| Epidemiological investigation found that the incidence of cryptorchidism,hypospadias and fertility decline in adult men were closely related to maternal smoking during pregnancy.Furthermore,animal studies have further shown that nicotine exposure during pregnancy can have adverse effects on the development and function of the reproductive system of male offspring,such as abnormal testosterone level and low sperm quality.It is suggested that nicotine exposure during pregnancy has testicular developmental toxicity.However,what is the mechanism of testicular developmental toxicity induced by prenatal nicotine exposure(PNE)? Is it heritable? These issues are still unclear.Based on this,the purpose of this study is to observe the changes of testicular morphology and function(testosterone synthesis and spermatogenesis)of F1 fetal rats and F1,F2 postnatal offspring rats,to explore the intrauterine origin and genetic effect of testicular developmental toxicity induced by PNE,and to analyze its potential mechanism.Further combined with cell experiments,the epigenetic molecular mechanism of nicotine induced low testosterone synthesis in Leydig cells was confirmed.This study will provide experimental and theoretical basis for a comprehensive and in-depth understanding of the testicular developmental toxicity of nicotine,and for exploring the early warning and prevention targets of male reproductive system related diseases.PART ONE: Testicular Developmental Toxicity and Its Mechanism in Fetal Rats Induced by Prenatal Nicotine ExposureObjective: To investigate the effects of PNE on testicular morphogenesis and functional differentiation in F1 fetal rats,to confirm the testicular development toxicity,and to explore its possible mechanism.Methods: Wistar rats were caged according to the ratio of male to female 2:1.After the success of pregnancy,pregnant rats were divided into two groups: control group and PNE group.From the second trimester(gestational day 9),the PNE group was given 2 mg/kg.d nicotine subcutaneously,and the control group was given the same volume of normal saline.On the gestational day 20,pregnant rats were killed after anesthesia with 2.5%~3% isoflurane.The fetuses were removed by caesarean section to identify the sex and record the body weight.The samples were collected and the relevant indicators were detected.Results: Compared with the control group,the birth weight of male fetal rats in PNE group decreased,as well as the mean area of testis.HE staining showed morphological damage,while electron microscopy showed vacuolation of mitochondria in PNE group.At the same time,the level of serum testosterone and intratesticular testosterone content in PNE group decreased significantly,the expression of St AR,P450 scc and 3β-HSD in fetal testis decreased,and the number of Leydig cells decreased too.Compared with the control group,the levels of H3K9 ac in the promoter region of St AR and 3β-HSD in PNE group decreased,while did no changes in the promoter region of P450 scc.The expression of HDAC4 increased in PNE group.The m RNA expression of n ACh R α4,α7,α10,β1,β2 and β4 was detected in the fetal testis of rats.Compared with the control group,the m RNA expression of n ACh R α4,α7,α10 and β2 of PNE group was significantly increased.Conclusion:PNE can cause testicular dysplasia in F1 male fetal rats,which may be related to the inhibited expression of testicular steroidogenic enzymes.Moreover,PNE-induced inhibition of steroidogenic enzymes expression may be directly regulated by n ACh R or indirectly caused by the abnormal histone modification of gene promoter region which might be mediated by HDAC4.PART TWO: Testicular Dysfunction and Its Heritability of Offspring Rats Induced by Prenatal Nicotine ExposureObjective: To investigate the effects of PNE on testicular morphological structure and function in F1 and F2 male offspring rats,to confirm the testicular development toxicity and its heritability,and to explore its possible mechanism.Methods: F0 generation: Pregnant Wistar rats were randomly divided into control and nicotine groups.Starting from the gestational day 9,the nicotine group was injected subcutaneously with nicotine 2 mg/kg.d,and the control group was given the same volume of normal saline.The pregnant rats were kept until normal delivery to produce the F1 offspring.F1 offspring: After birth,F1 offspring were fed with normal diet and weighed weekly.At postnatal week 6(PW6)and PW12,one subgroup of the F1 male offspring was randomly selected from each litter,which was anesthetized with 2.5%~3% isoflurane and then killed.Blood samples,hypothalamus,testis and epididymis were collected,and testis tissue weight was recorded.At PW12,one male offspring was randomly selected from each litter to mate with normal female rats to generate F2 offspring.F2 offspring: After birth,F2 offspring were fed with normal diet and weighed weekly.At PW12,one male offspring was randomly selected from each litter and killed as mentioned above.Blood samples,testis and epididymis were collected,and testis tissue weight was recorded.Results:(1)Compared with the control group,the body weight,testis weight and testis organ index of male offspring rats in the F1 PNE group decreased,and the testicular morphology and structure were abnormal,but there was no such change in F2 generation.(2)Compared with the control group,the testosterone synthetic function of testis in PNE group was inhibited continuously,whether in F1 or F2 generation,which showed that the level of serum testosterone and intratesticular testosterone was significantly reduced,the expression of St AR and 3β-HSD was decreased,as well as the number of Leydig cells.(3)Compared with the control group,the testicular spermatogenesis function of PNE group was damaged in different degrees in F1 and F2 generations,such as the decrease of sperm number and the increase of sperm deformity rate.(4)Compared with the control group,the level of H3K9 ac in the promoter region of St AR and 3β-HSD in testis of PNE group decreased continuously in F1 and F2 generations.(5)Compared with the control group,the HPT axis of PNE group was transiently activated in F1 adolescence,and inhibited in adulthood.Conclusion:The testicular dysplasia in F1 male fetal rats induced by PNE can continue to postnatal,resulting in testicular dysfunction.HPT axis may be involved in the regulation of postnatal testicular function.In addition,the testicular dysfunction of F1 generation caused by PNE can be partially inherited to F2 generation,which is manifested as the continuous decrease of testosterone synthesis from F1 to F2 generation,accompanied by a certain degree of inhibition of spermatogenesis.It may be related to the low level of histone modification in the gene promoter region and expression of steroidogenic enzymes.PART THREE: Nicotine-induced Inhibition of Testosterone Synthetic Function and Its Molecular Mechanism in Leydig CellsObjective: To investigate the effects of nicotine on testosterone synthetic function and the expression of n ACh R and HDAC4 in MLTC-1 cells by the treatment of different concentrations of nicotine,to confirm the mediating effect of n ACh R and HDAC4 on nicotine-induced inhibition of testosterone synthesis by intervening n ACh R and HDAC4 respectively.Methods: MLTC-1 cells were resuscitated and subcultured to obtain enough cell.The cells were treated with 0,0.1,1 and 10 μM nicotine solution for 72 h,or with 10 μM nicotine combined with 10 μM vecuronium bromide(n ACh R antagonist)and 1 μM LMK-235(HDAC4 inhibitor),respectively.Results: Compared with the control group,nicotine treatment could reduce the level of testosterone in the supernatant of MLTC-1 cells,and inhibited the expression of St AR,P450 scc and 3β-HSD in a concentration dependent manner.The level of H3K9 ac in the promoter region of St AR and 3β-HSD decreased significantly under the treatment of high dose(10 μM)nicotine,while the level of H3K9 ac in P450 scc was unchanged.At the same time,the expression of n ACh R α9 and β2 increased after nicotine treatment,as well as the expression of HDAC4.However,these changes were reversed after administration of n ACh R antagonist vecuronium bromide or HDAC4 inhibitor LMK-235.Conclusion:Nicotine can inhibit the synthesis of testosterone in MLTC-1 cells.The mechanism is that nicotine acts on its receptor n ACh R,and then directly induce the low expression of P450scc;at the same time,it can up-regulate HDAC4,and reduce the H3K9 ac level in St AR and 3β-HSD promoter region and gene expression,which eventually leads to the inhibition of testosterone synthesis. |
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