| In recent years,the incidence of infertility has been increasing worldwide,and about 10%-15%of couples of childbearing age are bothered by infertility.In China,the prevalence of infertility among couples of childbearing age is about 25%.With the development of industrialization,among many factors affecting infertility,environmental pollution is considered to be one of the important risk factors leading to infertility,and environmental pollutants have threatened human health.As a typical environmental endocrine disruptor,phthalates esters(PAEs)are widely used in the production of plastics.Di(2-ethylhexyl)phthalate(DEHP)is one of the most commonly used PAEs.It is widely used as a plasticizer in personal care products,daily chemicals,food packaging,and medical devices.At present,DEHP can be detected in the atmosphere,soil,waters,animals and plants around the world and is called "ubiquitous pollutants".Epidemiological studies have shown that DEHP exposure is closely related to the occurrence of various diseases such as obesity,breast cancer,and behavioral disorders,also including female reproductive impairment.Studies conducted in women seeking ART suggest that daily exposure to DEHP can cause problems such as decreased ovarian reserve,decreased egg retrieval rate,decreased oocyte quality,decreased clinical pregnancy rates and reduced live birth rates,and increased early abortion rates,which may cause female fertility to decline or cause infertility.Animal studies have shown that DEHP exposure causes reproductive toxicity by affecting ovarian function such as hormone synthesis and secretion,development of primitive and growing follicles,and egg maturation.Increasing DEHP pollution poses a serious threat to female reproductive health,and the decline in female fertility has become a problem that cannot be ignored worldwide.However,there are few studies on the reproductive toxicity of DEHP in females,and the toxicity characteristics and mechanism of action are not very clear.In recent years,assisted reproductive technology(ART)has become the main method of pregnancy aid for infertile people.Endometrial receptivity and embryo quality are key factors for the success of ART.The quality of oocytes is closely related to the quality of embryos.In this study,the impact of DEHP on the quality of oocytes was taken as the starting point.The effect of DEHP on the development potential of oocytes was mainly explored,and its mechanism was initially explored in order to provide some theoretical basis for the prevention and treatment of reproductive damage caused by DEHP.ObjectiveThe purpose of this study was to establish a model of DEHP exposure by administering DEHP to female mice by gavage,and to observe its effect on oocyte development potential and its mechanism.Materials and MethodsResearch subjects:In this experiment,clean-grade KM female mice aged 3 weeks and male KM mice aged 7-10 weeks were selected.The female rats were randomly divided into 4 groups:the control(corn oil)group and the DEHP low,medium,and high dose groups(300 mg/kg,600 mg/kg,1200 mg/kg).The 4 groups of mice were orally dosed with corn oil(control)or DEHP daily for 6 weeks(5 days per week),respectively.Dosing volumes were determined daily by corresponding mouse weight.Methods:After the treatment,some female mice in each group were given ovulation induction with PMSG and cooped up with male mice,and the fertilized eggs were collected to observe the cleavage rate and blastocyst formation rate.Some female mice were naturally caged with male mice to observe the number of embryo implantation sites.Some female mice were given ovulation induction with PMSG and the levels of Ca2+and reactive oxygen species(ROS)in oocytes were detected.Meanwhile,Ca2+fluorescence probe(Fluo-4 AM),ROS detection kits,Mito-Tracker Green,JC-10 fluorescent probe and qRT-PCR were respectively used to detect the levels of Ca2+and ROS,the distribution of mitochondria,mitochondrial membrane potential and DNA copy number in blastocysts.Then compared the differences between DEHP exposure group and control group.Statistical methods:Data analysis was performed using SPSS 22.0 statistical software.The measurement data are expressed as mean ± standard deviation(x±s).The comparison of measurement data among multiple groups uses single factor analysis of variance.When making pairwise comparisons between groups,the LSD test is used.Counting data is expressed as a percentage(%),using ?2 test or Fisher's exact test.P<0.05 was considered statistically significant.Results(1)Effect of DEHP on body weight of female miceThere was no statistically significant difference in body weight changes among the 4 groups of mice(P=0.067).(2)Effect of DEHP on early embryo developmentThe cleavage rates(69.09%,70.95%,60.47%)of mouse embryos in the DEHP low,medium and high dose groups were lower than those in the control group(87.12%),and the difference was statistically significant(P=0.000;P=0.000;P=0.000).There was no statistical difference among the low,medium and high dose groups of DEHP(P>0.05).The blastocyst formation rate of mouse embryos in the low,medium-and high dose groups of DEHP(64.04%,49.52%,32.69)%)were lower than the control group(86.62%),and the difference was statistically significant(P=0.000;P=0.000;P=0.000).There was also a statistically significant difference among the DEHP low,medium,and high dose groups(DEHP low vs medium P=0.030;DEHP low vs high P=0.000;DEHP medium vs high P=0.046).(3)Effect of DEHP on the number of implantation sitesThe number of embryo implantation sites[(13.80 ± 1.92),(13.80±1.79),(13.20±2.05)]of female mice in DEHP low,medium and high dose groups were lower than that of the control group(16.80 ± 0.84),and the difference was statistically significant(P=0.014;P=0.014;P=0.004).There was no statistical difference among DEHP exposure groups(P>0.05).(4)Effect of DEHP on the concentration of ROS and Ca2+in oocytesThe results of ROS detection in oocytes showed that the ROS level gradually increased with the increase of the DEHP dose.The levels of ROS in oocytes of mice in DEHP medium and high dose groups[(0.05±0.032),(0.066 ± 0.021)]were significantly higher than those in the control group(0.026±0.011),and the differences were statistically significant(P=0.000;P=0.000).The level of ROS in oocytes of DEHP low-dose group(0.028±0.012)was also higher than that in the control group,but the difference was not statistically significant(P>0.05,P=0.701).The results of Ca2+concentration detection showed that the level of Ca2+gradually increased with the increase of the DEHP dose.The levels of Ca2+ in oocytes of mice in the DEHP medium and high dose groups[(0.150 ± 0.024),(0.172±0.001)]were significantly higher than those in the control group(0.051 ± 0.039),and the differences were statistically significant(P=0.000;P=0.000).The level of Ca2+in oocytes of DEHP low-dose group(0.069±0.015)was also higher than that in the control group,but the difference was not statistically significant(P>0.05,P=0.104).(5)Effect of DEHP on mitochondrial function of blastocystThe results of ROS concentration in blastocysts showed that the ROS level gradually increased with the increase of the DEHP dose.The levels of ROS in embryos of mice in DEHP medium and high dose groups[(0.040±0.005),(0.047±0.007)]were significantly higher than those in the control group(0.028± 0.001),and the differences were statistically significant(P=0.009;P=0.000).The results of Ca2+concentration in the blastocyst showed that the Ca2+level gradually increased with the increase of the DEHP dose.The levels of Ca2+in the embryos of DEHP low,medium and high dose groups[(0.091±0.031),(0.162 ±0.016),(0.170 ± 0.004)]were higher than those in the control group(0.056 ± 0.035),and the differences were statistically significant(P=0.013;P=0.000;P=0.000).The results of blastocyst mitochondrial distribution showed that with the increase of the DEHP dose,the mitochondrial distribution was becoming more and more uneven.The results of blastocyst mitochondrial membrane potential showed that with the increase of the DEHP dose,the mitochondrial membrane potential gradually decreases.The mitochondrial membrane potentials of blastocysts in DEHP low,medium and high dose groups[(2.10 ± 1.22),(0.78±0.35),(0.36 ± 0.14)]were significantly lower than those in the control group(10.02 ± 4.97),and the differences were statistically significant(P=0.000;P=0.000;P=0.000).The results of blastocyst mitochondrial DNA copy number showed that with the increase of the DEHP dose,the mitochondrial DNA copy number tended to increase.The mitochondrial DNA copy number in the DEHP medium dose group(22018.53 ±8442.04)was significantly higher than the control,low and high dose groups[(11358.56 ± 1627.23),(11155.68± 1985.93),(13127.30 ± 2699.46)],the differences were statistically significant(P=0.000;P=0.000;P=0.001).The mtDNA copy number in the high-dose DEHP group was higher than that in the control group,but the difference was not statistically significant(P=0.505).Conclusions(1)DEHP exposure can decrease the cleavage rate and blastocyst formation rate of mouse embryos cultured in vitro,and reduce the number of embryo implantation sites.(2)DEHP exposure can impair mouse oocyte and embryo mitochondrial function and affect embryo quality. |
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