epiPIG

Several research groups, including members of the GECKO consortium, have shown that paternal diet prior to conception is associated with metabolic dysfunction in the offspring in several animal species. The epigenetic signature of spermatozoa from these species appeared to be amenable to nutritional challenges, such as under- or over-nutrition. Members of the GECKO consortium have also discovered that nutrition-mediated alteration of the sperm epigenome occurs in humans as well. Since the epigenetic signature of sperm plays a role in embryonic development, we hypothesise that diet-induced epigenetic changes to the spermatozoa can modulate health in the next generation offspring. While this phenomenon, called epigenetic inheritance, appears to be conserved from insects to humans, the specificity of the response in different species remains unknown.

To test if nutritional challenges have different impacts on the sperm epigenetic signature in various species, we will subject males of different species to dietary regimens, and examine the downstream epigenetic patterning in the spermatozoa. As well as offering a means to explore the taxonomy of sperm epigenetic signatures, testing the effect of nutritional stress in different animal species will allow us to conduct phylogenetically independent comparisons of the impacts of feeding ecology (e.g. trophic level – herbivore, omnivore, carnivore) and dietary composition on the sperm epigenome.

In epiPIG, we are interested in the mini-pigs as these animals share numerous similarities to human anatomy and whole-body metabolism. Even though mini pigs are somewhat distant to humans in the phylogenetic tree, mini-pigs are very prone to obesity after chronic feeding with a Western-style diet rich in fat, fructose and cholesterol.

We will conduct a dietary intervention trial utilising the mini-pig as a model organism to determine the role of chronic high-calorie dietary intake in males on the epigenetic programming of spermatozoa. We hypothesize that nutritional stress (overnutrition) modifies the sperm epigenome at genomic regions controlling embryo development, offspring metabolism, and cognitive function of the next generation.

Mini-pigs are stratified into one of two dietary groups, either a control diet or an experimental Western-Diet, enriched in saturated fat, cholesterol and refined carbohydrates. This Wester-type Diet is similar to those that have been administered to other species, and which trigger a variety of physiological responses including rapid weight gain, remodeling of epigenetic patterning in spermatozoa, and a modulation to the phenotype of downstream sired offspring. Diets used in this study were designed to be protein-matched in accordance with the protein leverage theory so that differential energy intake and weight gain is induced by an overconsumption of fat and carbohydrates.

The dietary intervention period lasts for 12 weeks, based on the duration of the spermatogenic cycle for mini-pigs and the expected time needed to induce a metabolic state of chronic nutritional stress. Mini-pigs are individually housed and receive fixed feeding rations. Following the 12-week intervention period, half of the animals are culled for tissue-specific epigenetic analyses, and the other half of the animals will undergo an additional 12-week washout period to determine, notably, if nutritionally induced epigenetic variation in sperm is reversible

Over the course of the intervention, biological samples and phenotypic information is collected at multiple time points. Semen and plasma samples are collected at baseline, three weeks into the intervention, after 12 weeks of intervention and at two time points during the washout period.

Fresh semen samples are analysed for standard semen quality parameters, such as sperm count and motility, then separated into fractions of mature spermatozoa and seminal plasma. Peripheral blood mononuclear cells (PBMCs) are isolated from whole blood. Plasma samples are used to assess the metabolic phenotype of the animals through analysis of a panels of biomarkers related to glucose metabolism (glucose, insulin, peptide-c, fructosamine) and lipid metabolism (total cholesterol, LDL, HDL, triglycerides, free fatty acids).

At the end of the intervention, tissue samples are collected for gene expression and histological analysis, including visceral and subcutaneous adipose tissue, liver, muscle, brain, pancreas, testicular and epididymal tissue. DNA methylation, small RNA profiling and chromatin structure analysis will be analysed to examine the impact of diet-induced nutritional stress on epigenetic information.