Effects Of Aromatase Inhibitor On Growth And CYP19A Gene Expression In Larvae Pufferfish, Takifugu Obscurus

Both sex determination and differentiation are important processes in individual development of vertebrate. The gender of Mammals and other higher vertebrates is mainly determined by gene, but in lower vertebrates, especially fish, the phenotypic sex is regulated by the sex determination gene and other factors. Studying on the process and rules of fish sex determination and differentiation can help us to understand the phylogeny of fish deeply, and to get the theoretical guidance about the unisexual fish population as well. The quality and yield may be improved dramatically by through the technology on domestication of unisexual fish in aquaculture production. The puffer fish Takifugu obscurus is aquatic products with high economic value. With the development of aquaculture, research on Takifugu obscures has covered development, nutrition, breeding, genetics and other related fields. It was discovered that there is a higher economic value in male Puffer fish than females during breeding process. So that finding a suitable sex control method for Takifugu obscurus is important. CYP19A mechanism of sex differentiation can give a reference for breading single-sex puffer fish and also provide the basis data of sex control in fish.In this paper, the effect of aromatase inhibitors (AI), letrozole on growth, development, sex differentiation and aromatase CYP19A gene expression in Takifugu obscurus larvae was investigated using morphology, histological sections and real-time qPCR methods. The results showed:the average body length of control puffer fish is linear with time during63dah, from3mm at2dah to35.5mm at63dah, nearly12times. The weight and length of puffer fish larva exposed to AI was less than the control one at the same day, and there is a trend that the earlier and longer exposure, the smaller weight and length are; the same trend was observed in concentration exposure group, fish had a smaller value of weight and length with the higher concentration.In the section of control puffer fish larva at63dah, a typical gonadal structure can be observed. The ovary is characterized by ovarian cavity; gonadal somatic cell was situated between the epithelial cells and germ cell, the germ cell formed layers fold, and extended to the ovary cavity. Testis has no cavity, germ cells arranged in a layer and closed to epithelial cells. The puffer fish exposed to AI was unable to find the normal ovarian structure, in addition to the normal development of testes, but also found some unusual gonadal structure. Common feature of these abnormal gonads was:scattered arrangement of germ cells, the location is not fixed. Some of them were suspected ovarian degeneration, we can see the shrinking of the cavities, and it may be inhibited during developing ovary or filled with other somatic cells. Some of them were suspected testis, germ cells can be found close to the gonadal epithelium and dispersed throughout the gonadal, vas deferens primordia was absent. There was a significant impact of AI on the sex ratio. Time exposed group begin with7dah,10dah have0%of female ratio at63dah. Exposed group begin with10dah have best inhibitory effect; In concentration group of exposure, females have no one at0.625mg/L concentration and females decreased as the concentration increased.We use real-time quantitative PCR method to detect the CYP19A expression after AI exposure. The results show that:CYP19A gene expression of puffer fish has sexual dimorphism after AI exposure. We divide into high expression A group and low expression B group. In A group of high expression of time exposure, CYP19A gene expression levels showed inhibition. At42dah, with the delay of exposure starting time, gene has low quantitative expression, the exposed group was begin with14dah has minimum gene expression; at63dah, gene expression was suppressed like42dah, we also found gene has minimum quantitative expression at14dah. Normal group and the exposed group have lower gene expression than42dah. At B group, CYP19A gene expression has no significant inhibitory effect at42dah and63dah, but normal group and exposure group have higher gene expression than42dah at63dah. In A group of high expression of concentration exposure, letrozole inhibited CYP19A gene expression. However, the inhibitory effect of concentrations exposure between the concentration groups has little difference; the rule of inhibition at63dah is consistent with42dah. At63dah, the normal group and exposed group have lower gene expression than42dah. In the low expression B group, CYP19A gene expression have no significant inhibitory effect at42dah and63dah, the different groups had no significantly difference at same day. Normal group and exposed group had high levels of gene expression than42dah at63dah.From experimental analysis, we obtain following conclusions:①AI exposure may change the CYP19A gene expression and affect larvae sex differentiation of Takifugu obscures. The final result is a reduction in the proportion of females.②Different starting times of exposure will affect different female sex ratio.10dah initial exposure group has the best results. Its female sex ratio was0%. ③Exposed to different concentrations group will also affect the female sex ratio; The female sex ratio of0.625mg/L concentration group was0%.