Peri-conceptional undernourishment perturbs offspring sperm methylome

Whereas an organism’s genotype is relatively static throughout life, the epigenome is highly dynamic and can adapt, or be altered, in response to external environment such as diet. Intrauterine exposure to nutrient availability can alter the establishment of epigenetic marks, not only in the exposed individuals, but also in their offspring. Inheritance of such environmentally acquired phenotype to the next generation occurs through epigenetic modifications in the germline. Here, we evaluated by genome wide approach how modification of the maternal diet pre (14 days )- and post (28 days) conception can affect methylation status of the offspring male gamete. In particular, using a sheep model, we focused on the effect of maternal undernutrition on adult sperm methylation and its long-term consequences on sperm physiology and quality. Moreover, we investigated if supplementation of folic acid, to increase the availability of methyl donors, could prevent or ameliorate the adverse uterine environment caused by maternal undernutrition. Male lamb obtained from mother subjected to different nutritional regimen (UND:undernutrition; FA:undernutrition and Folic acid supplementation) appear normal at birth, with a comparable body weight till day 30 post-partum. Sperm DNA methylation, obtained by Reduced Representation Bisulfite Sequencing, differed in offspring that experienced in utero undernutrition (UND and FA) compared to control group (CTR). In particular, the number of differentially methylated regions (DMRs) was lower when UND and FA groups were compared each other, whereas a higher number of DMRs was observed by comparison of CTR with both experimental groups. In addition, a high percentage of DMRs were shared between UND and FA groups when compared with CTR, clearly indicating a influence of maternal nutrition on the offspring sperm DNA methylation rearrangement. Gene ontology (GO) analysis showed variation in functional categories related to sperm functionality such as chondroitin sulfate synthesis, potassium ion import, and others related to metabolism (biotin and glucagon). Furthermore, using Computer Assisted Semen Analysis and flow cytometric measurement, we have found a significant alteration of the following sperm parameters: low sperm motility index and higher incidence of chromatin structure alterations was reported for spermatozoa collected from UND and FA groups compared to CTR. Finally, to verify the effect of such reported abnormality on lamb fertility we have used these semen for in vitro embryo production. While we have obtained good quality blastocyst from all three groups, a reduction in the percentage of embryo development, partially compensated in the FA group, was found using spermatozoa from UND rams. Taken together, our results confirm that a nutritional stress during early mammalian development can leads to epigenetic modification in the offspring. This damage can be partially ameliorated with folic acid supplementation, however some alteration still persists in the germline and could be passed to the next generation, with still little known consequence.