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

1.cAMP依赖蛋白酪氨酸激酶的鉴定：通过cAMP-PKA信号在精子连接部和主体部激活了一个脾酪氨酸激酶(SYK)。精子和淋巴细胞激活SYK的机制有很大不同，提示精子SYK的激活受一种新的信号系统调控。2.SYK激活与精子受精能力表达的关系观察：cAMP类似物处理的精子表现为鞭毛过度激活，与SYK激活一致。3.睾丸SYK基因序列分析：扩增产物(590bps)与猪脾SYK基因(SYK的C端催化结构域)序列一致，证实了SYK在睾丸中的表达。4.精子SYK底物的鉴定及功能分析：SYK底物为磷脂酶Cγ1(PLCγ1)。该酶在Y352残基被cAMP依赖的磷酸化，通过在Y783残基的磷酸化结合并激活PLCγ1。此外，U-73122对PLCγ1的化学抑制可有效抑制鞭毛过度激活的过度表达。5.尝试将PLCγ1肽片段导入精子：虽然本实验使用了两种商品化的试剂盒，但我们面临着试剂中包含的未知因素和导入方案严重损害精子活力的方法学问题。6.精子受精能力其他调节因子的鉴定和功能分析：精子蛋白激酶C是通过cAMP-PKA信号调控的。Ro-32-0432对精子蛋白激酶C的化学抑制可有效抑制鞭毛过度激活。

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.