Involvement Of Es-SOX8 In The Regulation Of Nuclear Morphogenesis Of Chinese Mitten Crab (Eriocheir Sinensis) Spermatogenic Cells

SOX transcription factors are involved in animal reproduction and have been implicated in spermatogenesis in multiple species.However,the relationship between SOX family and spermatogenesis in Eriocheir sinensis is rarely reported.In this subject,we investigated the function and possible molecular mechanism of Es-SOX8 during spermatogenesis in E.sinensis.Immunofluorescence results of testis tissue sections showed that Es-SOX8 was localized in the cytoplasm and nucleus of spermatogonia,spermatocytes and round spermatids,but was not expressed in mature sperm.In order to study the specific function of Es-SOX8 in spermatogenesis,we injected ds RNA of Es-Sox8 into E.sinensis for in vivo knockdown and detected the testis tissue sections of E.sinensis by H&E staining after knockdown.After Es-Sox8 knockdown,the spermatogenic cells in the seminiferous tubules at various developmental stages were obviously abnormal,mature sperms were blocked in the germinal zone and could not be discharged,and the mature spermatozoa in the lumen were absent.At the same time,we observed that the nuclear of spermatogenic cells were loose and hollow.Besides,the proportion of abnormally shaped nuclear in mature spermatozoa was significantly increased.Immunofluorescence showed that the Lamin protein was structurally disordered in Es-Sox8 knockdown tissues,indicating that sperm nucleus development was affected,suggesting that Es-SOX8 is likely to be involved in the sperm nucleus deformation process during spermatogenesis in E.sinensis.We analyzed the expression level of Es-BMP2,the potential target gene of Es-SOX8.The results of semiquantitative PCR indicated that the transcription level of this gene was significantly down-regulated.We used the dual-luciferase reporter gene experiment to find the promoter site of Es-BMP2.Ch IP experiment results indicated that the Es-SOX8 protein was bound to the promoter region of Es-BMP2.In summary,Es-SOX8 can directly bind to the promoter region of Es-BMP2 to regulate its transcription,thereby affecting the nuclear morphogenesis of mitten crab spermatogenesis.The down-regulation of the expression level of Es-KIFC1,a downstream protein of Es-BMP2,in the Es-Sox8 knockdown group indicated that the regulation of Es-SOX8 protein on sperm nuclear deformation is likely to be achieved by up-regulating Es-KIFC1.