Late gestation maternal overnutrition alters gene expression and histomorphology in neonatal foal testes

Abstract

Reproductive health of male offspring following perturbations in maternal nutrition is not well described in the horse; therefore, the objective of this study was to determine the effects of maternal overnutrition on neonatal foal testicular development. Sixteen Quarter Horse mares were used in a completely randomized design and stratified by expected foaling date, body weight, and body condition score into one of two dietary treatments beginning on gestational day 235: control (CON; n = 8) fed to meet nutrient requirements and overfed (HIGH; n = 8) fed 40% above CON. At 5 h postpartum, foals were euthanized, and testes from male foals were harvested, weighed, and stored for analyses. Samples were numerically coded in a blinded fashion and processed randomly to eliminate bias. Histomorphometric evaluations were performed following a hematoxylin & eosin stain and evaluated using ImageJ. All data were analyzed using the MIXED procedure in SAS v9.4. Horse within treatment serves as a random effect, with a main effect of treatment. For RNA sequencing, differential gene expression analysis used DESeq2 1.20.0 R package to compare gene expression levels between CON and HIGH, and P-values were adjusted using the Benjamini-Hochberg correction. The threshold for significance was set at P ≤ 0.05 with trends toward significance at 0.05 < P ≤ 0.10, with sequencing data using adjusted P-values. Stringency was set to include differentially expressed genes with an unadjusted P-value < 0.05 for pathway enrichment analysis. Testes from HIGH foals (n = 4) were 61% heavier than testes from CON foals (n = 4; P < 0.01). There was no difference in individual seminiferous tubule area (P = 0.76) or Sertoli cell number within tubule (P = 0.57); however, testes from HIGH foals had 2.3 times fewer tubules per 10× magnification field than CON foals (P = 0.01). Further, tubules represented a lesser percentage of image area relative to interstitial tissue in testes from HIGH foals (P = 0.05). RNA sequencing revealed 56 differentially expressed genes between HIGH and CON testes (P-adjusted < 0.05). Differentially expressed genes represented biological processes related to steroidogenesis, cell regulation, and energy metabolism. Thus, late gestation maternal overnutrition alters fetal testicular development with potential long-term implications to reproductive function.