Molecular approach to mammalian fertilization and its application to gene transfer into fertilized egg

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By means of monoclonal technology, we obtained six hybridoma cell lines producing mAbs to the porcine zona pellucida (ZP). The epitopes recognized by these mAbs were chemically characterized. Four of them recognized a porcine ZP-specific primary structure of protein and two of them recognized a steric structure of the protein similar in both porcine and human ZPs.Furthermore, the biochemical analysis of carbohydrate structure of porcine ZP revealed that considerable quantities of N-linked, neutral oligosaccharides in porcine ZP were terminated with N-acetylglucosamine residues, suggesting the possible roles of these sugar chains as the acceptors for the galactosyltransferase on sperm.Following the studies of sperm acceptor on ZP, we found the expression of MHC class II molecule on the posterior region of sperm and its possible roles of foreign DNA binding to sperm.Corresponding to the MHC class II molecule on sperm, CD4 molecule associated with p56^<lck> was definitely observed on the vitelline membrane of egg. Besides, the adherence between the posterior region of sperm head and the vitellus of egg was mediated by MHC class II molecule of sperm and CD4 molecule of egg and their signal into egg activation was transduced through p56^<lck> at the fusion step of murine fertilization.Actually, RNA transcript in murine eggs corresponding to parts of the CD4 gene and lck gene in thymocytes were obtained by using RT/PCR methods followed by the sequencing of PCR products. The deduced sequences potentially encode the aminoacid sequence of transmembrane to cytoplasmic domain of CD4 and that of N-terminal domain of p56^<lck> respectively. These results demonstrated that the signal transducing complex similar to that in immune T cells are expressed at the transcriptional levels in murine eggs.