| Optic atrophy 1(OPA1)and Dynamin-related protein 1(DRP1)with guanosine triphosphatase(GTPase)activity are members of the Dynamin superfamily.Members of the Dynamin superfamily proteins play key roles in cellular endocytosis,vesicle transport,membrane division,and fusion.In mammals,OPA1 is nuclear gene-encoded mitochondrial protein that targets the inner mitochondrial membrane and functions in the mitochondrial intermembrane space.DRP1 mainly exists in the cytoplasm.When mitochondria divided,DRP1 is recruited to the mitochondrial surface and participates in mitochondrial fission.Eriocheir sinensis belongs to Arthropod,Crustacea,Decapoda,Grapsidae,Eriocheir,and is an important economic crab in China.In this study,mitochondrial changes during E.sinensis spermatogenesis were investigated by tracking the distribution of mitochondrial protein OPA1 in spermatogenic cells and sperm after acrosome reaction.Identification of the distribution and function of DRP1 in germ cells lays a foundation for exploring mitochondrial fission during spermatogenesis in E.sinensis.In order to study the changes of mitochondria during the spermatogenesis of E.sinensis,this study prepared the polyclonal antibody Pc Ab-OPA1(Polyclonal antibody-OPA1)by using the mitochondrial protein OPA1 expressed in prokaryotic(the GTPase domain of OPA1 was selected).The results of immunofluorescence showed that in spermatogonia,OPA1 was concentrated in one pole,indicating main existence of mitochondria on one side of the cell;In spermatocytes,OPA1 was distributed in cytoplasm in a scattered pattern,and mitochondria changed from concentrated distribution to cytoplasm.In spermatids,cells began to deform,and the cytoplasm gradually gathered on one side of the cell.OPA1 localization found that mitochondria were present in the cytoplasm,but OPA1 was also observed in the preacrosomal sac;In mature sperm,OPA1 was localized in the nucleocup in the medial and acrosomal ducts.Combined with the results of immunoelectron microscopy,it was found that mitochondria existed in the inner side of the sperm nucleocup mouth in an atypical form without cristae,and OPA1 protein was present in the acrosome duct.Therefore,the mitochondrial protein OPA1 is not only involved in the remodeling of mitochondria,but also in the formation of sperm acrosomes during the process of E.sinensis sperm cells transforming into sperm.In addition,western blot analysis revealed that the OPA1 in the mature sperm of E.sinensis is S-OPA1(cleaved OPA1),not L-OPA1(intact OPA1),which may be the sperm mitochondria without cristae reason.In order to investigate the distribution and function of DRP1 protein in E.sinensis spermatogenic cells,the polyclonal antibody Pc Ab-DRP1(Polyclonal antibody-DRP1)was first prepared by using the prokaryotic expression of DRP1(the GTPase domain of DRP1 was selected).The results of immunofluorescence showed that in spermatogonia,spermatocytes,DRP1 was distributed in cytoplasm in scattered dots;In sperm cells,DRP1 mainly accumulated on the preacrosomal vesicles;In mature sperm,DRP1 protein was concentrated in the acrosome tube base and inner side of nuclear cup.Then,si RNA was injected in vivo to interfere the expression of DRP1 gene using RNA interference(RNAi)technology.After RNAi,using immunofluorescence technology to label mitochondria with the help of OPA1,it was found that after knocking down the expression of DRP1 gene,the mitochondrial morphology in the spermatogonia and spermatocytes of E.sinensis became longer,which proved that DRP1 was associated with mitochondrial fission function in spermatogonia and spermatocytes.Combined with mass spectrometry(LC-MS/MS)analysis,five proteins that may interact with DRP1 were obtained with co-immunoprecipitation technology.Among them,the interaction between Actin and DRP1 has been reported in the literature.During mitochondrial fission,actin filaments are rapidly assembled on mitochondria,which can recruit DRP1 to the cleavage site through direct binding.The functions of the remaining four proteins and their interaction with DRP1 need to be further confirmed.In summary,this study,preliminarily understood the changes of mitochondria in spermatogenic cells by analyzing the distribution characteristics of mitochondrial protein OPA1 during spermatogenesis of E.sinensis.Mitochondria in mature sperm have no cristae and are associated with the presence of a different form of OPA1(S-OPA1).OPA1 not only exists in mitochondria,but also in preacrosomal vesicle or acrosome in the process of sperm cell transformation into sperm,suggesting that OPA1 protein may be related to sperm acrosome formation.In spermatogonia and spermatocytes,DRP1 regulating mitochondrial fission obtained five proteins including Actin that may interact with DRP1,which may be related to the function of DRP1. |
