| In vertebrates, sex determination and differentiation are key events in the development of gonads. A variety of transcription factors, growth factors and steroid hormones are involved in these events. It is well documented that estrogens are the natural inducer for ovarian determination and differentiation in teleosts, while androgens, which are closely related to spermatogenesis, are not involved in the processes. Dmrtl which is a gene encoding a transcription factor with a DNA-binding. motif called DM domain has been suggested to be the first conserved gene involved in sex differentiation found from invertebrates to human. Amh (Anti-Mullerian Hormone) is a member of the transforming growth factor-β superfamily, which plays an important role in the regression of Mullerian ducts development in the male embryo of tetrapods. Unlike other vertebrates, fish do not have Mullerian ducts. In tilapia, Amh mRNA was detected in gonads of both sexes. Therefore, the possible role of Amh in sex differentiation in teleosts needs to be investigate. Cypllb2(11β-hydroxylase) is an enzyme responsible for the synthesis of11-ketotestosterone (11-KT, a potent androgen in fish), and it catalyzes testosterone into11β-hydroxy testosterone. Cyp19a1a (aromatase), the key catalyzes estradiol (E2) synthesis from androgens in teleosts. Dmrtl, Amh and Cyp11b2play important roles in testis development in tilapia as well as other teleosts. However, studies in protein level of these genes were scarce due to lack of antibodies. Therefore, the polyclonal antibody of Dmrt1, Amh and Cyp11b2of Nile tilapia are prepared, and expression profiles of Dmrtl,Amh and Cypllb2proteins in gonads during normal development and sex reversal were investigated in this study.The Nile tilapia (Oreochromis niloticus) is a good model for the study of sex determination and differentiation for its short reproductive cycle and availability of all-male (XY), all-female (XX) fry and artificial induction of sex-reversal (XY male-to-female reversal by exogenous estrogen treatment, and XX female-to-male reversal by estrogen synthetase inhibitors). Therefore, Nile tilapia was chosen as the experimental animal.First of all, the ORF(open reading frame) sequence of Dmrtl, Amh and Cypllb2were cloned by RT-PCR, and subcloned into pCold I by double digestion. Recombinant proteins were purified using Ni-NTA superflow cartridge. Then, the purified proteins were used to immunize New Zealand white rabbits for antibody production. Antibodies were purified using CNBr activated Sepharose4B. The availability of the produced antibodies were checked by Western blot analysis using Dmrtl, Amh and Cyp11b2recombinant proteins and further verified using one-year-old Nile tilapia gonads. Furthermore, these antibodies were used to investigate the expression profiles of Dmrtl, Amh and Cyp11b2proteins in gonads during normal development and sex reversal.Western blot analysis showed that the Dmrtl polyclonal antibody can specifically identify Dmrtl recombinant protein with a molecular weight of about35kD. Dmrt1antibody also identified single band in the testis (but not in the ovary) of Nile tilapia. These results verified the specity of the antibody and further confirmed that Dmrt1is specifically expressed in the testis. Immunohistochemistry showed that, Dmrtl was highly expressed in Sertoli cells and seminiferous epithelial cells from5dah. Dmrtl expression was down-regulated during E2induced XY male to female sex reversal; while up-regulated during Letrozole induced XX female to male sex reversal. Our results indicated that Dmrtl plays an important role in testicular differentiation and sex reversal in teleosts.At the same time, the expression of Cyp19ala in gonads was also examined during sex reversal. The result showed that Cyp19a1a expression was up-regulated E2induced XY male to female sex reversal; while down-regulated during Letrozole induced XX female to male sex reversal. Interestingly, both Dmrt1and Cyp19a1a were detected in gonads of E2and Letrozole treatment group at10-15dah. This result demonstrated the antagonism between male and female pathway during sex reversal.Western blot analysis showed that the Amh polyclonal antibody could specifically identify Amh recombinant protein about58kD and a single band wild protein in both testis and ovary of Nile tilapia. These results suggested that Amh polyclonal antibody is specific and Amh was expressed in both testis and ovary in Nile tilapia. Immunohistochemistry analysis showed that, Amh was highly expressed in myoid cells and Sertoli cells in the testis, and in granulose cell and thecal cell in the ovary. Ontogenically, the expression of Amh was detected from5dah, gradually increased in both ovary and testis, higher in testis, during normal development and Letrozole induced XX female to male sex reversal. Weak signal was detected until70dah in E2treated XY fish. These results suggested that Amh play an important role in the gonadal sex differentiation and sex reversal.Similarly, Western blot analysis showed that the Cypllb2polyclonal antibody could specifically identify Cypllb2recombinant protein about64kD, and identified a58kD single band in the testis of Nile tilapia. These results suggested that Cypllb2polyclonal antibody is specific and Cypllb2was specificall expressed in the testis of Nile tilapia. Immunohistochemistry showed that, Cyp11b2was expressed in Leydig cells of the testis. Unlike Dmrtl and Amh,the expression of Cyp11b2was started from30dah. In addition, Cyp11b2signal was detected until70dah in Letrozole treated XX gonads. These results further support that Cypllb2or11-KT might be involved in spematogenesis, but not be involved in sex determination.In summary, tilapia Dmrtl, Amh and Cypllb2polyclonal antibodies were prepared by immunizing New Zealand white rabbits with their recombinant proteins. They are specific, and can be used in Western blot analysis and immunohistochemistry study. Using these antibodies, we studied the expression profiles of these genes in gonads during normal development and sex reversal, and demonstrated that, Dmrtl, Amh may play important roles during the testicular development and maintenance in Nile tilapia; while Cyp11b2and11-KT may play important roles in spematogenesis, but not in sex determination. |
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