All mouse experiments were performed with C57BL/6J mice obtained from Charles River (Charles River Laboratories, France). All mice were housed in a temperature-controlled system and maintained on a 12 hr light/dark cycle (lights on at 7 a.m.). Experimental mice were given ad libitum access to either a high-fat high-sugar diet (WD) (U8954 version 205 HF 45% of energy from fat, SAFE, France) or a CD (SAFE A04, 5% of energy from fat, SAFE, France) and sterile water. To evaluate the impact of the diet of paternal ancestors on metabolic health, we developed two experimental models. On the one hand, WD feeding was maintained for five successive generations through the paternal line. Briefly, 3-week-old male mice were divided into two groups. Males from the first group were kept on CD, and the males of the other group were fed a WD for 3 months. This first generation of CD-fed and WD-fed males was named CD1 and WD1, respectively. At 4 months old, two independent males of WD1 group were then crossed with 7-week-old C57BL/6J female mice (CD-fed) obtained from Charles River (Charles River Laboratories, France) to generate WD2 offspring. One or two litters were obtained per male. The male progenies were kept and subjected to the same experimental procedure. Then, at least one male, selected randomly, fed a WD for 3 months from each family was mated with CD-fed females to generate WD3 offspring. The same procedure was repeated twice to generate the WD4 and WD5 offspring (Figure 1—figure supplement 1A). However, from the WD2 generation, a considerable heterogeneity with respect to total body weight mass was observed within the same litter (Figure 1—figure supplement 1C). For this reason and as illustrated in Figure 1—figure supplement 1A starting from this generation, more than one male per litter was chosen to mate. The complete experimental design was performed twice at approximately 6 months’ interval giving rise to four independent families. To demonstrate that we did not create significance with increasing the correlated sample, we selected a subset of 18 mice in WD4 and WD5 making sure that the four WD1 ancestors were equitably represented across the groups. Three combinations of 18 mice were selected and gave the same significant level with a p-value < 0.001 when total body weight mass or gWAT weight relative to total body weight of WD4 and WD5 groups was compared to the corresponding weight of CD group. One of these combinations is shown in Figure 1—figure supplement 2B. The same procedure was performed for the CD group (Figure 1—figure supplement 1B). However, in accordance with previous studies (Cropley et al., 2016; Massiera et al., 2010; Fullston et al., 2015; Zhou et al., 2018), body mass and gWAT weights of the CD-fed male progenies were very homogeneous (Figure 1—figure supplement 2D) and were combined in the same group.

Then, to determine whether the WD1 and WD5 phenotypes were paternally inter- and trans-generationally inherited in the absence of WD, four WD1 and nine WD5 males were crossed with a CD-fed female and their respective male and female progenies were fed a CD. The first generation was called F1-WD1 and F1-WD5, respectively. The F1 4-month-old male progenies were crossed with 7-week-old C57BL/6J female mice (CD-fed) to obtain the F2-WD1 and F2-WD5 progenies (Figure 3A). This experimental design was repeated once to obtain the F3-WD1 and F3-WD5 progenies. The control group of this experiment was obtained by crossing CD-fed males with 7-week-old C57BL/6J female mice (CD-fed) and maintained for four generations on CD.

To evaluate the role of sperm RNAs in transgenerational epigenetic inheritance of metabolic alterations, sperm RNAs extracted from two different CD, WD1, and WD5 males were microinjected into zygotes at the Center for Transgenic Models (University of Basel, Switzerland) following the same procedure as described in Gapp et al., 2014. The resulting progenies were called F1-RNA-CD, F1-RNA-WD1, and F1-RNA-WD5 progenies, respectively. F2-RNA and F3-RNA progenies were obtained after crossing F1-RNA and F2-RNA 4-month-old males, respectively, with 7-week-old C57BL/6J female mice (CD-fed) obtained from Charles River (Figure 5A).

All mouse experiments were conducted in accordance with the National and European legislations for the care and use of research animals.

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