Expression Of Foxl2 And Foxl3 During Gonadal Development And Sex Reversal Induced By MT In Grouper(Epinephelus Coioides )

Orange-spotted Grouper,Epinephelus coioides,is a marine protogynous hermaphrodite fish of commercial importance.Therefore,E.coioides was considered as a good model to study the development and reproduction.Due to the long reproduction cycle of male in nature,the lack of father in the breeding process has restricted the development of artificial breeding of grouper.The Fox genes code for a family of transcription factors characterized by a winged helix forkhead box DNA-binding domain of 80-100 bp.The family is organized into subfamilies(A–S)based on the amino acid sequence of this domain.Foxl2 is a forkhead transcription factor involved in mammalian development and regulation of reproduction.Recent researches have found that foxl2 is a basic regulatory factor in the development,maintenance and morphology of vertebrate ovaries.While,there is little research on foxl3.There are kinds of sex change species in marine fishes,while the underlying molecular basis is still unknown.In this study,we obtained the full lenth of foxl2 and foxl3,analyzed the two homologous genes by inference of protein sequence,amino acid alignment and phylogenetic tree,investigated gonadal histology,gene expression profiles of the gonadal development and MT induced sex reversal from female to male.The results were showed as follows:1.The complete cDNA of E.coioides is 1413 bp contains an open reading frame(ORF)of 921 bp which encoding a 306 animo acid protein.It preceds by 280 bp of 5’ UTR and the 3’ UTR is 212 bp.The foxl3 cDNA is 1260 bp long and contains a 795-bp ORF encoding a protein of 264 amino acids,with a 259-bp 5’ UTR and 602-bp 3’ UTR,containing a typical polyadenylation signal(AATAAA).2.Quantitative RT-PCR revealed that foxl2 is significantly higher than other tissues(p<0.05),while foxl3 is predominantly expressed in testis and ovary,with its higher leval in testis and low expression in ovary and other tissues(p<0.05).3.foxl2 was significantly upregulated in the process of ovarian maturation(p<0.05),suggesting that foxl2 might play an important role in the ovary.The expression level of foxl3 in the ovary was very low.With the gonadal development,the expression level rose in the bi-sex gonad and peaked in the testis(p<0.05).4.The estradiol-17b(E2)of grouper in reproduction was increased,and significantly decreased after reproduction(p<0.05).The expression level in testis recovered somewhat.5.We used 17α-methyltestosterone(MT)to induce the grouper in the gonadal differentiation period of 90 days after patching,which can develop into male directly.The expression of foxl2 in the experimental group was significantly lower than that in the control group(p<0.05),while the expression of foxl3 in the experimental group was significantly higher than that in the control group(p<0.05).Above all,we can deduce that foxl2 plays an important role in the development of overy.The role of foxl2 in reproduction may be quite conservation mainly by regulating the early differentiation of ovary by hypothalamus –pituitary-gonad axis.While foxl3 may participate in the development of testis,the role of foxl3 in reproduction may be regulated by testis and it was a potential male specific regulatory gene.The grouper of MT inducing in the gonadal differentiation period of 90 days after patching developed into male,which may be that MT inhibited the expression of aromatase gene and thus inhibited the synthesis of estrogen.Taken the expression profiles of foxl2 and foxl3 during gonadal development and MT inducing sex-reversal period and E2,we speculated that estrogen was the main regulator of ovarian differentiation in teleosts and androgen interfered ovarian development by inhibiting the synthesis of estrogen.Besides,the functions of foxl2 and foxl3 in the process of gonadal development and sex reversal in the grouper and the mechanism of sex reversal need further studying.