| Dynamin is a superfamily of proteins with the activity of guanosine triphosphatase(GTPase),which can affect membrane remodeling and plays a key role in cell endocytosis,membrane vesicle transport,membrane division / fusion and other processes.Eriocheir sinensis,as one of the most commercially important aquaculture species,belongs to Arthropod,Crustacea,Decapoda,Grapsidae.Spermatogenesis of male E.sinensis involves cytoplasmic exocytosis,nuclear deformation,acrosome formation and other events.The expression,distribution characteristics and functions of dynamin in this multi-step cell differentiation process are still unclear,and it is worthy of further discussion.In this study,the expression characteristics of dynamin in spermatogenesis of male E.sinensis was analyzed by means of molecular biology and cell biology,as well as the influence of this family proteins on sperm morphology and acrosome biogenesis.The results would provide theoretical data for the expression characteristics of dynamin in the spermatogenesis of E.sinensis,and laid a foundation for revealing their roles in the development of male germ cells of this species,and meanwhile,provided references for the related studies in other crustaceans.The expression and dynamic distribution of classical dynamin(Dyn1,Dyn2,Dyn3)in spermatogenesis of E.sinensis were investigated by western blotting and immunofluorescence.The results showed that classical dynamin was expressed in the germ cells of E.sinensis: Dyn2 and Dyn3 were always existed at different stages during spermatogenesis and sperm after acrosomal reaction,while Dyn1 was only localized in mature sperm and sperm after acrosomal reaction.Different from Dyn3,Dyn1 and Dyn2 not only exist in the apical cap structure of sperm after acrosomal reaction,but also in the tip of acrosomal tubule.Dynamin-like protein(DLP)sequence was obtained from the transcriptome database of E.sinensis testes,bioinformatics analysis confirmed that it belongs to the dynamin family.By molecular cloning,the expression systems and corresponding proteins of DLP were constructed and expressed,respectively.Polyclonal antibody DLP(PcAb-DLP)was prepared by antibody preparation technology.Immunofluorescence analysis showed that DLP was present in all levels of germ cells during spermatogenesis.The results of immunoelectron microscopy showed that DLP was distributed all the acrosome structure of the mature sperm,such as apical cap,fibrous layer,middle layer,lamellar structures and acrosomal tubule.Using RNA interference(RNAi)technology,siRNA was injected in vivo,and the statistical difference of DNM gene expression in E.sinensis was analyzed by real-time fluorescence quantitative PCR.It was found that DNM gene expression was significantly inhibited at 24 h after injection.Western blotting and immunofluorescence results showed that after RNAi,the expression level of dynamins decreased in the germ-line cells of E.sinensis.After the expression of DNM gene was knocked down,the semi-thin section analysis showed that the changes of chromatin from dispersion to agglutination could not be observed in spermatogonia,spermatocytes to spermatids;a large number of sperm were structurally "deformed".By transmission electron microscopy,the whole structure of spermatozoa was loose,the boundary between the membrane structures in the cells was unclear,and the nuclear membrane and membrane complex were destroyed;the form of acrosomal tubule was incomplete and the structure of apical cap was loose,all of these resulted that there was no obvious acrosomal structure in spermatozoa.This indicates that dynamins were involved in regulating the spermatogenesis process and stabilizing acrosomal structure of E.sinensis. |
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