Study On Zearalenone Induced Apoptotic Pathway Of Ovary Granulosa Cells

Zearalenone (ZEA), a phytoestrogen molecular, is an important member of biological“Resorcylic acid lactones”(RALs) family. ZEA widely distributes in many important crops,feed and food commodities. As an exogenous estrogen mycotoxin, ZEA shows high affinity toestrogen receptors. ZEA is hydroxylated and transformed rapidly into α-Zearalenol (α-ZOL)and β-Zearalenol (β-ZOL) in vivo. Estrogenic effects of these metabolites are enhancedsignificantly. It had shown that ZEA and its metabolites exist in biological system, not onlycaused reproductive disorders in livestock animals, led to high estrogen syndrome in humans,but also brought great harm to human and animal bodies. At present, there are many studieson the toxicity and mechanism of ZEA, and some of them have also demonstrated the toxiceffects on ovary. Due to the complexity metabolisms and interactions with other substances invivo, its mechanisms for ovary granulosa cells cytotoxicity and signaling pathway inducedapoptosis are limited. By detecting changes of apoptosis-related factors, this study exploredsignaling pathway which induced ovary granulosa cells apoptosis, and further evaluated toxicmechanisms of ZEA. Research results are as follow:1.36eight-week-old female SD rats were randomly divided into control groups and trialgroups, then treated with5mg/kg i.p. of ZEA (corn oil). Control groups were treated with thesame quantity of corn oil. At6h,12h,18h,24h,36h and48h after intraperitoneal injection,rats were sacrificed6(each groups for3). Ovarian tissues were taken for fixing in4%phosphate-buffered paraformaldehyde. HE staining were used to determine ovarianmorphological changes and Transmission electron microscope were used to observe theapoptosis of ovary granulosa cells in rats. Immunohistochemistry (SP) method results showed:①Fas in the trial group was significantly higher than that in the control group. The highestpeak of expression emerged at18h (p<0.01)and then was down-regulated.②FasL showed asignificant rise in the control group. The expression was significantly up-regulated in18h and24h.③FADD in trial group over expressed (p<0.05). The highest expression emerged at6h,The mean optical density value was0.2953±0.0096,later began to decline.④Caspase-10inthe trial group increased significantly. There were two peaks at12h and36h and distinctionwas statistically significant (p<0.05). 2. Porcine granulosa cells in primary culture as models were used to compare the effectsof different concentrations of ZEA. With increasing concentrations of ZEA, granulosa cellswere scattered, and morphological changed or quantity reduced by morphological observation.The vast majority of granulosa cells had died in the high-dose group (80μg/mL). MTT assayindicated that ZEA inhibited the growth of ovary granulosa cells. The results detected byimmunofluorescence showed that the apoptosis rate increased gradually with increasing ZEAconcentration. High concentration group cell proliferation rate was about1.01%. Theexpression of Fas, FasL, FADD and Caspase-10significantly increased by Western blotting in6concentration groups, in does-dependent manners.In conclusion, ZEA induced ovary granulosa cells apoptosis, and up-regulated theexpression of Fas, FasL, FADD, Caspase-10significantly. Preliminary results showed thatZEA could activate protein expression associating Fas pathway, and mediated ovary granulosacells apoptosis through the death receptor pathway. The results laid a foundation for furtherinvestigating ovary toxicity mechanism induced by ZEA.