Maternal peri-conceptional undernourishment perturbs offspring sperm methylome

in Reproduction

Correspondence should be addressed to P Toschi; Email:

*(P Toschi and E Capra contributed equally to this work)

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The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring. Sperm DNA methylation, measured by Reduced Representation Bisulfite Sequencing (RRBS), identified Differentially Methylated Regions (DMR) in offspring that experienced in utero undernutrition, both in UND (244) and FA (240), compared with CTR. Gene ontology (GO) analysis identified DMRs in categories related to sperm function, therefore we investigated whether the fertilizing capacity of the semen from the three groups differed in an in vitro fertilization assay. Spermatozoa from the undernourished groups showed lower motility and sperm chromatin structure abnormalities, represented by a higher percentage of DNA fragmentation and an increased number of immature cells, compared with CTR. While good quality blastocysts were obtained from all three groups, the proportion of embryos reaching the blastocyst stage was reduced in the UND vs CTR, an effect partially rescued by the FA treatment. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the semen of the resulting offspring, the effects of which in next generation remain to be elucidated.

Supplementary Materials

    • Supplementary file 1: Sequencing and methylation call statistics. For each sample (control (CTR1, 2, 3), utero undernourished (UND1, 2, 3) and utero undernourished with folic acid supplementation (FA1, 2, 3)), the sequence pairs analysed in total, the total number of cytosines analysed (Tot C), the percentage of cytosine methylated in CpG (mC in CpG), in CHG (mC in CHG), in CHH (mc in CHH) or other (CN) context are reported. H stands for A, T or C whereas N for any nucleotide.
    • Supplementary file 2: Venn diagrams representing the intersections between Highly Methylated Regions (HMRs) in control (CTR), undernutrition (UND) and undernutrition and Folic Acid supplementation (FA) animals in the three comparisons (CTR vs UND, CTR vs FA, UND vs FA) for reduced representation bisulfite sequencing (RRBS) and methylation enrichment and sequencing (Methyl-Seq) datasets. For each dataset, the intersect between 25th percentile of the HMRs was determined and calculated as a percentage.
    • Supplementary file 3: List of differentially methylated regions (DMRs) and DMR associated genes founded in the comparison between different experimental groups (CTR, UND, FA). We identified 244 DMRs in CTR vs UND, 240 DMRs in CTR vs FA and 106 DMRs in UND vs FA. Annotation of the 244, 240 and 106 DMRs identified 75, 69 and 38 DMR associated genes.


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