further analysis of the molecular mechanism of sperm-egg fusion.