水牛附睾不同部位精子超微结构及荧光标记差异分析

doi:10.11843/j.issn.0366-6964.2021.012.012

摘要/Abstract

摘要： 哺乳动物精子经过附睾成熟后才能获得运动及受精的能力，为解释水牛精子在附睾中的成熟过程，本研究选用性成熟期的沼泽型水牛附睾，利用乙烯吡咯烷酮包裹的硅胶微小颗粒（Percoll）梯度离心纯化分别提取附睾头、体和尾部精子，应用计算机辅助精子分析系统（CASA）检测精子活力，透射电镜观察附睾不同部位精子的超微结构，对精子进行荧光标记后，利用流式细胞仪和荧光显微镜观察检测不同部位精子质膜完整率、线粒体鞘膜电位和顶体差异。结果表明，Percoll分离得到附睾头、体和尾3部位精子的纯度达95%，不同部位精子活力分别为8.35%、20.21%和65.60%；附睾不同部位精子都存在着结构完整的精子以及相同的畸形类型，附睾尾部精子线粒体鞘高膜电位比率最高，精子质膜完整率从附睾头部到尾部逐渐升高，精子顶体完整率从附睾头部到尾部逐渐升高。本研究直观地展示了水牛附睾不同部位精子特征以及差异，为研究水牛精子成熟机理提供理论依据。

关键词: 附睾, 精子, 超微结构观察, 荧光分子探针, 顶体

Abstract: In mammals, spermatozoa acquired the ability of motility and fertilization only after being matured in the epididymis. To explain the maturation process of buffalo spermatozoa in the epididymis, sexually mature buffalo (Bubalus bubalis) epididymis were selected and the spermatozoa from the caput, corpus and cauda of the epididymis were purified by Polyvinylpyrrolidone coated silica (Percoll) gradient centrifugation. Computer-assisted sperm analysis system (CASA) was used to examine sperm motility, transmission electron microscopy to observe the ultrastructure of spermatozoa in different regions of the epididymis, and after fluorescent labeling of spermatozoa, flow cytometry and fluorescence microscopy were employed to determine the plasma membrane integrity, mitochondrial membrane potential and acrosome variations in different regions of the spermatozoa. As the result showed, the purity of spermatozoa obtained from the head, body and tail of epididymis by Percoll were 95%, whereas the motility of spermatozoa in various regions was 8.35%, 20.21% and 65.60%, respectively; All the spermatozoa in different regions of epididymis existed the same abnormality pattern, and the spermatozoa in the cauda of epididymis showed the highest ratio of mitochondrial membrane potential, the sperm plasma membrane integrity rate and and the sperm acrosome integrity rate increased gradually from the caput to the cauda region of epididymis. Overall, this study demonstrated the characterization and variation of spermatozoa in distinct regions of the buffalo epididymis which provides a theoretical basis for investigating the mechanism of sperm maturation.

Key words: epididymal, sperm, ultrastructure observation, fluorescent molecular probe, acrosome

中图分类号:

何翁潭, 张鹏飞, 黄愉淋, 肖凯, 黄良凤, 陆阳清, 张明. 水牛附睾不同部位精子超微结构及荧光标记差异分析[J]. 畜牧兽医学报, 2021, 52(12): 3449-3460.

HE Wengtan, ZHANG Pengfei, HUANG Yulin, XIAO Kai, HUANG Liangfeng, LU Yangqing, ZHANG Ming. Analysis of Ultrastructure and Fluorescent Markers of Sperm in Different Parts of Buffalo Epididymis[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3449-3460.

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