| ObjectivesBisphenol A(BPA),as A kind of environmental endocrine disruptor,is mainly used in industrial and daily necessities production.It can enter the body through contact channels such as digestive tract,respiratory tract and skin,causing certain harm to humans and animals.Due to its weak estrogen-like and strong anti-androgen-like effects,it can affect the male reproductive system,but the mechanism of damage is still unclear.Therefore,this study used early life en'vironmental exposure doses of BPA infected animal model,using miRNAs chip technology and bioinformatics methods for differences in gene prediction and signal pathways,and through the gene expression level,protein level detection,morphological analysis and other methods of differential expression of microRNAs(miRNA),sex hormone levels and the ultrastructural aspects and so on,morphological analysis and verification,to clarify the early life of BPA exposure on the reproductive system damage epigenetics miRNA regulation mechanism,It provides a theoretical basis for the early detection and prevention of BPA male injury.Methods1.SPF 10 week old healthy SD rats,including 30 male rats and 60 female rats.According to the ratio of male to female 2:1,the vaginal smear method was performed in the morning of the next day.The sperm was recorded as 0 days of pregnancy(GDO),and the males were recorded daily to avoid the father-derived factors.Individual differences ensure their balance.The pregnant female rats were randomly divided into five dose groups,and were intragastrically administered with GD5.The doses were 0,0.05,0.5,5,50 mg/kg,respectively,at a volume of 5 ml/kg.The rats were treated with gavage to GD19 once a day,and the rats were treated in three stages:GD20,PND21 and PND56.2.To observe the effects of prenatal BPA exposure on the general toxicity and the ratio of male to female in GD20 fetal mice.3.MicroRNA chip analysis was carried out on the testes of the GD20 stage offspring and the differential expression of miRNA in the testes of the three stage offspring was verified by real-time PCR,and the toxic signaling Pathway affected by them was predicted and analyzed by GO analysis and Pathway analysis.1.To observe the effects of prenatal BPA exposure on the total number of sperm,body weight,anal distance and organ coefficients of PND56 stage offspring;H&E staining and transmission electron microscopy were used to observe testicular tissue damage and fine structure damage.The expression levels of FSH,LH and T were determined by ELISA.Results1.Effects of prenatal BPA exposure on the general toxicity and miRNA expression profile of GD20 male fetal rats(1)Analysis of the general toxicity and sex ratio of fetal mice at GD20 stage showed that compared with the control group,the total weight of amniotic fluid in fetal mice exposed to BPA during pregnancy showed a certain degree of decline,but the effect was statistically significant only when the dose was 5mg/kg(P<0.05).As a result,BPA exposure during pregnancy did not affect placental weight in pregnant rats.However,low birth weight of fetal mice could be caused to a certain extent,and the decrease was statistically significant at doses of 0.5mg/kg,5mg/kg and 50mg/kg(P<0.05).However there was no significant difference in the ratio between males and females(P>0.05).(2)According to the analysis of microRNA chip results at the GD20 stage,compared with the control group,133 mimas were up-regulated and 136 mirnas were down-regulated in the 5mg/kg dose group,while 171 mirnas were up-regulated and 124 mirnas were down-regulated in the 0.05mg/kg dose group.Twenty-six mirnas were cross-linked by expression levels and differential expression multiples for real-time PCR verification.It was found that miR-361-5p and miR-19b-2-5p were up-regulated in the 0.05mg/kg and 5mg/kg dose groups,while miR-203a-3p was down-regulated in the 5mg/kg dose group,and the difference was statistically significant(P<0.05).Target gene prediction was carried out through TargetScan database,and the top three predicted signal pathways were Choline metabolism in cancer,MicroRNAs in caner and GABA signal pathways through Pathway analysis.2.Changes in the differential expression of miRNA during the growth and development of male offspring exposed to BPA during pregnancy(1)Real-time PCR was used to verify the expression of 26 mirnas in the testicles of the offspring at the stage of PND21.The expression of miR-409b was up-regulated in the group of 0.05mg/kg and the group of 5mg/kg,while the expression of miR-30b-3p was up-regulated in the group of 5mg/kg and the difference was statistically significant(P<0.05).(2)Real-time PCR was used to verify the expressions of 26 mirnas in the testicles of the offspring at the stage of PND56.From the results,compared with the control group,miR-203a-5p,miR-19b-2-5p,miR-671,miR-409b and miR-30b-3p in the 5mg/kg dose group showed increased relative expressions of miR-203a-5p,miR-19b-2-5p,miR-671,miR-409b and miR-30b-3p,and the differences were statistically significant(P<0.05).In the comparison between the 0.05mg/kg dose group and the control group,in addition to the corresponding increase in the relative expressions of miR-203a-5p,miR-19b-2-5p,miR-671,miR-409b and miR-30b-3p,miR-410-5p and miR-203a-3p were also increased,and the difference was statistically significant(P<0.05).3.Effects of prenatal BPA exposure on reproductive injury in male offspring rats(1)At the stage of PND56,the weight of male offspring in the 5mg/kg and 50mg/kg groups was significantly higher than that in the control group(P<0.05).The organ coefficient of testis and kidney decreased in different dose groups,but there was no statistical significance.The liver coefficient in the dose groups of 0.5mg/kg and 50mg/kg was significantly higher than that in the control group(P<0.05).The sperm count of each dose group was decreased to different degrees and the results were 0.05mg/kg,0.5mg/kg,and 5mg/kg(P<0.05).(2)The detection of the content levels of the three kinds of sex hormones showed that FSH,LH and T decreased in each exposure dose group,while LH and T decreased at 0.05mg/kg,which was statistically significant(P<0.05),and FSH decreased at 50mg/kg,which was statistically significant(P<0.05).(3)In the 0.05mg/kg group,H&E staining showed slight atrophy of the convoluted tubules and the disappearance of some spermatogenic cells.At the dose of 0.5mg/kg,the convoluted tubule atrophied,the spermatogenic cell layer thinned,and the spermatogenesis decreased.At the dose of 5mg/kg,spermatogenic cells were disordered and atrophied,the number of layers was changed,and spermatogenesis was reduced.When the dose reached 50mg/kg,a large number of multicellular giant cells could be seen under the testicular microscope,but there was no atrophy of the convoluted seminiferous duct,and the spermatogenesis was reduced to a certain extent.From the ultrastructure of testicular electron microscopy,mitochondria in the groups of 0.05mg/kg and 0.5mg/kg did not change significantly.When the dose increased to 5mg/kg,microscopically,the mitochondria showed broken ridges and vacuolar changes.At the dose of 50mg/kg,mitochondrial changes were more significant,most of which showed mitochondrial swelling,broken ridges and vacuolar changes.Conclusion1.Exposure to low doses of BPA during pregnancy resulted in decreased sperm count,abnormal sex hormone levels and testicular tissue damage in male offspring;2.Exposure to low doses BPA during pregnancy can lead to changes in the expression of miRNA in the testes of male offspring,and such changes may vary with growth and development.3.Through miRNA chip technology and bioinformatics prediction,the miRNA regulation mechanism of reproductive toxicity caused by exposure to low-dose BPA during pregnancy may be:Choline metabolism in cancer,MicroRNAs in caner,GABA Signaling pathways,Phosphatidylinostol Signaling pathways,Morphine addiction,FoxO signal pathway,Pancreatic secretion,Signaling pathways regulating pluripotency,VEGF Signaling pathways and Retrograde The endocannabinoid signaling pathway,however,needs to be further verified. |
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