Neonatal Bisphenol A exposure alters sexually dimorphic gene expression in the postnatal rat hypothalamus
Highlights
► The hypothalamus undergoes sexual dimorphism in neonatal life. ► Hormones are critical for this process therefore it may be vulnerable to endocrine disruption. ► Neonatal exposure to BPA altered the sex specific expression of estrogen receptors. ► Neonatal exposure to BPA altered the sex specific expression of Kiss1. ► These gene expression changes may underlie reproductive deficiencies that emerge later in life.
Introduction
BPA was initially developed as a possible synthetic estrogen (Dodds and Lawson, 1936), and is now a high volume production component of polycarbonate plastics, epoxy resins, dental sealants, thermal receipts and other products (Biedermann et al., 2010, Vandenberg et al., 2007). Human exposure is nearly ubiquitous, with urinary levels higher in children than adults (Calafat et al., 2008). The health impacts of BPA exposure remain controversial but growing evidence suggests that it has the potential to impose adverse outcomes on reproductive (Beronius et al., 2010, Cabaton et al., 2011, Howdeshell et al., 1999, Vandenberg et al., 2009) cardiovascular (Pant et al., 2011) and metabolic (Groff, 2010, Newbold, 2010) health. The hypothalamus is an integral part of each of these systems and thus conceivably a central target through which BPA could induce widespread effects across multiple organ systems. We and others have shown that BPA exposure can alter the sex specific organization of hypothalamic regions in the murine brain known to be important for coordinating gonadotropin release and sexual behavior, most notably the anteroventral periventricular nucleus of the hypothalamus (AVPV) (Patisaul et al., 2006, Patisaul et al., 2007, Rubin et al., 2006) and the arcuate nucleus (ARC) (Patisaul et al., 2009). Collectively these observations support the hypothesis that disruption of hypothalamic organization during critical windows of development could underlie a suite of neuroendocrine BPA effects.
Although long considered weakly estrogenic, the specific mechanisms by which BPA interacts with molecular and cellular targets within the hypothalamus and elsewhere are not clearly established (vom Saal et al., 2007). Classically, BPA is thought to disrupt genomic pathways mediated by the two primary forms of the nuclear estrogen receptor (ER); ER alpha (ERα) and ER beta (ERβ). Compared to estradiol, BPA has a binding affinity approximately 10,000–100,000 fold weaker for both ER isoforms (Andersen et al., 1999, Barkhem et al., 1998, Blair et al., 2000, Gould et al., 1998) but appears to bind each with relatively equal affinity (Kuiper et al., 1998) indicating that it has the potential to interact with either.
It is well established that estrogen is masculinizing during perinatal development in the murine brain, and that exposure to even low levels of estrogen or xenoestrogens during this time can permanently alter neuroendocrine pathways critical for mediating steroid negative feedback, gonadotropin release, energy homeostasis and sexual behavior (Amateau et al., 2004, Bader et al., 2011, Faulds et al., 2011, Gore, 2008, Simerly, 2002). We have recently shown that the expression of ERα and ERβ is sexually dimorphic within numerous subregions of the neonatal rat hypothalamus (Cao and Patisaul, 2011). For example, within the AVPV, ERβ expression is higher in males on the day of birth while ERα expression is higher in females, suggesting that the two isoforms may play different functional roles in the sexual differentiation of this region. This sex difference is transient, and eliminated within 48 h, suggesting that if BPA exposure during only the first two days of life alters expression levels, estrogen sensitivity during this critical period of sexual differentiation may be impacted. Here we tested the hypothesis that neonatal BPA exposure can affect the sex specific hypothalamic expression of ERα and ERβ across postnatal development, focusing specifically on the AVPV, ARC and two other subregions (the medial preoptic area (MPOA) and the ventrolateral division of the ventromedial nucleus (VMNvl)) known to be essential for the neuroendocrine control of gonadotropin release and other aspects of female reproductive physiology, metabolic regulation and behavior (Nance, 1976, Pfaff and Keiner, 1973, Pfaff et al., 1994, Simerly, 2002). Although previous studies have explored similar hypotheses in a variety of species, they primarily looked at adult expression in only a single sex and either employed a different exposure window (Ceccarelli et al., 2007) focused on only one hypothalamic area (Mahoney and Padmanabhan, 2010, Monje et al., 2007), or employed techniques with less anatomical resolution such as RT-PCR (Khurana et al., 2000, Monje et al., 2007). Thus, the present studies build on this prior work by (1) characterizing the specific hypothalamic subregions vulnerable to disruption in both sexes (2) identifying this disruption early in postnatal development and (3) more carefully identifing the critical window of exposure for ER disruption by BPA. Collectively, these studies test the hypothesis that perturbation of ER expression during the neonatal period of hypothalamic sexual differentiation could be a mechanism by which BPA induces a wide range of effects, some of which may not emerge until adulthood.
We also sought to determine if BPA exposure can disrupt the expression of Kiss1, a gene which codes for the kisspeptin family of peptides. Kisspeptin is now recognized to be the primary “gatekeeper” of gonadotropin release and essential for initiating pubertal onset (Oakley et al., 2009, Pineda et al., 2010). It also plays a role in energy homeostasis and is a putative effector of leptin actions on gonadotropin releasing (GnRH) neurons (Castellano et al., 2010). There are two populations of Kiss1 neurons in the murine brain, an anterior group which extends through the AVPV into the MPOA (a region now collectively referred to as the rostral periventricular area of the third ventricle (RP3V)) and a mediobasal hypothalamic group confined to the ARC. In adults, the RP3V population is sexually dimorphic, with females having higher expression levels than males (Kauffman et al., 2007), while the ARC population is not (Losa et al., 2010). These observations suggest that the anterior population participates in steroid positive feedback on gonadotropin release, while the posterior population plays a role in steroid negative feedback (Oakley et al., 2009, Pineda et al., 2010). Nearly all Kiss1 neurons in the RP3V and ARC co-express ERα, and a subset of RP3V Kiss1 neurons co-express ERβ (Smith et al., 2005, Smith et al., 2006) suggesting that disruption of ER expression within this neuronal phenotype could affect its sex specific ontogeny and function. Gestational and/or postnatal BPA exposure has been shown to alter Kiss1 mRNA expression within whole hypothalamic micropunches (Navarro et al., 2009, Xi et al., 2010), and RP3V Kiss1-immunoreactivity (Bai et al., 2011, Panzica et al., 2009) during peripuberty and adulthood. Although RP3V Kiss1 expression is not apparent in either sex until approximately the second week of life (Cao and Patisaul, 2011, Clarkson et al., 2009) we have shown that neonatal exposure to estrogen or an ERα selective agonist (Bateman and Patisaul, 2008, Patisaul et al., 2009) can defeminize kisspeptin signaling pathways in the adult RP3V, suggesting that the first few days of life may be a critical window of BPA vulnerability, and that this disruption may be apparent prior to puberty. In contrast to the RP3V, Kiss1 mRNA in the ARC is robustly expressed from PND 0 through PND 19, with higher levels in females than males within the first week of life (Cao and Patisaul, 2011). This indicates that Kiss1 expression may be influenced by neonatal estrogen and vulnerable to endocrine disruption by a chemical like BPA. Here we examined whether neonatal BPA exposure affects the sexual dimorphic ontogeny of Kiss1 mRNA expression in the RP3V and ARC.
The present study is the first to precisely characterize when, where and how neonatal BPA exposure affects sexual dimorphic ER and Kiss1 gene expression in the postnatal rat brain. Collectively, the results reveal the potential for BPA to disregulate the differential expression of ERα, ERβ and Kiss1 during a critical window of hypothalamic organization, findings which will help elucidate the mechanisms by which BPA can induce persistent effects across neuroendocrine systems.
Section snippets
Animal care, neonatal exposure and tissue collection
Pups were born to timed pregnant Long Evans rats (n = 13; Charles River, Raleigh, NC), housed at the Biological Resource Facility at North Carolina State University (NCSU) under a 14:10 h light:dark cycle (lights on at 07:00 h) at 23 °C and 50% average relative humidity. Animals were housed in thoroughly washed polysulfone caging with woodchip bedding, and fed a semi-purified, phytoestrogen-free diet ad libitum (AIN-93G, Test Diet, Richmond, IN) to minimize exposure to exogenous BPA, phytoestrogens,
Summary of results
A summary of the overall results is provided in Table 1. In the anterior hypothalamus, ER expression was higher in females than males by PND 10 in the MPOA but equivalent to male expression in the AVPV. Neonatal exposure to EB eliminated the sex differences primarily by reducing ER expression in females. BPA effects were dose and age dependent. On PND 4, ERα expression was augmented in both sexes of the HBPA group, but, by PND 10, female expression was lower compared to the unexposed controls.
Discussion
The present study provides comprehensive evidence that neonatal exposure to BPA alters early postnatal, sexually dimorphic expression of ERα, ERβ and Kiss1 mRNA in the rat hypothalamus, particularly in the RP3V region. Disruption of ER expression during this time period may have lifelong consequences because it is the age at which the hypothalamus is undergoing steroid hormone directed sexual differentiation. Perturbation of ER expression by BPA presumably alters sensitivity to endogenous
Conflict of interest statement
None.
Acknowledgements
This work is supported by NIEHS grant RO1 ES016001 to HBP. We thank Karina Todd for assistance with animal care and facilitating ISHH.
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