The Effects And Mechanisms Of Monocrotophos Pesticide On Sex Differentiation Of Zebrafish

Monocrotophos (MCP) is an organophosphorus pesticide that is highly active and widely used. Exposure to MCP pesticide could induce the hepatic mRNA expression and protein secretion of vitellogenin (VTG) in male goldfish, suggesting that MCP pesticide belongs to potential environmental estrogens. Previous researches have proved that MCP pesticide could induce imbalance of sex steroid levels by increasing levels of 17β-Estradiol(E2) and reducing levels of testosterone(T) in fish, which means MCP pesticide has the potential to adversely affect fish sex differentiation regulated by sex steroid. As an important model organism, zebrafish were chosen to investigate the effects and mechanisms of MCP pesticide on fish sex determination. They were exposed to 0.001, 0.010 and 0.100 mg/L MCP pesticide from fertilization until the end of sex differentiation (0~40dph). Sex ratio and gonad differentiation were recorded to evaluate the disruption of MCP pesticide on sex differentiation of zebrafish. To indicate mechanisms of MCP pesticide on sex differentiation of zebrafish, we also investigated the effects of MCP pesticide on P450arom genes (Cyp19a1a and Cyp19a1b) and sex-differentiating related transcription factor genes (Foxl2 and Dmrt1) during the stage of sex differentiation (23~40dph). The results below shed light on the further research about the effects of endocrine disrupting chemicals on sex differentiation of fish.The results indicated that:(1) Sex differentiation of zebrafish was disrupted by estrogenic property of MCP pesticide following a 0~40dph exposure to 0.001, 0.010 and 0.100 mg/L pesticide. There was a dose-dependent increase in sex ratio (female/male) after estrogenic property of MCP pesticide, and even 71% of fish were female in 0.100mg/L MCP treatment group, significantly different from the control group; Ovo-testis were observed in fish exposed to 0.001 and 0.100 mg/L MCP pesticide. The degenerative changes were observed from the sections of testis and ovaries after the toxicity of MCP pesticide, including the shape of oocyte irregular in the ovaries,increased acellularity and even large vacuoles observed in testes and ovaries.(2) Cyp19a1a(gonad P450arom gene) and Cyp19a1b(hypothalamus P450arom gene) mRNA expression levels were examined by real-time PCR in zebrafish following 0~23dph,0~30dph and 0~40dph exposure to 0.001, 0.010 and 0.100 mg/L MCP pesticide. Cyp19a1a and Cyp19a1b mRNA expression levels were upregulated after 0.010 or 0.100mg/L MCP pesticide exposure, and 0.100mg/L MCP pesticide exposure significantly enhanced Cyp19a1a and Cyp19a1b mRNA expression during the whole sex differentiation process of zebrafish. The paper hypothesized that MCP pesticide could increase plasma concentrations of E2 and reduce plasma levels of T by enhancing the conversion of T into E2 after elevating P450arom gene expression levels in gonad, and then cause further imbalance of estrogen and androgen levels by hypothalamic-pituitary-gonadal (HPG) axis after elevating P450arom gene expression levels in hypothalamus, leading to feminination and abnormal sex differentiation in zebrafish.(3) Foxl2 as one of ovaries-differentiating related transcription factor genes and Dmrt1 as one of testes-differentiating related transcription factor genes mRNA expression levels were examined by real-time PCR in zebrafish following 0~23dph,0~30dph and 0~40dph exposure to 0.001, 0.010 and 0.100 mg/L MCP pesticide. Foxl2 mRNA expression was enhanced after MCP pesticide exposure in a time-dependent manner. Concomitantly, Dmrt1 mRNA expression was inhibited and the suppressive effect was the most significant after 0~23dph exposure to MCP pesticide. The paper indicated that MCP pesticide could disrupt sex differentiation by activating ovaries-differentiating related transcription factor gene such as Foxl2 mRNA expression level and repressing testes-differentiating related transcription factor gene such as Dmrt1 mRNA expression level. However, detailed mechanisms need further study.In conclusion, MCP pesticide could disrupt sex differentiation of zebrafish mainly through estrogenic property and mRNA expression levels of sex-differentiating related genes were measured to provide the potential mechanisms of MCP pesticide, including: (1) Enhancing P450arom genes expression might increase E2 levels , decrease T levels ,and finally lead to feminination in zebrafish; (2) triggering feminination via activating ovaries-differentiating related transcription factor gene as Foxl2 mRNA expression level and repressing testes-differentiating related transcription factor gene as Dmrt1 mRNA expression level, will disrupt sex differentiation of zebrafish.