 generations. We identified common altered DNA methylation signatures and small non-coding RNA expression profiles in the spermatozoa from F0 and F1 males, providing a mechanism for the propagation of metabolic dysfunction to the next generation. The predicted pathways affected by these epigenetic marks were perturbed in metabolic tissues of the offspring. Our results support the existence of transgenerational reprogramming of the gametic epigenome and inheritance of diet-induced metabolic dysfunction throughout two generations.

Animal care
Male and female SpragueeDawley founder rats were obtained from Charles River Laboratories (Germany). At 4 weeks of age, F0 male breeders were fed either with a high-fat diet (HFD; TD.88137/ TD.08811, 42/45% energy from fat, Harlan Laboratories, USA) or a control chow diet (R36-Lab For Lactamin, Sweden) for 12 weeks ( Figure 1A). Water and food were provided ad libitum. To obtain the F1 offspring, one F0 male breeder was housed together with a 12 week-old female rat, with free access to chow diet from 7:00 to 18:00, for 8 consecutive days. Male F0 breeders returned to their respective cages with the original diet, while F0 female rats consumed only chow diet throughout mating, gestation and lactation. To assure that there were no differences between female breeders, body and tissue weight, as well as blood glucose levels were evaluated (Table T2). To control for postnatal nutrition, litter sizes were standardized to 12 pups at day-1 after birth. At day-3, all pups were weighed and at day-5 litter sizes were reduced to 8 pups (4 males and 4 females). The pups were weaned from mothers at 21 days of age, and male or female siblings were housed together and fed a chow diet. At week 10 of age, one of the F1 siblings was subjected to a HFD for 12 weeks, and another sibling was kept on chow diet (control group) ( Figure 1A). To generate F2 offspring, only chow-fed F1 male rats were mated with 12 week-old females from an independent line. During mating, one F1 male was housed with one female for 10 consecutive days. Exactly as for F1, the F2 offspring litter size was measured and pups were weaned to a chow diet at 21 days of age. At week 10, a subgroup of the F2 offspring was also subjected to a HFD treatment for 12 weeks ( Figure 1A). Body weight and food intake of F0 male breeders and F1 and F2 offspring were monitored weekly by collecting and weighing offered and remaining food. All the rats were maintained under a temperature-controlled environment and 12:12-h light:dark cycle at the animal facility at the Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. All the experimental procedures were approved by the animal ethical committee, Stockholm North, and conducted in accordance with regulations for protection of laboratory animals. All the experiments were repeated using two independent cohorts of F0 breeders and F1 and F2 offspring. Statistical analysis related to transgenerational effects was made considering one sibling per litter. If more than one sibling per litter was used, the number is stated in the figure legend.

Intraperitoneal glucose tolerance test (ipGTT)
The ipGTT was performed in the F0 male breeders and F1 and F2 offspring after 12 weeks under the assigned diet (control or HFD). Rats underwent an overnight fast (15 h) prior to the ipGTT. On the day of the experiment, the animals received an intraperitoneal injection of 2 g glucose/kg body weight. The blood collected from the tail was used to measure glucose levels at basal (0), 15, 30, 60, 90 and 120 min after glucose injection. Blood glucose levels were measured using Accu Check Advantage Glucometer (Roche) and insulin levels were measured using an ELISA kit (see details below).

Intraperitoneal insulin tolerance test (ipITT)
The ipITT was performed in the F0 male breeders after 12 weeks on the assigned diet. On the day of the experiment, food was removed from the cages 3 h prior to the ipITT. Insulin (Actrapid, Novo Nordisk; 1 U/kg) was administrated intraperitoneally. The blood collected from the tail was used to measure the blood glucose levels at basal (0), 10, 30, 45, 60 and 90 min after insulin injection. A different subset of animals underwent the glucose tolerance test or the insulin tolerance test to avoid stress response associated to injections and blood sampling.

Endpoint
Rats were subjected to food deprivation from 4 h prior to the termination. Animals were anesthetized with sodium pentobarbital (100 mg/ kg, ip). Blood was collected and the plasma was stored at À20 C until use. Tissues were harvested, snap-frozen in liquid nitrogen and stored at À80 C until use.

Blood analysis
Blood insulin and leptin levels were measured using the ultrasensitive rat ELISA kits (#90060 and #90040, respectively, Crystal Chem Inc., USA). Blood triglycerides and free fatty acid levels were determined by colorimetric assays (#ab65336 and #ab65341, Abcam, UK). The assays were performed according to manufacturer's instructions.
2.6. Pancreas optical projection tomography (OPT) The entire pancreas from 5 day-old F1 female offspring was fixed in 4% paraformaldehyde (PFA) for 2 h at 4 C, followed by a subsequent dehydration in methanol 33%, 66% and 100% for 15 min at each step. To quench the autofluorescence, tissues were incubated for 24 h in a methanol:DMSO:H2O2 (2:1:3) solution, followed by two washes in methanol for 30 min. Samples were brought to À80 C for 1 h and brought back to room temperature 3 times to ensure that antigens in the deeper parts of the tissue were rendered accessible. Samples were rehydrated in Tris-Buffered Saline and Tween 20 buffer (TBST) 33%, 66% and 100% for 15 min at each step, and blocked in 10% rat serum in TBST for 24 h. Samples were incubated for 48 h with primary insulin antibody, followed by 48 h incubation with secondary antibody. Finally, tissues were mounted in 1% low melting agarose and dehydrated in 100% methanol for 24 h. Samples were washed in BABB solution (1:2 Benzyle alcohol, Benzyle benzoate) for 24 h, scanned, visualized and quantified in a stereomicroscope equipped for epifluorescence fluorochrome (Alexa 488, Alexa 594, Cy 3).

Isolation of motile spermatozoa
The cauda epididymis was dissected from the anesthetized animal and punctured in a Petri dish containing sperm isolation buffer (Earle's Balanced Salt Solution, 25 mM Hepes, 48.5 mM bovine serum albumin) pre-warmed to 37 C. Samples were transferred to a 14 ml round bottom tube, overlaid with isolation buffer and subjected to a swim-up

Analysis of sequencing data
Analysis was performed in R using Bioconductor packages. Preprocessed reads of at least 15 nt were aligned to rat genome (rn4) with Bowtie [10]. Methylated DNA peaks were called by MACS2 peakcall [11]. Regions of differential methylation were identified using DiffBind. Rat mature miRNAs and precursor sequences were obtained from miRBase [12] version 20. piRNA sequences were downloaded from piRNABank [13]. Rat tRNAs were retrieved from UCSC genome browser [14]. tRFs were defined when more than 10% of all reads mapping to a particular sncRNA were identical to a tRNA and the overlapping sequence was used as a tRF. Before measuring differential expression, lowly expressed (less than one count per million reads) or inadequately represented (less than half of the samples) sncRNAs were excluded from the tests. Differential sncRNA expression was calculated by edgeR [15] with a p-value cut-off of 0.05 for miRNAs, and 0.05 or 0.01 for piRNAs and tRFs, in F0 and F1 respectively.
2.11. DNA methylation in WAT DNA was extracted using the DNeasy blood & tissue kit (Qiagen; Hilden, Germany). Fragmented DNA obtained by sonication was enriched for methylation by (MBD)-capture or used as input control for qPCR analysis. Specific primers for each target are described in supplementary Table T7. 2.12. Transcriptomic and correlation analysis Total RNA from white adipose tissue was isolated using the RNeasy Lipid Tissue kit (Qiagen). RNA quality was assured by the integrity of ribosomal subunits and lack of degradation by the Experion Automated Electrophoresis System (Bio-Rad Laboratories, CA, USA). Affymetrix whole-transcript expression analysis (GeneChip Rat Gene 1.1 ST) was used to measure transcripts differentially expressed between chowand HFD-fed F2 females from F0-CD and F0-HF fathers (n ¼ 6e7 per group; 1 sibling per litter). Analysis were performed at the Bioinformatics and Expression Analysis core facility (BEA) at Karolinska Institutet, Huddinge, Sweden, Data were normalized using the expression console plier method, followed by two-way ANOVA statistical analysis (p-values were corrected with the Benjamini and Hochberg method). Correlation analysis of predicted rat let-7c target genes found to be expressed in the gene array data was performed using R Project for statistical computing analysis. Gene ontology analysis of the genes inversely correlated with let-7c expression was performed using DAVID bioinformatics