Study on the mechanisms of oocyte-sperm interaction in fertilization process

通过对精子核蛋白的研究，揭示了人类精子解聚的过程。使用体外解聚系统，可以清楚地观察到精子的核转化。解聚体系为0.05%溶血磷脂-5 mM二硫苏糖醇-1.1NaC l溶液。在该系统中，大多数精子核完全解聚。用35%的冰醋酸或0.4%的H_2SO_4从去凝集的细胞核中提取碱性核蛋白，用十二烷基硫酸钠凝胶电泳法分离并转移到硝酸纤维素膜上。与抗人组蛋白H_2B抗体和HRP标记的二抗孵育后，观察到印迹反应。该斑点显示出66 kDa的迁移率。用抗组蛋白H_2B抗体和FITC标记的二抗，在玻片上观察了逐渐解聚的精子核的形态和荧光变化。这些结果表明，在精子核成熟过程中，核蛋白被鱼精蛋白取代后，人精子核中含有大量的组蛋白。

Sperm nuclear proteins were studied to reveal the process of human sperm decondensation. Using in vitro decondensing system sperm nuclear transformation were clearly observed. This decondensing system was 0.05% lysolecithin - 5mM dithiothreitol - 1.OM NaCl solution. Most of sperm nuclei were decondensed completely in this system. Basic nuclear proteins were extracted from decondensed nuclei with 35% acetic acid or O.4N H_2S0_4, and were separated on SDS-PRGE and transferred onto a nitrocellulose membrane. Following incubation with anti-human histone H2B antibody and HRP-labelled secondary antibody, a blot was observed. This blot exhibited mobilities of 66 kDa. Furthermore another experiment showed the existence of histone H2B.Using anti- histone H2B antibody and FITC-labelled secondary antibody, the morphological and fluorescent changes of gradually decondensing sperm nuclei were observed on a glass slide. These results suggested that human sperm nuclei contain histones considerably after nucleoproteins were replaced by protamines in the process of sperm nuclear maturation.