Exposure To Ethephon Compromises Endometrial Decidualization In Mice During Early Pregnancy

Objective:The incidence of human infertility has greatly increased in the last few decades in many countries,including the United States and the People’s Republic of China,and >10% of infertile couples suffer from idiopathic infertility.Many studies have found that some infertility are may originate from exposure(s)to chemicals.Ethephon is one of the most commonly used plant growth regulators.Studies have found that ETH can cause damage to various of tissues and organs,have hepato toxicity,immune toxicity,and nephro toxicity.However,the toxic effects of ETH on the female reproductive system during early pregnancy have rarely been reported.In rodents,uterine decidualization is a critical event during early pregnancy.Aberrant decidualization has been linked to numerous pregnancy complications,including infertility,recurrent miscarriage,preeclampsia,intrauterine growth restriction,and preterm delivery.Therefore,the aim of this study was to determine whether ETH could compromise endometrial decidualization during the early stages of pregnancy.Importantly,our research could provide a new direction to illustrate the reproductive toxicity of ETH.Methods:1.ETH pregnant mouse model: From the gestational day 1(GD1)of pregnancy,the ETH group was given 71.25﹑142.5 and 285mg/kg ETH by oral gavage and the uteri tissue were immediately stored at-80 ℃.Conducting the following experiments:(1)Weighing the weight of pregnant mice.(2)Counting the number of embryo implantation(3)RT-PCR and Western-blot were used to detect the m RNA and protein expression of COX2、HOXA10、MMP9、BMP2 and PR.(4)Western blot analysis of GPR120 protein expression in the endometrium on GD7,GD8.(5)Enzyme-linked immunosorbent assay(ELISA)was applied to test serum E2 and P4 levels.(6)Triglyceride levels were determined on GD6,GD7,and GD8 during decidualization using the triglyceride assay kit.(7)Real-time PCR and western blot were performed to determine the expression of genes responsible for fatty acid synthesis,fatty acid uptake and transport,fatty acid β-oxidation in the endometrial tissue.2.Establishment of artificial induced decidualization of pseudopregnant mice: Mature female CD1 mice were mated with vasectomized male CD1 mice to produce a pseudopregnancy model.The day on which a vaginal plug was observed was designated as day 1 of pseudopregnancy(PPD1).Artificial decidualization was induced by injecting corn oil into one side of the uterine horn on the morning of PPD4(stimulated),with the other side of the uterine horn receiving no infusion(unstimulated).Beginning from PPD1,the mice were treated with 285mg/kg ETH daily via oral gavage.Finally,uteri were collected on PPD8.Performing the following experiments:(1)weighing the stimulated and unstimulated uteri weight on the control group and ETH exposure group(2)Real-time PCR and western blot analysis of decidualization markers m RNA expression in the decidual stimulated endometrium.(3)Triglyceride levels were determined in the decidual stimulated endometrium using the triglyceride assay kit(4)Real-time PCR and western blot were performed to determine the expression of genes responsible for fatty acid synthesis,fatty acid uptake and transport,fatty acid β-oxidation in the decidual stimulated endometrium3.ETH cell model: The stromal cells were induced decidualization and treated with ETH in vitro.Performing the following experiments:(1)Real-time PCR analysis of dtprp and HOXA10 m RNA expressions after experimentally induced decidualization(ID)using E2 and P4 in vitro.(2)triglyceride levels were determined in ETH exposed m ESCs decidualized with E2 and P4 using the triglyceride assay kit.(3)The lipid deposition of m ESCs cultured in control and ETH group were observed by using BODIPY probe.(4)Western blot analysis of GPR120 protein expression.Real-time PCR analysis of dtprp m RNA expression after transfection with GPR120 c DNA plasmid in the ETH treated m ESCs decidualized with E2 and P44.Establishment of breeding experimental model: From the gestational day 1(GD1)of pregnancy until gestational day 9(GD9)of pregnancy,the ETH group was given 285mg/kg ETH by oral gavage.Stop the poisoning on GD10,and record number of newborn rats after the pregnant rats give birth.Results:1.The effect of ETH exposure on endometrial decidualization :Compared to control group,285 mg/kg body weight ETH treatment significantly reduced the number of implantation on GD6,GD7 and GD8.Furthermore,285 mg/kg body weight ETH treatment significantly decreased the absolute and relative uterine weights on GD7,with identical results observed on GD6 and GD8.Real-time PCR results showed that m RNA levels of BMP2 and HOXA10 were significantly downregulated,whereas the m RNA level of COX2 was significantly upregulated in mice exposed to 285 mg/kg body weight ETH.Western blotting revealed a significant increase in COX2,MMP9,and PR expressions and decreased HOXA10 expression in the endometrial tissue on GD6,GD7,and GD8.2.The effect of ETH exposure on artificial decidualization in vivo and in vitro: In control mice,the stimulated uterine horns formed robust deciduoma after decidualization was induced;however,in the 285 mg/kg body weight ETH treated mice,the deciduoma was significantly reduced.Additionally,the fold change in stimulated/unstimulated uteri weight was significantly reduced in mice exposed to 285 mg/kg body weight ETH when compared with control mice.Real-time PCR results showed that m RNA levels of HOXA10,and BMP2 were markedly reduced in ETH-exposed mice than in control mice.The m RNA levels of both dtprp and HOXA10 were significantly reduced after ETH treatment in m ESC.3.The effect of ETH exposure on serum steroid levels: ELISA results showed the serum E2 level was significantly reduced on GD8 in the ETH treated group when compared with that determined in the control group.On GD7 and GD8,the serum P4 level in the ETH treated group was also remarkably reduced when compared with that observed in the control group.4.The effect of ETH exposure on endometrial lipid metabolism: The data revealed that triglyceride levels were significantly increased on GD6 and GD8 after ETH exposure.With identical results observed on artificial decidualization in vivo and in vitro.Real-time PCR results demonstrated that genes responsible for fatty acid synthesis,fatty acid uptake and transport,fatty acid β-oxidation were significantly disordered following ETH exposure both on GD7 and the decidual stimulated endometrial tissue.Green fluorescrence in bodipy staining demonstrates the enhanced accumulation of lipid droplets in the ETH treated m ESCs decidualized with E2 and P4.5.The effect of ETH exposure on GPR120: Western blotting results showed GPR120 expression was considerably reduced after ETH exposure both in vitro and in vivo.and the compromised decidualization induced by ETH treatment was rescued by GPR120 overexpression.6.The effect of ETH exposure on later pregnancy outcomes:The number of fetuses per female was decreased in the first litter after ETH exposure,indicating abnormal fertility after exposure to ETH.Conclusion:ETH inhibited the decidualization and lipid metabolism of the mouse endometrium in early pregnancy,GPR120 expression was crucial for the impaired decidualization induced by ETH.