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Germline transmission of epigenetic information is a critical component of epigenetic inheritance. Previous studies have suggested that an erasure of DNA methylation is required to develop stem cells in the morula embryo. An exception involves imprinted genes that escape this DNA methylation erasure. Transgenerational differential DNA methylation regions (DMRs) have been speculated to be imprinted-like and escape this erasure. The current study was designed to assess if morula embryos escape the erasure of dichlorodiphenyltrichloroethane-induced transgenerational sperm DMR methylation. Observations demonstrate that the majority (98%) of transgenerational sperm DMR sites retain DNA methylation and are not erased, so appearing similar to imprinted-like sites. Interestingly, observations also demonstrate that the majority of low-density CpG genomic sites had a significant increase in DNA methylation in the morula embryo compared to sperm. This is in contrast to the previously observed DNA methylation erasure of higher-density CpG sites. The general erasure of DNA methylation during embryogenesis appears applicable to high-density DNA methylation sites (e.g. CpG islands) but neither to transgenerational DMR methylation sites nor to low-density CpG deserts, which constitute the vast majority of the genome's DNA methylation sites. The role of epigenetics during embryogenesis appears more dynamic than the simple erasure of DNA methylation., Competing Interests: The authors have declared that no competing interests exist., (© The Author(s) 2023. Published by Oxford University Press.)

Abnormal penile foreskin development in hypospadias is the most frequent genital malformation in male children, which has increased dramatically in recent decades. A number of environmental factors have been shown to be associated with hypospadias development. The current study investigated the role of epigenetics in the etiology of hypospadias and compared mild (distal), moderate (mid shaft), and severe (proximal) hypospadias. Penile foreskin samples were collected from hypospadias and non-hypospadias individuals to identify alterations in DNA methylation associated with hypospadias. Dramatic numbers of differential DNA methylation regions (DMRs) were observed in the mild hypospadias, with reduced numbers in moderate and low numbers in severe hypospadias. Atresia (cell loss) of the principal foreskin fibroblast is suspected to be a component of the disease etiology. A genome-wide (> 95%) epigenetic analysis was used and the genomic features of the DMRs identified. The DMR associated genes identified a number of novel hypospadias associated genes and pathways, as well as genes and networks known to be involved in hypospadias etiology. Observations demonstrate altered DNA methylation sites in penile foreskin is a component of hypospadias etiology. In addition, a potential role of environmental epigenetics and epigenetic inheritance in hypospadias disease etiology is suggested., (© 2023. The Author(s).)

The current study was designed to use a rodent model to determine if exposure to the chemotherapy drug ifosfamide during puberty can induce altered phenotypes and disease in the grand-offspring of exposed individuals through epigenetic transgenerational inheritance. Pathologies such as delayed pubertal onset, kidney disease, and multiple pathologies were observed to be significantly more frequent in the F1 generation offspring of ifosfamide lineage females. The F2 generation grand-offspring ifosfamide lineage males had transgenerational pathology phenotypes of early pubertal onset and reduced testis pathology. Reduced levels of anxiety were observed in both males and females in the transgenerational F2 generation grand-offspring. Differential DNA methylated regions (DMRs) in chemotherapy lineage sperm in the F1 and F2 generations were identified. Therefore, chemotherapy exposure impacts pathology susceptibility in subsequent generations. Observations highlight the importance that prior to chemotherapy, individuals need to consider cryopreservation of germ cells as a precautionary measure if they have children., Competing Interests: The authors declare no competing interests., (© 2022.)

Genome-wide DNA methylation analysis is one of the most common epigenetic processes analysed for genome characterization and differential DNA methylation assessment. Previous genome-wide analysis has suggested an important variable in DNA methylation methods involves CpG density. The current study was designed to investigate the CpG density in a variety of different species genomes and correlate this to various DNA methylation analysis data sets. The majority of all genomes had >90% of the genome in the low density 1-3 CpG/100 bp category, while <10% of the genome was in the higher density >5 CpG/100 bp category. Similar observations with human, rat, bird, and fish genomes were observed. The methylated DNA immunoprecipitation (MeDIP) procedure uses the anti-5-methylcytosine antibody immunoprecipitation followed by next-generation sequencing (MeDIP-Seq). The MeDIP procedure is biased to lower CpG density of <5 CpG/100 bp, which corresponds to >95% of the genome. The reduced representation bisulphite (RRBS) protocol generally identifies DMRs in higher CpG density regions of ≥3 CpG/100 bp which corresponds to approximately 20% of the genome. The whole-genome bisulphite (WGBS) analyses resulted in higher CpG densities, often greater than 10 CpG/100bp. WGBS generally identifies ≥2 CpG/100bp, which corresponds to approximately 50% of the genome. Limitations and potential optimization approaches for each method are discussed. None of the procedures can provide complete genome-wide assessment of the genome, but MeDIP-Seq provides coverage of the highest percentage. Observations demonstrate that CpG density is a critical variable in DNA methylation analysis, and different molecular techniques focus on distinct genomic regions.

Environmental toxicants have been shown to promote the epigenetic transgenerational inheritance of disease through exposure specific epigenetic alterations in the germline. The current study examines the actions of hydrocarbon jet fuel, dioxin, pesticides (permethrin and methoxychlor), plastics, and herbicides (glyphosate and atrazine) in the promotion of transgenerational disease in the great grand-offspring rats that correlates with specific disease associated differential DNA methylation regions (DMRs). The transgenerational disease observed was similar for all exposures and includes pathologies of the kidney, prostate, and testis, pubertal abnormalities, and obesity. The disease specific DMRs in sperm were exposure specific for each pathology with negligible overlap. Therefore, for each disease the DMRs and associated genes were distinct for each exposure generational lineage. Observations suggest a large number of DMRs and associated genes are involved in a specific pathology, and various environmental exposures influence unique subsets of DMRs and genes to promote the transgenerational developmental origins of disease susceptibility later in life. A novel multiscale systems biology basis of disease etiology is proposed involving an integration of environmental epigenetics, genetics and generational toxicology., (© 2022. The Author(s).)

Many environmental toxicants have been shown to be associated with the transgenerational inheritance of increased disease susceptibility. This review describes the generational toxicity of some of these chemicals and their role in the induction of epigenetic transgenerational inheritance of disease. Epigenetic factors include DNA methylation, histone modifications, retention of histones in sperm, changes to chromatin structure, and expression of non-coding RNAs. For toxicant-induced epigenetic transgenerational inheritance to occur, exposure to a toxicant must result in epigenetic changes to germ cells (sperm or eggs) since it is the germ cells that carry molecular information to subsequent generations. In addition, the epigenetic changes induced in transgenerational generation animals must cause alterations in gene expression in these animals' somatic cells. In some cases of generational toxicology, negligible changes are seen in the directly exposed generations, but increased disease rates are seen in transgenerational descendants. Governmental policies regulating toxicant exposure should take generational effects into account. A new approach that takes into consideration generational toxicity will be needed to protect our future populations., (© The Author(s) 2022. Published by Oxford University Press.)

Because epigenetics is a critical component for gene expression, the hypothesis was tested that DNA methylation alterations are dynamic and continually change throughout gametogenesis to generate the mature sperm. Developmental alterations and stage-specific DNA methylation during gametogenesis from primordial germ cells (PGCs) to mature sperm are investigated. Individual developmental stage germ cells were isolated and analyzed for differential DNA methylation regions (DMRs). The number of DMRs was highest in the first three comparisons with mature PGCs, prospermatogonia, and spermatogonia. The most statistically significant DMRs were present at all stages of development and had variations involving both increases or decreases in DNA methylation. DMR-associated genes were identified and correlated with gene functional categories, pathways, and cellular processes. Observations identified a dynamic cascade of epigenetic changes during development that is dramatic during the early developmental stages. Complex epigenetic alterations are required to regulate genome biology and gene expression during gametogenesis., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

The herbicide glyphosate has been shown to promote the epigenetic transgenerational inheritance of pathology and disease in subsequent great-grand offspring (F3 generation). This generational toxicology suggests the impacts of environmental exposures need to assess subsequent generations. The current study was designed to identify epigenetic biomarkers for glyphosate-induced transgenerational diseases using an epigenome-wide association study (EWAS). Following transient glyphosate exposure of gestating female rats (F0 generation), during the developmental period of gonadal sex determination, the subsequent transgenerational F3 generation, with no direct exposure, were aged to 1 year and animals with specific pathologies identified. The pathologies investigated included prostate disease, kidney disease, obesity, and presence of multiple disease. The sperm were collected from the glyphosate lineage males with only an individual disease and used to identify specific differential DNA methylation regions (DMRs) and the differential histone retention sites (DHRs) associated with that pathology. Unique signatures of DMRs and DHRs for each pathology were identified for the specific diseases. Interestingly, at a lower statistical threshold overlapping sets of DMRs and DHRs were identified that were common for all the pathologies. This is one of the first observations that sperm histone retention can potentially act as a biomarker for specific diseases. The DMR and DHR associated genes were identified and correlated with known pathology specific-associated genes. Observations indicate transgenerational epigenetic biomarkers of disease pathology can be identified in the sperm that appear to assess disease susceptibility. These biomarkers suggest epigenetic diagnostics could potentially be used to facilitate preventative medicine.

One of the most important developing cell types in any biological system is the gamete (sperm and egg). The transmission of phenotypes and optimally adapted physiology to subsequent generations is in large part controlled by gametogenesis. In contrast to genetics, the environment actively regulates epigenetics to impact the physiology and phenotype of cellular and biological systems. The integration of epigenetics and genetics is critical for all developmental biology systems at the cellular and organism level. The current review is focused on the role of epigenetics during gametogenesis for both the spermatogenesis system in the male and oogenesis system in the female. The developmental stages from the initial primordial germ cell through gametogenesis to the mature sperm and egg are presented. How environmental factors can influence the epigenetics of gametogenesis to impact the epigenetic transgenerational inheritance of phenotypic and physiological change in subsequent generations is reviewed., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Plastic-derived compounds are one of the most frequent daily worldwide exposures. Previously a mixture of plastic-derived toxicants composed of bisphenol A, bis(2-ethylhexyl) phthalate, and dibutyl phthalate at low-dose exposures of a gestating female rats was found to promote the epigenetic transgenerational inheritance of disease to the offspring (F1 generation), grand-offspring (F2 generation), and great-grand-offspring (F3 generation). Epigenetic analysis of the male sperm was found to result in differential DNA methylation regions (DMRs) in the transgenerational F3 generation male sperm. The current study is distinct and was designed to use an epigenome-wide association study to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations indicate disease-specific DMRs called epimutations in the transgenerational F3 generation great-grand-offspring of rats ancestrally exposed to plastics. The epigenetic DMR biomarkers were identified for testis disease, kidney disease, and multiple (≥2) diseases. These disease sperm epimutation biomarkers were found to be predominantly disease-specific. The genomic locations and features of these DMRs were identified. Interestingly, the disease-specific DMR-associated genes were previously shown to be linked with each of the specific diseases. Therefore, the germline has ancestrally derived epimutations that potentially transmit transgenerational disease susceptibilities. Epigenetic biomarkers for specific diseases could be used as diagnostics to facilitate clinical management of disease and preventative medicine., (© The Author(s) 2021. Published by Oxford University Press.)

Background: Environmentally induced epigenetic transgenerational inheritance of pathology and phenotypic variation has been demonstrated in all organisms investigated from plants to humans. This non-genetic form of inheritance is mediated through epigenetic alterations in the sperm and/or egg to subsequent generations. Although the combined regulation of differential DNA methylated regions (DMR), non-coding RNA (ncRNA), and differential histone retention (DHR) have been shown to occur, the integration of these different epigenetic processes remains to be elucidated. The current study was designed to examine the integration of the different epigenetic processes., Results: A rat model of transiently exposed F0 generation gestating females to the agricultural fungicide vinclozolin or pesticide DDT (dichloro-diphenyl-trichloroethane) was used to acquire the sperm from adult males in the subsequent F1 generation offspring, F2 generation grand offspring, and F3 generation great-grand offspring. The F1 generation sperm ncRNA had substantial overlap with the F1, F2 and F3 generation DMRs, suggesting a potential role for RNA-directed DNA methylation. The DMRs also had significant overlap with the DHRs, suggesting potential DNA methylation-directed histone retention. In addition, a high percentage of DMRs induced in the F1 generation sperm were maintained in subsequent generations., Conclusions: Many of the DMRs, ncRNA, and DHRs were colocalized to the same chromosomal location regions. Observations suggest an integration of DMRs, ncRNA, and DHRs in part involve RNA-directed DNA methylation and DNA methylation-directed histone retention in epigenetic transgenerational inheritance.

Previous studies have demonstrated that exposure to environmental factors can cause epigenetic modifications to germ cells, particularly sperm, to promote epigenetic and transcriptome changes in the embryo. These germ cell and embryo cell epigenetic alterations are associated with phenotypic changes in offspring. Epigenetic inheritance requires epigenetic changes (i.e. epimutations) in germ cells that promote epigenetic and gene expression changes in embryos. The objective of this perspective is to examine the evidence that germ cell epigenome modifications are associated with embryo cell epigenetic and transcriptome changes that affect the subsequent development of all developing somatic cells to promote phenotype change. Various epigenetic changes in sperm, including changes to histone methylation, histone retention, non-coding RNA expression and DNA methylation, have been associated with alterations in embryo cell epigenetics and gene expression. Few studies have investigated this link for oocytes. The studies reviewed herein support the idea that environmentally induced epigenetic changes in germ cells affect alterations in embryo cell epigenetics and transcriptomes that have an important role in the epigenetic inheritance of pathology and phenotypic change.

Methylated DNA immunoprecipitation is a large scale purification technique. It enables the isolation of methylated DNA fragments for subsequent locus-specific or genome-wide analysis. Here we describe an immunoprecipitation protocol using a monoclonal mouse anti 5-methyl-cytidine antibody followed by next-generation sequencing (MeDIP-Seq).

Dioxin was historically one of the most common industrial contaminants with several major industry accidents, as well as governmental actions involving military service, having exposed large numbers of the worldwide population over the past century. Previous rat studies have demonstrated the ability of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) exposure to promote the epigenetic transgenerational inheritance of disease susceptibility in subsequent generations. The types of disease previously observed include puberty abnormalities, testis, ovary, kidney, prostate and obesity pathologies. The current study was designed to use an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Therefore, the transgenerational F3 generation dioxin lineage male rats with and without a specific disease were compared to identify differential DNA methylation regions (DMRs) as biomarkers for disease. The genomic features of the disease-specific DMRs were characterized. Observations demonstrate that disease-specific epimutation DMRs exist for the transgenerational dioxin lineage rats that can potentially be used as epigenetic biomarkers for testis, kidney, prostate and obesity diseases. These disease-specific DMRs were associated with genes that have previously been shown to be linked with the specific diseases. This EWAS for transgenerational disease identified potential epigenetic biomarkers and provides the proof of concept of the potential to develop similar biomarkers for humans to diagnose disease susceptibilities and facilitate preventative medicine., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Environmental exposures such as chemical toxicants can alter gene expression and disease susceptibility through epigenetic processes. Epigenetic changes can be passed to future generations through germ cells through epigenetic transgenerational inheritance of increased disease susceptibility. The current study used an epigenome-wide association study (EWAS) to investigate whether specific transgenerational epigenetic signatures of differential DNA methylation regions (DMRs) exist that are associated with particular disease states in the F3 generation great-grand offspring of F0 generation rats exposed during gestation to the agricultural pesticide methoxychlor. The transgenerational epigenetic profiles of sperm from F3 generation methoxychlor lineage rats that have only one disease state were compared to those that have no disease. Observations identify disease specific patterns of DMRs for these transgenerational rats that can potentially serve as epigenetic biomarkers for prostate disease, kidney disease, obesity, and the presence of multiple diseases. The chromosomal locations, genomic features, and gene associations of the DMRs are characterized. Disease specific DMR sets contained DMR-associated genes that have previously been shown to be associated with that specific disease. Future epigenetic biomarkers could potentially be developed and validated for humans as a disease susceptibility diagnostic tool to facilitate preventative medicine and management of disease., (© The Author(s) 2020. Published by Oxford University Press.)

Atrazine is a common agricultural herbicide previously shown to promote epigenetic transgenerational inheritance of disease to subsequent generations. The current study was designed as an epigenome-wide association study (EWAS) to identify transgenerational sperm disease associated differential DNA methylation regions (DMRs) and differential histone retention regions (DHRs). Gestating female F0 generation rats were transiently exposed to atrazine during the period of embryonic gonadal sex determination, and then subsequent F1, F2, and F3 generations obtained in the absence of any continued exposure. The transgenerational F3 generation males were assessed for disease and sperm collected for epigenetic analysis. Pathology was observed in pubertal onset and for testis disease, prostate disease, kidney disease, lean pathology, and multiple disease. For these pathologies, sufficient numbers of individual males with only a single specific disease were identified. The sperm DNA and chromatin were isolated from adult one-year animals with the specific diseases and analyzed for DMRs with methylated DNA immunoprecipitation (MeDIP) sequencing and DHRs with histone chromatin immunoprecipitation (ChIP) sequencing. Transgenerational F3 generation males with or without disease were compared to identify the disease specific epimutation biomarkers. All pathologies were found to have disease specific DMRs and DHRs which were found to predominantly be distinct for each disease. No common DMRs or DHRs were found among all the pathologies. Epimutation gene associations were identified and found to correlate to previously known disease linked genes. This is one of the first observations of potential sperm disease biomarkers for histone retention sites. Although further studies with expanded animal numbers are required, the current study provides evidence the EWAS analysis is effective for the identification of potential pathology epimutation biomarkers for disease susceptibility., Competing Interests: The authors declare no competing interests.

Jet fuel hydrocarbons is the generic name for aviation fuels used in gas-turbine engine powered aircraft. The Deepwater Horizon oil rig explosion created the largest environmental disaster in U.S. history, and the second largest oil spill in human history with over 800 million liters of hydrocarbons released into the Gulf of Mexico over a period of 3 months. Due to the widespread use of jet fuel hydrocarbons, this compound mixture has been recognized as the single largest chemical exposure for military personnel. Previous animal studies have demonstrated the ability of jet fuel (JP-8) exposure to promote the epigenetic transgenerational inheritance of disease susceptibility in subsequent generations. The diseases observed include late puberty, kidney, obesity and multiple disease pathologies. The current study is distinct and was designed to identify potential sperm DNA methylation biomarkers for specific transgenerational diseases. Observations show disease specific differential DNA methylation regions (DMRs) called epimutations in the transgenerational F3 generation great-grand-offspring male rats ancestrally exposed to jet fuel. The potential epigenetic DMR biomarkers were identified for late puberty, kidney, obesity, and multiple diseases, and found to be predominantly disease specific. These disease specific DMRs have associated genes that were previously shown to be linked with each of these specific diseases. Therefore, the germline (i.e. sperm) has environmentally induced ancestrally derived epimutations that have the potential to transgenerationally transmit disease susceptibilities to subsequent generations. Epigenetic biomarkers for specific diseases could be developed as medical diagnostics to facilitate clinical management of disease, and allow preventative medicine therapeutics., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Permethrin and N,N-diethyl-meta-toluamide (DEET) are the pesticides and insect repellent most commonly used by humans. These pesticides have been shown to promote the epigenetic transgenerational inheritance of disease in rats. The current study was designed as an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation epimutation biomarkers for specific transgenerational disease., Methods: Outbred Sprague Dawley gestating female rats (F0) were transiently exposed during fetal gonadal sex determination to the pesticide combination including Permethrin and DEET. The F3 generation great-grand offspring within the pesticide lineage were aged to 1 year. The transgenerational adult male rat sperm were collected from individuals with single and multiple diseases and compared to non-diseased animals to identify differential DNA methylation regions (DMRs) as biomarkers for specific transgenerational disease., Results: The exposure of gestating female rats to a permethrin and DEET pesticide combination promoted transgenerational testis disease, prostate disease, kidney disease, and the presence of multiple disease in the subsequent F3 generation great-grand offspring. The disease DMRs were found to be disease specific with negligible overlap between different diseases. The genomic features of CpG density, DMR length, and chromosomal locations of the disease specific DMRs were investigated. Interestingly, the majority of the disease specific sperm DMR associated genes have been previously found to be linked to relevant disease specific genes., Conclusions: Observations demonstrate the EWAS approach identified disease specific biomarkers that can be potentially used to assess transgenerational disease susceptibility and facilitate the clinical management of environmentally induced pathology.

Numerous environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Alterations in the germline epigenome are necessary to transmit transgenerational phenotypes. In previous studies, the pesticide DDT (dichlorodiphenyltrichloroethane) and the agricultural fungicide vinclozolin were shown to promote the transgenerational inheritance of sperm differential DNA methylation regions, non-coding RNAs and histone retention, which are termed epimutations. These epimutations are able to mediate this epigenetic inheritance of disease and phenotypic variation. The current study was designed to investigate the developmental origins of the transgenerational differential histone retention sites (called DHRs) during gametogenesis of the sperm. Vinclozolin and DDT were independently used to promote the epigenetic transgenerational inheritance of these DHRs. Male control lineage, DDT lineage and vinclozolin lineage F3 generation rats were used to isolate round spermatids, caput epididymal spermatozoa, and caudal sperm. The DHRs distinguishing the control versus DDT lineage or vinclozolin lineage samples were determined at these three developmental stages. DHRs and a reproducible core of histone H3 retention sites were observed using an H3 chromatin immunoprecipitation-sequencing (ChIP-Seq) analysis in each of the germ cell populations. The chromosomal locations and genomic features of the DHRs were analyzed. A cascade of epigenetic histone retention site alterations was found to be initiated in the round spermatids and then further modified during epididymal sperm maturation. Observations show that in addition to alterations in sperm DNA methylation and ncRNA expression previously identified, the induction of differential histone retention sites (DHRs) in the later stages of spermatogenesis also occurs. This novel component of epigenetic programming during spermatogenesis can be environmentally altered and transmitted to subsequent generations through epigenetic transgenerational inheritance., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetic transgenerational inheritance potentially impacts disease etiology, phenotypic variation, and evolution. An increasing number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease. Previous observations have demonstrated that the agricultural fungicide vinclozolin and pesticide DDT (dichlorodiphenyltrichloroethane) induce transgenerational sperm epimutations involving DNA methylation, ncRNA, and histone modifications or retention. These two environmental toxicants were used to investigate the impacts of parent-of-origin outcross on the epigenetic transgenerational inheritance of disease. Male and female rats were collected from a paternal outcross (POC) or a maternal outcross (MOC) F4 generation control and exposure lineages for pathology and epigenetic analysis. This model allows the parental allelic transmission of disease and epimutations to be investigated. There was increased pathology incidence in the MOC F4 generation male prostate, kidney, obesity, and multiple diseases through a maternal allelic transmission. The POC F4 generation female offspring had increased pathology incidence for kidney, obesity and multiple types of diseases through the paternal allelic transmission. Some disease such as testis or ovarian pathology appear to be transmitted through the combined actions of both male and female alleles. Analysis of the F4 generation sperm epigenomes identified differential DNA methylated regions (DMRs) in a genome-wide analysis. Observations demonstrate that DDT and vinclozolin have the potential to promote the epigenetic transgenerational inheritance of disease and sperm epimutations to the outcross F4 generation in a sex specific and exposure specific manner. The parent-of-origin allelic transmission observed appears similar to the process involved with imprinted-like genes., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

A number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. This requires alterations in the germline (sperm or egg) epigenome. Previously, the agricultural fungicide vinclozolin was found to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs) termed epimutations that help mediate this epigenetic inheritance. The current study was designed to investigate the developmental origins of the transgenerational DMRs during gametogenesis. Male control and vinclozolin lineage F3 generation rats were used as a source of embryonic day 13 (E13) primordial germ cells, embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus vinclozolin lineage samples were determined for each developmental stage. The top 100 statistically significant DMRs for each stage were compared. The developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were investigated. In addition, the DMR associated genes were identified. Previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs). Interestingly, the majority of the DMRs identified in the current study for the caudal sperm originated during the spermatogenic process in the testis. A cascade of epigenetic alterations initiated in the PGCs appears to be required to alter the epigenetic programming during spermatogenesis to modify the sperm epigenome involved in the transgenerational epigenetic inheritance phenomenon.

Epigenetic alterations in the germline can be triggered by a number of different environmental factors from diet to toxicants. These environmentally induced germline changes can promote the epigenetic transgenerational inheritance of disease and phenotypic variation. In previous studies, the pesticide DDT was shown to promote the transgenerational inheritance of sperm differential DNA methylation regions (DMRs), also called epimutations, which can in part mediate this epigenetic inheritance. In the current study, the developmental origins of the transgenerational DMRs during gametogenesis have been investigated. Male control and DDT lineage F3 generation rats were used to isolate embryonic day 16 (E16) prospermatogonia, postnatal day 10 (P10) spermatogonia, adult pachytene spermatocytes, round spermatids, caput epididymal spermatozoa, and caudal sperm. The DMRs between the control versus DDT lineage samples were determined at each developmental stage. The top 100 statistically significant DMRs at each stage were compared and the developmental origins of the caudal epididymal sperm DMRs were assessed. The chromosomal locations and genomic features of the different stage DMRs were analyzed. Although previous studies have demonstrated alterations in the DMRs of primordial germ cells (PGCs), the majority of the DMRs identified in the caudal sperm originated during the spermatogonia stages in the testis. Interestingly, a cascade of epigenetic alterations initiated in the PGCs is required to alter the epigenetic programming during spermatogenesis to obtain the sperm epigenetics involved in the epigenetic transgenerational inheritance phenomenon., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetic transgenerational inheritance of disease and phenotypic variation can be induced by several toxicants, such as vinclozolin. This phenomenon can involve DNA methylation, non-coding RNA (ncRNA) and histone retention, and/or modification in the germline (e.g. sperm). These different epigenetic marks are called epimutations and can transmit in part the transgenerational phenotypes. This study was designed to investigate the vinclozolin-induced concurrent alterations of a number of different epigenetic factors, including DNA methylation, ncRNA, and histone retention in rat sperm. Gestating females (F0 generation) were exposed transiently to vinclozolin during fetal gonadal development. The directly exposed F1 generation fetus, the directly exposed germline within the fetus that will generate the F2 generation, and the transgenerational F3 generation sperm were studied. DNA methylation and ncRNA were altered in each generation rat sperm with the direct exposure F1 and F2 generations being distinct from the F3 generation epimutations. Interestingly, an increased number of differential histone retention sites were found in the F3 generation vinclozolin sperm, but not in the F1 or F2 generations. All three different epimutation types were affected in the vinclozolin lineage transgenerational sperm (F3 generation). The direct exposure generations (F1 and F2) epigenetic alterations were distinct from the transgenerational sperm epimutations. The genomic features and gene pathways associated with the epimutations were investigated to help elucidate the integration of these different epigenetic processes. Our results show that the three different types of epimutations are involved and integrated in the mediation of the epigenetic transgenerational inheritance phenomenon.

A variety of environmental toxicants and factors have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Epigenetic alterations in the germline (sperm or egg) are required to transmit transgenerational phenotypes. The current study was designed to investigate the potential role of histones in sperm to help mediate the epigenetic transgenerational inheritance. The agricultural fungicide vinclozolin and the pesticide DDT (dichlorodiphenyltrichloroethane) were independently used to promote the epigenetic transgenerational inheritance of disease. Purified cauda epididymal sperm were collected from the transgenerational F3 generation control and exposure lineage male rats for histone analysis. A reproducible core of histone H3 retention sites was observed using an H3 chromatin immunoprecipitation (ChIP-Seq) analysis in control lineage sperm. Interestingly, the same core group of H3 retention sites plus additional differential histone retention sites (DHRs) were observed in the F3 generation exposure lineage sperm. Although new histone H3 retention sites were observed, negligible change in histone modification (methylation of H3K27me3) was observed between the control and exposure lineages. Observations demonstrate that in addition to alterations in sperm DNA methylation and ncRNA previously identified, the induction of differential histone retention sites (DHRs) also appear to be involved in environmentally induced epigenetic transgenerational inheritance.

Background: Environmental toxicants such as DDT have been shown to induce the epigenetic transgenerational inheritance of disease (e.g., obesity) through the germline. The current study was designed to investigate the DDT-induced concurrent alterations of a number of different epigenetic processes including DNA methylation, non-coding RNA (ncRNA) and histone retention in sperm., Methods: Gestating females were exposed transiently to DDT during fetal gonadal development, and then, the directly exposed F1 generation, the directly exposed germline F2 generation and the transgenerational F3 generation sperm were investigated., Results: DNA methylation and ncRNA were altered in each generation sperm with the direct exposure F1 and F2 generations being predominantly distinct from the F3 generation epimutations. The piRNA and small tRNA were the most predominant classes of ncRNA altered. A highly conserved set of histone retention sites were found in the control lineage generations which was not significantly altered between generations, but a large number of new histone retention sites were found only in the transgenerational generation DDT lineage sperm., Conclusions: Therefore, all three different epigenetic processes were concurrently altered as DDT induced the epigenetic transgenerational inheritance of sperm epimutations. The direct exposure generations sperm epigenetic alterations were distinct from the transgenerational sperm epimutations. The genomic features and gene associations with the epimutations were investigated to help elucidate the integration of these different epigenetic processes. Observations demonstrate all three epigenetic processes are involved in transgenerational inheritance. The different epigenetic processes appear to be integrated in mediating the epigenetic transgenerational inheritance phenomenon.

Among pregnant women ibuprofen is one of the most frequently used pharmaceutical compounds with up to 28% reporting use. Regardless of this, it remains unknown whether ibuprofen could act as an endocrine disruptor as reported for fellow analgesics paracetamol and aspirin. To investigate this, we exposed human fetal testes (7-17 gestational weeks (GW)) to ibuprofen using ex vivo culture and xenograft systems. Ibuprofen suppressed testosterone and Leydig cell hormone INSL3 during culture of 8-9 GW fetal testes with concomitant reduction in expression of the steroidogenic enzymes CYP11A1, CYP17A1 and HSD17B3, and of INSL3. Testosterone was not suppressed in testes from fetuses younger than 8 GW, older than 10-12 GW, or in second trimester xenografted testes (14-17 GW). Ex vivo, ibuprofen also affected Sertoli cell by suppressing AMH production and mRNA expression of AMH, SOX9, DHH, and COL2A1. While PGE2 production was suppressed by ibuprofen, PGD2 production was not. Germ cell transcripts POU5F1, TFAP2C, LIN28A, ALPP and KIT were also reduced by ibuprofen. We conclude that, at concentrations relevant to human exposure and within a particular narrow 'early window' of sensitivity within first trimester, ibuprofen causes direct endocrine disturbances in the human fetal testis and alteration of the germ cell biology.

Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5-17.5 days post-coitum) and humans (8-12 gestational weeks) and under different culture conditions. BPA concentrations of 10(-8)M and 10(-5)M for 72 h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10-5M suppressed it in the Wistar strain. The suppressive effects at 10-5M were seen as early as 24h and 48 h in both strains. BPA at 10(-7)-10(-5)M for 72 h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10(-8)M, 10(-7)M, and 10(-5)M for 72 h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.

Context: Masculinization depends on the fetal testis. Exposure of the human fetus during pregnancy to paracetamol and/or to other mild analgesics is associated with an increased risk of cryptorchidism., Objective: We aimed to determine whether mild analgesics disrupted the morphology and endocrine function of the human testis., Design: We used an in vitro system based on the culture of human fetal testes exposed or not to paracetamol, its metabolite N-(4-hydroxyphenyl)-arachidonoylethanolamide (AM404), aspirin, indomethacin, and ketoconazole at 10(-4) to 10(-7) M., Setting: The study was conducted at the University of Rennes I., Patients/participants: Human fetal testes were from pregnant women after induced abortion, between 7 and 12 weeks of gestation (GW)., Main Outcome Measures: Testosterone (RIA), anti-Müllerian hormone (ELISA), insulin-like factor 3 (RIA), and prostaglandin (PG) D2 and PGE2 (ELISA) were assayed in the medium. Testicular cells were counted using histology and image analysis. The possible nuclear receptor-mediated activities of the analgesics were investigated using reporter cell lines expressing estrogen, androgen, and peroxisome proliferator-activated γ receptors., Results: Indomethacin and aspirin stimulated testosterone production, particularly by the younger testes (8-9 GW vs 10-12 GW). Paracetamol, AM404, and ketoconazole decreased insulin-like factor 3 levels. Aspirin stimulated whereas ketoconazole inhibited AMH production. PGE2 levels were inhibited by paracetamol and aspirin in the 7 to 12 GW testes and by indomethacin but only in 7 to 9.86 GW testes. The inhibitory trends seen for PGD2 were not statistically significant., Conclusions: Analgesics at concentrations relevant to human exposure cause endocrine disturbances in the fetal testis. We suggest that the fetal human testis displays slight critical age windows for sensitivity to direct exposure to aspirin, indomethacin, and paracetamol. The analgesic-induced inhibition of INSL3 may be the mechanism by which analgesics increase the risk of cryptorchidism. Greater caution is required concerning consumption of analgesics during pregnancy.