DNA methylation-independent growth restriction and altered developmental programming in a mouse model of preconception male alcohol exposure

Abstract

The preconception environment is a significant modifier of dysgenesis and the development of environmentally-induced disease. To date, fetal alcohol spectrum disorders (FASDs) have been exclusively associated with maternal exposures, yet emerging evidence suggests male-inherited alterations in the developmental program of sperm may be relevant to the growth-restriction phenotypes of this condition. Using a mouse model of voluntary consumption, we find chronic preconception male ethanol exposure associates with fetal growth restriction, decreased placental efficiency, abnormalities in cholesterol trafficking, sex-specific alterations in the genetic pathways regulating hepatic fibrosis, and disruptions in the regulation of imprinted genes. Alterations in the DNA methylation profiles of imprinted loci have been identified in clinical studies of alcoholic sperm, suggesting the legacy of paternal drinking may transmit via heritable disruptions in the regulation of imprinted genes. However, the capacity of sperm-inherited changes in DNA methylation to broadly transmit environmentally-induced phenotypes remains unconfirmed. Using bisulphite mutagenesis and second-generation deep sequencing, we find no evidence to suggest that these phenotypes or any of the associated transcriptional changes are linked to alterations in the sperm-inherited DNA methylation profile. These observations are consistent with recent studies examining the male transmission of diet-induced phenotypes and emphasize the importance of epigenetic mechanisms of paternal inheritance beyond DNA methylation. This study challenges the singular importance of maternal alcohol exposures and suggests paternal alcohol abuse is a significant, yet overlooked epidemiological factor complicit in the genesis of alcohol-induced growth defects, and may provide mechanistic insight into the failure of FASD children to thrive postnatally.

Intergenerational Effects of Alcohol: A Review of Paternal Preconception Ethanol Exposure Studies and Epigenetic Mechanisms in the Male Germline

“…In addition to these effects demonstrating vulnerability of the adult sperm methylome to alcohol, animals exposed to prenatal alcohol exhibited increased CpG methylation at the pro‐opiomelanocortin (POMC) gene promoter in sperm that is associated with reduced POMC expression and suppression of its HPA axis mediating function in offspring (Govorko et al., ). Not all results support methylation changes in response to ethanol, as one recent study found that paternal voluntary alcohol consumption, which altered cholesterol trafficking and hepatic fibrosis markers in offspring, had no effect on DNA methylation in sperm (Chang et al., ). In addition, despite a number of studies demonstrating effects of environmental perturbations on methylation in sperm, the effect sizes of significant changes in methylation at individual cytosines are often small (e.g., ~10 to 20%), and thereby, the significance for cross‐generational inheritance is hard to discern, given the high penetrance observed in many preclinical paternal epigenetic inheritance models (Shea et al., ).…”

“…Thus, to test the hypotheses inspired by cross‐generational epidemiological data, preclinical investigators have utilized isogenic rodent strains under controlled laboratory conditions to directly test the effects of various paternal preconception exposures to a vast range of environmental insults on an equally expansive number of biological and behavioral measures in offspring. Most prominently featuring studies pertaining to dietary change, chronic stress, and various drugs of abuse, many paternal preconception exposures with rodents have now been found to directly impart complex physiological and behavioral phenotypes to offspring (for excellent reviews, see Rando and Simmons, , Chang et al., ; Goldberg and Gould, ). For instance, obese fathers confer deficits in glucose metabolism to offspring (Chen et al., ; Cropley et al., ; de Castro Barbosa et al., ; Fullston et al., ).…”

“…To date, there have been over 40 published paternal preconception alcohol exposure studies in rodents (for review, see Finegersh et al., ), most varying in species, route of administration, and duration. Despite these differences, some intergenerational (F1) effects have been consistent such as low fetal and birthweight (Bielawski et al., ; Chang et al., , ; Ledig et al., ), altered organ weights (Abel, ; Chang et al., ; Ledig et al., ; Lee et al., ), and increased number of runts (Bielawski and Abel, ; Bielawski et al., ). Additional phenotypes reported include increased cortical thickness (Jamerson et al., ), reduced testosterone (Abel and Lee, ), and altered neurotransmitter levels (Nelson et al., ).…”

Who's your daddy? Behavioral and epigenetic consequences of paternal drug exposure

“…Bdnf methylation and mRNA changes in VTA associate with lower sensitivity to alcohol‐induced anxiolysis and lower alcohol consumption in male offspring (Finegersh and Homanics, 2014). However, a recent study in mice found no changes in sperm‐inherited DNA methylation in sires after voluntary alcohol consumption (Chang et al, 2017). Thus, in some instances, alcohol‐induced changes to the sperm epigenome can have long‐term functional consequences in male offspring.…”

“…Paternal alcohol exposure induces several developmental aberrations. It reduces litter sizes (Emanuele et al, 2001a; Cicero et al, 1994a, b; Tanaka et al, 1982) and increases the number of runts (Bielawski et al, 2002; Meek et al, 2007; Bielawski and Abel, 1997; Abel, 1993; Chang et al, 2017), malformations (Mankes et al, 1982; Bielawski and Abel, 1997), and pup mortality (Cicero et al, 1994a, b; Meek et al, 2007) in rats and mice. Litters from alcohol‐exposed sires also exhibit increased (Emanuele et al, 2001b; Abel, 1995) or decreased male‐to‐female ratios (Abel, 1993).…”