| Objective Toxoplasma gondii is an obligate intracellular parasitic protozoan with which the pregnant women infected may transport the tachyzoite into the fetus and lead to grave health problems,including abortion,fetal death and congenital toxoplasmosis.However,the mechanisms still were not elucidated.In the present study,we built a model of pregnant mice infected with Toxoplasma gondii in order to study①the effect of Toxoplasma gondii on embryonic growth and development,②the effect of Toxoplasma gondii on the apoptosis and the expression of bcl-2 and bax protein in placental trophoblastic cell of pregnant mice,③the effect of Toxoplasma gondii on the progesterone and estriol(P,E3)of pregnant mice,④the effect of Toxoplasma gondii on hematology and serum ferritin concentration of pregnant mice,and to elucidate the mechanisms of developmental toxicity of Toxoplasma gondii to the BALB/c embryonic mice.Methods 160 BALB/c pregnant mice(9-10 week)were randomly divided into 2 groups: the infection group(100 mice)and the control group(60 mice).On the 8th day of pregnancy,the mice of infection group were treated with 100 tachyzoites by intraperitoneal injection individually,while the mice of control group were treated with PBS.20 infected mice and 12 control mice were killed on the 10th,12th,14th,16th and 18th day respectively.①the blood of a Half of mice was collected,and the serum was separated by centrifugalization.The concentration of serum E3,P and Ferritin of pregnant mice in different pregnancy period was determined by Radioimmunoassay.②The blood of another half of mice was collected for blood cell analysis.③All of the pregnant mice were killed and the fetal mice were taken out of the uterus.To observe the appearance of the fetus,to count quantity of the dead fetus,the live fetus and total fetus,to measure weight,body length and tail length of fetus mice.Half of fetus were embedded with paraffin and sliced,another fetus were kept in a deep freeze refrigerator. The content of Zn,Cu and Fe of fetal mice were determined by flames atomic absorption spectrometry(FAAS).Iron distribution in the section of the whole fetal mice,liver and brain of fetal mice was shown by DAB enhanced Pert's reaction.The total content of protein of fetal mice was determined by colorimetric method.④All of the placentas were taken out of the uterus.Some were used to make tissue tablettings of placentas,which were stained with Wright-Giemsa Solution and examined microscopically.Other placentas were fixed in Bouin's solution,embedded in paraffin and sliced(4μm).Hematoxylin and eosin(HE)staining showed pathological change of placenta.Apoptosis was shown by TUNEL,and the expressions of TOX, bcl-2 and bax protein in placental cell was shown by immunohistochemistry.Results(1)Quantity of the live fetus and total fetus and mean weight of fetus in infection group significantly decreased compared with the control group(P＜0.05)on the 14th,16th,18th day of pregnancy.Mean body length and tail length of fetus in infection group significantly decreased compared with the control group on the 16th,18th day of pregnancy,but not on 14th day of pregnancy(P＞0.05).(2)Flames atomic absorption spectrometry(FAAS)showed the contents of Zn and Fe of fetal mice in control group gradually increased during pregnancy,but the content of Cu gradually decreased.The content of Zn of fetal mice in infection group decreased compared with the control group on the 12th,14th,16th,18th day of pregnancy (P＜0.05),but not on the 10th day of pregnancy.The content of Cu of fetal mice in infection group decreased compared with the control group on the 16th,18th day of pregnancy(P＜0.05), but not on the 10th,12th,14th day of pregnancy.The content of Fe of fetal mice in infection group was not different with the control group on the 10th day of pregnancy,but decreased on 16th,18th day of pregnancy and decreased more significantly on 12th,14th day of pregnancy.(3) By DAB enhanced Perl's reaction,iron staining was shown in the section of whole fetal mice, liver and brain of fetal mice.Iron staining in the infection group was significant weak compared with the control group.(4)The total protein content of fetal mice in the infection group significantly decreased on the 16th,18th day of pregnancy compared with the control group (P＜0.01),but not on the 14th day.(5)By Wright-Giemsa staining,tachyzoites of Toxoplasma gondii were shown in placentas of the infection group,and the quantity of tachyzoite gradually increased during pregnancy.HE staining showed inflammatory reaction,apoptotic body in the villus of placentas.(6)By TUNEL,apoptosis was shown in placental tissue of infection and control group,and increased gradually during pregnancy.Apoptosis index(AI)of placentas of the infection group was significant higher than that of the control group on 14th,16th,18th day of pregnancy(P＜0.01),but not on 10th,12th day of pregnancy.(7)By immunohistochemistry, the expression of TOX antigen was shown in placental syncytiotrophoblast cell of the infection group,but not in the control group.The expression of Bax was shown in placental villus and decidua of the infection and the control group,and gradually increased during pregnancy. Whereas the expression of Bcl-2 was shown in placental syncytiotrophoblast cell,and gradually decreased during pregnancy.In the late pregnancy,the expression of Bax in placentas of the infection group was apparent higher than that of the control group,whereas the expression of Bcl-2 was less than that of the control group(P＜0.01).The ratio of Bax:Bcl-2 increased approximately 2-3 folds in placenta of infection group compared with that of control group.(8) The concentrations of serum E3 and P of pregnant mice in the infection group were not different with that of the control group on the 10th,12th,14th day of pregnancy(P＞0.05),but significantly decreased on the 16th,18th day of pregnancy(P＜0.01).(9)Blood cell analysis demonstrated that there was not significant difference in RBC,HGB,HCT and MCH of the infected pregnant mice and control group during pregnancy(P＞0.05).RDW of the infected pregnant mice lightly increased compared with that of control group,but it was not significant (P＞0.05).MCV of infected pregnant mice decreased compared with that of control group on the 12th day of pregnancy(P＜0.05),and decreased more significantly on the 16th and 18th day of pregnancy(P＜0.01),but not on the 10th and 14th day of pregnancy(P＞0.05).MCHC of infected pregnant mice significantly decreased compared with that of control group on the 14th, 16th and 18th day of pregnancy(P＜0.01),but not on the 10th and 12th day of pregnancy (P＞0.05).(10)By radioimmunoassay,the concentration of serum ferritin of the infected pregnant mice lightly decreased compared with that of control group on the 10th,12th,and 14th day of pregnancy,but it was not significant(P＞0.05).However,the concentration of serum ferritin of the infected pregnant mice was significantly less than that of control group on the 16th and 18th day of pregnancy(P＜0.01).Conclusions(1)Infection with Toxoplasma gondii during pregnancy not only induced the decreases of quantity of the live fetus and total fetus,mean weight of fetus,mean body length and tail length of fetus,but also induced the decreases of the contents of Zn,Cu and Fe of fetal mice.This change demonstrated that infection with Toxoplasma gondii during pregnancy caused fetus growth retardation,and the decrease of the contents of Zn,Cu and Fe of fetal mice may be involved in the mechanisms.(2)Infection with Toxoplasma gondii during pregnancy caused the increase of apoptosis in placental syncytiotrophoblast cell.The increase of Bax,the decrease of Bcl-2 and disbalance of Bax/Bcl-2 ratio may be involved in the mechanisms.Infection with Toxoplasma gondii during pregnancy induced inflammatory reaction and enhanced apoptosis of placentas,which hindered placental material and nutrition transport and caused the breakdown of placental endocrine secretion.This change may be involved in the mechanism of fetus growth retardation.(3)Infection with Toxoplasma gondii during pregnancy induced the decrease of the concentrations of serum E3 and P of pregnant mice,which may interact with fetus growth retardation and placental inflammatory reaction and enhanced apoptosis.(4)Infection with Toxoplasma gondii during pregnancy induced slightly change of blood cell and the decrease of the concentration of serum ferritin of pregnant mice.This change demonstrated the pregnant mice infected with Toxoplasma gondii were slender hypoferremia,which may one of causes of the decrease of fetal iron content.
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