| The sex related genes as the foundation of sex differentiation provide the possibility of sex differentiation, but the sex differentiation is the reciprocity of sex related genes and the environmental factors. With the sex reversal obtained by MT immersion and temperature treatment during sex differentiation, it will be helpful to understand the mechanism of sex determination to analyze the sex-related gene expression in sex reversal. Half-smooth tongue-sole (Cynoglossus semilaevis) is a newly exploited and commercially important cultured marine flatfish, in which females grow 2-3 times faster than males. Thus, the development of all-female tongue sole stock with temperatures and MT would be of significant benefit for aquaculture. 1 Gonadal differentiation and effects of temperature and MT on sex determination in C. semilaevisGonadal differentiation of C. semilaevis was observed by histological sectioning. The histological differentiation occurred firstly in 30 days post hatching(dph). There were two types of gonad. The one with cavity will develop into ovary, and the other one without cavity will develop into testis. By histological sectioning to distinguish the phenotypic sex and by female-specific marker to determination the genetic sex, effects of temperature and MT on gonad differentiation in tongue sole was addressed in a separate experiment. Juvenile tongue sole were grown at 16℃, 20℃, 24℃,28℃or 32℃from 25 to 100dph, respectively. High temperatures(28 or 32℃) induced phenotypic sex reversal in juvenile tongue sole producing a higher proportion of males ( 69.2% males at 28℃, 0.01 0.05; 57.1% males at 20℃, P>0.05). Raising tongue sole at the temperature(24℃) held sex ratios close to 1:1. Juvenile tongue sole were immersed with MT of 20μg/L·H2O,30μg/L·H2O,50μg/L·H2O,80μg/L·H2O,100μg/L·H2O from 25 to 100dph, respectively. High and low concentration of MT induced phenotypic sex reversal in juvenile tongue sole producing a significantly higher proportion of males (100%(P<0.01),97.7%(P<0.01),100%(P<0.01),97.4%(P<0.01),100%(P<0.01), respectively). The female-specific marker also suggested that high temperature and MT immersion induced the sex reversal from the female to male. These findings indicate that sex differentiation in tongue sole are affected by temperature and MT.2 Molecular cloning, characterization and expression analysis of ovarian P450arom(tsP450aromA) and brain P450arom(tsP450aromB) in C. semilaevis The 2266 bp full-length ovarian P450 aromatase (termed tsP450aromA) cDNA encoding a deduced protein of 526 residues was isolated from the ovary of the C. semilaevis by using homologous cloning and RACE. Sequence and phylogenic analyses showed that the tongue sole P450aromA belonged to ovarian P450arom subfamilies. The putative tsP450aromA amino acid residues shared 59～77% sequence identify with other fish ovarian P450arom, and shared 56～60.7% sequence identify with brain P450arom of other fish. The promoter region (5'- flanking region) of tsP450aromA gene was cloned from tongue sole containing two Ad4 binding motifs and a half site of the palindrome sequences of estrogen-responsive element (ERE half). The RT-PCR analysis reveals that tsP450aromA mRNA was expressed highly in the ovary and weakly in the testis, but not other tissues. The P450aromA mRNA increased with the gonad development, but it is less abundant in testis in all phase. However, the tsP450aromA decreased in sex reversal male treatment by MT or high temperature. These results suggested that the tsP450aromA is involved in course of the gonad development and sex determination in tongue sole.The 2184 bp full-length brain P450 aromatase (termed tsP450aromB) cDNA encoding a protein of 498 amino acid residues was isolated from the brain of the C. semilaevis by using homologous cloning and RACE. Sequence and phylogenic analyses showed that the tsP450aromB belonged to brain P450arom subfamilies. The putative tsP450aromB amino acid residues shared 48.3～66.1% sequence identify with other fish brain P450arom, and shared 34.2～49.9% sequence identify with ovarian P450arom of other fish. The sequence identify between the tsP450aromA and tsP450aromB is 45.1%. The RT-PCR analysis reveals that tsP450aromB mRNA was expressed highly in the brain and gill and weakly in the gonad, but not in other tissues. The tsP450aromB decreased in sex-reversed male treatment by MT or high temperature. These results suggested that the tsP450aromB is involved in course of the gonad development and sex determination in tongue sole.3 Molecular cloning, characterization and expression analysis of FTZ-F1 homologue(tsFTZ-F1) in C. semilaevisThe full-length cDNA of the C. semilaevis FTZ-F1 homologue (tsFTZ-F1) was isolated from the testis by using homologous cloning, and the cDNA is 3143 bp in length, including the 5'terminal UTR of 66 bp, the encoding region of 1458 bp and the 3'terminal UTR of 1619 bp. Sequence, tissue distribution, function and phylogenic analyses of the FTZ-F1 showed that tsFTZ-F1belonged to SF-1/Ad4BP group. The RT-PCR analysis reveals that tsFTZ-F1 mRNA was highly expressed in the gonad, kidney, brain, head kidney and weakly in other tissues. However, the expression level in the brain and head kidney of female was much higher than those of male. It suggested that the tsFTZ-F1 involve in the organogenesis for the tsFTZ-F1 expression in embryo of different phase was higher aboundantly than that of larvae from hatching to 25 days post hatching. The expression of tsFTZ-F1 and tsP450aromA in the testis of sex-reversal decreased significantly in response to MT immersion, and it suggested the tsFTZ-F1 involve in the course of sex reversal. The tsFTZ-F1 probably acts as a transcriptional modulator by binding to the promoter of tsP450aromA to implement the expression of tsP450aromA.4 Molecular cloning, characterization and expression analysis of DMRT1 (tsDMRT1) in C. semilaevisThe 608 bp cDNA of the partial C. semilaevis DMRT1 homologue (tsDMRT1) was isolated from the testis by using homologous cloning and 5'-RACE, and including the 5'terminal UTR of 45 bp and starting coden. It code a deduced protein of 187 residues, including DM dormain. The RT-PCR analysis reveals that tsDMRT1 was only expressed in the testis, but not in other tissues. The DMRT1 also expressed in sex-reversed male treatment by MT or high temperature. These results suggested that the DMRT1 is involved in course of the gonad development and testis differentiation in tongue sole.
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