| Orange-spotted grouper(Epinephelus coioides),a protogynous hermaphroditic seawater fish with high nutritional value,belongs to Perciformes,Percoidei,Serranidae,Epinephelinae,Epinephelus,is commonly used as a model for the study of gonadal sex differentiation in vertebrate.Anti-Mullerian hormone(AMH)and anti-Mullerian hormone receptor II(AMHRII)were glycoprotein,belongs to the TGF-? superfamily.In mammals,Anti-Mullerian hormone was termed as it was able to cause the degeneration of Mullerian ducts and also involved in preventing female reproductive organs from developing.In teleosts,none report showing the existence of Mullerian ducts in any species,while,so far,AMH and its receptor AMHRII gene have been cloned and identified in many fish species,as well as their high expression in the gonad,indicating that AMH/AMHRII system may play some roles in sex differentiation or during the sex reversal processes.In the present study,using RACE,we have cloned the full length cDNA sequence of both AMH and AMHRII;RT-PCR was performed to show the expression of AMH and AMHRII in different tissues and organs;localization of the AMH and AMHRII in the gonad was detected by in situ hybridization;according the the above results,prokaryotic expression of AMH was performed to produce and purification the recombinant protein.As a whole,our study will increase our knowledge to further investigating the role that AMH and AMHRII played in both sex differentiation and sex reversal.All the results we got are list as below:(1)In Orange-spotted grouper(Epinephelus coioides),the full length of AMH cDNA was 1726 bp,containing 1557bp open reading frame(ORF),coding of 518 amino acids;the full length of AMHRII cDNA was 1822bp,containing 1497bp open reading frame(ORF),coding 498 amino acids.In multiple sequence alignment,the grouper AMH sequence showed the highest homology with sea bass,approximating 83%;similarly,the grouper AMHRII also showed the highest homology with sea bass,reaching 85%,indicating the AMH and AMHRII system in grouper and sea bass have similar functions.(2)RT-PCR results showed that both AMH and AMHRII were detected in kidney,heart,brain,pituitary gland and skin in low expression,while in gonad,the gene showed an extremely high expression in testis and comparatively lower in the ovary,and the expression in testis to far higher than that in ovarian;almost no expression in the liver,stomach,gills,muscle and spleen.Regarding to these results,AMH/AMHRII system plays an important role sex determination and sex differentiation.(3)In situ hybridization results showed that both AMH and AMHIIare mainly expressed in the supporting cells in testis,and mainly expressed in follicular cells surrounding the mature oocytes in the ovary.Because both AMH and AMHRII were present in the follicular cell of the ovary,indicating that AMH/AMHRII system may have a certain effect on the development of ovary and female germ cells.Accordingly,AMH/AMHRII system could be an important regulator of the sex differentiation.(4)Finally,we produced and purified the recombinant protein of AMH using prokaryotic expressionsystem.The protein was verified by Western Blot and according to the specific band we purified the recombinant AMH production.We also prepared the specific polyclonal antibodies by immune rabbit.Overall,in the present study,we employed the grouper,a protogynous hermaphroditic seawater fish,to further investigate the pertential role of AMH/AMHRII system in sex differentiation and sex reversal.As a whole,our study will increase our knowledge to the whole reproductive endocrinology network,and will provide a solid theoretical foundation for our understanding of teleost sex differentiation and sex reversal. |
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