Studies On The Function And Signaling Pathway Of Amh Gene In Orange-spotted Grouper (Epinephelus Coioides)

A variety of mechanisms are involved in sex determination in vertebrates.Because of the variety of teleosts,which lead the sex development and sex differentiation mechanisms in fishes are diversed.Especially in sex reversal fishes,information on gonad development and differentiation remains limited（researchs are limited to estrogen、androgen and aromatase,etc.）.As we all known that endogenous estrogen is a natural inducer of ovarian differentiation in teleost.However,other factors than sex hormones can also influence sex differentiation in fish,such as anti-Müllerian hormone（amh）.In female mammals,AMH not only acts by decreasing aromatase（CYP19A1）biosynthesis,it also effect recruitment of primitive folliclar and female follicμLar development.Compared with mammals,there are few studies on amh in teleosts.The orange-spotted grouper（Epinephelus coioides）is a protogynous hermaphroditic fish,which undergo sexual reversal during development,thus represents an ideal model for investigating gonad development and differentiation in vertebrates.Aiming at reversing the gaps in the study of gonad development and differentiation in grouper,the overexpression model of amh in vivo was successfully constructed,and the function of amh in gonadal development and sexual differentiation was explored.At the same time,the regulation of amh on the corresponding signaling pathways was preliminarily studied.The Results are as follows:（1）After prokaryotic expression and purification,we prepared the polyclonal antibody amh.Western blot and immunohistochemical detection were performed to verify the specificity of the antibody.Western blot analysis of gonads at different stages of differentiation and in various tissues revealed that during the development from ovary to testis,amh signal gradually shows a trend of enhancement,and amh signal was only detected in the gonads（high in testes and low in the ovary）and were not found in other tissues.Immunohistochemistry also proved the specificity of the prepared polyclonal antibody amh.（2）By constructed the overexpression model of amh in vivo,the experiment of feeding amh plasmid in juvenile grouper was carried out.The experimental Results show that compared with the control group,the structure of the gonad changed in the feeding group,which ovarian degeneration and appeared of spermatogonia and spermatocytes.Real-time PCR result also show that increased male-pathway gene expression and reduced female-pathway gene expression in vivo.SimμLtaneous determination of hormones found that compared with the control group,in the amh plasmid feeding group,the content of E₂ in serμm decreased significantly,but the content of 11-KT in serμm increased significantly.The Results showed that amh played an important role in sex reversal of grouper,which could lead to ovarian degeneration and gonad development to testis.（3）By constructed the overexpression model of amh in vivo,the experiment of feeding amh plasmid in undifferentiated juvenile grouper was carried out.The experimental Results show that compared with the control group,the structure of the gonad changed in the feeding group,which ovarian apoptosis and appeared a greater nμmber of spermatogonia.Real-time PCR result also show that increased male-pathway gene expression and reduced female-pathway gene expression in vivo.SimμLtaneous determination of hormones found that compared with the control group,in the amh plasmid feeding group,the content of E₂ in serμm decreased significantly,but the content of 11-KT in serμm increased significantly.The Results showed that amh played an important role in sex differentiation of grouper,which induced the development to male gonads,but that this male tendency was preceded by female differentiation.（4）Finally,based on the above in vivo Results,we speculate the possible signaling pathways in the upstream and downstream of amh and conduct in vitro experiments.The Results showed that the Amh protein expressed and purified by cell lines had biological activity,and proved that Amh in grouper could activate the downstream Smad phosphorylation pathway.Two-molecule fluorescence complementation experiments in cell lines showed that target protein Smad5 and downstream Smad4 could interact.Co-transfection of amh expression plasmid could significantly inhibit the promoter activity of cyp19a1a.It was also found that the specific binding regions of Smad on cyp19a1a promoter were-2257/-2246 bp and-1834/-1823 bp.In conclusion,in vitro experiments can preliminarily prove that there is amh-Smads-cyp19a1a signaling pathways in orange-spotted grouper.