| Centropristis striata, a kind of valuable marine-cultured fishes, is favored by consumers in general because of fleshy muscle and delicious taste. However, some limiting factor were found in artificial breeding, such as advanced age of male parent fish, male and female reproductive asynchrony and lack of male fish, which seriously restricted the development of artificial breeding work. Therefore, it is the key to large-scale artificial reproduction to investigate the mechanism of sex reversal and accelerate sex reversal of females through artificial methods. In this study, the female Centropristis striata was induced into male through embedding exogenous material, furthermore, technique of histology and molecular biology was adopted to analyze the inducing effect, Which would provid necessary theoretical basis for research on mass seed production of groupers.1 Histological and physiological change of Centropristis striata in the process of sex reversal The present study treated female Centropristis striata with 17α-amethyl testosterone(17α-MT) and aromatase inhibitor(AI), to explore the endocrine controlling mechanism in protogynous sex change, including observing and analyzing histological and physiological change. The results show that the gonads of treated fish had already developed into testes at the end of the experiment, which produced sperm capable of fertilization; control fish had ovaries undergoing active vitellogenesis except for a few testes. First of all, the gonadosomatic index(GSI) of treated fish decreased firstly and increased subsequently, whereas untreated control fish showed no significant change. Obvious marriage color and fat protuberant head were observed in treated fish during sex change, and the yellow cystic gonads had already developed into milky testes, while gonads of control fish most present bright dark gray, gonad development of sex-reversed males can be divided into four main stages: ovary development period, ovary period with little sperm, spermary period with little ovary, and spermary development period(complete sex reversal). Furthermore, the plasma androgen(testosterone) levels were increased and maintained at a high level in the 17α-MT-treated fish, while estradiol-17β(E2) levels were low compared to controls, but no significant differences were observed in aromatase activity of brain between 17α-MT-treated and control group. Similarily, the testosterone levels of AI-treated fish was significantly higher than that of control fish, and AI treatments caused a significant drop in E2 levels similarly, aromatase activity in the brain had little change compared with the control group. In contrast, the testosterone levels of co-treatment fish was higher than that of 17α-MT-treated and AI-treated fish alone, and E2 levels of co-treatment fish was very low compared to other groups. In addition, the sex-reversed males treated with a combination of letrozole and 17α-MT resulted in significantly higher sperm motility and lifespan than the sex-reversed male treated with only 17α-MT and AI.2 Molecular cloning and spatial expression patterns of Dmrt1 and Foxl2 of Centropristis striata In order to address the role of Dmrt1 gene and Foxl2 gene during gonadal differentiation period of Centropristis striata, Dmrt1 gene and Foxl2 gene were isolated and characterized from the Centropristis striata by RT-PCR and subsequent RACE. The isolated Dmrt1 c DNA is 1426-bp long, including a 903-bp ORF encoding a protein of 300 amino acids and a special DM domain. The isolated Foxl2 c DNA is 1985-bp long, including a 918-bp ORF encoding a protein of 306 amino acids and a special FH domain. The tissue distribution pattern of dmrt1 and Foxl2 was analyzed in adult male and female Centropristis striata by q PCR. The analysis results demonstrated that the expression of the Dmrt1 was confined to the the testis, while a very low level in other tissues. Expression pattern of Foxl2 m RNA in females presented a high level in ovary, pituitary, brain and gill, but a very low level in other tissues. The expression level of Foxl2 m RNA in males presented a high level in pituitary, brain and gill, whereas the expression levels were low in testis, and the expression levels were very low in other tissues.3 Analysis of gene expression of Dmrt1 and Foxl2 m RNAs during sex reversal of Centropristis striata Expression pattern of Dmrt1 and Foxl2 was examined in gonad tissues by RT-q PCR assays to elucidate the expression regular pattern during sex reversal process after embedding exogenous 17α-MT and aromatase inhibitors in muscle. The expression of Dmrt1 gene in the gonad of experimental and control group showed different degrees of upward trend, while the upward trend was more significant in the experimental group(P<0.05). The expression of Foxl2 gene in the gonad of experimental and control group showed different degrees of downward trend, while the downward trend was more significant in the experimental group(P<0.05). |
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