Study On The Function And Regulatory Mechanism Of Transcription Factor Sox30 In Spermatogenesis Of Nile Tilapia

Animal spermatogenesis is a biological process in which males produce mature sperms.The abmormality of spermatogenesis directly affects the fertility of male.The regulatory mechanism underlying animal spermatogenesis is one of the hotspots and difficulties of animal reproductive biology research.Sox30 is a member of Sox transcription factor family.The previous study has showed that Sox30 plays a key role in spermiogenesis in mice.However,there is no research about the function of Sox30 in other vertebrates including fish.The previous study has suggested that Sox30 is highly expressed in the testis of the male Nile tilapia.In the present study,by using Western blot and immunofluorescence,we analyzed the expression pattern and cellular location of Sox30 in the gonads of Nile tilapia at different developmental stages.By using CRISPR/Cas9 technology,we analyzed the functions of Sox30 in testis development and spermatogenesis of Nile tilapia.We also explored the regulatory mechanism of Sox30 during testis development and spermatogenesis and transcription regulation by Dmrt1 on the Sox30 gene in Nile tilapia.The main results of this study are as follows.1.The expression pattern and cellular localization of Sox30 in the gonads of Nile tilapia at different developmental stageBy using Western blot with anti-Sox30 antibody,the expression of Sox30 protein in the gonads of Nile tilapia at 30 days after hatching?dah?,75 dah,90 dah and 180 dah was investigated.The results demonstrated that Sox30 was expressed in the testis of Nile tilapia at 75 dah and its expression level increased at 90 dah and 180 dah.But there was no expression of Sox30 in the ovary of Nile tilapia at different stages.Immunofluorescence staining with anti-Sox30 antibody revealed that Sox30 was localized in the spermatocytes,the round spermatids and the epithelial cells of efferent duct in the testis of Nile tilapia at 90 dah,and the spermatocytes,round spermatids,spermatozoa and the epithelial cells of the efferent duct at 180 dah and mature sperms.However,the positive signal of Sox30 was not found in the ovary of Nile tilapia.2.The function of Sox30 in testis development and spermatogenesis of Nile tilapiaWe constructed Sox30 homozygous mutant line of Nile tilapia by CRISPR/Cas9technology.The morphological and histological analysis of the testis in Sox30^+/+and Sox30^-/-males at 90 dah were performed.The results showed that there were both spermatogonia and spermatocytes in the testis of both wild-type males and Sox30homozygous mutant males,but the number of the spermatocytes in the testis of mutant Nile tilapia was less than that of wild-type males.The morphological analysis of the testis in Sox30^+/+and Sox30^-/-males at 180 dah were also performed.The results showed that compared with wild-type males,the GSI of Sox30 homozgous mutant males was significantly decreased.By Pap staining,sperm morphological analysis revealed that some sperms of Sox30 homozygous mutant males have abnormal tails,such as curl tail,bent tail and shortened tail.The computer-assisted sperm analysis system was used to evaluate parameters of sperm motility such as progression motility?PR?,average linear velocity?VSL?,average path velocity?VAP?,and wobble?WOB?.We found that compared with the wild-type,the motility parameters,such as PR,VSL,VAP and WOB,of sperm from Sox30-deficient male fish significantly decreased;but the ratio of non-progression motility sperms was significantly increased.The fertilization rate of homozygous mutant males was significantly decreased compared to that of wild-type males.These results indicate that the mutant of Sox30 can result in abnormal sperm tail development in males,which can affect the fertility of male fish.3.The mechanism of Sox30 in the spermatogenesis of Nile tilapia?1?The potential target genes of Sox30 were screened by ChIP-seqWe performed Ch IP-seq analysis to identify the potential downstream target genes of Nile tilapia Sox30 at the whole-genome level.According to the principle of Ch IP-seq technology,we collected the testes of Nile tilapia at 180 dah into single cells and fixed them.The genome of the cells treated with nuclease enzyme was cut into DNA fragments of 100 bp?900 bp.Then the anti-Sox30 antibody was used to precipitate the complexes of Sox30 and its binding DNA,and finally the genomic DNA fragments bound by Sox30 were extracted to perform high-throughput sequencing.Analysis of the sequence data showed that 20,812,218 raw reads and 20,635,915 clean reads were obtained in the Nile tilapia Sox30 immunoprecipitation product,and 25,604,443 raw reads and 25,316,577 clean reads were obtained in the input control group that did not undergo Sox30 immunoprecipitation.The results revealed that the main motif for Sox30binding in Nile tilapia is"C?T/G/A?C?G/A/T?T?A/C/G?A?T/G/C?G?T/C?G?T/C/A?G?T/A/C?C?G/T/A?".\We identified 5093 specific peaks in the Sox30 immunoprecipitation.Among them,2746 peaks were located in the intergenic region,1738 and 72 peaks were located in the intron and exon regions,respectively,and 537 peaks were located in the promoter region 2.0 kb upstream of the transcription start site?TSS?.A genomic alignment revealed that 499 potential target genes of Sox30,including Ift140,Rab3,Ccdc180 and Pgr related to spermatogenesis,were associated with 537 peaks.The GO function annotation demonstrated that these genes are mainly involved in biological processes related to spermtogenesis and fertilization,and so on.?2?The changes of proteome of semen in Sox30 homozygous mutant male fishTo elucidate the regulatory mechanism of Sox30 involving in spermiogenesis,we performed label-free quantitative proteomic analysis of semen from wild-type and Sox30 homozygous mutant males to investigate the change of proteome.Collected the semen from 3 wild-type fish and 3 Sox30 homozygous mutant fish,respectively,and extracted the total protein to perform the proteome sequencing.The analysis of the proteome data demonstrated that compared with the control,the expression of 21proteins was upregulated,and the expression of 48 proteins,including Rab-5a,Rab-5c,Rab7 and vesical transport protein Sec22b which may involve in the flagellum formation of spermatozoa,was downregulated.GO functional annotation analysis showed that the differentially expressed proteins were mainly involved in the biological processes,such as protein localization,ribonucleoprotein complex biogenesis,protein transport and vesicle organization,and molecular functions,including RNA binding,structural molecule activity,ribonucleoprotein complex and carbohydrate kinase activity.Based on ChIP-seq and proteome data,we analyzed whether 499 potential target genes of Sox30 identified by Ch IP-seq contained the genes encoding the differentially expressed proteins from proteomes data.Interestingly,we found that Ccdc28a,Terf2ip and LOC109202134 are both Sox30 potential target genes identified by ChIP-seq,and the encoded products Ccdc28a,Terf2ip and LOC109202134 are differentially expressed proteins in the semen proteomes of wild-type and Sox30 homozygous mutant males.This result indicates that Sox30 may affect sperm function by directly regulating the expression of Ccdc28a,Terf2ip and LOC109202134.These potential target genes of Sox30 obtained from proteomic analysis and Ch IP-seq can lay the foundation for the in-depth study of the molecular mechanism of Sox30.4.Transcription of the Sox30 gene is positively regulated by Dmrt1 in Nile tilapia.Because doublesex and mab-3 related transcription factor 1?Dmrt1?is highly expressed in the testis of Nile tilapia and involved in the male sex differentiation,we analyzed that Dmrt1 may regulate the transcription of the Sox30 gene.The analysis of transcriptome data from Nile tilapia gonads revealed that Sox30 and Dmrt1 similarly exhibited a high expression in Nile tilapia testes from 90 dah to 300 dah,and the transcription of the Sox30 gene was reduced to about 50%in the testes of male tilapia with Dmrt1 knockdown.We cloned the Sox30 promoter with 1,982 bp.The sequence analysis showed that there was a putative cis-regulatory element?CRE?for Dmrt1.Dual-luciferase reporter assay confirmed that Dmrt1 overexpression significantly promoted transcriptional activity of the Sox30 promoter and this promotion was decreased following the mutation of putative CRE for Dmrt1 within the Sox30 promoter.Chromatin immunoprecipitation-based PCR?ChIP-PCR?and electrophoretic mobility shift assay?EMSA?demonstrated that Dmrt1 directly binds to the putative CRE within the Sox30 promoter.These results together indicate that Dmrt1 positively regulates the transcription of the tilapia Sox30 gene by directly binding to the specific CRE within the Sox30 promoter.In conclusion,Sox30 was mainly expressed in spermatocytes,round spermatids,spermtozoa and epithelial cells of efferent duct of the testis and mature sperms in Nile tilapia.Sox30 homozygous mutant resulted in the abnormal development of sperm tail,the decreased sperm mobility,and reducing male fertility.Sox30 may regulate the expression of intraflagellar transport proteins and spermatogenesis-related genes Ift140,Rab3,Ccdc180 and Pgr to affect spermatogenesis of Nile tilapia,and the transcription of Sox30 was positively regulated by Dmrt1.This study will not only elucidate the function and signaling pathway of the Sox30 gene during spermatogenesis in the Nile tilapia,but also provide new insight into the regulatory mechanism of male substerility or sterility in animals.