The Effect And Mechanism Of Tributyltin (TBT) Exposure On Growth And Development Of Placenta And The Function Of Corpus Luteum In Pregnant Mice

Background and objectives:Tributyltin（TBT）is a toxic organotin compound,which is widely used in ship antifouling paint to prevent hull fouling because of its bactericidal effect,but it also causes pollution to marine aquatic organisms and water.Eating seafood contaminated by TBT is the main way for the human exposure to TBT.It can be detected in human blood,liver and placenta.Present studies have shown that TBT has a variety of toxic effects,such as hepatotoxicity,neurotoxicity,immunotoxicity,reproductive toxicity and so on,among which reproductive toxicity has aroused widespread concern.In male animals,TBT reduces the weight of testis and epididymis and affects the quantity and quality of sperm.In female animals,TBT reduces the weight of ovary and uterus,damages the development and reserve of follicles,inhibits the development of uterine glands,and interferes with the maturation of oocytes.TBT can prevent embryo implantation and reduce pregnancy rate,fetal weight and survival rate of offspring.In addition to damaging gonadal organs,TBT can change the activity of steroid synthase and interfere with hormone homeostasis in both sexes.In addition,TBT have a toxic effect on fetus through the placental barrier.Oxidative damage and apoptosis are mainly involved in the toxigenic mechanism of TBT.Normal ovarian corpus luteum and placental function play an important role in embryo implantation and pregnancy maintenance.However,in the study of reproductive toxicity of TBT,there is no study on the relationship between the growth and development of embryos and the function of placenta and corpus luteum.Therefore,the purpose of this study is to explore the effects of TBT exposure on placental development and ovarian corpus luteum function in pregnant mice and its mechanism,and to provide basic support for the theory that TBT can affect embryonic development through placental and corpus luteum dysfunction.Methods:1)Establishment of TBT-treated animal model:The pregnant mice were continuously fed with different doses of TBT（0,0.2,2mg/kg/day）for 8 or 13 days from gestational day 1（GD1）,and the samples of uterus,placenta,ovary,liver and kidney were collected,weighed and photographed.We calculated the numbers of implanted site and resorbed embryo and levels of serum progesterone and estradiol were measured by radioimmunoassay.2)Hematoxylin-eosin（H&E）staining:We observed the histomorphology of ectoplacental cone（EPC）on GD8 and placenta on GD13 and analyzed the areas of total placenta,spongiotrophoblast and labyrinth on GD13 by H&E staining.We also observed the histomorphology of corpus luteum of ovary and analyzed the number and area of corpus luteum.3)Quantitative real-time PCR（q PCR）:The m RNA expression of placental key developmental genes（Fra1,Eomes,Hand1 and Ascl2）and ovary steroidogenic enzymes genes（STAR,HSD3B1,CYP11A1 and CYP19A1）was detected by q PCR.4)Immunohistochemistry:We detected the expression of Laminin and proliferating cell nuclear antigen（PCNA）in placenta and the immunoreactivity of estrogen receptor 1（ESR1）and cytochrome P450 aromatase（P450 arom）in ovarian corpus luteum.5)TUNEL（Td T-mediated d UTP Nick-End Labeling,TUNEL）staining:The number of TUNEL positive cells was detected and statistically analyzed.6)Western blotting:The expression of apoptosis-related proteins p53,Bcl-2 and cleaved-caspase-3 in placenta and the expression of apoptosis-related proteins P53,Bcl-2 and Bax in ovarian luteum were detected.7)The detection of oxidative stress index:The levels or activity of indicators of oxidative stress including malondialdehyde（MDA）,hydrogen Peroxide（H₂O₂）,catalase（CAT）and superoxide dismutase（SOD）in placenta and ovary were measured using specific commercial kits.Results:Part one:Effects of TBT exposure on placenta growth and development in mice during early pregnancy and its mechanism1)There was no significant difference in the maternal weight gain,the weight of liver and kidney and organ coefficient between control and TBT-treated groups on GD8 and GD13（P>0.05）.2)There was no significant difference in the number of embryo implantation between control and TBT-treated groups on GD8 and GD13（P>0.05）.On GD8,there was no obvious sign of embryo absorption in each group.On GD13,compared with the control group,there was no significant difference in the number of embryo abortion in 0.2mg/kg/day concentration TBT group（P>0.05）,but the number of resorbed embryo in 2mg/kg/day concentration TBT group was significantly higher than that of control group（P<0.01）.In addition,the absolute and relative weight of embryos treated with TBT decreased significantly（P<0.01 or 0.001）.3)Compared with the control group,there was no significant difference in the weight of placenta in 0.2mg/kg/day concentration TBT group（P>0.05）,but the weight of placenta decreased significantly in 2mg/kg/day concentration TBT group（P<0.05）.The placenta organ coefficients in the TBT groups were significantly lower than that in the control group（P<0.01 or 0.001）.4)The EPC was significantly atrophied on GD8 in TBT treatment groups.Compared with the control group,there was no significant difference in areas of total placenta,spongiotrophoblast and labyrinth in 0.2mg/kg/day concentration TBT group on GD13（P>0.05）,but these parameters in the group treated with 2mg/kg/day concentration TBT were significantly lower than those in the control group（P<0.05or 0.001）.In addition,there was no significant difference in the ratio of spongiotrophoblast/labyrinth area in group treated by 0.2mg/kg/day TBT,but the ratio increased significantly in group treated with 2mg/kg/day TBT（P<0.001）.5)The branches of fetal blood vessels in the labyrinth of placenta in control group were abundant on GD13,but the branches of fetal vessels decreased and were shortened in the TBT treatment group.Especially in the 2mg/kg/day concentration TBT-treated group,the fetal vascular network could not be formed.The results of statistical analysis showed that areas of laminin-positive regions of placenta in TBT treated groups decreased significantly,compared with the control group（P<0.001）.6)Compared with the control group,there was no significant difference in the expression level of Fra1 m RNA in 0.2mg/kg/day TBT treated group on GD8（P>0.05）.The expression level of Fra1 m RNA was significantly down-regulated in2mg/kg/day concentration TBT treated group on GD8（P<0.001）.The expression level of Fra1 m RNA was significantly down-regulated in the two TBT treated groups on GD13（P<0.001）.The expression level of Eomes m RNA had the same trend as Fra1.The expression level of Hand1 m RNA was significantly down-regulated in TBT treated groups on GD8（P<0.01 or 0.001）.The expression level of Hand1m RNA did not significantly change in the group treated with 0.2mg/kg/day TBT（P>0.05）and was significantly down-regulated in the group treated with 2mg/kg/day TBT on GD13（P<0.01）.The expression level of Ascl2 m RNA significantly decreased on GD8 and GD13 in TBT treated groups（P<0.001）,but the expression of Ascl2 m RNA in 0.2 mg/kg/day concentration TBT group significantly increased on GD8（P<0.01）and decreased on GD13（P<0.001）.7)Compared with the control group,the number of PCNA positive cells in placental cells on GD8 and GD13 in TBT treated groups decreased significantly（P<0.01 or 0.001）.8)Compared with the control group,there was no significant difference in the expression of p53 protein in 0.2mg/kg/day concentration TBT treatment group on GD8（P>0.05）,but the expression of p53 protein was up-regulated in 2mg/kg/day concentration TBT treatment group（P<0.01）.On GD13,the expression of p53protein was up-regulated in TBT treated groups（P<0.05 or 0.001）.On GD8 and GD13,the expression of Bcl-2 protein was down-regulated in TBT treated groups（P<0.05 or 0.001）.On GD8,there was no significant difference in the expression of cleaved caspase-3 protein between control and 0.2mg/kg/day concentration TBT treatment group,but the expression of cleaved caspase-3 protein in 2mg/kg/day concentration TBT treatment group was up-regulated（P<0.001）.In addition,the expression of cleaved caspase-3 protein on GD13 was up-regulated in TBT treatment groups（P<0.01 or 0.001）.9)Compared with the control group,on GD8,there was no significant difference in MDA level and SOD activity in the group treated with 0.2mg/kg/day TBT（P>0.05）,but the level of MDA increased significantly and the activity of SOD decreased significantly in the group treated with 2mg/kg/day TBT（P<0.05 or 0.001）.There was no significant difference in the level of H₂O₂and the activity of CAT between control and TBT treated groups on GD8.Compared with the control group,on GD13,the level of MDA in TBT groups increased significantly（P<0.001）and the level of H₂O₂ in the group treated with 0.2mg/kg/day TBT group had no significant change（P>0.05）and increased significantly in the group treated with 2mg/kg/day TBT（P<0.001）.There was no significant difference in the activities of CAT and SOD in the group treated with 0.2mg/kg/day TBT,while the activities of CAT and SOD in the group treated with 2mg/kg/day TBT decreased significantly（P<0.05 or 0.001）.Part two:Effects of TBT exposure on corpus luteum function in mice and its mechanism10)On GD8,there was no significant difference in the levels of serum P and E₂between control and the TBT treated groups（P>0.05）.On GD13,but there was no significant difference in the levels of serum P and E₂ in the group treated with0.2mg/kg/day TBT（P>0.05）,but the levels of serum P and E₂ in 2mg/kg/day TBT treated group were significantly lower than those of the control group（P<0.01 or0.001）.11)There was no significant difference in ovary weight and organ coefficient on GD8 between control and TBT treated groups,but ovary weight and organ coefficient in TBT treated groups on GD13 significantly decreased compared with the control group（P<0.05,0.01 or 0.001）.In addition,there was no significant difference in the number and relative area of corpus luteum on GD8 between control and TBT-treated groups.On GD13,compared with the control group,there was no significant difference in the number and relative area of corpus luteum in the group treated with0.2mg/kg/day TBT,but the number and relative area of corpus luteum decreased significantly in in the group treated with 2mg/kg/day TBT（P<0.05）.12)Compared with the control group,the expression levels of STAR,HSD3B1and CYP19A1 on GD8 and GD13 in TBT groups decreased significantly（P<0.001）.On GD 8,there was no significant difference in the expression of CYP11A1 m RNA in the group treated with 0.2 mg/kg/day TBT（P>0.05）,but the expression of CYP11A1m RNA in the group treated with 2mg/kg/day TBT on GD 8 and the two TBT groups on GD13 was significantly down-regulated（P<0.001）.13)On GD8,there was no significant difference in the average optical density（AOD）of ESR1 among the three groups（P>0.05）.Compared with the control group,on GD13,there was no significant difference in AOD of ESR1 in the group treated with 0.2mg/kg/day TBT,but AOD of ESR1 decreased significantly in the group treated with 2mg/kg/day TBT（P<0.01）.Compared with the control group,on GD8,there was no significant difference in the AOD of Aromatase in the TBT group in the treatment group with 0.2mg/kg/day TBT,but the AOD of Aromatase in the group treated with 2mg/kg/day TBT on GD8 and the two TBT treated groups on GD13 decreased significantly（P<0.05 or 0.01）.14)Compared with the control group,the level of MDA in ovarian tissue on GD8 increased significantly in TBT treated groups（P<0.05 or 0.01）.There was no significant difference in H₂O₂ level and CAT activity on GD8 among the three groups（P>0.05）.Compared with the control group,on GD8,there was no significant difference in the activity of SOD in TBT group with 0.2mg/kg/day concentration（P>0.05）,but the activity of SOD in TBT group with 2mg/kg/day concentration decreased significantly（P<0.0l）.On GD13,the levels of MDA and H₂O₂ in TBT groups increased significantly,while the activities of CAT and SOD decreased significantly（P<0.05,0.01 or 0.001）.15)Compared with the control group,there was no significant difference in the number of TUNEL positive cells in ovarian corpus luteum among the three groups on GD 8,but the number of TUNEL positive cells on GD13 in TBT treatment group increased significantly（P<0.01 or 0.001）.16)Western blotting showed that the expression of p53 was up-regulated on GD8and GD13 in TBT groups,compared with the control group（P<0.01 or 0.001）.On GD8,there was no significant difference in Bcl-2 Protein in the group treated with0.2mg/kg/day TBT.And the expression of Bcl-2 Protein was down-regulated in group treated with 2mg/kg/day TBT on GD8 and the TBT groups on GD13（P<0.05or 0.001）.There was no significant difference in Bax protein in the group treated with0.2mg/kg/day TBT on GD8.But the expression of Bax protein in the group treated with 2mg/kg/day TBT on GD8 and TBT treated groups on GD13 was up-regulated（P<0.001）.Conclusions:In early pregnancy,maternal exposure to TBT led to adverse pregnancy outcomes possibly by impairing placental development and downregulating the endocrine function of corpus luteum.The mechanism may be involved in abnormal expression of key genes in placental development,down-regulation of proliferation,up-regulation of apoptosis and imbalance of oxidative stress.In addition,it also affected the endocrine function of corpus luteum by downregulating the number and area of corpus luteum,the expression of key enzymes of steroid hormone synthesis,and inducing ovarian oxidative stress and apoptosis.