Paternal over- and under-nutrition programme fetal and placental development in a sex-specific manner in mice
- Lifespan and Population Health, School of Medicine, University of Nottingham, United Kingdom
- Maternal and Fetal Health Research Centre, Faculty of Biology Medicine and Health, University of Manchester, St Mary’s Hospital, Oxford Road, United Kingdom
- Deep Seq, School of Life Sciences, Queen's Medical Centre, University of Nottingham, United Kingdom
- Nottingham Arabidopsis Stock Centre (NASC), University of Nottingham, Sutton Bonington Campus, Plant Science Building, School of Biosciences, United Kingdom
- Centre for Reproductive Health, Institute for Regeneration and Repair, University of Edinburgh, United Kingdom
- School of Medicine, University of Dundee, United Kingdom
- School of Veterinary Medicine and Science, University of Nottingham, United Kingdom
- Faculty of Health and Life Sciences, University of Bristol, United Kingdom
- Division of Clinical Medicine, School of Medicine and Population Health, University of Sheffield, United Kingdom
Figures
Figure 1
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 are presented as mean ± SEM and were analysed using a one-way ANOVA with Holm-Sidak post hoc tests for multiple comparison. *p<0.05, **p<0.01.
Figure 2
Impact of diet on male non-fasting metabolic status.
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. N=8 males in each group. Data are presented as mean ± SEM and were analysed using a one-way ANOVA with Holm-Sidak post hoc tests for multiple comparison. *p<0.05, **p<0.01.
Figure 3
Impact of diet on male gut microbiota.
(A) Faith’s phylogenetic diversity and (B) Pielou’s measure of species evenness. (C) Overall bacterial abundance at the phylum level and relative abundance of (D) Deferribacteres, (E) Protobacteria, and (F) TM7 phylum. (G) Relative (Z-score) abundance of bacteria at the family level 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). N=8 males in each group. Data are presented as box plots in A, showing trhe mean (solid line) and 95% data ranges, and as mean ± SEM in C–F. Data were analysed using either a one-way ANOVA (panels D, E, F, and G) or Kruskal-Wallis test (panels A, B, and C) with Holm-Sidak or Dunn’s post hoc tests for multiple comparison, respectively. *p<0.05, **p<0.01.
Figure 4 with 1 supplement
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 are presented as mean ±S EM in C and E–H. Data were analysed using either a one-way ANOVA (panels E, F, G, and H) or Kruskal-Wallis test (panel C) with Holm-Sidak or Dunn’s post hoc tests for multiple comparison, respectively. *p<0.05, **p<0.01.
Figure 4—figure supplement 1
Representative negative control images of testes showing (A) no primary antibody controls for Plzf (green) and Dddx4 (red) with DAPI (nuclei) shown in blue.
(B) No secondary controls for Plzf (green) and Dddx4 (red) with DAPI (nuclei) shown in blue. (C) No primary and (D) no secondary controls for Sox9 (green) with DAPI (nuclei) shown in blue.
Figure 5
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 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.
Figure 6
Impact of paternal diet on early (embryonic day [E]8.5) 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 haematoxylin and eosin (H&E)-stained whole E8.5 conceptuses. Ectoplacental cone (EPC) (C) invasion depth, (D) area, and (I) alignment. (F) Representative EPC outgrowth after 48 hr in culture, stained for α-tubulin. (G) Trophoblast and (H) EPC area at 24 and 48 hr time points. 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 are presented as mean ± SEM in A, C–E and G–H. 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.
Figure 7
Impact of paternal diet on fetal and placental weight in late (embryonic day [E]17.5) 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 are presented as mean ± SEM andwere analysed using a generalised linear mixed model analysis with paternal origin of litter and duration on respective diet incorporated as random effects (panels A, B, C, E, F, G, I, J, and K) or t-test (panels D, H, and L) following assessment for normality using a Shapiro-Wilk test. *p<0.05, **p<0.01, ***p<0.001.
Figure 8
Analysis of late gestation (embryonic day [E]17.5) 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-sectional 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 an E17.5 placenta at ×20 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 separate litters and stud males. Data are presented as mean ± SEM and were analysed using a one-way ANOVA (panels B, G–L) or Kruskal-Wallis test (panel B) with post hoc correction where appropriate.
Figure 9
Late gestation (embryonic day [E]17.5) 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) Heatmap 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.
Additional files
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Supplementary file 1
Ingredients and nutritional information of diets fed to male mice.
- https://cdn.elifesciences.org/articles/109392/elife-109392-supp1-v1.docx
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Supplementary file 2
Sexing PCR primer sequences.
- https://cdn.elifesciences.org/articles/109392/elife-109392-supp2-v1.docx
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Supplementary file 3
Late gestation placental RNA-seq data showing sexually dimorphic genes between males and females of the same parental diet group.
- https://cdn.elifesciences.org/articles/109392/elife-109392-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/109392/elife-109392-mdarchecklist1-v1.docx
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Paternal over- and under-nutrition programme fetal and placental development in a sex-specific manner in mice
eLife 15:RP109392.
https://doi.org/10.7554/eLife.109392.4
