epigenetic transgenerational inheritance of testis …...male reproductive health has been in...

R E S E A R C H A R T I C L E

Epigenetic transgenerational inheritance of testis

pathology and Sertoli cell epimutations generational

origins of male infertilityIngrid Sadler-Riggleman1dagger Rachel Klukovich 2dagger Eric Nilsson1Daniel Beck1 Yeming Xie2 Wei Yan2Dagger and Michael K Skinner 1Dagger

1Center for Reproductive Biology School of Biological Sciences Washington State University Pullman WA99164-4236 USA 2Department of Physiology and Cell Biology University of Nevada Reno School of MedicineReno NV 89557 USA

Correspondence address Center for Reproductive Biology School of Biological Sciences Washington State University Pullman WA 99164-4236 USA Telthorn1-509-335-1524 Fax thorn1-509-335-2176 E-mail skinnerwsuedudaggerCo-first authorsDaggerCo-senior authorsManaging editor Dana Dolinoy

Abstract

Male reproductive health has been in decline for decades with dropping sperm counts and increasing infertility which hascreated a significant societal and economic burden Between the 1970s and now a general decline of over 50 in sperm con-centration has been observed in the population Environmental toxicant-induced epigenetic transgenerational inheritancehas been shown to affect testis pathology and sperm count Sertoli cells have an essential role in spermatogenesis by pro-viding physical and nutritional support for developing germ cells The current study was designed to further investigate thetransgenerational epigenetic changes in the rat Sertoli cell epigenome and transcriptome that are associated with the onsetof testis disease Gestating female F0 generation rats were transiently exposed during the period of fetal gonadal sex deter-mination to the environmental toxicants such as dichlorodiphenyltrichloroethane (DDT) or vinclozolin The F1 generationoffspring were bred (ie intercross within the lineage) to produce the F2 generation grand-offspring that were then bred toproduce the transgenerational F3 generation (ie great-grand-offspring) with no sibling or cousin breeding used The focusof the current study was to investigate the transgenerational testis disease etiology so F3 generation rats were utilized TheDNA and RNA were obtained from purified Sertoli cells isolated from postnatal 20-day-old male testis of F3 generation ratsTransgenerational alterations in DNA methylation noncoding RNA and gene expression were observed in the Sertoli cellsfrom vinclozolin and DDT lineages when compared to the control (vehicle exposed) lineage Genes associated with abnor-mal Sertoli cell function and testis pathology were identified and the transgenerational impacts of vinclozolin and DDTwere determined Alterations in critical gene pathways such as the pyruvate metabolism pathway were identifiedObservations suggest that ancestral exposures to environmental toxicants promote the epigenetic transgenerational inheri-tance of Sertoli cell epigenetic and transcriptome alterations that associate with testis abnormalities These epigenetic

Received 11 January 2019 revised 28 August 2019 accepted 19 July 2019

VC The Author(s) 2019 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc40) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly citedFor commercial re-use please contact journalspermissionsoupcom

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Environmental Epigenetics 2019 1ndash18

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alterations appear to be critical factors in the developmental and generational origins of testis pathologies and maleinfertility

Key words Sertoli cell male infertility testis pathology transgenerational vinclozolin DDT

Introduction

Epimutations were originally defined as lsquoheritable epigeneticchanges in chromosomes which do not involve changes in theDNA sequence itselfrsquo [1 2] A more updated definition of epige-netics is lsquomolecular factors and processes that regulate genomeactivity independent of DNA sequence and are mitoticallystablersquo and for epimutations is lsquoenvironmentally induced differ-ential presence of epigenetic alterations that can influence ge-nome activity compared to organisms not having the exposurersquo[3] A number of environmental factors can alter epigenetic pro-cesses such as DNA methylation histone modifications (egmethylation and acetylation) chromosome structure noncod-ing RNA (ncRNA) and RNA methylation to influence gene ex-pression and genome activity Epigenetic transgenerationalinheritance describes a germline transmission of epimutationsthrough generations without continued exposure to or presenceof the original exposure beyond the F0 generation [3] In caseswhere the germline is exposed during fetal gonadal sex deter-mination the epigenome in the germline can be modifiedThese epimutations appear to become lsquoimprinted-likersquo and po-tentially escape post-fertilization methylation erasure to allowtransmission to the subsequent generations [4 5] The hypothe-sis is that this promotes stem cells in the embryo to develop analtered epigenome and transcriptome Somatic cells and tissuesderived from these epigenetically altered germline and stemcells will potentially develop alterations in cell type-specific epi-genomes and transcriptomes [6] These cell specific altered epi-genomes will impact the fate of organs and tissues and may beearly indicators and initiators of the development of diseasesusceptibility later in life [7ndash9] Previous examples of this phe-nomenon have been observed in prostate epithelial cellsfor prostate disease [10 11] and granulosa cells for ovarian dis-ease [12]

Numerous studies have shown epigenetic transgenerationalinheritance to occur in different species involving a wide varietyof different environmental exposures Epigenetic transgenera-tional inheritance has been shown in plants [13] flies [14]worms [15] fish [16] birds [17 18] rodents [5 19] pigs [20] andhumans [21] Environmentally induced transgenerational dis-eases and abnormalities in mammals have been observed suchas testis disease [8 22 23] prostate disease [9 24] kidney dis-ease [9 24 25] obesity [26] ovarian and uterine disease [22 2527ndash30] tumor development [9] and behavioral changes [31ndash36]A large number of environmental toxicants have been shown tobe involved in triggering these transgenerational diseases andabnormalities that include the agricultural fungicide vinclozolin[5 37ndash39] the herbicide atrazine [31 40] plasticizers such asbisphenol A [22 41] and phthalates [22] insect repellant diethyl-toluamide (DEET) with the insecticide permethrin [27] pesticidemethoxychlor [25] hydrocarbons (jet fuel) [28] and industrialcompounds such as benzo[a]pyrene [42] the biocide tributyltin[43] mercury [16] dioxins [24 29 44] and the herbicide glypho-sate [45]

The current study was designed to examine the ability ofvinclozolin and dichlorodiphenyltrichloroethane (DDT) to in-duce transgenerationally affected Sertoli cell epigenome and

transcriptome alterations that associate with testis pathologySertoli cells play a critical role in spermatogenesis providingstructural and nutritional support for the developing germ cellsand are involved in the formation of the bloodndashtestis barrierwhich creates a serum and pathogen free environment for thespermatogenic cells within the seminiferous tubules [46]Therefore Sertoli cells synthesize a number of transport bind-ing proteins [47 48] and provide the primary energy metabolites(ie pyruvate and lactate) for the developing germ cells whichare sequestered within the bloodndashtestis barrier and unable toacquire glucose themselves [49] A disruption in normal Sertolicell development and function can affect spermatogenesis andpromote testis pathology Abnormal spermatogenesis and lowsperm count are often linked to infertility which can involvetestis diseases like cryptorchidism hypospadias and testicularcancer [50ndash52] Sperm counts have been declining significantlybetween 1973 and 2011 as shown in a 2017 meta-regressionanalysis [53] Environmental factors such as endocrine disrupt-ing chemicals pesticides heat diet stress and smoking havebeen shown to be associated with this sperm count drop andgeneral testis health problems [53] The molecular basis forthese testis pathologies and generational impacts are investi-gated in the current study

Exposure of gestating rats to the environmental toxicantssuch as DDT and vinclozolin leads to the epigenetic transge-nerational inheritance of adult onset diseases including testisdisease and a decrease in sperm count andor motility [26 54]Originally vinclozolinrsquos involvement in epigenetic transgenera-tional inheritance of disease was observed in 2005 by Anwayet al [5] who found that vinclozolin exposure of gestating ratsleads to epigenetic transgenerational inheritance of uniqueDNA methylation changes (epimutations) in sperm Subsequentstudies involving vinclozolin and DDT among other environ-mental toxicants confirmed these findings [26 39 55 56]Vinclozolin is an agricultural fungicide used in fruit and vegeta-ble production and is an anti-androgenic compound that acts asa competitive antagonist of the androgen receptor [57] DDT is apesticide which was widely used through the 1950s and 1960sin the USA until banned in 1972 It continues to be used in manyparts of the world for insect and malaria control DDT accumu-lates in the environment and in fatty tissue and is an estrogenreceptor agonist that has estrogenic effects in animals [58]

The epigenetic transgenerational inheritance phenomenonrequires the germline transmission of altered epigenetic infor-mation between generations [59] A variety of different environ-mental factors promoting epigenetic transgenerationalinheritance were found to induce exposure specific alterationsin sperm DNA methylation [5 59] Subsequently vinclozolinwas found to promote alterations in sperm ncRNA transgenera-tionally [60] This supported previous studies indicating ncRNAgermline alterations are important factors in epigenetic trans-generational inheritance [61 62] Recently we observed thatboth vinclozolin and DDT cause concurrent alterations in caudaepididymal rat sperm DNA methylation ncRNA and histone re-tention [55 56] Therefore several different epigenetic processesare likely integrated in epigenetic transgenerational inheri-tance A 2013 study using vinclozolin determined that

2 | Environmental Epigenetics 2019 Vol 5 No 3

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vinclozolin impacts the epigenetic transgenerational inheri-tance of Sertoli cell DNA methylation and gene expression alter-ations [8] This current study extends these findings withgenome-wide analyses of DNA methylation and ncRNA altera-tions and associated gene expression changes These transge-nerational alterations in Sertoli cell epigenetics correlate tocorresponding alterations in testis pathology

ResultsExperimental Design and Testis Pathology

The F0 generation gestating female rats were exposed at ap-proximately 90 days of age to DDT or vinclozolin during gesta-tional days E8ndashE14 which corresponds to fetal gonadal sexdetermination and the germline differentiation period of devel-opment The toxicants were dissolved in dimethylsulfoxide(DMSO) and administered by daily intraperitoneal injection dur-ing the transient exposure time frame A separate group of con-trol females was injected on the same schedule with only DMSOas a vehicle control Each exposure lineage involved six differ-ent F0 generation females and was referred to as DDT vinclozo-lin and control lineages respectively This study andexperimental approach were not designed for risk assessmentbut to investigate the transgenerational phenomenon The F1generation animals were raised to 90 days of age and then bredwithin each lineage to obtain the F2 generation animals The F2generation animals were bred in the same manner to obtain theF3 generation animals The only animals directly exposed werethe F0 generation females No sibling or cousin breeding wasperformed to avoid inbreeding artifacts The male F3 generationpups were raised to 1 year of age for testis pathology analysis orto 18ndash22 days of age for isolation of Sertoli cells The 20-day-oldmale pups were randomly divided into 3 different groups fromdifferent litters for each lineage with each group comprising 6ndash11 animals depending on litter sizes obtained Within eachgroup the testis tissues were combined into one pool for isola-tion of Sertoli cells The total Sertoli cell pools from each groupwere divided into two aliquots and stored as cell pellets at80C for DNA and RNA preparations

The F3 generation Sertoli cells were isolated at 18ndash22 days ofage to obtain optimally purified populations of cells The purityof the cell preparations is routinely monitored and has previ-ously been shown to be gt98 Sertoli cells by histology and sub-sequent culture purity determined with immunocytochemistryfor fibronectin containing cells such as peritubular cells as pre-viously described [8 63ndash65] A cell fraction at this stage of 18ndash22 days of development allows for efficient analysis andreduces the impact of testis pathology on the analysis since themajority of pathologies develop between 6 and 12 months ofage Therefore at this early stage of development the study isnot confounded by the presence of disease Another group of F3generation animals was aged to 1 year and used to assess testispathology Testes were evaluated by microscopic examinationof testis sections Testis pathology was determined by the pres-ence of atrophy of seminiferous tubules vacuoles in the semi-niferous epithelium and sloughed spermatogenic cells in thetubule lumens as described in Methods Representative histo-pathologies are presented in Supplementary Fig S1 In the cur-rent study DDT was found to promote testis disease in 46 ofthe males compared to 8 in the control lineage animals(Fig 1a) Vinclozolin promoted a 44 frequency of testis pathol-ogy in the males (Fig 1a) Therefore both vinclozolin and DDTpromoted the epigenetic transgenerational inheritance of testis

pathology as previously described [66 67] The group of animalssacrificed at 18ndash22 days of age for Sertoli cell preparations wereused to investigate the molecular mechanisms in part involvedin this transgenerational testis pathology

Genomic DNA and RNA were isolated separately from theSertoli cell pool aliquots from each group and then used formethylated DNA immunoprecipitation (MeDIP) sequencing orRNA sequencing as described in the Methods Each of the poolscontained 6ndash11 different animals per pool from different littersThe 9 MeDIP samples (3 control lineage pools 3 DDT lineagepools and 3 vinclozolin lineage pools) were enriched for meth-ylated DNA using an MeDIP procedure employing a 5-methylcy-tosine specific antibody and antibody specific magnetic beadsor were processed to isolate the different RNA categories (totalRNA for mRNA and long ncRNA or small ncRNA) Sequencinglibraries were created for each MeDIP or RNA pool for next-generation sequencing (NGS) and run on the Illumina 2500 se-quencer for MeDIP-Seq or RNA-Seq by the WSU Genomics Core

Figure 1 transgenerational induced testis disease frequency Environmentally

induced transgenerational testis disease occurrence in several separate experi-

ments (a) Current study vinclozolin and DDT-induced transgenerational testis

disease with associated control [66 67] (b) Previous reports of transgenerational

pathology for plastics [22 41] dioxin [24] pesticides [27] and jet fuel hydrocar-

bons [28] with associated control frequency Plt 005 Plt001 Plt0001

Epigenetic transgenerational inheritance of testis pathology and Sertoli cell epimutations | 3

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Laboratory The procedures and bioinformatics details are de-scribed in the Methods section

Sertoli Cell DNA Methylation Analysis

The MeDIP-Seq information was used to identify differentialDNA methylated regions (DMRs) based on comparisons betweencontrol and DDT or vinclozolin lineage sequencing data Readdepth comparisons of the NGS reads identified DMRs for each ofthe analyses with a range of threshold P-values (Fig 2) The DDTlineage Sertoli cell DMRs were abundant and a Plt 1e06 wasselected for further analysis At Plt 1e06 5359 DMRs werefound with the majority of them having a single 100 bp statisti-cally significant window however 533 DMRs had 2 significantwindows (Fig 2a) The corresponding false discovery rate (FDR)analysis of 0001 correlated to the edgeR value of Plt 1e06 iden-tified The vinclozolin lineage Sertoli cell DMRs were less abun-dant and a Plt 1e06 was selected for further analysis At this P-value 122 DMRs were found with the majority of 108 having asingle 100 bp significant window DMR (Fig 2b) All the DMR hadan FDR adjusted P-value of less than 005 The DDT and vinclo-zolin lineage Sertoli cell DMRs were primarily distinct from eachother with only 29 DMRs in common (Fig 2c) This is notablesince one-fourth of the small number of DMRs in the vinclozolinlineage overlapped with the DDT lineage DMRs The lists of DDTSertoli cell DMRs are presented in Supplementary Table S1 andthe vinclozolin Sertoli cell DMRs in Supplementary Table S2The 29 overlapping DMRs are presented in SupplementaryTable S3 The increase or decrease in DNA methylation is pre-sented for each DMR in Supplementary Tables S1 and S2 with alog fold change indicated (exposurecontrol) A positive foldchange is an increase in DNA methylation and negative value adecrease in DNA methylation For the DDT lineage Sertoli cellDMRs 70 had an increased log fold change in methylationwhile 30 had a decrease in methylation For the vinclozolinlineage Sertoli cell DMRs 41 increased and 59 decreased inmethylation The MeDIP procedure does not identify individualCpG site DNA methylation changes but the mean change forthe DMR regions The majority of the DMRs had 1ndash3 CpGs per100 bp and were 1 kb in length so the analysis provides a meanchange across the CpGs within the DMR

The chromosomal locations of the DDT and vinclozolintransgenerational affected Sertoli cell DMRs are presented inFig 3 The DDT Sertoli cell DMRs are presented with the 2 win-dows number in Fig 3a due to the inability to present the verylarge all window number of DMRs The vinclozolin Sertoli cellDMRs for all windows are presented in Fig 3b All chromosomescontain DMRs except the Y chromosome in the vinclozolinSertoli cell lineage Further analysis of the DMRs identified thepercentage of intergenic gene-associated and repeat element-associated DMRs in regard to chromosomal locations For DDTDMRs 49 were apparently intergenic 51 were associatedwith genes and 84 were associated with a short lt100 bp re-peat element within the full DMR length For vinclozolin DMRs50 were intergenic 50 associated with genes and 80 asso-ciated with a short lt100 bp repeat elements within the DMRregion

The numbers of DMRs at different CpG densities (CpG per100 bp) covering all DMRs at a P-value of Plt 1e06 for DDT(Supplementary Fig S2A) and vinclozolin (Supplementary FigS2C) demonstrate a density of 1ndash4 CpG per 100 bp over the entireDMR length (eg 1ndash2 kb) The predominant density is 1 CpG per100 bp which is a low density CpG region across the DMR totallength and has previously been termed a CpG desert [68]

The length of the DMRs are presented for DDT lineage DMRs inSupplementary Fig S2B and vinclozolin lineage DMRs inSupplementary Fig S2D The DMR length is assessed by extend-ing the 100 bp window at Plt 1e06 out at 100 bp intervals untilthe Plt 005 stringency is lost as described in the Methods Thepredominant DMR length is 1 kb and all of them cover a rangeof 1ndash5 kb The numbers of DMRs beyond 5 kb DMR length arefew but some DDT lineage DMRs appear at 10 kb DMR lengthSimilar observations have been made in previous studies usingDDT and vinclozolin lineage sperm DMRs [55 56]

Sertoli Cell Coding and Noncoding RNA Analysis

Expression profiles of Sertoli cell noncoding RNAs both longand small were determined using RNA-Seq and the differentialexpression was analysed between the control and vinclozolin orDDT exposure lineages A variety of P-values were used to deter-mine a significance threshold and Plt 1e04 was used for

Figure 2 transgenerationally affected Sertoli cell DMRs The number of DMRs

found using different P-value cutoff thresholds The All Window column shows

all DMRs The Multiple Window column shows the number of DMRs containing

at least two significant windows The number of DMRs with different P-value

thresholds presented and the P-value threshold of 1e06 highlighted (a) DDT

lineage F3 generation Sertoli cells (b) Vinclozolin lineage F3 generation Sertoli

cells (c) Venn diagram of the DDT and vinclozolin lineage DMRs at P-value

1e06


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Figure 3 Sertoli cell DMR and sncRNA chromosomal locations (a) The DMR locations on the individual chromosomes Multiple window DDT lineage DMRs at a P-value

threshold of 1e06 are shown here (b) The DMR locations on the individual chromosomes All vinclozolin lineage DMRs at a P-value threshold of 1e06 are shown

here The red arrowheads identify the DMR sites and the black boxes the clusters of DMRs Chromosomal locations of differentially expressed small noncoding RNAs

(sncRNAs) (c) Vinclozolin lineage differentially expressed sncRNAs while 220 sncRNAs have an unknown location and are not shown (d) DDT lineage differentially

expressed sncRNAs while 31 sncRNAs have an unknown location and are not shown Black boxes represent clusters while red arrowheads represent individual

sncRNAs Plt1 x 104


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subsequent analyses (Fig 4) The vinclozolin lineage containedthe highest number of differentially expressed mRNAs com-pared to the DDT lineage at 1705 and 1050 mRNAs respectively(Fig 4a) The differentially expressed long noncoding RNAs(lncRNAs) were almost half that number while the sncRNAsdisplayed extensive differential expression The vinclozolin lin-eage contained 2920 differentially expressed sncRNAs whilethe DDT lineage had 489 These sncRNAs were separated intosncRNA categories by type and both lineages had piRNAs as themost affected small noncoding RNA class (Fig 4b) The increaseor decrease in RNA expression is presented in SupplementaryTables S4ndashS12 with a log fold change indicated for each differen-tially expressed RNA The DDT differential expressed RNA hadfor mRNA a 49 increase and 51 decrease for lncRNA a 16increase and 84 decrease and for sncRNA 62 increase and38 decrease expression The vinclozolin differential expressedRNA had for mRNA a 57 increase and 43 decrease forlncRNA a 13 increase and 87 decrease and for sncRNA a 69increase and 31 decrease in expression

The differentially expressed RNAs were mapped to theirchromosomal locations and the differentially expressedlncRNAs and mRNAs were found on all chromosomes exceptfor the mitochondrial genome (Fig 5andashd) In addition only theDDT lineage showed a differentially expressed mRNA on the Ychromosome (Fig 5d) The sncRNAs were found on the majorityof chromosomes in both lineages The vinclozolin lineage haddifferential expression on all chromosomes except for the Y

chromosome while the DDT lineage had differential expressionon all chromosomes except for the Y chromosome and chromo-some 16 (Fig 3c and d) The widespread chromosomal locationsof these altered RNAs indicate that ancestral exposure to an en-vironmental toxicant can have genome-wide effects Finally anoverlap between DMRs and differentially expressed RNAs isshown as a Venn diagram (Fig 6) In the vinclozolin lineageonly three mRNAs are linked to the DMRs while the differen-tially expressed mRNAs only overlapped with two differentiallyexpressed lncRNAs These weak links suggest a lack of associa-tions among DMRs and altered RNA expression profiles (Fig 6a)The sncRNAs displayed a sizeable overlap with both the mRNAsand lncRNAs with two sncRNAs being common to both mRNAand lncRNA suggesting a potential causative relationshipamong lncRNAs sncRNAs and mRNAs This is in contrast tothe DDT lineage which had the most overlap between theDMRs and mRNAs as well as lncRNAs In addition there wasalso some overlap between the sncRNAs and the mRNAs orlncRNAs (Fig 6b) These observations support the proposal thatthe exposure-induced epigenetic transgenerational inheritanceinvolves both DMRs and ncRNAs

Sertoli Cell Gene Association Analysis

DMRs for both DDT and vinclozolin F3 generation lineage Sertolicells were associated with genes that were within 10 kb distance(ie include promoter regions) These DMR-associated genes arepresented in Supplementary Tables S1 and S2 and sorted intogene categories in Fig 7a and b The overlapping DMR-associated genes between the DDT and vinclozolin F3 genera-tion lineage Sertoli cells are presented in Supplementary TableS3 The main categories for the DDT Sertoli DMR-associatedgenes are signaling transcription metabolism and receptorwhile for the vinclozolin lineage they are signaling metabolismand transcription The differential expressed RNAs for bothDDT and vinclozolin F3 generation lineage Sertoli cells are pre-sented in Supplementary Tables S4ndashS11 and are sorted by genecategories in Fig 7c and d The main gene categories for bothDDT and vinclozolin altered mRNAs are metabolism signalingtranscription cytoskeleton and receptor The DMR- and mRNA-associated genes (Supplementary Tables S1 S2 S10 S11) wereanalysed with a KEGG pathway analysis and the top gene path-ways are presented in Supplementary Fig S3 Six gene path-ways were in common between the vinclozolin DMR-associatedgene pathways (Supplementary Fig S3A) and DDT DMR-associated gene pathways (Supplementary Fig S3B) They arethe cAMP signaling pathway endocytosis viral carcinogenesispathways in cancer MAPK signaling pathway and Ras signalingpathway Fourteen pathways were in common betweenthe vinclozolin and DDT mRNA-associated gene pathways(Supplementary Fig S3C and D)

Sertoli Cell Testis and Infertility Pathology Associations

Pyruvate and lactate produced by Sertoli cells are essential en-ergy metabolites for spermatogenic cells sequestered within thebloodndashtestis barrier A previous study identified the pyruvatepathway [69 70] to be associated with vinclozolin-inducedSertoli cell transgenerational DMRs [8] In the current study thepyruvate pathway was also found to be affected by both theDDT and vinclozolin Sertoli cell DMR-associated genes andmRNA as shown in Fig 8 As shown the potentially alteredDMR-associated genes and RNA may influence pyruvate

Figure 4 differentially expressed RNAs (a) The number of differentially

expressed mRNAs long noncoding RNAs (lncRNAs) and small noncoding RNAs

(sncRNAs) at different P-value thresholds for vinclozolin (Vin) and DDT lineage

F3 generation Sertoli cells (b) Differentially expressed sncRNA gene categories

at Plt1 x 104


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production which would directly impact spermatogenesis inthe testis

The Sertoli cell DMRs and altered mRNAs were examined forpotential overlap with previously identified genes shown to be

involved with Sertoli cell testis and infertility pathology Thegenes previously identified to be associated with abnormal tes-tis pathology were obtained from several exhaustive reviews[71ndash74] A list of all 362 genes is presented in Supplementary

Figure 5 chromosomal locations of differentially expressed lncRNAs and mRNAs (a) Vinclozolin lncRNA locations and (c) mRNA locations (b) DDT lncRNAs and (d)

mRNA locations Black boxes represent clusters while red arrowheads represent individual RNAs Plt1 x 104


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Table S13 The transgenerationally altered ncRNA had limitedgene associations and no overlap observed The Sertoli cellDMR-associated genes and mRNAs were compared and the listsof overlapping pathology associated genes presented inSupplementary Fig S4 These associated genes were categorizedas Sertoli cell testis or male infertility linked pathology genes inFig 9c The vinclozolin DMR-associated genes were not found tooverlap with the known pathology genes but the altered mRNAdid contain overlap with the pathology genes (Fig 9a and b) Incontrast the DDT DMR-associated genes had 40 overlaps(Fig 9a and Supplementary Fig S4C) and mRNAs had 28 over-laps (Supplementary Fig S4B) Interestingly a large percentageof the DDT and vinclozolin pathology genes were in common(Supplementary Fig S4D) The DDT mRNA overlaps were dis-tinct from the DMR overlaps The DDT and vinclozolin mRNAshad Sertoli cell genes such as Daz1 Stra8 Crem Sox8 and DMRTgenes involved The DDT DMR overlaps had Adam2 Tex14Acur1 and Csf1 genes involved Therefore the DDT lineageSertoli cell DMR-associated genes and altered mRNA involved anumber of genes previously shown to be involved in Sertoli celltestis and infertility pathology (Fig 9c)

Discussion

The experimental design used the IP injection of a gestating fe-male rat to high environmentally relevant dose exposures forDDT and vinclozolin [25 26] Generally an oral exposure to alower dose would be used for risk assessment analysis The cur-rent study was not designed for risk assessment but to furtherinvestigate the transgenerational phenomenon The observa-tions demonstrate an environmental exposure can promote the

epigenetic transgenerational inheritance of disease susceptibil-ity in the testis through molecular alterations in the Sertolicells This should not be considered a direct exposure risk as-sessment study but further elucidates the epigenetic transge-nerational impacts of ancestral environmental exposures

Environmental toxicants such as DDT and vinclozolin havebeen shown to promote the epigenetic transgenerational inheri-tance of adult onset disease [59] Epigenetic transgenerationalinheritance starts with epigenetic modifications in the germlinethrough exposure to environmental factors which can be trans-mitted through the sperm and egg to subsequent generationsThis germline transmission potentially leads to an altered epi-genome in the stem cell of the embryo and subsequently all so-matic cells and tissues [6] The altered epigenomes appear toinfluence the susceptibility to disease later in life [12] Thisstudy investigates the transgenerational effects of DDT and vin-clozolin on the Sertoli cell epigenome and transcriptome Theseand a number of other environmental toxicants have beenshown previously to affect testis health and spermatogenesis[5 59] In the current study DDT and vinclozolin were bothfound to promote a transgenerational testis pathology fre-quency of approximately 45 of the male populations exam-ined compared to 8 in the control population (Fig 1a) Thiscompares with the 25ndash35 testis pathology frequency inducedby several other toxicants as observed in previous studies(Fig 1b) These studies showed the impacts of plastics (bisphe-nol A and phthalates) [22 41] dioxin [24] pesticides (permethrinand DEET) [27] and hydrocarbons (jet fuel) [28] on the inductionof transgenerational testis pathology (Fig 1b) The testis diseasefrequency in these previous studies ranged between 20 and 35compared to 13 in the independent control The dramatic de-cline in human sperm concentrations of 50 over the past50 years and corresponding increase in male infertility [53] sug-gest an environmental and generational aspect to male testispathology should be considered The current study wasdesigned to examine a molecular mechanism for this potentialenvironmental and generational origin of male infertility

Sertoli cells are an essential somatic cell population in thetestis supporting spermatogenesis nutritionally and structur-ally Although other testis somatic cell types such as Leydigcells and peritubular cells have critical functions the Sertoli cellis the most integrated with spermatogenesis Abnormal Sertolicell function can affect testis health and fertility as has beenshown in previous studies [75] Sertoli cells from prepubertalrats (18ndash22 days old) can be isolated as a highly purified popula-tion of cells In addition at this age there is in general negligibledisease present in the animals which would be a confounder inthe study Since the altered Sertoli cell epigenome has alreadybeen programmed earlier in development analysis of the 18ndash22days old Sertoli cell will provide insights into the molecularaspects of testis disease susceptibility later in life Thereforethe late pubertal Sertoli cell is a good model to study the effectsof environmental toxicant exposure on epigenetics and transge-nerational inheritance of testis pathology The F0 generationgestating female rats were exposed to DDT or vinclozolin duringfetal days E8ndash14 of development which is the time of gonadalsex determination The offspring from each generation are bredthrough the third generation without any further exposure tothe toxicants The F3 generation is the first true transgenera-tional group since during toxicant exposure of the F0 pregnantfemale the fetus (F1 generation) as well as the fetusrsquos germline(F2 generation) are exposed [76] Since the F1 and F2 generationphenotypes and molecular actions can be due to direct expo-sure the current study focused only on transgenerational

Figure 6 Venn diagram of differentially expressed epigenetic modifications (a)

Vinclozolin lineage (b) DDT lineage


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aspects of the F3 generation The 18ndash22 days old F3 generationmales were sacrificed and Sertoli cells were isolated for prepa-ration of DNA and RNA for further study by MeDIP-Seq andRNA-Seq

The experimental design used an intercross within the line-age to allow both maternal and paternal allelic contributions forpathology and epigenetics to be considered in the transgenera-tional animals Since epigenetics involves a parent of origin al-lelic transmission an outcross to wild type animals caneliminate the paternal or maternal contribution Previous stud-ies have demonstrated the outcross of the F3 generation to theF4 generation will promote the loss or reduction of a specific pa-thology [25 26] For example pesticides induced pathology inan outcrossed F4 generation demonstrated that the paternal al-lelic outcross promoted female obesity while the maternal out-cross promoted male obesity [25 26] Since biological

populations are generally an intercross within a specific ex-posed population and not a true outcross the intercross also isconsidered a more normal biology Therefore the current studyfocused on the F3 generation from an intercross However fu-ture studies need to investigate the impacts of an outcross toassess sex specific allelic transmission

Transgenerationally Affected Sertoli Cell DMR Analysis

DMRs were found for both the F3 generation DDT and vinclozo-lin lineages through read depth comparisons between controland DDT or vinclozolin sequencing data Although a potentiallimitation is the use of three pools of 6ndash11 animals each versusindividual animal analysis the statistical analysis with anedgeR P-value lt1e06 and FDR lt005 suggests there is a lowvariability in the data and that analysis of pooled samples yields

Figure 7 gene category frequency plots (a) DDT Sertoli cell DMR and (b) vinclozolin Sertoli cell DMR gene categories (c) DDT Sertoli cell mRNA and (d) vinclozolin

Sertoli cell mRNA gene categories The categories and numbers of DMRs or mRNAs are presented


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adequate statistical power The number of DMRs found in theDDT lineage F3 generation Sertoli cells is much higher than thenumber of DMRs found in the vinclozolin lineage at the same P-value lt1e06 The numbers are 5359 for DDT versus 122 for vin-clozolin Although it is uncertain why DDT DMRs are higher innumber the estrogenic actions of DDT versus the anti-androgenic actions of vinclozolin may be a factor to considerThe developing male fetus may be particularly sensitive to DDTpromoting a more profound effect on the sperm epigenome andmale somatic cells like Sertoli cells

The chromosomal distribution for both exposures is quiteeven with DMRs on all chromosomes for the DDT lineage andon all except Y for the vinclozolin lineage The Y chromosomecontains large regions of repeat elements that are highly

methylated as heterochromatin so they are anticipated to notbe sensitive to DNA methylation alteration Previous studieshave demonstrated that the CpG density of the DMRs is 1ndash2 CpGper 100 bp within the entire 1ndash2 kb DMR which is considered aCpG desert [68] Very few DMRs have a density of 3 and 4 per 100bps with close to zero for even higher CpG densities This is con-sistent with the observation that genes subject to epigenetictransgenerational inheritance are mainly regulated via ldquoCpGdesertsrdquo whereas high density CpG regions ldquoCpG islandsrdquo havea housekeeping or maintenance role in tissue-specific gene reg-ulation Gene associations for the DMRs found in the F3 genera-tion DDT and vinclozolin lineage Sertoli cells were determinedand the genes were sorted into categories Signaling transcrip-tion and metabolism were the main gene categories observed

Figure 8 KEGG pathway pyruvate metabolism [69 70] The genes correlated with altered DMR and mRNA are indicated in the insert legend with the colored circled

genes


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ctober 2019

Transgenerationally Affected Sertoli Cell ncRNAAnalysis

Being derived from the noncoding portion of the genome non-coding RNAs were once considered to be ldquojunk RNArdquo TheseRNAs are transcribed but are not protein coding and can be di-vided into two categories of long (gt200 nt) and small (lt200 nt)ncRNA It has since been observed that long noncoding RNAsare differentially expressed in certain diseases such as cancer[77] and after ancestral exposure to the environmental toxicantDDT [55] This led to the proposal that ncRNAs may actually befunctional and may maintain epigenetic memory using post-transcriptional mechanisms In addition lncRNAs can regulateDNA methylation remodel chromatin and regulate histonemodifications [61] Small noncoding RNAs have been shown tobe differentially expressed throughout spermatogenesis [78]such that their expression is regulated At estimates of over 20000 long and small ncRNAs in a single spermatozoon [79 80]the impact of these noncoding RNAs on any offspring is poten-tially very significant Observations indicate differential

expression of ncRNA is a component of transgenerationally af-fected Sertoli cell functions

Transgenerationally Affected Sertoli Cell TranscriptomeAnalysis

The vinclozolin and DDT lineages both displayed differentialcoding and noncoding RNA expression profiles Interestinglythe vinclozolin lineage had more differentially expressed RNAsin all categories including sncRNAs lncRNAs and mRNAs Themost significant difference was found in the sncRNAs categorywith the vinclozolin lineage having 2920 altered sncRNAs andDDT having only 489 sncRNAs When the sncRNAs were sepa-rated by small ncRNA categories the pattern between the vin-clozolin and DDT lineages was surprisingly similar Bothoverwhelmingly had piRNAs as the most differentially alteredclass followed by miRNAs Chromosomal locations of the dif-ferentially expressed RNAs indicated that although the majorityof the genome was affected by differential expression some of

Figure 9 Sertoli cell testis and infertility-associated genes The previously identified pathology-associated genes [71ndash74] in Supplementary Table S13 were compared to

the Sertoli cell DMR and mRNA-associated genes (a) DMR-associated gene overlap with pathology genes (b) mRNA overlap with pathology genes (c) Testis disease as-

sociated genes within the gene lists in Supplementary Fig S4 in the overlapping associated genes Plt1e05 The various testis disease parameters have the associated

genes linked


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the clusters were in similar chromosomal locations by type ofdifferentially expressed large RNAs between the different line-ages The ncRNAs are anticipated to play a critical role in theepigenetic transgenerational inheritance of testis pathologiesThe similarities of the DDT and vinclozolin transgenerationaldifferentially expressed ncRNAs and mRNAs were dramaticand future studies should further investigate the similarities ofthe exposure lineage transgenerational effects on the Sertolicells

An analysis of overlapping differential epigenetic modifica-tions between the vinclozolin and DDT lineages revealed simi-larities in some RNA expression For the vinclozolin lineage thesmall noncoding RNAs had a substantial overlap with both themRNAs with 77 and the lncRNAs with 347 In addition out ofthe 122 DMRs in the vinclozolin Sertoli cells only 3 overlappedwith any RNAs specifically the mRNA In contrast the DDT lin-eage Sertoli cells had a greater overlap of 84 of the DMRs withthe mRNAs In addition the DMRs had a significant overlapwith the lncRNAs with 26 The sncRNAs also overlapped withthe lncRNAs and the mRNAs indicating that although theDMRs may be an effector of altered RNA expression thesncRNAs may also play a part Observations suggest all differen-tially expressed epimutations may affect gene expression in anintegrated manner

Analysis of the vinclozolin and DDT actions on the transge-nerational mRNA expression demonstrates a significant overlapof the altered gene expression and nearly all the major genepathways identified for both were the same (SupplementaryFig S3) Interestingly similar KEGG pathways were identified inall the epimutation data sets with metabolism pathways incancer cAMP signaling pathway P13K-Act signaling pathwayand MAPK pathway in common The pyruvate metabolismpathway that is essential for spermatogenesis through the pro-duction of pyruvate and lactate by Sertoli cells also appears tobe transgenerationally impacted (Fig 8) as previously described[8]

Transgenerational Epimutation Associations withSertoli Cell Testis and Infertility Pathology

As previously described for vinclozolin lineage animals [8] thepyruvate metabolism pathway (Fig 8) was affected in the DDTand vinclozolin lineage animals with both the DMR and mRNA-associated genes This suggests a deficiency in an energy sourcefor the developing germ cells and correlates with the spermato-genic cell apoptosis and testis pathology observed (Fig 1) [8]Since the primary energy source (metabolite) for developingspermatogenic cells is pyruvate or lactate produced by Sertolicells alterations would directly impact germ cell survival

Previous studies have identified a large number of genes as-sociated with Sertoli cell testis and male pathology andinfertility (Supplementary Table S13) This list of genes wasobtained from several exhaustive reviews [71ndash74] The previ-ously identified pathology-associated genes were compared tothe current study epimutation-associated genes for DMR anddifferentially expressed mRNA A number of interesting over-laps were observed for the DDT- and vinclozolin-associatedgenes (Fig 9 and Supplementary Fig S4) Interestingly 20 of thegenes that overlapped with the pathology genes were the samebetween the DDT and vinclozolin altered mRNA These includedGata4 Sox8 Crem Dmrt1 and Inha This supports the conclusionthat the transgenerational epigenetic impacts on the Sertolicells may be a component of the testis pathology observed

Conclusions

A number of environmental toxicants have been shown to pro-mote the epigenetic transgenerational inheritance of testis dis-ease and male infertility [5 8 37 81] This involves epigeneticalterations in the germline (eg sperm) to impact the early em-bryonic stem cells epigenome and transcriptome Subsequentlyall cell types and tissues will have altered epigenomes and tran-scriptomes The current study demonstrates that the transge-nerationally affected Sertoli cells following ancestral vinclozolinor DDT exposure have alterations in DMRs ncRNA and mRNAThe correlations in the DMR ncRNA and mRNA support abnor-mal Sertoli cell functions that can impact spermatogenic celldevelopment and subsequent male infertility Since the currenthuman male population has a dramatic increase in infertilityand a decrease in sperm numbers [44 50ndash53] observations sug-gest ancestral exposures promoting the epigenetic transgenera-tional inheritance of testis disease may be an importantcomponent of the etiology of male infertility Therefore ances-tral exposures and generational impacts on male infertilityneed to be seriously considered in testis disease etiology

MethodsAnimal Studies and Breeding

Female and male rats of an outbred strain Hsd Sprague DawleySDVR TM (Harlan) at about 70 and 100 days of age were fed ad libwith a standard rat diet and received ad lib tap water for drink-ing To obtain time-pregnant females the female rats in proes-trus were pair-mated with male rats The sperm-positive (day 0)rats were monitored for diestrus and body weight On days8 through 14 of gestation [82] the females received daily intra-peritoneal injections of vinclozolin (100 mgkg BWday) DDT(25 mgkg BWday) or DMSO The doses of DDT and vinclozolinused are anticipated environmental exposure [83 84] The vin-clozolin and DDT were obtained from Chem Service Inc (WestChester PA) and were injected in a 20 ml DMSO vehicle as previ-ously described [24 81] Treatment lineages are designatedldquocontrolrdquo ldquoDDTrdquo or ldquovinclozolinrdquo lineages The treated gestatingfemale rats were designated as the F0 generation The offspringof the F0 generation rats were the F1 generation Non-littermatefemales and males aged 70ndash90 days from the F1 generation ofcontrol DDT or vinclozolin lineages were bred to obtain F2 gen-eration offspring The F2 generation rats were bred to obtain F3generation offspring F3 generation males were euthanized at18ndash22 days for testis collection and Sertoli cell isolation or at1 year of age for testis pathology analysis The F1ndashF3 generationoffspring were not themselves treated directly with vinclozolinor DDT The control DDT and vinclozolin lineages were housedin the same room and racks with lighting food and water aspreviously described [24 59 81] The diet consisted of freechoice Teklad 2020X rodent chow (Envigo Inc) All experimentalprotocols for the procedures with rats were pre-approved by theWashington State University Animal Care and Use Committee(IACUC approval 6252)

Testis Pathology Analysis

The Washington Animal Disease Diagnostic Laboratory(WADDL) at the Washington State University College ofVeterinary Medicine has board certified veterinary pathologiststhat assisted in initially establishing the criteria for the pathol-ogy analyses and identifying testis parameters to assess [9]


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WADDL performed full necropsies as required on animals thatdied prior to the time of scheduled sacrifice at one year andperformed tumor classifications in the current study

Testis were evaluated for pathologies as previously de-scribed [24] Testis histopathology criteria included the presenceof (i) Atrophic seminiferous tubules showing reduced Sertoliand germ cells (ii) Round smooth-edged basally locatedvacuoles in the seminiferous epithelium (iii) Sloughed sperma-togenic cells in the tubule lumen Representative pathologiesare presented in Supplementary Fig S1 Testis sections werealso examined by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (In situ celldeath detection kit Fluorescein Sigma St Louis MO) A cut-offwas established to declare a tissue ldquodiseasedrdquo based on themean number of histopathological abnormalities of each type(atrophy vacuoles sloughed cells in lumen or TUNEL positive)plus two standard deviations from the mean of control tissuesby each of the three individual evaluators blinded to the treat-ment groups This number was used to classify rats into thosewith and without testis disease in each lineage Two of threeevaluators would have to score an animalrsquos testes as having anabove-cutoff quantity of a specific histopathological abnormal-ity (atrophy vacuoles or sloughed cells in lumen) in order for arat tissue section to finally be declared ldquodiseasedrdquo

Testis Tissue Collection and Isolation of Sertoli Cells

The 20-day-old rats were sacrificed and the testes were dis-sected Sertoli cells were prepared as described in Tung et al[85] Briefly testes were washed two times with HBSS The tu-nica albuginia was removed from each testis and discarded Thetissue was then chopped with a razor blade until it appeared ho-mogeneous and then underwent sequential enzymatic diges-tion and wash steps to isolate the purified Sertoli cells The firststep uses trypsin (025) and DNase (03 mgml) followed by atrypsin inhibitor (03 mgml) incubation and washes The nextdigestion step involves collagenase (05 mgml) and DNase fol-lowed by the final digestion with hyaluronidase (1 mgml) andDNase The final pellet will contain the purified Sertoli cellswhich can be used for isolation of DNA and RNA Enzymes usedwere DNase I (Sigma DN-25) Collagenase (Sigma C1639)Hyaluronidase (Sigma H3757) and Trypsin Inhibitor Type I-S(Sigma T-6522) The purity of the Sertoli cell populations areroutinely monitored with microscopy and has been previouslyestablished with fibronectin immunocytochemistry to be gt98pure as described [8 63ndash65]

DNA and RNA Isolation

Sertoli cell pellets were resuspended in 100 ll 1 PBS then com-bined with 820 ll DNA extraction buffer (50 mM Tris HCl 10 mMEDTA pH 80 05 SDS) and 80 ll proteinase K (20 mgml) Thesample was incubated at 55C for at least 2 hours under con-stant rotation Then 300 ll of protein precipitation solution(Promega A7953) was added the sample mixed thoroughly andincubated for 15 minutes on ice The sample was centrifuged at14 000 rpm for 30 minutes at 4C One ml of the supernatant wastransferred to a 2 ml tube and 2 ll of glycoblue and 1 ml of cold100 isopropanol were added The sample was mixed well byinverting the tube several times then left in 20C freezer for atleast one hour After precipitation the sample was centrifugedat 14 000 rpm for 20 minutes at 4C The supernatant was takenoff and discarded without disturbing the (blue) pellet The pelletwas washed with 70 cold ethanol and centrifuged for

10 minutes at 4C at 14 000 rpm and the supernatant was dis-carded The tube was spun again briefly to collect residual etha-nol to bottom of tube and then as much liquid as possible wasremoved with gel loading tip Pellet was air-dried at RT until itlooked dry (about 5 minutes) Pellet was then resuspended in100 ll of nuclease free water

Total RNA Isolation

Trizol reagent (Thermo Fisher) or mirVana miRNA isolation kit(Life Technologies) was used to extract total RNA from purifiedSertoli cells with some modifications to the manufacturerrsquos pro-tocol The mirVana kit was used to extract control lineageSertoli cells stored as pellets at 80C until use Lysis buffer wasadded to the cell pellets which were heated to 65C for10 minutes and manually homogenized The default protocolwas used for the remainder of the extraction The vinclozolinand DDT lineage cells were stored in 12 ml Trizol at 80C untiluse To recover small RNA at the RNA precipitation step theamount of isopropanol was increased to 1 ml then the defaultprotocol was resumed All RNA was eluted in 50 ll of water with05 ll murine RNase inhibitor (NEB) RNA concentration was de-termined using the Qubit RNA HS Assay Kit (Thermo Fisher) AnRNA 6000 Pico chip was run on the Agilent 2100 Bioanalyzer forquality control analysis

Methylated DNA Immunoprecipitation (MeDIP)

MeDIP with genomic DNA was performed as follows DNA iso-lated from Sertoli cell pools (three pools each of control DDTand vinclozolin lineage animals) was sonicated using theCovaris M220 the following way the genomic DNA was dilutedto 130 ll with TE buffer into the appropriate Covaris tubeCovaris was set to 300 bp program and the program was run foreach tube in the experiment About 10 ll of each sonicated DNAwas run on 15 agarose gel to verify fragment size The soni-cated DNA was transferred from the Covaris tube to a 17 mlmicrofuge tube and the volume was measured The sonicatedDNA was then diluted with TE buffer (10 mM Tris HCl pH 751 mM EDTA) to 400 ll heat-denatured for 10 minutes at 95Cthen immediately cooled on ice for 10 minutes Then 100 ll of5 IP buffer and 5 lg of antibody (monoclonal mouse anti 5-methyl cytidine Diagenode C15200006) were added to the de-natured sonicated DNA The DNA-antibody mixture was incu-bated overnight on a rotator at 4C The following day magneticbeads (Dynabeads M-280 Sheep anti-Mouse IgG 11201 D) werepre-washed as follows The beads were resuspended in the vialthen the appropriate volume (50 ll per sample) was transferredto a microfuge tube The same volume of Washing Buffer (atleast 1 ml 1 PBS with 01 BSA and 2 mM EDTA) was addedand the bead sample was resuspended Tube was then placedinto a magnetic rack for 1ndash2 minutes and the supernatant wasdiscarded The tube was removed from the magnetic rack andthe beads were washed once The washed beads wereresuspended in the same volume of 1 IP buffer (50 mM sodiumphosphate pH 70 700 mM NaCl 025 TritonX-100) as the initialvolume of beads 50 ll of beads were added to the 500 ll of DNAndashantibody mixture from the overnight incubation then incu-bated for 2 hours on a rotator at 4C After the incubation thebeadndashantibodyndashDNA complex was washed three times with 1IP buffer as follows The tube was placed into magnetic rack for1ndash2 minutes and the supernatant discarded then washed with1 IP buffer three times The washed beadndashDNA solution is thenresuspended in 250 ll digestion buffer with 35 ll Proteinase K


Dow




ctober 2019

(20 mgml) The sample was then incubated for 2ndash3 hours on arotator at 55C and then 250 ll of buffered phenolndashchloroformndashisoamylalcohol solution was added to the supe and the tubewas vortexed for 30 seconds then centrifuged at 14 000 rpm for5 minutes at room temperature The aqueous supernatant wascarefully removed and transferred to a fresh microfuge tubeThen 250 ll chloroform were added to the supernatant from theprevious step vortexed for 30 seconds and centrifuged at14 000 rpm for 5 minutes at room temperature The aqueous su-pernatant was removed and transferred to a fresh microfugetube To the supernatant 2 ll of glycoblue (20 mgml) 20 ll of 5 MNaCl and 500 ll ethanol were added and mixed well then pre-cipitated in 20C freezer for 1 hour to overnight The precipi-tate was centrifuged at 14 000 rpm for 20 minutes at 4C and thesupernatant was removed while not disturbing the pellet Thepellet was washed with 500 ll cold 70 ethanol in 20C freezerfor 15 minutes then centrifuged again at 14 000 rpm for5 minutes at 4C and the supernatant was discarded The tubewas spun again briefly to collect residual ethanol to bottom oftube and as much liquid as possible was removed with gel load-ing tip Pellet was air-dried at RT until it looked dry (about5 minutes) then resuspended in 20 ll H2O or TE DNA concentra-tion was measured in Qubit (Life Technologies) with ssDNA kit(Molecular Probes Q10212)

MeDIP-Seq Analysis

The MeDIP DNA samples were used to create libraries for NGSusing the NEBNextVR UltraTM RNA Library Prep Kit for IlluminaVR

(NEB San Diego CA) starting at step 14 of the manufacturerrsquosprotocol to generate double stranded DNA After this step themanufacturerrsquos protocol was followed Each pool received a sep-arate index primer NGS was performed at WSU SpokaneGenomics Core using the Illumina HiSeq 2500 with a PE50 appli-cation with a read size of approximately 50 bp and approxi-mately 100 million reads per pool Five to six libraries were runin one lane MeDIP analysis data validation has previously beenshown with mass-spectrometry sequencing and bisulfite se-quencing [84 86]

Statistics and Bioinformatics

The basic read quality was verified using summaries producedby the FastQC [Simon Andrews httpwwwbioinformaticsbabrahamacukprojectsfastqc] program The new data wascleaned and filtered to remove adapters and low quality basesusing Trimmomatic [87] The reads for each MeDIP sample weremapped to the Rnor 60 rat genome using Bowtie2 [88] with de-fault parameter options The mapped read files were then con-verted to sorted BAM files using SAMtools [89] To identifyDMRs the reference genome was broken into 100 bp windowsThe MEDIPS R package [90] was used to calculate differentialcoverage between control and exposure sample groups TheedgeR P-value [91] was used to determine the relative differencebetween the two groups for each genomic window Windowswith an edgeR P-value less than an arbitrarily selected thresholdwere considered DMRs The DMR edges were extended until nogenomic window with an edgeR P-value less than 01 remainedwithin 1000 bp of the DMR CpG density and other informationwas then calculated for the DMR based on the reference ge-nome Since sequencing reads overlap for the entire DMR lengthsome DMR with CpGs outside the DMR length are listed as 0CpG but when flanking regions are considered contain CpG

This allowed the DMR to be precipitated with the antibody inthe MeDIP procedure

DMRs were annotated using the biomaRt R package [92] toaccess the Ensembl database [93] The genes that overlappedwith DMR were then input into the KEGG pathway search [6970] to identify associated pathways The DMR-associated geneswere manually then sorted into functional groups by consultinginformation provided by the DAVID [94] Panther [95] andUniprot databases incorporated into an internal curated data-base (wwwskinnerwsuedu ldquounder genomic datardquo) DMR con-taining repeat elements were identified using the RepeatMaskeroutput file provided by NCBI for the Rnor 60 reference genomeAll molecular data have been deposited into the public databaseat NCBI (GEO GSE118306 SRA PRJNA480508) and R code com-putational tools available at GitHub (httpsgithubcomskinnerlabMeDIP-seq) and wwwskinnerwsuedu

mRNA and ncRNA Analysis

KAPA RNA HyperPrep Kit with RiboErase was used to constructmRNA and lncRNA libraries following the manufacturerrsquos proto-col with the following modifications the adaptor and barcodesused were from NEBNext Multiplex Oligos for Illumina Beforethe final amplifications libraries were incubated with the USERenzyme (NEB) for 15 minutes at 37C PCR cycle number was de-termined using qPCR with the KAPA RealTime LibraryAmplification Kit KAPA Pure beads were used for size selection(200ndash700 bp) Agilent DNA High Sensitivity chips were used forquality control analysis and the Qubit dsDNA high sensitivityassay (Thermo Fisher) was used to determine concentrationLibraries were pooled and sequenced with the Illumina HiSeq4000 sequencer (PE100 sequencing)

The NEBNext Multiplex Small RNA Library Prep Set forIllumina was used to construct small RNA libraries with theNEBNext Multiplex Oligos for Illumina as barcodes KAPA Purebeads were used at 13 and 37 ratios for purification and sizeselection followed by the Pippin Prep 3 gel with marker P(Sage Science) for final size selection (115ndash160 bp) Qubit dsDNAhigh sensitivity assay (Thermo Fisher) was used to determineconcentration and the Agilent DNA High Sensitivity Chips wereused for quality control analysis Following pooling and concen-tration with 22 KAPA Pure beads libraries were sequencedwith the Illumina HiSeq 4000 sequencer (single-end 50 bp) witha customized sequencing primer 50-ACA CGT TCA GAG TTCTAC AGT CCG A-30

ncRNA Bioinformatics and Statistics

The small ncRNA data were annotated as follows low-qualityreads and reads shorter than 15 nt were discarded byTrimmomatics (v033) The remaining reads were matched toknown rat sncRNA consisting of mature miRNA (miR-Base re-lease 21) precursor miRNA (miRBase release 21) tRNA(Genomic tRNA Database rn5) piRNA (piRBase) rRNA (Ensemblrelease 76) and mitochondrial RNA (Ensembl release 76) usingAASRA pipeline with default parameters Read counts gener-ated by AASRA were statistically normalized by DESeq2

The long ncRNA and mRNA data were annotated as followstrimmomatics (v033) was used to remove adaptor sequencesand the low-quality reads from the RNA sequencing data of thelarge RNA libraries To identify all the transcripts we usedHiSAT2 (v210) and StringTie (v134d) to assemble the sequenc-ing reads based on the Ensembl_Rnor_60 The differential ex-pression analyses were performed by Cuffdiff The coding and


Dow




ctober 2019

the noncoding genes were primarily annotated through rat CDSdata ensembl_Rnor_60 The non-annotated genes wereextracted through our in-house script and then analysed byCPAT indicating the true noncoding RNAs

Ethics Statement

All experimental protocols for the procedures with rats werepre-approved by the Washington State University Animal Careand Use Committee (IACUC approval 6252)

Authors Roles

MKS conceived the study and obtained fundingMKS amp WY supervised the study ISR RK EN DB YX performedtechnical analysis and obtained the dataDB amp YX performed bioinformatics and statistical analysis ISRand RK wrote the first draft of the manuscriptAll authors edited and revised the manuscript

Acknowledgements

We acknowledge Ms Jayleana Barton Ms Hannah Kimbeland Mr Hayden McSwiggin for technical assistance MsAmanda Quilty for editorial assistance and Ms HeatherJohnson for assistance in preparation of the manuscript Wethank the Genomics Core laboratory at WSU Spokane Thiswork was supported by the NIH NIEHS under GrantES012974 The funders had no role in the conceptualizationpreparation or decision to publish this manuscript Thiswork was supported by the NIH NIEHS under GrantES012974 to MKS and John Templeton Foundation grant61174 to MKS The funders had no role in the conceptualiza-tion preparation or decision to publish this manuscript

Data Availability

All molecular data has been deposited into the public data-base at NCBI (GEO GSE118306 SRA PRJNA480508) and Rcode computational tools available at GitHub (httpsgithubcomskinnerlabMeDIP-seq) and wwwskinnerwsuedu

Supplementary Data

Supplementary data are available at EnvEpig online

Conflict of interest statement None declared

References1 Holliday R The inheritance of epigenetic defects Science 1987

238163ndash702 Berger Sl Kouzarides T Shiekhattar R Shilatifard A An oper-

ational definition of epigenetics Genes Dev 200923781ndash33 Nilsson E Sadler-Riggleman I Skinner MK Environmentally

induced epigenetic transgenerational inheritance of diseaseEnviron Epigenet 201841ndash13

4 Jirtle Rl Skinner MK Environmental epigenomics and diseasesusceptibility Nat Rev Genet 20078253ndash62

5 Anway MD Cupp AS Uzumcu M Skinner MK Epigenetictransgenerational actions of endocrine disruptors and malefertility Science 20053081466ndash9

6 Skinner MK Environmental epigenetic transgenerational in-heritance and somatic epigenetic mitotic stability Epigenetics20116838ndash42

7 Nilsson E Larsen G Manikkam M Guerrero-Bosagna CSavenkova M Skinner M Environmentally induced epige-netic transgenerational inheritance of ovarian disease PLoSOne 20127e36129

8 Guerrero-Bosagna C Savenkova M Haque MM Nilsson ESkinner MK Environmentally induced epigenetic transge-nerational inheritance of altered Sertoli cell transcriptomeand epigenome molecular etiology of male infertility PLoSOne 20138e59922

9 Anway MD Leathers C Skinner MK Endocrine disruptor vin-clozolin induced epigenetic transgenerational adult-onsetdisease Endocrinology 20061475515ndash23

10Anway MD Skinner MK Transgenerational effects of the en-docrine disruptor vinclozolin on the prostate transcriptomeand adult onset disease Prostate 200868517ndash29

11Klukovich R Nilsson E Sadler-Riggleman I Beck D Xie Y YanW Skinner MK Environmental toxicant induced epigenetictransgenerational inheritance of prostate pathology andstromal-epithelial cell epigenome and transcriptome altera-tions ancestral origins of prostate disease Sci Rep 201991ndash17

12Nilsson E Klukovich R Sadler-Riggleman I Beck D Xie Y YanW Skinner MK Environmental toxicant induced epigenetictransgenerational inheritance of ovarian pathology and gran-ulosa cell epigenome and transcriptome alterations ances-tral origins of polycystic ovarian syndrome and primaryovarian insufiency Epigenetics 201813875ndash95

13Quadrana L Colot V Plant transgenerational epigeneticsAnnu Rev Genet 201650467ndash91

14Brookheart RT Duncan JG Drosophila melanogaster anemerging model of transgenerational effects of maternal obe-sity Mol Cell Endocrinol 201643520ndash8

15Kelly WG Multigenerational chromatin marks no enzymesneed apply Dev Cell 201431142ndash4

16Carvan MJ Kalluvila TA Klingler RH Larson JK Pickens MMora-Zamorano FX Connaughton VP Sadler-Riggleman IBeck D Skinner MK Mercury-induced epigenetic transge-nerational inheritance of abnormal neurobehavior is corre-lated with sperm epimutations in zebrafish PLoS One 201712e0176155

17Leroux S Gourichon D Leterrier C Labrune Y Coustham VRiviere S Zerjal T Coville Jl Morisson M Minvielle F et alEmbryonic environment and transgenerational effects inquail Genet Sel Evol 20174914

18Guerrero-Bosagna C Morisson M Liaubet L Rodenburg TB deHaas EN Kostal L Pitel F Transgenerational epigenetic inher-itance in birds Environ Epigenet 20184dvy008

19Padmanabhan N Jia D Geary-Joo C Wu X Ferguson-SmithAC Fung E Bieda MC Snyder FF Gravel RA Cross JC et alMutation in folate metabolism causes epigenetic instabilityand transgenerational effects on development Cell 201315581ndash93

20Braunschweig M Jagannathan V Gutzwiller A Bee GInvestigations on transgenerational epigenetic responsedown the male line in F2 pigs PLoS One 20127e30583

21Northstone K Golding J Davey Smith G Miller Ll Pembrey MPrepubertal start of fatherrsquos smoking and increased body fatin his sons further characterisation of paternal transgenera-tional responses Eur J Hum Genet 2014221382ndash6

22Manikkam M Tracey R Guerrero-Bosagna C Skinner MPlastics derived endocrine disruptors (BPA DEHP and DBP)


Dow




ctober 2019

induce epigenetic transgenerational inheritance of obesityreproductive disease and sperm epimutations PLoS One 20138e55387

23Doyle TJ Bowman JL Windell VL McLean DJ Kim KHTransgenerational effects of di-(2-ethylhexyl) phthalate ontesticular germ cell associations and spermatogonial stemcells in mice Biol Reprod 201388112

24Manikkam M Tracey R Guerrero-Bosagna C Skinner MKDioxin (TCDD) induces epigenetic transgenerational inheri-tance of adult onset disease and sperm epimutations PLoSOne 20127e46249

25Manikkam M Haque MM Guerrero-Bosagna C Nilsson ESkinner MK Pesticide methoxychlor promotes the epigenetictransgenerational inheritance of adult onset disease throughthe female germline PLoS One 20149e102091

26Skinner MK Manikkam M Tracey R Guerrero-Bosagna CHaque MM Nilsson E Ancestral dichlorodiphenyltrichloro-ethane (DDT) exposure promotes epigenetic transgenera-tional inheritance of obesity BMC Med 201311221ndash16

27Manikkam M Tracey R Guerrero-Bosagna C Skinner MPesticide and insect repellent mixture (permethrin and DEET)induces epigenetic transgenerational inheritance of diseaseand sperm epimutations Reprod Toxicol 201234708ndash19

28Tracey R Manikkam M Guerrero-Bosagna C Skinner MHydrocarbons (jet fuel JP-8) induce epigenetic transgenera-tional inheritance of obesity reproductive disease and spermepimutations Reprod Toxicol 201336104ndash16

29Bruner-Tran KL Ding T Yeoman KB Archibong A Arosh JAOsteen KG Developmental exposure of mice to dioxin pro-motes transgenerational testicular inflammation and an in-creased risk of preterm birth in unexposed mating partnersPLoS One 20149e105084

30Bruner-Tran KL Osteen KG Developmental exposure toTCDD reduces fertility and negatively affects pregnancy out-comes across multiple generations Reprod Toxicol 201131344ndash50

31McBirney M King SE Pappalardo M Houser E Unkefer MNilsson E Sadler-Riggleman I Beck D Winchester P SkinnerMK Atrazine induced epigenetic transgenerational inheri-tance of disease lean phenotype and sperm epimutation pa-thology biomarkers PLoS One 201712e0184306

32Crews D Gore AC Hsu TS Dangleben NL Spinetta MSchallert T Anway MD Skinner MK Transgenerational epi-genetic imprints on mate preference Proc Natl Acad Sci USA20071045942ndash6

33Crews D Gillette R Scarpino SV Manikkam M Savenkova MISkinner MK Epigenetic transgenerational inheritance of al-tered stress responses Proc Natl Acad Sci USA 20121099143ndash8

34Skinner MK Anway MD Savenkova MI Gore AC Crews DTransgenerational epigenetic programming of the brain tran-scriptome and anxiety behavior PLoS One 20083e3745

35 Jawaid A Roszkowski M Mansuy IM Transgenerational epi-genetics of traumatic stress Prog Mol Biol Transl Sci 2018158273ndash98

36Metz GA Ng JW Kovalchuk I Olson DM Ancestral experienceas a game changer in stress vulnerability and disease out-comes Bioessays 201537602ndash11

37Anway MD Memon MA Uzumcu M Skinner MKTransgenerational effect of the endocrine disruptor vinclozo-lin on male spermatogenesis J Androl 200627868ndash79

38Guerrero-Bosagna C Covert T Haque MM Settles M NilssonEE Anway MD Skinner MK Epigenetic transgenerational in-heritance of vinclozolin induced mouse adult onset disease

and associated sperm epigenome biomarkers Reprod Toxicol201234694ndash707

39Beck D Sadler-Riggleman I Skinner MK Generational com-parisons (F1 versus F3) of vinclozolin induced epigenetictransgenerational inheritance of sperm differential DNAmethylation regions (epimutations) using MeDIP-Seq EnvironEpigenet 201731ndash12 dvx016

40Hao C Gely-Pernot A Kervarrec C Boudjema M Becker EKhil P Tevosian S Jegou B Smagulova F Exposure to thewidely used herbicide atrazine results in deregulation ofglobal tissue-specific RNA transcription in the third genera-tion and is associated with a global decrease of histone trime-thylation in mice Nucleic Acids Res 2016449784ndash802

41Wolstenholme JT Edwards M Shetty SR Gatewood JD TaylorJA Rissman EF Connelly JJ Gestational exposure to bisphe-nol A produces transgenerational changes in behaviors andgene expression Endocrinology 20121533828ndash38

42Mohamed el SA Song WH Oh SA Park YJ You YA Lee S ChoiJY Kim YJ Jo I Pang MG The transgenerational impact ofbenzo(a)pyrene on murine male fertility Hum Reprod 2010252427ndash33

43Chamorro-Garcia R Diaz-Castillo C Shoucri BM Kach HLeavitt R Shioda T Blumberg B Ancestral perinatal obesogenexposure results in a transgenerational thrifty phenotype inmice Nat Commun 201782012

44Sanabria M Cucielo MS Guerra MT Dos Santos Borges CBanzato TP Perobelli JE Leite GA Anselmo-Franci JA DeGrava Kempinas W Sperm quality and fertility in rats afterprenatal exposure to low doses of TCDD a three-generationstudy Reprod Toxicol 20166529ndash38

45Kubsad D Nilsson EE King SE Sadler-Riggleman I Beck DSkinner MK Assessment of glyphosate induced epigenetictransgenerational inheritance of pathologies and sperm epi-mutations generational toxicology Sci Rep 201996372

46Dym M Fawcett DW The blood-testis barrier in the rat andthe physiological compartmentation of the seminiferous epi-thelium Biol Reprod 19703308ndash26

47Skinner MK Griswold MD Sertoli cells synthesize and secretetransferrin-like protein J Biol Chem 19802559523ndash5

48Griswold MD Interactions between germ cells and Sertolicells in the testis Biol Reprod 199552211ndash6

49Griswold MD Protein secretions of Sertoli cells Int Rev Cytol1988110133ndash56

50Skakkebaek NE Rajpert-De Meyts E Buck Louis Gm ToppariJ Andersson A-M Eisenberg ML Jensen TK Joslashrgensen NSwan SH Sapra KJ et al Male reproductive disorders and fer-tility trends influences of environment and genetic suscepti-bility Physiol Rev 20169655ndash97

51 Jensen TK Jacobsen R Christensen K Nielsen NC Bostofte EGood semen quality and life expectancy a cohort study of43277 men Am J Epidemiol 2009170559ndash65

52Eisenberg ML Li S Cullen MR Baker LC Increased risk of inci-dent chronic medical conditions in infertile men analysis ofUnited States claims data Fertil Steril 2016105629ndash36

53Levine H Jorgensen N Martino-Andrade A Mendiola JWeksler-Derri D Mindlis I Pinotti R Swan SH Temporaltrends in sperm count a systematic review and meta-regression analysis Hum Reprod Update 201723646ndash59

54Anway MD Rekow SS Skinner MK Comparative anti-androgenic actions of vinclozolin and flutamide on transge-nerational adult onset disease and spermatogenesis ReprodToxicol 200826100ndash6

55Skinner MK Ben Maamar M Sadler-Riggleman I Beck DNilsson E McBirney M Klukovich R Xie Y Tang C Yan W


Dow




ctober 2019

Alterations in sperm DNA methylation non-coding RNA andhistone retention associate with DDT-induced epigenetictransgenerational inheritance of disease Epigenet Chromatin2018111ndash24

56Ben Maamar M Sadler-Riggleman I Beck D McBirney MNilsson E Klukovich R Xie Y Tang C Yan W Skinner MKAlterations in sperm DNA methylation non-coding RNA ex-pression and histone retention mediate vinclozolin-inducedepigenetic transgenerational inheritance of disease EnvironEpigenet 201841ndash19 dvy010

57Kelce WR Monosson E Gamcsik MP Laws SC Gray LEnvironmental hormone disruptors evidence that vinclozo-lin developmental toxicity is mediated by antiandrogenicmetabolites Toxicol Appl Pharmacol 1994126276ndash85

58Mrema EJ Rubino FM Brambilla G Moretto A Tsatsakis AMColosio C Persistent organochlorinated pesticides and mech-anisms of their toxicity Toxicology 201330774ndash88

59Skinner MK Endocrine disruptor induction of epigenetictransgenerational inheritance of disease Mol Cell Endocrinol20143984ndash12

60Schuster A Skinner MK Yan W Ancestral vinclozolin expo-sure alters the epigenetic transgenerational inheritance ofsperm small noncoding RNAs Environ Epigenet 201621ndash10dvw001

61Yan W Potential roles of noncoding RNAs in environmentalepigenetic transgenerational inheritance Mol Cell Endocrinol201439824ndash30

62Zhao Y Sun H Wang H Long noncoding RNAs in DNA meth-ylation new players stepping into the old game Cell Biosci2016645

63Boucheron C Baxendale V Isolation and purification of mu-rine male germ cells Methods Mol Biol 201282559ndash66

64Marzowski J Sylvester SR Gilmont RR Griswold MDIsolation and characterization of Sertoli cell plasma mem-branes and associated plasminogen activator activity BiolReprod 1985321237ndash45

65Tung PS Skinner MK Fritz IB Fibronectin synthesis is amarker for peritubular cell contaminants in Sertoli cell-enriched cultures Biol Reprod 198430199ndash211

66Nilsson E King SE McBirney M Kubsad D Pappalardo MBeck D Sadler-Riggleman I Skinner MK Vinclozolin inducedepigenetic transgenerational inheritance of pathologies andsperm epimutation biomarkers for specific diseases PLoS One201813e0202662

67King SE McBirney M Beck D Sadler-Riggleman I Nilsson ESkinner MK Sperm epimutation biomarkers of obesity andpathologies following DDT induced epigenetic transgenera-tional inheritance of disease Environ Epigenet 201951ndash15dvz008

68Skinner MK Guerrero-Bosagna C Role of CpG deserts in theepigenetic transgenerational inheritance of differential DNAmethylation regions BMC Genomics 201415692

69Kanehisa M Goto S KEGG Kyoto encyclopedia of genes andgenomes Nucleic Acids Res 20002827ndash30

70Kanehisa M Goto S Sato Y Kawashima M Furumichi MTanabe M Data information knowledge andprinciple back to metabolism in KEGG Nucl Acids Res 201442D199ndash205

71Matzuk MM Lamb DJ The biology of infertility researchadvances and clinical challenges Nat Med 2008141197ndash213

72Kovac JR Pastuszak AW Lamb DJ The use of genomics pro-teomics and metabolomics in identifying biomarkers ofmale infertility Fertil Steril 201399998ndash1007

73 Jiang XH Bukhari I Zheng W Yin S Wang Z Cooke HJ ShiQH Blood-testis barrier and spermatogenesis lessons fromgenetically-modified mice Asian J Androl 201416572ndash80

74Manku G Culty M Mammalian gonocyte and spermatogoniadifferentiation recent advances and remaining challengesReproduction 2015149R139ndash157

75Sharpe RM Skakkebaek NE Testicular dysgenesis syndromemechanistic insights and potential new downstream effectsFertil Steril 200889e33ndash38

76Skinner MK What is an epigenetic transgenerational pheno-type F3 or F2 Reprod Toxicol 2008252ndash6

77Kung JT Colognori D Lee JT Long noncoding RNAs past pre-sent and future Genetics 2013193651ndash69

78Chen Q Yan W Duan E Epigenetic inheritance of acquiredtraits through sperm RNAs and sperm RNA modificationsNat Rev Genet 201617733ndash43

79Krawetz SA Kruger A Lalancette C Tagett R Anton EDraghici S Diamond MP A survey of small RNAs in humansperm Hum Reprod 2011263401ndash12

80Zhang X Gao F Fu J Zhang P Wang Y Zeng X Systematicidentification and characterization of long non-coding RNAsin mouse mature sperm PLoS One 201712e0173402

81Manikkam M Guerrero-Bosagna C Tracey R Haque MMSkinner MK Transgenerational actions of environmentalcompounds on reproductive disease and identification of epi-genetic biomarkers of ancestral exposures PLoS One 20127e31901

82Nilsson EE Anway MD Stanfield J Skinner MKTransgenerational epigenetic effects of the endocrine disrup-tor vinclozolin on pregnancies and female adult onset dis-ease Reproduction 2008135713ndash21

83Guillette LJ Jr Gross TS Masson GR Matter JM Percival HFWoodward AR Developmental abnormalities of the gonadand abnormal sex hormone concentrations in juvenile alliga-tors from contaminated and control lakes in Florida EnvironHealth Perspect 1994102680ndash8

84Guerrero-Bosagna C Settles M Lucker B Skinner MEpigenetic transgenerational actions of vinclozolin on pro-moter regions of the sperm epigenome PLoS One 20105e13100

85Tung PS Skinner MK Fritz IB Cooperativity between Sertolicells and peritubular myoid cells in the formation of the basallamina in the seminiferous tubule Ann NY Acad Sci 1984438435ndash46

86Lienhard M Grasse S Rolff J Frese S Schirmer U Becker MBorno S Timmermann B Chavez L Sultmann H et al QSEA-modelling of genome-wide DNA methylation from sequenc-ing enrichment experiments Nucleic Acids Res 201745e44

87Bolger AM Lohse M Usadel B Trimmomatic a flexible trim-mer for Illumina sequence data Bioinformatics 2014302114ndash20

88Langmead B Salzberg SL Fast gapped-read alignment withBowtie 2 Nat Methods 20129357ndash9

89Li H Handsaker B Wysoker A Fennell T Ruan J Homer NMarth G Abecasis G Durbin R 1000 Genome Project DataProcessing Sungroup The sequence alignmentmap formatand SAMtools Bioinformatics 2009252078ndash9

90Lienhard M Grimm C Morkel M Herwig R Chavez L MEDIPSgenome-wide differential coverage analysis of sequencingdata derived from DNA enrichment experimentsBioinformatics 201430284ndash6

91Robinson MD McCarthy DJ Smyth GK edgeR a Bioconductorpackage for differential expression analysis of digital geneexpression data Bioinformatics 201026139ndash40


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ctober 2019

92Durinck S Spellman PT Birney E Huber W Mapping identi-fiers for the integration of genomic datasets with the RBioconductor package biomaRt Nat Protoc 200941184ndash91

93Cunningham F Amode MR Barrell D Beal K Billis K Brent SCarvalho-Silva D Clapham P Coates G Fitzgerald S et alEnsembl 2015 Nucleic Acids Res 201543D662ndash669

94Huang da W Sherman BT Lempicki RA Systematic and inte-grative analysis of large gene lists using DAVID bioinformat-ics resources Nat Protoc 2009444ndash57

95Mi H Muruganujan A Casagrande JT Thomas PD Large-scalegene function analysis with the PANTHER classification sys-tem Nat Protoc 201381551ndash66


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alterations appear to be critical factors in the developmental and generational origins of testis pathologies and maleinfertility

Key words Sertoli cell male infertility testis pathology transgenerational vinclozolin DDT

Introduction

Epimutations were originally defined as lsquoheritable epigeneticchanges in chromosomes which do not involve changes in theDNA sequence itselfrsquo [1 2] A more updated definition of epige-netics is lsquomolecular factors and processes that regulate genomeactivity independent of DNA sequence and are mitoticallystablersquo and for epimutations is lsquoenvironmentally induced differ-ential presence of epigenetic alterations that can influence ge-nome activity compared to organisms not having the exposurersquo[3] A number of environmental factors can alter epigenetic pro-cesses such as DNA methylation histone modifications (egmethylation and acetylation) chromosome structure noncod-ing RNA (ncRNA) and RNA methylation to influence gene ex-pression and genome activity Epigenetic transgenerationalinheritance describes a germline transmission of epimutationsthrough generations without continued exposure to or presenceof the original exposure beyond the F0 generation [3] In caseswhere the germline is exposed during fetal gonadal sex deter-mination the epigenome in the germline can be modifiedThese epimutations appear to become lsquoimprinted-likersquo and po-tentially escape post-fertilization methylation erasure to allowtransmission to the subsequent generations [4 5] The hypothe-sis is that this promotes stem cells in the embryo to develop analtered epigenome and transcriptome Somatic cells and tissuesderived from these epigenetically altered germline and stemcells will potentially develop alterations in cell type-specific epi-genomes and transcriptomes [6] These cell specific altered epi-genomes will impact the fate of organs and tissues and may beearly indicators and initiators of the development of diseasesusceptibility later in life [7ndash9] Previous examples of this phe-nomenon have been observed in prostate epithelial cellsfor prostate disease [10 11] and granulosa cells for ovarian dis-ease [12]

Numerous studies have shown epigenetic transgenerationalinheritance to occur in different species involving a wide varietyof different environmental exposures Epigenetic transgenera-tional inheritance has been shown in plants [13] flies [14]worms [15] fish [16] birds [17 18] rodents [5 19] pigs [20] andhumans [21] Environmentally induced transgenerational dis-eases and abnormalities in mammals have been observed suchas testis disease [8 22 23] prostate disease [9 24] kidney dis-ease [9 24 25] obesity [26] ovarian and uterine disease [22 2527ndash30] tumor development [9] and behavioral changes [31ndash36]A large number of environmental toxicants have been shown tobe involved in triggering these transgenerational diseases andabnormalities that include the agricultural fungicide vinclozolin[5 37ndash39] the herbicide atrazine [31 40] plasticizers such asbisphenol A [22 41] and phthalates [22] insect repellant diethyl-toluamide (DEET) with the insecticide permethrin [27] pesticidemethoxychlor [25] hydrocarbons (jet fuel) [28] and industrialcompounds such as benzo[a]pyrene [42] the biocide tributyltin[43] mercury [16] dioxins [24 29 44] and the herbicide glypho-sate [45]

The current study was designed to examine the ability ofvinclozolin and dichlorodiphenyltrichloroethane (DDT) to in-duce transgenerationally affected Sertoli cell epigenome and

transcriptome alterations that associate with testis pathologySertoli cells play a critical role in spermatogenesis providingstructural and nutritional support for the developing germ cellsand are involved in the formation of the bloodndashtestis barrierwhich creates a serum and pathogen free environment for thespermatogenic cells within the seminiferous tubules [46]Therefore Sertoli cells synthesize a number of transport bind-ing proteins [47 48] and provide the primary energy metabolites(ie pyruvate and lactate) for the developing germ cells whichare sequestered within the bloodndashtestis barrier and unable toacquire glucose themselves [49] A disruption in normal Sertolicell development and function can affect spermatogenesis andpromote testis pathology Abnormal spermatogenesis and lowsperm count are often linked to infertility which can involvetestis diseases like cryptorchidism hypospadias and testicularcancer [50ndash52] Sperm counts have been declining significantlybetween 1973 and 2011 as shown in a 2017 meta-regressionanalysis [53] Environmental factors such as endocrine disrupt-ing chemicals pesticides heat diet stress and smoking havebeen shown to be associated with this sperm count drop andgeneral testis health problems [53] The molecular basis forthese testis pathologies and generational impacts are investi-gated in the current study

Exposure of gestating rats to the environmental toxicantssuch as DDT and vinclozolin leads to the epigenetic transge-nerational inheritance of adult onset diseases including testisdisease and a decrease in sperm count andor motility [26 54]Originally vinclozolinrsquos involvement in epigenetic transgenera-tional inheritance of disease was observed in 2005 by Anwayet al [5] who found that vinclozolin exposure of gestating ratsleads to epigenetic transgenerational inheritance of uniqueDNA methylation changes (epimutations) in sperm Subsequentstudies involving vinclozolin and DDT among other environ-mental toxicants confirmed these findings [26 39 55 56]Vinclozolin is an agricultural fungicide used in fruit and vegeta-ble production and is an anti-androgenic compound that acts asa competitive antagonist of the androgen receptor [57] DDT is apesticide which was widely used through the 1950s and 1960sin the USA until banned in 1972 It continues to be used in manyparts of the world for insect and malaria control DDT accumu-lates in the environment and in fatty tissue and is an estrogenreceptor agonist that has estrogenic effects in animals [58]

The epigenetic transgenerational inheritance phenomenonrequires the germline transmission of altered epigenetic infor-mation between generations [59] A variety of different environ-mental factors promoting epigenetic transgenerationalinheritance were found to induce exposure specific alterationsin sperm DNA methylation [5 59] Subsequently vinclozolinwas found to promote alterations in sperm ncRNA transgenera-tionally [60] This supported previous studies indicating ncRNAgermline alterations are important factors in epigenetic trans-generational inheritance [61 62] Recently we observed thatboth vinclozolin and DDT cause concurrent alterations in caudaepididymal rat sperm DNA methylation ncRNA and histone re-tention [55 56] Therefore several different epigenetic processesare likely integrated in epigenetic transgenerational inheri-tance A 2013 study using vinclozolin determined that


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sncRNAs Plt1 x 104


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at Plt1 x 104


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Discussion








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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Discussion








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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Discussion








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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Discussion








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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Discussion








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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Discussion








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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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ctober 2019






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genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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ctober 2019






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ctober 2019





genes


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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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genes linked


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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Conclusions







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Total RNA Isolation





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MeDIP-Seq Analysis














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Ethics Statement


Authors Roles


Acknowledgements


Data Availability


Supplementary Data



























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Total RNA Isolation





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Acknowledgements


Data Availability


Supplementary Data



























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MeDIP-Seq Analysis














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Supplementary Data



























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epigenetic transgenerational inheritance of testis …...male reproductive health has been in...

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