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1.  Effects of chronic NMDA-NR2b inhibition in the median eminence of the reproductive senescent female rat 
Journal of neuroendocrinology  2013;25(10):887-897.
Gonadotrophin-releasing hormone (GnRH) neurones of the hypothalamic-pituitary-gonadal (HPG) axis drive reproductive function and undergo age-related decreases in activation during the transition to reproductive senescence. Decreased GnRH secretion from the median eminence (ME) partially arises from attenuated glutamatergic signaling via the NMDA receptor (NMDAR), and may be due to changing NMDAR stoichiometry to favor NR2b over NR2a subunit expression with aging. We have previously shown that the systemic inhibition of NR2b-containing receptors with ifenprodil, an NR2b-specific antagonist, stimulates parameters of luteinising hormone (used as a proxy for GnRH) release in both young and middle-aged females. Here, we chronically administered ifenprodil, an NR2b-specific antagonist, at the site of GnRH terminals in the median eminence (ME) or at GnRH perikarya in the preoptic area, in reproductively senescent middle-aged female rats to determine whether NR2b antagonism could restore aspects of reproductive functionality. Effects on oestrous cyclicity, serum hormones, and protein expression of GnRH, NR2b, and phosphorylated NR2b (Tyr-1472) in the ME were measured. Chronic ifenprodil treatment in the ME, but not the preoptic area, altered oestrous cyclicity by increasing the percentage of days spent in pro-oestrus. This was accompanied by increased GnRH fluorescence intensity in the external ME zone and a greater proportion of GnRH terminals that co-labelled with pNR2b with treatment. We also observed changes in the relationships between protein immunofluorescence, serum hormone levels, and other aspects of reproductive physiology in acyclic females, as revealed by bionetwork analysis. Together, these data support the hypothesis that NMDAR-NR2b expression and phosphorylation state play a role in reproductive senescence and highlight the ME as a major player in reproductive aging.
PMCID: PMC3800684  PMID: 23957788
Median Eminence; Menopause; Reproductive senescence; NMDA receptor; GnRH
2.  Neuroendocrine control of the transition to reproductive senescence: Lessons learned from the female rodent model 
Neuroendocrinology  2012;96(1):1-12.
The natural transition to reproductive senescence is an important physiological process that occurs with aging, resulting in menopause in women and diminished or lost fertility in most mammalian species. This review focuses on how rodent models have informed our knowledge of age-related changes in GnRH neurosecretory function and the subsequent loss of reproductive capacity. Studies in rats and mice have shown molecular, morphological and functional changes in GnRH cells. Furthermore, during reproductive aging altered sex steroid feedback to the hypothalamus contributes to a decrease of stimulatory signaling and increase in inhibitory tone onto GnRH neurons. At the site of the GnRH terminals where the peptide is released into the portal vasculature, the cytoarchitecture of the median eminence becomes disorganized with aging, and mechanisms of glial-GnRH neuronal communication may be disrupted. These changes can result in the dysregulation of GnRH secretion with reproductive decline. Interestingly, reproductive aging effects on the GnRH circuitry are observed in middle age even prior to any obvious physiological changes in cyclicity. We speculate that the hypothalamus may play a critical role in this mid-life transition. Because there are substantial species differences in these aging processes, we also compare and contrast rodent aging to that in primates. Work discussed herein shows that in order to understand neuroendocrine mechanisms of reproductive senescence, further research needs to be conducted in ovarian-intact models.
PMCID: PMC3574559  PMID: 22354218
female reproductive aging; hypothalamus; neuroendocrine; reproductive senescence; menopause; ovarian intact
3.  Prenatal PCBs Disrupt Early Neuroendocrine Development of the Rat Hypothalamus 
Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague-Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans: PCB138, PCB153 and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD) and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells, or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the AVPV of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABAB receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.
PMCID: PMC3060304  PMID: 21277884
Polychlorinated biphenyls (PCBs); endocrine disruption; hypothalamus; sexual differentiation; neuroendocrine development; estrogen receptor; developmental apoptosis; anteroventral periventricular nucleus; medial preoptic nucleus
5.  Changes in Androgen Receptor, Estrogen Receptor alpha, and Sexual Behavior with Aging and Testosterone in Male Rats1 
Hormones and behavior  2010;58(2):306-316.
Reproductive aging in males is characterized by a diminution in sexual behavior beginning in middle age. We investigated the relationships among testosterone, androgen receptor (AR) and estrogen receptor alpha (ERα) cell numbers in the hypothalamus, and their relationship to sexual performance in male rats. Young (3 months) and middle-aged (12 months) rats were given sexual behavior tests, then castrated and implanted with vehicle or testosterone capsules. Rats were tested again for sexual behavior. Numbers of AR and ERα immunoreactive cells were counted in the anteroventral periventricular nucleus and the medial preoptic nucleus, and serum hormones were measured. Middle-aged intact rats had significant impairments of all sexual behavior measures compared to young males. After castration and testosterone implantation, sexual behaviors in middle-aged males were largely comparable to those in the young males. In the hypothalamus, AR cell density was significantly (5-fold) higher, and ERα cell density significantly (6-fold) lower, in testosterone- than vehicle-treated males, with no age differences. Thus, restoration of serum testosterone to comparable levels in young and middle-aged rats resulted in similar preoptic AR and ERα cell density concomitant with a reinstatement of most behaviors. These data suggest that age-related differences in sexual behavior cannot be due to absolute levels of testosterone, and further, the middle-aged brain retains the capacity to respond to exogenous testosterone with changes in hypothalamic AR and ERα expression. Our finding that testosterone replacement in aging males has profound effects on hypothalamic receptors and behavior has potential medical implications for the treatment of age-related hypogonadism in men.
PMCID: PMC2879440  PMID: 20223236
androgen receptor; estrogen receptor alpha; aging; male rat; anteroventral periventricular nucleus (AVPV); medial preoptic nucleus (MPN)
Experimental neurology  2007;210(2):339-348.
Hippocampal dendritic spine and synapse numbers in female rats vary across the estrous cycle and following experimental manipulation of hormone levels in adulthood. Based on behavioral studies demonstrating that learning patterns are altered following puberty, we hypothesized that dendritic spine number in rat hippocampal CA1 region would change post-pubertally. Female Sprague-Dawley rats were divided into prepubertal (postnatal day (P) 22), peripubertal (P35) and post-pubertal (P49) groups, with the progression of puberty evaluated by vaginal opening, and estrous cyclicity subsequently assessed by daily vaginal smears. Spinophilin immunoreactivity in dendritic spines was used as an index of spinogenesis in area CA1 stratum radiatum (CA1sr) of hippocampus. First, electron microscopy analyses confirmed the presence of spinophilin specifically in dendritic spines of CA1sr, supporting spinophilin as a reliable marker of hippocampal spines in young female rats. Second, stereologic analysis was performed to assess the total number of spinophilin-immunoreactive puncta (i.e. spines) and CA1sr volume in developing rats. Our results indicated that the number of spinophilin-immunoreactive spines in CA1sr was decreased 46% in the post-pubertal group compared to the two younger groups, whereas the volume of the hippocampus underwent an overall increase during this same developmental time frame. Third, to determine a potential role of estradiol in this process, an additional group of rats was ovariectomized (OVX) prepubertally at P22, then treated with estradiol or vehicle at P35, and spinophilin quantified as above in rats perfused on P49. No difference in spinophilin puncta number was found in OVX rats between the two hormone groups, suggesting that this developmental decrease is independent of peripheral estradiol. These changes in spine density coincident with puberty may be related to altered hippocampal plasticity and synaptic consolidation at this phase of maturity.
PMCID: PMC3075007  PMID: 18096161
Puberty; dendritic spine; hippocampus; CA1; spinophilin; estrogen; synaptic plasticity; spinogenesis
7.  The Hypothalamic Median Eminence and its Role in Reproductive Aging 
The median eminence at the base of the hypothalamus serves as an interface between the neural and peripheral endocrine systems. It is the site where hypothalamic releasing hormones are released into the portal capillary bed to be transported to the anterior pituitary, which provides further signals to target endocrine systems. Of specific relevance to reproduction, a group of about 1000 neurons in mammals release the gonadotropin-releasing hormone (GnRH) peptide from neuroterminals in the median eminence. During the life cycle, there are dramatic changes in reproductive demands, and we focus this review on how GnRH terminals in the median eminence change during reproductive senescence. We discuss morphological and functional properties of the median eminence, and how relationships among GnRH terminals and their microenvironment of nerve terminals, glial cells, and the portal capillary vasculature determine the ability of GnRH peptide to be secreted and to reach its target in the anterior pituitary gland.
PMCID: PMC2929984  PMID: 20738281
median eminence; hypothalamus; GnRH; glia; tanycyte; reproductive aging
8.  Sexual Experience Changes Sex Hormones But Not Hypothalamic Steroid Hormone Receptor Expression in Young and Middle-aged Male Rats 
Hormones and behavior  2009;56(3):299-308.
Testosterone is well known to regulate sexual behavior in males, but this is dependent upon prior sexual experience. Aging is associated with decreased libido and changes in testosterone, but the role of experience in these age-related processes has not been systematically studied. We examined effects of age and sexual experience on serum hormones (total testosterone, free testosterone, estradiol, LH) and on numbers of androgen receptor (AR) and estrogen receptor α (ERα) immunoreactive cells in the hypothalamus. Extensive sexual experience was given to male rats at 4 months of age. Rats were euthanized at either 4 months (young) or 12 months (middle-aged (MA)). Comparable sexually naïve male rats were handled and placed into the testing arena but did not receive any sexual experience. Thus, we had four groups: young-naïve, young-experienced, MA-naïve and MA-experienced. Serum hormone levels were assayed, and numbers of AR and ERα cells were quantified stereologically in the medial preoptic nucleus (MPN) and the anteroventral periventricular nucleus (AVPV). Sexually experienced males had significantly elevated serum testosterone and free testosterone in both age groups. Both total and free testosterone were higher, and estradiol lower, in middle-aged than young rats. Experience did not alter either AR or ERα expression in the preoptic brain regions studied. Aging was associated with increased expression of AR, but no change in ERα. These results show that sexual experience can induce short-term and long-term alterations in serum hormones but these effects are not manifested upon their receptors in the hypothalamus.
PMCID: PMC2739266  PMID: 19559704
androgen receptor; estrogen receptor α; sexual experience; aging; anteroventral periventricular nucleus (AVPV); medial preoptic nucleus (MPN)
9.  Cell death mechanisms in GT1-7 GnRH cells exposed to polychlorinated biphenyls PCB74, PCB118, and PCB153 
Toxicology and applied pharmacology  2009;237(2):237-245.
Exposure to endocrine-disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) causes functional deficits in neuroendocrine systems. We used an immortalized hypothalamic GT1-7 cell line, which synthesizes the neuroendocrine peptide gonadotropin-releasing hormone (GnRH), to examine the neurotoxic and endocrine disrupting effects of PCBs and their mechanisms of action. Cells were treated for 1, 4, 8, or 24 h with a range of doses of a representative PCB from each of three classes: coplanar (2,4,4′,5-tetrachlorobiphenyl: PCB74), dioxin-like coplanar (2′,3,4,4′,5′ pentachlorobiphenyl: PCB118), non-coplanar (2,2′,4,4′,5,5′-hexachlorobiphenyl: PCB153), or their combination. GnRH peptide concentrations, cell viability, apoptotic and necrotic cell death, and caspase activation were quantified. In general, GnRH peptide levels were suppressed by high doses and longer durations of PCBs, and elevated at low doses and shorter time points. The suppression of GnRH peptide levels was partially reversed in cultures co-treated with the estrogen receptor antagonist ICI 182,780. All PCBs reduced viability and increased both apoptotic and necrotic cell death. Although the effects for the three classes of PCBs were often similar, subtle differences in responses, together with evidence that the combination of PCBs acted slightly differently from individual PCBs, suggest that the three tested PCB compounds may act via slightly different or more than one mechanism. These results provide evidence that PCB congeners have endocrine disrupting and/or neurotoxic effects on the hypothalamic GnRH cell line, a finding that has implications for environmental endocrine disruption in animals.
PMCID: PMC2702519  PMID: 19362103
PCB; GnRH; GT1 cells; estrogen receptor; apoptosis; necrosis; endocrine disruption
10.  Three-dimensional properties of GnRH neuroterminals in the median eminence of young and old rats1 
The decapeptide GnRH that regulates reproduction in all vertebrates is stored in, and secreted from, large dense-core secretory vesicles in nerve terminals in the median eminence. GnRH is released from these terminals with biological rhythms that are critical for the maintenance of normal reproduction. During reproductive aging in female rats, there is a loss of GnRH pulses and a diminution of the GnRH surge. However, information about the specific role of GnRH nerve terminals is lacking, particularly in the context of aging. We sought to gain novel ultrastructural information about GnRH neuroterminals by performing three-dimensional (3D) reconstructions of GnRH neuroterminals and their surrounding microenvironment in the median eminence of young (4-5 month) and old (22-24 month) ovariectomized Sprague-Dawley female rats. Median eminence tissues were freeze-plunge embedded, and serial ultrathin sections were collected on slot grids for immunogold labeling of GnRH immunoreactivity. Sequential images were used to create 3D models of GnRH terminals. These reconstructions provided novel perspectives into the morphological properties of GnRH terminals, and their neural and glial environment. We also noted that the cytoarchitectural features of the median eminence became disorganized with aging. Quantitative measures showed a significant decrease in the apposition between GnRH terminal membranes and glial cells. Our data suggest reproductive aging in rats is characterized by structural organizational changes to the GnRH terminal microenvironment in the median eminence.
PMCID: PMC2821827  PMID: 19757493
three-dimensional (3D) reconstruction; gonadotropin-releasing hormone (GnRH); glia; median eminence; serial electron microscopy; reproductive aging
11.  Hypothalamic neural systems controlling the female reproductive life cycle: Gonadotropin-releasing hormone, glutamate, and GABA 
The hypothalamic-pituitary-gonadal (HPG) axis undergoes a number of changes throughout the reproductive life cycle that are responsible for the development, puberty, adulthood, and senescence of reproductive systems. This natural progression is dictated by the neural network controlling the hypothalamus including the cells that synthesize and release gonadotropin-releasing hormone (GnRH) and their regulatory neurotransmitters. Glutamate and GABA are the primary excitatory and inhibitory neurotransmitters in the central nervous system, and as such contribute a great deal to modulating this axis throughout the lifetime via their actions on receptors in the hypothalamus, both directly on GnRH neurons as well as indirectly though other hypothalamic neural networks. Interactions among GnRH neurons, glutamate, and GABA, including the regulation of GnRH gene and protein expression, hormone release, and modulation by estrogen, are critical to age-appropriate changes in reproductive function. Here, we present evidence for the modulation of GnRH neurosecretory cells by the balance of glutamate and GABA in the hypothalamus, and the functional consequences of these interactions on reproductive physiology across the life cycle.
PMCID: PMC2821833  PMID: 19349036
GnRH; NMDA receptor; glutamate; GABA; hypothalamus; reproductive aging; puberty
12.  Neuroendocrine systems as targets for environmental endocrine-disrupting chemicals 
Fertility and sterility  2008;89(2 Suppl):e101-e102.
PMCID: PMC2753451  PMID: 18308047
endocrine disruption; neuroendocrine; hypothalamus; GnRH; polychlorinated biphenyl; PCB; reproduction
13.  The Recreational Drug Ecstasy Disrupts the Hypothalamic-Pituitary-Gonadal Reproductive Axis in Adult Male Rats 
Neuroendocrinology  2008;88(2):95-102.
Reproductive function involves an interaction of three regulatory levels: hypothalamus, pituitary, and gonad. The primary drive upon this system comes from hypothalamic gonadotropin-releasing hormone (GnRH) neurosecretory cells, which receive afferent inputs from other neurotransmitter systems in the central nervous system to result in the proper coordination of reproduction and the environment. Here, we hypothesized that the recreational drug ±-3,4-Methylenedioxymethamphetamine (MDMA; “ecstasy”), which acts through several of the neurotransmitter systems that affect GnRH neurons, suppresses the hypothalamic-pituitary-gonadal (HPG) reproductive axis of male rats. Adult male Sprague-Dawley rats self-administered saline or MDMA or saline either once (acute) or for 20 days (chronic), and were euthanized 7 days following last administration. We quantified hypothalamic GnRH mRNA, serum luteinizing hormone (LH) concentrations, and serum testosterone levels, as indices of hypothalamic, pituitary, and gonadal functions, respectively. The results indicate that the hypothalamic and gonadal levels of the HPG axis are significantly altered by MDMA, with GnRH mRNA and serum testosterone levels suppressed in rats administered MDMA compared to saline. Furthermore, our finding that hypothalamic GnRH mRNA levels are suppressed in the context of low testosterone concentrations suggests that the central GnRH neurosecretory system may be a primary target of inhibitory regulation by MDMA usage.
PMCID: PMC2753463  PMID: 18309234
MDMA; ecstasy; neuroendocrinology; GnRH; testosterone; endocrine disruption; male reproduction
14.  Developmental Programming and Endocrine Disruptor Effects on Reproductive Neuroendocrine Systems 
Frontiers in neuroendocrinology  2008;29(3):358-374.
The ability of a species to reproduce successfully requires the careful orchestration of developmental processes during critical time points, particularly the late embryonic and early postnatal periods. This article begins with a brief presentation of the evidence for how gonadal steroid hormones exert these imprinting effects upon the morphology of sexually differentiated hypothalamic brain regions, the mechanisms underlying these effects, and their implications in adulthood. Then, I review the evidence that aberrant exposure to hormonally-active substances such as exogenous endocrine-disrupting chemicals (EDCs), may result in improper hypothalamic programming, thereby decreasing reproductive success in adulthood. The field of endocrine disruption has shed new light on the discipline of basic reproductive neuroendocrinology through studies on how early life exposures to EDCs may alter gene expression via non-genomic, epigenetic mechanisms, including DNA methylation and histone acetylation. Importantly, these effects may be transmitted to future generations if the germline is affected via transgenerational, epigenetic actions. By understanding the mechanisms by which natural hormones and xenobiotics affect reproductive neuroendocrine systems, we will gain a better understanding of normal developmental processes, as well as to develop the potential ability to intervene when development is disrupted.
PMCID: PMC2702520  PMID: 18394690
imprinting; neuroendocrinology; endocrine disruption; hypothalamus; preoptic area; fetal basis of adult disease; sexual dimorphism; reproduction; epigenetic
15.  Effects of Perinatal Polychlorinated Biphenyls on Adult Female Rat Reproduction: Development, Reproductive Physiology, and Second Generational Effects 
Biology of reproduction  2008;78(6):1091-1101.
Perinatal exposures to endocrine-disrupting chemicals such as polychlorinated biphenyls (PCBs) can cause latent effects on reproductive function. Here, we tested whether PCBs administered during late pregnancy would compromise reproductive physiology in both the fetally-exposed female offspring (F1 generation), as well as in their female offspring (F2 generation). Pregnant Sprague-Dawley rats were treated with the PCB mixture Aroclor (A) 1221 (0, 0.1, 1 or 10 mg/kg) on embryonic days 16 and 18. Somatic and reproductive development of F1 and their F2 female offspring were monitored, including ages of eye opening, pubertal landmarks, and serum reproductive hormones. The results showed that low doses of A1221 given during this critical period of neuroendocrine development caused differential effects of A1221 on F1 and F2 female rats. In both generations, litter sex ratio was skewed towards females. In the F1 generation, additional effects were found including a significant alteration of serum luteinizing hormone (LH) in the 1 mg/kg A1221 group. The F2 generation showed more profound alterations, particularly with respect to fluctuations in hormones and reproductive tract tissues across the estrous cycle. On proestrus, the day of the preovulatory GnRH/gonadotropin surge, F2 females whose mothers had been perinatally exposed to A1221 exhibited substantially suppressed LH and progesterone concentrations, and correspondingly smaller uterine and ovarian weights on estrus, compared to F2 decendants of control rats. These latter changes suggest a dysregulation of reproductive physiology. Thus, low levels of exposure to PCBs during late fetal development cause significant consequences on the maturation and physiology of two generations of female offspring. These findings have implications for reproductive health and fertility of wildlife and humans.
PMCID: PMC2692589  PMID: 18305224
Aroclor 1221; PCB; reproduction; transgenerational; endocrine disruption; LH; progesterone; estradiol
16.  The NMDA Receptor Subunit NR2b: Effects on LH Release and GnRH Gene Expression in Young and Middle-aged Female Rats, with Modulation by Estradiol 
Neuroendocrinology  2007;87(3):129-141.
The loss of reproductive capacity during aging involves changes in the neural regulation of the hypothalamic gonadotropin-releasing hormone (GnRH) neurons controlling reproduction. This neuronal circuitry includes glutamate receptors on GnRH neurons. Previously, we reported an increase in the expression of the NR2b subunit protein of the NMDA receptor on GnRH neurons in middle-aged compared to young female rats. Here, we examined the functional implications of the NR2b subunit on the onset of reproductive aging, using an NR2b-specific antagonist ifenprodil. Young (3–5 mos.) and middle-aged (10–13 mos.) female rats were ovariectomized (OVX), 17β-estradiol (E2) or vehicle (cholesterol) treated, and implanted with a jugular catheter. Serial blood sampling was undertaken every 10 minutes for 4 hours, with ifenprodil (10mg/kg) or vehicle injected (i.p.) after one hour of baseline sampling. The pulsatile release of pituitary LH and levels of GnRH mRNA in hypothalamus were quantified as indices of the reproductive axis. Our results showed effects of ifenprodil on both endpoints. In OVX rats given cholesterol, neither age nor ifenprodil had any effects on LH release. In E2-treated rats, aging was associated with significant decreases in pulsatile LH release. Additionally, ifenprodil stimulated parameters of pulsatile LH release in both young and middle-aged animals. Ifenprodil had few effects on GnRH mRNA; the only significant effect of ifenprodil was found in the middle-aged, cholesterol group. Together, these findings support a role for the NR2b subunit of the NMDAR in GnRH/LH regulation. Because most of these effects were exhibited on pituitary LH release in the absence of a concomitant change in GnRH gene expression, it is likely that NMDA receptors containing the NR2b subunit plays a role in GnRH-induced LH release, independent of de novo GnRH gene expression.
PMCID: PMC2671961  PMID: 18025808
Luteinizing hormone (LH); gonadotropin-releasing hormone (GnRH); glutamate; N-methyl-D-Aspartate receptor (NMDAR); NR2b; reproductive aging; reproductive senescence; estrogen; GnRH mRNA; ifenprodil
17.  Age-related Changes in Hypothalamic Androgen Receptor and Estrogen Receptor α in Male Rats# 
The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereological analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERα) cells in three POA nuclei of male Sprague-Dawley rats [anteroventral periventricular nucleus (AVPV), median preoptic area (MePO), and medial preoptic nucleus (MPN)], at young (3 mo), middle-aged (12 mo) and old (20 mo) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle-aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR-immunoreactive cell numbers and density in the AVPV, MePO and MPN were significantly higher in old compared with young or middle-aged rats. No change in the total number or density of ERα immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily-labeled ERα cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory up-regulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age.
PMCID: PMC2671934  PMID: 19051266
androgen receptor; estrogen receptor; aging; anteroventral periventricular nucleus (AVPV); median preoptic area (MePO); medial preoptic nucleus (MPN)
18.  Gene bionetworks involved in the epigenetic transgenerational inheritance of altered mate preference: environmental epigenetics and evolutionary biology 
BMC Genomics  2014;15(1):377.
Mate preference behavior is an essential first step in sexual selection and is a critical determinant in evolutionary biology. Previously an environmental compound (the fungicide vinclozolin) was found to promote the epigenetic transgenerational inheritance of an altered sperm epigenome and modified mate preference characteristics for three generations after exposure of a gestating female.
The current study investigated gene networks involved in various regions of the brain that correlated with the altered mate preference behavior in the male and female. Statistically significant correlations of gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified gene networks (bionetworks) involved in sex-specific mate preference behavior. Observations demonstrate the ability of environmental factors to promote the epigenetic transgenerational inheritance of this altered evolutionary biology determinant.
Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-377) contains supplementary material, which is available to authorized users.
PMCID: PMC4073506  PMID: 24885959
Epigenetics; Brain; Networks; Evolution; Behavior
19.  Transgenerational neuroendocrine disruption of reproduction 
Nature reviews. Endocrinology  2011;7(4):197-207.
Exposure to endocrine disrupting chemicals (EDCs) is associated with dysfunctions of metabolism, energy balance, thyroid function and reproduction, and an increased risk of endocrine cancers. These multifactorial disorders can be ‘programmed’ through molecular epigenetic changes induced by exposure to EDCs early in life, the expression of which may not manifest until adulthood. In some cases, EDCs have detrimental effects on subsequent generations, which indicates that traits for disease predisposition may be passed to future generations by nongenomic inheritance. This Review discusses current understanding of the epigenetic mechanisms that underlie sexual differentiation of reproductive neuroendocrine systems in mammals and summarizes the literature on transgenerational epigenetic effects of representative EDCs: vinclozolin, diethylstilbesterol, bisphenol A and polychlorinated biphenyls. The article differentiates between context-dependent epigenetic transgenerational changes—namely, those that require environmental exposure, either via the EDC itself or through behavioral or physiological differences in parents—and germline-dependent epigenetic mechanisms. These processes, albeit discrete, are not mutually exclusive and can involve similar molecular mechanisms including DNA methylation and histone modifications and may predispose exposed individuals to transgenerational disruption of reproductive processes. New insights stress the crucial need to develop a clear understanding of how EDCs may program the epigenome of exposed individuals and their descendants.
PMCID: PMC3976559  PMID: 21263448
20.  Social transmission of Pavlovian fear: fear-conditioning by-proxy in related female rats 
Animal Cognition  2013;17(3):827-834.
Pairing a previously neutral conditioned stimulus (CS; e.g., a tone) to an aversive unconditioned stimulus (US; e.g., a foot-shock) leads to associative learning such that the tone alone will elicit a conditioned response (e.g., freezing). Individuals can also acquire fear from a social context, such as through observing the fear expression of a conspecific. In the current study, we examined the influence of kinship/familiarity on social transmission of fear in female rats. Rats were housed in triads with either sisters or non-related females. One rat from each cage was fear conditioned to a tone CS+ shock US. On day two, the conditioned rat was returned to the chamber accompanied by one of her cage mates. Both rats were allowed to behave freely, while the tone was played in the absence of the foot-shock. The previously untrained rat is referred to as the fear-conditioned by-proxy (FCbP) animal, as she would freeze based on observations of her cage-mate’s response rather than due to direct personal experience with the foot-shock. The third rat served as a cage-mate control. The third day, long-term memory tests to the CS were performed. Consistent with our previous application of this paradigm in male rats (Bruchey et al. in Behav Brain Res 214(1):80–84, 2010), our results revealed that social interactions between the fear conditioned and FCbP rats on day two contribute to freezing displayed by the FCbP rats on day three. In this experiment, prosocial behavior occurring at the termination of the cue on day two was significantly greater between sisters than their non-sister counterparts, and this behavior resulted in increased freezing on day three. Our results suggest that familiarity and/or kinship influences the social transmission of fear in female rats.
PMCID: PMC3984423  PMID: 24310150
Social transmission; Fear-conditioning; Observational learning; Indirect conditioning
21.  Anxiogenic Effects of Developmental Bisphenol A Exposure Are Associated with Gene Expression Changes in the Juvenile Rat Amygdala and Mitigated by Soy 
PLoS ONE  2012;7(9):e43890.
Early life exposure to Bisphenol A (BPA), a component of polycarbonate plastics and epoxy resins, alters sociosexual behavior in numerous species including humans. The present study focused on the ontogeny of these behavioral effects beginning in adolescence and assessed the underlying molecular changes in the amygdala. We also explored the mitigating potential of a soy-rich diet on these endpoints. Wistar rats were exposed to BPA via drinking water (1 mg/L) from gestation through puberty, and reared on a soy-based or soy-free diet. A group exposed to ethinyl estradiol (50 µg/L) and a soy-free diet was used as a positive estrogenic control. Animals were tested as juveniles or adults for anxiety-like and exploratory behavior. Assessment of serum BPA and genistein (GEN), a soy phytoestrogen, confirmed that internal dose was within a human-relevant range. BPA induced anxiogenic behavior in juveniles and loss of sexual dimorphisms in adult exploratory behavior, but only in the animals reared on the soy-free diet. Expression analysis revealed a suite of genes, including a subset known to mediate sociosexual behavior, associated with BPA-induced juvenile anxiety. Notably, expression of estrogen receptor beta (Esr2) and two melanocortin receptors (Mc3r, Mc4r) were downregulated. Collectively, these results show that behavioral impacts of BPA can manifest during adolescence, but wane in adulthood, and may be mitigated by diet. These data also reveal that, because ERβ and melanocortin receptors are crucial to their function, oxytocin/vasopressin signaling pathways, which have previously been linked to human affective disorders, may underlie these behavioral outcomes.
PMCID: PMC3434201  PMID: 22957036
22.  Epigenetic synthesis: a need for a new paradigm for evolution in a contaminated world 
Epigenetics is a perspective, not a set of techniques. Molecular biology and genetics are the dominant disciplines in biology today, but practitioners of these fields have only recently ‘rediscovered’ the importance of the environment. This has led to increasing research into molecular epigenetics and the interface between the environment and gene regulation. Beyond the study of epigenetic mechanisms at the level of the gene, more investigation of epigenetic outcomes at the level of both the individual organism and the evolution of the population is needed.
PMCID: PMC3434969  PMID: 22991583
23.  Age- and hormone-regulation of opioid peptides and synaptic proteins in the rat dorsal hippocampal formation 
Brain research  2010;1379:71-85.
Circulating estrogen levels and hippocampal-dependent cognitive functions decline with aging. Moreover, the responses of hippocampal synaptic structure to estrogens differ between aged and young rats. We recently reported that estrogens increase levels of post-synaptic proteins, including PSD-95, and opioid peptides leu-enkephalin and dynorphin in the hippocampus of young animals. However, the influence of ovarian hormones on synaptic protein and opioid peptide levels in the aging hippocampus is understudied. Here, young (3–5 mo old), middle-aged (9–12 mo old), and aged (about 22 mo old) female rats were ovariectomized for 4 weeks and then subcutaneously implanted with a silastic capsule containing vehicle or 17β-estradiol. After 48 hours, rats were subcutaneously injected with progesterone or vehicle and sacrificed one day later. Coronal sections through the dorsal hippocampus were processed for quantitative peroxidase immunohistochemistry of leu-enkephalin, dynorphin, synaptophysin, and PSD-95. With age, females showed opposing changes in leu-enkephalin and dynorphin levels in the mossy fiber pathway, particularly within the hilus, and regionally specific changes in synaptic protein levels. 17β-estradiol, with or without progesterone, altered leu-enkephalin levels in the dentate gyrus and synaptophysin levels in the CA1 of young but not middle-aged or aged females. Additionally, 17β-estradiol decreased synaptophysin levels in the CA3 of middle-aged females. Our results support and extend previous findings indicating 17β-estradiol modulation of hippocampal opioid peptides and synaptic proteins while demonstrating regional and age-specific effects. Moreover, they lend credence to the “window of opportunity” hypothesis during which hormone replacement can modulate hippocampal structure and circuitry to improve cognitive outcomes.
PMCID: PMC3020269  PMID: 20828542
aging; estrogen; hippocampus; opioids; synaptic protein
24.  Life Imprints: Living in a Contaminated World 
Environmental Health Perspectives  2011;119(9):1208-1210.
Background: The links between nature and nurture need to be redefined to accommodate anthropogenic chemical contamination. Although some local remediation of contamination has occurred, at the global level this is simply not possible. Contaminants are spread by population migration, by introduction via the food chain, and through air and water currents, even to regions that were never exposed directly to these environmental insults. In recognizing and accepting this worldwide change, we must move on and consider the types of adaptations that could occur as a consequence.
Objectives: We propose a paradigm shift in the field that integrates various disciplines involved in the study of environmental contamination to recognize that contamination is widespread and cannot be remedied at the global level.
Discussion: Greater effort must be placed on integrative and interdisciplinary studies that explicitly illuminate how the causal mechanisms and functional outcomes of related processes operate at each level of biological organization while at the same time revealing the relations among the levels.
Conclusions: To anticipate and understand the future, we must devote more study to what is likely to happen and less to what has happened. Only then will we begin to understand how ancestral environmental exposures act at both the level of the individual and the level of their descendants to influence all aspects of life history.
PMCID: PMC3230404  PMID: 21571618
endocrine-disrupting chemicals; epigenetics; evolutionary biology; heritable; proximate effect; ultimate effect
25.  Neuroendocrine targets of endocrine disruptors 
Hormones (Athens, Greece)  2010;9(1):16-27.
The central neuroendocrine systems are responsible for the control of homeostatic processes in the body, including reproduction, growth, metabolism and energy balance, and stress responsiveness. These processes are initiated by signals in the central nervous system, specifically the hypothalamus, and are conveyed first by neural and then by endocrine effectors. The neuroendocrine systems, as the links between the brain and peripheral endocrine systems, play critical roles in the ability of an organism to respond to its environment under normal circumstances. When neuroendocrine homeostasis is disrupted by environmental endocrine-disrupting chemicals, a variety of perturbations can ensue, particularly when endocrine disruption occurs during critical developmental time periods. This article will discuss the evidence for environmental endocrine disruption of neuroendocrine systems, and the sequelae on endocrine and reproductive functions.
PMCID: PMC2896297  PMID: 20363718
GnRH; hypothalamus; endocrine disruption; fetal basis of adult disease; transgenerational; epigenetic; sex differences; thyroid

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