| Objective:Tri-ortho-cresyl phosphate?TOCP?is one of the three isomers of triresyl phosphate?TCP?,and it is an organophosphate?OP?.TOCP has the characteristics of good compatibility,flame retardancy,mildew resistance and abrasion resistance,and has been widely used in modern industry as a plasticizer.TOCP could be absorbed into the body through the respiratory tract,digestive tract,and skin,and damage multiple systems in the body,such as acute and delayed neurotoxicity.In recent years,some studies have shown that TOCP exposure could also cause reproductive toxicity,such as reducing the number of sperm and vitality in males.There are also a reduction in the number of primordial follicles,preantral follicles and mature follicles in females.TOCP exposure could lead to decrease the number of litters,the weight of fetal,but increase the fetal abortion rate during pregnancy.TOCP could be enriched in some organs such as lung,gallbladder and liver after it entering the body.It could also enter the fetus through the placenta.As we know the placenta and corpus luteum plays an important role during the pregnancy.However,there is still no relevant research on the effects of TOCP exposure on placental growth and development,pregnancy corpus luteum function,and its intergenerational genetic effects during pregnancy being reported.And in this study,we intend to observe the effects on growth of mouse placenta,ovarian corpus luteum functions,transgenerational genetic effects,and their mechanisms during TOCP exposure in pregnancy.Method:We selected 6-8 weeks old,28-30 grams of healthy adult female Kunming mice,and given them different doses of TOCP?100,200,400 mg/kg/day?by gavage from the first day of pregnancy?gestational day 1,GD1?.The mice in the control group were given equal amounts of vegetable oil.On the 8th day of pregnancy?GD8?and the 13th day?GD 13?,the animals were sacrificed,and the blood,the uterus,placenta and ovarian tissues were collected for photographing,weighing,and counting the number of embryo implantation.We observed the fine structure of placenta by HE staining,detected the mRNA of Ascl-2,Esx1,Hand1 and Hand2about placental growth and development by real-time quantitative PCR,detected the expression of PCNA,a placental cell proliferation-related antigen,by immunohistochemistry.And western-blotting was used to detect the expressions of placental cell apoptosis related proteins,such as Bcl-2,Bax,P53,and autophagy related proteins,such as Becline-1,Atg5,LC3.The biochemical detection was used to detect placental oxidative stress related indicators,such as malondialdehyde?Malondialdehyde,MDA?,Catalase?CAT?,Superoxide Dismutase?SOD?,Hydrogen Peroxide?H2O2?.Radioimmunoassay was used for the determination of estrogen and progesterone in serum.And we also observed and compared the changes in the number and area of ovarian corpus luteum,detected the expressions of ovarian steroid hormone synthase associated gene StAR,Cyp11a1 and Hsd3b1 mRNA by f real-time quantitative PCR,observed the oxidative stress related indicators in ovarian tissue by biochemical detection.And we also observed the expression of apoptosis and autophagy related proteins by Western blotting.In order to observe the effect of TOCP on offspring,we administrated the pregnant mice by gavage with the dosage of each experimental group?1,10,100 mg/kg/day?from GD 1 until delivery.The mice in the control group were given equal amounts of vegetable oil.And we collected the required specimens in PND 49 of F1,F2 and F3 generations,and observed the weight,anogenital distance?AGD?,the number of litters,the weight of each organ and the coefficient of organs,including changes in the weight of the reproductive system.The changes in the mRNA of DNA methyltransferase in the ovary in F3 generation female mice were observed by qPCR.And we obtained the information about preliminary effects of TOCP exposure before delivery on their offspring and their epigenetic changes.Results:Part one:The effect of TOCP exposure during pregnancy on the growth and development of mouse placenta and its mechanism?1?Both in GD 8 and GD 13,there was no significant difference between each dose of TOCP exposure groups?100,200,400 mg/kg/day?and the control group in the weight of uterus,ovary,liver,kidney,or organs coefficient?P>0.05?.?2?Compared with the control group,there was no significantly different number of embryos in the 100 mg/kg/day group in GD 8?P>0.05?,but the number in the 200 mg/kg/day group and the 400 mg/kg/day group decreased significantly?P<0.05?.And embryo absorption could be seen.In GD 13,there was no significant difference between the 100mg/kg/day group and the control group in the number of embryos?P>0.05?,but the number in 200mg/kg/day group and400mg/kg/day group decreased significantly compared with the control group?P<0.05?,and embryo absorption was visible.The embryo weight in each TOCP exposure groups?100,200,400 mg/kg/day?was significantly higher than that of the control group?P<0.001?.The placenta weight in the 100 mg/kg/day group was significantly higher than that in the control group?P<0.05?.There was no significant difference between the other TOCP exposure groups and the control group?P>0.05?.The residual placental tissue after embryo absorption was also seen in each experimental group.?3?In GD8,compared with the control group,the development of the ectoplacental cone?EPC?was significantly worse in the TOCP exposure groups,especially in the 200mg/kg/day group and 400mg/kg/day group.The EPC structure was significantly damaged,and there was no obvious EPC structure.In GD13,the total placental area of TOCP exposure groups?100,200,400 mg/kg/day?decreased significantly?P<0.001?.In addition,TOCP exposure also significantly reduced the area of sponge trophoblasts?P<0.001?and labyrinth trophoblasts?P<0.001?.?4?In GD 8,compared with the control group,exposure to different doses of TOCP?100,200,400 mg/kg/day?significantly inhibited the expression of Ascl2and Esx1 mRNA in the placenta?P<0.01?,and Hand1 mRNA expression was inhibited in the 400 mg/kg/day group?P<0.05?,and Hand2 mRNA expression was inhibited in the 100 mg/kg/day group?P<0.001?,but increased in the 400 mg/kg/day group High?P<0.01?.In GD 13,compared with the control group,the mRNA expressions of Ascl2,Esx1,Hand1,and Hand2 in the each TOCP exposure groups?100,200,400 mg/kg/day?were significantly down-regulated?P<0.001?.?5?Compared with the control group,the number of PCNA-positive cells in the placenta decreased significantly in a dose-dependent manner after exposure to TOCP both in GD 8 and GD 13?P<0.05 or P<0.001?.?6?In GD 8,compared with the control group,the expression of P53 did not change significantly?P>0.05?,while the expression of Bcl-2 in the 100 mg/kg/day and 400 mg/kg/day groups decreased significantly?P<0.05 or P<0.01?,and that of Bax was significantly increased in the 400 mg/kg/day group?P<0.001?.In GD13,compared with the control group,P53 expression increased significantly in the400 mg/kg/day group?P<0.05?,and that of Bcl-2 in the 100 mg/kg/day,200 mg/kg/day,and 400 mg/reduced significantly?P<0.001?.The expression of Bax increased significantly in the 200 mg/kg/day and 400 mg/kg/day groups?P<0.001?.?7?In GD 8,compared with the control group,expression of Beclin-1 was significantly increased in the 400 mg/kg/day group?P<0.05?,and that of Atg5 was significantly increased in 100 mg/kg/day,200 mg/kg/day,and 400 mg/kg/day group?P<0.05 or P<0.01?.LC3-II/LC3-I was significantly increased in the 400 mg/kg/day group?P<0.01?.In GD 13,compared with the control group,the expression of Beclin-1 was significantly increased in the 200 mg/kg/day and 400mg/kg/day groups?P<0.05 or P<0.01?.Atg5 was significantly increased in the100 mg/kg/day,200mg/kg/day and 400 mg/kg/day groups?P<0.05?.LC3-II/LC3-I was significantly increased in the 100mg/kg/day,200mg/kg/day and 400mg/kg in the/day groups?P<0.05 or P<0.001?.?8?In GD 8,compared with the control group,the oxidative stress marker,MDA was significantly increased in the 100mg/kg/day and 400mg/kg/day groups?P<0.05?;H2O2 was significantly increased in 100mg/kg/day,200mg/kg/day and400mg/kg/day groups?P<0.05 or P<0.01 or P<0.001?.The antioxidant stress marker CAT was significantly decreased in the 100mg/kg/day group?P<0.01?,however,it was significantly increased in the 400 mg/kg/day group?P<0.01?.SOD decreased significantly in 100 mg/kg/day and 200 mg/kg/day groups?P<0.001 or P<0.05?,but increased significantly in 400 mg/kg/day group?P<0.001?.In GD 13,compared with the control group,the oxidative stress marker MDA was significantly increased in the 200mg/kg/day and 400 mg/kg/day groups?P<0.05?.H2O2 was significantly increased in 200mg/kg/day and 400 mg/kg/day group?P<0.05 or P<0.001?.The antioxidant stress marker CAT was significantly decreased in the 100mg/kg/day,200mg/kg/day and 400 mg/kg/day groups?P<0.05 or P<0.01?.SOD decreased significantly in the 100 mg/kg/day,200 mg/kg/day and 400 mg/kg/day groups?P<0.001?.Part two:the effects of TOCP exposure on corpus luteum function and its mechanism?9?In GD 8,compared with the control group,the serum level of estradiol was not significantly different in the 100 mg/kg/day group?P>0.05?,and decreased significantly in the 200 mg/kg/day and 400 mg/kg/day groups?P<0.05?.But in GD 8,there was no significant difference between the control group and other TOCP exposure groups in serum progesterone?P>0.05?.In GD13,the serum level of estradiol was not significantly different between control group and other TOCP exposure groups?P>0.05?.In GD 13,compared with the control group,the serum levels of progesterone decreased extremely significantly in the 100mg/kg/day group and 400mg/kg/day group?P<0.001?,and also decreased significantly in the200mg/kg/day group compared with the control group?P<0.05?.Whether in GD 8or GD 13,there was no significant difference in ovarian weight between in the control group and in all the TOCP exposure groups?P>0.05??10?Both in GD 8 or GD 13,there was no statistical difference in the TOCP exposure group?100,200,400 mg/kg/day?compared with the control group in the ovarian corpus luteum?P>0.05?.In GD 8,the relative area of the ovarian corpus luteum decreased significantly in the 400 mg/kg/day group?P<0.05?,and in GD13,the relative area of the ovarian corpus luteum decreased significantly in the 100mg/kg/day and 400 mg/kg/day groups?P<0.01 or P<0.05?.Compared with the control group,the expressions of ovarian steroid hormone synthase associated gene were significantly down-regulated in each group of TOCP exposure?100,200,400mg/kg/day??P<0.001?.?11?In GD 8,compared with the control group,the expression of P53 and Bcl-2did not change significantly?P>0.05?.But the expression of Bax increased significantly in the 200 mg/kg/day and 400 mg/kg/day groups?P<0.05?.In GD13,compared with the control group,the expression of P53 was significantly increased in the 100mg/kg/day,200mg/kg/day,and 400mg/kg/day groups?P<0.01?,and the expressions of Bcl-2 were significantly reduced in the 200mg/kg/day and 400 mg/kg/day groups?P<0.01 or P<0.05?.The expressions of Bax was significantly increased in the 100mg/kg/day,200mg/kg/day and 400 mg/kg/day groups?P<0.05 or P<0.001?.?12?In GD 8,compared with the control group,the expression of Beclin-1 was significantly increased in the 100mg/kg/day,200mg/kg/day and 400 mg/kg/day groups?P<0.01 or P<0.001?.The expressions of Atg5 and LC3-II/LC3-I were not significantly different from the control group to the other TOCP exposure groups?P>0.05?.In GD 13,compared with the control group,the expression of Beclin-1was significantly increased in the 100 mg/kg/day,200 mg/kg/day,and 400 mg/kg/day groups?P<0.001,P<0.05,or P<0.01?.The expressions of Atg5 were significantly increased in the 100mg/kg/day and 400mg/kg/day groups?P<0.01or P<0.05?,and there was no significant difference of the LC3-II/LC3-I between TOCP exposure groups and the control group?P>0.05?.?13?In GD 8,compared with the control group,the oxidative stress marker MDA was significantly increased in the 100mg/kg/day,200mg/kg/day and400mg/kg/day groups?P<0.01,or P<0.001?.H2O2 showed a significant dose-dependent increase in the 100mg/kg/day,200mg/kg/day and 400mg/kg/day groups?P<0.05 or P<0.01 or P<0.001,?.The antioxidative stress marker CAT decreased significantly in the 100mg/kg/day,200mg/kg/day and 400mg/kg/day groups?P<0.001?.SOD was also decreased significantly in 100mg/kg/day,200mg/kg/day and 400mg/kg/day groups?P<0.001?.In GD 13,compared with the control group,the oxidative stress marker MDA was significantly increased in the100 mg/kg/day,200 mg/kg/day and 400 mg/kg/day groups?P<0.01 or P<0.001?.H2O2 was significantly increased in the 100 mg/kg/day,200 mg/kg/day and 400 mg/kg/day groups?P<0.05 or P<0.01?;the antioxidant stress marker CAT was significantly decreased in the 400 mg/kg/day group.?P<0.01?;SOD decreased significantly in the 100mg/kg/day,200mg/kg/day,and 400mg/kg/day groups?P<0.05 or P<0.001?.Part three:the effect of TOCP exposure before delivery on transgenerational inheritance and its mechanism in mice?14?In the F1 generation,the body weight of newborn pups increased significantly in the 1 mg/kg/day group compared with the control group?P<0.01?.And the AGD decreased significantly in the 100 mg/kg/day group?P<0.05?.The number of litters decreased significantly in the 100 mg/kg/day group?P<0.01?.In the F2 generation,the number of litters per litter in the TOCP exposure groups?1,10,100 mg/kg/day?were significantly decreased?P<0.05 or P<0.01?;in the F3generation,the AGD of newborn pups decreased significantly in the 1 mg/kg/day group and 100 mg/kg/day group compared with the Control group?P<0.01 or P<0.001?.The number of litters per fetus significantly decreased in the 100mg/kg/day group?P<0.05?,the rest were not statistically different?P>0.05?.?15?In female mice,compared with the control group,the body weight of the F1generation was significantly decreased in the 100 mg/kg/day group?P<0.05?.In male mice,compared with the control group,the body weight of the F1 generation was 10 mg/kg/day group increased significantly?P<0.01?.There were no statistical difference between the control group and the remaining TOCP exposure groups?P>0.05?.?16?In the F1 generation of female mice,the ovarian weight was significantly reduced in the 10mg/kg/day and 100mg/kg/day groups?P<0.01 or P<0.05?,but there was no statistical difference in the 1mg/kg/day group?P>0.05?.In the F3generation,the uterine weight and uterine coefficient were significantly lower in the10 mg/kg/day and 100 mg/kg/day groups compared with the control group?P<0.05 or P<0.01?.In male mice,testicular weight,testicular coefficient,epididymal weight,and epididymal coefficient were not statistically different in all TOCP exposure groups compared with the control group?P>0.05?.?17?The ovary mRNA expressions of Dnmt1,Dnmt3a and Dnmt3b were significantly up-reguated in the 100 mg/kg/day group compared with the control group?P<0.01 or P<0.001?.And the rest of the TOCP exposure groups not were significant different compared with the control group?P>0.05?.Conclusion:1.The growth and development of mouse placenta is significantly inhibited by the TOCP exposure during pregnancy.The mechanism may be included in oxidative stress injury,proliferation decrease,increased apoptosis and autophagy in placenta cells.2.TOCP exposure during pregnancy affects the secretory function of ovarian corpus luteum significantly in mice.The mechanism may be included in oxidative stress injury,proliferation decrease,increased apoptosis and autophagy in ovary cells.3.TOCP exposure before birth affects on the reproductive system of offspring in mice.The possible mechanism may be achieved by changing the DNA methylation level in the offspring ovary. |
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