International Journal of Aquaculture, 2025, Vol.15, No.4, 184-196 http://www.aquapublisher.com/index.php/ija 190 This means that its gene regulatory network remains responsive to environmental and endocrine signals in the adult stage, and its epigenetic state is not an unshakable endpoint, but more like a switchable reversible switch. Some scholars have suggested that "episogenetic sensitivity" may be one of the key factors in the ability of hermaphrodite fish to evolve gender conversion. Specifically, in the grouper genome, the epigenetic status of certain important sites associated with gender decisions may be more likely to be remodeled than in the general species (Nakamura et al., 2021). For example, endocrine changes caused by external factors (temperature, social stress, drugs) can trigger changes in DNA methylation or histone modification at these key sites, thereby reversing their expression status and thus reversing the gender function of the gonad (Soyano et al., 2022). New technologies in recent years, such as ATAC-seq, ChIP-seq, etc., can be used to analyze chromatin accessibility and histone marker distribution. If applied to sample comparison of grouper gonad differentiation/reversal, it will inevitably reveal the contribution of epigenetics to gender plasticity in a deeper way. 5 Regulatory Mechanism of Sex Hormones and Their Signaling Pathways 5.1 Estrogen and androgen synthesis and its metabolic genes (such as cyp19a1a, hsd17b) The anabolic genes of sex steroids play a central role in gender regulation, and the aromatases Cyp19a1a (P450arom) and 17β-hydroxysteroid dehydrogenase (HSD17 family) are key enzymes responsible for estrogen synthesis and androgen conversion, respectively. cyp19a1a aromatizes androgens such as androgens such as androsene and testosterone into estradiol, and is therefore a gene necessary for ovarian differentiation and maintenance. During the gender determination of grouper, cyp19a1a is expressed at high levels in female gonads to ensure a steady stream of sufficient estrogen production, thereby promoting follicle development and inhibiting the misinitiation of male pathways (Cheng et al., 2020). Studies have confirmed that knocking out cyp19a1a (ovarian aromatase) in zebrafish and tilapia by gene editing will cause partial or even complete sexual reversal in XX females, further demonstrating their function in female differentiation. In grouper, there has been no reports of cyp19a1a knockdown, but evidence through transcriptome and epistemological regulation has shown that cyp19a1a downregulation is one of the early molecular events of sexual reversal. There are many subtypes of 17β-HSD in fish, among which 17β-HSD type 3 is responsible for converting androstenedione to testosterone in male testes; while 17β-HSD type 1 mainly converts estrone to estradiol in the ovary (Liu et al., 2017). Another related gene, cyp11b (11β-hydroxylase), catalyzes the conversion of testosterone to 11-ketotestosterone, which is the synthesis step of fish's main androgens. Studies on orange-spot grouper show that cyp11b is highly expressed in male semen and sexual reversal individuals, while almost non-expressed in female ovaries. This means that the expression of cyp11b can be used as one of the molecular markers of male tissues. In fact, in some early stages of sexual reversal (such as after artificial MT treatment), upregulation of cyp11b often precedes complete schizophrenia. 5.2 Effects of changes in hormone levels on gonadal remodeling The most direct manifestation of grouper gender reversal is the rebalancing of sexual steroid hormone levels: estrogen falls from its peak, and androgen rises significantly. This transition of the endocrine environment plays a decisive role in the remodeling of gonadal tissue. During the process of female fish turning into male fish, the continuous decline in estradiol (E2) levels will cause follicle cells to lose support signals and a large number of oocytes enter atresia and apoptosis, thus making room for sperm hyperplasia (Wu et al., 2020). Meanwhile, androgens, especially 11-ketotestosterone (11-KT), are significantly elevated, which is the main hormone driver that promotes schizobulin formation and spermatogenesis. Some studies have quantified the changes in hormones in artificially induced sexual reversal experiments: after injecting a certain dose of cortisol intraperitoneal cavity, it was found that the serum 11-KT concentration increased significantly within a few weeks. At the same time, oocytes in ovarian tissue began to degenerate, spermatogonia appeared and developed in large quantities, and finally the gonads completed semenization (Soyano et al., 2022). If cortisol treatment is stopped at this time, the 11-KT level will gradually decrease, the developed semen tissue will stop maturing and partially degenerate, and some individuals may even restore part of the ovarian structure, indicating that the sexual reversal results may also be unstable when hormones are insufficient. This suggests that our gonadal remodeling requires sustained androgenic signals to consolidate. At the population level, sex hormone changes are also associated with gender
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