| The sox gene family is a general name for a series of genes that are highly homologous to the sry(sex determination region of Y chromosome)gene of male mammals.Up to now,more than 40 sox genes have been found in different species and performed different functions in organism,many studies have reported that sox genes are involved in a wide range of development processes,especially in sex determination.Compared with other genes,sox3 was a single-exon gene which located on the X chromosome and expressed by progenitor cells.In mice,sox3 has been proved to be related to the gonadal development and gametogenesis.In fish,the various expression profiles of sox3 reveal distinct roles,mainly in testicular or ovarian development.However,the accurate expression pattern is still unclear in fish.Therefore,it is necessary to countersign the expression pattern of sox3 gene for further study of its function.Medaka is a small size freshwater fish that an excellent model for the study of developmental biology and cell biology.Medaka was the first non-mammal which found the male sex determining gene dmy,and the only vertebrate which had the female sex determining gene foxl3.There are many transgenic stains have been established and the genes were expressed in the gonads specifically,which provides a good platform for studying the function of genes related to reproduction and gonadal development.In addition,many scientists have devoted themselves to the research of reproductive genes in medaka,and many reproductive cell marker genes,such as dazl,dnd,piwi and vasa have been found and transgenic strains established successfully.Cas9 and other knockout techniques have also been used in medaka successfully,hence the medaka is a good model species for studying gonadal development and sex differentiation.In this study,the sox3 gene was cloned with a cDNA length of 1786 bp,including 5 'UTR region of 191 bp,CDS region of 912 bp that encoding 303 amino acids,and 3' UTR region of 683 bp.Bioinformatics analysis showed that sox3 was highly conserved among different species,which was more than 70% similar to the sox3 gene in mammals.Amino acid sequence alignment and phylogenetic tree showed that sox3 was much conservative in evolutionary position.The results of chromosome linear analysis showed that the position of medaka sox3 was consistent with that of other vertebrates,including human and zebrafish.Multiple analysis results showed that sox3 was highly conserve in evolution.RT-PCR results showed that sox3 was expressed in brain,eyes and gonads,brain and eyes were the tissues which rich of the nervous system,that was consistent with the results of embryos,indicating that sox3 played an important role in the nervous system.The expression of sox3 in ovary was higher than that in testis.In situ hybridization showed that sox3 was highly expressed in oogonia and early oocytes.However,sox3 was expressed in granulosa cells and theca cells later in oocytes different from vasa.sox3 was not obviously detected in spermatogonia cells,but was expressed in spermatogonia cells at the later stage of development,and was also specifically expressed in the location of chromatid body of sperm.All results were indicated that sox3 played an important role in gametogenesis and maturation.The results of embryo indicated that sox3 was involved in the development of nervous system.Luciferase report showed that miR-375-3p analogues could effectively inhibit the activity of luciferase,indicated that sox3 had the binding site of miR-375-3p.The study on the expression pattern of sox3 in medaka,combined with miRNA,helps to understand the molecular mechanism and role of sox3 in the reproductive development of vertebrates,and also provide a good foundation for further understanding of the gonadal development of medaka and related gene functions. |
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