An invention of method to suppress expression of sperm fertilizing ability for the purpose of establishment of production system of porcine embryos

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1.Identification of cAMP-dependent protein tyrosine kinase : A spleen tyrosine (Y) kinase (SYK) was activated via the cAMP-PKA signaling in the sperm connecting and principal pieces. The mechanism for SYK activation is quite different between spermatozoa and lymphocytes, suggesting that the activation of sperm SYK is controlled by a novel signaling system.2.Observation of relationship between SYK activation and expression of sperm fertilizing ability : The cAMP analog-treated spermatozoa exhibited flagellar hyperactivation coincidently with the SYK activation. This indicates a role of SYK as a regulator for sperm fertilizing ability.3.Analyses of cDNA sequence of testicular SYK : The sequence of PCR product (590 bps) amplified from testicular cDNA was same as that of pig spleen syk gene (the C-terminal catalytic domain of SYK), confirming production of SYK in the testis.4.Identification and functional analyses of sperm SYK substrate : The SYK substrate was a phospholipase Cγ1 (PLCγ1). This enzyme, which was cAMP-dependently phosphorylated at the Y352 residue, bound to and activated PLCγ1 by the phosphorylation at the Y783 residue. Additionally, chemical inhibition of PLCγ1 with U-73122 was valid to the suppression of over-expression of flagellar hyperactivation.5.Trial to introduce PLCγ1 peptide fragments into spermatozoa : Although two kinds of commercial kits were used in this experiment, we faced problems of methodology that unknown factors included in the reagents and introduction protocol severely damaged sperm motility. A possible method for peptide introduction remains to be improved.6.Identification and functional analyses of other regulatory factors of sperm fertilizing ability : Sperm PKC was controlled via the cAMP-PKA signaling. The chemical inhibition of sperm PKC with Ro-32-0432 was valid to the suppression of over-expression of flagellar hyperactivation.