International Journal of Aquaculture, 2025, Vol.15, No.4, 184-196 http://www.aquapublisher.com/index.php/ija 188 3.3 The correlation between spatiotemporal expression patterns and gender transformation stages Different spatiotemporal expression patterns of gender-related genes are of great significance to understanding the stages of grouper gender transition. The expression of many key genes in different stages of development and in different tissues is strictly regulated. In the embryonic/littlefish stage, some gender-related genes may show male-female expression differences early, indicating the initial differentiation direction of the gonad; while others only initiate expression and regulate gonadal function when the gonad develops rapidly during puberty. In grouper, since their gender can be changed acquired, it is necessary to focus not only on gene expression in the early stage of gender differentiation (the male-female-determining phase), but also on reprogramming when sexual reversal occurs after maturation. Dmrt1 and cyp19a1a can be regarded as "opposite indicators" of gonad fate: in the mature ovaries of female fish, cyp19a1a is highly expressed while DMrt1 is nearly silent; while in the male fish's semen, DMrt1 maintains a high level while cyp19a1a is suppressed for a long time. At the initiation of sexual reversal, the expression of both increases and decreases: the significantly increased DNA methylation level in the promoter region of the aromatase gene leads to a decrease in its transcription, while the de-repression of the DMRT1 promoter achieves a large amount of transcription. This suggests that the molecular switches of grouper females to males are directly related to the transition of these key gene expression states (Figure 2) (Wang et al., 2018). For example, Foxl2 and amh genes: Foxl2 is continuously expressed in female ovaries to maintain follicles, while amh is mainly expressed by male sperm support cells to inhibit excessive primitive germ cells; during sexual reversal, Foxl2 is gradually downregulated and amh begins to appear in the newly formed sperm tissue. The expression changes of these genes correspond to the histological process of follicle reduction and spermatogenesis (Lyu et al., 2019). In addition, hormone receptors and signaling pathway components also show phase-specific expression patterns. The gonadotropin receptor fshr is relatively low in the ovaries in the pre-sexual reversal stage, but it is enhanced in residual follicles and newly formed semen tissue at the beginning of the reversal, suggesting that FSH signal is involved in the trigger of reversal; recent brain and pituitary transcriptome analysis of oblique band grouper under natural social reversal conditions showed that after determining a new social status (preparing for sexual transition), the clock genes and melatonin synthesis-related genes in the brain were rapidly upregulated after the dominant female fish were determined to have a new social status (preparing for sexual transition). This may mean that circadian signaling and light sensing are involved in the initiation of socially induced gender-transforming behaviors (Wang et al., 2018). This spatiotemporal expression association of brain-godal axis genes provides clues for us to understand how environmental factors act on the gonad through the central role. 4 The Role of Non-Coding Rna and Epigenetic Mechanisms in Gender Regulation 4.1 Regulatory functions of miRNA and lncRNA in gender decisions With the development of genomics, it has been recognized that a large number of non-coding RNAs (ncRNAs) play an important role in the regulation of gene expression, including during the process of gender determination and differentiation in fish. In the gonadal tissues of fish such as groupers, abundant microRNAs (miRNAs) and long-chain non-coding RNAs (lncRNAs) were detected, and their expressions often differ between male and female individuals, suggesting that they are involved in the regulation of the gender pathway. Some miRNAs have been found to have sex-biased expression: specifically high in male or female gonads, thereby targeting genes that regulate gender-related. Studies have shown that miR-26a-5p is upregulated during androgen-induced sexual transition, which can inhibit the expression of the aromatase cyp19a1a, leading to a decrease in estrogen synthesis, thereby promoting the maleization process. In a study on sexual reversal of turtles, dozens of miRNAs significantly related to gender differences were identified, among which miR-26a-5p, miR-212-5p, miR-202-5p, etc. play a role in the sexual reversal process and are predicted to target multiple gender-related genes (Zhang et al., 2021). Together with the target genes, these miRNAs form a network that regulates gonad development. Long-chain non-coding RNA (lncRNA) affects gene expression through various mechanisms: some lncRNAs can act as "sponges" of miRNAs, binding and isolating miRNAs to relieve their inhibition of target genes; others can directly bind to transcription factors or chromatin modification complexes to regulate gene transcription. In the study of fish gender differentiation, lncRNA is an emerging field but has shown importance. Reports show that in the gonads of species such as carp and raw fish, many lncRNAs have co-expression patterns with adjacent gender-related genes, suggesting that they may participate in regulation in concert (Yuan et al., 2019).
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