获能处理对猪精子质量及泛素对顶体酶抑制剂水平调控的影响

doi:10.16431/j.cnki.1671-7236.2023.01.018

摘要/Abstract

摘要： 【目的】探究猪精子体外获能前后顶体酶抑制剂（AI）表达量以及AI的泛素化水平的变化，了解AI与泛素-蛋白酶体系统（UPS）间的联系，为深入研究泛素-蛋白酶体途径（UPP）在猪精子获能过程中的作用提供参考。【方法】选择18~24月龄的成年健康长白公猪，使用手握法采集公猪精液，将一部分精子进行获能处理，一部分作为对照（鲜精），使用计算机辅助精子分析系统（CASA）、低渗肿胀法（HOST）、考马斯亮蓝染色、Western blotting和锌离子（Zn²⁺）标记检测获能前后精子的动力学参数、质量参数、酪氨酸磷酸化水平和Zn²⁺含量；Western blotting检测获能前后精子中AI和泛素（Ub）的表达量；免疫荧光法检测AI和Ub在精子中的定位；免疫共沉淀分析AI和Ub的结合情况。【结果】与新鲜精子相比，获能精子动力学参数VSL和BCF极显著升高（P<0.01），获能精子的活力、质膜完整性、顶体膜完整性、活率均极显著降低（P<0.01）；获能精子蛋白酪氨酸磷酸化水平极显著升高（P<0.01），Zn²⁺极显著外流（P<0.01）。Western blotting检测结果表明，与新鲜精子相比，获能精子AI表达量极显著降低（P<0.01），Ub表达量显著升高（P<0.05）；免疫荧光结果表明，AI和Ub均定位于精子头部顶体区域；免疫共沉淀分析结果显示，获能精子AI发生了泛素化，在抑制泛素激活酶（E1）后，泛素化的AI极显著减少（P<0.01）。【结论】在猪精子体外获能期间，精子蛋白酪氨酸磷酸化水平极显著提高、泛素化水平增强，且AI被降解，从而释放有活性的顶体酶，为后续精子穿透透明带（ZP）与卵母细胞结合完成受精做准备。

关键词: 猪; 精子; 获能; 顶体酶抑制剂(AI); 泛素化

Abstract: 【Objective】 The aim of this study was to investigate the changes of acrosin inhibitor (AI) expression and AI ubiquitination level before and after boar sperm capacitation in vitro, and to understand the relationship between AI and ubiquitin-proteasome system (UPS), so as to provide a reference for further study of the role of ubiquitin-proteasome pathway (UPP) in boar sperm capacitation.【Method】 Adult healthy Landwhite boars aged 18-24 months were selected, and boar semen was collected by hand-holding method, a part of sperm was capacitated, and a part of sperm was as control (fresh sperm).The kinetic parameters, quality parameters, tyrosine phosphorylation level and zinc ions (Zn²⁺) content of sperm were detected by computer assisted sperm analysis system (CASA), hypotonic swelling method (HOST), Coomassie brilliant blue staining, Western blotting and Zn²⁺ labeling, respectively.Western blotting was used to detect the expression of AI and ubiquitin (Ub) in sperm before and after capacitation.The localization of AI and Ub in sperm was detected by immunofluorescence.The binding of AI and Ub was analyzed by immunoprecipitation.【Result】 Compared with fresh sperm, the kinetic parameters VSL and BCF of capacitated sperm were extremely significantly increased (P<0.01), and the motility, plasma membrane integrity, acrosomal membrane integrity and viability of capacitated sperm were extremely significantly decreased (P<0.01).The tyrosine phosphorylation level of capacitated sperm protein was extremely significantly increased (P<0.01), and Zn²⁺ efflux was extremely significantly increased (P<0.01).The results of Western blotting showed that compared with fresh sperm, the expression of AI in capacitated sperm was extremely significantly decreased (P<0.01), and the expression of Ub was significantly increased (P<0.05).Immunofluorescence results showed that both AI and Ub were located in the acrosome region of sperm head.The results of immunoprecipitation analysis showed that AI was ubiquitinated in capacitated sperm, and the ubiquitinated AI was extremely decreased (P<0.01).【Conclusion】 During in vitro capacitation of boar sperm, the tyrosine phosphorylation level of sperm protein was extremely significantly increased and the ubiquitination level was enhanced, and AI was degraded, thereby releasing active acrosomal enzymes, in order to prepare for the subsequent sperm penetration through the zona pellucida (ZP) of oocytes to complete fertilization.

Key words: pigs; sperm; capacitation; acrosin inhibitor (AI); ubiquitination

中图分类号:

S828

尹伊, 吕艳秋, 陈璇, 曹立朋, 张君正, 金一. 获能处理对猪精子质量及泛素对顶体酶抑制剂水平调控的影响[J]. 中国畜牧兽医, 2023, 50(1): 174-185.

YIN Yi, LYU Yanqiu, CHEN Xuan, CAO Lipeng, ZHANG Junzheng, JIN Yi. Effects of Capacitation on Sperm Quality and Regulation of Acrosin Inhibitor Levels by Ubiquitin in Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 174-185.

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