Regulation of Leydig Cell Differentiated Function by the Industrial Chemical Bisphenol A (BPA)

Abstract

Exposure of the population to bisphenol A (BPA) is significant due to its extensive use in the manufacture of a wide variety of consumer products, including polycarbonate plastics and epoxy resins. However, there is concern that early-life exposure to BPA may alter developmental programming and predispose individuals to adverse health outcomes such as infertility and obesity in later life. This dissertation describes the adverse effects of early-life exposure to BPA on Leydig cell function using the rat model. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone, which supports the male phenotype. The objectives were to investigate the effects of developmental BPA exposure on (i) Leydig cell development; (ii) adipose tissue derived molecules (e.g., adiponectin) and their regulation of Leydig cells; and, (iii) estrogen metabolism and antioxidant capacity in Leydig cells. Exposure of male rats to BPA by maternal gavage at doses of 2.5 and 25 µg/kg body weight once a day from gestational day 12 to day 21 postpartum stimulated Leydig cell division in the prepubertal period and increased Leydig cell numbers in the testes of adult male rats at 90 days. BPA-induced proliferative activity in the prepubertal period was associated with induction of cell cycle protein expression, up-regulation of growth factor receptors (e.g., insulin-like growth factor 1 receptor and epidermal growth factor receptors), activation of mitogen activated protein kinase 3/1 and possibly decreased anti-Mullerian hormone (AMH)-mediated signaling. Furthermore, in vitro experiments confirmed that BPA acted directly as a mitogen in Leydig cells. On the other hand, developmental BPA exposure had an inhibitory effect on Leydig cell androgen secretion by decreasing protein expression of the luteinizing hormone receptor (LHR) and the 17beta-hydroxysteroid dehydrogenase 3 (HSD17B3) enzyme. A separate study investigated the interaction of developmental BPA exposure with high fat diet (HFD) feeding from postnatal day 70 to 98 on adipose tissue and Leydig cell function. This is important because use of high fat diets and the incidence of obesity in the population are on the increase. Following perinatal BPA exposure, i.e., gestational day 12 to postnatal day 21, male rats were maintained on normal or high fat diet from PND 70 to 90 days of age. Animals were sacrificed within 48 h of day 98 postpartum when serum, gonadal fat tissue and Leydig cells were obtained for further analysis. Results showed that BPA exposure alone and/or in combination with HFD feeding decreased serum adiponectin levels compared to control animals on normal fat diet (NFD) (P<0.01). Similarly, BPA exposure and HFD feeding decreased Leydig cell adiponectin and its receptor (AdipoR2) expression. However, serum testosterone levels were paradoxically greater in HFD-fed male rats exposed to the 25 μg/kg dose of BPA compared to control HFD animals (P<0.05). The decrease in androgen secretion was associated with reduced Leydig cell HSD17B3 enzyme protein expression. In contrast, HFD feeding had the effect of up-regulating HSD17B3 enzyme expression in Leydig cells. Finally, the effects of developmental BPA exposure and HFD feeding on estrogen metabolism and antioxidant capacity in Leydig cells were investigated. In general, maintenance of male rats on HFD decreased serum 17β-estradiol levels in all groups compared to age-mates on NFD (P<0.05). BPA exposure increased estrogen sulfotransferase expression in adipose tissue and Leydig cells but this effect was reversed by maintenance on the HFD. Also, HFD feeding increased serum levels of thiobarbituric acid reactive substance, a marker for oxidative stress, in male rats exposed to BPA. Exposure to BPA at the 25 µg/kg dose and/or HFD feeding increased reactive oxygen species generation in Leydig cells. Analysis of enzyme antioxidant capacity showed that superoxide dismutase activity was decreased in Leydig cells in the HFD groups, whereas catalase and glutathione peroxidase activities were increased by exposure to BPA and HFD feeding. In vitro experiments confirmed that BPA acts directly to induce oxidative stress in Leydig cells. This is important because oxidative stress is known to inhibit Leydig cell androgen biosynthesis. In conclusion, developmental exposure to BPA disrupted Leydig cell development and function. Further, the effects of BPA in Leydig cells were influenced by a HFD. Altogether, the present results showed that BPA causes biological effects at environmentally relevant exposure levels with implication for public health.