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The mean library insert size for the WGBS was 195.9 bp (SD 8.3); mean coverage was 20.2 (2.1); and the mean GC content was 22.7 (0.2). We compared methylation differences from the late exposed (LE) versus control dataset to the methylation differences in the early exposed (EE) versus control dataset to determine the role of the 56-day wash-out period on sperm DNA methylation changes. No CpG sites remained significant following conservative Bonferroni correction, so we imposed a methylation difference threshold on the top 10 K sites, retaining only those with a > 10% methylation difference in the LE relative to control datasets. This resulted in 3321 nominally significantly differentially methylated CpG (dmCpG) sites. We then analyzed methylation at those same 3321 dmCpG sites in the EE relative to control dataset. Regardless of the exposure timing, the direction of methylation change at these sites is largely the same. Linear regression of the data showed significant correlation between LE and EE mean methylation differences relative to controls (Fig. 1A, p  < 0.0001, R² = 0.82).

We separated the data based on the direction of methylation change present at the 3321 CpG sites from the LE compared to control dataset. This resulted in 1844 hypomethylated CpG sites and 1477 hypermethylated CpG sites in the exposed animals relative to the controls. Comparison of the methylation change at each CpG site shows that the magnitude of the methylation difference relative to controls is greater in the LE sperm than it is at the same CpG sites for the EE sperm for both the hypomethylated (Fig. 1B) and hypermethylated CpGs (Fig. 1C).

The mean methylation difference relative to controls across the 1844 hypomethylated CpG sites was 15.5% in the LE dataset which contrasts significantly with the 8.5% difference at the same CpG sites in the EE dataset (Fig. 1B, p < 0.0001). Similarly, there was a 13.9% mean methylation difference relative to controls across the 1477 hypermethylated CpG sites in the LE dataset as compared to a 7.5% difference at the same CpG sites in the EE dataset (Fig. 1C, p < 0.0001). These differences remained significant following Bonferroni correction for the number of CpG sites analyzed.

Ingenuity Pathway Analysis (IPA) software was used to interrogate the genes associated with the top dmCpGs from both datasets. Briefly, for those CpG sites with a greater than 10% methylation difference relative to controls, an unadjusted p-value threshold of 5.0X10⁻⁵ was implemented in both the LE and EE datasets to restrict the number to those with the greatest significance. This resulted in a list of 744 CpG sites from the LE dataset and a distinct list of 317 CpG sites from the EE dataset. Notably, only five CpG sites were in common between these two lists. IPA recognized and included 492 and 203 LE and EE dataset genes, respectively, in the downstream analyses.

We began with the IPA-generated list of 500 significant functions categories and their associated annotations for the LE dataset. Given the overrepresentation of background information associated with cancer in the IPA Knowledge Base, we removed categories associated with cancer from our analysis, as has been reported previously [19]. We also removed terms associated with only a single gene. This left 289 categories and their associated disease or function annotations. We then analyzed the data by first looking at categories with ten or more annotations (Fig. 2A, top). These categories included “cardiovascular system development, function, and disease”, “embryonic development”, “nervous system development, function, and disease”, and “developmental disorder”. We also identified the top ten most significant disease or function annotations (Fig. 2A, bottom).

From both the LE and EE datasets, distinct genes of interest were identified for independent validation. Sites were selected based on > 10% methylation difference, having ≥ 2 significantly dmCpG sites within a 100 bp region, the associated gene having a role in early life developmental processes, and the CpG site being located within the body of the gene. The following genes were selected: Homeobox Protein Hox-B9 (Hoxb9), Methyltransferase Like 11B (Mettl11b), Slit Guidance Ligand 2 (Slit2), LDL Receptor Related Protein 1 (Lrp1), Citron Rho-Interacting Serine/Threonine Kinase (Cit), Synaptotagmin (Syt17), Synaptonemal Complex Protein 3 (Sycp3), Gamma-Aminobutyric Acid Type A Receptor Subunit Beta2 (Gabrb2), Oncostatin M (Osm), 2-Phosphoxylose Phosphatase 1 (Pxylp1), Glutamate Ionotropic Receptor NMDA Type Subunit 2A (Grin2a), Synapsin III (Syn3), Netrin G1 (Ntng1), and Metastasis Suppressor 1-Like Protein 1 (Mtss1l). We confirmed performance of each of the pyrosequencing assays using defined mixtures of unmethylated and methylated DNAs (R² = 0.85–0.99, p < 0.0001–0.025, Additional file 1: Fig. S1).

Two-tailed t-tests for Osm, Gabrb2, Pxylp1, Ntng1, and Mtss1l, confirmed significant methylation changes relative to controls by pyrosequencing, in the same direction as the WGBS for the LE (Fig. 3A-E top, p < 0.05–0.005). We then broadened our analysis to assess methylation at all the CpG sites captured by the pyrosequencing assay to see if CE exposure similarly affected neighboring sites. We included sperm from both exposure groups to determine how the timing of the exposure impacted DNA methylation changes. A two-factor ANOVA—one factor being exposure status and one being CpG site—showed a significant main effect of exposure for Osm, Gabrb2, Pxylp1, Ntng1, and Mtss1l (p < 0.0001 for all genes, Fig. 3A-E bottom). Post hoc tests revealed no additional significant methylation changes for Osm.

To determine whether methylation changes in sperm of CE-exposed males were detectable in the next generation, we analyzed F1 tissues for methylation at CpG sites that were validated by pyrosequencing. We first examined Pxylp1 in F1 sperm by pyrosequencing of both exposure groups given the abundant levels of this gene’s protein product in the epididymis and in mature spermatids [25]. A two-factor ANOVA revealed a significant effect of exposure on DNA methylation in F1 sperm (p < 0.0001). Post hoc analysis revealed a significant loss of methylation in F1 sperm from animals born to LE cannabis fathers compared to controls (p < 0.05, Fig. 4A), and the difference approached significance when comparing the F1 sperm from EE and control offspring (p = 0.06).

We then analyzed methylation changes at Mtss1l in offspring hippocampus (Fig. 4B) and NAc (Fig. 4C) tissues. We observed a significant effect of paternal exposure on DNA methylation in both F1 tissues (p < 0.0001). Like the methylation changes present at these CpG sites in F0 sperm, post hoc tests revealed significant losses of methylation present in EE offspring relative to control offspring. In the hippocampus, four of the nine CpG sites analyzed were significantly hypomethylated relative to controls in the EE group (p < 0.05–0.01). This included one of the CpG sites (CpG site 1) that was initially identified via WGBS as being hypomethylated in the EE paternal sperm. In the NAc, EE offspring were hypomethylated relative to controls for four of the nine CpG sites analyzed (p < 0.05). This included the other CpG site that was initially identified via WGBS (CpG site 5) as being hypomethylated in the EE paternal sperm.

Our finding that there are heritable changes in DNA methylation for Mtss1l in brain tissues prompted us to investigate whether those methylation changes are functionally related to changes in gene expression. We first examined this relationship in hippocampal tissue of the EE and control offspring at Mtss1l CpG site 1—one of the two sites initially identified via WGBS as being affected in paternal sperm. Pearson correlations showed no significant relationship between DNA methylation and gene expression for control or exposed offspring (Additional file 3: Fig. S3A). We stratified this analysis by sex (Additional file 3: Fig. S3B and S3C) given the known sex differences in hippocampal function [26, 27] and still observed no significant relationships between DNA methylation and gene expression.

In the NAc, we examined the relationship between DNA methylation at CpG site 5 (the second site initially identified via WGBS in sperm) and gene expression for the control and EE offspring. There were no significant relationships between methylation and expression when both sexes were analyzed together (Fig. 5A). However, known sex differences in the NAc [28, 29] led us to again stratify the analysis by sex. In males (Fig. 5B), control offspring showed a significant inverse methylation-expression relationship (p < 0.05, R² = 0.61), while exposed offspring had a significant positive methylation-expression relationship (p < 0.05, R² = 0.53). In females (Fig. 5C), control offspring showed a significant positive methylation-expression relationship (p < 0.05, R² = 0.52), while exposed offspring showed an inverse though non-significant relationship.

There is growing evidence associating prenatal cannabis exposure and teratologies in babies and children and early life exposure to cannabis has been associated with cardiovascular defects in epidemiologic and animal studies [14, 30, 31]. We measured heart weights and body weights of each of the F1 offspring from the EE, LE and control fathers and normalized heart weight to body weight. One-factor ANOVA revealed a significant effect of paternal exposure on offspring heart weight (p = 0.0039). Post hoc tests showed significant increased heart weight relative to controls for both the EE offspring (p = 0.0013), and the LE offspring (p = 0.0099) (Fig. 6A). Based on physiological differences in cardiovascular function and disease in males and females [32, 33], we separated this analysis by sex. A one-factor ANOVA in females (Fig. 6B) showed a significant effect of paternal exposure on offspring heart weight (p < 0.05). Post hoc tests showed significant increases in heart weights for both EE (p < 0.05) and LE (p < 0.05) offspring. In males (Fig. 6C), a one-factor ANOVA just approached significance for the effect of paternal exposure status on offspring heart weight (p = 0.05). Post hoc t-tests showed a significant increase in heart weight relative to controls only in the EE offspring (p < 0.05).

The broad goals of the present study were to characterize the effects of paternal CE exposure on the entirety of the sperm DNA methylome in a rat model of exposure, and to determine if such effects are heritable. We did not detect significant methylation differences via WGBS after implementing a conservative Bonferroni p-value threshold. This may have been due to insufficient sample size and/or insufficient coverage. As a result, we used unadjusted p values, prioritizing based on stringent thresholds to enrich for bona fide differences.

The results showed that, regardless of the timing of the exposure, most of the methylation changes occurred in the same direction in both groups relative to the controls. We next addressed the impact of the EE-specific “washout” period on the magnitude of the methylation changes present between exposed and control animals. We found that these methylation differences were dramatically diminished in the EE animals relative to the LE animals. This supports the hypothesis that exposure cessation for the duration of at least one spermatogenic cycle is effective at minimizing many of the CE-induced epigenetic effects in sperm.

However, a distinct group of CpG sites were significantly differentially methylated relative to controls in the EE dataset that were not present in the LE dataset. These results suggest that some early exposure changes persist during the wash-out period when most other changes were minimized, and that these particular changes were specific to the early exposure period. Thus, it is possible that the spermatogonial stem cells themselves were the target of this exposure and sperm derived from these particular progenitors harbor the epigenetic modifications. Any changes that may have affected only the post-spermatogonial sperm from the early exposure (and thus might resemble changes in the late exposed sperm) might no longer have been present at the time of WGBS given the resorption of sperm over the 56-day wash-out period. Importantly, if this is true, then the persistent EE-associated changes are possibly permanent, as the progenitor pool undergoes self-renewal throughout the reproductive life of a male [34]. Different epigenetic architectures and different patterns of gene expression in these cell types and phases of spermatogenesis may have further resulted in distinct CpG sites being differentially methylated.

We used IPA to determine functional relevance of the genes with the dmCpG sites. We analyzed disease and function annotations associated with the input gene lists for both datasets. There were some notable commonalities across the LE and EE analyses, including “nervous system development, function, and disease”, “developmental disorder”, and “embryonic development”. There were also notable differences. In the LE results, “cardiovascular system development, function and disease” was among the top annotations, while for the EE results there were multiple annotations associated with behavior, including spatial learning, exploratory behavior, and cognition, as well as functions associated with embryonic development including development of sensory organ, formation of eye, and lens formation.

It is interesting that, regardless of the timing of the CE exposure, there were shared functions of the genes that were significantly differentially methylated. That we saw significant terms associated with early developmental processes supports our previous findings from studies assessing the effect of THC exposure on sperm DNA methylation [18]. In that study, we found significant KEGG pathway terms associated with early developmental processes including the hippo signaling pathway. Further, we previously provided initial evidence in support of the hypothesis that genes involved in early developmental processes are increasingly sensitive to environmentally induced epigenetic disruptions [35]. The IPA findings from this study provide additional evidence in support of this hypothesis.

We next sought to validate methylation changes at select genes identified via WGBS using bisulfite pyrosequencing. At those genes that did validate WGBS findings, we asked whether methylation changes were heritable. Whether DNA methylation changes in sperm are heritable and detectable in the next generation is one of the most critical and yet incredibly challenging questions to address. We assume that a subset of sperm is randomly impacted by preconception cannabis exposure, since methylation changes in the sperm population are not universal at any given CpG site. For detection in the next generation, one of the affected sperm must successfully fertilize the egg. Further, the methylation change(s) would need to resist post-fertilization reprogramming to persist in the developing embryo where the change is then expected to be propagated across the three germ layers. This also requires that the exposure-related methylation change does not impede the sperm’s ability to successfully fertilize an egg or the viability of the embryo in order to be detectable. Of particular relevance, studies from De Domenico [36] and Innocenzi [37] have demonstrated that in vivo exposure to the synthetic cannabinoid receptor agonist JWH-133, induced altered DNA methylation at paternally imprinted genes in sperm. These changes were subsequently detectable in placental tissues and were associated with placental and embryonic defects [36, 37].

We examined F1 sperm for Pxylp1 methylation changes given the high levels of expression of this gene in the epididymis and its enhanced presence in mature spermatids [25]. Pxylp1 is involved in the synthesis of mature glycosaminoglycan chains and possesses hydrolase and phosphatase activity [38]. It has been associated with rare diseases such as nemaline myopathy, a hereditary congenital neuromuscular disorder characterized by muscle weakness, impaired speech abilities, and hypoventilation [39]. There was significant hypomethylation in sperm from the F1 offspring of the LE animals compared to controls; a methylation change that remarkably resembles that observed in the F0 LE paternal sperm. These data are consistent with intergenerational transmission of this paternal preconception exposure. It further demonstrates that the F1 generation’s sperm population is primed to pass this epigenetic alteration to the F2 generation, indicating the potential for transgenerational inheritance.

Given the involvement of Ntng1, Gabrb2, and Mtss1l in neuronal processes, we examined F1 hippocampal and NAc tissues to determine if the sperm methylation alterations are transmissible to the F1 generation. We observed changes in Mtss1l DNA methylation in both the F1 hippocampus and NAc comparable to the father’s sperm, consistent with intergenerational transmission of this epigenetic effect. There was an interesting temporal observation for this gene, where in both generations, only the EE fathers and their offspring exhibited methylation changes while there were no meaningful changes in the LE paternal sperm or F1 tissues. The most parsimonious explanation for these results is that the F0 spermatogonial stem cells were altered, since these changes were only evident in the exposed fathers in which there was sufficient time for the sperm derived from the spermatogonia to have been produced and matured. Methylation changes present in their offspring, led us to next assess the relationship between DNA methylation and Mtss1l gene expression in the F1 brains.

Analysis in the hippocampus by F0 exposure group did not reveal significant relationships between methylation and expression, and no meaningful differences were found between offspring of exposed versus control animals. However, there are sex differences in hippocampal function, which prompted us to separate male and female offspring for analysis. Control males showed a positive correlation between methylation and expression, while in the females, there was an inverse relationship. Interestingly, paternal preconception exposure to cannabis—followed by a wash-out period—caused these relationships to reverse, where male offspring now displayed an inverse relationship and females had a positive relationship between methylation and expression. These relationships were not statistically significant; however, given that Mtss1l plays a role in regulating synaptic plasticity, even slight methylation-mediated changes in expression could have important functional consequences. Indeed, we have previously shown that a 1% change in methylation can double or halve the level of transcription of the gene encoding IGF2 [40].

The NAc and hippocampus exhibited similar patterns of methylation-expression relationships. When we stratified analysis by sex, control males had an inverse methylation-expression relationship that was significant, while control females had a significant positive relationship. Paternal preconception cannabis exposure resulted in a significant positive Mtss1l methylation-expression relationship in the male EE offspring in the NAc, and an inverse relationship in the EE female offspring. This was evident despite the wash-out period following the paternal exposure.

Literature on Mtss1l is sparse, but recent work has focused on the role of this I-BAR domain-containing gene in regulation of plasma membrane dynamics, dendritic spine formation, and postsynaptic currents [41]. Studies have demonstrated that exercise alters expression regulation of Mtss1l and have identified this gene as a novel effector involved in synaptic rearrangement in an exercise-dependent manner [41]. The ability of the Mtss1l function to be influenced by exercise raises questions about its vulnerability to other types of influences, as we have found here with cannabis. In other studies, Mtss1l mutations have been associated with severe neurodegeneration and brain iron accumulation (NBIA) [42]. While this area of research is still growing, the role of epigenetics in this syndrome is not yet understood [42]. However, abundant literature suggests a role of the environment in neurodegenerative disorders, which may be mediated in part via epigenetic mechanisms [43]. There is also a role for mitochondrial dysfunction in NBIA [42]. This presents another possible avenue for future study with regard to epigenetics, since cofactors necessary for the proper establishment and maintenance of the epigenome are supplied by the mitochondria [44].

We were especially intrigued by the effect of paternal preconception CE exposure on sex-specific relationships between Mtss1l DNA methylation and gene expression. Increasing numbers of studies report sex differences across the epigenome, particularly in brain tissues [45,46,47,48,49]. Epigenetic mechanisms play important roles in maintaining these innate sexual dimorphisms, and disruption of these epigenetic regulatory states can alter brain development and sexualization [50]. Rat models of early life exposure to bisphenol A have shown altered brain sexualization [50], and preconception and perinatal exposures to multiple chemicals and drugs have sex-specific behavioral effects on offspring [51]. In fact, paternal exposure to CE results in impaired working memory in female but not male offspring, and decreased attention behaviors in males but not females [24]. Though we did not previously know if there are inherent epigenetically regulated sexual dimorphisms in these regions of the brain that regulate Mtss1l expression, it was a surprising finding to see the relationships between methylation and expression become inverted and reflective of the normal patterns seen in the opposite sex, as a result of paternal preconception cannabis exposure.

The final endpoint that we assessed in this study was physical abnormality in F1 offspring. Studies have demonstrated an association between perinatal cannabis use and cardiovascular teratologies in offspring [14, 30, 31]. One Hawaiian study found prenatal cannabis exposure was associated with multiple cardiovascular defects in babies [30]. An epidemiologic study by Wilson et al. found that paternal cannabis use is associated with a congenital heart defect in which the positions of the aorta and pulmonary artery are interchanged in offspring [31].

We observed a significant increase in heart weights after adjustment for body weight of offspring born to fathers exposed to CE prior to mating. This effect was significant for offspring born to both EE and LE fathers for females. Surprisingly in males this phenotype was only significant in offspring born to the EE fathers. While we do not understand the mechanism, this cardiomegaly finding is in line with studies associating paternal preconception and perinatal cannabis exposures with cardiovascular defects in offspring. Future studies examining morphological, histological, and expression changes in heart tissues are needed to confirm these findings.

It is curious that our cardiovascular findings and the heritable changes in DNA methylation and gene expression were mostly in offspring of fathers exposed to CE with a 56-day wash-out period prior to mating. This raises important questions about the mechanisms underlying heritability—whether attributable to DNA methylation alone, or if other epigenetic mechanisms might also be at play. One study that might provide additional clues examined paternal preconception exposure to stress, with one week or 12 weeks between the end of the stress and the initiation of mating [52]. Authors found that offspring neurodevelopment and adult stress reactivity were impacted by paternal preconception exposure to stress when there was a 12-week recovery period following stress, compared to those animals born to mice with just a one-week recovery [52]. The authors further demonstrated that this was mediated by stress-induced changes present in the epididymal epithelial cell extracellular vesicle cargo, including noncoding RNAs, that influence sperm maturation [52]. Thus, future studies of paternal preconception exposure to cannabis should investigate the interactions between DNA methylation and other epigenetic mechanisms, as well as extracellular vesicles, and how this might influence offspring outcomes.

One additional mechanism that should be investigated in future studies is the role of the placenta in mediating the effects of paternal exposure on offspring epigenetic and phenotypic outcomes. Gene expression in the placenta is enriched for paternally expressed imprinted genes, whose methylation may be altered following a paternal exposure such as CE [53]. Given that the placenta plays critical roles in regulating fetal growth and development and that the placental methylome changes in response to environmental cues [54], the interaction between the sperm epigenome and the placental epigenome should be investigated and any downstream effects on fetal developmental processes should be studied further.

Our study has several limitations. First, sample sizes were somewhat small for our paternal exposure model—there were 12 animals per group. Second, it is possible that a 56-day wash-out period was not sufficient for the effects of the CE exposure to be fully resolved. Longer wash-out periods should be examined to determine epigenetic mitigation effects. Third, while we targeted 60X coverage for our WGBS, the effective coverage averaged 20X for our study. Thus, we may not have had sufficient depth to detect all meaningful changes or to achieve significance following Bonferroni correction. Future work is needed to investigate mechanisms underlying the observed cardiomegaly phenotype, and to determine whether any of the epigenetic and gene expression changes observed in offspring correspond to any changes in offspring neurodevelopment or behavior.