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Although male infertility is currently diagnosed when abnormal sperm parameters are found, the poor predictive ability of sperm parameters on natural fecundity and medically assisted reproduction outcome poses the need for improved diagnostic techniques for male infertility. The accumulating evidence about the role played by the sperm epigenome in modulation of the early phases of embryonic development has led researchers to focus on the epigenetic mechanisms within the sperm epigenome to find new molecular markers of male infertility. Indeed, sperm epigenome abnormalities could explain some cases of unexplained male infertility in men showing normal sperm parameters and were found to be associated with poor embryo development in IVF cycles. The present mini-review summarizes the current knowledge about this interesting topic, starting from a description of the epigenetic mechanisms of gene expression regulation (i.e. DNA methylation, histone modifications, and non-coding RNAs' activity). We also discuss possible mechanisms by which environmental factors might cause epigenetic changes in the human germline and affect embryonic development, as well as subsequent generations' phenotypes. Studies demonstrating sperm epigenome abnormalities in men with male infertility are reviewed, with particular emphasis on those with the more severe form of spermatogenic dysfunction. Observations demonstrate that the diagnostic and prognostic efficacy of sperm epigenome evaluation will help facilitate the management of men with male factor infertility. [ABSTRACT FROM AUTHOR]

Three successive multiple generations of rats were exposed to different toxicants and then bred to the transgenerational F5 generation to assess the impacts of multiple generation different exposures. The current study examines the actions of the agricultural fungicide vinclozolin on the F0 generation, followed by jet fuel hydrocarbon mixture exposure of the F1 generation, and then pesticide dichlorodiphenyltrichloroethane on the F2 generation gestating females. The subsequent F3 and F4 generations and F5 transgenerational generation were obtained and F1–F5 generations examined for male sperm epigenetic alterations and pathology in males and females. Significant impacts on the male sperm differential DNA methylation regions were observed. The F3–F5 generations were similar in ∼50% of the DNA methylation regions. The pathology of each generation was assessed in the testis, ovary, kidney, and prostate, as well as the presence of obesity and tumors. The pathology used a newly developed Deep Learning, artificial intelligence-based histopathology analysis. Observations demonstrated compounded disease impacts in obesity and metabolic parameters, but other pathologies plateaued with smaller increases at the F5 transgenerational generation. Observations demonstrate that multiple generational exposures, which occur in human populations, appear to increase epigenetic impacts and disease susceptibility. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Exposure to vinclozolin has been shown to induce the epigenetic transgenerational inheritance of increased susceptibility to disease, and to induce transgenerational changes to the epigenome. In the current study, gestating F0 generation rats were exposed to vinclozolin, and the subsequent F1, F2 and transgenerational F3 generations were evaluated for diseases and pathologies. F1 and F2 generation rats exhibited few abnormalities. However, F3 generation rats showed transgenerational increases in testis, prostate, and kidney disease, changes in the age of puberty onset in males, and an increased obesity rate in females. Overall there was an increase in the rate of animals with disease, and in the incidence of animals with multiple diseases. The objective of the current study was to analyze the sperm epigenome of F3 generation rats with specific abnormalities and compare them to rats without those abnormalities, in an effort to find epigenetic biomarkers of transgenerational disease. Unique signatures of differential DNA methylation regions (DMRs) in sperm were found that associated with testis disease, prostate disease and kidney disease. Confounding factors identified were the presence of multiple diseases in the analysis and the limited number of animals without disease. These results further our understanding of the mechanisms governing epigenetic transgenerational inheritance, and may lead in the future to the use of epigenetic biomarkers that will help predict an individual’s susceptibility for specific diseases. [ABSTRACT FROM AUTHOR]

Two of the most prevalent ovarian diseases affecting women’s fertility and health are Primary Ovarian Insufficiency (POI) and Polycystic Ovarian Syndrome (PCOS). Previous studies have shown that exposure to a number of environmental toxicants can promote the epigenetic transgenerational inheritance of ovarian disease. In the current study, transgenerational changes to the transcriptome and epigenome of ovarian granulosa cells are characterized in F3 generation rats after ancestral vinclozolin or DDT exposures. In purified granulosa cells from 20-day-old F3 generation females, 164 differentially methylated regions (DMRs) (P < 1 x 10−6) were found in the F3 generation vinclozolin lineage and 293 DMRs (P < 1 x 10−6) in the DDT lineage, compared to controls. Long noncoding RNAs (lncRNAs) and small noncoding RNAs (sncRNAs) were found to be differentially expressed in both the vinclozolin and DDT lineage granulosa cells. There were 492 sncRNAs (P < 1 x 10−4) in the vinclozolin lineage and 1,085 sncRNAs (P < 1 x 10−4) in the DDT lineage. There were 123 lncRNAs and 51 lncRNAs in the vinclozolin and DDT lineages, respectively (P < 1 x 10−4). Differentially expressed mRNAs were also found in the vinclozolin lineage (174 mRNAs at P < 1 x 10−4) and the DDT lineage (212 mRNAs at P < 1 x 10−4) granulosa cells. Comparisons with known ovarian disease associated genes were made. These transgenerational epigenetic changes appear to contribute to the dysregulation of the ovary and disease susceptibility that can occur in later life. Observations suggest that ancestral exposure to toxicants is a risk factor that must be considered in the molecular etiology of ovarian disease. [ABSTRACT FROM AUTHOR]

Ancestral environmental exposures to a variety of environmental toxicants and other factors have been shown to promote the epigenetic transgenerational inheritance of adult onset disease. The current study examined the potential transgenerational actions of the herbicide atrazine. Atrazine is one of the most commonly used herbicides in the agricultural industry, in particular with corn and soy crops. Outbred gestating female rats were transiently exposed to a vehicle control or atrazine. The F1 generation offspring were bred to generate the F2 generation and then the F2 generation bred to generate the F3 generation. The F1, F2 and F3 generation control and atrazine lineage rats were aged and various pathologies investigated. The male sperm were collected to investigate DNA methylation differences between the control and atrazine lineage sperm. The F1 generation offspring (directly exposed as a fetus) did not develop disease, but weighed less compared to controls. The F2 generation (grand-offspring) was found to have increased frequency of testis disease and mammary tumors in males and females, early onset puberty in males, and decreased body weight in females compared to controls. The transgenerational F3 generation rats were found to have increased frequency of testis disease, early onset puberty in females, behavioral alterations (motor hyperactivity) and a lean phenotype in males and females. The frequency of multiple diseases was significantly higher in the transgenerational F3 generation atrazine lineage males and females. The transgenerational transmission of disease requires germline (egg or sperm) epigenetic alterations. The sperm differential DNA methylation regions (DMRs), termed epimutations, induced by atrazine were identified in the F1, F2 and F3 generations. Gene associations with the DMRs were identified. For the transgenerational F3 generation sperm, unique sets of DMRs (epimutations) were found to be associated with the lean phenotype or testis disease. These DMRs provide potential biomarkers for transgenerational disease. The etiology of disease appears to be in part due to environmentally induced epigenetic transgenerational inheritance, and epigenetic biomarkers may facilitate the diagnosis of the ancestral exposure and disease susceptibility. Observations indicate that although atrazine does not promote disease in the directly exposed F1 generation, it does have the capacity to promote the epigenetic transgenerational inheritance of disease. [ABSTRACT FROM AUTHOR]

Abstract: Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs]) and neurobehavior (i.e., visual startle and spontaneous locomotion) in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans. [ABSTRACT FROM AUTHOR]

Background: The potential that adolescent chemotherapy can impact the epigenetic programming of the germ line to influence later life adult fertility and promote epigenetic inheritance was investigated. Previous studies have demonstrated a number of environmental exposures such as abnormal nutrition and toxicants can promote sperm epigenetic changes that impact offspring. Methods: Adult males approximately ten years after pubertal exposure to chemotherapy were compared to adult males with no previous exposure. Sperm were collected to examine differential DNA methylation regions (DMRs) between the exposed and control populations. Gene associations and correlations to genetic mutations (copy number variation) were also investigated. Methods and Findings: A signature of statistically significant DMRs was identified in the chemotherapy exposed male sperm. The DMRs, termed epimutations, were found in CpG desert regions of primarily 1 kilobase size. Observations indicate adolescent chemotherapy exposure can promote epigenetic alterations that persist in later life. Conclusions: This is the first observation in humans that an early life chemical exposure can permanently reprogram the spermatogenic stem cell epigenome. The germline (i.e., sperm) epimutations identified suggest chemotherapy has the potential to promote epigenetic inheritance to the next generation. [ABSTRACT FROM AUTHOR]

Exposure to environmental factors can induce the epigenetic transgenerational inheritance of disease. Alterations to the epigenome termed “epimutations” include “primary epimutations” which are epigenetic alterations in the absence of genetic change and “secondary epimutations” which form following an initial genetic change. To determine if secondary epimutations contribute to transgenerational transmission of disease following in utero exposure to the endocrine disruptor vinclozolin, we exposed pregnant female rats carrying the lacI mutation-reporter transgene to vinclozolin and assessed the frequency of mutations in kidney tissue and sperm recovered from F1 and F3 generation progeny. Our results confirm that vinclozolin induces primary epimutations rather than secondary epimutations, but also suggest that some primary epimutations can predispose a subsequent accelerated accumulation of genetic mutations in F3 generation descendants that have the potential to contribute to transgenerational phenotypes. We therefore propose the existence of “tertiary epimutations” which are initial primary epimutations that promote genome instability leading to an accelerated accumulation of genetic mutations. [ABSTRACT FROM AUTHOR]

For the past 120 years, the Weismann barrier and associated germ plasm theory of heredity have been a doctrine that has impacted evolutionary biology and our concepts of inheritance through the germline. Although August Weismann in his 1872 book was correct that the sperm and egg were the only cells to transmit molecular information to the subsequent generation, the concept that somatic cells do not impact the germline (i.e., the Weismann barrier) is incorrect. However, the doctrine or dogma of the Weismann barrier still influences many scientific fields and topics. The discovery of epigenetics, and more recently environmentally induced epigenetic transgenerational inheritance of phenotypic variation and pathology, have had significant impacts on evolution theory and medicine today. Environmental epigenetics and the concept of epigenetic transgenerational inheritance refute aspects of the Weismann barrier and require a re-evaluation of both inheritance theory and evolution theory. [ABSTRACT FROM AUTHOR]

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. • Environmental induction of new transgenerational sperm histone retention sites. • Identification of a developmental cascade of histone retention. • Potential role of novel sperm histone retention in epigenetic inheritance. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Identifies the developmental origins of transgenerational sperm epimutations. • Demonstrates a developmental cascade of epigenetic alterations in the germline is required. • Demonstrates epimutations are acquired at all spermatogenic and epididymal stages. • Elucidates the germline epigenetic programming for environmentally induced epigenetic transgenerational inheritance. [ABSTRACT FROM AUTHOR]

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. Image 1 • Dioxin promotes the epigenetic transgenerational inheritance of disease. • Sperm epimutation biomarkers identified for testis, prostate, kidney, and obesity disease. • Disease specific epigenetic biomarkers identified with negligible overlap. [ABSTRACT FROM AUTHOR]