Department of Obstetrics and Gynecology, West Virginia University, School of Medicine, Morgantown, WV 26505-9186, USA.
J Reprod Immunol. 2010 Jan;84(1):24-31. doi: 10.1016/j.jri.2009.10.006. Epub 2009 Nov 27.
Sperm proteins that interact with zona pellucida 3 (ZP3) have not been clearly identified in humans. In the present study, the yeast two-hybrid (Y2H) system was used to identify human sperm proteins that interact with human ZP3. Human ZP3 cDNA was cloned into pAS2-1 yeast vector and used as bait to find reactive proteins in the human testis cDNA library. Six specific clones were obtained that were further confirmed for interaction using the mammalian two-hybrid system. These six clones showed homologies with several proteins in the GenBank database. Of these, the strongest ZP3-interacting protein, that shows 97% homology with ubiquitin associated protein-2 like (UBAP2L), was tested in the hemizona assay. UBAP2L antibodies significantly (p<0.001) inhibited human sperm-zona binding in this assay. We conclude that the Y2H system is a useful strategy for identifying novel genes encoding proteins that interact with ZP proteins. To our knowledge, this is the first study using the Y2H system to identify sperm proteins that interact with human oocyte ZP3. Novel proteins identified using this system may find applications in elucidating the fertilization cascade, development of a new generation of non-steroidal contraceptives, and specific diagnosis and treatment of human infertility.
在人类中，与透明带 3（ZP3）相互作用的精子蛋白尚未被明确鉴定。在本研究中，使用酵母双杂交（Y2H）系统来鉴定与人类 ZP3 相互作用的人类精子蛋白。将人类 ZP3 cDNA 克隆到 pAS2-1 酵母载体中，并用作诱饵，以在人睾丸 cDNA 文库中寻找反应性蛋白。获得了六个特异性克隆，进一步使用哺乳动物双杂交系统证实了它们的相互作用。这些六个克隆与 GenBank 数据库中的几种蛋白质具有同源性。其中，与泛素相关蛋白-2 样（UBAP2L）具有 97%同源性的最强 ZP3 相互作用蛋白，在半透明带试验中进行了测试。UBAP2L 抗体在该试验中显著（p<0.001）抑制了人精子与透明带的结合。我们得出结论，Y2H 系统是鉴定与 ZP 蛋白相互作用的新基因编码蛋白的有效策略。据我们所知，这是首次使用 Y2H 系统鉴定与人类卵透明带 3 相互作用的精子蛋白的研究。使用该系统鉴定的新蛋白可能有助于阐明受精级联、开发新一代非甾体避孕药以及人类不育症的特异性诊断和治疗。
