Molecular Mechanism Of Oocyte Development Regulated By Sox3 In Zebrafish

SOX transcription factors refer to a class of HMG box gene family,which participate a variety of biological processes,such as cell fate decision,tissue homeostasis and organ development,are important for the embryonic development.More than 30 SOX transcription factors have been identified in vertebrates so far.According to homology of the HMG box and structures of the proteins,they can be divided into 9 subgroups.Sox3 as a member of SOXB1 family,acts as a transcription activating factor and involves in neural,gonadal development and tumorigenesis.But the functions of Sox3 in gonadal development and its molecular mechanisms have not yet been elucidated.In this study,RT-PCR and immunofluorescence were used to detect sox3expression,which showed that sox3 was widely expressed,with especially high expression in ovary,brain and eye.High expression has also observed in theca cells and granulosa cells in ovary.sox3 knockout zebrafish lines were generated by CRISPR/Cas9 technology.I found that follicle development in sox3^-/-females was retardant and the numbers of stage III and stage IV follicles were significantly lower than those in sox3^+/+.Moreover,sox3^-/-female zebrafish fecundity was reduced,while the sox3^-/-male zebrafish was fertile.These data suggested that Sox3 plays an important role in ovary development in zebrafish.To investigate molecular mechanisms of sox3 in ovary development in zebrafish,both ovaries and testes of sox3^+/+and sox3^-/-were further analyzed by RNA-seq.About 23512900 clean reads were generated,the average ratio of mapping to reference genes was 81.61%and the average ratio of genome mapping was 91.11%.In sox3^+/+,there were 21023 genes in ovary and 24111 genes in testis,while in sox3^-/-,there were 20608 genes in ovary and 23998 genes in testis.Differential expression were analyzed through group comparisons and the results were as follows.Firstly,both sox3^+/+and sox3^-/-showed more numbers of expressed genes in testis than in ovary.Secondly,in sox3^-/-ovary,the expressed gene number was reduced when compaired with sox3^+/+ovary.The same results were also observed in tesits.Thirdly,KEGG pathway analysis showed that the apoptosis signaling pathway was significantly up-regulated while the ovarian steroidogenesis showed significantly down-regulated in sox3^-/-,which suggested that sox3 might regulate gonadal development through these pathways.Ovary-like undifferentiated gonad could develop into testis with the disappearance of oocytes,which was caused by apotosis in zebrafish.This study showed that apoptosis can be inhibited by sox3 in ovary of zebrafish.Expression of Cleaved-caspase 3 in the sox3^-/-zebrafish ovary was higher than wild types and TUNEL detection in the sox3^-/-ovary section showed more apoptotic cells in stage III and stage IV follicles in comparsion with wild types.Over-expression of Sox3 in HEK293T cell leaded to an increase of anti-apoptosis protein Bcl-2,and the flow cytometry showed that apoptosis was down-regulated.In addition,the expression levels of both 17?-E2 and cyp19a1a were down-regulated in the sox3^-/-zebrafish ovary.Sox3 can regulate cyp19a1a expression and influenced the expression level of17?-E2,thus changed the apoptosis levels of ovaries.These data provide new insights into sex differentiation and development in zebrafish.