Effect And Mechanism Of Intrauterine Exposure Of TCDD On Reproductive Function And Testicular Cell Apoptosis In Male Offspring

ObjectivesTo investigate the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)in intrauterine exposure on progeny reproductive function and testicular cell apoptosis.To further explore the impact and possible mechanism of TCDD on the occurrence and development of the reproductive system,so as to provide theoretical support for the prevention of related diseases.MethodsThe sexual maturity Sprague-Dawley male rats and female rats were mated overnight at a ratio of 1:2.The female rats were observed sperm-positive vaginal plug as mating success.The day of sperm-positive vaginal plug observed was noted as gestation day 0(GD0),the number of mating success of rats to meet the needs of the experiment,the pregnant rats were randomly divided into:control(corn oil)group,low dose of TCDD(100 ng/kg)and high dose group TCDD(500 ng/kg),with 6 rats in each group and exposed to corn oil(control)or TCDD by gavage from gestation day(GD)8 to 14.The pregnant rats were given birth naturally.On postnatal day21(PND21),the Anal to genital distance(AGD)was measured.Two male young adult rats per litter randomly were selected to weighing,anesthesia,blood,put to death,testes were weighed and testicular organ coefficients were calculated,Put one of testes in fixed in 4%paraformaldehyde fixed fluid,then embedding,sectioning,put another one immediately after in vitro in RNA store preservation solution,and then put it into-80?freezer.Serum testosterone(T)was measured by Enzyme linked immunosorbent assay(ELISA).Sperm quality and fertility were evaluated in adult male offspring Histopathological changes in testicular tissues were observed by HE stain.The mRNA levels of Bax,Bcl-2,Caspase-3 in testes were determined by RT-PCR.The expressions of Bax,Bcl-2,Caspase-3 in testes were determined by western blot.Statistical analysis was performed on the data obtained from the experiment using SPSS 25.0 software,and the significance level was?=0.05.ResultsThe 100 ng/kg TCDD group and the 500 ng/kg TCDD group compared with the control group,the AGDs((1.49±0.04)cm,(1.32±0.01)cm and(1.21±0.02)cm)were shortened(P<0.05),the number of sperm((72.33±4.46)×10~6/mL,(63.67±3.44)×10~6/mL,(45.33±5.47)×10~6/mL)and sperm viability((78.53±1.26)%,(64.40±3.14)%,(53.87±3.65)%)were significantly decreased,sperm abnormality rates((2.17±0.75)%,(4.83±0.75)%,(8.00±1.10)%)were increased(P<0.05),serum T Levels((2.68±0.06)ng/mL,(2.38±0.08)ng/mL,and(2.10±0.09)ng/mL)were significantly decreased(P<0.05),and the differences were statistically significant.Compared with the control group,the differences between testicular weight(P<0.05)and testicular organ coefficient(P<0.05)in the 500 ng/kg TCDD group were statistically significant.The results of HE staining showed that the testicular tissue morphology was abnormal in the exposed group compared with the control group.The relative expression of Bax protein in testicular tissue cells(0.836±0.004,1.185±0.004,1.414±0.001)increased with increasing dose(P<0.05).The relative expression of Bax protein in testicular tissue cells(0.836±0.004,1.185±0.004,1.414±0.001)increased with increasing dose(P<0.05),then the expression of Bcl-2(1.368±0.053,1.108±0.040,0.751±0.022)opposite(P<0.05),while the expression of Pro-Caspase-3 was significantly decreased in the 500 ng/kg TCDD group,but the expression of Cleaved-Caspase-3 protein was significantly increased(P<0.05).Compared with the control group,the relative expression of Bcl-2(1.126±0.109,0.809±0.114,0.680±0.721)mRNA was significantly decreased,the relative expression of Bax(0.942±0.052,1.093±0.097,1.281±0.078)mRNA increased with increasing dose,and the relative expression of Caspase-3(1.056±0.080,1.189±0.043,1.307±0.029)mRNA decreased with increasing dose,and the ratio of Bcl-2/Bax was(1.239±0.210,0.850±.0.120,0.633±0.054)all decreased significantly(P<0.05)in the TCDD groups.Conclusions1.Intrauterine exposure of TCDD can lead to decreased reproductive function in male rats;2.Intrauterine exposure to TCDD can inhibit the proliferation and induce apopt-osis of testicular tissue cells,leading to imbalance of testicular tissue cell prolifera-tion-apoptosis;3.Testicular tissue cell proliferation-apoptosis imbalance is related to the activa-tion of the mitochondrial pathway of apoptosis.