| Objective:Di-(2-ethylhexyl) phthalate(DEHP) is used as a plasticizer and widely dispersed in the environment. DEHP is non-covalently bound to plastics, and therefore, it will leach out of these products after repeated use, heating, and/or cleaning of the products,allowing the toxicant to be ingested through inhalation, ingestion and dermal contact on a daily basis, and harming to animal and human reproductive system. Studies have shown that maternal exposure to DEHP reduces endometrial receptivity and embryo implantations, increases resorptions, and decreases fetal weights of offspring.However, no detailed information is available about the effect of DEHP on the placentation during the pregnancy. Therefore, this work was designed to evaluate the impact of DEHP on the placenta and embryo development by gavage in vivo.Methods:Mice were administered DEHP by gavages at 125, 250, 500 mg/kg/day from gestational days(GD) 1. Treated mice were sacrificed for uterus, embryo and placenta collection at GD 9 and 13. Hematoxylin and eosin staining was observed the effect of DEHP on the histopathology of placenta. Immunohistochemistry was used to examine the effect of DEHP on the proliferation of placenta and labyrinth vascularization;The expressions of marker genes in placenta were analyzed by real-time quantitative PCR; Western blot was to investigate the effects of DEHP the apoptosis of placenta and related signaling pathways.Results:(1) At GD 9, treatment with DEHP did not significantly affect the weight of embryo enclosed by deciduae and the ratio of embryo vs. body weight compared to control group(P>0.05). However, at GD 13, DEHP treatment significantly reduced the weight of embryo plus placenta and the ratio of embryo plus placental weight to body weight(P<0.05 or 0.01),and placental weight and the ratio of placental weight to body weight compared to control group(P<0.01).(2) At GD 9, DEHP treatment significantly inhibited growth and development of ecoplacental cone. Total area of placenta and area of spongiotrophoblast were significantly reduced compared to control group at GD 13(P<0.05 or 0.01).(3) Expression levels of Ascl2 and Esx1 m RNA is significantly lower in the DEHP-treated group than in the control group at GD 9 and 13(P<0.05 or 0.01). At GD 9, there was no significant difference in the expression of Fosl1 m RNA between the DEHP-treated group and control group(P>0.05). However, on GD 13, treatment with DEHP significantly inhibited Fosl1 m RNA level compared to control(all P<0.01),and significantly increased level of Eomes and hand1 m RNA(P<0.05 or0.01).(4) DEHP treatment significantly disrupts labyrinth vascularization of placentas compared to control group at GD13.(5) Immunohistochemical results showed that a significant decrease in Ki67-positive cells was detected in the EPC, the labyrinth and spongiotrophoblast zone of placenta compared to control group at GD 9 and 13.(6) DEHP treatment significantly increased levels of Bax, caspase-3 and Caspase-8 m RNA, and decreased Bcl-2 m RNA at GD13(P<0.05 or 0.01). However,At GD9, there are no significant differences between DEHP-treated group and control group(P>0.05). Western blot results showed that intact caspase-3 and-8 were cleaved to active fragments after DEHP treatment at GD9 and GD13. DEHP treatment significantly increased Bax protein levels, but declined Bcl-2 protein levels at GD13(all P<0.01).(7) Western blot analysis showed that phospho-Erk1/2 levels significantly increased in the 250 and 500 mg DEHP/kg/day dose group compared with control group at GD 9(P<0.01). At GD 13, administration of different dose DEHP all significantly increased level of phosphor-Erk1/2 relative to control group(P<0.05 or0.01).Conclusions:(1) DEHP significantly inhibited the growth and development of ectoplacental cone and placenta during pregnancy.(2) DEHP disrupts the formation of labyrinth vascularization during pregnancy.(3) DEHP significantly inhibited the proliferation of ectoplacental cone and placenta during pregnancy.(4) DEHP induces the apoptosis of placenta via dysregulation of the MAPK signaling pathway and the activation of caspase-3 and-8, up-regulation of Bax and down-regulation of Bcl-2 m RNA and protein. |
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