## 4 Discussion  
### Effect of Methomyl on Sex Steroid Hormone Synthesis Substrates  
CHO is a precursor of the sexual steroid hormones testosterone (T), 11-ketotesosterone (11-KT), and 17\(\beta\) -estradiol (E2). The basic process of CHO synthesis in fish is shown in Figure 2[18]. CHO is transported from the mitochondrial outer membrane to the inner membrane under the action of steroidogenic acute regulatory protein (StAR) [19]. CHO is converted to PREG by the action of the CHO side-chain cleavage enzyme (CYP11A1) [20]. PREG is converted into PROG catalyzed by 3\(\beta\)-hydroxysteroid dehydrogenase (3\(\beta\)-HSD) in the smooth endoplasmic reticulum [18], and 17\(\alpha\) hydroxylase (P45017\(\alpha\)) catalyzes the conversion of PREG and PROG to androgen [18]. Androgen is converted into estradiol under the catalysis of aromatase [21]. In this study, methyl had no significant effect on CHO content in serum and testes at low concentrations (0.2 and 2 ug L\({}^{-1}\)) (\(p\) > 0.05), but a significant (\(p\) < 0.05) decrease in CHO content in serum and testes at high methanol concentrations (20 and 200 ug\(\cdot\)L\({}^{-1}\)) was found. [PERSON] et al. [22] studied the effect of difenoconazole on CHO in zebrafish and found that low concentrations of difenoconazole (0.1% and 10 ug L\({}^{-1}\)) had no significant effect on CHO content (\(p\) > 0.05). High concentrations (500 ug L\({}^{-1}\)) significantly reduced CHO content (\(p\) < 0.05), which was consistent with the change trends of CHO in our study. Other studies have also shown that environmental estrogen could alter gonadal steroid production at specific sites within the steroidogenic pathway, that is, downstream of CHO mobilization to side-chain cleavage of P450 scc and/or conversion of CHO to PREG [23]. The decreased levels of PREG produced by the testes of methyl-treated tilapia suggest that methyl may affect CHO availability or reduce the activity of the side-chain cleavage enzyme P450 scc, which mediates the conversion of CHO to PREG [8]. Similarly, the response of PREG and PROG to methyl in serum and testes showed no effect at low concentrations (0.2 and 2 ug L\({}^{-1}\)) (\(p\) > 0.05) and significantly decreased at high concentrations (20 and 200 ug L\({}^{-1}\)). However, a significant (\(p\) < 0.05) decrease in the present experiment, consistent with that of CHO in the serum and testes, was ob  
Figure 2: The sex hormone biosynthesis pathway in fish. (Note: CHO (Cholesterol), PREG (Pregnancy, PROG (Progesterone), P450 scc (Cholesterol side chain cleavage enzyme/CYP11A1), 3\(\beta\)-HSD (3\(\beta\)-hydroxysteroid dehydrogenase), CYP17 (Cytochrome P450 17\(\alpha\)-hydroxylease), 17\(\alpha\)-HPREG (17\(\alpha\)-Hydroxygreenolone), 17\(\alpha\)-HPROG (17\(\alpha\)-Hydroxy progesterone), DHEA (Dehydroepiandrostore), ADSD (Androstenedione), 17\(\beta\)-HSD1 (17\(\beta\)-hydroxysteroid dehydrogenase I), 17\(\beta\)-HSD2 (17\(\beta\)-hydroxysteroid dehydrogenase type II), 17\(\beta\)-HSD2 (17\(\beta\)-hydroxysteroid dehydrogenase type III), E1 (Estrone), E2 (17\(\beta\)-estradiol), T (Testosterone), 11-KT (11-ketotesosterone), 11\(\beta\)-HT (Hydroxy testosterone), DHP(17\(\alpha\),209-dihydroxy Progesterone)). served. PREG is transformed from CHO under the catalysis of CYP11A1 [20], and PROG is transformed from PREG under the catalysis of 3\(\beta\)-HSD in the smooth endoplasmic reticulum [18]. Therefore, a decrease in CHO content inevitably leads to a decrease in PREG and PROG content. At the same time, our previous study showed that the expression of testes 3\(\beta\)-HSD mRNA was significantly inhibited at high concentrations (20 and 200 \(\mu\)g L\({}^{-1}\)) (\(p<0.05\)), and the inhibition intensity increased with an increase in methyl concentration [5]. This suggests that decreased levels of 3\(\beta\)-HSD in the testes further inhibited the conversion of PREG to PROG, thereby exacerbating the decline in PROG levels.