Studies On Acroframosome And Potential Functional Analysis Of Spermatogenesis Related Proteins In Decapoda Crustaceans

The reproduction of commercial Crustacean species is a hot issue in the field of aquaculture in China. The mechanism of spermatogenesis, however, is one of the most important scientific problems in Reproductive Biology. It is also the basis of artificial fertilization of aquatic species. During spermatogenesis, the reconstruction of cytoskeleton and molecular regulation mechanism are the major focus in the field. Significantly different from mammalian spermatogenesis, Crustacean sperms do not possess flagellum. Their spermatogenesis process mainly contains acrosome formation and nucleus reshaping. Here, we studied the cytoskeleton structure, kinetics and physiology during spermatogenesis of Crustacean Decapoda species.First part:Cytoskeleton and its kinetic mechanism in spermatogenesis of Decapoda Natantia Eucarida Exopalaemon modestus Acroframosome (AFS), a cytoskeleton structure formed during spermiogenesis of Eucarida species, was first reported in spermatogenesis of Macrobrachium nipponense. However, its constitutions and functions have not yet been reported. Based on the model of E. modestus which is a major commercial shrimp species in Tai Lake, we explored whether AFS is the universally existed cytoskeleton structure in spermatogenesis of Eucarida. The immunofluorescence, which located Tubulin in different stages during spermiogenesis, proved that AFS structure also existed in Eucarida spermiogenesis. AFS is mainly constituted of microtubulin, and as predicted by its structure, it also contains a few microfilaments to sustain the cellular flexibility. This result shows that AFS is the cytoskeleton structure that exists in all Eucarida species.In order to study the motor protein that co-function with AFS, we cloned the kinesin protein kifc1 gene, and studied its structure, gene and protein distribution, and carried cargoes. Through immunofluorescence, we tried to co-localize organelle Golgi apparatus marker GM130 and mitochondria marker with KIFC1.We found that kinesin protein KIFC1 co-localized with the above organelles in the acrosome formation site and the edge of one side of the nucleus, indicating that these cellular components are cargoes transported by KIFC1. In sum, kinesin protein KIFC1, which is AFS cytoskeleton-dependent, forms a temporary organelle called the lamellar complex (LCx) in Eucarida spermatogenesis, and assists the formation of evaginable umbrella-like acrosome and the reshaping of disk-like nucleus in mature sperm.Second part:p53’role in germ cell apoptosis in spermatogenesis of Decapoda Reptantia Eriocheir sinensis In order to study the defense mechanism in Decapoda Crustacean animal’s normal spermatogenesis process, we cloned the homologous gene of p53 in the testis of E. sinensis. The sequence analysis shows that the p53 homolog has conservative DNA-binding domain and Zinc-binding domain. The semi-quantitative analysis of p53 gene in different tissues indicates that p53 is universally existed and is indispensable for life. Through in situ hybridization, we examined the expression pattern of p53 gene in each stage of spermiogenesis. Its gene expression pattern suggests that p53 might repair the impaired DNA to guarantee the normal spermatogenesis, or induce apoptosis to eliminate the abnormal spermatid. However, how p53 functions still require future examination.Third part:PHB mediated mitochonrial ubiquitination in spermatogenesis of Decapoda Reptantia Eriocheir sinensis The sperm of E. Sinensis has cup-shaped nucleus, which embedded several mitochondria at the opening of the cup. The acrosome vesicle also contains the derivants of mitochondria. The formation of mitochondria distribution pattern contains the processes of mitochondria number decrease, structure change, and transportation. The number decrease of sperm mitochondria can be achieved through autophagy or ubiquitination pathway. In order to study the mitochondria ubiquitination and degradation mechansim during spermatogensis of crustacean animals, we focus on a mitochondria inner membrane protein Prohibitin (PHB). PHB is an evolutionally highly conserved protein, and is closely associated with mammalian spermatogenesis and sperm quality control. It is discovered as a substrate of ubiquitin, and might function through ubiquitin-proteasome pathway (UPP). So as to study whether PHB protein mediate the ubiquitination pathway of sperm mitochondria, we prepared the specific antibody with the techniques including prokaryotic expression, recombinant protein purification, rabbit immunization and antibody purification. Western blot result shows that PHB protein is highly expressed in all the tested tissues. Through immunofluorescence, we examined PHB protein distribution in each stage of spermiogenesis. The result shows that PHB expression is high during acrosome formation and nucleus reshaping in early and middle stages in spermiogenesis, indicating that PHB might act through sustaining mitochondria’s function. The co-localization experiment of PHB and polyuiquitin FK2, as well as mitochondria and FK2 reveals that in the early and middle stages of spermiogenesis, PHB, polyubiquitin FK2 and mitochondria all distributed at one side of the cell. The signal of FK2 was quite week, showing that the ubiquitination level of PHB and mitochondria at that moment is low. In the middle and late stage, PHB, mitochondria maker and FK2 signals mainly co-localized at the acrosomal cap and one side of the nucleus. FK2 signal was gradually enhanced, indicating that polyubiquitination increased in the middle and late stages. In the mature sperm, PHB, mitochondria marker and FK2 co-localized at the acrosomal cap. The results suggest that during spermiogenesis, PHB participates in the mitochondria ubiquitination and degradation as the substrate of ubiquitin. In order to further prove the above conclusion, we used shRNA interference technique to reduce PHB expression and overexpression technique to increase PHB expression. The interference experiment shows that the reduced PHB expression directly affected polyubiquitin level and mitochondria status. Overexpression experiment reveals that PHB overexpression greatly increased polyubiquitination level. In vitro experiment also proves that PHB mediates mitochondrial ubiquitination as a substrate of polyubiquitin.