The Research Of Immature Testicular Injury Induced By Di-(2-Ethylhexyl) Phthalate And The Long-Term Effects

PART1 STEREOLOGICAL ANALYSIS AND MECHANISM RESEARCH OF IMMATURE TESTICULAR INJURY INDUCED BY DI-(2-ETHYLHEXYL)PHTHALATEObjective: To investigate the effects of Di-(2-ethylhexyl)phthalate(DEHP),an environmental endocrine disruptor,on the reproductive function of immature testes in prepubertal rats and its underlying mechanism.Methods: Prepubertal male Sprague-Dawley(SD)rats were randomly divided into two groups and modeled by intragastric administration from postnatal day(PND)22 to PND35;the control group were administrated with corn oil,and the DEHP group were intragastric administration of DEHP(500mg/kg/d).The rats were sacrificed and samples were taken at PND36 for further research.Rat body weight,testicular weight,testicular organ coefficient,serum sex hormone levels were measured;the morphological changes of the testes by PAS staining were observed;Stereology methods were used to study the quantitative indicators of different structures in the testis and the quantitative data of the spermatogenetic cells;At the same time,the transcriptome sequencing and bioinformatics analysis were conducted to screen out related pathways and related genes to reveal the potential mechanism of the damage.The RNA-seq data were validated by quantitative real-time polymerase chain reaction(q RT-PCR).Results: Compared with the control group,DEHP caused a decrease of5.4%,29.1%,and 33.0% of the body weight,testicular weight and testicular organ coefficient of prepubertal rats,respectively.The serum levels of testosterone and luteinizing hormone(LH)were decreased,in contrast to the increased follicle-stimulating hormone(FSH)level;DEHP caused immature testes obvious atrophy of seminiferous tubules accompanied by pathological features such as vacuolation of seminiferous epithelium,loose arrangement of spermatogenic cells,and spermatogenic cells sloughing.Stereological quantitative research found that,compared to the control group,the volume of the testis decreased by 29.2%,the volume of the seminiferous tubules and the seminiferous epithelium in the testis decreased by 37.3% and 38.4%,respectively.There was no significant change in the volume of the interstitial tissue,but the volume fraction increased by 48.6%;the thickness of the seminiferous epithelium was reduced by 21.2%,and the diameter of seminiferous tubules was reduced by 16.3%,and the length of seminiferous tubules was reduced by 12.1%;the number of type B spermatogonia,?-?stage pachytene spermatocytes,?-?? stage spermatocytes,round spermatids,and elongating spermatids in the seminiferous epithelium was significantly reduced.There was no significant change in the number of A type spermatogonia,preleptotene spermatocyte,leptotene spermatocyte,zygotene spermatocyte,Sertoli cells and Leydig cells.Transcriptome sequencing identified 5,548 differentially expressed genes(DEGs)upon DEHP exposure,among which 540 genes were up-regulated and 5,008 genes were down-regulated.Bioinformatics analysis revealed that the reproductive toxicity of DEHP to immature testes might be mediated through steroid hormone synthesis,retinol metabolism,CAMs,and oxidative phosphorylation signaling pathways,and affected by the changes of genes expression related to the pathways.DEGs selected from RNA-seq data were validated by q RT-PCR,and the results showed the same trends as the RNA-seq results.Conclusion: Prepubertal DEHP exposure could significantly affect the growth and development of rat,resulting in lower body weight,testicular weight and lower serum testosterone and LH levels.It also could lead testicular morphological damage and influence the spermatogenesis in the testis,resulting in the number of type B spermatogonia,?-? stage pachytene spermatocytes,?-?? stage spermatocytes,round spermatids and elongating spermatids decreased.Through transcriptome sequencing and bioinformatics analysis,it is speculated that the testicular reproductive function damage might be mediated by steroid hormone synthesis,retinol metabolism,CAMs,and oxidative phosphorylation signaling pathways.PART 2 THE LONG-TERM EFFECTS OF DEHP EXPOSURE ON IMMATURE TESTICULAR INJURY AND THE DYNAMIC EVALUATION OF GENE EXPRESSION LEVELSObjective: The first part of the research showed that prepubertal DEHP exposure could cause significant damage to the reproductive function of immature testes in rats.In this part,we will continue to follow up to further explore the long-term effects of prepubertal DEHP exposure on rat reproductive function and the dynamic evaluation of gene expression levels.Methods: The same SD rats with grouping strategy as the first part were followed up to adulthood after DEHP exposure in prepubertal stage,and the long-term effects of the two key periods such as sexual maturity(PND52)and adulthood(PND90)in of reproductive development were clarified.Rat body weight,testicular weight,testicular organ coefficient,sex hormone levels were measured in each stage.The mating experiment was conducted,and epididymal sperm count and malformation rate were measured in adulthood.The morphological characteristics of testicular tissue in different periods were observed qualitatively,and the different structures in testis and spermatogenic cells were measured quantitatively in different periods with stereological methods.At the same time,the transcriptome sequencing and bioinformatics analysis along the follow-up stages were dynamically evaluated.The RNA-seq data were validated by qRT-PCR.Results: The body weight,testicular weight,and testicular organ coefficients of rats were still lower than those in the control group in sexual maturity period and the indexes could return to normal levels in adulthood.The levels of serum testosterone,LH,and FSH were still at the abnormal levels in sexual maturity period,and testosterone and FSH returned to normal levels in adulthood,but LH level was still lower than normal.The morphological and pathological damage of testis relieved during sexual maturity period,and recovered significantly in adulthood.Stereological quantitative research found that the volume of the testis and different testicular structures could quickly restore during sexual maturity period,and reached to the normal levels in adulthood.The thickness of seminiferous epithelium,the diameter of the seminiferous tubular lumen,the diameter and length of seminiferous tubules showed lower levels in sexual maturity,and returned to normal levels in adulthood.The number of type B spermatogonia,?-? stage pachytene spermatocytes,?-?? stage spermatocytes returned to the normal level during sexual maturity period and stayed normal in adulthood.Round spermatids,elongating spermatids,and elongated spermatids were still lower than normal during sexual maturity period,but returned to the normal in adulthood.In adulthood,there was no significant difference in sperm count,sperm malformation rate and mating experiment compared with the control group,suggesting that fertility might not be significantly affected.Transcriptome sequencing revealed that there were 2006 and 222 DEGs at the two stages,among which 1192 and 130 genes were up-regulated,and 814 and 92 genes were down-regulated,respectively.Bioinformatics analysis revealed that the recovery mechanism might be mediated by DNA replication,mismatch repair,homologous recombination,MAPK signaling pathway in sexual maturity period,and activated relaxin and ribosomal signaling pathways in adulthood might play an important role in the recovery of reproductive injury.DEGs selected from RNA-seq data were validated by q RT-PCR,and the results showed the same trends as the RNA-seq results.Conclusion: The effects of reproductive toxicity upon DEHP exposure in prepubertal stage was still partially residual during sexual maturation,and basically returned to normal in adulthood.Transcriptome sequencing speculated the recovery of reproductive injuries were related to the change of the genes expression levels in signaling pathways related to reproduction.