| Zearalenone(ZEA),one of the most prevalent estrogenic mycotoxin,is widely exists in animal feed.Previous work showed that ZEA might cause liver,reproductive and developmental toxicity.So far,the report of ZEA on porcine reproductive performance mostly fixed on its estrogenic activity.However,the adverse effects and the mechanisms of ZEA on the development and steroidogenesis of porcine granulosa cells,also including the mammalian ovarian folliculogenesis remain largely unknown.To further elucidate the pathways and mechanisms of ZEA exposure on the porcine reproductive system,our research based on the culture system in vitro of porcine granulosa cells or mouse ovaries,and mice were introduced as an animal model.Utilizing advanced technologies in reproductive biology and the transcriptome we investigated the molecular mechanisms of ZEA exposure on the ovarian development and steroidogenesis.This study aimed to provide a theoretical basis for solving porcine endocrine dyscrasia,premature ovarian failure and infertility caused by environmental estrogen.The adverse effects of ZEA on porcine granulosa cells were evaluated in experiment one.The granulosa cells were administered different concentrations of ZEA during cultured in vitro.We found that 10 ?M ZEA treatment groups had significantly increased apoptosis rate of the granulosa cells compared with the control group.(8.8%: 29.1%)RNA-seq analysis indicated that the gene expression pattern of porcine granulosa cells were changed by ZEA exposure.Compared with control group,10 ?M and 30 ?M ZEA treatment groups showed that the expression level of 209 and 271 DEGs significantly up-regulated,while 1098 and 1305 DEGs significantly down-regulated GO-enriched functions suggested that the granulosa cells treated with ZEA were significantly enriched in apoptosis and steroidogenesis process.A protein-protein interaction(PPI)network was visualized from the information of DEGs,which were verified by Q-PCR.Then we identified the core protein coded by TGF-?,IGF1 and IER3 involved in ZEA induced granulosa cells dysfunction.ZEA significantly decreased the expression of genes essential for estrogen synthesis by granulosa cells including FSHR,CYP19A1 and HSD17?.Furthermore,there was a significant reduction in the concentrations of 17?-estradiol in the cultured medium of granulosa cells.Collectively,these results demonstrate a concretely deleterious effect of ZEA exposure on the development and production of steroid hormones in porcine ovarian follicular granulosa cells in vitro.The adverse effects of ZEA on porcine granulosa cells were evaluated by comparison of mouse granulosa cells in experiment two.The granulosa cells were administered different concentrations of ZEA during cultured in vitro.We found that 10 ?M ZEA treatment groups had no significantly increased apoptosis rate of the mouse granulosa cells compared with the control group.(8.1%: 11.8%)RNA-seq analysis indicated that the gene expression pattern of mouse granulosa cells were unchanged by ZEA exposure.Compared with 10 ?M ZEA treatment porcine groups and 30 ?M ZEA treatment mouse groups showed that there was 209 DEGs.In addition,there was 619 DEGs in 10 ?M ZEA treatment porcine groups and 30 ?M ZEA treatment mouse groups.GO-enriched functions suggested that the granulosa cells treated with ZEA were significantly enriched in apoptosis and steroidogenesis process.A protein-protein interaction(PPI)network was visualized from the information of DEGs,which were verified by Q-PCR.Then we identified the core protein coded by TGF-?,IGF1 and IER3 involved in ZEA induced granulosa cells dysfunction.ZEA significantly decreased the expression of genes essential for estrogen synthesis by granulosa cells including FSHR,CYP19A1 and HSD17?.Furthermore,there was a significant reduction in the concentrations of 17?-estradiol in the cultured medium of granulosa cells.Collectively,these results demonstrate a concretely deleterious effect of ZEA exposure on the development and production of steroid hormones in porcine ovarian follicular granulosa cells in vitro.The protein-protein interaction(PPI)network constructed by 619 DEGs indicated that inflammation-related proteins significantly interacted with other proteins in porcine granulosa cells,while cancer-related proteins significantly interacted with other proteins in mouse granulosa cells.The results suggested that porcine granulosa cells were more sensitive than mouse granulosa cells in 10 ?M ZEA treatment.In addition,there might be potential hazard of the inflammatory response and the cancer to porcine granulosa cells under the 30 ?M ZEA treatment.The adverse effects of ZEA exposure on mammalian primordial follicle formation were evaluated in experiment three.Using the culture system in vitro of mouse ovaries,we investigated the biological effects of ZEA exposure on murine ovarian germ cell cyst breakdown and primordial follicle assembly.Our results demonstrated that newborn mouse ovaries exposed to 10 or 30 ?M ZEA in vitro had significantly decreased germ cell cyst breakdown and primordial follicle assembly compared to the control group.Moreover,the presence of ZEA in vitro increased the numbers of TUNEL and ?H2AX positive cells within mouse ovaries and the ratio of mRNA levels of the apoptotic genes Bax/Bcl-2.Furthermore,exposure to ZEA led to remarkable changes in the Lhx8 3?-UTR DNA methylation dynamics in oocytes and severely reduced the mRNA of oocyte specific genes.In conclusion,ZEA exposure impairs mouse primordial follicle formation in vitro.In conclusion,ZEA exposure significantly changed the gene expression pattern of porcine granulosa cells,including the increased apoptosis-related and decreased steroidogenesis-related genes.Porcine granulosa cells were more sensitive than mouse granulosa cells with the ZEA exposure.ZEA exposure might lead to the potential cancer hazard of granulosa cells.Furthermore,exposure to ZEA led to remarkable changes of DNA methylation dynamics,significantly decreased germ cell cyst breakdown and primordial follicle assembly. |
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