Duncan A E, Fraser L R
Biomedical Sciences Division, King's College London, UK.
J Reprod Fertil. 1993 Jan;97(1):287-99. doi: 10.1530/jrf.0.0970287.
Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.
在旨在调控获能以及与完全受精能力相关参数（即超活化运动和顶体反应）表达的培养基中，研究了附睾小鼠精子悬液中依赖环磷酸腺苷（cAMP）的磷酸化变化。在使用外源底物初步评估冻融和冻干精子悬液中cAMP依赖性蛋白激酶活性后，使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳，随后进行放射自显影或免疫印迹，检测内源性精子磷蛋白的磷酸化情况。在未获能和获能的悬液中均检测到大量磷蛋白，其中大多数可能与运动有关；从这些悬液中32P掺入量的减少推断，受精能力的完全表达似乎涉及内源性磷酸化量的增加。添加cAMP依赖性蛋白激酶抑制剂H8和PKI(6 - 22)酰胺表明，检测到的大多数磷蛋白是以cAMP依赖性方式磷酸化的。特别令人感兴趣的是一种分子量约为95,000的磷蛋白，在获能悬液中一直能观察到。有证据表明，该蛋白可能在酪氨酸残基上发生磷酸化，因为加入磷酸酪氨酸磷酸酶抑制剂原钒酸盐会改变该蛋白的磷酸化。此外，使用抗磷酸酪氨酸抗体PY - 20进行免疫检测，鉴定出了五种分子量约为116,000、105,000、95,000、86,000和76,000的蛋白，可能还有一种分子量为54,000的第六种蛋白。在经离子载体处理的精子中，95,000蛋白一直减少，表明该蛋白位于顶体帽区域。这些结果表明，该蛋白可能与Leyton和Saling(1989)描述的含磷酸酪氨酸蛋白相同，后者被认为在顶体胞吐作用中发挥作用。
