| ObjectiveTo explore the toxicological effects and biochemical molecular changes of Leydig cells when exposed to environmentally-related low concentrations of bisphenol A,nonylphenol,and their mixtures,so as to explore the multi-and trans-generational effects induced by bisphenol A and nonylphenol.And then the study further disscuse the multi-and trans-generational genetic mechanism of rodent male reproductive toxicity caused by bisphenol A and nonylphenol.MethodsSeventy-two females and 24 males 28-day-old SD rats were randomly divided into 4 groups according to the ratio of male and female 3:1,and the male and female were fed separately.One week after acclimatization to the animal room,we started gavage of the exposure solution.Rats were caged and mated at 56 days old(excluding littermates)and continued to receive exposure solution during this period until delivery.Parental female rats continued to be exposed after delivery until the end of the lactation period.Parental male rats continued to be exposed to 90 days old;they were named F0+♂.The exposure period covered the critical stages of testicular development and the spermatogenic cycle in rats.The first generation to which the female rats gave birth after exposure was stopped were named F1-.On the contrary,the first generation to which the female rats gave birth while exposure continued were named F1+.The male rats of F1-were fed normally to 90 days old;they were named F1-♂.The rats of F1+ started to receive gavage with exposure solution after weaning,continued to receive exposure solution until 56 days old,then were caged and mated(excluding littermates).The female rats of F1+ continued to be exposed during pregnancy until delivery(no exposure during lactation).The male rats of F1+ continued exposure to 90 days old;they were named F1+♂.The first and second generations of F1+ rats were not exposed,and they were named F2-and F3-,respectively.Then the male rats were fed to 90 days old and were named F2-♂ and F3-♂,respectively.The 90-day-old male rats in F0+,F1+,F1-,F2-and F3-generations were taken the testes and serum,then the testis were weighed to calculate the organ coefficient.The testis was used to make paraffin sections and stained with H&E,to observed the changes in the morphological structure of interstitial tissues.Testes were made OCT frozen sections for oil O red staining to observed the changes in lipid droplet content in interstitial tissues.The transmission electron microscopy was used to detect the changes of ultrastructure in testicular Leydig cells.FTIR spectroscopy was used to detect spectral data and analysis.The serum were used ELESA kit to measures the level of testosterone.Results1.After treatment with BPA,NP and BPA&NP,the weight coefficient of the testis was significantly decreased compared with the control group in generation F1+(P < 0.05).In generations F0+,F1-,F2-and F3-,the weight coefficient of the testis from BPA-,NP-and BPA&NP-treated rats showed no significant difference compared with the corresponding control group.BPA and NP had an interactive effect on the testicular coefficient in generation F0+(two-way ANOVA,Pinteraction = 0.012).However,there were no interaction effects between BPA and NP on the organ coefficient of the testis in other generations(two-way ANOVA,Pinteraction > 0.05).2.In generations F0+ and F1+,the morphology of interstitial tissue from BPA-,NP-and BPA&NP-treated rats exhibited an increase in the number of vacuoles compared to control rats.Specifically,the BPA&NP group revealed more vacuoles in interstitial tissue than the BPA or NP groups.But in generations F1-,F2-and F3-,the histopathologic examination of interstitial tissue did not reveal any distinct changes among the control,BPA,NP and BPA&NP groups.Interestingly,Oil Red O staining showed that the number of lipid droplets was reduced in BPA,NP and BPA&NP groups compared with the control group in F2-and F3-generations.3.In generation F0+,the mitochondria in LCs of the BPA,NP and BPA&NP groups were slightly swollen compared to the control group.Nuclear deformation in the BPA,NP and BPA&NP groups showed swollen endoplasmic reticulum compared to the control group.Moreover,combined exposure to BPA and NP induced the formation of autophagosomes with a vacuolar water-like structure and increased the number of lysosomes.In generation F1+,it seems that the ultrastructure changes such as mitochondrial swelling and endoplasmic reticulum swelling in BPA,NP and BPA&NP groups had become more obvious than that of the control group.Notably,the number of lysosomes in exposure groups of generation F1+ was increased compared to the exposure groups of generation F0+.In addition,severe interstitial oedema was observed in the BPA&NP group of generation F1+.The BPA&NP group of generation F2-showed nucleus invagination,and the BPA or NP groups of generation F2-showed interstitial oedema.In generation F3-,mitochondrial swelling,interstitial oedema and lysosomes were frequently observed in BPA,NP and BPA&NP groups.4.Cluster segregations between exposed and control groups were observed based on the fingerprint region of 1800–900 cm-1in the F0+,F1+,F1-,F2-and F3-generations.The main biochemical alterations for segregation were amide I,amide II and nucleic acids.BPA and NP single and co-exposure induced significant differences in the ratio of amide I to amide II compared to the corresponding control group in all generations.BPA exposure resulted in remarkable changes of cellular gene transcription and DNA oxidative damage across all generations.Direct exposure to BPA,NP and BPA&NP of F0+ and F1+ generations could significantly decrease lipid accumulation in LCs in the F2-and F3-generations.ConclusionsIn summary,this study is the first to investigate the multi-and transgenerational effects of parental BPA and NP exposure on male reproductive health with a focus on the biochemical features of testicular interstitial(Leydig)cells.Our data demonstrate that chronic exposure to low doses of BPA,NP and BPA&NP is potentially associated with changes of the metabolism and protein secondary structures of LCs in multiple generations.These findings suggest that the effects of BPA and NP on LC structure and function possibly occur multigenerationally,and even transgenerationally.The major limitation of this study is still a lack of explorations into the mechanisms underlying epigenetic transgenerational inheritance.For this,in future studies,we suggest taking a special interest in the mechanisms by which these EDCs affect male reproduction,especially in a transgenerational manner. |
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