Functions Of PIWIs In Spermatogonium Maintenance And Quality Control Of Mature Sperm In Eriocheir Sinensis

Spermatogenesis is an important process in developmental biology,which lays the foundation of the produce of mature sperm.PIWIs have important roles in spermatogenesis,such as spermatogonia stem cell proliferation,meiosis and spermiogenesis.As a crustacean species,who produces the non-flagellar sperm,the regulation of spermatogenesis in Chinese mitten crab(Eriocheir sinensis)is largely unknown.Whether or how PIWI proteins participate in regulation of spermatogenesis in E.sinensis still need to be further studied.In the present study,we investigate the roles of PIWI proteins in spermatogenesis of E.sinensis.In the present study,we analyzed the expression pattern of PIWI proteins in testis of E.sinensis.We cloned and characterized three Piwis from testis of E.sinensis.Both of three Piwis were highly expressed in the testis.The proteins encoded by these three Piwis were specifically expressed in testis.Fluorescence in situ hybridization(FISH)of three Piwis showed that they were expressed in spermatogonia and spermatocytes,only Piwi1 was expressed in spermatid and mature sperm.Immunofluorescence showed that three PIWI proteins located in the cytoplasm of spermatocyte and spermatid,PIWI1 and PIWI3 located in the nucleus of mature sperm.The localization of three PIWI proteins in testis of E.sinensis indicated that they might have important roles in regulation of spermatogenesis.We found that PIWIs had roles in quality control of mature sperm by increasing apoptosis in abnormal germ cell.Double strands RNA(ds RNA)was used to knockdown Piwis in E.sinensis,which could significantly reduce both the transcriptional and protein level of Piwi genes and PIWI proteins.The knockdown of PIWI proteins significantly increased the ratio of abnormal mature sperm in testis of E.sinensis,but significantly inhibited the apoptosis and the transcriptional level of apoptotic genes in germ cell.To investigate the mechanism about the effects of PIWIs knockdown on the produce of abnormal sperm,we did overexpression in HEK293 cells.Cisplatin was used to induce DNA damage in HEK293 cells which could mimic the produce of abnormal germ cell.We found that PIWIs significantly promoted apoptosis in HEK293 cell with DNA damage induced by cisplatin and had significant effects on the protein level of apoptotic proteins.In conclusion,we speculate that the knockdown of PIWI may increase the produce of abnormal sperm by inhibiting apoptosis in abnormal germ cell,which is important for the elimination of abnormal germ cell.We also found that PIWIs might maintain spermatogonium by regulating Wnt signaling pathway.The knockdown of Piwis in E.sinensis significantly promoted the transformation of spermatogonium to spermatocyte.To investigate the mechanism about the effects of PIWIs knockdown on the transformation of spermatogonium to spermatocyte,we expressed PIWIs in HEK293 cells and found that PIWIs significantly inhibited the cell proliferation and increased the cell proportion in G0/G1 phase.The inhibition of cell proliferation induced by PIWIs was due to the inhibition of Wnt signaling pathway.PIWIs could significantly reduced the expression level of β-catenin,so that inhibited Wnt signaling pathway and decreased the transcriptional level of Cyclin D and Cyclin E.PIWIs regulated Wnt signaling pathway by inhibiting the phosphorylation of β-catenin at Ser552.The down-regulated of β-catenin by PIWIs significantly changed the adherens junction by decreasing the protein level of α-catenin and ZO1 and changing the localization of these proteins in cell junction.In conclusion,this study reveals the roles of PIWIs in spermatogenesis of E.sinensis.Lack of PIWIs significantly increases abnormal mature sperm.We propose “apoptosis” mechanism,which shows that abnormal germ cell can be produced during the development of germ cell in E.sinensis,but PIWIs can induce apoptosis in abnormal germ cell,by doing so to remove abnormal germ cells and ensure the quality of mature sperm.At the same time,PIWIs regulate the transformation of spermatogonia to spermatocyte by inhibiting Wnt signaling pathway to reduce the Cyclins and cell adherens junction.The roles of PIWIs in the produce of nonflagellar sperm enhance our knowledges about the roles of PIWIs in spermatogenesis,as well as the regulation of spermatogenesis in crustacean species.