Expression of CD4/p56lck Complex on Murine Egg Vitelline Membrane and Its Role in Fertilization

Abstract

Corresponding to the presence of major histocompatibility complex (MHC) class II on murine sperm, CD4 on the murine egg vitelline (plasma) membrane was clearly shown by indirect immunofluorescence (IIF) test with anti CD4 monoclonal antibody (mAb). By means of immunoprecipitation, CD4 from murine eggs was found to have the same size as that of the authentic CD4 from T-cells. Furthermore, the full-length of CD4 cDNA from eggs was synthesized by RT/PCR-Southern blot hybridization. The sequence of CD4 cDNA from eggs was found to be identical with that of T cells. In addition, src-related tyrosine protein kinase (p56lck) was demonstrated to be associated with CD4 under neath the plasma membrane of eggs by IIF with anti-p56lck. The autophosphorylation of p56lck after cross-linking of CD4 with anti CD4 mAb has been shown in eggs as well as in T cells by immunocomplex kinase assay. The transcriptional expression of the signal transducing region of lck gene in eggs was further confirmed by RT-PCR method. The binding between eggs labeled with anti-CD4 mAb and sperm labeled with anti-monomorphic region of class II mAb was completely blocked. In addition, sperm cells adhered extensively to CD4-expressed Spodoptera frugiperda (Sf9) cells and the adhesion between both cells was specifically blocked by labeling Sf9-CD4 cells with anti-CD4 mAb and sperm cells with anti-class II mAb. Considering these findings together with the fact that an interspecies’ heterogeneity is present in CD4 amino acid sequence at the interactive site with class II, we strongly suggested that one of species’ specific intercellular adhesion between two gametes at the fusion step in fertilization is definitely mediated by class II located on the posterior region of sperm head and CD4 on the plasma membrane of egg associated with p56lck.