Neonatal genistein or bisphenol-A exposure alters sexual differentiation of the AVPV☆
Introduction
Endocrine-active compounds (EACs) are a growing concern among policy-makers and the public at large, and are also a subject of intense debate. To date, hundreds of synthetic compounds have been identified as potential EACs by the United States Environmental Protection Agency, largely because they are capable of binding to estrogen receptors (ER) in vitro and initiating transcription [15], [16], [17]. In previous studies, the relative binding affinities (RBAs) of most EACs for estrogen receptors have been found to be extremely low (about 1/1000 to 1/1,000,000th that of 17β-estradiol). Despite these low RBAs, both natural and synthetic EACs have been shown to evoke biological responses in a number of animal species [6], [20], [25], [41] prompting concern that they may interfere with hormone action in humans. Here, we compared the effects of the synthetic EAC bisphenol-A (BIS) and the naturally occurring EAC genistein (GEN) on the development of a sexually dimorphic nucleus in the rodent hypothalamus.
The neural circuitry that coordinates reproductive function is sexually differentiated by exposure to gonadal hormones during a perinatal critical period. In mammals, the ovaries of developing females are generally quiescent, thus normal development of the female brain and reproductive tract occurs in the absence of estrogen. In contrast, the perinatal male brain is exposed to high levels of estrogen, synthesized locally through the aromatization of testicular testosterone. These sex differences in hormone synthesis and exposure result in the development of distinct male and female neuroanatomical circuits, neuroendocrine functions, and reproductive behaviors.
The anteroventral periventricular nucleus (AVPV) of the hypothalamus is a sexually dimorphic nucleus that is thought to convey hormonal and environmental signals to gonadotropin-releasing hormone (GnRH) neurons to trigger the preovulatory surge of LH [9], [29]. The female AVPV is nearly twice the size of the male AVPV [5] and contains approximately three times as many dopaminergic neurons [34]. Neurons that express tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis, are therefore more abundant in the female AVPV, and also have a topographic distribution in females that is distinct from that of males [22]. Both the number and distribution of dopaminergic neurons in the female AVPV can be masculinized by the neonatal administration of testosterone or estrogen [5], [33], [34], [35]. Similarly, male mice lacking ERα have dopaminergic cell populations in the AVPV akin to those seen in wild-type females, suggesting that the sexual differentiation of TH expression in the AVPV is estrogen-dependent and mediated at least partly through ERα [36].
In the present study, we sought to determine if exposure to BIS or GEN during the first 72 h of life could affect the expression patterns of TH and ERα in the AVPV at weaning. BIS entered commercial development in the 1950s and is an industrial chemical used primarily to make polycarbonate plastic and epoxy resins. GEN is an isoflavone phytoestrogen common to legumes and soy-based foods. BIS and GEN have been shown to bind to both major forms of the estrogen receptor (ERα and ERβ) in vitro, albeit with low RBAs compared to 17β-estradiol (E2). BIS has an RBA of less than 0.4 for both ERα and ERβ, while GEN has an RBA of less than 5 for ERα and an RBA between 13 to 87 for ERβ, depending on the assay [16], [17]. Despite these low RBAs, both compounds have been found to influence reproductive physiology and behavior when administered during development. For example, exposure to BIS during the pre- or postnatal period increases ERα mRNA expression in the female mediobasal hypothalamus and the male anterior pituitary [13], disrupts the sexual differentiation of the locus coeruleus [14], and results in early, persistent estrus in females [30]. Perinatal exposure to GEN can impede male reproductive behavior and function in adulthood [40], alter the volume of the sexually dimorphic nucleus of the preoptic area in both males and females [7], [19] and ultimately result in persistent estrus in mature females [18]. These data suggest that both compounds are capable of disrupting estrogen-dependent processes in the developing brain, including sexual differentiation.
If our chosen EACs are estrogenic, they should masculinize the female AVPV by decreasing the number of TH-expressing neurons. By contrast, if BIS and GEN are anti-estrogenic, they should demasculinize the male AVPV by increasing the number of TH-expressing neurons. Because the number of AVPV neurons expressing ERα is also higher in females than in males [22], we also hypothesized that BIS and GEN might affect the overall number of ERα-expressing neurons in the AVPV. Lastly, we looked for co-expression of TH and ERα within AVPV neurons and sought to determine whether this neuronal phenotype is affected by neonatal exposure to E2, BIS and GEN.
Section snippets
Animal care
Animal care and maintenance were conducted in accordance with the applicable portions of the Animal Welfare Act and the U.S. Department of Health and Human Services “Guide for the Care and Use of Laboratory Animals”. The experimental protocol was approved by the CIIT Centers for Health Research Institutional Animal Care and Use Committee, and supervised by animal care personnel and the attending veterinarian. Five timed-pregnant dams (Sprague Dawley, Charles River, Raleigh, NC) arrived at the
TH immunoreactive cells
Consistent with previous reports, TH expression patterns were sexually dimorphic in the AVPV [33], [36]. There was a significant effect of both sex [F(1,45) = 14.442, p ≤ 0.001] and treatment [F(3,45) = 7.200, p ≤ 0.001] on the number of TH-ir cells (Fig. 2). Within the males, there was a significant effect of treatment [F(3,22) = 9.567, p ≤ 0.001] on the number of AVPV TH-ir cells (Fig. 3A). The total number of TH-ir cells was significantly higher in males treated with either BIS [p ≤ 0.008] or GEN [p ≤
Discussion
The AVPV contains multiple sexually dimorphic cell types that are sensitive to hormones during development. The male AVPV is normally masculinized by estrogen, aromatized from testicular testosterone. This process results in fewer TH-expressing cells, particularly in the more medial region of the AVPV [33], [35], [36]. Postnatal exposure to BIS and GEN significantly increased the total number of TH-ir neurons in the male AVPV. These data indicate that BIS and GEN effectively interfered with
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2020, Reproductive ToxicologyCitation Excerpt :Available human data corroborate animal data and link prenatal exposure to heightened risk of deleterious childhood behaviors including anxiety [116–119]. Further, our work shows that the AVPV is particularly sensitive, with perinatal BPA altering its physical size and its sex-specific dimorphism of dopaminergic and kisspeptin neurons, among other outcomes [111,120,121]. Of most relevance to CLARITY-BPA, work in multiple rat strains shows that developmental BPA exposure can alter expression of ERs in multiple brain regions, including the AVPV, amygdala, and surrounding structures, that coordinate reproductive and other sexually dimorphic behaviors [115,121].
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Funded by ACC IMFOB0034 to EKP.