| Sex determination and sex differentiation were two important processes of the phylogeny in vertebrates. In the higher vertebrates, the genetic material plays a conclusive role of sex determination. However, in the lower vertebrates, such as fish, the sex differentiation was affected by the external factor except for genetic material. Research on the rule of fish sex differentiation has important significance in the evolution of vertebrates. Takifugu obscures belongs to the genus Takifugu, is one of the most important cultured fish in our country. This species of fish has high value of medicine production and edibleness. Methods of histological research were used for researching the gonad development of Takifugu obscures in alevin stage. The time nodes of sex differentiation were identified. The real-time PCR was operated for determining the function of CYP19A gene in sex differentiation,Gonadal development and sex differentiation of Takifugu obscures were observed by using histological techniques. The results showed that at lldah (day after hatching) primordial germ cell (PGC) appeared at the coelarium which was between bladder and rectum. The diameter of PGC was about 7μm and poor cytoplasm staining contains a big hyperchromatic nucleolus. The cell unclear accounted for 1/2 or 2/3 of the PGC. At 14dah, a pair of gonad anlag (GA) contains several PGCs appeared from the coelarium, and then developed into primary gonads (PG) at 29dah. Differentiation of gonads could be observed at 35dah, primary ovarian cavity (Oc) appeared in a part of primary gonads. Spermatogonia cell and spermaduct anlag could be observed in the rest part of the primary gonads at 40dah. Primitive spermary lobule and spermaduct appeared at 47dah, and oogonia could be observed at the germinal epithelium. Oogonium was a kind of round cell and its diameter was about 17～20μm. Appearance of auxocyte is the marker of sex differentiation. Spermatocytes and oocytes could be observed at 81dah and 115dah respectively.Aromatase (P450arom) is a member of the cytochrome P450 superfamily, and plays a crucial role in sex differentiation of fish. P450arom can catalyze the conversion of androstendione and testosterone to estrone and estradiol respectively. P450arom was encoded by CYP19A gene. To obtain partial CYP19A gene fragments, PCR was performed using genomic DNA derived from 2 years old Takifugu obscures ovary tissue as template and primers designed against other fishes CYP19A gene sequences. This gene fragment isolated was 1446bp length. The CYP19A gene fragment sequence shows 86% identity with that of other fish species, but low with the sequence of CYP19B. Showed that CYP19A gene was a conserved gene in fishes.The results of the quantitative real-time PCR analysis showed that the expression of CYP19A in the female was significantly higher than male. It may be because of CYP19A gene's physiological action. The expression of CYP19A measured in every stage of female samples increased significant at 44dah. At a later time about 47dah, proliferation of PGC and appearance of oogonia were observed by histological research. Synthesized the two results showed that the high expression of CYP19A translated abundant of P450arom which can catalyzes the conversion of androgen to estrogen and participated in the gonad structure building. On the other hand, the expression of CYP19A in male was also increased gradually. This phenomena showed that P450arom maybe have some functions in spermary develops.Gonad development and CYP19A expression during the period of Takifugu obscures sex differentiation were researched by histology and quantitative real-time PCR. The time nodes and the regulating procession of CYP19A gene in sex differentiation were identified. The results can help us to study the mechanism of gonad development, sex differentiation and sexual inversion further. |
