| The Chinese giant salamander (Andrias davidianus), which belongs to the order Caudata in the family Cryptobranchidae, is one of the largest extant amphibian species. And it has been included as a national class II protected species by the Chinese government. Thus, as a special species, the Chinese giant salamander has the high medicinal, nutritional and scientific value. However, during the past several years, the population of the Chinese giant salamander has declined dramatically due to the reduction of suitable habits, overharvesting, and increasing prevalence of infectious diseases. In order to protect this valuable species, the researchers started to study the domestication and breeding of the Chinese giant salamander. With the development of the artificial breeding technology, the population of the Chinese giant salamander has increased fast. However, the higher temperature in the mesocosms led to much more male salamanders. In order to solve this problem, the Chinese giant salamanders were exposed to the heterogeneous estradiol at a certain concentration during the sensitive time for sexual differentiation, intermittently, for forty days. At last, the salamanders all developed to the females. The phenomenon above indicated that the water temperature and estradiol both play an important role in the gonad differentiation of the Chinese giant salamander. In the present study, we have cloned the ERa and P450arom sequences for the first time, and have also studied the ERa, P450arom, Sox9 and Dmrt2 expressions during the development to provide the basic information for the further research.Firstly, primers were designed on the known ERa and P450arom sequences of some Urodela on Genbank. The full length of ERa and P450arom coding region were obtained by RT-PCR and Race technology. In addition, the tissue distribution pattern of ERa and P450arom mRNA in various tissues including kidney, liver, heart, lung, muscle, small intestine, skin, eye, brain and ovary from the female Chinese giant salamander was determined by RT-PCR. Secondly, the salamanders at 12months, 24months and 36months were chosen to analysis the head-body length, tail length and body weight during development. Additionally, the development of the gonads was also observed by histological analysis. Finally, the relative expressions of ERa, P450arom, Sox9 and Dmrt2 mRNAs in the livers, brains and gonads at 12,24 and 36 months have been analyzed by quantitative Real-time PCR. The main results were as follows:1. The full length of ERa coding region is 1 755 bp, which includes the ATG start site and the TGA termination signal, and it predicts a protein of 584 amino acids. The ERa shows high similarity to the other 7 species in the DNA binding domain. The N-terminal sequence for the Chinese giant salamander is MPLH, lacking the first MT sequence. According to the reported ERa full amino acid sequences of amphibians, we suggested that the lack of the N-terminal MT sequence may be a common molecular characteristic in the Urodela.2. The full length of P450arom coding region is 1 509 bp, including the ATG start site and the TGA termination signal, and it predicts a protein of 502 amino acids. The amino acid sequence of the Chinese giant salamander P450arom showed high similarity with other vertebrate species. Especially in the amphibians, they were all above 75%. It revealed that the P450arom protein of the Chinese giant salamander is conserved in the evolutionary history of the P450arom protein.3. Tissue-specific expression of ERa and P450arom mRNA was also examined by the conventional RT-PCR. The results showed ERa expressed in the kidney, ovary, and liver in the female 7-year-old salamander. In addition, the P450arom mRNA was found in the brain and ovary, but not in other tissues.4. To better understand the relationship of the Chinese salamander ERa and P450arom protein with the other vertebrates in the evolutionary history, phylogenetic trees were constructed using MEGA5.0 software using numerous ERa and P450arom proteins from various vertebrates. The results showed that the Chinese salamander ERa and P450arom were similar to the amphibians, but not so similar to the reptilians, birds and mammalians.5. The morphometric analysis showed that the female and male ones had significant differences in head-body length (P<0.05) and body weight (P<0.01) at 24 months, but there was no difference seen in tail length. At 36 months, there existed significant (P<0.01) differences in head-body length, tail length and body weight between the female and male Chinese giant salamander. And the male is much bigger than the female ones.6. The histological analysis of the gonads showed that the gonad was undifferentiated at 12 months. At 24 months, the gonads have obviously differentiated to the ovary and the testis, and the oocytes and the seminiferous lobule can be observed clearly. At 36 months, the ovarian cavity at the central region became much bigger. In addition, the ovocyte was also much larger. In the testis, we can see the tubular lacuna.7. Quantitative real time PCR analysis of the P450arom showed that P450arom was expressed at a significantly (P<0.01) higher level in the ovaries than in the testis after differentiation. In the brain, P450arom expressed at a higher level when it was undifferentiated and a lower expression was observed after differentiation. At 36 months, the brain P450arom was at a significantly (P<0.05) higher level in the male than the female. The result revealed that P450arom may be involved in the development of the ovarian and the brain.8. Quantitative real-time PCR analysis of the ERa showed that the gonad ERa transcript became significantly (P<0.01) lower after differentiation. And it was higher in the testis than the ovaries at both 24 and 36 months. In addition, there existed a significant (P<0.01) intersexual difference at 36 months. In the brain, the significant (P<0.01) intersexual difference have also occurred at 36 months. However, the ERa expressions in the female livers were higher than that in the male after differentiation. Moreover, there was a significant (P<0.05) difference at 24 months. The results revealed that ERa expression in the liver may be necessary for the vitellogenesis. In addition, the endogenous estrogen may be involved in testis development.9. Quantitative real-time PCR analysis of the Sox9 showed that the Sox9 transcript has been detected in the gonads during the development. And the Sox9 transcript was significantly (P<0.01) higher in the ovaries than in the testis at both 24 and 36 months. The result revealed that Sox9 gene may be involved in the gonadal development of both the male and female. In addition, we have detected Sox9 transcript in the liver and brain during different development stages.10. Quantitative real-time PCR analysis of the Dmrt2 showed that the Dmrt2 mRNA has been detected in the gonads at both 24 and 36 months, and there existed significant (P<0.05) intersexual differences at 36 months. The result indicated that the Dmrt2 gene mainly involved in the testis development of the Chinese giant salamander. In addition, we have only detected the Dmrt2 transcript in the brain and liver of the Chinese giant salamander at 24 months. |
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