【作者】 何莹；

【导师】 侯林； 杨万喜；

【作者基本信息】 辽宁师范大学 ， 海洋生物学， 2009， 硕士

【摘要】 本文采用免疫荧光、免疫电镜和RT-PCR的方法,对肌球蛋白Myosin Va在中华绒螯蟹精子发生过程中的空间分布与表达进行研究。精子发生是一个复杂的细胞改变的过程,从干细胞、精母细胞转化成成熟的精子,包括精子形成阶段有丝分裂和减数分裂中的纺锤体的形成,细胞骨架的改变,膜区域的形成和重组。这些改变和细胞的重组是理解马达分子蛋白在细胞改变中机制的理想模型。哺乳动物的精子发生一直是近年来国内外研究的热点问题,但较低等动物如甲壳动物(Crustacea)中,精子结构、形态及精子发生调控的分子机制都跟哺乳动物的精子存在显著差别,它们的精子不具有微管套和精子尾部。而这恰恰是研究的薄弱环节。目前,学术界对肌球蛋白在十足类甲壳动物精子形成过程中作用的分子机制研究尚属空白。因此研究肌球蛋白Myosin Va在中华绒螯蟹精子发生过程中的空间分布与表达具有重要意义。肌动蛋白是马达分子蛋白之一。其在精子发生过程中改变精细胞形状,运输线粒体,参与顶体发生过程。许多肌动蛋白微丝的末端结合蛋白(包括myosin蛋白)在肌动蛋白的聚合和解聚方面起到重要的作用。肌动蛋白在精子细胞中存在多种结构,肌动蛋白和它相关的蛋白家族myosin的关系及它们在精子发生过程中的作用越来越引起人们的重视。所有的肌动蛋白的马达蛋白都属于肌球蛋白(myosin)家族。Myosin Va是在脊椎动物中表达的三种肌球蛋白V(myosins V)之一,是最具典型特征的myosins V。Myosin-Va参与小泡的运输,参与在肌动蛋白-微管的交叉处的从肌动蛋白(actin)到微管(microtubule)上的转换,以及在微管上短距离运输。本研究首次确定myosin Va在中华绒螯蟹精巢中表达,且其与哺乳动物精子中的acroplaxome结构类似,并首次观察到该物种的精子形成过程及肌球蛋白Va在精子发生过程中的空间分布。将对其进一步研究提供有价值参考,并对未来进一步研究myosin Va在其它较低等动物的精子发生中的机制奠定了基础。本研究的实验结果是myosin Va在中华绒螯蟹的精子发生过程中表达,并且存在着规律的空间分布模式:(1)Myosin Va在精原细胞和精母细胞时期主要分布在细胞核周围的细胞质中;(2)Myosin Va在精细胞早期时分布在细胞核的前极性端;(3)Myosin Va在精细胞分化过程的分布:只在成熟精子中表达;(4)Myosin Va分布在成熟精子的细胞核内和顶体与细胞核中间的核内膜处。我们还利用免疫电镜的方法,发现了中华绒螯蟹精子形成的过程及该过程中myosin Va的表达情况。更多还原

【Abstract】 The paper used the methods of Immunofluorescence, Immunogold electron microscopy and RT-PCR to study myosin Va in Chinese mitten crab, Eriocheir sinensis, and variational distribution during spermatogenesis.Spermatogenesis involves intricate cellular change in order to transform the precursor germ cell, the spermatogonium, into a mature spermatozoon. Including formation of mitotic and meiotic spindles, changes in the cytoskeleton, formation and redistribution of membrane domains occur during spermiogenesis. The nature of these changes and cellular rearrangements makes an ideal model to spermatogenesis for the understanding the role of molecular motor proteins in cellular change. It has been a hot research about spermatogenesis in mammals in recent years, but there was a significant difference between the sperm structure, morphology and the molecular regulation mechanism in lower animals such as crustaceans (Crustacea)and that in mammalian sperm.It is known too little about spermatogenesis in lower animals.So it has great significance to study myosin Va in Chinese mitten crab, Eriocheir sinensis, and variational distribution during spermatogenesis.Actins provide diverse structures in sperm cells, and actins have been shown to play major roles during spermatogenesis, changing cell shapes, transporting mitochondria, participating in acrosomal biogenesis. Many actin filament end-binding proteins play essential roles in polymerization and depolymerization of actin which occur only at the filament ends. Meanwhile, actin-based motor proteins, myosin, have implicated diverse functions during spermatogenesis. The molecular relationships between actin and its related myosin, as well as their functions during spermatogenesis attract much attention in recent years.All actin filament motor proteins belong to the myosin family. Myosin Va, one of three myosins V expressed in vertebrates, is the best characterized of the Class V myosins. all actin filament motor proteins belong to the myosin family.Myosin Va has been shown to be involved in transporting cargo to the cell periphery and can switch from actin to microtubules at an actin–microtubule intersection and diffuses along the microtubule for a short distance.The study will provide valuable references for the further studies.The results of this study is myosin Va is expressed, localized and regulated during spermatogenesis of Chinese mitten crab: (1) The predominant localization of myosin Va around the nucleus in the cytoplasm of in early spermatogenesis;(2) Myosin Va mainly localizes on one side of early spermatid nuclei;(3) Myosin Va only expressed in the mature sperms after the early spermatid differentiation. (4) Myosin Va is expressed in inner side of nuclei and within nuclei of spermatozoa。Also, we used Immunogold electron microscopy to find the process of spermiogenesis and localisation of myosin Va in spermiogenesis.更多还原