Preliminary Studies On Expression And Function Of Cypl9Ala And Cypl9Alb In Nile Tilapia(Oreochromis Niloticus)

Most studies have shown that estrogens play a pivotal role in sex determination, ovarian differentiation and maintenance of fish. Two kinds of aromatase (encoded by cyp19a1a and cyp19a1b), the key enzymes for estrogen synthesis, existed in teleost. However, previous researches mainly aimed at the expression and function of cyp19a1b in brain and cyp19a1a in the gonad while less in cyp19a1b in the gonad and cyp19a1a in the brain. Additionally, the function of gonadal development and ovary maintenance in female fish was mostly demonstrated by indirect approaches such as sex reverse from female to male induced via treatment of aromatase inhibitor and estrogen-receptor antagonist while by loss of function and gain of function studies. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with a stable XX/XY sex determination system, has become one of the most important species in global aquaculture. Moreover, it is a good experimental model for understanding the developmental genetic basis of sex determination because of the availability of monosex populations and the whole genome sequences. In this study, the difference and relationship betwen the two aromatases in the expression pattern and cellular localization in gonad and brain were analysized by Real-time PCR, immunohistochemistry and the function by gene knockout. The main results are listed as follows:1) The expression of cyp19a1b displayed sex difference in the brain at30and180dah, higher in male compared to female as indicated by Real-time PCR. Gonadal transcriptome data sequenced by our laboratory demonstrated that both of the two aromatases were expressed in gonads, higher in the female compare to the male. The expression of cyp19a1a and cyp19a1b were detected from5dah, the critical period of sex determination and differentiation, and strongest at30dah among all developmental stages. Additionally, the expression of cyp19a1a was higher than cypl9alb at30dah. Expression of both aromatases were found to be sexual dimorphic in all developmental stages of brain by immunohistochemistry. The expression of Cyp19a1b was observed in radial glial cells (RGCs) from3dah of brain while expression of Cyp19a1a was only detected in the180dah of brain. The immunohistochemistry results of both Cypl9ala and Cypl9alb were consistent with those from transcriptome analysis. The signal of Cyp19a1b was also detected in the theca cell of XX gonad and in the Ledig cell of XY gonad and co-expressed with Cypl9ala in interstitial cells of the XX gonad. However, no signal of Cypl9ala was detected in the testis.2) Multiple of mutant types including in-frame and frame-shift deletions were obtained by knockout of cyp19a1a and/or cyp19a1b using CRISPR/Cas9system in XX fish. Deletion of Cyp19a1a caused sex reverse from female to male while the4-month-old XX control ovary was full of Ⅰ and Ⅱ oocytes. In Cyp19a1a-deficient XX gonad, sex reverse was further proved by immunohistochemistry. The expression of Dmrt1(usually expressed specifically in the control testis) and Cyp11b2(11β-hydroxylase, the key enzyme for11-KT synthesis) were detected; while no signal in XX control and that the signal of Cypl9ala was not detected while expressed in interstitial cells of XX control. Phenotypes of meiosis arrest and somatic cell proliferation were observed in Cyp19a1b-deficient5-month-old gonad. Similar to deletion ofcyp19a1a, lower Cyp19a1a and stronger Dmrt1and Gsdf (expressed only in somatic cells surrounding the spermatogonia and oogonia) were detected in Cyp19a1b-deficient XX gonad, compared with the XX control gonad. Meiosis was blocked. Cyp19a1b-deficient gonad of7-months-old developed as ovotestis, in which Dmrt1expressed in somatic cells (Sertoli-like cells) and Cyp19a1a expressed in interstitial cells surrounding the oocyte of the ovarian tissue while Cyp11b2in the Ledig cells of the testicular tissue of ovotestis. These data showed that Cyp19a1a was involved in ovary determination and differentiation and Cyp19a1b was involved in ovary maintenance.In summary, both aromatases were expressed in XX gonads stronger than XY gonads. Cyp19a1b was co-expressed with Cyp19a1a in ovary interstitial cells and it was also expressed in theca cells of the ovary and in Ledig cells of the testis. Knockout of cyp19a1a and cyp19a1b revealed that cyp19a1a was involved in ovarian determination and differentiation and cyp19a1b was involved in ovarian maintenance in teleosts.