Effects Of MT And E2 On Sex Differentiation Of Large Yellow Croaker(Larimichthys Crocea)

Large yellow croaker(Larimichthys crocea)is an important marine economic fish in China.Its sex-determined type is female homologous and male heterozygous XX♀-XY♂ type.There are obvious male-female differences in growth,and females have faster growth rate,when the sexual maturity is reached,the individual is usually larger than the male,but the male body shape and color are better than the female,and the net weight rate(the visceral/weight)is higher.Therefore,the aquaculture industry has a demand for whole female and whole male single seedlings of large yellow croaker.In this study,we used estrogen-induced,paraffin tissue sections,real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR),transcriptome sequencing(RNA-seq)and other techniques to develop the sex of estrogen and androgen on large yellow croaker.The study of differentiation aims to lay the foundation for elucidating the genetic control of sex differentiation in large yellow croaker.The main results are as follows.(1)Under the condition of water temperature of 24～26℃ and salinity of 23～25,starting from 50dph,the concentration gradient of 17α-methyltestosterone(MT)and 17β-estradiol(E2)solution at 0,1,10,50μg/L were respectively prepared and applied separately to 50 dph larval large yellow croaker,treated with medicated bath for two hours every day,and sampled at 115 dph to measure the growth of each group of large yellow croaker.The results showed that both MT and E2 inhibited the growth of large yellow croaker to a certain extent,and its inhibition was dose-dependent and increased with the increase of MT or E2 concentration.Compared with MT,the same concentration of E2 inhibited the growth of large yellow croaker more strongly.(2)Through the production of paraffin sections,the gonad differentiation was observed and the statistical reversal ratio was counted.The results showed that in the individuals who were genetically female after sex identification,the development of some ovaries was significantly inhibited by the action of exogenous androgens compared with the control group,in some genetic female gonads,the ovarian-specific tissue structure such as the ovary cavity and the testis-specific tissue structure such as seminal lobule,efferent duct anlage were observed.Sperm cells were observed in the high concentration treatment group.In the three treatment groups of 1,10,and 50μg/L,the sexual reversal ratios were 15%,44%,and 53%,respectively.In individuals who were identified as genetic males by sex identification,no sexual reversal was observed.(3)qRT-PCR was used to detect the expression changes of sex determination and gonadal development genes(amh,dmrt1,gsdf,foxl2,cyp19a)in the gonads of large yellow croakers in each treatment group after exogenous hormone induction.The results showed that MT could down-regulate the expression of male related amh,dmrt1 and gsdf in the genetic female individual,and the expression of female related foxl2 and cyp19a also played a downward role.E2 can down-regulate the expression of amh,dmrt1 and gsdf genes in the male individuals of large yellow croaker,and up-regulate the expression of the aromatase cyp19a,but inhibit the expression of the upstream regulatory foxl2.(4)The gonads of the large yellow croaker juveniles in each treatment group and the control group were sequenced by transcriptome.The sequencing results showed that the number of differentially expressed genes in the gonads of each group of large yellow croakers after MT treatment were 608,249 and 351,respectively.The volcano map was drawn and found that the fold change of the differentially expressed genes became significantly smaller as the concentration of MT treatment increased.The number of differentially expressed genes obtained by E2 treatment was 378,282 and 458,respectively.From the number or volcano map,any change with the concentration of E2 treatment was found.Principal component analysis of each gonad transcriptome after induction revealed that both the induced genetic female and the normal male or the induced genetic male and normal female did not get together well.