Epigenetic changes brought about by perinatal stressors: A brief review of the literature

Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

Contents lists available at SciVerse ScienceDirect

Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox

Epigenetic changes brought about by perinatal stressors: A brief review of the literature Blase Billack a,⁎, Ryan Serio b, Ilton Silva c,⁎⁎, 1, Craig H. Kinsley c,⁎⁎⁎ a b c

Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Jamaica, NY 11439, USA Institute for Biotechnology, St. John's University, Jamaica, NY 11439, USA Department of Psychology-Center for Neuroscience, University of Richmond, Richmond, VA 23173, USA

a r t i c l e

i n f o

Article history: Received 28 February 2012 Accepted 28 August 2012 Keywords: Gonadal hormones Epigenetics Heterosexuality Homosexuality Postnatal stress Prenatal stress Perinatal stress Sexual differentiation Social behaviors Stress DNA methylation Hormones

a b s t r a c t Introduction: Numerous studies employing various animal models have found that perinatal stress, encountered in utero during sensitive developmental stages or shortly after birth, disrupts both sexual differentiation and sexual behavior in offspring. The biochemical, cellular, genetic and epigenetic events which are involved in the organismal response to perinatal stress are currently under investigation. Methods, Results and Discussion: In this review, the reader is introduced to perinatal stressors as a toxicological phenomenon, and several recently characterized epigenetic responses to said stressors are discussed. © 2012 Elsevier Inc. All rights reserved.

1. Introduction: perinatal stressors as toxicological phenomenon Prenatal development of the mammalian brain requires a significant amount of energy, time and care. The ramifications of disruptions of any of the above features results in an organ that is less efficient or under-functions to a point that may compromise the survival of its owner. More subtle influences are potentially disruptive, too, as they may produce modifications in behavior that are deleterious. Such are the effects described here of stressors administered during the prenatal or gestational time or shortly after birth, collectively referred to here as perinatal stressors, which, through basic, toxicological actions exert effects upon early brain development in a variety of ways,

⁎ Correspondence to: B. Billack, Department of Pharmaceutical Sciences, St. John's University, 8000 Utopia Parkway, Jamaica, NY 11439, USA. ⁎⁎ Correspondence to: I. Silva, Department of Physiology, Bioscience Institute, University of Sao Paulo, SP 05508‐900, Brazil. ⁎⁎⁎ Correspondence to: C.H. Kinsley, Department of Psychology, Center for Neuroscience, B-326/328, Gottwald Science Center, University of Richmond, 28 Westhampton Way, Richmond, VA 23173, USA. E-mail addresses: [email protected] (B. Billack), [email protected] (C.H. Kinsley). 1 Present address: Department of Psychology, University of Richmond, Richmond, VA, USA. 1056-8719/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.vascn.2012.08.169

including sexual differentiation. Various perinatal stressors include, but are not limited to, those which are listed in Table 1. Though beyond the scope of the current review, the process of sexual differentiation is both delicate and long-lasting; variations in its development lead to significant permanent modifications of sexrelated behaviors. Prenatal and early postnatal sexual differentiation of the brain ultimately dictates the many adult sex differences in behavior, physiology and anatomy that are referred to as sexually dimorphic. Gonadal steroids (Ward & Weisz, 1984) such as testosterone, its metabolite estradiol (Patchev, Hayashi, Orikasa, & Almeida, 1995), and estradiol-induced increases in prostaglandin-E(2) (Amateau & McCarthy, 2004), all exert strong effects on organizing the early male brain (Wright, Burks, & McCarthy, 2008); female brain development appears to occur in the virtual absence of gonadal and other steroids; though a role for small quantities of estradiol in the development of the female brain has been postulated. Therefore, an active hormonal stimulation is necessary for normal sexual development in males, whereas in females, it is the relative absence of such effects which contributes to their development. Interference with this delicate process can result in life-long modifications of sexually dimorphic brain function and behavioral outcomes. For example, it is well known that a variety of prenatal stressors significantly modify the basic organizational program of sexual differentiation, due

222

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

Table 1 A partial list of epigenetic mechanisms observed in animals exposed to perinatal or other related stressors. Stressor

Organism

Epigenetic mechanism

Consequence

Reference(s)

IUGRa Prenatal Hypoxia

Rats Rats

Histone modification/chromatin remodeling Altered DNA methylation

Park et al., 2008 Patterson et al., 2010

Prenatal Exposure to Insecticide or Fungicide

Rats

Altered DNA methylation in the male germ line

Reduced expression of Pdx1b Decreased PKC {epsilon} in the hearts of male offspring Spermatogenic cell defects, increased incidence of male infertility, cancers

Postnatal Exposure to Excess Glucocorticoid Postnatal Exposure to Excess Estradiol

Mice

Poor Postnatal Maternal Care of Pupsd

Rats

Prenatal Stress

Mice

Prenatal Stress

Mice

Acute Restraint Stress

Rats

3rd Trimester Depression

Humans

Neglect or abuse Maternal Separation After Birth

Rats Mice

Hypomethylation of Fkbp5c gene in the hypothalamus and hippocampus Increased methylation of the progesterone receptor gene promoter in the preoptic area and mediobasal hypothalamus of females Altered DNA methylation of exon 17 in GR receptor promoter of hippocampus; altered histone acetylation and dysregulation of acetylation-dependent transcriptional activation Altered DNA methylation of exon 17 in GR receptor promoter of hypothalamus; decreased methylation of CRF promoter in amygdala and hypothalamus; elevations of DNMT1 activity in female, but not male, placentas Reduced miRNAs against Tgfbr3f transcripts in whole brain Increased levels of H3K9 tri-methylation in the dentate gyrus (DG) and CA1; Reduced levels of H3K9 mono-methylation and H3K27 tri-methylation in the same regions; no effect on levels of H3K4 tri-methylation Altered DNA methylation of a CpG site proximal to exon 1-F of the NR3C1g gene in neonates Altered DNA methylation of Bdnfh Crfr2i found to be hypomethylated in brains of male mouse pups; increased methylation of Mecp2j

a b c d e f g h i j

Rats

Increased expression of FKBP5 Authors did not attempt to correlate the percent change in CpG methylation with the expression levels of PR Decreased GR activity in hippocampus in pups; lowered expression of ATRXe

Decreased GR activity in hippocampus of pups; Increased transcription of CRF in amygdala; global changes in DNA methylation

Anway et al., 2005; Nilsson, Anway, Stanfield, & Skinner, 2008 Lee et al., 2010 Schwarz et al., 2010

Weaver et al., 2004; Meaney & Szyf, 2005; Weaver et al., 2006; Weaver, 2007 Mueller & Bale, 2008

Increased betaglycan expression

Morgan & Bale, 2011

Findings of this study suggest positive and negative effects on gene transcription.

Hunter et al., 2009

Increased infant cortisol stress reactivity

Oberlander et al., 2008

Increased anxiety-related behaviors Reduced expression of CRFR2 receptor and MeCP2

Roth et al., 2009 Franklin et al., 2010

Intrauterine growth retardation. Pdx1 is a transcription factor that regulates pancreas development and B-cell differentiation. Fkbp5 encodes a protein that functions as a co-chaperone for androgen, glucocorticoid, mineralocorticoid and progesterone receptors. As evidenced by low licking and grooming of pups. ATRX is an ATP-dependent chromatin remodeling helicase expressed in the hippocampus and involved with neurogenesis. Tgfbr3 encodes the TGF-β family type 3 receptor, also known as betaglycan. NR3C1 is the gene for the human glucocorticoid receptor. Bdnf encodes a brain-derived neurotrophic factor involved in stress responses. Crfr2 is a receptor for corticotropin releasing factor. Mecp2 is a the gene for methyl CpG-binding protein 2.

primarily to disruption of fetal testosterone levels and their synchronization with neurodevelopmental epochs such as general brain and nuclear maturation (Alonso, Castellano, & Rodriguez, 1991; Barbazanges, Piazza, Le, & Maccari, 1996; Grisham, Kerchner, & Ward, 1991; Humm, Lambert, & Kinsley, 1995; Keshet & Weinstock, 1995; McEwen, 1994; Ward, 1972; Ward & Weisz, 1980, 1984). Such alterations are telling, as they produce significant and long-lasting effects on physiology and behavior. Further attesting to the toxicological facets of prenatal stress, both sexes are sensitive to it. For example, prenatally stressed males display reductions in aggressive behaviors and shorter latencies to respond parentally to pups (Harvey & Chevins, 1985; Kelley, 1988; Kinsley & Bridges, 1988; Kinsley & Svare, 1986), forms of demasculinization and feminization. Similarly, prenatally stressed females display reductions in maternal and aggressive behavior, fertility and fecundity (Herrenkohl, 1979; Kerchner, Malsbury, Ward, & Ward, 1995; Kinsley, Mann, & Bridges, 1988b; Kinsley & Svare, 1988; Kinsley, Mann, & Bridges, 1988a). Further, as a consequence of prenatal stress, both sexes display alterations in estradiol-induced (Kinsley & Bridges, 1987) and stress-induced prolactin secretion (Kinsley, Mann, & Bridges, 1989), nitric oxide synthases activity (Miller, Mueller, Gifford, & Kinsley, 1999), as well as a differential sensitivity to opiates (Insel, Kinsley, Mann, & Bridges, 1990; Kinsley et al., 1988a). It is, however, the marked reductions in male-typical sexual behavior that are the most prevalent, the most striking, and the best documented of the many effects on behavior and physiology of exposure

to stress in utero primarily in rodents. For instance, the sexual behavior of prenatally stressed males is demasculinized, as they display little copulatory behavior (Ward, 1972), significantly fewer ejaculatory responses and a diminished luteinizing hormone surge (Kinsley, Mann, & Bridges, 1992) when exposed to sexually-receptive females. Their sexual behavior is also feminized, as measured by high rates of lordotic responsiveness to mounting by another male (in estrogen-treated males; Ward, 1972; Ward & Weisz, 1984). These effects have been extensively replicated and remain valuable measures of prenatal stress effects (Ward & Ward, 1985). Dörner et al. (1991) have suggested that prenatal stress may operate in male-typical sexual behavior in a similar manner in humans. Sexual dimorphisms have been identified in the size of the preoptic nucleus, stria terminalis, amygdala, hippocampus (Kawashima & Takagi, 1994), anterior commissure (Jones et al., 1997) and certain nuclei of the ventromedial nucleus of the hypothalamus (VMH) (Flanagan-Cato, 2000), all of which suggests numerous structural sexual dimorphisms. Certain other cell types in the brain exhibit dimorphic properties as well. For example, in the medial preoptic area (mPOA), a region important for sexual behavior and physiology, there are sex differences in the morphology of astrocytes (Amateau & McCarthy, 2002). The toxicological effects of prenatal stress may be wrought by interference with the wide-ranging but otherwise normal effects of hormones on organization and activity of regional groups of neurons/ nuclei, effects which regulate normal sexually dimorphic behavior. Estrogen promotes excitatory effects in both male and female neurons

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

by enhancing neuronal firing rates (Beyer & Feder, 1987), increasing the number of processes of nerve fibers (Kawata, Yuri, & Morimoto, 1994), enhancing neuritic outgrowth and elongation both in vitro (Diaz, Lorenzo, Carrer, & Caceres, 1992), and in vivo (Cooke & Woolley, 2005; Gerrits et al., 2008), and promoting the formation of synapses in the CA1 region of the hippocampus over the course of the estrous cycle (McEwen, 1994; Yankova, Hart, & Woolley, 2001). Thus, because neuronal networks and activity are regulated by steroid hormones, and prenatal stress alters the prenatal endocrine environment, such events are likely sensitive to disruption by the toxicology of prenatal stress. Moreover, the activity of sex behavior-regulating neurons is altered by prenatal stress, as prenatally stressed males show marked reductions in the number and intensity of neurons expressing the immediate early gene protein c-fos (Humm et al., 1995), as well as NMDA receptor phosphorylation (Dominguez et al., 2007), subsequent to exposure to sexually-receptive females. In addition to effects on cellular structure and biochemical processes of regional groups of neurons/nuclei, prenatal stress may affect the basic shape and hence, the function, of individual neurons. Adult neurons are sensitive to, and modify their structure following, stress hormone exposure (Lambert et al., 1998, 2000). Also, the effects of steroid hormone exposure on the cytoarchitecture of neurons in several hypothalamic brain nuclei have been reported; these effects, however, may be specific for discrete sub-regions. For instance, estradiol alone stunts the elongation of primary dendrites in ventrolateral VMH whereas the dorsomedial subdivision is largely unaffected (Griffin & Flanagan-Cato, 2008). Interestingly, a sub-population of neurons of the VMH that are involved in the rat's lordosis response (Daniels, Miselis, & Flanagan-Cato, 1999) did not express receptors for estrogen. These data suggest that the effects of the hormone on circuit modulation were via unknown indirect mechanisms (Daniels & FlanaganCato, 2000). As steroid hormones normally induce significant behavioral, physiological and neuroanatomical sexual dimorphisms, and given the types of behaviors affected by prenatal stress, it follows that there may also be sex differences in the morphology of mPOA neurons. Further, given the mPOA's role in regulating parental responses, which are significantly affected in both prenatally stressed males and females (Harvey & Chevins, 1985; Kelley, 1988; Kinsley & Bridges, 1988; Kinsley et al., 1988a), potential neuronal structural effects of prenatal stress in the mPOA are worth examining. Sexual dimorphisms in the cytoarchitecture of neurons, including size of neuronal cell soma and dendritic length, have been reported in the mPOA (Madeira et al., 1999, 2001). Additionally, it has been shown that prenatal stress can alter the dendritic morphology of neurons in several mesocorticolimbic structures including the nucleus accumbens and hippocampus (Martinez-Tellez, Hernandez-Torres, Gamboa, & Flores, 2009); however, the effects of prenatal stress on neuronal cytoarchitecture of the mPOA in adult offspring have not been described. Recently, Gerecke et al. (2012) reported that prenatal stress has significant effects on the structure of mPOA neurons. Hence, the toxicology of prenatal stress extends from hormone to macro to micro levels. To what extent does it equate with epigenetic actions in the organism? 2. Dysregulation of HPA axis in prenatal stress The most well-characterized stress response mechanism transmitted from mother to fetus is that mediated by hypothalamic–pituitary– adrenal (HPA) axis stimulation (reviewed in Viltart & VanbesianMailliot, 2007; Cottrell & Seckl, 2009). In this system, corticotrophinreleasing factor (CRF), also known as corticotrophin-releasing hormone (CRH), is initially released from the hypothalamus and travels to the anterior pituitary where it binds to receptors and stimulates the synthesis of a precursor protein known as pro-opiomelanocortin (POMC). The POMC precursor is post-translationally modified to adrenocorticotropic hormone (ACTH) which enters the systemic circulation and

223

travels to the adrenal glands. Once there, ACTH binds to receptors in the adrenals, which, in turn, leads to the synthesis and release of glucocorticoids, primarily cortisol in humans and corticosterone in rodents, into the systemic circulation. These glucocorticoid hormones then bind to receptors on metabolically active peripheral organs to exert their functional effects, which include increased resistance to stress, increased intermediary metabolism with a release of fat from body stores and an increase in gluconeogenesis, anti-inflammatory actions and, in the case of the hippocampus and hypothalamus, inhibitory feedback effects on CRF production. Behavioral and metabolic changes can be attributed to an excess of glucocorticoid exposure which occurs during periods of prolonged stress and is governed to a large extent by the autonomic nervous system (reviewed in McEwen et al., 1997). The stress response of an organism is triggered when there is a threat to its homeostatic balance. The HPA axis primarily mediates this response by ensuring that energy stores are mobilized in a manner that adequately provides physical protection from those alterations in homeostasis. As a result, the β-adrenergic branch of the autonomic nervous system is activated, leading to changes favoring the well-known “fight-or-flight” adaptive responses. Increases in blood pressure accompany a decrease in secretions to shunt blood away from digestive organs and toward vital and highly metabolic organs, and alterations in emotional patterns of behavior become evident. In many ways, prenatal stress in human and rodent models recapitulates glucocorticoid overexposure in pregnancy via increased HPA stimulation and leads to metabolic, cardiovascular and psychiatric disorders in adulthood (Cottrell & Seckl, 2009; Heim & Nemeroff, 2001; Lesage et al., 2004). Feedback circuits are used extensively by endocrine systems to regulate hormone secretion. It would follow suit that any disruption to a critical negative feedback control circuit would have far-reaching consequences on the biochemical constitution of an affected organism, particularly during the pliable developmental stages. Much of the negative feedback response to HPA activation is conducted within the hippocampus, and is mediated by glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Low expression of hippocampal GR has been observed in a rat model of kindling-induced fear (Kalynchuk & Meaney, 2003). Due in part to inhibition of negative feedback circuitry, a decrease in hippocampal GR expression in rats exposed to either exogenous glucocorticoids or prenatal stress has been reported and is accompanied by an elevation in CRH and altered corticosterone levels (Mueller & Bale, 2008; Welberg, Seckl, & Holmes, 2001). The behavioral and metabolic consequences of increased serum glucocorticoid concentrations are well-characterized, as major sequelae of Cushing syndrome, a disease of excess cortisol secretion, include hypertension, diabetes, obesity, irritability, cognitive difficulties, depression and anxiety. In a normal physiological stress response, the placental barrier effectively protects the fetus from bursts of maternal glucocorticoid release via metabolic breakdown by 11-β-hydroxysteroid dehydrogenase (11β-HSD) (Benediktsson, Calder, Edwards, & Seckl, 1997). However, prenatal stress is sufficient in dampening the protective effect of this enzyme and results in excess glucocorticoid exposure in rodent models, which occurs, at least in part, through the downregulation of corticosterone binding globulin (Takahashi et al., 1998). This could very well be an adaptive response, as sustained stress can be a cue for the developing fetus that danger is prevalent or food is scarce. The idea that the developing organism undergoes adaptive responses in its metabolic phenotype in the presence of suboptimal conditions, such as undernutrition or gestational stress, to give it an increased chance of postnatal survival is called the Predictive Adaptive Response hypothesis (Gluckman & Hanson, 2004). This hypothesis opens the door to investigate the epigenetic mechanisms of toxicity resulting from perinatal stressors which alter the HPA axis and/or other targets, particularly at different stages of fetal growth (Grace, Kim, & Rogers, 2011).

224

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

3. Epigenetic mechanisms Epigenetics is a branch of molecular biology that describes heritable changes in gene expression independent of changes in nucleotide sequence (reviewed in Holliday, 1994; Watson & Goodman, 2002). Moreover, epigenetic alterations are stable in nature yet reversible. A simple yet powerful example of epigenetic regulation of gene expression is the addition of a bulky methyl group obtained from a methyl donor such as S-adenosylmethionine (SAM) to a cytosine residue in the promoter region of a gene by a DNA methyltransferase (DNMT) (Bird, 2002; Szyf, 2009). This method of modification often has repressive effects on gene expression by impeding access of the binding domain to activating transcription factors (Bird, 2002). Another frequent yet more complex type of epigenetic control is histone modification. Histones are nuclear proteins that assemble into octameric complexes consisting of two H2A–H2B dimers and a H3–H4 tetramer that together form a nucleosome, with approximately 147 base pairs of DNA which wrap around the octamer (Jiang & Pugh, 2009). The histone code hypothesis, proposed over a decade ago, eloquently describes the post-translational modifications of epigenetic components acting upon histones to produce downstream effects in regulation of gene expression (Strahl & Allis, 2000). Such modifications serve as the fundamental letters that spell out a vast, enriched molecular language. The diversity of modifications and their functional consequences on gene expression are dependent on several key structural alterations, primarily on the N-terminal extensions, or “tails”, of histones (Gardner, Allis, & Strahl, 2011). These reversible histone protein modifications include: (i) the addition of methyl groups by histone methyltransferases (HMTs) or the removal of these groups by histone demethylases, (ii) acetylation modifications, by either histone acetyltransferases (HAT) or histone deacetylases (HDAC), (iii) the addition of phosphate groups by serine/threonine kinases and removal by phosphatases, and (iv) additional modifications such as sumoylation, glycosylation, poly-ADP-ribosylation, and ubiquitination (Bártová, Krejcí, Harnicarová, Galiová and Kozubek, 2008). In addition, chromatin architecture is formed and maintained by ATP-dependent chromatin remodeling factors, chromodomain-containing proteins that interact with methylated histones, bromodomain-containing proteins that interact with acetylated histones, and scaffolding proteins (Jenuwein & Allis, 2001). In general, methylation marks on histone proteins are transcriptionally repressive while acetylation marks are transcriptionally active. There are certainly exceptions to this. For example, trimethylation of lysine 9 of histone 3 (H3K9me3) is typically indicative of epigenetic silencing while H3K4me3 is normally part of a signature for transcriptional activation (Lachner & Jenuwein, 2002; Li et al., 2008). Likewise, chromodomain-containing proteins are usually co-repressors while proteins having bromodomains are transcriptional co-activators, with notable exceptions. As a general rule, transcriptionally active regions of chromatin are dubbed euchromatin while repressive regions are called heterochromatin (Jenuwein & Allis, 2001; Li et al., 2008). The chromatin remodeling machinery is under tight regulation as well. Some repressive marks such as those present on centromeric regions are maintained for life (constitutive heterochromatin), while others are de-repressed under environmental conditions amiable for enhanced gene expression (facultative heterochromatin). The stable yet reversible nature of facultative heterochromatin makes it particularly relevant to this discussion. The role of microRNAs (miRNAs), particularly with respect RNAassociated gene silencing, is also an important component in epigenetic responses (Morris, 2008; Zhou, Hu, & Lai, 2010). miRNAs are short, 21–23 nucleotide hairpin-like structures that provide an epigenetic mechanism for gene silencing by binding to mRNA transcripts and targeting them for degradation prior to translation (Bartel, 2009). In at least some cases, multiple miRNAs may target a single transcript or a group of related transcripts at different regions, leading to a redundant

suppressant activity (Malumbres, 2012; Shirdel, Xie, Mak, & Jurisica, 2011). While very little concerning the role of miRNAs in the toxicology of perinatal stress is currently known, these small RNAs with an important regulatory role will undoubtedly be an intense area of study in the coming years. 3.1. Epigenetic control of the HPA axis in prenatal and perinatal stress The idea that epigenetic alterations were involved in stress response programming began when it was noticed that pup handling of offspring born under prenatal stress environments displayed a reversal in their behavior with accompanying changes in HPA activity (Vallee et al., 1997; Wakshlak & Weinstock, 1990). In observing maternal care measured by amount of licking and grooming (LG) performed by mothers, it has been shown that hippocampal GR mRNA and protein expression increases in pups exposed to higher levels of maternal care (Weaver, 2007; Weaver, Cervoni, Champagne, et al., 2004). The reversal of HPA overstimulation and behavioral defects associated with prenatal stress is observed in cases of foster parenting as well (Darnaudery et al., 2004; Maccari et al., 1995), indicating a role for epigenetic programming in this process. Indeed, epigenetic changes regulating HPA activity have been elucidated (reviewed in Grace et al., 2011; Meaney, Szyf, & Seckl, 2007). One mechanism by which hippocampal GR activity is diminished is via DNA methylation of a cytosine residue at a 5′ CpG dinucleotide site of exon 17 in the promoter region of the gene that encodes GR (Meaney & Szyf, 2005; Weaver et al., 2004). This methylation mark was frequently observed in offspring in low LG (LLG) dams compared to high LG (HLG) dams exhibiting greater maternal care. Exon 17 methylation of the hypothalamic GR promoter has also been observed in male offspring of rats exposed to prenatal stress (Mueller & Bale, 2008) With this modification, the transcription factor NGFI-A cannot bind to the GR promoter to induce transcription (Weaver et al., 2007). A region encompassing exon 1-F of the NR3C1 gene, identified as the human homolog of the rat GR 17 promoter region (Cottrell & Seckl, 2009; Turner, Schote, Macedo, Pelascini, & Muller, 2006), was also found to be methylated in human newborns delivered by mothers with third trimester depression and correlated significantly with increased salivary cortisol stress responses, indicating HPA disruption (Oberlander et al., 2008). Additional changes in methylation patterns have been evinced in rodent brains of offspring of mothers exposed to prenatal stress. Microarray analyses of the frontal cortex and hippocampus are consistent with high degree of global DNA hypermethylation and transcriptional repression, even in cases of bystander stress where mothers were not directly stressed (Mychasiuk, Schmold, et al., 2011; Mychasiuk, Gibb and Kolb, 2011). This phenomenon is recapitulated by neonatal maternal care, where offspring of mothers in HLG dams display greater stimulation of gene expression compared to subjects in LLG dams (Weaver, Meaney, & Szyf, 2006). Strikingly, the reverse has been observed in pups of rats fostered for a considerable amount of time prenatally (and preconceptionally in the case of fathers) in enrichment condos designed for stress minimization (Mychasiuk et al., 2012). Under such enrichment conditions, analysis of the frontal cortex and hippocampus of pups revealed significant global hypomethylation, which corresponded to greater exploratory behavior and less time spent in the upward direction during a negative geotaxis task (Mychasiuk et al., 2012). When analyzing specific gene promoter regions, several changes have been identified as a result of perinatal stress in regions of the brain responsible for behavior, cognition and sexual differentiation. For example, a decrease in DNA methylation of the CRF promoter has been observed in the amygdalas of male offspring of prenatally stressed rats (Mueller & Bale, 2008). It is noteworthy that release of CRF from the amygdala occurs during stress (Cook, 2004) which, in turn, may exert receptor-mediated effects in this region of the brain in rat pups exposed to perinatal stress (Brunton, Donadio, & Russell, 2011). As hypomethylation is often associated with increased transcriptional

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

expression by removing the bulky methyl barrier that restricts access to transcription factors, this modification possibly reveals an increased stress response mediated by the amygdala that may drive additional glucocorticoid secretion. Another recent study on maternal deprivation from postnatal (PN) days 2 through 13 detected promoter hypomethylation in the hypothalamic paraventricular nucleus (PVN) which led to similar upregulation of CRF (Chen et al., 2012). In a study of corticosterone-treated 4 week old mice, significant loss of methylation was observed in the Fkbp5 gene in the hypothalamus and hippocampus. This gene encodes a GR chaperone that has been implicated in post-traumatic stress, depression and bipolar disorder (Lee et al., 2010). A transcript for a CRF-responsive receptor, Crfr2, was also shown to be hypomethylated in brains of male mouse pups in response to maternal separation for 2 weeks following birth, and this hypomethylation was linked to reduced gene expression (Franklin et al., 2010). The same study revealed transgenerational depressive symptoms, monitored through the F3 generation, which correlated to DNA hypermethylation and subsequent downregulation of methyl CpG-binding protein 2 (MeCP2), a methyl-binding epigenetic regulatory protein by which deficiency has been implicated in male-specific social and neurobiological disorders (McCarthy et al., 2009; Nagarajan, Hogart, Gwye, Martin, & LaSalle, 2006). Interestingly, in mice, control male placentas have been found to exhibit significantly lower expression of DNMT1 than control female placentas while early prenatal stress significantly increases DNMT1 expression in female placentas, but not male placentas (Mueller & Bale, 2008). These observations suggest that a gender-specific level of maternal–fetal placental regulation via epigenetic modifications exists during prenatal stress, perhaps via the redistribution of enzyme reservoirs necessary for epigenetic remodeling in a sex-dependent manner. It is also important to note that the type and intensity of perinatal stress can elicit dramatically different epigenetic responses in rats; e.g., mild prenatal stress leads to hypermethylation of DNA while high prenatal stress results in decreased methylation of DNA (Mychasiuk, Ilnytskyy, Kovalchuk, Kolb, & Gibb, 2011). 3.2. Non-HPA-mediated stress-induced epigenetic alterations in brain development Aside from its crucial role in HPA negative feedback, the hippocampus is involved in several other key developmental processes. Owing to the structural plasticity of its neural circuitry, the hippocampus is an important regulator of neurogenesis, spatial processing, neuroendocrine control, pain perception, emotional control, and other processes involved in learning, memory, and behavior (Leuner & Gould, 2010). The ventral hippocampus is postulated to be involved with brain processes associated with anxiety-related behaviors, while the dorsal hippocampus has a role in certain forms of learning and memory, particularly spatial learning (Bannerman et al., 2004). Another brain region implicated in prenatal stress alterations is the amygdala, which is involved in fear and anxiety programming in association with decreases in GR and elevations in CRF, respectively (Cratty, Ward, & Johnson, 1995; Kalynchuk & Meaney, 2003; Seckl & Meaney, 2004). The hippocampus exhibits a great deal of plasticity in response to stress, as it harbors an elevated number of steroid receptors and chromatin remodeling enzymes (Hunter, McCarthy, Milne, Pfaff, & McEwen, 2009). Mutation or altered expression of a number of chromatin remodeling genes in the hippocampus has been linked to various mental disorders (Lagali, Corcoran, & Picketts, 2010). These genes include several acetyl- and methyltransferases, binding proteins for epigenetic machinery, KRAB-ZFPs, and ATP-dependent remodeling factors. 3.3. KRAB-ZFP-dependent chromatin remodeling Epigenetically modulated stress response mechanisms by the hippocampus are multifarious. They involve transcriptional repression

225

mediated by the interplay of several factors; among them the Krüppelassociated box domain zinc finger proteins (KRAB-ZFP) and their cofactors (Groner et al., 2010; Tian et al., 2009; Wang, Rauscher, Cress, & Chen, 2007). KRAB-associated protein-1 (KAP1) is a co-repressor for epigenetic silencing that is highly expressed in the forebrain region, including the hippocampus (Jakobsson et al., 2008; Lein et al., 2007). KAP1 interacts with several KRAB-ZFPs and is instrumental in the recruitment of histone lysine N-methyltransferase (SETDB1), heterochromatin protein-1 (HP1), and the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex for facultative heterochromatin formation and spreading in response to stress (Groner et al., 2010; Sripathy, Stevens, & Schultz, 2006). HP1 isoforms are chromodomain containing proteins that interact with H3K9me3 to form a complex that stabilizes the epigenetic silencing effects of histone methylation for the induction of heterochromatin. The NuRD complex is a massive, multi-subunit complex involved in HDAC as well as ATP-dependent remodeling functions (Zhang et al., 1999). The net effect of this assembly is the formation of a bulky complex that restricts access to key binding regions for transcription factors involved in gene expression and prevents structural alterations such as acetylation, via HDAC activity, which would favor a more transcriptionally “open” chromatin structure. Acute restraint stress in rats has been shown to dramatically increase global hippocampal histone methylation at the repressive H3K9me3 mark (Hunter et al., 2009), implying the activation of a rapidly acting transcriptional repression program. One such program has been identified in which H3K9me3 and HP1β become extended along target gene strands in a 3′ to 5′ direction in a model of KRABmediated repression (Groner et al., 2010). Further research will reveal whether this mechanism plays a role in the epigenetic silencing response witnessed in the offspring of prenatally stressed or acutely stressed rodents. It is possible that epigenetic silencing mechanisms in the offspring of prenatally stressed mothers exhibit a more coordinated, less global facultative response involving genes specific for behavioral and metabolic activity. Interestingly, mice deleted for KAP1 in the adult forebrain reveal an anxious phenotype, with spatial memory-related learning impairment only in the presence of a stressor (Jakobsson et al., 2008). Although the functions of most of the KRABZFPs expressed in the hippocampus have not been elucidated, mutations in at least three genes (ZNF41, ZNF81, ZNF674) with known KRAB domains are implicated in X-linked mental retardation (Lagali et al., 2010). 3.4. ATP-dependent chromatin remodeling factors Chromatin remodeling factors often use the energy produced from ATP hydrolysis to induce dynamic changes in the structure of chromatin (Travers, 1999). A large eukaryotic family of ATP-dependent chromatin remodeling factors belong to SWI/SNF and related complexes. The SWI/SNF protein complex is thought to disrupt normal DNA/histone packaging to facilitate transcription factor binding via induction of topological stress on DNA strands to render the macromolecule more accessible (Aoyagi & Hayes, 2002). The SWI/SNF family member Alpha-thalassemia/mental retardation X-linked (ATRX, Rad54) is an ATP-dependent chromatin remodeling helicase expressed in the hippocampus that has a prominent role in neurogenesis (Seah et al., 2008). Severe mental retardation syndrome occurs from mutation of this gene on the X chromosome. Weaver et al. (2006) identified ATRX as one of three selected hippocampal genes from a panel of over 900 that were stably regulated by maternal care, as determined by the degree of licking-grooming and arched back nursing (LG-ABN) on rat pups. Low gene expression of ATRX was observed in pups of low LG-ABN dams compared to their high LG-ABN counterparts. This effect was reversed by administration of the HDAC inhibitor, trichostatin A (TSA). It can be speculated that the decreased expression was a result of dysregulation of acetylation-

226

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

dependent transcriptional activity, plausibly on the nucleosomes of those genes that are normally bound to and regulated by this remodeling factor. Reversal of expression profiles by administration of either TSA or a methyl donor, methionine, was mirrored in a significant portion of the genes differentially expressed based on LG-ABN profiles, showing clear widespread involvement of epigenetic regulatory mechanisms in conjunction with different levels of maternal care during the perinatal stage (Weaver et al., 2006). 3.5. Metabotropic glutamate receptors and the stress response Metabotropic glutamate receptors (GRM) are 7-transmembrane domain-containing G-protein-coupled receptor ion channels that are highly plastic in that they exhibit differential methylation patterns in human brain development from gestation to adulthood (Stadler et al., 2005). Highest expression levels are observed in the brain, particularly in the hippocampus, cerebellum, thalamus and cerebral cortex. Group I receptors (GRM1/5) are involved in excitatory neurotransmission and are connected to the phosphatidylinositol (PI)-calcium signaling pathway. Group II receptors (GRM2/3) and Group III receptors (GRM4/6/7/8) are often inhibitory and negatively regulate cAMP formation and adenylyl cyclase-dependent signal transduction. Group II receptors have been associated with psychosis (Harrison, Lyon, Sartonus, Burnet, & Lane, 2008; Tsunoka et al., 2010). Group I receptors are involved in fear conditioning and in plasticity at synapses involved in fear conditioning (Rodrigues, Bauer, Farb, Schafe, & LeDoux, 2002). In addition, alterations of GRM expression due to epigenetic changes may account for the inhibitory effects of prenatal stress upon neurogenesis in the hippocampus and spatial learning (Coe et al., 2003; Lemaire, Koehl, Le Moal, & Abrous, 2000). Future studies aimed at exploring this possibility will be telling. A downregulation of metabotropic L-glutamate receptor (GRM5), also known as mGluR5, has been reported during the perinatal period in response to low LG-ABN (Weaver et al., 2006). This effect has been reproduced in a model of prenatal restraint stress (Zuena et al., 2008) which found that hippocampal GRM expression was regulated in a gender-specific manner by which lower GRM5 expression was observed in males than in females while Group II (GRM2/3) receptors were demonstrated to be downregulated in both sexes. Sex-specific alterations in GRM expression were also observed by Mychasiuk, Gibb, et al. (2011) upon microarray analysis of the frontal cortex and hippocampus of PN21-sacrificed pups of mothers subjected to prenatal stress involving platform elevation at critical periods of gestation. Unlike Zuena et al. (2008), an increase in receptor expression from Groups I and II were observed in males compared to females. Although the changes in expression were inconsistent between studies, results from both point to the importance of GRMs in shaping sexually dimorphic events in response to perinatal stressors. 3.6. Epigenetic modifications associated with GRM Regulation Alterations in GRMs provide a link between epigenetic regulation and transcriptional control of processes involved in stress response. Blockade of Group I and II receptors by antagonists stimulates the HPA axis in vivo (Bradbury et al., 2003; Scaccianoce et al., 2003). In contrast, administration of a GRM2/3 agonist to mice promotes an epigenetic response of DNA demethylation of both brain-derived neurotrophic factor (Bdnf) and reelin (Reln) via a mechanism that involves GADD45b binding to the promoters of these genes (Matrisciano, Dong, Gavin, Nicoletti, & Guidotti, 2011); moreover, this effect was also induced by valproic acid and clozapine in the same study. GADD45b does not appear to act as a ubiquitous demethylating agent, but rather has its activity confined to specific promoter regions, including the promoters of growth factors modulating neurogenesis (Ma, Jang, Guo, et al., 2009). The DNA demethylating activity of valproic acid is well-characterized and is at least partially responsible for producing behavioral modifications in

schizophrenic and bipolar patients (Detich, Bovenzi, & Szyf, 2003; Milutinovic, D'Alessio, Detich, & Szyf, 2007). It is also worthy of mention that reelin has been found to be regulated by BDNF during brain development (Ringstedt, Linnarsson, & Wagner, 1998). Decreased expression of reelin due to hypermethylation of its gene promoter is associated with psychiatric diseases, including schizophrenia and bipolar disorder (Chen, Sharma, Costa, Costa, & Grayson, 2002; Costa et al., 2002; Grayson et al., 2005). BDNF is a secreted neurotrophin involved in the stress response mechanism. Expression is either up- or downregulated by alterations in histone acetylation patterns depending on the type of stress encountered (Fuchikami, Yamamoto, Morinobu, Takei, & Yamawaki, 2010). In a manner akin to agonist activation of GRM2/3, perinatal stress has been shown to affect the methylation status of BDNF (Roth, Lubin, Funk, & Sweatt, 2009). To this end, DNA methylation is observed at the promoter region of the BDNF gene in infant rodents that were neglected or abused by stressed parents within their first week following birth, and this epigenetic mark was conserved into the next generation (Roth et al., 2009). These observations, taken together, suggest that GRMs provide a link between epigenetic regulation and transcriptional control in the stressed brain. 3.7. Epigenetic regulation of sexually dimorphic traits Sexual differentiation is characterized by a combination of genetic and hormonal changes during embryonic and fetal development. Sex chromosome inheritance and gonadal hormone signaling are responsible for sexually dimorphic characteristics. Epigenetic regulation is being revealed as a robust mechanism for controlling many of these processes. For example, genomic imprinting of genes from parental sex chromosomes onto their progeny is tightly regulated by methylation events which allow silencing of certain genes and expression of others in a sex-specific manner (Gabory, Attig, & Junien, 2009). Additionally, gonadal hormone surges that occur during a temporal window in a perinatal-sensitive period lead to sexually dimorphic organizational changes in the brain (Arnold & Gorski, 1984). Alterations in hormonal signaling and resultant gene expression changes have been studied extensively in perinatal stress models. The perinatal window in which rodents are most sensitive to phenotypic changes brought about by hormonal secretion has been welldefined. Sexual differentiation is affected from embryonic day 18 (E18) to PN10 (Arnold & Gorski, 1984; Rhees, Shryne, & Gorski, 1990). In males, a testosterone surge is coincident with the onset of this period on E18 and a second peak occurs at PN1 merely 2 hours after birth (Corbier, Edwards, & Roffi, 1992; Weisz & Ward, 1980). Testosterone is metabolized to dihydrotestosterone by 5α-dihydrotestosterone reductase while estradiol is produced by aromatase activity. Metabolism to estradiol is a key step in male sexual differentiation (Bale, 2011; McEwen, Lieberburg, Chaptal, & Krey, 1977), and inhibition of aromatase leads to a feminized phenotype (Amateau, Alt, Stamps, & McCarthy, 2004; Nugent & McCarthy, 2011). Estradiol exerts its pharmacologic activities primarily via estrogen receptors (ERs)- such as ERα and ERβ, which are known to act in the nucleus of target cells. Activation of ERβ is involved in defeminization of brain and behavior (Kudwa, Bodo, Gustafsson, & Rissman, 2005). It is also noteworthy that estradiol can directly affect ion channels and G protein coupled receptors, independent of the estrogen receptor (ER), but the degree to which such effects are involved in sexual dimorphic brain development remains to be determined (McCarthy, 2008). The hypothalamus is important for female sexual behavior while male sexual behavior is controlled to a large degree by the preoptic area (POA) (McCarthy et al., 2009). In the first 10 days following birth, ERα expression is typically elevated in the POA of females and found to be decreased in the POA of male rats coincident with promoter hypermethylation (DonCarlos & Handa, 1994; Kurian, Olesen, & Auger, 2010). To further illustrate the complexity of

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

estradiol in determining sex-specific events via epigenetic regulation, Schwarz, Nugent, and McCarthy (2010) demonstrated that exogenous estradiol administration to female newborn rats at birth leads to increased methylation of the gene promoter for progesterone receptor (PR) in both the POA (at PN1) and in the mediobasal hypothalamus (at PN1, PN20 and PN60), while the same estradiol treatment in female pups resulted in an increased methylation of the promoter for ERβ in the POA (at PN20). Not surprisingly, methylation marks such as these are also affected by perinatal maternal care. Female rats raised by LLG dams for 7 days postpartum displayed increased cytosine methylation of the 1b ERα promoter region in the POA compared to their counterparts raised by HLG dams, which prevented Stat5b binding and activation of the promoter (Champagne et al., 2006). A greater extent of promoter methylation of ERα in the POA has been observed in females compared to males at PN1 and PN60 (Schwarz et al., 2010). Whether such methylation marks correlate with masculine sexual behavior in females is yet to be determined but an interesting speculation nonetheless. Prenatal stress applied to different time points either early or late in the gestation period has been shown to cause phenotypes consistent with low testosterone activity, including shortened anogenital distance, small testis size, and reduced total testosterone concentrations (Dunn, Morgan, & Bale, 2011; Reznikov, Nosenko, & Tarasenko, 1999; Ward & Weisz, 1980). Reduction in testosterone secretion can have direct epigenetic consequences. Murray, Hien, de Vries, and Forger (2009) used a neonatal mouse model to show that treatment of both males and females administered exogenous testosterone with the HDAC inhibitor valproic acid effectively reversed the masculinizing effects of testosterone in the bed nucleus of the stria terminalis. While the mechanisms of testosterone reduction during the perinatal sensitive period are still largely undetermined, new clues have emerged from miRNA profiling. Morgan and Bale (2011) analyzed the whole-brain miRNA signatures of second generation offspring of prenatally stressed mice and observed a dysmasculinized phenotype of males characterized by shortened anogenital distances, low testis weights, and a distinct miRNA profile that included significant reductions in 3 miRNAs that converged on the common target transcript of the Tgfbr3 gene encoding betaglycan. This TGF-β family type 3 receptor was subsequently found to be significantly more highly expressed in F2 prenatally stressed male offspring compared to second generation control male and female pups (Morgan & Bale, 2011). Betaglycan participates in multiple aspects of TGF-β family ligand signaling, with its principal function purported as being a transmembrane coreceptor for inhibin A/B binding to type 2 activin receptors (ActRII) (Wiater & Vale, 2003; Wiater et al., 2009). Inhibin/betaglycan/ ActRII complexes primarily antagonize the activity of activins (Wiater et al., 2009), dimeric proteins formed by disparate arrangements of inhibin β-type subunits (Vale et al., 1988). While activins are broadly expressed in most tissues and serve a wide variety of functions involved in reproduction, immunity, inflammation, wound repair and cell cycle regulation, inhibins (and betaglycan) are more specifically expressed locally in gonads, adrenals and placental tissue, and are associated predominantly with reproductive effects and sex hormone regulation (Hedger & Winnall, 2011). Abrogation of activin A-mediated follicle-stimulated hormone (FSH) secretion is a major function of inhibin signaling through the betaglycan coreceptor (Lewis et al., 2000). Important functions of FSH in males include testicular growth and stimulation of germ cell differentiation in Sertoli cells, and reduced levels would be consistent with abnormal testis development and low weight. Indeed, CRF released from stress-related glucocorticoid signaling has an inhibitory effect on FSH and luteinizing hormone (LH) and chronic immobilization stress has been shown to reduce plasma levels of both hormones (Collu, Taché, & Ducharme, 1979). However, a direct correlation between perinatal stress and reduced FSH secretion has thus far not been identified. This may be due to time-dependent and sex-specific effects on hormonal regulation or there may be an as

227

yet undefined mechanism by which betaglycan exerts its hormonal effects. Increased inhibin/betaglycan activity has been demonstrated to have inhibitory effects on several bone morphogenetic proteins (BMPs) (Farnsworth et al., 2006; Wiater & Vale, 2003). Considering several BMPs are known to have a role in modulating gonadal hormone synthesis and secretion in developmental processes (Dooley, Attia, Rainey, Moore, & Carr, 2000; Glister, Satchell, & Knight, 2010), betaglycan interactions with these ligands allow for several additional potential points of regulatory activity amenable to alterations in perinatal stress models. Clearly, with the identification of betaglycan as a novel receptor affected by perinatal stress-induced epigenetic dysregulation via disruption of miRNA-mediated transcript degradation, it can be assumed that further employment of miRNA-based technology to probe perinatal stress events will fructify with recognition of additional transcriptional targets that better clarify the connection between altered gene expression and phenotypic changes. 4. The relevance of understanding the epigenetic basis of prenatal stress to human diseases Over the past few years, much effort has been directed at understanding epigenetics, the alterations that occur in utero, and their association with diseases during the adult life. In fact, epigenetic programming is a delicate regulatory process that occurs during the initial steps of embryonic development; superimposing a stressor agent onto the vulnerable genetic substrate during this phase could lead to a defective epigenetic alteration and, ultimately, to a future pathological condition in humans, such as type 2 diabetes, stroke, heart diseases and cancer (Barker et al., 1993; Lesage et al., 2004; Louey & Thornburg, 2005; Tamashiro & Moran, 2010). Further, there is speculation that assisted reproductive technology (ART) might be associated with birth defects, through epigenetic alterations (Maher, Afnan, & Barratt, 2003). In a prospective study, DeBaun, Niemitz, and Feinberg (2003) found that a group of ARTconceived children with Beckwith-Wiedemann syndrome (a congenital disorder characterized by overgrowth and neoplasia) had aberrant methylation and imprinting in genes LIT1 and/or H19. Specific epigenetic alterations also were found in two ART-children who developed Angelman syndrome, which is characterized by severe mental retardation and motor disorders (Cox et al., 2002). Although there is strong evidence linking epigenetic disturbances and diseases later in life in human populations, most of the molecular data come from animal models, mainly demonstrating how altered nutritional states during pregnancy introduce epigenetic mechanisms involved in human disease susceptibility. For instance, prenatal stress effects in mice are associated with significant effects on food and water intake in the gravid female (Kinsley & Svare, 1986). Rats submitted to intrauterine growth retardation (IUGR) evince reduced expression of Pdx1 (a transcription factor that regulates pancreas development and β cell differentiation), and is an effect mediated by modifications in histones and consequent chromatin remodeling (Park, Stoffers, Nicholls, & Simmons, 2008). Consequently, Pdx1 reduction caused by epigenetic alterations may contribute to the development of type 2 diabetes and β cell dysfunction in both humans (Stoffers, Zinkin, Stanojevic, Clarke, & Habener, 1997) and experimental animals (Brissova et al., 2005). A recent study showed that hypoxia in pregnant rats results in decreased PKC epsilon protein expression in the heart of adult offspring, which is mediated by epigenetic modifications in DNA methylation (Patterson, Chen, Xue, Xiao, & Zhang, 2010). Because PKC epsilon has a protective effect in heart ischemia and reperfusion injuries, its reduction may thus increase the susceptibility to cardiac dysfunction (Murriel & Mochly-Rosen, 2003). More to the point of the current review, exposure to toxic substances during gestation has been shown to affect offspring development. For example, Anway, Cupp, Uzumcu, and Skinner (2005) treated independent groups of pregnant rats with methoxychlor or

228

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

vinclozolin (two environmental toxicants commonly used as insecticide and fungicide, respectively). They demonstrated spermatogenic cell defects and subfertility in the male F1 generation and, surprisingly, all the subsequent generations analyzed (F1–F4) showed the same phenotype. The authors suggest that the mechanism involved in this transgenerational effect appears to be related to altered DNA methylation of the male germ line. Additional observations revealed that these animals also developed diseases such as cancer, kidney and prostate diseases during adulthood, indicating ancillary effects connected to the main ones. These data clearly show that in utero exposure to a stressor can lead to a disease later in life, possibly through defective epigenetic modifications. We know that aberrant epigenetic alterations are involved in the development of many different types of human tumors. Growths such as lymphomas, gliomas, ovarian, prostate and colon cancers (Esteller, 2008) are prevalent. These abnormal alterations include, mainly, changes in DNA methylation, histone modifications, nucleosome positioning, and non-coding mRNA expression (Sharma, Kelly, & Jones, 2010). The role of prenatal stress in tumor development, however, remains unclear, although it makes sense that perturbations in utero could contribute in some way similar as in other diseases with a fetal origin. Nonetheless, further investigation is necessary to support this assumption. Lastly, remarkable efforts have been directed in the development of a new therapeutic approach, the so-called “epigenetic therapy”. Unlike mutations, epigenetic modifications are potentially reversible; and the development of drugs that act as histone deacetylase (HDAC) or DNA methyltransferase (DNMT) inhibitors have been shown to be a promising approach in the treatment of diseases associated with epigenetic defects (Egger, Liang, Aparicio, & Jones, 2004). Moreover, these drugs have the potential to reactivate silenced tumor-suppressor genes, which leads to their utility in clinical cancer drug trials (Egger et al., 2004; Laird, 2005).

5. Summary and conclusions The rodent prenatal stress model is highly amenable to the study of epigenetic mechanisms perturbed by stress during pregnancy. To the present date, a handful of epigenetic effects have been identified to result as a consequence of prenatal or early postnatal stressors (Table 1). A better understanding of the pathophysiological mechanisms involved in perinatal stress-induced epigenetic defects, therefore, could provide the basis for the development of diagnostic and treatment strategies in the early stages of gestation, reducing the possibility of diseases during adult life. A large battery of experimental tools to study the epigenetic phenomena which result as a consequence of perinatal stress have been developed over the past 10 years. As we have seen here, methods have been developed which allow for the easy measurement of methylation events on DNA or histone proteins. Histone acetylation and deacetylation can also be easily quantified. Various other methods have been procured allowing for the measurement of miRNAs which can alter the expression of important target genes sensitive to perinatal stressors. In addition, pharmacological inhibitors of HMTs, HATs and HDACs will make the study of epigenetic changes resulting from perinatal stress one which is less technically challenging and more hypothesis-driven. Overall, a better understanding of such myriad and multi-faceted effects is warranted.

Acknowledgements The authors are extremely grateful to Marina Guralnik (Masters candidate, St. John's University), Grant sponsor: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil.

References Alonso, J., Castellano, M. A., & Rodriguez, M. (1991). Behavioral lateralization in rats: Prenatal stress effects on sex differences. Brain Research, 539, 45–50. Amateau, S. K., Alt, J. J., Stamps, C. L., & McCarthy, M. M. (2004). Brain estradiol content in newborn rats: Sex differences, regional heterogeneity, and possible de novo synthesis by the female telencephalon. Endocrinology, 145, 2906–2917. Amateau, S. K., & McCarthy, M. M. (2002). Sexual differentiation of astrocyte morphology in the developing rat preoptic area. Journal of Neuroendocrinology, 14, 904–910. Amateau, S. K., & McCarthy, M. M. (2004). Induction of PGE2 by estradiol mediates developmental masculinization of sex behavior. Nature Neuroscience, 7, 643–650. Anway, M. D., Cupp, A. S., Uzumcu, M., & Skinner, M. K. (2005). Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science, 308, 1466–1469. Aoyagi, S., & Hayes, J. J. (2002). hSWI/SNF-catalyzed nucleosome sliding does not occur solely via a twist-diffusion mechanism. Molecular and Cellular Biology, 22, 7484–7490. Arnold, A. P., & Gorski, R. A. (1984). Gonadal steroid induction of structural sex differences in the central nervous system. Annual Review of Neuroscience, 7, 413–442. Bale, T. (2011). Sex differences in prenatal epigenetic programing of stress pathways. Stress, 14, 348–356. Bannerman, D. M., Rawlins, J. N., McHugh, S. B., Deacon, R. M., Yee, B. K., Bast, T., Zhang, W. N., Pothuizen, H. H., & Feldon, J. (2004). Regional dissociations within the hippocampus— Memory and anxiety. Neuroscience and Biobehavioral Reviews, 28, 273–283. Barbazanges, A., Piazza, P. V., Le, M. M., & Maccari, S. (1996). Maternal glucocorticoid secretion mediates long-term effects of prenatal stress. Journal of Neuroscience, 16, 3943–3949. Barker, D. J. P., Gluckman, P. D., Godfrey, K. M., Hardind, J. E., Owens, J. A., & Robinson, J. S. (1993). Fetal nutrition and cardiovascular disease in adult life. Lancet, 341, 938–941. Bartel, D. P. (2009). MicroRNAs: Target recognition and regulatory functions. Cell, 136, 215–233. Bártová, E., Krejcí, J., Harnicarová, A., Galiová, G., & Kozubek, S. (2008). Histone modifications and nuclear architecture: A review. The Journal of Histochemistry and Cytochemistry, 56, 711–721. Benediktsson, R., Calder, A. A., Edwards, C. R., & Seckl, J. R. (1997). Placental 11 betahydroxysteroid dehydrogenase: A key regulator of fetal glucocorticoid exposure. Clinical Endocrinology, 46, 161–166. Beyer, C., & Feder, H. H. (1987). Sex steroids and afferent input: Their roles in brain sexual differentiation. Annual Review of Physiology, 49, 349–364. Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes & Development, 16, 6–21. Bradbury, M. J., Giracello, D. R., Chapman, D. F., Holtz, G., Schaffhauser, H., Rao, S. P., Varney, M. A., & Anderson, J. J. (2003). Metabotropic glutamate receptor 5 antagonist-induced stimulation of hypothalamic–pituitary–adrenal axis activity: Interaction with serotonergic systems. Neuropharmacology, 44, 562–572. Brissova, M., Blaha, M., Spear, C., Nicholson, W., Radhika, A., Shiota, M., et al. (2005). Reduced PDX-1 expression impairs islet response to insulin resistance and worsens glucose homeostasis. American Journal of Physiology. Endocrinology and Metabolism, 288, E707–E714. Brunton, P. J., Donadio, M. V., & Russell, J. A. (2011). Sex differences in prenatally programmed anxiety behavior in rats: Differential corticotropin-releasing hormone receptor mRNA expression in the amygdaloid complex. Stress, 14, 634–643. Champagne, F. A., Weaver, I. C., Diorio, J., Dymov, S., Szyf, M., & Meaney, M. J. (2006). Maternal care associated with methylation of the estrogen receptor-alpha1b promoter and estrogen receptor-alpha expression in the medial preoptic area of female offspring. Endocrinology, 147, 2909–2915. Chen, J., Evans, A. N., Liu, Y., Honda, M., Saavedra, J. M., & Aguilera, G. (2012). Maternal deprivation in rats is associated with corticotrophin-releasing hormone (CRH) promoter hypomethylation and enhances CRH transcriptional responses to stress in adulthood. Journal of Neuroendocrinology, 24, 1055–1064. Chen, Y., Sharma, R. P., Costa, R. H., Costa, E., & Grayson, D. R. (2002). On the epigenetic regulation of the human reelin promoter. Nucleic Acids Research, 30, 2930–2939. Coe, C. L., Kramer, M., Czéh, B., Gould, E., Reeves, A. J., Kirschbaum, C., & Fuchs, E. (2003). Prenatal stress diminishes neurogenesis in the dentate gyrus of juvenile rhesus monkeys. Biological Psychiatry, 54, 1025–1034. Collu, R., Taché, Y., & Ducharme, J. R. (1979). Hormonal modifications induced by chronic stress in rats. Journal of Steroid Biochemistry, 11, 989–1000. Cook, C. J. (2004). Stress induces CRF release in the paraventricular nucleus, and both CRF and GABA release in the amygdala. Physiology & Behavior, 82, 751–762. Cooke, B. M., & Woolley, C. S. (2005). Gonadal hormone modulation of dendrites in the mammalian CNS. Journal of Neurobiology, 64, 34–46. Corbier, P., Edwards, D. A., & Roffi, J. (1992). The neonatal testosterone surge: A comparative study. Archives Internationales de Physiologie, de Biochimie et de Biophysique, 100, 127–131. Costa, E., Chen, Y., Davis, J., et al. (2002). REELIN and schizophrenia: A disease at the interface of the genome and the epigenome. Molecular Interventions, 2, 47–57. Cottrell, E. C., & Seckl, J. R. (2009). Prenatal stress, glucocorticoids and the programming of adult disease. Frontiers in Behavioral Neuroscience, 3, 19. Cox, G. F., Bürger, J., Lip, V., Mau, U. A., Sperling, K., & Wu, B. L. (2002). Intracytoplasmic sperm injection may increase the risk of imprinting defects. American Journal of Human Genetics, 71, 162–164. Cratty, M. S., Ward, H. E., & Johnson, E. A. (1995). Prenatal stress increases corticotropinreleasing factor (Crf) content and release in rat amygdala minces. Brain Research, 675, 297–302. Daniels, D., & Flanagan-Cato, L. M. (2000). Functionally-defined compartments of the lordosis neural circuit in the ventromedial hypothalamus in female rats. Journal of Neurobiology, 45, 1–13.

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231 Daniels, D., Miselis, R. R., & Flanagan-Cato, L. M. (1999). Central neuronal circuit innervating the lordosis-producing muscles defined by transneuronal transport of pseudorabies virus. Journal of Neuroscience, 19, 2823–2833. Darnaudery, M., Koehl, M., Barbazanges, A., Cabib, S., Le, M. M., & Maccari, S. (2004). Early and later adoptions differently modify mother–pup interactions. Behavioral Neuroscience, 118, 590–596. DeBaun, M. R., Niemitz, E. L., & Feinberg, A. P. (2003). Association of in vitro fertilization with Beckwith–Wiedemann syndrome and epigenetic alterations of LIT1 and H19. American Journal of Human Genetics, 72, 156–160. Detich, N., Bovenzi, V., & Szyf, M. (2003). Valproate induces replication-independent active DNA demethylation. The Journal of Biological Chemistry, 278, 27586–27592. Diaz, H., Lorenzo, A., Carrer, H. F., & Caceres, A. (1992). Time lapse study of neurite growth in hypothalamic dissociated neurons in culture: Sex differences and estrogen effects. Journal of Neuroscience Research, 33, 266–281. Dominguez, J. M., Balfour, M. E., Lee, H. S., Brown, J. L., Davis, B. A., & Coolen, L. M. (2007). Mating activates NMDA receptors in the medial preoptic area of male rats. Behavioral Neuroscience, 121, 1023–1031. DonCarlos, L. L., & Handa, R. J. (1994). Developmental profile of estrogen receptor mRNA in the preoptic area of male and female neonatal rats. Brain Research. Developmental Brain Research, 79, 283–289. Dooley, C. A., Attia, G. R., Rainey, W. E., Moore, D. R., & Carr, B. R. (2000). Bone morphogenetic protein inhibits ovarian androgen production. The Journal of Clinical Endocrinology and Metabolism, 85, 3331–3337. Dörner, G., Poppe, I., Stahl, F., Kölzsch, J., & Uebelhack, R. (1991). Gene- and environment-dependent neuroendocrine etiogenesis of homosexuality and transsexualism. Experimental and Clinical Endocrinology, 98(2), 141–150. Dunn, G. A., Morgan, C. P., & Bale, T. L. (2011). Sex-specificity in transgenerational epigenetic programming. Hormones and Behavior, 59, 290–295. Egger, G., Liang, G., Aparicio, A., & Jones, P. A. (2004). Epigenetics in human disease and prospects for epigenetic therapy. Nature, 429, 457–463. Esteller, M. (2008). Epigenetics in cancer. The New England Journal of Medicine, 358, 1148–1159. Farnsworth, P. G., Stanton, P. G., Wang, Y., Escalona, R., Findlay, J. K., & Ooi, G. T. (2006). Inhibins differentially antagonize activin and bone morphogenetic protein action in a mouse adrenocortical cell line. Endocrinology, 147, 3462–3471. Flanagan-Cato, L. M. (2000). Estrogen-induced remodeling of hypothalamic neural circuitry. Frontiers in Neuroendocrinology, 21, 309–329. Franklin, T. B., Russiq, H., Weiss, I. C., et al. (2010). Epigenetic transmission of the impact of early stress across generations. Biological Psychiatry, 68, 408–415. Fuchikami, M., Yamamoto, S., Morinobu, S., Takei, S., & Yamawaki, S. (2010). Epigenetic regulation of BDNF gene in response to stress. Psychiatry Investigation, 7, 251–256. Gabory, A., Attig, L., & Junien, C. (2009). Sexual dimorphism in environmental epigenetic programming. Molecular and Cellular Endocrinology, 304, 8–18. Gardner, K. E., Allis, C. D., & Strahl, B. D. (2011). OPERating ON chromatin, a colorful language where context matters. Journal of Molecular Biology, 409, 36–46. Gerecke, K. M., Kishore, R., Jasnow, A., Quadros-Menella, P., Parker, S., Kozub, F. J., Lambert, K. G., & Kinsley, C. H. (2012). Alterations of sex-typical microanatomy: Prenatal stress modifies the structure of medial preoptic area neurons in rats. Developmental Psychobiology, 54, 16–27. Gerrits, P. O., Kortekaas, R., Veening, J. G., de Weerd, H., Algra, A., Mouton, L. J., & van der Want, J. J. L. (2008). Estrous cycle-dependent neural plasticity in the caudal brainstem in the female golden hamster: Ultrastructural and immunocytochemical studies of axo-dendritic relationships and dynamic remodeling. Hormones and Behavior, 54, 627–639. Glister, C., Satchell, L., & Knight, P. G. (2010). Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin coreceptor betaglycan during bovine antral follicle development: Inhibin can antagonize the suppressive effect of BMPs on thecal androgen production. Reproduction, 140, 699–712. Gluckman, P. D., & Hanson, M. A. (2004). The developmental origins of the metabolic syndrome. Trends in Endocrinology and Metabolism, 15, 183–187. Grace, C. E., Kim, S. -J., & Rogers, J. M. (2011). Maternal influences on epigenetic programming of the developing hypothalamic–pituitary–adrenal axis. Birth Defects Research. Part A, Clinical and Molecular Teratology, 91, 797–805. Grayson, D. R., Jia, X., Chen, Y., et al. (2005). Reelin promoter hypermethylation in schizophrenia. Proceedings of the National Academy of Sciences of the United States of America, 102, 9341–9346. Griffin, G. D., & Flanagan-Cato, L. M. (2008). Estradiol and progesterone differentially regulate the dendritic arbor of neurons in the hypothalamic ventromedial nucleus of the female rat (Rattus norvegicus). The Journal of Comparative Neurology, 510, 631–640. Grisham, W., Kerchner, M., & Ward, I. L. (1991). Prenatal stress alters sexually dimorphic nuclei in the spinal cord of male rats. Brain Research, 551, 126–131. Groner, A. C., Meylan, S., Ciuffi, A., et al. (2010). KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading. PloS One, 6, e1000869. Harrison, P. J., Lyon, L., Sartonus, L. J., Burnet, P. W., & Lane, T. A. (2008). The group II metabotropic glutamate receptor 3 (mGluR3, mGlu3, GRM3): Expression, function and involvement in schizophrenia. Journal of Psychopharmacology, 22, 308–322. Harvey, P. W., & Chevins, P. F. (1985). Crowding pregnant mice affects attack and threat behavior of male offspring. Hormones and Behavior, 19, 86–97. Hedger, M. P., & Winnall, W. R. (2011). Regulation of activin and inhibin in the adult testis and the evidence for functional roles in spermatogenesis and immunoregulation. Molecular and Cellular Endocrinology, 359, 30–42. Heim, C., & Nemeroff, C. B. (2001). The role of childhood trauma in the neurobiology of mood and anxiety disorders: Preclinical and clinical studies. Biological Psychiatry, 49, 1023–1039.

229

Herrenkohl, L. R. (1979). Prenatal stress reduces fertility and fecundity in female offspring. Science, 206, 1097–1099. Holliday, R. (1994). Epigenetics: An overview. Developmental Genetics, 15, 453–457. Humm, J. L., Lambert, K. G., & Kinsley, C. H. (1995). Paucity of c-fos expression in the medial preoptic area of prenatally stressed male rats following exposure to sexually receptive females. Brain Research Bulletin, 37, 363–368. Hunter, R. G., McCarthy, K. J., Milne, T. A., Pfaff, D. W., & McEwen, B. S. (2009). Regulation of hippocampal H3 histone methylation by acute and chronic stress. Proceedings of the National Academy of Sciences of the United States of America, 106, 20912–20917. Insel, T. R., Kinsley, C. H., Mann, P. E., & Bridges, R. S. (1990). Prenatal stress has long-term effects on brain opiate receptors. Brain Research, 511, 93–97. Jakobsson, J., Cordero, M. I., Bisaz, R., Groner, A. C., Busskamp, V., Bensadoun, J. C., Cammas, F., Losson, R., Mansuy, I. M., Sandi, C., & Trono, D. (2008). KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress. Neuron, 60, 818–831. Jenuwein, T., & Allis, C. D. (2001). Translating the histone code. Science, 293, 1074–1080. Jiang, C., & Pugh, B. F. (2009). Nucleosome positioning and gene regulation: Advances through genomics. Nature Reviews. Genetics, 10, 161–172. Jones, H. E., Ruscio, M. A., Keyser, L. A., Gonzalez, C., Billack, B., Rowe, R., Hancock, C., Lambert, K. G., & Kinsley, C. H. (1997). Prenatal stress alters the size of the rostral anterior commissure in rats. Brain Research Bulletin, 42, 341–346. Kalynchuk, L. E., & Meaney, M. J. (2003). Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 27, 1225–1234. Kawashima, S., & Takagi, K. (1994). Role of sex steroids on the survival, neuritic outgrowth of neurons, and dopamine neurons in cultured preoptic area and hypothalamus. Hormones and Behavior, 28, 305–312. Kawata, M., Yuri, K., & Morimoto, M. (1994). Steroid hormone effects on gene expression, neuronal structure, and differentiation. Hormones and Behavior, 28, 477–482. Kelley, D. B. (1988). Sexually dimorphic behaviors. Annual Review of Neuroscience, 11, 225–251. Kerchner, M., Malsbury, C. W., Ward, O. B., & Ward, I. L. (1995). Sexually dimorphic areas in the rat medial amygdala: Resistance to the demasculinizing effect of prenatal stress. Brain Research, 672, 251–260. Keshet, G. I., & Weinstock, M. (1995). Maternal naltrexone prevents morphological and behavioral alterations induced in rats by prenatal stress. Pharmacology, Biochemistry, and Behavior, 50, 413–419. Kinsley, C. H., & Bridges, R. S. (1987). Prenatal stress reduces estradiol-induced prolactin release in male and female rats. Physiology & Behavior, 40, 647–653. Kinsley, C. H., & Bridges, R. S. (1988). Prenatal stress and maternal behavior in intact virgin rats: Response latencies are decreased in males and increased in females. Hormones and Behavior, 22, 76–89. Kinsley, C. H., Mann, P. E., & Bridges, R. S. (1988a). Prenatal stress alters morphine- and stress-induced analgesia in male and female rats. Pharmacology, Biochemistry, and Behavior, 30, 123–128. Kinsley, C. H., Mann, P. E., & Bridges, R. S. (1988b). Prenatally stressed females and fertility and fecundity: Involvement of postcoital prolactin surges. Society for Neuroscience Abstracts, 14(281). Kinsley, C. H., Mann, P. E., & Bridges, R. S. (1989). Alterations in stress-induced prolactin release in adult female and male rats exposed to stress, in utero. Physiology & Behavior, 45, 1073–1076. Kinsley, C. H., Mann, P. E., & Bridges, R. S. (1992). Diminished luteinizing hormone release in prenatally stressed male rats after exposure to sexually receptive females. Physiology & Behavior, 52, 925–928. Kinsley, C., & Svare, B. (1986). Prenatal stress reduces intermale aggression in mice. Physiology & Behavior, 36, 783–786. Kinsley, C., & Svare, B. (1988). Prenatal stress alters maternal aggression in mice. Physiology & Behavior, 42, 7–13. Kudwa, A. E., Bodo, C., Gustafsson, J. A., & Rissman, E. F. (2005). A previously uncharacterized role for estrogen receptor beta: Defeminization of male brain and behavior. Proceedings of the National Academy of Sciences of the United States of America, 102, 4608–4612. Kurian, J. R., Olesen, K. M., & Auger, A. P. (2010). Sex differences in epigenetic regulation of the estrogen receptor-alpha promoter within the developing preoptic area. Endocrinology, 151, 2297–2305. Lachner, M., & Jenuwein, T. (2002). The many faces of histone lysine methylation. Current Opinion in Cell Biology, 14, 286–298. Lagali, P. S., Corcoran, C. P., & Picketts, D. J. (2010). Hippocampus development and function: role of epigenetic factors and implications for cognitive disease. Clinical Genetics, 78, 321–333. Laird, P. W. (2005). Cancer epigenetics. Human Molecular Genetics, 14, R65–R76. Lambert, K. G., Buckelew, S. K., Staffiso-Sandoz, G., Gaffga, S., Carpenter, W., Fisher, J., & Kinsley, C. H. (1998). Activity-stress induces atrophy of apical dendrites of hippocampal pyramidal neurons in male rats. Physiology & Behavior, 65, 43–49. Lambert, K. G., Gerecke, K. M., Quadros, P. S., Doudera, E., Jasnow, A. M., & Kinsley, C. H. (2000). Activity-stress increases density of GFAP-immunoreactive astrocytes in the rat hippocampus. Stress, 3, 275–284. Lee, R. S., Tamashiro, K. L., Yang, X., Purcell, R. H., Harvey, A., Willour, V. L., Huo, Y., Rongione, M., Wand, G. S., & Potash, J. B. (2010). Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice. Endocrinology, 151, 4332–4343. Lein, E. S., Hawrylycz, M. J., Ao, N., Ayres, M., Bensinger, A., Bernard, A., Boe, A. F., Boguski, M. S., Brockway, K. S., Byrnes, E. J., Chen, L., Chen, L., Chen, T. M., Chin, M. C., Chong, J., Crook, B. E., Czaplinska, A., Dang, C. N., Datta, S., Dee, N. R., Desaki, A. L., Desta, T., Diep, E., Dolbeare, T. A., Donelan, M. J., Dong, H. W.,

230

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231

Dougherty, J. G., Duncan, B. J., Ebbert, A. J., Eichele, G., Estin, L. K., Faber, C., Facer, B. A., Fields, R., Fischer, S. R., Fliss, T. P., Frensley, C., Gates, S. N., Glattfelder, K. J., Halverson, K. R., Hart, M. R., Hohmann, J. G., Howell, M. P., Jeung, D. P., Johnson, R. A., Karr, P. T., Kawal, R., Kidney, J. M., Knapik, R. H., Kuan, C. L., Lake, J. H., Laramee, A. R., Larsen, K. D., Lau, C., Lemon, T. A., Liang, A. J., Liu, Y., Luong, L. T., Michaels, J., Morgan, J. J., Morgan, R. J., Mortrud, M. T., Mosqueda, N. F., Ng, L. L., Ng, R., Orta, G. J., Overly, C. C., Pak, T. H., Parry, S. E., Pathak, S. D., Pearson, O. C., Puchalski, R. B., Riley, Z. L., Rockett, H. R., Rowland, S. A., Royall, J. J., Ruiz, M. J., Sarno, N. R., Schaffnit, K., Shapovalova, N. V., Sivisay, T., Slaughterbeck, C. R., Smith, S. C., Smith, K. A., Smith, B. I., Sodt, A. J., Stewart, N. N., Stumpf, K. R., Sunkin, S. M., Sutram, M., Tam, A., Teemer, C. D., Thaller, C., Thompson, C. L., Varnam, L. R., Visel, A., Whitlock, R. M., Wohnoutka, P. E., Wolkey, C. K., Wong, V. Y., Wood, M., Yaylaoglu, M. B., Young, R. C., Youngstrom, B. L., Yuan, X. F., Zhang, B., Zwingman, T. A., & Jones, A. R. (2007). Genome-wide atlas of gene expression in the adult mouse brain. Nature, 445, 168–176. Lemaire, V., Koehl, M., Le Moal, M., & Abrous, D. N. (2000). Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus. Proceedings of the National Academy of Sciences of the United States of America, 97, 11032–11037. Lesage, J., Del-Favero, F., Leonhardt, M., Louvart, H., Maccari, S., Vieau, D., & Darnaudery, M. (2004). Prenatal stress induces intrauterine growth restriction and programmes glucose intolerance and feeding behaviour disturbances in the aged rat. The Journal of Endocrinology, 181, 291–296. Leuner, B., & Gould, E. (2010). Structural plasticity and hippocampal function. Annual Review of Psychology, 61, 111–140. Lewis, K. A., Gray, P. C., Blount, A. L., MacConell, L. A., Wiater, E., Bilezikjian, L. M., & Vale, W. (2000). Betaglycan binds inhibin and can mediate functional antagonism of activin signalling. Nature, 404, 411–414. Li, F., Huarte, M., Zaratiegui, M., Vaughn, M. W., Shi, Y., Martienssen, R., & Cande, W. Z. (2008). Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin. Cell, 135, 272–283. Louey, S., & Thornburg, K. L. (2005). The prenatal environment and later cardiovascular disease. Early Human Development, 81, 745–751. Ma, D. K., Jang, M. H., Guo, J. U., Kitabatake, Y., Chang, M. L., Pow-Anpongkul, N., Flavell, R. A., Lu, B., Ming, G. L., & Song, H. (2009). Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science, 323, 1074–1077. Maccari, S., Piazza, P. V., Kabbaj, M., Barbazanges, A., Simon, H., & Le, M. M. (1995). Adoption reverses the long-term impairment in glucocorticoid feedback induced by prenatal stress. Journal of Neuroscience, 15, 110–116. Madeira, M. D., Ferreira-Silva, L., & Paula-Barbosa, M. M. (2001). Influence of sex and estrus cycle on the sexual dimorphisms of the hypothalamic ventromedial nucleus: Stereological evaluation and Golgi study. The Journal of Comparative Neurology, 432, 329–345. Madeira, M. D., Leal, S., & Paula-Barbosa, M. M. (1999). Stereological evaluation and Golgi study of the sexual dimorphisms in the volume, cell numbers, and cell size in the medial preoptic nucleus of the rat. Journal of Neurocytology, 28, 131–148. Maher, E. R., Afnan, M., & Barratt, C. L. (2003). Epigenetic risks related to assisted reproductive technologies: Epigenetics, imprinting, ART and icebergs? Human Reproduction (Oxford, England), 18, 2508–2511. Malumbres, M. (2012). miRNAs versus oncogenes: The power of social networking. Molecular Systems Biology, 8, 569. Martinez-Tellez, R. I., Hernandez-Torres, E., Gamboa, C., & Flores, G. (2009). Prenatal stress alters spine density and dendritic length of nucleus accumbens and hippocampus neurons in rat offspring. Synapse, 63, 794–804. Matrisciano, F., Dong, E., Gavin, D. P., Nicoletti, F., & Guidotti, A. (2011). Activation of group II metabotropic glutamate receptors promote DNA demethylation in the mouse brain. Molecular Pharmacology, 80, 174–182. McCarthy, M. M. (2008). Estradiol and the developing brain. Physiological Reviews, 1, 91–124. McCarthy, M. M., Auger, A. P., Bale, T. L., De Vries, G. J., Dunn, G. A., Forger, N. G., Murray, E. K., Nugent, B. M., Schwarz, J. M., & Wilson, M. E. (2009). The epigenetics of sex differences in the brain. Journal of Neuroscience, 29, 12815–12823. McEwen, B. S. (1994). How do sex and stress hormones affect nerve cells? Annals of the New York Academy of Sciences, 743, 1–16. McEwen, B. S., Biron, C. A., Brunson, K. W., Bulloch, K., Chambers, W. H., Dhabhar, F. S., Goldfarb, R. H., Kitson, R. P., Miller, A. H., Spencer, R. L., & Weiss, J. M. (1997). The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions. Brain Research Reviews, 23, 79–133. McEwen, B. S., Lieberburg, I., Chaptal, C., & Krey, L. C. (1977). Aromatization: Important for sexual differentiation of the neonatal rat brain. Hormones and Behavior, 3, 249–263. Meaney, M. J., & Szyf, M. (2005). Maternal care as a model for experience-dependent chromatin plasticity? Trends in Neurosciences, 28, 456–463. Meaney, M. J., Szyf, M., & Seckl, J. R. (2007). Epigenetic mechanisms of perinatal programming of hypothalamic–pituitary–adrenal function and health. Trends in Molecular Medicine, 13, 270–277. Miller, S. D., Mueller, E., Gifford, G. W., & Kinsley, C. H. (1999). Prenatal stress-induced modifications of neuronal nitric oxide synthase in amygdala and medial preoptic area. Annals of the New York Academy of Sciences, 877, 760–763. Milutinovic, S., D'Alessio, A. C., Detich, N., & Szyf, M. (2007). Valproate induces widespread epigenetic reprogramming which involves demethylation of specific genes. Carcinogenesis, 28, 560–571. Morgan, C. P., & Bale, T. L. (2011). Early prenatal stress epigenetically programs dysmasculinization in second-generation offspring via the paternal lineage. Journal of Neuroscience, 31, 11748–11755.

Morris, K. V. (2008). RNA Mediated Transcriptional Gene Silencing. In K. V. Morris (Ed.), RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Norfolk, UK: Caister Academic Press. Mueller, B. R., & Bale, T. L. (2008). Sex-specific programming of offspring emotionality after stress in early pregnancy. Journal of Neuroscience, 28, 9055–9065. Murray, E. K., Hien, A., de Vries, G. J., & Forger, N. G. (2009). Epigenetic control of sexual differentiation of the bed nucleus of the stria terminalis. Endocrinology, 150, 4241–4247. Murriel, C. L., & Mochly-Rosen, D. (2003). Opposing roles of delta and epsilon PKC in cardiac ischemia and reperfusion: Targeting the apoptotic machinery. Archives of Biochemistry and Biophysics, 420, 246–254. Mychasiuk, R., Gibb, R., & Kolb, B. (2011a). Prenatal stress produces sexually dimorphic and regionally specific changes in gene expression in hippocampus and frontal cortex of developing rat offspring. Developmental Neuroscience, 33, 531–538. Mychasiuk, R., Ilnytskyy, S., Kovalchuk, O., Kolb, B., & Gibb, R. (2011b). Intensity matters: Brain, behaviour and the epigenome of prenatally stressed rats. Neuroscience, 180, 105–110. Mychasiuk, R., Schmold, N., Ilnytskyy, S., Kovalchuk, O., Kolb, B., & Gibb, R. (2011c). Prenatal bystander stress alters brain, behavior, and the epigenome of developing rat offspring. Developmental Neuroscience, 33, 159–169. Mychasiuk, R., Zahir, S., Schmold, N., Ilnytskyy, S., Kovalchuk, O., & Gibb, R. (2012). Parental enrichment and offspring development: Modifications to brain, behavior and the epigenome. Behavioural Brain Research, 228, 294–298. Nagarajan, R. P., Hogart, A. R., Gwye, Y., Martin, M. R., & LaSalle, J. M. (2006). Reduced MeCP2 expression is frequent in autism frontal cortex and correlates with aberrant MECP2 promoter methylation. Epigenetics, 1, e1–e11. Nilsson, E. E., Anway, M. D., Stanfield, J., & Skinner, M. K. (2008). Transgenerational epigenetic effects of the endocrine disruptor vinclozolin on pregnancies and female adult onset disease. Reproduction, 135, 713–721. Nugent, B. M., & McCarthy, M. M. (2011). Epigenetic underpinnings of developmental sex differences in the brain. Neuroendocrinology, 93, 150–158. Oberlander, T. F., Weinberg, J., Papsdorf, M., Grunau, R., Misri, S., & Devlin, A. M. (2008). Prenatal exposure to maternal depression, neonatal methylation of human glucocorticoid receptor gene (NR3C1) and infant cortisol stress responses. Epigenetics, 3, 97–106. Park, J. H., Stoffers, D. A., Nicholls, R. D., & Simmons, R. A. (2008). Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1. The Journal of Clinical Investigation, 118, 2316–2324. Patchev, V. K., Hayashi, S., Orikasa, C., & Almeida, O. F. (1995). Implications of estrogen-dependent brain organization for gender differences in hypothalamo– pituitary–adrenal regulation. The FASEB Journal, 9, 419–423. Patterson, A. J., Chen, M., Xue, Q., Xiao, D., & Zhang, L. (2010). Chronic prenatal hypoxia induces epigenetic programming of PKC epsilon gene repression in rat hearts. Circulation Research, 107, 365–373. Reznikov, A. G., Nosenko, N. D., & Tarasenko, L. V. (1999). Prenatal stress and glucocorticoid effects on the developing gender-related brain. The Journal of Steroid Biochemistry and Molecular Biology, 69, 109–115. Rhees, R. W., Shryne, J. E., & Gorski, R. A. (1990). Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area in female rats. Journal of Neurobiology, 5, 781–786. Ringstedt, T., Linnarsson, S., & Wagner, J. (1998). BDNF regulates reelin expression and Cajal–Retzius cell development in the cerebral cortex. Neuron, 21, 305–315. Rodrigues, S. M., Bauer, E. P., Farb, C. R., Schafe, G. E., & LeDoux, J. E. (2002). The group I metabotropic receptor Grm5 is required for fear memory formation and long-term potentiation in the lateral amygdala. Journal of Neuroscience, 22, 5219–5229. Roth, T. L., Lubin, F. D., Funk, A. J., & Sweatt, J. D. (2009). Lasting epigenetic influence of early-life adversity on the BDNF gene. Biological Psychiatry, 65, 760–769. Scaccianoce, S., Matrisciano, F., Del Bianco, P., Caricasole, A., Di Giorgi Gerevini, V., Cappuccio, I., Melchiorri, D., Battaglia, G., & Nicoletti, F. (2003). Endogenous activation of group-II metabotropic glutamate receptors inhibits the hypothalamic–pituitary– adrenocortical axis. Neuropharmacology, 44, 555–561. Schwarz, J. M., Nugent, B. M., & McCarthy, M. M. (2010). Developmental and hormone-induced epigenetic changes to estrogen and progesterone receptor genes in brain are dynamic across the life span. Endocrinology, 151, 4871–4881. Seah, C., Levy, M. A., Jiang, Y., Mokhtarzada, S., Higgs, D. R., Gibbons, R. J., & Bérubé, N. G. (2008). Neuronal death resulting from targeted disruption of the Snf2 protein ATRX is mediated by p53. Journal of Neuroscience, 28, 12570–12580. Seckl, J. R., & Meaney, M. J. (2004). Glucocorticoid programming. Annals of the New York Academy of Sciences, 1032, 63–84. Sharma, S., Kelly, T. K., & Jones, P. A. (2010). Epigenetics in cancer. Carcinogenesis, 31, 27–36. Shirdel, E. A., Xie, W., Mak, T. W., & Jurisica, I. (2011). NAViGaTing the micronome—Using multiple microRNA prediction databases to identify signalling pathway-associated microRNAs. PloS One, 6, e17429. http://dx.doi.org/10.1371/journal.pone.0017429. Sripathy, S. P., Stevens, J., & Schultz, D. C. (2006). The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression. Molecular and Cellular Biology, 26, 8623–8638. Stadler, F., Kolb, G., Rubusch, L., Baker, S. P., Jones, E. G., & Akbarian, S. (2005). Histone methylation at gene promoters is associated with developmental regulation and region-specific expression of ionotropic and metabotropic glutamate receptors in human brain. Journal of Neurochemistry, 94, 324–336. Stoffers, D. A., Zinkin, N. T., Stanojevic, V., Clarke, W. L., & Habener, J. F. (1997). Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nature Genetics, 15, 106–110.

B. Billack et al. / Journal of Pharmacological and Toxicological Methods 66 (2012) 221–231 Strahl, B. D., & Allis, C. D. (2000). The language of covalent histone modifications. Nature, 403, 41–45. Szyf, M. (2009). Epigenetics, DNA methylation, and chromatin modifying drugs. Annual Review of Pharmacology and Toxicology, 49, 243–263. Takahashi, L. K., Turner, J. G., & Kalin, N. H. (1998). Prolonged stress-induced elevation in plasma corticosterone during pregnancy in the rat: Implication for prenatal stress studies. Psychoneuroendocrinology, 23, 571–581. Tamashiro, K. L., & Moran, T. H. (2010). Perinatal environment and its influences on metabolic programming of offspring. Physiology & Behavior, 100, 560–566. Tian, C., Xing, G., Xie, P., Lu, K., Nie, J., Wang, J., Li, L., Gao, M., Zhang, L., & He, F. (2009). KRAB-type zinc-finger protein Apak specifically regulates p53-dependent apoptosis. Nature Cell Biology, 11, 580–591. Travers, A. (1999). An engine for nucleosome remodeling. Cell, 96, 311–314. Tsunoka, T., Kishi, T., Kitajima, T., Okochi, T., Okumura, T., Yamanouchi, Y., Kinoshita, Y., Kawashima, K., Naitoh, H., Inada, T., Ujike, H., Yamada, M., Uchimura, N., Sora, I., Iyo, M., Ozaki, N., & Iwata, N. (2010). Association analysis of GRM2 and HTR2A with methamphetamine-induced psychosis and schizophrenia in the Japanese population. Progress in Neuro-Psychopharmacology & Biological Psychiatry, 34, 639–644. Turner, J. D., Schote, A. B., Macedo, J. A., Pelascini, L. P., & Muller, C. P. (2006). Tissue specific glucocorticoid receptor expression, a role for alternative first exon usage? Biochemical Pharmacology, 72, 1529–1537. Vale, W., Rivier, C., Hsueh, A., Campen, C., Meunier, H., Bicsak, T., Vaughan, J., Corrigan, A., Bardin, W., & Sawchenko, P. (1988). Chemical and biological characterization of the inhibin family of protein hormones. Recent Progress in Hormone Research, 44, 1–34. Vallee, M., Mayo, W., Dellu, F., Le, M. M., Simon, H., & Maccari, S. (1997). Prenatal stress induces high anxiety and postnatal handling induces low anxiety in adult offspring: Correlation with stress-induced corticosterone secretion. Journal of Neuroscience, 17, 2626–2636. Viltart, O., & Vanbesian-Mailliot, C. C. A. (2007). Impact of prenatal stress on neuroendocrine programming. Scientific World Journal, 7, 1493–1537. Wakshlak, A., & Weinstock, M. (1990). Neonatal handling reverses behavioral abnormalities induced in rats by prenatal stress. Physiology & Behavior, 48, 289–292. Wang, C., Rauscher, F. J., III, Cress, W. D., & Chen, J. (2007). Regulation of E2F1 function by the nuclear corepressor KAP1. The Journal of Biological Chemistry, 282, 29902–29908. Ward, I. L. (1972). Prenatal stress feminizes and demasculinizes the behavior of males. Science, 175, 82–84. Ward, I. L., & Ward, O. B. (1985). Sexual befavior differentiation: Effects of prenatal manipulation in rats. In D. Pfaff, R. W. Goy, & N. Adler (Eds.), Handbook of Behavioural Physiology: Reproduction (pp. 77–98). New York: Plenum Press. Ward, I. L., & Weisz, J. (1980). Maternal stress alters plasma testosterone in fetal males. Science, 207, 328–329. Ward, I. L., & Weisz, J. (1984). Differential effects of maternal stress on circulating levels of corticosterone, progesterone, and testosterone in male and female rat fetuses and their mothers. Endocrinology, 114, 1635–1644.

231

Watson, R. E., & Goodman, J. I. (2002). Epigenetics and DNA methylation come of age in toxicology. Toxicological Sciences, 67, 11–16. Weaver, I. C. G. (2007). Epigenetic programming by maternal behavior and pharmacological intervention. Nature versus nurture: Let's call the whole thing off. Epigenetics, 2, 22–28. Weaver, I. C. G., Cervoni, N., Champagne, F. A., et al. (2004). Epigenetic programming by maternal behavior. Nature Neuroscience, 7, 847–854. Weaver, I. C., D'Alessio, A. C., Brown, S. E., Hellstrom, I. C., Dymov, S., Sharma, S., Szyf, M., & Meaney, M. J. (2007). The transcription factor nerve growth factor-inducible protein A mediates epigenetic programming: Altering epigenetic marks by immediate-early genes. Journal of Neuroscience, 27, 1756–1768. Weaver, I. C. G., Meaney, M. J., & Szyf, M. (2006). Maternal care effects on the hippocampal transcriptome and anxiety-mediated behaviors in the offspring that are reversible in adulthood. Proceedings of the National Academy of Sciences of the United States of America, 103, 3480–3485. Weisz, J., & Ward, I. L. (1980). Plasma testosterone and progesterone titers of pregnant rats, their male and female fetuses, and neonatal offspring. Endocrinology, 106, 306–316. Welberg, L. A. M., Seckl, J. R., & Holmes, M. C. (2001). Prenatal glucocorticoid programming of brain corticosteroid receptors and corticotrophin-releasing hormone: Potential implications for behavior. Neuroscience, 104, 71–79. Wiater, E., Lewis, K. A., Donaldson, C., Vaughan, J., Bilezikjian, L., & Vale, W. (2009). Endogenous betaglycan is essential for high-potency inhibin antagonism in gonadotropes. Molecular Endocrinology, 23, 1033–1042. Wiater, E., & Vale, W. (2003). Inhibin is an antagonist of bone morphogenetic protein signaling. The Journal of Biological Chemistry, 278, 7934–7941. Wright, C. L., Burks, S. R., & McCarthy, M. M. (2008). Identification of prostaglandin E2 receptors mediating perinatal masculinization of adult sex behavior and neuroanatomical correlates. Developmental Neurobiology, 68, 1406–1419. Yankova, M., Hart, S. A., & Woolley, C. S. (2001). Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: A serial electron-microscopic study. Proceedings of the National Academy of Sciences of the United States of America, 98, 3525–3530. Zhang, Y., Ng, H. H., Erdjument-Bromage, H., Tempst, P., Bird, A., & Reinberg, D. (1999). Analysis of the NuRD subunits reveal a histone deacetylase core complex and a connection with DNA methylation. Genes & Development, 13, 1924–1935. Zhou, H., Hu, H., & Lai, M. (2010). Non-coding RNAs and their epigenetic regulatory mechanisms. Biology of the Cell, 102, 645–655. Zuena, A. R., Mairesse, J., Casolini, P., Cinque, C., Alemà, G. S., Morley-Fletcher, S., Chiodi, V., Spagnoli, L. G., Gradini, R., Catalani, A., Nicoletti, F., & Maccari, S. (2008). Prenatal restraint stress generates two distinct behavioral and neurochemical profiles in male and female rats. PloS One, 3, e2170.