Estradiol-Induced Sexual Reversal And Its Effects On Gonadal Differentiation And Development In Mandarin Fish(Siniperca Chuatsi)

Female mandarin fish(Siniperca chuatsi)grows faster compared to the male,highlighting the importance of sex-controlled breeding.Achieving this objective will enhance germplasm,accumulate breeding materials,and improve production.Although studies have reported the production of pseudo-male S.chuatsi(XX phenotype males),the effect of methyltestosterone on gonadal development,and potential genes associated with sex determination of S.chuatsi,while researchers have not yet explored the production of pseudo-female S.chuatsi(XY phenotype females)and the effect of estradiol on gonadal differentiation and development in S.chuatsi.In this study,sexreversed pseudo-female S.chuatsi was produced by feeding 17β-estradiol-treated diets during the sexually undifferentiated stage.The gonadal transcriptomes of the female,male,and pseudo-female S.chuatsi were subsequently analyzed to determine the expression levels of key genes and signaling pathways that are associated with sex differentiation.Additionally,intraperitoneal injections of 17β-estradiol were administered to male S.chuatsi that had already completed gonadal differentiation.The resulting histological changes in the testis of S.chuatsi,variations of sex steroid hormone level in serum,and alterations of gene expression related to gonadal development were then examined to determine the effects of estradiol on the gonadal differentiation and development of S,chuatsi and its underlying causes.The primary findings of the study are as follows:(1)The pseudo-female S.chuatsi was produced using 17β-estradiol.Sex-reversed pseudo-female S.chuatsi was produced by administering 100 mg/kg 17β-estradiol for 60 days prior to sexually undifferentiated stage(6 days post hatching).The gonads of the pseudo-female exhibited the morphological and histological characteristics of ovaries,and had a similar period of development to that of the female,with a substantial number of oocytes in phase II observed.Differential gene expression levels were analyzed in the gonads of the female,male,and pseudo-female.The result of hierarchical clustering indicated that the pseudo-female clustered together with the normal female.(2)Transcriptome analysis and comparison of gonads in female,male and pseudofemale S.chuatsi were performed.The expression of the long-chain acyl coenzyme A synthase gene acl3 a was down-regulated in the pseudo-female compared to the normal female,suggesting that there are differences in lipid synthesis and accumulation in the ovaries between them.Moreover,the expression of the male sex-determining genes dmrt1 and amh is down-regulated and the expression of the female sex-determining gene sox3 is up-regulated during 17β-estradiol-induced sex reversal of S.chuatsi,and these genes may be potential sex-determining genes of S.chuatsi.Additionally,the sex reversal of S.chuatsi is mediated by several signaling pathways,for example,the steroid hormone biosynthesis signaling pathway,in which estrogen synthesis is activated,androgen synthesis is inhibited,and the TGF-β signaling pathway is inhibited.These pathways may affect sex differentiation and maintenance of S.chuatsi by regulating the number of germ cells,sex hormone level and early gonad development.(3)This study explored the effects of exogenous 17β-estradiol on the testis development of S.chuatsi.The exogenous 17β-estradiol injection had an estrogenic effect on male S.chuatsi.Testis development was inhibited,in which the number of spermatocytes was reduced and spermatocyte development was degraded.The testosterone of serum level was significantly reduced,while the estradiol level of serum was significantly increased.The expression of vtg was significantly up-regulated in both liver and testis,the expression of the estrogen receptor gene Erα was up-regulated in liver and the expression of the androgen receptor gene Arα was down-regulated,and genes related to sex determination and differentiation in fish,dmrt1 and foxl2,were significantly down-regulated and up-regulated,respectively.The above results suggest that 17β-estradiol inhibits testis development of S.chuatsi by up-regulating or down-regulating the expression of genes related to sex differentiation,gonadal development,steroid hormone synthesis and interfering with the original estrogen and androgen balance.In summary,continuous treatment with 17β-estradiol for 60 d before sex differentiation of S.chuatsi resulted in sex-reversed the pseudo-female(XY phenotype female);17β-estradiol influenced the direction of sex differentiation in S.chuatsi by regulating sex-determining genes such as dmrt1,amh and sox3,as well as signaling pathways such as the steroid hormone biosynthesis,TGF-β.Additionally,exogenous 17β-estradiol can inhibit the development of S.chuatsi testis by down-regulating the gene expression of androgen signaling pathway.The results of this study can enrich our understanding of the mechanism related to sex determination and gonad development of S.chuatsi,and provide material and technical reference for sex control breeding of S.chuatsi.