Rajeev S K, Reddy K V R
Division of Immunology, National Institute for Research in Reproductive Health, Indian Council of Medical Research, Mumbai, India.
Hum Reprod. 2004 Feb;19(2):234-42. doi: 10.1093/humrep/deh066.
Male infertility is the major cause of conception failure in about 25% of all infertile couples. Understanding the causes of male infertility depends to a certain extent on the proteins present on the spermatozoa. The study aim was to investigate first, whether there is any difference in the expression of sperm membrane proteins between fertile and infertile males; and second, whether there is any functional significance of these proteins in the spermatozoa.
Six different protocols were employed to extract sperm membrane proteins. A 57 kDa protein was identified and purified using different chromatographic techniques. The homogeneity and isoelectric point of the protein was confirmed by 2D-electrophoresis. The protein was characterized by immunofluorescence, ELISA, flow cytometry, SDS-PAGE and Western blot analysis. The role of 57 kDa protein in sperm-oocyte binding was studied in vitro.
All six sperm extracts of normozoospermic and infertile subjects showed 16-18 major and 12-15 minor protein bands. However, in one of the methods, the lysis buffer containing N-octyl-beta-D-glycopyranoside (NOG) resulted in an additional protein band at the 57 kDa region in 95% of normozoospermic samples. The protein was either absent (approximately 80%) or negligible (approximately 20%) in infertile subjects. The protein was localized to the head of non-acrosome-reacted spermatozoa (NAR), and shifted to the equatorial segment in acrosome-reacted (AR) spermatozoa. The antibody directed against purified 57 kDa protein inhibited binding of human sperm to zona-free hamster oocytes in a dose-dependent manner.
The results suggest that lack and/or low expression of 57 kDa protein may be one of the reasons for infertility in men. Therefore, the protein could be used as a marker for sperm quality in men.
男性不育是约25%不育夫妇受孕失败的主要原因。了解男性不育的原因在一定程度上取决于精子上存在的蛋白质。本研究的目的首先是调查生育男性和不育男性精子膜蛋白的表达是否存在差异；其次是这些蛋白质在精子中是否具有任何功能意义。
采用六种不同方案提取精子膜蛋白。使用不同的色谱技术鉴定并纯化了一种57 kDa的蛋白质。通过二维电泳确认该蛋白质的同质性和等电点。通过免疫荧光、酶联免疫吸附测定、流式细胞术、十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和蛋白质印迹分析对该蛋白质进行表征。在体外研究了57 kDa蛋白质在精子 - 卵母细胞结合中的作用。
正常精子症患者和不育患者的所有六种精子提取物均显示出16 - 18条主要蛋白带和12 - 15条次要蛋白带。然而，在其中一种方法中，含有N - 辛基 - β - D - 吡喃葡萄糖苷（NOG）的裂解缓冲液在95%的正常精子症样本中导致在57 kDa区域出现一条额外的蛋白带。在不育患者中，该蛋白质要么缺失（约80%），要么含量极少（约20%）。该蛋白质定位于未发生顶体反应的精子（NAR）头部，并在发生顶体反应的（AR）精子中转移至赤道段。针对纯化的57 kDa蛋白质的抗体以剂量依赖方式抑制人精子与去透明带仓鼠卵母细胞的结合。
结果表明57 kDa蛋白质的缺乏和/或低表达可能是男性不育的原因之一。因此，该蛋白质可作为男性精子质量的标志物。
