Preconceptional and in utero exposure of sheep to a real-life environmental chemical mixture disrupts key markers of energy metabolism in male offspring.

Over recent decades, an extensive array of anthropogenic chemicals have entered the environment and have been implicated in the increased incidence of an array of diseases, including metabolic syndrome. The ubiquitous presence of these environmental chemicals (ECs) necessitates the use of real-life exposure models to the assess cumulative risk burden to metabolic health. Sheep that graze on biosolids-treated pastures are exposed to a real-life mixture of ECs such as phthalates, per- and polyfluoroalkyl substances, heavy metals, pharmaceuticals, pesticides, and metabolites thereof, and this EC exposure can result in metabolic disorders in their offspring. Using this model, we evaluated the effects of gestational exposure to a complex EC mixture on plasma triglyceride (TG) concentrations and metabolic and epigenetic regulatory genes in tissues key to energy regulation and storage, including the hypothalamus, liver, and adipose depots of 11-month-old male offspring. Our results demonstrated a binary effect of EC exposure on gene expression particularly in the hypothalamus. Principal component analysis revealed two subsets (B-S1 [n = 6] and B-S2 [n = 4]) within the biosolids group (B, n = 10), relative to the controls (C, n = 11). Changes in body weight, TG levels, and in gene expression in the hypothalamus, and visceral and subcutaneous fat were apparent between biosolid and control and the two subgroups of biosolids animals. These findings demonstrate that gestational exposure to an EC mixture results in differential regulation of metabolic processes in adult male offspring. Binary effects on hypothalamic gene expression and altered expression of lipid metabolism genes in visceral and subcutaneous fat, coupled with phenotypic outcomes, point to differences in individual susceptibility to EC exposure that could predispose vulnerable individuals to later metabolic dysfunction.

Immunohistochemical evidence for an endocrine/paracrine role for ghrelin in the reproductive tissues of sheep.

BACKGROUND: The gut hormone, ghrelin, is involved in the neuroendocrine and metabolic responses to hunger. In monogastric species, circulating ghrelin levels show clear meal-related and body weight-related changes. The pattern of secretion and its role in ruminant species is less clear. Ghrelin acts via growth hormone secretagogue receptors (GHSR-1a) to alter food intake, fat utilization, and cellular proliferation. There is also evidence that ghrelin is involved in reproductive function. In the present study we used immunohistochemistry to investigate the presence of ghrelin and GHSR-1a in sheep reproductive tissues. In addition, we examined whether ghrelin and GHSR-1a protein expression is developmentally regulated in the adult and fetal ovine testis, and whether there is an association with markers of cellular proliferation, i.e. stem cell factor (SCF) and proliferating cell nuclear antigen (PCNA). METHODS: Antibodies raised against ghrelin and its functional receptor, GHSR-type 1a, were used in standard immunohistochemical protocols on various reproductive tissues collected from adult and fetal sheep. GHSR-1a mRNA presence was also confirmed by in situ hybridisation. SCF and PCNA immunoexpression was investigated in fetal testicular samples. Adult and fetal testicular immunostaining for ghrelin, GHSR-1a, SCF and PCNA was analysed using computer-aided image analysis. Image analysis data were subjected to one-way ANOVA, with differences in immunostaining between time-points determined by Fisher's least significant difference. RESULTS: In adult sheep tissue, ghrelin and GHSR-1a immunostaining was detected in the stomach (abomasum), anterior pituitary gland, testis, ovary, and hypothalamic and hindbrain regions of the brain. In the adult testis, there was a significant effect of season (photoperiod) on the level of immunostaining for ghrelin (p < 0.01) and GHSR-1a (p < 0.05). In the fetal sheep testis, there was a significant effect of gestational age on the level of immunostaining for ghrelin (p < 0.001), GHSR-1a (p < 0.05), SCF (p < 0.05) and PCNA (p < 0.01). CONCLUSION: Evidence is presented for the presence of ghrelin and its receptor in various reproductive tissues of the adult and fetal sheep. In addition, the data indicate that testicular expression of ghrelin and its receptor is physiologically regulated in the adult and developmentally regulated in the fetus. Therefore, the ghrelin ligand/receptor system may have a role (endocrine and/or paracrine) in the development (cellular proliferation) and function of the reproductive axis of the sheep.