Andrea C. Gore - US grants

gonadotropin releasing factor; molecular biology; hormone regulation /control mechanism; gene expression; messenger RNA; preoptic areas; gene induction /repression; neurons; in situ hybridization; laboratory rat;

The goal of the proposed studies is to determine effects of ovarian steroid hormones, aging, and their interactions on the function, neuroanatomy, and neuropharmacology of GnRH neurons, and their regulation by NMDA receptors (NMDAR). GnRH neurons are the key cells regulating reproduction, and inputs from glutamate to GnRH perikarya and neuroterminals, mediated by the NMDAR, play important regulatory roles in GnRH synthesis and secretion. These processes are altered by age-related changes in circulating gonadal steroid hormones, which in turn have profound effects on neuroendocrine brain regions. The proposed studies will utilize ovariectomized female rats (young, middle-aged and old) and monkeys (young and old), given hormone (estrogen +/- progesterone) or vehicle replacement. The reproductive status of all animals is characterized prior to ovariectomy, and circulating estradiol and progesterone concentrations will be monitored during (in monkeys) or at the termination (rats) of each experiment. We will test the hypotheses that GnRH neurons express NMDAR subunits, that this expression is altered by hormones and/or aging, and that this has functional consequences. Aim 1 will utilize light microscopy to examine effects of hormones and aging on the anatomical relationships between NMDAR subunits and GnRH perikarya in rat and monkey hypothalamus preoptic area. In Aim 2, we will examine effects of hormones and aging on GnRH neuroterminats and their expression of NMDAR subunits in the median eminence of rats and monkeys, using light and electron microscopy. Data from Aims 1 and 2 will provide a comprehensive understanding of the interactions of GnRH neurons and NMDAR, and their hormonal and age regulation, at both GnRH pedkarya and terminals. In Aim 3 we will utilize a neuropharmacological approach to examine effects of NMDAR activation or antagonism on the ultrastructure of GnRH neuroterminals, and on GnRH gene expression. As a whole, these three Specific Aims will provide novel information on effects of hormones, aging, and their interactions on GnRH neurons. The experimental design will facilitate comparisons between female rats and monkeys. The outcomes of these studies will have considerable relevance to understanding the causes and consequences of menopause in women and in informing future studies on postmenopausal hormone replacement therapy.

DESCRIPTION: (Adapted from the Investigator's Abstract) The overall goal of this research proposal is to examine the mechanisms by which environmental toxicants adverse;y affect reproductive neuroendocrine development, using the rat as a model. There are four specific aims: The goal of specific aim I is to establish am experimental model for evaluating development. Specific aim II will test the hypothesis that the GnRH neurosecretory system plays a critical role in environmental toxicant induced stimulation or delay of reproductive development; characterize the mechanisms and compare the effects of the different toxicants. Specific aim III will test the hypothesis that exposure to environmental toxicants affects the development of neurotransmitter/growth factor system inputs to GnRH neurons, hypothalamine GT1-7 neuronal cell line, which synthesizes and secretes GnRH, methoxychlor, Aroclor 1221, Aroclor 1254, lead and chlorpyrifos affect the GnRH neurosecretory system.

[unreadable] DESCRIPTION (provided by applicant): Exposure to environmental toxicants can profoundly affect neurological and reproductive development, function and behavior, particularly when the exposure occurs during the susceptible prenatal period. The overall goal of this research proposal is to study the molecular targets at which environmental toxicants act to disrupt reproductive neuroendocrine function in adulthood. It is our working hypothesis that in utero exposure to endocrine disrupting chemicals (EDCs) has long-term consequences on reproductive outcomes, manifested as alterations in reproductive behavior and function, diminished fertility and fecundity, and a hastening of reproductive aging. These changes are mediated at least in part by effects of EDCs on neuroendocrine regions of the brain responsible for the regulation of reproduction, namely the hypothalamus and preoptic area. Our goal is to establish a model for investigating how one class of EDCs, polychlorinated biphenyls (PCBs) causes these long-term consequences, and to identify the molecular and cellular targets of the EDCs, using female Sprague-Dawley rats as a model. In Specific Aim 1, we will establish dose-response models to test effects of prenatal PCBs on reproductive neuroendocrine function in adulthood (fertility, fecundity, behavior, premature reproductive aging). In Specific Aim 2, we will identify target genes acted upon by prenatal PCBs in the hypothalamus using novel gene technologies (differential display PCR and microarrays). Finally, in Specific Aim 3, we will examine the neuroanatomical expression and regulation of the target molecules on which PCBs act in neuroendocrine brain regions. These studies should provide novel information on the possible mechanisms by which prenatal exposure to PCBs interferes with reproductive neuroendocrine function of adult female mammals. The results should have important implications for understanding consequences of, and developing therapies for, exposures of wildlife and humans to EDCs such as PCBs. [unreadable] [unreadable]

gonadotropin releasing factor; postmenopause; hormone regulation /control mechanism; Macaca mulatta; animal colony;

DESCRIPTION (provided by applicant) We propose to organize a one-day Forum on Endocrine Disrupting Chemicals (EDCs) on June 3, 2005, the day before the Endocrine Society Annual Meeting in San Diego, California. The major goals of this meeting are fourfold: first, to educate and update endocrinologists on endocrine disruption research; second, to make this currently under-represented discipline at Endocrine Society meetings a strong and regular presence in the future; third, to provide a forum for students and fellows to participate actively in a poster session and discussion; and fourth, to publish results as a special edition of a journal. The Endocrine Society is equally represented by basic scientists and clinicians, and its expertise is extremely well-suited to perform research in the field of endocrine disruption. Nevertheless, to date there is a lack of presence of the endocrine disruption field at the Endocrine Society meeting that we believe we can remedy through this conference. To achieve these goals, the conference is organized into five sessions. 1) A keynote address (50 minutes) given by Dr. Kenneth Korach to provide a broad perspective to the field of EDCs as an introduction to the Endocrine community. 2) A morning symposium (2 hours) entitled "Early Exposure to EDCs and Long-Term Endocrine Consequences." 3) A poster session focusing on students and fellows (1.5 hours) to encourage participation of young scientists. 4) The afternoon symposium (2 hours) entitled "Hot Topics in Endocrine Disruption" is biomedically-relevant and will discuss mechanisms of EDC actions and their relevance to human health and disease. 5) The meeting will end with a panel discussion (50 minutes) led by leaders in the Endocrine Society including President Andrea Dunaif, who will discuss: "Formulating an Endocrine Society Agenda for EDCs Research and Public Awareness." As a workshop at the Endocrine Society Annual Meeting, our proposed Forum is ideally suited to provide public health information on EDCs to the basic, translational and clinical participants. In order to limit future contaminations and to mitigate existing exposures, it is critical that we educate the endocrine community and make this subject a permanent part of the Endocrine Society's agenda.

[unreadable] DESCRIPTION (provided by applicant): It is becoming increasingly clear that the mechanisms for reproductive aging involve a complex interaction of the three levels of the hypothalamic-pituitary-gonadal (HPG) axis. In rats and other rodents, reproductive cycles become irregular at middle age and acyclicity ensues shortly thereafter, although the ovaries still contain functional follicles. These observations support the role of the hypothalamus and/or pituitary in the transition to acyclicity. My laboratory has been studying a role in reproductive senescence for GnRH neurons, the primary cells controlling reproductive function. Recent evidence suggests that a major mechanism for compromised HPG function during aging is due to age-related alterations in regulatory inputs to GnRH cells. Here, I will investigate the neuroendocrine mechanisms that underlie the transition to acyclicity at middle age, focusing on how the NMDA receptor (NMDAR), which mediates effects of the neurotransmitter glutamate, interacts with GnRH cells at their perikarya in the preoptic area (POA) and neuroterminals in the median eminence (ME), and the consequences of these effects on reproductive decline. My model is young (regular estrous cycles) and middle-aged (regular or irregular cycles, or acyclic) Sprague-Dawley rats; therefore, my analyses will take both age (young vs. middle-aged) and reproductive status (cyclic, irregularly cycling, or acyclic) into consideration as independent variables. Three Specific Aims are proposed to test our hypotheses. Aim 1 will investigate effects of age-related changes in NMDARs in POA, on GnRH cells specifically and in POA nuclei in general. Aim 2 will examine similar relationships in the ME. Aim 3 will study how NMDARs and estrogen receptors (ERs) may be expressed in the same target cells in POA and ME, including GnRH cells, and how these change during reproductive aging. Together, these studies will link GnRH neurons, NMDARs, and ERs, to elucidate how these systems act coordinately to result in the transition to acyclicity at middle age, and to provide insight into their mechanisms. My three Specific Aims form the basis for a model of the perimenopausal transition in women, an area relevant to improving healthy aging, and will provide fundamental and functional insights into the role of the hypothalamus in reproductive senescence. These experiments have clinical implications for postmenopausal hormone replacement therapy, for identifying non- hormonal approaches to treating menopausal symptoms, and for potentially expanding the reproductive lifespan, which is becoming more important as women continue to postpone childbearing until later in life. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): Translational Science and specific Challenge Topic, 15- ES-101*: Effects of Environmental Exposures on Phenotypic Outcomes Using Non-human Models. The overall goal of this research proposal is to study transgenerational, epigenetic effects of endocrine- disrupting chemicals (EDCs) on neuroendocrine function. Environmental EDC exposures can result in permanent dysfunctions in reproductive development, impaired fertility, and hormonally-related disease states. Developing organisms, particularly fetuses and infants, are especially vulnerable to endocrine disruption. During these early life periods, hormones are responsible for normal development of the hypothalamus, the brain region that regulates endocrine and hormonal systems and is responsible for the control of adult reproductive physiology and behavior. Disruptions of these systems by environmental EDCs can interfere with the acquisition and maintenance of these processes, resulting in aberrant neuroendocrine development and a compromised adult phenotype. We propose that EDCs such as polychlorinated biphenyls (PCBs) can effect these changes in the fetally exposed (F1) individuals through actions on steroid hormone receptor gene and protein expression in the hypothalamus. Additionally, effects may be transmitted to future generations (F2, F3) through transgenerational, epigenetic mechanisms. The proposed studies seek to understand the mechanisms by which fetal exposures to PCBs cause permanent imprinting changes on gene expression in the hypothalamus to cause adult dysfunction. We will also investigate how these effects are transmitted to subsequent generations. Our goals in the fetally-exposed (F1) generation are to measure effects of expression of genes for steroid hormone receptors in the hypothalamus of PCB vs. control animals (Aim 1);and to explore and identify how PCBs may cause epigenetic modifications to these genes through analyses of DNA methylation and histone modifications (Aim 2). Then, comparisons will be made among the F1, F2 and F3 generations of rats to determine the manifestation of transgenerational, epigenetic effects and to ascertain the mechanism for transmission (Aim 3). As a whole, these experiments are designed to provide mechanistic insight into endocrine disruption of hypothalamic function during critical developmental life stages, the latent manifestation of a disrupted adult phenotype, and the transmission of this trait to subsequent generations. PUBLIC HEALTH RELEVANCE: The proposed studies on endocrine disruption are highly relevant to humans. The EDC chosen for the current proposal, PCBs are a persistent and continuing problem, as virtually all living humans have a detectable body burden of PCBs. Therefore, understanding the transgenerational epigenetic effects of PCBs in a rat model can provide information about public policy, prevention, and medical interventions in humans.

DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals (EDCs) are compounds in the environment that perturb endocrine systems. EDCs are ubiquitous in the modern world, and detectable in virtually all humans and wildlife. Many effects of EDCs are mediated by hormone receptors such as estrogen receptors (ERs) that are widely distributed in the brain, and thereby EDCs perturb neurobiological functions. The hypothalamus, hippocampus and amygdala are brain regions with high expression of ERs, and are proven targets for endocrine disruption. However, the cellular, molecular and physiological processes for these effects, and the functional behavioral implications for exposures of these brain regions to EDCs, are not well explored. Furthermore, the brain has a number of structural and functional sexual dimorphisms that develop early in life, a process that is sculpted by actions of estrogens on ERs, and disrupted by EDCs. Therefore, it is the overarching goal of this research to determine the molecular and cellular mechanisms by which exposure of developing fetuses to ecologically-relevant levels of EDCs perturb the sexual differentiation of specific brain areas, and the consequences on behaviors regulated by these regions. The proposed studies will fill a gap in knowledge by testing effects of perinatal exposure to a class of endocrine-disrupting chemicals, specifically polychlorinated biphenyls (PCBs), on sexually dimorphic neurobiological processes. PCBs are a ubiquitous and persistent environmental contaminant, and while banned for decades, they are still prevalent in soil and groundwater, leach into food and water, and are detectable in tissues of virtually all humans. The proposed studies will use a well-established rat model already in use in the PIs'labs to test effects of exposure to PCBs during a critical hormone-sensitive developmental window of late gestation, a critical period for brain sexual differentiation during which the developing nervous system is exquisitely sensitive to both endogenous and exogenous hormones, particularly estrogens. The experiments will quantify expression of a network of estrogen-regulated genes (Aim 1);elucidate some potential epigenetic mechanisms for regulation of identified targets (Aim 2);determine the manifestation of gene expression changes through protein immunohistochemistry (Aim 3);and ascertain the final outcome as a behavioral phenotype (Aim 4). These experiments have broad implications for humans as exposure to PCBs is universal, persistent, and has wide-ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform public policy, medical interventions, and prevention. In addition, results on PCBs, used as a "model" EDC for decades, can help us better understand effects of other estrogenic EDCs still in common use such as those in plastics, pesticides, and beyond. PUBLIC HEALTH RELEVANCE: Exposures of humans to PCBs and other estrogenic EDCs are universal, and these compounds have wide- ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform medical interventions and prevention, and guide public health policy. The rat model is highly conserved with humans, and it enables us to test the cause-and- effect relationship between prenatal PCBs, the development of adult disease, and the neural mechanisms underlying these biomedical processes.

DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals (EDCs) are compounds in the environment that perturb endocrine systems. EDCs are ubiquitous in the modern world, and detectable in virtually all humans and wildlife. Many effects of EDCs are mediated by hormone receptors such as estrogen receptors (ERs) that are widely distributed in the brain, and thereby EDCs perturb neurobiological functions. The hypothalamus, hippocampus and amygdala are brain regions with high expression of ERs, and are proven targets for endocrine disruption. However, the cellular, molecular and physiological processes for these effects, and the functional behavioral implications for exposures of these brain regions to EDCs, are not well explored. Furthermore, the brain has a number of structural and functional sexual dimorphisms that develop early in life, a process that is sculpted by actions of estrogens on ERs, and disrupted by EDCs. Therefore, it is the overarching goal of this research to determine the molecular and cellular mechanisms by which exposure of developing fetuses to ecologically-relevant levels of EDCs perturb the sexual differentiation of specific brain areas, and the consequences on behaviors regulated by these regions. The proposed studies will fill a gap in knowledge by testing effects of perinatal exposure to a class of endocrine-disrupting chemicals, specifically polychlorinated biphenyls (PCBs), on sexually dimorphic neurobiological processes. PCBs are a ubiquitous and persistent environmental contaminant, and while banned for decades, they are still prevalent in soil and groundwater, leach into food and water, and are detectable in tissues of virtually all humans. The proposed studies will use a well-established rat model already in use in the PIs' labs to test effects of exposure to PCBs during a critical hormone-sensitive developmental window of late gestation, a critical period for brain sexual differentiation during which the developing nervous system is exquisitely sensitive to both endogenous and exogenous hormones, particularly estrogens. The experiments will quantify expression of a network of estrogen-regulated genes (Aim 1); elucidate some potential epigenetic mechanisms for regulation of identified targets (Aim 2); determine the manifestation of gene expression changes through protein immunohistochemistry (Aim 3); and ascertain the final outcome as a behavioral phenotype (Aim 4). These experiments have broad implications for humans as exposure to PCBs is universal, persistent, and has wide-ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform public policy, medical interventions, and prevention. In addition, results on PCBs, used as a model EDC for decades, can help us better understand effects of other estrogenic EDCs still in common use such as those in plastics, pesticides, and beyond.

DESCRIPTION (provided by applicant): The world is contaminated, never to return to conditions that existed prior to the chemical revolution. Although some local remediation of contamination has occurred, at a global level this is simply not possible. A class of contaminants is known as Endocrine Disrupting Chemicals (EDC) because of their ability to perturb the body's hormone systems. Poor storage, spills, deliberate and accidental dispersal have had well-document effects on wildlife and human health. However, the field of EDC research remains highly controversial and polarized. Such compounds are now a permanent part of our environment and creating previously unknown evolutionary pressures. We must transcend traditional toxicological testing to develop new methods and perspectives if we are to anticipate and understand EDCs' impact on the future. Life in this new world is a combination of ancestral exposures due to heritable epigenetic modifications to DNA in germ cells (transgenerational), together with exposures experienced during the individual's own lifetime (body burden) that cause molecular epigenetic changes to that individual. These processes are an underappreciated force in driving evolutionary change in all species, including humans. The challenge is how to model the cumulative and progressive changes both across lifespans and within generations. We propose a unique study of multigenerational exposures to sequential environmental toxicants, each known to perturb hormones, brain and behavior. Our proposed model investigates interactions of ancestral and immediate epigenetic modifications, factors that have never been studied together. Over the course of 6 generations, we make the iconoclastic prediction that individuals will evolve in unique ways due to contemporary environmental driving forces (chemical contamination), with descendants responding differently to proximal chemical stimuli than their ancestors. To do this work, we propose to model two exposures separated by 3 generations; different EDC classes will be used at environmentally relevant levels, each with a different mode of action. Endpoints will be body weight, physiological parameters, neurobiological gene expression and molecular epigenetic assays, together with behavioral characterization of the animals in a suite of behaviors involved in social, anxiety, and cognitive function. Realistically simulating the nature of life challenges across and within generations will provide the framework for understanding and anticipating how environmental contamination will affect the evolution of all species.

ABSTRACT Every human has a body burden of endocrine-disrupting chemicals (EDCs), and levels of EDCs correlate with reproductive, endocrine, and neurobehavioral deficiencies. As environmental stressors, EDCs interact with other types of stressors to increase chronic disease and impair the quality of life. This proposal seeks to understand how the developmental trajectory of an individual is shaped by the interaction of behavioral stress during two life stages in the context of contamination. We focus on the social behavioral phenotype, as shifts in the reaction norms of social behavior can profoundly change an individual?s relationship to its community, his/her reproductive success, and mental health. We postulate that neurodevelopment in a contaminated world changes an individual?s baseline social phenotype, and that further life stressors overlaid upon the EDC phenotype create greater deflections from the behavioral norm. We will approach this question in several novel ways. First, we will model constant low-level exposure to a mixture of common-use EDCs throughout life, and assess emotional reactivity and sociality. Second, we will examine the effects of a typical life challenge (mild stress) during two critical life periods: to the mother (during pregnancy), to the individual during adolescence, or both. Third, we will compare males and females; this enables us to identify susceptibilities that may relate to well-established gender differences in disease and neurobehavioral dysfunction. Finally, by measuring changes in neuromolecular activity and neuroanatomical organization in a defined network of interconnected limbic and forebrain nuclei regulating the social phenotype, we can gain mechanistic insights into these processes. Thus, our overarching hypothesis is that each life stressor (lifelong EDC exposures, mild prenatal stress, and mild stress during adolescence) concatenates to shift the reaction norms for neurodevelopment and social behavior, and that the combination of stressors exacerbates adverse outcomes for neurobiological health. Mechanistically, we further propose that EDCs and stressors modulate this phenotype through perturbing the normal complementarity of estrogen and androgen signaling in the social decision-making network of the brain. There are 3 Specific Aims. Aim I will establish the sexually dimorphic behavioral phenotype of a lifetime of exposure to low levels of a mixture of common-use EDCs, upon which is superimposed mild stress during critical life stages (gestational, adolescent, or both). Aim II will determine underlying neuromolecular mechanisms for the changes caused by lifelong EDC exposures and gestational/adolescent stressors, focusing on estrogen/androgen-sensitive circuits. Aim III will identify neuroanatomical and cytoarchitectural substrates for the changes caused by EDCs and stressors, prioritizing estrogen-androgen signaling pathways. Proposed work has the potential to have a broad impact, ranging from societal and government policies, the health crisis caused by increasing chronic disease, and understanding fundamental biological principles at the molecular, cellular and organismal levels.

ABSTRACT Exposures to environmental endocrine-disrupting chemicals (EDCs), especially during early life, are strongly linked to adverse reproductive outcomes. Polychlorinated biphenyls (PCBs) are a well-established family of EDCs, used for decades in industrial applications. While banned decades ago, humans are exposed to PCBs today primarily through the food chain. In both humans and animals, higher PCB body burdens are associated with endocrine and reproductive dysfunctions. However, there are several important gaps in knowledge in this field that we propose to fill. First, while PCBs perturb each level of the hypothalamic-pituitary-gonadal (HPG) axis when assessed individually, the cross-talk among these levels has not been explored. Second, while early development (fetus, infant) is recognized as a period of vulnerability to EDCs, the preconceptional period is another critical window that has not been studied for PCB actions on HPG systems, especially in females, something we propose to redress. Third, the vast majority of phenotyping following EDC exposures is conducted at a single life stage, typically in the adult. Yet, the reproductive system undergoes enormous dynamic change in structure and function throughout life, underscoring the need for a lifecycle approach to phenotyping. The fourth and perhaps largest gap is whether and how preconceptional exposures reprogram the germline epigenome, and if that leads to somatic effects. In this proposal, we will develop and utilize a novel intergenerational model of preconceptional exposure to PCBs. We will feed pregnant Sprague-Dawley rat dams (F0 grandmothers) human-relevant levels of a PCB mixture, or vehicle, from E8-18, when germ cell epigenetic marks are being erased in the gonads of the F1 fetuses; and/or during lactation when F1 pups are P1-21 and when germ cell epigenetic marks are re-established. The F1 females are bred with untreated male rats to generate the F2 generation. The F2 female offspring (grandchildren) will be phenotyped at 3 reproductive life stages: neonatal, pubertal, and adult. Physiological outcomes, expression of key HPG genes and proteins involved in the control of reproductive competence, and underlying molecular epigenetic marks (DNA and histone methylation) will be assessed. In addition, we will use a novel method to isolate primordial germ cells (PGCs) of newborn F1 and F2 females rats of the PCB and vehicle lineage, to quantify gene expression and methylation marks. This allows us to relate HPG phenotypes to prior germ cell programming. Thus, this proposal is anticipated to provide novel insights into the effects of preconceptional PCB exposures on female HPG physiology, function, and underlying epigenetic mechanisms of regulation, results of which are directly relevant to women?s environmental health.