Effects Of Monocrotophos Pesticide On The Reproductive Axis In Goldfish (Carassius Auratus)

Monocrotophos (MCP) is an organophosphorus pesticide that is highly toxic, and exposure to MCP-based pesticides significantly induced the hepatic mRNA expression and protein secretion of vitellogenin (VTG) in male goldfish, suggesting that MCP has significant estrogenic properties. It is hypothesized that MCP induces VTG production by interfering with hormone secretion regulated by the reproductive axis (hypothalamic-pituitary-gonadal axis). To better characterize MCP induction of VTG expression, we investigated the effects of MCP on sex steroid levels, and examined potential underlying mechanisms by determining the changes in endocrine-mediated responses along the reproductive axis in male goldfish, including the expression of P450arom isoforms, the synthesis and secretion of GtHs (gonadotropins) and the regulation of GnRHs (gonadotropin-releasing hormones). Furthermore, as female reproduction is also under the control of the reproductive axis, with a similar regulation mechanism with the males, to elucidate the influence of MCP on reproductive and reproductive endocrine systems, the effects of MCP pesticide on the reproductive axis in the female goldfish is also conducted.The results indicated that:(1) Plasma sex steroid levels were examined by radioimmunoassay (RIA) in goldfish(Carassius auratus) following a 21-day exposure to 0.01,0.10 and 1.00 mg·L-1 40% MCP-based pesticide.In the males, T basal levels in the the plasma were relatively high, while E2 concentrations in the plasma were as extremely low; in the femles, E2 basal concentrations in the plasma were relatively high, while T levels were relatively low. MCP exposure reduced plasma levels of T and increased plasma concentrations of E2, and there appeared to be a dose-related increase in the ratio of E2/T after MCP treatment. This provided convincing evidence for disruption of MCP on sex steroids secretion, by which VTG was induced in males and ovarian growth was increased in females. (2) P450arom mRNA expression levels in gonad and hypothalamus were examined by real-time PCR in goldfish (Carassius auratus) following a 21-day exposure to 0.01,0.10 and 1.00 mg·L-1 40% MCP-based pesticide. P450aromA mRNA expression in the gonad was upregulated after MCP pesticide exposure in a dose-dependent manner. However, P450aromB mRNA levels in the hypothalamus were not changed by any treatment. Our study indicated that gonadal P450aromA, not brain P450aromB, is the specific target of MCP. Thus, we hypothesize that MCP treatment elevates gonadal aromatase expression and causes the conversion of T into E2 and thus causes the significant elevation of E2 levels and the E2/T ratio.(3)FSH-βand LH-βmRNA expression levels in pituitary were examined by real-time PCR in goldfish (Carassius auratus) following a 21-day exposure to 0.01, 0.10 and 1.00 mg·L-1 40% MCP-based pesticide, and we also investigated the potential impacts of MCP treatment on plasma levels of GtHs by RIA.Treatment with MCP resulted in increased FSH-βmRNA levels and FSH plasma levels, while it caused a decrease in LH-βmRNA expression and LH plasma levels. One could deduce that the increase in FSH synthesis and secretion by MCP could lead to the stimulation of E2 production, while the decrease in LH synthesis and secretion could lead to the inhibition of T production, thus enhancing the imbalance of estrogen and androgen caused by increased P450aromA gene expression. The data suggested that reproductive endocrine function was also important target of MCP interference with the reproductive axis in goldfish, indirectly impacting sex steroid synthesis and secretion.(4) sGnRH,cGnRH-ⅡmRNA expression levels in hypothalamus were examined by real-time PCR in goldfish (Carassius auratus) following a 21-day exposure to 0.01, 0.10 and 1.00 mg·L-1 40% MCP-based pesticide. The mRNA levels of sGnRH and cGnRH-Ⅱin the hypothalamus of goldfish were not affected by any of the MCP pesticide treatments. The potential effects of MCP pesticide on neuroendocrine regulation need further study.In the present study, the endocrine disruption of the reproductive axis by MCP was investigated in male and female goldfish by evaluating the potential impact on synthesis and conversion of gonadal sex steroids, synthesis and secretion of pituitary GtHs, and regulation of hypothalamic GnRHs. The results provided the potential mechanisms underlying estrogenic effects of MCP. Compared to compounds such as E2, MCP did not seem to exert its estrogenic effects in goldfish via ER-mediated mechanisms, but rather at other points along the reproductive axis. MCP induced VTG production in male goldfish via (a) increasing P450aromA gene expression in the gonad, leading to the increased E2 levels in the plasma; and (b) interfering with GtHsβsubunit expression and secretion at the pituitary level, resulting in a disruption of reproductive endocrine control and an indirect impact on sex steroids. This study also provided convincing evidence for reproductive toxicology of MCP by disrupting of the HPG axis at multiple sites in female goldfish, resulting in induced plasma E2 levels and thus enhanced ovarian growth. Through systematic perturbation of the reproductive axis, we are developing a greater understanding of systemic responses to chemical stressors at multiple levels of biological organization, ranging from molecular responses to whole-animal outcomes, that are needed to support the scientifically credible use of biomarkers and other endpoints in ecological risk assessments for pesticides.