The Functional Mode And Localization Of Acrosin In Chinese Mitten Crab, Eriocheir Sinensis

Chinese mitten crab, Eriocheir sinensis, is an important kind of crab with high economical and nutritional value. It provide a good model for the research in fields like developmental biology, evolutionary biology and reproductive biology. The current cultivation of Chinese mitten crab in China witness the increase in production as well as the decline in quality and promiscuity in species. To solve this problem, pivotal is to pay attention on the reproductive research of E.sinensis.Spermatogenesis and fertilizaiton are two important issues in reproductive biology. The most serious obscurity in fertilization is the complexity in the recognization, penetration and fusion of gametes. In E.sinensis, the spermatogenesis, including speriogenesis, is just as complex as that in mammals. Acrosin is an important serine protease in mammalian fertilizaiton. It is of great significance to explore the function of acrosin in inferior animals like crab, so as to unveil the complexity of their reproduction. This current project, taking the spermatogenesis in crab for the model, contribute to expand our understanding of the mechanism underlying their reproduction. We cloned the full-length cDNA of acrosin in Chinese mitten crab, and employ techniques including western blotting and immunofluorescence, to investigate the important role of acrosin in the spermatogenesis and fertilization of E.sinensis.A. The expression specificity of acroisn in E.sinensis. Initially, the cDNA cloning of acrosin in E.sinensis demonstrate its expression. Moreover, the RT-PCR and western blotting results indicate the expression specificity of acrosin in testis, on the level of mRNA and protein relatively. Besides, qRT-PCR result reveals the elevating expression of acrosin in mature crab in comparison to the young induviduals. This also implicates the requirement of acrosin for a crab individual, and the significance of acrosin to reproduction.B. The prediction of tertiary structure of E.sinensis acrosin. The prediction of tertiary structure illustrates the zymogen domain and catalytic domain of E.sinensis acrosin protein. The relative function of these two domains, may reveal the role of acrosin during fertilization.C. Localization of acrosin during spermatogenesis in E.sinensis. Acrosin is expressed and accumulated initially in spermatocytes and early spermatids. In these cells, acrosin was distributed on one side of the nucleus, revealing the probable sites of proacrosomal vesicles. Since accumulated in middle spermatids, in late or final spermatids, acrosin is distributed specifically in the acrosomal tubule and inner acrosomal membrane, which reveal the two main roles of acrosin. Localizing in acrosomal tubule, acrosin will be delivered upon the recognition of two gametes during fertilization. Acrosin functions as a serine protease and contributes to the penetration of zona pellucida; Localizing on the inner acrosomal membrane, acrosin functions as an activator and hydrolyze other important proteases in acrosme, thus promote their participation in fertilization.D. Co-localization of acrosin and GM130revealing the origination of acrosome. Acrosin is an important component and well demonstrated to be localized in the acrosome with the evidence provided in this project. In this context, acrosin can be a promising marker for acrosome. On the other hand, GM130is a marker of Golgi’s apparatus. The results from co-localization of acrosin and GM130, illustrate remanent GM130signal in the acrosome of final spermatids. As a vesicle organelle, acrosome may be demonstated to be originated from Golgi’s apparatus in this context.