Es-SOX2 Is Involved In The Regulation Of Spermatogenesis Of Eriocheir Sinensis Via Directly Regulating Of Kruppel-like Factor Es-klf4

KLF4 and SOX2 are important cellular transcription factors,but they have never been reported in Eriocheir sinensis(E.sinensis),and their functions during spermatogenesis in Eriocheir sinensis have not been explored.In this subject,the CDS sequences of es-klf4 and es-sox2 of E.sinensis were cloned for the first time.Preliminary studies showed that Es-KLF4 and Es-SOX2 m RNA and protein levels were highly expressed in testis,spermaducts and seminal vesicles.The results of fluorescence in situ hybridization showed that es-sox2 and es-klf4 m RNA signals were strongly expressed in the nucleus and cytoplasm of germ cells at all stages.After the targeted knockdown of Es-KLF4 and Es-SOX2 by intracavitary injection of ds RNA,the DNA of a large number of germ cells was damaged,the apoptosis level and proliferation ability were significantly increased,along with nuclear abnormalities in a large number of Stage II and Stage III sperm cells and spermatozoa.In order to investigate whether Es-KLF4 and Es-SOX2 would be affected by the microenvironment and thus alter their functional performance,Es-KLF4 and Es-SOX2 were overexpressed in HEK293 T cell line.After overexpression of Es-KLF4,the proportion of cells in the proliferation state was significantly reduced,the cells were arrested in the G1/S phase,and the expression of cell cycle-related proteins was significantly decreased.In contrast to Es-KLF4 overexpression,after overexpression of Es-SOX2,the proportion of proliferating HEK293 T cells was significantly increased,the proportion of cells in the S phase was significantly increased,and the expression levels of cell cycle-related proteins were significantly increased.In addition,we were surprised to find that the transcript level and protein expression of Es-KLF4 were significantly decreased in the testis of the ds SOX2 group,while the transcript level and protein expression of Es-SOX2 were not significantly changed in the testis of the ds KLF4 group.The results of promoters and DNA binding sites prediction showed that there was a sequence containing both the Es-SOX2 DNA binding site and the es-klf4 promoter in the es-klf4 promoter region.Further chromatin co-immunoprecipitation results showed that this sequence naturally binds to Es-SOX2 protein in the testis of E.sinensis,and the dual-luciferase reporter gene assay showed that Es-SOX2 has a significant transcriptional regulation effect on this sequence.In summary,in the testis of E.sinensis,Es-SOX2 can directly regulate the transcription of Es-KLF4 gene,thereby participating in the regulation of physiological processes such as cell cycle,cell apoptosis and sperm nucleus remodeling during spermatogenesis of E.sinensis.It is worth mentioning that the functions of Es-SOX2 and Es-KLF4 may be related to the microenvironment,and their functions may be reversed in different cellular contexts.