International Journal of Aquaculture, 2025, Vol.15, No.4, 184-196 http://www.aquapublisher.com/index.php/ija 187 endocrine changes cause the Foxl2/cyp19a1a pathway to weaken, the dmrt1/sox9 pathway may have relatively upper hand, thereby driving gonads towards male development. In addition to the Dmrt1-Foxl2 pair, there are other similar antagonist groups in fish gender decision-making. For example, the Wnt4/β-catenin signaling pathway tends to support ovarian development, while Amh/Amhr2 signaling promotes schizophrenia differentiation (Figure 1) (Wu et al., 2017); the Cyp19a1a product estradiol maintains ovarian structural integrity, while androgen (11-KT) drives schizophrenia development (Peng et al., 2020). These factors form key nodes in the gender regulatory network through complex regulatory relationships. Figure 1 Location of Amh in the testes (Adopted from Wu et al., 2017) Image caption: (A) Western blotting of grouper testicular and ovarian protein extracts using anti-Amh antiserum and anti-Actin antiserum. The black arrowhead denotes Amh protein. (B and C) Immunohistochemical staining of testes using anti-Amh antiserum. Black arrowheads indicate the positive signal (brown color) of anti-Amh antiserum. (D and E) Immunofluorescent staining of testes using anti-Amh antiserum and anti-Vasa antiserum. Vasa is a germ cell marker. White arrowheads indicate a positive signal (green color) of anti-Amh antiserum. OT, ovarian tissue; RO, regressed oocyte, SC, spermatocyte; SG, spermatogonia, SP, sperm; TT, testicular tissue (Adopted from Wu et al., 2017) 3.2 Differential gene groups revealed by expression profiling analysis With the help of high-throughput sequencing technology, the analysis of the transcriptomes of grouper's different gender and sexual transition stages can reveal a large number of gender-related differentially expressed gene populations. These genes include transcription factors, hormone synthetases, receptors, signaling pathway molecules, and metabolic and structural proteins (Li, 2024). By comparing the gene expression profiles of female, primary male and secondary male, the researchers found that a considerable portion of gene expression showed significant differences between male and female. Genes related to ovarian function such as cyp19a1a, Foxl2, bmp15, figla, etc. are highly expressed in female samples, but are low and even non-expressed in male samples (Peng et al., 2020). Some cell proliferation and differentiation factors also show dynamic changes during sexual reversal. For example, the spatiotemporal expression changes of genes such as Wnt4, igf1, FST (ovarian-related) and cxcl12, gsdf (spermally related) reflect the process of ovarian tissue regression and sperm occurrence. In addition to conventional protein-encoding genes, transcriptome analysis also reveals the potential role of many non-coding RNAs in gender differences: dozens of microRNAs and long-chain non-coding RNAs are significantly differentially expressed between male and female gonads, which may be involved in gender fate decisions by influencing post-transcriptional regulation of key genes. The gender transition process may vary slightly between different grouper individuals, so screening of different genes requires stricter criteria and sufficient repetition. Through time series transcriptome data, a series of gender-related genes can be sorted in order of expression peaks, and their order and causal relationship in gender reversal can be inferred. Studies have conducted multi-stage sampling and sequencing of grouper induced by artificial androgen treatment and found that first responded to some stress and metabolic genes, followed by reproductive axis-related genes (upregulation, ultimately, the gonad direct target gene is expressed in large quantities, thus delineating a top-down timeline of molecular events (Han et al., 2018).
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