Exposure to a complex cocktail of environmental endocrine-disrupting compounds disturbs the kisspeptin/GPR54 system in ovine hypothalamus and pituitary gland

Abstract

Background: Ubiquitous environmental chemicals, including endocrine-disrupting chemicals (EDCs), are associated with declining human reproductive health, as well as an increasing incidence of cancers of the reproductive system. Verifying such links requires animal models exposed to "real-life," environmentally relevant concentrations/mixtures of EDC, particularly in utero, when sensitivity to EDC exposure is maximal.

Objectives: We evaluated the effects of maternal exposure to a pollutant cocktail (sewage sludge) on the ovine fetal reproductive neuroendocrine axes, particularly the kisspeptin (KiSS-1)/GPR54 (G-protein-coupled receptor 54) system.

Methods: KiSS-1, GPR54, and ERalpha (estrogen receptor alpha) mRNA expression was quantified in control (C) and treated (T) maternal and fetal (110-day) hypothalami and pituitary glands using semiquantitative reverse transcription polymerase chain reaction, and colocalization of kisspeptin with LHbeta (luteinizing hormone beta) and ERalpha in C and T fetal pituitary glands quantified using dual-labeling immunohistochemistry.

Results: Fetuses exposed in utero to the EDC mixture showed reduced KiSS-1 mRNA expression across three hypothalamic regions examined (rostral, mid, and caudal) and had fewer kisspetin immunopositive cells colocalized with both LHbeta and ERalpha in the pituitary gland. In contrast, treatment had no effect on parameters measured in the adult ewe hypothalamus or pituitary.

Conclusions: This study demonstrates that the developing fetus is sensitive to real-world mixtures of environmental chemicals, which cause significant neuroendocrine alterations. The important role of kisspeptin/GPR54 in regulating puberty and adult reproduction means that in utero disruption of this system is likely to have long-term consequences in adulthood and represents a novel, additional pathway through which environmental chemicals perturb human reproduction.

Keywords: GPR54; environmental chemicals; hypothalamus; kisspeptin; pituitary; prenatal exposure; sheep.

Figure 1

KiSS-1 mRNA expression (mean ± SE) in fetal hypothalamus (A) and pituitary (B) after in utero exposure to sewage sludge chemicals (treated). *p < 0.05, and **p < 0.02 compared with respective control value.

Figure 2

Maternal ERα mRNA expression (mean ± SE) in the hypothalamus (A) and the pituitary (B) of T sheep were not significantly affected by exposure to sewage sludge relative to control sheep.

Figure 3

Representative photomicrographs of staining for kisspeptin (A, D) and LHβ (B, E), and merged images (C, F), in fetal pituitaries from control (top) and treated (bottom) animals. Cells showing colocalization are stained yellow (indicated by arrows). Bar = 20 μm. (G–I) Quantification per field of view of the mean LHβ-immunopositive cell number (G), the mean number of cells immunopositive for both LHβ and kisspeptin cells (H), and percentage of kisspeptin/LHβ immunopositive cells as a proportion of the total number of LHβ-immunopositive cells (I) in fetal pituitary tissue sections from control and treated animals. *p < 0.05, and ***p < 0.001, compared with respective control value.

Figure 4

Representative photomicrographs of staining for kisspeptin (A, D) and ERα (B, E), and merged images (C, F), in fetal pituitaries from control (top) and treated (bottom) animals. Examples of cells showing colocalization are indicated by arrows. Bar = 20 μm. (G–I) Quantification per field of view of the mean ERα-immunopositive cell number (G), the mean kisspeptin/ERα cells immunopositive for both ERα and kisspeptin (H), and percentage of kisspeptin/ERα immunopositive cells as a proportion of the total number of ERα immunopositive cells (I) in fetal pituitary tissue sections from control and treated animals. *p < 0.01, and ***p < 0.001, compared with respective control value.

Figure 5

Representative photomicrographs of ERα (blue-black) and LHβ (brown) double immunohistochemistry in fetal pituitaries from control (A) and treated (B) animals. Colocalized cells are indicated by arrows; bar = 20 μm. (C) Quantification per field of view of the mean of LHβ, ERα, and LHβ/ERα immunopositive cells in fetal pituitary tissue sections from control and treated animals. *p < 0.05 compared with respective control value.