The effect of cannabis exposure on the entire sperm DNA methylome in rats

A new study examines how cannabis use among men could cause heritable effects on male fertility.

Introduction

Earlier research showed how heritable epigenetic changes occur following toxic exposures. The best known of these is sperm DNA modification, which is a heritable stable covalent change in gene expression without any change in the actual codons. DNA methylation is a key part of sperm formation and maturation but also offers an opportunity for potentially detrimental effects due to environmental exposures.

Cannabis use in pregnancy has been linked to neurodevelopmental, musculoskeletal, and cardiovascular problems in the offspring. Earlier research showed methylation changes in sperms following male cannabis use, with partial reversal once the drug was stopped. This is supported by animal experiments using the most potent cannabis derivative, tetrahydrocannabidiol (THC), demonstrating heritable methylation effects at a candidate autism gene, as well as issues with synaptic transmission, voluntary movements, and impaired cognition.

The current study, published online in the journal Epigenetics and Chromatin, focuses on the effects of cannabis use by the father in the preconceptional period in the form of persistent methylation changes in the whole epigenome of the offspring.

Using whole genome bisulfite sequencing (WGBS), the rat study looked at cannabis extract (CE) exposure for 28 days followed by 56 days washout (early exposure, EE); 28-day exposure to CE after the first 56 days of vehicle (late exposure, LE); and a control group. All were followed up for the same period. The 56 day period corresponds to a single spermatogenesis cycle.

What did the study show?

Late exposure, persistent changes

The study demonstrated differences in the extent of epigenetic modification via methylation with the time of exposure, even though the direction of change in both cases was the same. At both exposures, however, the average differences in methylation showed a strong correlation.

The 56-day washout period in the EE group of rats was associated with a marked decrease in methylation changes. “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.”

The magnitude of change was greater with late exposure compared to early exposure, whether hypo- or hyper-methylation. The average difference of 15.5% at the hypomethylated sites in the LE group contrasts with the 8.5% difference at the same sites in the EE group. For the hypermethylated sites, the corresponding differences were 14% vs. 7.5%, respectively.

The EE group showed methylation changes in some genes in the sperm and brain DNA, relative to the controls, that were not found in the LE group.

The damage may be to the spermatogonial stem cells, which pass on the resulting methylation change to the sperm derived from them. Sperms derived from spermatogonia at a later stage of development might have been resorbed during the washout period, which accounts for the above finding.

This could indicate persistent or even permanent changes following early exposure of the self-renewing progenitor pool of spermatogonia stem cells, despite the washout period. The detection of strongly similar methylation patterns in the sperm and some brain tissues of the CE-exposed rat offspring might indicate that paternal preconceptional CE exposure-related methylation changes can be passed down through generations.

Interestingly, most of the genes affected were those implicated in development.

Heritable changes

These changes in the methylation of sperm DNA were confirmed to be heritable. The sperm of the offspring born to CE-exposed males (LE group) showed a marked loss of methylation compared to control offspring. With the EE group, the results just fell short of significance.

The same methylation losses were also found in the Mtss1 gene in the hippocampus and nucleus accumbens (NAc), compared to the control offspring, with hypomethylation in 4/9 sites analyzed. This gene controls changes in synaptic transmission, which indicates the importance of even slight changes in methylation due to the resulting enormously magnified effects on gene expression.

Sex-dependent changes in gene expression

However, this did not reflect in a uniform effect on the expression of these genes. When analyzed by sex, male control offspring showed a negative association, while females showed a positive association. In CE-exposed offspring, males showed a positive and females a non-significant negative association.

Interestingly, the inverted patterns of association between gene encoding and methylation levels seen between male and female offspring of CE-exposed male rats reflect the actual physiologic differences in patterns of behavior and cognition between the offspring of either sex following paternal exposure to CE.

Cardiomegaly

CE-exposed offspring showed increased heart weight in both groups, but when separately analyzed, females showed significant increases in both groups, whereas males showed an increase of significance only in the EE group. Overall, the effect was significant only in females.

What are the conclusions?

The study demonstrates heritable epigenetic changes in DNA methylation of sperms in CE-exposed male rats. Secondly, it shows such changes being passed on to the offspring, in the sperm as well as brain areas involved in development. Despite a washout period corresponding to one spermatogenesis cycle, some of these changes persisted. Earlier research suggests this could be due to stress-induced changes in the sperms that affect their differentiation and maturation.

The study also showed cardiomegaly in the male and female offspring of preconceptionally CE-exposed male rats. 

Taken together, these results demonstrate that paternal preconception exposure to cannabis affects intergenerational outcomes. As cannabis legalization expands and consumption increases, it is imperative that we improve understanding of how exposure in one generation can shape health and disease of future generations.”

Further research is required in many areas touched upon by this study, such as the mechanism of heritability, how the placenta plays a role in transmitting the effects of this paternal exposure to the offspring, the mechanism of cardiomegaly in the offspring of the exposed rats, and whether these offspring show any other neurodevelopmental or behavioral effects in keeping with the modifications in gene expression.  

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.