| PART Ⅰ:Effect of Di-b-utyl Phthalate on Pregnant Rats’ chorionic gonadotropin and Induction of Fetal Rat HypospasiaObjectiveTo study the key factor of wnt/beta-catenin pathway in the testis of hypospadias rat,explore the change of chorionic gonadotropin secreted by the placentae of pregnane rat that exposures in DBP,and it’s affection to the expression of StAR and testosterone in the fetal rat testis,irnvestigate the possible mechanism hypospadias in SD rats induced by DBP.Methods(1)45 pregnant SD rats were randomly divided into two groups(15 in the control group and 30 in DBP group)and fed under the same conditions.The rats were feeding on the 13-19 day of it’s gestation,The control group ware feeding 3ml/kg corn oil per day.The DBP group was feeding by DBP 800mg/kg and admix with com oil total dose 3ml/kg.(2)Get the pregnancy rats’ serum and detected the level of chorionic gonadotropin on the 13-19 day of gestation.(3)Take the fetus out on day 19 of pregnancy,identify the male fetal mice,find out the hypospadias and divided into,DBP treatment normal group,DBP treatment of hypospadias group and normal control group,take the pictures of the genital tubercle each group,measurement the AGD,and statistics the incidence of hypospadias,and take the testical,genital tubercle and shstained with haematoxylin&eosin then photographed.(4)Take the blood from fetal rats and teat the testosterone in serum.The expression of StAR,GSK-3beta,p-GSK-3beta and beta-catenin in the testical of each group were detected by western blot.(5)analyze all the dates.Results(1)the incidence of hypospadias after DBP treatment was 41.9%.the urethral orifice in the DBP treatment hypospadias group was all on the ventral side of the genital tubercle.In the control group and DBP treatment normal group,the urethral orifice in the normal group was at the top of the genital tubercle.The AGD of the fetal rats in the normal group(2.93±0.44mm)was higher then DBP treatment hypospadias group(1.61±0.31mm)and DBP treatment normal group(2.09±0.55mm)(p<0.05).(2)There was no significant difference in the level of chorionic gonadotropin between the DBP group and the control group at the 13 day of pregnancy.The level of chorionic gonadotropin in the DBP group was lower than the control geoup in the 14&15 day of pregnancy,but the different were no statistically significant.After 16 days of pregnancy,the level of chorionic gonadotropin in the DBP group decreased and the data were statistically significant compared with the control group(P<0.05).(3)After treatment with DBP,the testosterone content in the hypospadias group(1.74±0.31ng/ml)was significantly lower than the testosterone content in the control group(2.97±0.70ng/ml),and the difference between this two data was statistically visible(p<0.05).After treatment with DBP,the testosterone level in the hypospadias group was slightly lower than the normal group(2.16±0.44 ng/ml),but there was no visible difference in statistics between those two groups.(4)The level of StAR,beta-catenin and GSK-3beta in DBP treatment hypospadias group,DBP treated normal group were significantly lower than control grou.On the contrary,the brightness of p-GSK-3beta(Ser9)was significantly higher than that of the other two groups.ConclusionDBP can reduce the chorionic gonadotropin in serum secreted by the placenta during SD rat’s pregnancy,and affect the expression of the key factor in wnt/beta-catenin pathway which can reduce the transcription of StAR,and decreased the synthesis of testosterone in the testis to interrupt the genital development in male fetal rats.PART Ⅱ The significance and effect of di-n-butyl phthalate on the differentiation of Leydig cell extracted from the testicles of fetal miceObjectiveStudy the effects of di-n-butyl phthalate on the differentiation of fetal rat Leydig cell,and explore the mechanisms how di-n-butyl phthalate leading hypospadias in rats.Methods(1)45 pregnant SD rats were randomly divided into two groups(15 in the control group and 30 in DBP group)and fed under the same conditions.The rats were gavagev on the 13-19 day of gestation,The control group ware gavagev 3ml/kg corn oil per day.The DBP group was fed with DBP 800mg/kg and admix with corn oil total dose 3ml/kg.(2)Get the pregnancy rats’ serum and detected the level of chorionic gonadotropin at 13-19 days of gestation.(3)The testicular tissues from two groups of fetuses were extracted respectively,and obtained the primary Leydig cells by differential adherence method.Tested the cell viability and secretion function by detect the 3beta-hydroxyl steroid dehydrogenase(3beta-HSD)in different culture stages(after 2hours、12hours&48hours).(4)Get the testis of fetal male mice from each group,obtain the primary Leydig cells by differential adherence method.Cultured for 12 hours with DBP in different control(0,62.5,125,250,500,1000 mol/L)after 2 hours cultured for primary Leydig cells.Tested the cell viability and secretion function by Detect the 3beta-hydroxyl steroid dehydrogenase(3beta-HSD)in different culture stages.Results(1)The result of the modeling is referring to the first part.(2)All the cells were growing well.Primary culture for 2 hours,the leydin cells were all round.The hypospadias group had a long spindle shape after 12 hours of culture,and gathered into a cord shape.After 48 hours,it was completely spread and showed a net pull.After 12 hours of primary culture,the control group began to spread out,showing a mesh like growth and a long spindle shape in some cells.There was no obvious change in cell morphology during the process of culture.(3)After 24 hours of culture,the differentiation of the Leydig cells exposure from DBP in 62.5,125 and 250 mol/L were no significantly different from those that grew in the control group.Cell differentiation were significantly affected at 500,1000 mol/L.ConclusionDBP can affect the transformation of fetal rat Leydig cells from fetal type to maturity,thereby affecting testosterone secretion.It may affect the development of genital organs and lead to hypospadias. |
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