Development of diagnosis and therapy for male infertility ; The infertility-related gene approach including seminal plasma motility inhibitor

我们的目的是确定Sg对获能性的影响，这是精子必须经历的一系列转化才能成为可育的。我们用胎儿脐带血的超滤液诱导能化。我们观察到，Sg的存在抑制了人精子的过度活化。但由于CASA对精子运动模式的分析非常不稳定，因此没有发现Sg浓度对剂量的依赖性。以顶体反应为指标，我们发现Sg对人精子获能的抑制呈剂量依赖性。通过对Xantine氧化酶的化学发光测量，发现Sg抑制该过程的机制之一是干扰O_2的生成。我们还发现，经洗涤的精子孵育后，Sg被降解，降解产物也抑制了人精子的能化。这些数据表明，Sg和/或其降解产物可能是精子获能的天然调节剂。我们研究了sgi突变是否会导致不孕。我们之前报道了5名不育男性和8名可育男性的Sg I第二外显子缺失180bp，我们也在其中一名可育男性中发现了更小的Sg I缺失产物（比正常产物）。目前正在对突变形式蛋白进行纯化和分析，以确定蛋白质功能的差异。虽然Sg基因的SNP分析是通过DNA二维电泳方法进行的，但目前尚未得到证实。成功地表达了具有高活性和高纯度的r-Sg蛋白。产生这种r-Sg作为抗原的抗体与天然Sg和SPMI特异性反应。

Our aim was to determine the effect of Sg on capacitation, which is the series of transformations that spermatozoa must undergo to become fertile. We induced capacitation by ultrafiltrates of fetal cord blood. It was observed that hyperactivation of the human spermatozoa was suppressed by the existence of Sg. But the dose dependence on the concentration of Sg could not be found because the analysis on the motility pattern of spermatozoa by CASA was very unstable. Using the acrosome reaction as an index, we revealed that Sg suppressed capacitation of the human spermatozoa in the dose dependent manner. By the measurement of chemiluminescence using Xantine oxidase, one mechanism of suppressed capacitation by Sg was interference with O_2 generation during this process. We also found that Sg was degraded by the incubation with the washed spermatozoa and the degradation products also inhibited capacitation of the human spermatozoa. These data suggest that Sg and/or its degradation products may be natural regulators of sperm capacitation.We investigated whether the mutation of the Sg I causes infertility. We have previously reported a deletion of 180 bp in the second exon of the Sg I in 5 infertile men and 8 fertile men, and we also found the smaller products of deleted Sg I(than normal one)in one of these fertile men. The mutation form protein is currently being purified and analyzed to determine the difference in the protein function. Though the SNP analysis of the Sg gene is carried out by the DNA two-dimensional electrophoretic method, presently, it has not been confirmed. It succeeded in the expression of the r-Sg protein with much high activity and high-purity. The antibody that produced this r-Sg as an antigen peculiarly reacted with native Sg and SPMI.