Sperm DNA Methylation Changes Research Articles

Preconceptional capsaicin intervention mitigates negative effects of paternal obesity on metabolic characteristics in male offspring upon high-fat diet challenge

The activation of brown adipose tissue by cold exposure in paternal mice has been reported to improve the metabolic health of offspring. Notably, dietary components for activating BAT have drawn considerable attention owing to their high feasibility for human trials. Therefore, here the potential metabolic improving effects of dietary capsaicin intervention on diet-induced obese paternal mice and their offspring were measured. The results support that paternal preconceptional capsaicin intervention not only elicited the anticipated enhancement of brown fat activity in obese paternal mice, but also improved the metabolic profiles in male offspring evidenced by their elevated resistance to high-fat diet challenge. Moreover, changes in sperm DNA methylation and small non-coding RNA profile were noted in paternal mice with capsaicin intervention, though additional investigation is needed to further delve into the mechanisms. In summary, paternal dietary intervention activating BAT could represent a promising strategy for enhancing the metabolic health of offspring.

Age-related changes in sperm DNA methylation and their forensic and clinical implications.

As a link between a stable genome and a dynamic environment, epigenetics is a promising tool for mapping age-related changes in human DNA. Methylated cytosine changes at specific loci are generally less studied in sperm DNA than in somatic cell DNA. Age-related methylation changes can be connected to various reproductive health problems and multiple disorders in offspring. In addition, they can be helpful in forensic fields, where testing of specific loci in semen samples found at sexual assault crime scenes can predict a perpetrator's age and narrow down the police investigation. This review focuses on age-related methylation changes in sperm. It covers the biological role of methylation, methylation testing techniques and the implications of methylation changes in forensics and clinical practice.

Testicular histone hyperacetylation in mice by valproic acid administration affects the next generation by changes in sperm DNA methylation.

Various studies have described epigenetic inheritance through sperms. However, the detailed mechanisms remain unclear. In this study, we focused on DNA methylation in mice treated with valproic acid (VPA), an inducer of epigenomic changes, and analyzed the treatment effects on the sperm from the next generation of mice. The administration of 200 mg/kg/day VPA to mice for 4 weeks caused transient histone hyperacetylation in the testes and DNA methylation changes in the sperm, including promoter CpGs of genes related to brain function. Oocytes fertilized with VPA-treated mouse sperm showed methylation fluctuations at the morula stage. Pups that were fathered by these mice also showed behavioral changes in the light/dark transition test after maturation. Brain RNA-seq of these mice showed that the expression of genes related to neural functions were altered. Comparison of the sperm DNA methylation status of the next generation of mice with that of the parental generation revealed the disappearance of methylation changes observed in the sperm of the parental generation. These findings suggest that VPA-induced histone hyperacetylation may have brain function-related effects on the next generation through changes in sperm DNA methylation.

Cannabis alters DNA methylation at maternally imprinted and autism candidate genes in spermatogenic cells

Cannabis use in the United States is increasing, with highest consumption among men at their peak reproductive years. We previously demonstrated widespread changes in sperm DNA methylation with cannabis exposure in humans and rats, including genes important in neurodevelopment. Here, we use an in vitro human spermatogenesis model to recapitulate chronic cannabis use and assess DNA methylation at imprinted and autism spectrum disorder (ASD) candidate genes in spermatogonial stem cell (SSC)- and spermatid-like cells. Methylation at maternally imprinted genes SGCE and GRB10 was significantly altered in SSC- and spermatid-like cells, respectively, while PEG3 was significantly differentially methylated in spermatid-like cells. Two of ten randomly selected ASD candidate genes, HCN1 and NR4A2, had significantly altered methylation with cannabis exposure in SSC-like cells. These results support our findings in human cohorts and provide a new tool with which to gain mechanistic insights into the association between paternal cannabis use and risk of ASD in offspring.

Paternal diet induces transgenerational epigenetic inheritance of DNA methylation signatures and phenotypes in sheep model.

Transgenerational epigenetic inheritance (TEI) requires transmission of environmentally induced epigenetic changes and associated phenotypes to subsequent generations without continued exposure to the environmental factor that originated the change. TEI is well-established in plants and Caenorhabditis elegans; however, occurrence in mammals is debated and poorly understood. Here, we examined whether paternal diet from weaning to puberty-induced changes in sperm DNA methylation that were transmitted to subsequent generations. Over 100 methylated cytosines, environmentally altered in the F0 generation, were inherited by the F1 and F2 generations. Furthermore, the F0 paternal diet was associated with growth and male fertility phenotypes in subsequent generations. Differentially methylated cytosines were correlated with gene expression. Our results demonstrate that some sperm methylation sites may escape DNA methylation erasure and are transmitted to subsequent generations despite the 2 waves of epigenetic programming: in primordial germ cells and in embryos after fertilization. These results advance our understanding of the complex relationships between nature and nurture.

Simulated Wildfire Smoke Significantly Alters Sperm DNA Methylation Patterns in a Murine Model.

Wildfires are now a common feature of the western US, increasing in both intensity and number of acres burned over the last three decades. The effects of this changing wildfire and smoke landscape are a critical public and occupational health issue. While respiratory morbidity due to smoke exposure is a priority, evaluating the molecular underpinnings that explain recent extrapulmonary observations is necessary. Here, we use an Apoe−/− mouse model to investigate the epigenetic impact of paternal exposure to simulated wildfire smoke. We demonstrate that 40 days of exposure to smoke from Douglas fir needles induces sperm DNA methylation changes in adult mice. DNA methylation was measured by reduced representation bisulfite sequencing and varied significantly in 3353 differentially methylated regions, which were subsequently annotated to 2117 genes. The differentially methylated regions were broadly distributed across the mouse genome, but the vast majority (nearly 80%) were hypermethylated. Pathway analyses, using gene-derived and differentially methylated region-derived gene ontology terms, point to a number of developmental processes that may warrant future investigation. Overall, this study of simulated wildfire smoke exposure suggests paternal reproductive risks are possible with prolonged exposure.

Sperm DNA methylation mediates the association of male age on reproductive outcomes among couples undergoing infertility treatment

Parental age at time of offspring conception is increasing in developed countries. Advanced male age is associated with decreased reproductive success and increased risk of adverse neurodevelopmental outcomes in offspring. Mechanisms for these male age effects remain unclear, but changes in sperm DNA methylation over time is one potential explanation. We assessed genome-wide methylation of sperm DNA from 47 semen samples collected from male participants of couples seeking infertility treatment. We report that higher male age was associated with lower likelihood of fertilization and live birth, and poor embryo development (p < 0.05). Furthermore, our multivariable linear models showed male age was associated with alterations in sperm methylation at 1698 CpGs and 1146 regions (q < 0.05), which were associated with > 750 genes enriched in embryonic development, behavior and neurodevelopment among others. High dimensional mediation analyses identified four genes (DEFB126, TPI1P3, PLCH2 and DLGAP2) with age-related sperm differential methylation that accounted for 64% (95% CI 0.42–0.86%; p < 0.05) of the effect of male age on lower fertilization rate. Our findings from this modest IVF population provide evidence for sperm methylation as a mechanism of age-induced poor reproductive outcomes and identifies possible candidate genes for mediating these effects.

Chronic Exposure to Cadmium Induces Differential Methylation in Mice Spermatozoa.

Cadmium exposure is ubiquitous and has been linked to diseases including cancers and reproductive defects. Since cadmium is nonmutagenic, it is thought to exert its gene dysregulatory effects through epigenetic reprogramming. Several studies have implicated germline exposure to cadmium in developmental reprogramming. However, most of these studies have focused on maternal exposure, while the impact on sperm fertility and disease susceptibility has received less attention. In this study, we used reduced representation bisulfite sequencing to comprehensively investigate the impact of chronic cadmium exposure on mouse spermatozoa DNA methylation. Adult male C57BL/J6 mice were provided water with or without cadmium chloride for 9 weeks. Sperm, testes, liver, and kidney tissues were collected at the end of the treatment period. Cadmium exposure was confirmed through gene expression analysis of metallothionein-1 and 2, 2 well-known cadmium-induced genes. Analysis of sperm DNA methylation changes revealed 1788 differentially methylated sites present at regulatory regions in sperm of mice exposed to cadmium compared with vehicle (control) mice. Furthermore, most of these differential methylation changes positively correlated with changes in gene expression at both the transcription initiation stage as well as the splicing levels. Interestingly, the genes targeted by cadmium exposure are involved in several critical developmental processes. Our results present a comprehensive analysis of the sperm methylome in response to chronic cadmium exposure. These data, therefore, highlight a foundational framework to study gene expression patterns that may affect fertility in the exposed individual as well as their offspring, through paternal inheritance.

Aging-induced changes in sperm DNA methylation are modified by low dose of perinatal flame retardants.

Aims: Paternal age is increasing in developed countries. Understanding of aging-related epigenetic changes in sperm is needed as well as factors that modify such changes. Materials & methods: Young pubertal and mature rats were exposed perinatally to vehicle or environmental xenobiotic 2,2',4,4'-tetrabromodiphenyl ether. Epididymal sperm was reduced representation bisulfite sequenced. Differentially methylated regions (DMRs) were identified via MethPipe. Results: In control animals, 5319 age-dependent DMRs were identified. Age-related DMRs were enriched for embryonic development. In exposed rats, DNA methylation was higher in young and lower in mature animals then in controls. Conclusions: Sperm methylome undergoes significant age-dependent changes, which may represent a causal link between paternal age and offspring phenotype. Environmental xenobiotics can interfere with the natural process of epigenetic aging.

Sperm DNA methylation changes after short-term nut supplementation in healthy men consuming a Western-style diet.

Many environmental and lifestyle factors have been implicated in the decline of sperm quality, with diet being one of the most plausible factors identified in recent years. Moreover, several studies have reported a close association between the alteration of specific sperm DNA methylation signatures and semen quality. To evaluate the effect of tree nut consumption on sperm DNA methylation patterns in healthy individuals reporting eating a Western-style diet. This is a post hoc analysis conducted in a subset of participants (healthy, non-smoking, and young) from the FERTINUTS 14-wk randomized-controlled, parallel trial, recruited between December 2015 and February 2017. The participants included in the current study (n=72) were randomly selected in a proportion 2:1 from the original FERTINUTS trial between the 98 participants that completed the entire dietary intervention (nut group, n=48; control group, n=24). Sperm DNA methylation patterns were examined at baseline and after 14weeks in 48 individuals consuming 60g/d of mixed nuts (nut group) and in 24 individuals following the usual Western-style diet avoiding consumption of nuts (control group). Over the course of the trial, no significant changes in global methylation were observed between groups. However, in the nut group, we identified 36 genomic regions that were significantly differentially methylated between the baseline and the end of the trial and 97.2% of the regions displayed hypermethylation. We identified no such change in the control group over the same period of time. We also utilized the recently developed germ line age calculator to determine if nut consumption resulted in alterations to the epigenetic age of cells and no significant differences were found. Adding nuts to a regular Western-style diet subtly impacts sperm DNA methylation in specific regions, demonstrating that there are some sperm epigenome regions that could respond to diet.

A germ cell-specific ageing pattern in otherwise healthy men.

Life‐long sperm production leads to the assumption that male fecundity remains unchanged throughout life. However, recently it was shown that paternal age has profound consequences for male fertility and offspring health. Paternal age effects are caused by an accumulation of germ cell mutations over time, causing severe congenital diseases. Apart from these well‐described cases, molecular patterns of ageing in germ cells and their impact on DNA integrity have not been studied in detail. In this study, we aimed to assess the effects of ‘pure’ ageing on male reproductive health and germ cell quality. We assembled a cohort of 198 healthy men (18–84 years) for which end points such as semen and hormone profiles, sexual health and well‐being, and sperm DNA parameters were evaluated. Sperm production and hormonal profiles were maintained at physiological levels over a period of six decades. In contrast, we identified a germ cell‐specific ageing pattern characterized by a steady increase of telomere length in sperm and a sharp increase in sperm DNA instability, particularly after the sixth decade. Importantly, we found sperm DNA methylation changes in 236 regions, mostly nearby genes associated with neuronal development. By in silico analysis, we found that 10 of these regions are located in loci which can potentially escape the first wave of genome‐wide demethylation after fertilization. In conclusion, human male germ cells present a unique germline‐specific ageing process, which likely results in diminished fecundity in elderly men and poorer health prognosis for their offspring.

Paternal THC exposure in rats causes long-lasting neurobehavioral effects in the offspring

Developmental neurotoxicity of a wide variety of toxicants mediated via maternal exposure during gestation is very well established. In contrast, the impacts of paternal toxicant exposure on offspring neurobehavioral function are much less well studied. A vector for paternal toxicant exposure on development of his offspring has been identified. Sperm DNA can be imprinted by chemical exposures of the father. Most but not all of the epigenetic marks in sperm are reprogrammed after fertilization. The persisting epigenetic marks can lead to abnormal genetic expression in the offspring. We have found that paternal delta-9-tetrohydrocannabinol (THC) exposure in rats causes changes in methylation of sperm (Murphy et al., 2018). This is similar to cannabis-associated changes in sperm DNA methylation we found in human males who smoke cannabis (Murphy et al., 2018). In the current study we investigated the intergeneration effects of THC exposure of young adult male rats (0 or 2 mg/kg/day orally for 12 days) to the neurobehavioral development of their offspring. This paternal THC exposure was not found to significantly impact the clinical health of the offspring, including litter size, sex ratio, pup birth weight, survival and growth. However, it did cause a long-lasting significant impairment in attentional performance in the offspring relative to controls when they were tested in adulthood. There was also a significant increase in habituation of locomotor activity in the adult offspring of the males exposed to THC prior to mating. This study shows that premating paternal THC exposure even at a modest dose for a brief period can cause deleterious long-term behavioral effects in the offspring, notably significant impairment in an operant attention task. Further research should be conducted to determine the degree to which this type of risk is seen in humans and to investigate the mechanisms underlying these effects and possible treatments to ameliorate these long-term adverse behavioral consequences of paternal THC exposure.

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation

BackgroundSeveral independent research groups have shown that alterations in human sperm methylation profiles correlate with decreased fecundity and an increased risk of poor embryo development. Moving these initial findings from the lab into a clinical setting where they can be used to measure male infertility though requires a platform that is stable and robust against batch effects that can occur between sample runs. Operating parameters must be established, performance characteristics determined, and guidelines set to ensure repeatability and accuracy. The standard for technical validation of a lab developed test (LDT) in the USA comes from the Clinical Laboratory Improvement Amendments (CLIA). However, CLIA was introduced in 1988, before the advent of genome-wide profiling and associated computational analysis. This, coupled with its intentionally general nature, makes its interpretation for epigenetic assays non-trivial.ResultsHere, we present an interpretation of the CLIA technical validation requirements for profiling DNA methylation and calling aberrant methylation using the Illumina Infinium platform (e.g., the 450HM and MethylationEPIC). We describe an experimental design to meet these requirements, the experimental results obtained, and the operating parameters established.ConclusionsThe CLIA guidelines, although not intended for high-throughput assays, can be interpreted in a way that is consistent with modern epigenetic assays. Based on such an interoperation, Illumina’s Infinium platform is quite amenable to usage in a clinical setting for diagnostic work.

Perinatal exposure to low dose 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) alters sperm DNA methylation in adult rats

Polybrominated diphenyl ethers (PBDEs) are a group of ubiquitous reproductive toxins. Given that spermatogenesis requires extensive epigenetic changes, we hypothesize that PBDEs impact sperm DNA methylation. Pregnant Wistar rats were exposed perinatally to 0.2mg/kg 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and caudal epididymal sperm were collected from offspring on postnatal days (PNDs) 65 and 120. Libraries were prepared from sperm DNA and sequenced with an average of 18.0 million unique reads per sample. Differential methylated regions (DMRs) were identified via MethPipe package. BDE-47 exposure increased DNA methylation of epididymal sperm on PND 65 in genes, promoters and intergenic regions; however, on PND120 methylation decreased in these genomic elements. We identified 21 and 9 exposure-related DMRs in sperm collected on PND65 and PND120, respectively. Two DMRs overlapped between the two time-points. This is the first study to demonstrate that environmentally-relevant perinatal exposure to PBDE results in long-lasting changes in sperm DNA methylation.

Cooled-storage of equine semen does not induce major changes in sperm DNA methylation

A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 106 and 100 × 106 cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensation, immediately after processing and at 24 h-intervals until 72 h of storage. DNA cytosine methylation was assessed by ELISA after DNA extraction and denaturation. DNA methylation did neither change over time nor was affected by semen concentration. Total motility, progressive motility, membrane integrity, PST, mitochondrial membrane potential and chromatin condensation changed over storage time, but no differences between semen concentrations could be demonstrated. The results demonstrate that cooled-storage of equine semen does not induce changes in sperm DNA cytosine methylation. In cooled-semen of good quality, a concentration of 100 × 106 sperm/mL does not affect semen longevity. It can be concluded that a better fertility of cooled-stored than cryopreserved stallion semen is at least in part a result of only minor influences of cooled-storage on DNA integrity and methylation.

Cooled-storage of equine semen does not induce major changes in sperm DNA methylation

Cooled-storage of equine semen does not induce major changes in sperm DNA methylation

From the Cover: Sperm Molecular Biomarkers Are Sensitive Indicators of Testicular Injury following Subchronic Model Toxicant Exposure.

Traditional testis histopathology endpoints remain the gold standard for evaluating testicular insult and injury in a non-clinical setting, but are invasive and unfeasible for monitoring these effects clinically in humans. Assessing testicular injury in humans relies on semen and serum hormone analyses, both of which are insensitive and poor indicators of effect. Therefore, we hypothesized that sperm messenger RNA (mRNA) transcripts and DNA methylation marks can be used as translatable and sensitive indicators or testicular injury. Dose-response studies using adult male Fischer 344 rats subchronically exposed to model Sertoli cell toxicants (0.14, 0.21, and 0.33% 2,5-hexanedione, and 30, 50, and 70 mg/kg/day carbendazim), and a model germ cell toxicant (1.4, 3.4, and 5.1 mg/kg/day cyclophosphamide) for 3 months were evaluated for testicular injury by traditional histopathological endpoints, changes in sperm mRNA transcript levels using custom PCR arrays, and alterations in sperm DNA methylation via reduced representation bisulfite sequencing. Testis histopathological evaluation and PCR array analysis of the sperm transcriptome identified dose-dependent changes elicited by toxicant exposure (P < 0.05). Global sperm DNA methylation analysis of subchronic 0.33% 2,5-hexandione and 5.1 mg/kg/day cyclophosphamide exposure using a Monte Carlo approach did not identify differentially methylated regions (methylation difference > 10% and q < 0.05) with robust signatures. Overall, these results suggest that sperm mRNA transcripts are sensitive indicators of low dose toxicant-induced testicular injury in the rat, while sperm DNA methylation changes are not. Additionally, the Monte Carlo analysis is a powerful approach that can be used to assess the robustness of signals resulting from -omic studies.

14 EPIGENETIC CHANGES IN EQUINE SPERMATOZOA DURING CRYOPRESERVATION: A PRELIMINARY STUDY

The use of cryopreserved semen for insemination of mares facilitates breeding management but often results in reduced conception rates. This has been mainly attributed to changes in sperm membrane function caused by the freezing-thawing procedure. However, semen processing may also contribute to epigenetic changes in spermatozoa. In the present study, we therefore addressed changes in sperm DNA-methylation induced by cryopreservation of stallion semen. We hypothesised that the cryoprotectant may influence the DNA-methylation level of frozen-thawed semen. For this purpose, semen was collected from fertile Shetland pony stallions. Global DNA-methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) with a monoclonal antibody sensitive and specific for 5-methylcytosin after DNA extraction and denaturation (100 ng of DNA per sample). The level of 5-methylcytosin in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). Statistical analysis was done with the SPSS Statistics 21 software. Values are means ± standard error of the mean. In Experiment 1, 1.5 mL of raw semen (n = 6 stallions, 1 ejaculate each) was shock-frozen at –196°C for 15 min and thawed at 38°C for 60 s. Semen motility and membrane integrity were completely absent, while DNA-methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In Experiment 2, 3 ejaculates per stallion (n = 6) were included. Semen quality and DNA-methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (Minitube, Tiefenbach, Germany; cryoprotectant: 5% glycerol) or BotuCrio (Nidacon, Mölndal, Sweden; cryoprotectants: 1% glycerol and 4% methylformamid) extender. Semen was frozen in 0.5-mL straws in a computer-controlled rate freezer (IceCube 14 M; Sylab, Purkersdorf, Austria, cooling rates: 20°C to 5°C: 0.3°C min–1, 5°C to 25°C: 10°C min–1, –25°C to –140°C: 25°C min–1). Semen motility, morphology, and membrane integrity were significantly reduced (e.g. total motility before freezing: 88.8 ± 1.4%) by cryopreservation but not influenced by the extender used (e.g. total motility: Ghent 69.5 ± 2.0, BotuCrio 68.4 ± 2.2%; P &lt; 0.001 v. nonfrozen semen). Cryopreservation significantly (P &lt; 0.01) increased the level of DNA-methylation (before freezing: 0.6 ± 0.1%, Ghent 6.4 ± 3.7, BotuCrio 4.4 ± 1.5%; P &lt; 0.01), but no differences between the freezing extenders were seen. The level of DNA-methylation was not correlated with semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA-methylation level in stallion semen. In the present study, this effect occurred irrespective of the cryoprotectant but was not seen after shock-freezing in the absence of cryoprotectants. The reduced fertility of mares after insemination with frozen-thawed semen may at least in part be explained by methylation of sperm DNA, which occurs in response to the cryopreservation procedure.

Obesity-induced sperm DNA methylation changes at satellite repeats are reprogrammed in rat offspring.

There is now strong evidence that the paternal contribution to offspring phenotype at fertilisation is more than just DNA. However, the identity and mechanisms of this nongenetic inheritance are poorly understood. One of the more important questions in this research area is: do changes in sperm DNA methylation have phenotypic consequences for offspring? We have previously reported that offspring of obese male rats have altered glucose metabolism compared with controls and that this effect was inherited through nongenetic means. Here, we describe investigations into sperm DNA methylation in a new cohort using the same protocol. Male rats on a high-fat diet were 30% heavier than control-fed males at the time of mating (16–19 weeks old, n = 14/14). A small (0.25%) increase in total 5-methyl-2’-deoxycytidine was detected in obese rat spermatozoa by liquid chromatography tandem mass spectrometry. Examination of the repetitive fraction of the genome with methyl-CpG binding domain protein-enriched genome sequencing (MBD-Seq) and pyrosequencing revealed that retrotransposon DNA methylation states in spermatozoa were not affected by obesity, but methylation at satellite repeats throughout the genome was increased. However, examination of muscle, liver, and spermatozoa from male 27-week-old offspring from obese and control fathers (both groups from n = 8 fathers) revealed that normal DNA methylation levels were restored during offspring development. Furthermore, no changes were found in three genomic imprints in obese rat spermatozoa. Our findings have implications for transgenerational epigenetic reprogramming. They suggest that postfertilization mechanisms exist for normalising some environmentally-induced DNA methylation changes in sperm cells.

Genome-wide sperm DNA methylation changes after 3 months of exercise training in humans.

DNA methylation programs gene expression and is involved in numerous biological processes. Accumulating evidence supports transgenerational inheritance of DNA methylation changes in mammals via germ cells. Our aim was to determine the effect of exercise on sperm DNA methylation. Twenty-four men were recruited and assigned to an exercise intervention or control group. Clinical parameters were measured and sperm samples were donated by subjects before and after the 3-month time-period. Mature sperm global and genome-wide DNA methylation was assessed using an ELISA assay and the 450K BeadChip (Illumina). Global and genome-wide sperm DNA methylation was altered after 3 months of exercise training. DNA methylation changes occurred in genes related to numerous diseases such as schizophrenia and Parkinson's disease. Our study provides the first evidence showing exercise training reprograms the sperm methylome. Whether these DNA methylation changes are inherited to future generations warrants attention.