Impact of diet on male growth.

(A) Mean weekly body weight of males fed either a control diet (CD), low protein diet (LPD, methyl donor-supplemented LPD (MD-LPD), Western diet (WD) or methyl donor-supplemented Western Diet (MD-WD). Mean (B) testicular weight, (C) seminal vesicle weight (D) total fat weight (combined weight of individual fat pads) and (E) ratio of white adipose tissue (WAT) to brown adipose tissue (BAT). Mean serum (F) Tnf and (G) inhibin β-A chain levels. N = 6-8 males in each group. Data were analysed using a one-way ANOVA with Holm-Sidak post hoc tests for multiple comparison. * P <0.05, ** P <0.01.

Impact of diet on male non-fasting metabolic status and gut microbiota.

Serum (A) glucose and (B) insulin in males fed either a control diet (CD), low protein diet (LPD, methyl donor-supplemented LPD diet (MD-LPD), Western diet (WD) or methyl donor-supplemented WD (MD-WD). Hepatic (C) cholesterol, (D) free fatty acids (FFAs) and (E) triglyceride (TG) concentrations. (F) Faith’s phylogenetic diversity and (G) Pielou’s measure of species evenness. (H) Overall bacterial abundance at the phylum level and relative abundance of (I) Deferribacteres, (J) Protobacteria and (K) TM7 phylum. (L) Relative (Z-score) abundance of bacteria at the family level. N = 8 males in each group. Data were analysed using either a one-way ANOVA or Kruskal–Wallis test with Holm-Sidak or Dunn’s post hoc tests for multiple comparison respectively. * P <0.05, ** P <0.01.

Impact of diet on male testicular morphology.

(A) Representative images showing tubule morphology in males fed either a control diet (CD), low protein diet (LPD, methyl donor-supplemented LPD (MD-LPD), Western diet (WD) or methyl donor-supplemented WD (MD-WD). (B) Examples of tubule anomalies identified in WD and MD-WD males including separation of the epithelium from the tubule basement membrane (as indicated by an arrow in i), appearance of vacuoles (as indicated by an arrow in ii), and loss of the germinal epithelium (as indicated by arrows in iii and iv). (C) Frequency of abnormal tubules per testis in males fed either CD, LPD, MD-LPD, WD or MD-WD. (D) Representative staining patterns for DAPI (nuclear counterstain), Sox9 (marker of Sertoli cells), Ddx4 (marker of spermatocytes and spermatids) and Plzf (marker of spermatogonial stem cells) in testes from males fed CD, LPD, MD-LPD, WD and MD-WD. (E) Number of DAPI, (F) Sox9, (G) Ddx4 and (H) Plzf+ cells. N = 7-8 males in each group. Data were analysed using either a one-way ANOVA or Kruskal–Wallis test with Holm-Sidak or Dunn’s post hoc tests for multiple comparison respectively. * P <0.05, ** P< 0.01.

Impact of diet on testicular gene expression.

Volcano plots comparing differential gene expression between males fed a control diet (CD) and males fed (A) a low protein diet (LPD), (B) methyl donor-supplemented LPD (MD-LPD) or (C) Western diet (WD). (D) Pathway and (E) network analysis of differentially expressed genes between CD and WD fed males. (F) Volcano plots comparing differential gene expression between males fed a control diet (CD) and males fed a methyl donor-supplemented WD (MD-WD). (G) Pathway and (H) network analysis of differentially expressed genes between CD and MD-WD fed males. N = 8 males in each group.

Impact of paternal diet on early (E8.5) placental development.

(A) Litter size at E8.5 from males fed either a control diet (CD), low protein diet (LPD), methyl donor-supplemented LPD (MD-LPD), Western diet (WD) or methyl-donor supplemented WD (MD-WD) prior to mating. (B) Representative images of H&E stained whole E8.5 conceptuses. Ectoplacental cone (EPC) (C) invasion dept, (D) area and (I) alignment. (F) Representative EPC outgrowth after 48 hours in culture, stained for alpha-tubulin. (G) Trophoblast and (H) EPC area at 24 and 48 hour timepoints. N = 10-13 litters in A, derived from a minimum of 8 separate stud males per group, and 4-9 conceptuses in C-H, each from a separate litter and stud male. Data were analysed using either a one-way ANOVA or Kruskal–Wallis test with Holm-Sidak or Dunn’s post hoc tests for multiple comparison respectively. * P <0.05.

Impact of paternal diet on fetal and placental weight in late (E17.5) gestation.

(A) Late gestation fetal weight and distributions above the 90th centile and below the 10th centile of control diet (CD) fetal weights from males fed either a CD, low protein diet (LPD), methyl donor-supplemented LPD (MD-LPD), Western diet (WD) or methyl donor-supplemented WD (MD-WD) prior to mating. Late gestation (B) female, (C) male and (D) intra-diet comparison of fetal weight. (E) Late gestation placental weight and distributions above the 90th centile and below the 10th centile of CD placental weights. Late gestation (F) female, (G) male and (H) intra-diet comparison of placental weight. (I) Late gestation fetal:placental ratio and distributions above the 90th centile and below the 10th centile of CD fetal:placental ratio. Late gestation (G) female, (H) male and (I) intra-diet comparison of fetal:placental weight ratio. N = 10-13 litters, derived from a minimum of 8 separate stud males per group. Data are presented as mean ± SEM or as box plots with the mean and individual data points outside of the 10-90th percentile. Data were analysed using a generalised linear mixed model analysis with paternal origin of litter incorporated as a random effect or t-test following assessment for normality using a Shapiro-Wilk test. * P <0.05, ** P <0.01, ***P <0.001.

Analysis of late gestation (E17.5) placental morphology in response to paternal diet.

(A) Representative image of a cross section through the late gestation (E17.5) placenta highlighting the decidua, junctional zone, labyrinth zone and chorionic plate regions. (B) Cross section area and (C) relative regional composition of placentas derived from males fed either a control diet (CD), low protein diet (LPD), methyl donor-supplemented LPD (MD-LPD), Western diet (WD) or methyl-donor supplemented WD (MD-WD) prior to mating. (D) Representative images of a E17.5 placenta at 20X magnification prior to the application of (E) estimation of compartment global volumes using Cavalieri’s Principle with a grid comprising a test system of points and (F) stereological method for estimating the surface density of maternal blood vessels and fetal capillaries using a grid comprising isotropic lines. E17.5 placental (G) volume, (H) labyrinth zone volume, (I) junctional zone volume, (J) maternal blood space volume, (K) maternal blood surface area and (L) fetal capillary surface area. N = 5 placentas per group, each from a separate litters and stud males. Data are presented as mean ± SEM and were analysed using a one-way ANOVA or Kruskal–Wallis test with post-hoc correction where appropriate.

Late gestation (E17.5) placental gene expression is altered in a diet and sex-specific manner.

(A) Differential gene expression and (B) pathway analysis from male placentas derived from males fed either a control diet (CD), low protein diet (LPD), methyl donor-supplemented LPD (MD-LPD), Western diet (WD) or methyl-donor supplemented WD (MD-WD) prior to mating. (C) Differential gene expression and (D) pathway analysis from female placentas. (E) Principal component analysis comparing CD male and female placental gene expression profiles. (F) Heat map of the 301 sexually dimorphic genes between male and female CD placentas and (G) the pathway analysis. Heatmaps showing the relative level of expression and clustering of the same 301 genes in (H) LPD, (I) MD-LPD, (J) WD and (K) MD-WD male and female placentas. Volcano plots highlighting the significantly (pAdj <0.05) upregulated (red) and downregulated (blue) differentially expressed genes between male and female (L) LPD, (M) MD-LPD, (N) WD and (O) MD-WD placentas. (P) Overlap of sexually dimorphic genes between male and female CD, LPD, WD and MD-WD placentas. (Q) Chromosomal locations of common X and Y chromosome sexually dimorphic genes between CD, LPD, WD and MD-WD placentas. N = 4 male and 4 female placentas, each from separate litters and stud males.