| ZEA(Zearalenone)is a nonsteroidal fungal toxin with estrogen activity produced by fusarium.ZEA and its metabolites are widely found in grain raw materials and feeds such as barley,sorghum,corn and wheat,which can directly or indirectly contaminate animal products and pose a great threat to human health.The estrogen-like effects of ZEA can disrupt hormone levels in the body,causing great harm to the reproductive system of animals,leading to a series of reproductive diseases and bringing huge economic losses to livestock breeding and production industries.Through in vivo and in vitro experiments,this study investigated the effects of ZEA at different concentrations on weaned piglets’ uterus and endometrial epithelial cells,in order to provide theoretical basis for solving the reproductive toxicity mechanism of ZEA and the reproductive diseases caused during production.In this study,immunofluorescence,immunohistochemistry,qRT-PCR,transcriptomics and Western Blot were used to compare the distribution and expression differences of estrogen and progesterone receptor,proliferation and apoptosis related genes and genes related to Wnt/β-catenin signaling pathway in the uterus of the post-weaning piglets and endometrial epithelial cells.Forty weaned piglets with no significant difference in age and initial weight between 34 and 36 days were randomly divided into four treatments,each with 10 pigs.The control treatment was fed the basic diet,while the other treatments were basal diet supplemented with ZEA at the level of 0.5,1.0 and 1.5 mg/kg.All pigs were fed individually in a metabolic cage for 35 days after 10-d adaptation.ZEA(0,5,20 and 80 μmol/L)were added for continuous culture.And the cells were collected after 24 h for subsequent experiments.Research on the relationship between estrogen progesterone receptor pathway and ZEA-induced uterine hypertrophy: The immunohistochemical results show that ER-α,ER-β and PR immunoreactive substance were mainly localized in the luminal epithelium,lamina propria,uterine glands,stromal cells and the vessels,and there was no significant change i n the distribution of the immunoreactive substance,due to the presence of ZEA.The relative mRNA and protein expressions of ER-α,ER-β and PR in uteri increased linearly(P < 0.01)with an increasing level of ZEA,while the expression of ER-α and ER-β in the endometrial epithelial cells increased linearly(P < 0.01).Research on the relationship between proliferation and apoptosis pathway and ZEA-induced uterine hypertrophy: The relative mRNA and protein expressions of BCL-2 and PCNA in porcine endometrial epithelial cells increased linearly(P < 0.05),while the expressions of BAX and Caspase 3 decreased linearly(P < 0.05)with an increasing of ZEA.Research on the relationship between Wnt/β-catenin pathway related genes and ZEA-induced uterine hypertrophy: ZEA at the concentration of 20 and 80 μmol/L could alter 25 and 34 genes of the 36 genes closely related to Wnt signaling pathway,respectively.And the relative mRNA and protein expressions of Wnt1,β-catenin and GSK-3β in porcine endometrial epithelial cells all increased linearly(P < 0.05),while the expressions of CCND1 decreased linearly(P < 0.05),with an increasing of ZEA.In addition,the immunofluorescence showed that β-catenin accumulated in the nucleus with the increasing of ZEA,and signs of invading the nucleus in the 80 μmol/L ZEA treatment was observed.Overall,(1)ZEA could promote abnormal uterine development through estrogen and progesterone receptor pathways(ER-α,ER-β and PR);(2)ZEA could inhibit the occurrence of apoptosis through the proliferation pathway(BCL-2,PCNA),promote the proliferation of endometrial epithelial cells,and lead to uterine hypertrophy;(3)ZEA could activate the Wnt/β-catenin signaling pathway and participate in the uterine development by altering the expression of related genes in the pathway. |
