Effect Of Dmrt1 Gene On Sex Determination In Medaka

Gender is the foundation of sexual reproductive behavior and plays a pivotal role in the survival and reproduction of species.Multiple sex determination mechanisms,ranging from genetic to environmental variables,are present in teleosts.The Japanese medaka（Oryzias latipes）is an intriguing and useful model organism for research on the genetic and environmental impacts on vertebrate sex determination because its sex determination mechanism is a complex interaction process of genetic and environmental factors.The dmrt1 gene is a highly conserved gene that plays a key role in sex determination and differentiation in various vertebrates.This study focuses on the developmentally important model organism,the medaka fish,by constructing dmrt1 mutant and transgenic medaka expressing exogenous dmrt1.The study investigates the effect of the dmrt1 gene on medaka sex determination by analyzing the sex reversal phenotypes of the mutants and transgenic fish.The main research findings are as follows:1.Using the CRISPR/Cas9 gene editing system,we successfully targeted and knocked out the dmrt1 gene in medaka,obtaining homozygous mutants in the F3 generation.By combining the appearance characteristics of the mutants with the identification of their genetic sex,we found that all XY mutants exhibited female external characteristics,implying that sex reversal occurred in the XY homozygous mutants.Further gonadal dissection and histological analysis of the mutants revealed that the gonadal tissues of the XY mutants were underdeveloped ovaries.Using quantitative real-time PCR,we examined the expression of sex-related genes in the ovaries of the XY mutant medaka,finding significant downregulation of male sex-related genes dmrt1,tex9,myb,and protamin and upregulation of female sex-related genes cyp19a,foxl2,bouncer.2,42sp50,and nanos3 compared to wild-type XY medaka.In addition,the expression of female development-related genes in XX dmrt1^-/-was altered,leading to abnormal ovary development in XX dmrt1^-/-.2.The dmrt1 gene of Chinese medaka（Oryzias sinensis）was cloned and identified.Through protein sequence homology analysis and evolutionary analysis,it was found that the dmrt1 of Chinese medaka is highly homologous to that of Japanese medaka,with a close evolutionary relationship.We further constructed the eukaryotic expression vector pTol2-CMV-Osdmrt1-3×HA for the Chinese medaka dmrt1 gene and microinjected it into fertilized medaka eggs.PCR identification confirmed that the Chinese medaka dmrt1 gene was successfully integrated into the medaka genome,and Western blot analysis verified the expression of exogenous dmrt1 in medaka at the protein level,indicating successful construction of transgenic medaka.Phenotypic analysis of the transgenic medaka revealed that XX transgenic medaka exhibited male external characteristics,and gonadal dissection and histological analysis showed the presence of normally developed testes in XX transgenic medaka.When XX transgenic males were mated with wild-type XX females,the females produced normal eggs,and there was no significant difference in fertility or fertilization rate compared to the control group.Quantitative real-time PCR results showed significant upregulation of male development-related genes dmrt1,drc1,tex9,myb,and protamin and downregulation of female development-related genes cyp19a,foxl2,42sp50,and nanos3 in the testes of XX transgenic medaka.In summary,this study revealed the important role of the dmrt1 gene in the medaka sex determination cascade.The dmrt1 gene promotes and maintains male development while simultaneously suppressing female development in medaka.The dmrt1 gene is a key factor in medaka sex determination,and its increased expression can promote the reversal of female medaka to male.Mutations in the dmrt1 gene cause XY male medaka to exhibit female characteristics,and XX mutant female medaka display abnormal ovary development.These findings provide a scientific basis and reference material for the production of monosex aquaculture and gene-edited breeding in economically important fish species.