Estrogen and environmental estrogenic chemicals exert developmental effects on rat hypothalamic neurons and glias

Abstract

We investigated effects of 17β-estradiol (E₂) and endocrine disrupters, nonylphenol (NP) and bisphenol-A (BPA), focusing on the neuronal development in cultures of fetal rat hypothalamic cells. We applied different concentrations of E₂, NP or BPA to the cultured hypothalamic cells and observed their effects on dendritic and synaptic development by immunocytochemistry using anti-microtubule associated protein-2 (MAP2) and anti-synapsin I antibodies, respectively. Administration of E₂ for 7 days affected MAP2-positive area as well as synapsin I-positive area. NP and BPA also influenced neuronal developments. The significant increase both in MAP2- and synapsin I-positive areas was observed at 10 and/or 100 nM of them, while 1 μM of them reduced the positive areas. Synaptic densities calculated from synapsin I-positive area/MAP2-positive area were not constant among different doses of three chemicals, but increased at 10 and/or 100 nM and decreased at 1 μM. Furthermore, immunostaining of NP-treated cells with the antibody against glial fibrillary acidic protein (GFAP) revealed that glial development was similarly influenced by NP. Therefore, the present results demonstrated that not only E₂ but also the environmental estrogenic chemicals, NP and BPA, affect development of fetal rat hypothalamic cells in vitro.

Introduction

During the brain development, estrogen participates in the organization of neural circuits that control a broad spectrum of neuroendocrine and behavioral functions. Many works published for these two decades have demonstrated that alterations in the estrogenic milieu of the developing CNS influence various aspects of cellular differentiation including neurite extension and branching, synaptic formation, myelination, expressions of neurotransmitter and neuropeptides, cell death and/or survival and glial development (MacLusky and Naftolin, 1981, Arnold and Gorski, 1984, Bloch and Gorski, 1988, Beyer, 1999, Carrer and Cambiasso, 2002, Simerly, 2002, Forger, 2006). Through those neurotrophic and/or differentiation-promoting effects, estrogen appears to be crucial for the sexual differentiation of CNS structures and functions. As it has widely known in rodents, estrogen is effective on sexual differentiation only during the “critical period” of the brain development which extends from late embryonic days to the first week after birth (MacLusky and Naftolin, 1981, McEwen, 1981). Among different brain regions affected by estrogen, hypothalamic regions such as the medial preoptic area (mPOA) and the ventromedial nucleus (VMN) contain sexually differentiated neuronal circuits responsible for controlling reproductive functions (reviewed in Flanagan-Cato, 2000, Flanagan-Cato et al., 2001, Flanagan-Cato et al., 2006, Simerly, 2002, Simerly, 2005, Ohtani-Kaneko, 2006). Indeed, various methods revealed the presence of estrogen bindings, expressions of estrogen receptor (ER) genes and their proteins in this restricted area of the rodent brain during the “critical period” (Sheridan et al., 1974, MacLusky et al., 1979a, MacLusky et al., 1979b, DonCarlos and Handa, 1994, Yokosuka et al., 1997).

On the other hand, in the last decade, there is a growing concern that exposure to environmental chemicals which mimic the action of estrogen has the potential to disrupt endocrine functions and poses growing threat to health. Many studies now document the ability of environmental chemicals to interfere with the endocrine system, and behaviors in wildlife and experimental animals. Environmental chemicals with estrogenic activities are found in many synthetic products; insecticides, fungicides, household products, products of plastics, and so forth (Tapiero et al., 2002). Bispenol-A (BPA), a monomer of polycarbonate plastics, and nonylphenol (NP), a detergent for breaking down plastic products, both of which are widely used in industrial compounds and consumer products, exhibit estrogenic activities promoting transcriptional activation and cellular proliferation (Soto et al., 1991, Krishnan et al., 1993). Furthermore, BPA and NP have been found to affect the sexual differentiation of the brain and behavior in vivo (Nagao et al., 2000, Kubo et al., 2003, Rubin et al., 2006). However, evidence of the direct effects of these chemicals on hypothalamic neurons during the “critical period” is still lacking. In the present study, we removed hypothalamic cells from rat fetuses at embryonic day 15, because gonadal steroid hormones start to be secreted on the 17th day of gestation in rats (Cseruns, 1986). We cultivated and treated them with E₂, BPA and NP to mimic the “critical period” of the sexual differentiation in vitro. We then investigated their direct effects on the dendritic, synaptic and glial development of the hypothalamic cells.