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：脾脏酪氨酸激酶（SYK）通过cAMP-PKA信号在精子连接和主要片段中被激活。精子和淋巴细胞之间SYK的激活机制有很大不同，提示精子SYK的激活受一种新的信号系统控制。SYK激活与精子受精能力表达关系的观察：cAMP类似物处理的精子鞭毛过度激活与SYK激活一致。这表明SYK在精子受精能力中起调节作用。睾丸SYK基因cDNA序列分析：从睾丸cDNA中扩增出的PCR产物（590bps）序列与猪脾脏SYK基因（SYK的c端催化结构域）序列一致，证实了睾丸中有SYK的产生。精子SYK底物鉴定及功能分析：SYK底物为磷脂酶c - γ1 （plc - γ1）。该酶在Y352残基上被camp依赖磷酸化，通过Y783残基的磷酸化结合并激活plc - γ - 1。此外，U-73122对plc - γ - 1的化学抑制对鞭毛过度激活的过表达有抑制作用。将plc γ - 1肽片段导入精子的试验：本实验虽然使用了两种市售试剂盒，但在方法学上存在试剂中未知因素和导入方案严重损害精子活力的问题。一种可能的引入多肽的方法仍有待改进。精子受精能力其他调控因子的鉴定及功能分析：精子PKC是通过cAMP-PKA信号调控的。Ro-32-0432对精子PKC的化学抑制作用可有效抑制鞭毛过度活化的过表达。

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.