Expression characteristics of the cyp19a1b aromatase gene and its response to 17ß-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-ß-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Transcriptome analysis of four types of gonadal tissues in largemouth bass (Micropterus salmoides) to reveal its sex-related genes.

The sex determination system of largemouth bass (Micropterus salmoides, LMB) is XX/XY; however, the underlying molecular mechanisms involved in early sex differentiation, gonadal development, and exogenous hormone-induced sex reversal remain unknown. In this study, LMB at 15 days post-hatching (dph) were fed diets containing 20 mg/kg of 17α-methyltestosterone (17α-MT) or 30 mg/kg of 17ß-estradiol (17ß-E2) for 60 days, respectively. Serum steroid levels, histological observations of the gonads, and identification of sex-specific markers were employed to screen the gonads of 60-day-old normal female fish (XX-F), normal male fish (XY-M), 17ß-E2 induced pseudo-female fish (XY-F), and 17α-MT-induced pseudo-male fish (XX-M) for transcriptome sequencing in order to uncover genes and pathway involved in the process of sexual reversal. The results from histology and serum sex steroid hormone analysis showed that both 17α-MT and 17ß-E2 were capable of inducing sex reversal of LMB at 15 dph. Transcriptome results revealed a total of 2,753 genes exhibiting differential expression, and the expression pattern of these genes in the gonads of XX-M or XY-F resembled that of normal females or males. The male sex-biased genes that are upregulated in XX-M and downregulated in XY-F are referred to as key genes for male reversal, while the female sex-biased genes that are upregulated in XY-F and downregulated in XX-M are referred to as key genes for female reversal. Finally, 12 differentially expressed genes (DEGs) related to male sex reversal were screened, including star2, cyp17a, cyp11b1, dmrt1, amh, sox9a, katnal1, spata4, spata6l, spata7, spata18 and foxl3. 2 DEGs (foxl2a and cyp19a1b) were found to be associated with female sex reversal. The changes in these genes collectively influence the direction of sex differentiation of LMB. Among them, star2, dmrt1 and cyp19a1b with significantly altered expression levels may play potentially crucial role in the process of gender reversal. The expression patterns of 21 randomly selected genes were verified using qRT-PCR which confirmed the reliability and accuracy of the RNA-seq results. These findings not only enhance our understanding of the molecular basis underlying sex reversal but also provide crucial data support for future breeding research on unisexual LMB.

Effects of 17α-methyltestosterone and letrozole on growth and gonadal development in largemouth bass (Micropterus salmodies).

In order to optimize the parameters for reversing masculinization and establish the techniques for sex induction of pseudo-males and creation of all-female fry in largemouth bass (Micropterus salmodies, LMB), 15-day-old LMB (1.00 ± 0.10 cm in length, 0.10 ± 0.01 g in weight) were fed a diet supplemented with either 17α-methyltestosterone (MT) or letrozole (LE) and their combination. The experimental groups were M20 (20 mg/kg MT), L20 (20 mg/kg LE) and M10L10 (10 mg/kg MT and 10 mg/kg LE). The control group, named C, was not feed MT or LE. After 60 days, exogenous hormone in the diets was stopped and the effects of MT and LE on growth, male ratio, and gonadal development in LMB were evaluated. At 12-month-old, blood and gonadal tissue samples were collected to measure sex steroid hormones levels, analyze expression levels of dmrt1 and cyp19a1a genes, as well as examine the gonads morphology. The results showed no significant differences in growth between the experimental groups and the control group after a 60-day feeding period with the formulated diet (p > 0.05). The sex reversal ratio of M20, L20, M10L10 were 95.00%, 80.00%, 76.47%, respectively. The gonadal tissue sections showed that the gonadal structure of masculinized fish morphologic resembled that of control male fish. At 12-month-old, the sex reversal ratio in M20, L20, M10L10 and C groups were 100%, 86.67%, 73.33% and 50.00%, respectively. The testicular of pseudo-male fish in the M20 group exhibited well-developed morphology similarities to that of the control group males. However, the testes of pseudo-male fish in the L20 and M10L10 groups were smaller size Estradiol (E2) levels in the experimental groups was significantly lower than those in the control group females (p < 0.05), while testosterone (T) levels were significantly higher than that of the control group (p < 0.05). Compared to the female fish in the control group, pseudo-male fish from all experimental groups showed significantly upregulated expression of dmrt1 (p < 0.05), and significantly downregulated expression of cyp19a1a (p < 0.05). Pseudo-males selected from group M20 exhibited a significantly higher proportion of female offspring (92.00%) compared to the control group (46.50%). In summary, 20 mg/kg MT was the optimal inducing concentration.