大嘴鲈鱼的性别决定系统(Micropterussalmoides,LMB)为XX/XY;然而,参与早期性别分化的潜在分子机制,性腺发育,和外源性激素诱导的性逆转仍然未知。在这项研究中,孵化后15天(dph)的LMB饲喂含有20mg/kg17α-甲基睾酮(17α-MT)或30mg/kg17β-雌二醇(17β-E2)的饮食60天,分别。血清类固醇水平,性腺的组织学观察,和性别特异性标记的鉴定被用来筛选60天大的正常雌性鱼(XX-F)的性腺,正常雄性鱼(XY-M),17β-E2诱导的假雌性鱼(XY-F),和17α-MT诱导的假雄性鱼(XX-M)进行转录组测序,以揭示参与性逆转过程的基因和途径。组织学和血清性类固醇激素分析的结果表明,17α-MT和17β-E2均能够在15dph下诱导LMB的性逆转。转录组结果显示,共有2,753个基因表现出差异表达,这些基因在XX-M或XY-F性腺中的表达模式与正常雌性或雄性相似。在XX-M中上调并在XY-F中下调的男性性别偏向基因被称为男性逆转的关键基因,而在XY-F中上调而在XX-M中下调的女性性别偏见基因被称为女性逆转的关键基因。最后,筛选12个与男性性别逆转相关的差异表达基因(DEGs),包括star2cyp17a,cyp11b1,dmrt1,amh,sox9a,katnal1,spata4,spata6l,spata7,spata18和foxl3.发现2个DEGs(foxl2a和cyp19a1b)与女性性别逆转有关。这些基因的变化共同影响LMB的性别分化方向。其中,star2,dmrt1和cyp19a1b表达水平显着改变,可能在性别逆转过程中起着潜在的关键作用。使用qRT-PCR验证了21个随机选择的基因的表达模式,这证实了RNA-seq结果的可靠性和准确性。这些发现不仅增强了我们对性别逆转的分子基础的理解,而且为未来单性LMB的育种研究提供了重要的数据支持。
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 17β-estradiol (17β-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), 17β-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 17β-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.