Vasantha Padmanabhan - US grants

The objective of this proposal is to test the hypothesis that important mechanisms by which GnRH and gonadal secretions induce and maintain sexual maturation involve changes in the biopotency as well as the quantity and pattern of the secreted gonadotropins. In this regard, the relative roles of GnRH and gonadal secretions will be separated by the use of the nutritionally restricted castrate lamb model in which endogenous GnRH secretion can be altered by nutritional modification or increased by administration of GnRH of varying frequencies and amplitudes. Comparative information will be obtained from spontaneously maturing lambs. In each of the experimental protocols we will examine the secretory profiles of gonadotropins (bioactive and immunoreactive) and correlate the changes in the B/I ratios with the relative distributions of gonadotropin isoforms following chromatofocusing. The emphasis will be on oFSH. The oFSH isohormones will be characterized for their in vitro biopotency, immunoassayability, receptor binding ability, plasma disappearance rates, and carbohydrate content. The physiological relevance of the oFSH biopotency changes will be further examined by the extraction of oFSH from various serum pools and reconstitution of oFSH free sera derived from different experimental protocols. These reconstituted sera will be tested for their ability to induce maturational indices such as induction of LH receptors in immature granulosa cells, 3H-thymidine incorporation and, if feasible, ovarian follicle growth by means of in vivo assays. Similar physiologic studies will be used to test various oFSH isoforms (of varying degees of glycosylation) derived from oFSH pituitary standards. To complement the above studies the neuroendocrine regulation of oFSH secretion will be further studied in in vitro ovine pituitary culture systems. Therefore, taken together, the proposed studies will provide an unique opportunity to dissect the mechanisms of the neuroendocrine regulation of oFSH heterogeneity at the pituitary level (post- translational modifications) and in the serum (post-secretion alterations). It will be possible to examine whether the various oFSH isoforms in the circulation during pubertal development in the lamb influence physiologic events such as organ growth and function and which FSH isohormones play key roles in target cells responsiveness.

The objective of this proposal is to test the hypothesis that important mechanisms by which GnRH and gonadal secretions induce and maintain sexual maturation involve changes in the biopotency as well as the quantity and pattern of the secreted gonadotropins. In this regard, the relative roles of GnRH and gonadal secretions will be separated by the use of the nutritionally restricted castrate lamb model in which endogenous GnRH secretion can be altered by nutritional modification or increased by administration of GnRH of varying frequencies and amplitudes. Comparative information will be obtained from spontaneously maturing lambs. In each of the experimental protocols we will examine the secretory profiles of gonadotropins (bioactive and immunoreactive) and correlate the changes in the B/I ratios with the relative distributions of gonadotropin isoforms following chromatofocusing. The emphasis will be on oFSH. The oFSH isohormones will be characterized for their in vitro biopotency, immunoassayability, receptor binding ability, plasma disappearance rates, and carbohydrate content. The physiological relevance of the oFSH biopotency changes will be further examined by the extraction of oFSH from various serum pools and reconstitution of oFSH free sera derived from different experimental protocols. These reconstituted sera will be tested for their ability to induce maturational indices such as induction of LH receptors in immature granulosa cells, 3H-thymidine incorporation and, if feasible, ovarian follicle growth by means of in vivo assays. Similar physiologic studies will be used to test various oFSH isoforms (of varying degees of glycosylation) derived from oFSH pituitary standards. To complement the above studies the neuroendocrine regulation of oFSH secretion will be further studied in in vitro ovine pituitary culture systems. Therefore, taken together, the proposed studies will provide an unique opportunity to dissect the mechanisms of the neuroendocrine regulation of oFSH heterogeneity at the pituitary level (post- translational modifications) and in the serum (post-secretion alterations). It will be possible to examine whether the various oFSH isoforms in the circulation during pubertal development in the lamb influence physiologic events such as organ growth and function and which FSH isohormones play key roles in target cells responsiveness.

Using female sheep as the experimental model, this amended proposal will test the hypothesis that normally occurring changes in FSH heterogeneity are of functional significance , with increases in biopotency associated with the onset of puberty and the preovulatory period. The goals are to: (1) establish the modulatory role of estradiol in mediating FSH heterogeneity, (2) determine nature of FSH isoforms secreted during different phases of puberty and ovulatory cyclicity. (3) reveal the role of FSH heterogeneity in the initiation of the pubertal process through administration of different isoform mixes of recombinant-produced FSH to immature GnRH antagonist-treated lambs and (4) establish if freshly secreted non-metabolized, FSH isoforms in hypothalamo-pituitary portal plasma can effect different biological responses at the target site.

DESCRIPTION (Adapted from applicant's abstract): Recent studies in the PI's laboratory have provided evidence that FSH secretion can be characterized by episodic and basal secretion modes. The basal component of FSH secretion appears to be the dominant mode. While basal FSH secretion is not under acute control of GnRH, the level of basal FSH secretion can be altered by changes in activin, inhibin, and follistatin tone. These pituitary regulatory proteins appear to be synthesized and act probably via paracrine/autocrine modes to modulate basal FSH secretion. When the hypothalamus is disconnected from the pituitary, basal FSH secretion is significantly reduced which suggests that hypothalamic input is a necessary component in setting basal FSH tone. The longterm goal of this proposal is to investigate the mechanisms that regulate the release of basal FSH secretion. The overall hypothesis to be tested is that the level of FSH secretion during any physiologic state is primarily determined by the bioavailability of activin, a potent stimulator of FSH biosynthesis and secretion. Activin bioavailablity is in turn, determined by the prevailing tone of inhibin and follistatin and the available pituitary activin receptors. Integrative studies are proposed to dissect the complex functional overlaps among these FSH regulatory proteins at the pituitary level. Techniques such as hypophyseal-portal sampling, hypothalamo-pituitary disconnect techniques, in situ hybridization, antisense technology, and long-term pituitary cell perifusion studies will be utilized to determine how the hypothalamus and pituitary interact in controlling the mechanisms which govern basal FSH release.

DESCRIPTION: (Adapted from the applicant's description) The long-term goal of this project is to dissect out the mechanisms which govern the release of follicle-stimulating hormone (FSH). The recent studies utilizing sheep as a model has provided compelling evidence that: 1) FSH secretion is episodic; 2) each gonadotropin releasing hormone (GnRH) pulse is associated with an FSH pulse; 3) GnRH-independent episodes of FSH occur; 4) Factor(s) present in the hypophyseal-pituitary portal blood can selectively regulate FSH secretion; and 5) the FSH-releasing activity in the hypophyseal portal blood is not due to activin. In this proposal the researchers will determine 1) the biochemical nature of the FSH-releasing activity; and 2) whether FSH-releasing factors with similar chemical characteristics such as those present in the hypothalamic portal blood can be obtained from hypothalamic extracts.

In mammals a key hormone involved in reproduction is follicle-stimulating hormone, FSH. This hormone is released by the pituitary gland, which is connected to part of the brain called the hypothalamus. The mechanisms that regulate the timed release of this hormone are largely unknown. Recently it has been shown that the blood circulating to the pituitary from the brain can include an unknown compound that stimulates FSH release (FSH-releasing factor, or FSH-RF) in a pulsatile fashion. This action seems to be independent from the release regulated by the known contribution of the compound activin, or of another releasing hormone called GnRH. This project utilizes biochemical, pharmacological and anatomical techniques, with a novel model system to separate the neural supply from the blood supply to the pituitary, to localize the timing and source of this novel releasing factor as a step towards identifying it.
Results will be very important for neuroendocrinology by clarifying release mechanisms for a major hormone involved in puberty as well as reproduction, and will have a likely impact also in veterinary and agricultural fields.

Polycystic ovarian syndrome (PCOS) is the most common endocrinopathy and it affects 10 percent of reproductive-aged women. The etiology of chronic hyperandrogenic anovulations, such as PCOS, may have genetic underpinnings. Although the underlying mechanisms are unknown, PCOS is now recognized as hyperandrogenism accompanied by anovulation. Polycystic ovarian morphology is highly correlated with conditions in which the fetus has been exposed to high amounts of sex steroids before birth. For example, women with classical 21-hydroxylase deficiency mimic PCOS, exhibit anovulation, ovarian hyperandrogenism, and LH hypersecretion. Perhaps excess sex steroids early in life may provide a hormonal "insult" that results in manifestation of PCOS later in adulthood. This proposal aims to use a new model, the prenatally-androgenized sheep (long gestation, mono-ovular species), to investigate causal mechanisms for the developmental origins of PCOS. Our preliminary studies indicate that these sheep develop ovulatory defects during adulthood similar to those of women with PCOS: anovulation, elevated LH levels, hyperandrogenemia, hyperinsulinemia, and multifollicular ovaries. In this proposal, we will test the following hypothesis: prenatal exposure to androgens disrupts adult reproductive function culminating in hyperandrogenic anovulation and that this disruption is mediated via reduced sensitivity to the positive feedback actions of estradiol, abnormal gonadotropic drive and/or altered ovarian sensitivity to FSH. The specific Aims of the proposed research are to determine 1) the extent to which fetal exposure to androgens disrupts reproductive cyclicity, ovarian function, ovulatory capacity and fertility in adulthood, (2) if reduced sensitivity to estradiol stimulatory feedback of gonadotropin secretion contributes to the disruptive effects of prenatal- androgenization on postnatal reproductive cyclicity, and (3) if abnormal gonadotropic drive and/or reduced ovarian sensitivity to FSH contributes to the disruptive effects of prenatal- androgenization on postnatal reproductive cyclicity. If our hyposthesis proves to be correct, this would form the basis for a distinct developmental origin of an important reproductive disease in adulthood. Specifically it will establish that discrete, experimentally induced androgen excess of fetal sheep provides the first clear etiology for hyperandrogenic anovulation in adulthood.

DESCRIPTION (provided by applicant): The susceptibility of the reproductive system to early exposure to steroid hormones has become a major concern in our modern societies. At risk is the fetus whose mother has been exposed to exogenous steroids for a variety of reasons: she has had failed contraception and continued exposure to contraceptive steroids, she is on anabolic steroids, or she is inadvertently being exposed to environmental compounds that have estrogenic or androgenic activity. Experimental manipulation of the prenatal steroid environment provides a powerful investigative tool for unraveling mechanisms that underlie programming of the reproductive axis. Findings from the current funding period found prenatal testosterone (T) excess disrupts the developmental trajectory of the fetus culminating in reproductive neuroendocrine, ovarian, metabolic and behavioral perturbations and implicates ovarian hyperandrogenism and hyperinsulinemia in the development of the pathology. This proposal aims to capitalize on this recently validated sheep model to test a novel, unifying hypothesis for the developmental origins of infertility disorders namely Hyperinsulinemia and functional hyperandrogenism resulting from metabolic perturbations and/or increased steroid drive to the fetal neuroendocrine-ovarian system plays a key role in prenatal T-induced reproductive dysfunctions. The specific goals of the P01 proposal are to 1) use an insulin-sensitizing agent postnatally to ameliorate hyperinsulinemia and determine if it rescues reproductive function in prenatal T-treated sheep, 2) use an anti-androgen postnatally and determine if it rescues reproductive function in prenatal T-treated sheep, 3) determine if co-treatment with T and an androgen antagonist during fetal life will prevent metabolic and reproductive pathology from developing in the adult, and 4) determine the neuroanatomical, neuroendocrine, ovarian, behavioral and metabolic mechanisms underlying restoration of function. The proposal targets key elements of strategic plans that emanated from workshops convened by the NICHD in 2000-01 From Cells to Selves and focuses on the following areas: fetal antecedents of disease, reproductive health for the 21st Century, developmental biology and biobehavioral development.

Each woman is endowed with a finite number of primordial follicles at birth. Most of this initial endowment is depleted during normal reproductive life, mainly through atresia. The length of reproductive life is largely defined by the rate of follicular depletion. The variability in the length of reproductive life suggests variability in the initial endowment of primordial follicles and/or rate of follicle loss. Reproductive life span can be shortened by factors that reduce primordial follicular endowment, accelerate the rate of recruitment/atresia of follicles, and/or induce follicular deficiencies leading to developmental arrest of follicles. Increasing evidence suggests that the environment to which the fetus is exposed can alter the path of reproductive organ differentiation. The use of animal models offers exciting potential for delineating the mechanisms by which human fertility disorders can be programmed in fetal life. Our studies found that prenatal exposure of sheep to testosterone results in altered neuroendocrine feedback, hyperandrogenism, multifollicular ovarian morphology, increased follicular atresia and reproductive cycle defects culminating in early reproductive failure. The objective of this proposal is to delineate the ovarian programming that occurs as a consequence of in utero sex steroid exposure that is responsible for reproductive failure later in life. The working hypothesis is as follows: Exposure of fetuses to excess testosterone during critical stages of ovarian follicular development alters normal developmental trajectories by changing the expression patterns of key growth factors, androgen/estrogen receptors, and ratio of anti- to proapoptotic Bcl-2 members, resulting in accelerated recruitment of primordial to primary follicles, accelerated growth of primary follicles to antral stage and enhanced incidences of atresia or arrest of antral follicles. These alterations eventually, lead to premature depletion of ovarian follicular reserve and ovarian failure. The deleterious effects of testosterone are mediated either by its androgenic action, estrogenic action due to its conversion to estradiol, or both. Further, prenatal exposure to steroids alters ovarian follicular susceptibility to postnatal steroids leading to premature ovarian failure. The outcome of these studies is of relevance to 3 of the targeted NIH missions, fetal antecedents of disease, developmental biology and reproductive health for the 21st century.

SUMMARY Endocrine disrupting compounds (EDC) are hormonally active, synthetic or natural chemicals that interfere with normal functioning of the endocrine system, most notably the reproductive endocrine axis. Concern about EDC has mainly been fueled by studies that point to likely effects of EDC exposure on humans including dramatic increases in estrogen sensitive cancers, decline in human sperm quality and quantity, a notable rise in endometriosis, and early puberty in women. Because of the universal and crucial role estradiol plays in reproduction and other biologic processes, estrogenic pollutants in the environment are of particular concern. Given that sex steroids play a crucial role in organ differentiation during development, it is reasonable to expect in utero exposure to exogenous steroid mimics may alter the developmental trajectory of the fetus culminating in adult reproductive dysfunction. Our preliminary studies using sheep as a model revealed that prenatal exposure to the plasticizer bisphenol A (BPA), an environmentally relevant EDC, at levels approaching that found in human maternal blood and amniotic fluids, resulted in low birth weight female offspring, early postnatal hypergonadotropism, and cycle defects manifested as severe dampening of the LH surge. In this proposal, we will test the hypothesis that prenatal exposure to BPA, an environmental estrogen mimic, at levels similar to what human fetuses are exposed to, will disrupt adult reproductive function by disrupting the mechanisms controlling postnatal neuroendocrine feedback controls of GnRH/LH secretion and ovarian sensitivity to gonadotropins. Further, prenatal exposure to BPA will exacerbate postnatal reproductive susceptibility to steroid exposure and culminate in reproductive failure. Three Specific Aims will test these hypotheses. Specific Aim 1 will determine if unconjugated BPA in the maternal circulation reaches the fetus and at levels seen by the human fetus disrupt adult reproductive function. Specific Aim 2 will determine if prenatal BPA effect is mediated at the neuroendocrine or ovarian level and involves disruption of the mechanisms controlling postnatal neuroendocrine feedback controls of GnRH/LH secretion and/or ovarian sensitivity to gonadotropins. Specific Aim 3 will determine if prenatal exposure to BPA exacerbates reproductive susceptibility to postnatal estradiol exposure culminating in reproductive failure. The proposal targets key elements of strategic plans that emanated from workshops convened by the NICHD in 2000-01 by focusing on three targeted areas: fetal antecedents of disease, reproductive health for the 21st century, and developmental biology. The findings will be relevant to research on fetal origin of infertility and the threat estrogenic environmental disruptors at current exposure levels pose to human health.

Abnormal hormonal, nutritional, metabolic or environmental conditions during pregnancy alter the developmental trajectory of the fetus culminating in adult diseases. Exposure of sheep to excess testosterone (T) during fetal life leads to neuroendocrine, ovarian and metabolic defects that parallel the reproductive and metabolic phenotype of women with polycystic ovary syndrome, with postnatal obesity amplifying these defects. Ovarian defects involve increased follicular recruitment and follicular persistence. Ovarian disruptions are evident during fetal life and include increased androgen receptor expression, imbalance in the ratio of estrogen receptor alpha and beta, and altered insulin signaling. Metabolic defects, mediated by androgenic actions ofT, are also evident eariy in fetal life culminating in insulin resistance. Postpubertal treatment with an insulin sensitizer improves insulin sensitivity and prevents further deterioration of reproductive axis implicating a role for insulin. Our recent findings suggest that gestational co-treatment with an androgen antagonist overcomes disruptive effects of prenatal T at the neuroendocrine and ovarian level. Proposed studies will determine the contributing role of prenatal androgen in the programming of prenatal T-induced reproductive pathology and delineate the relative role of postnatal hyperinsulinemia and functional ovarian hyperandrogenism in facilitating the expression of cyclic ovarian disruptions. Specifically, studies will address if attenuation of hyperinsulinemic status beginning before puberty with insulin sensitizers or ovarian hyperandrogenism with an androgen antagonist would overcome follicular persistence and rescue cyclic function in prenatal T-treated sheep and if prevention of androgen action by co-treatment with androgen antagonist during fetal life would prevent programming of metabolic and reproductive pathologies. Ovaries from these interventional studies will be used to 1) address the role of intraovarian steroid and insulin receptor signaling, 2) identify protein clusters mediating prenatal T-induced ovarian dysfunction;and 3) delineate the role of epigenetic modifications in mediating differential protein expressions. Findings from these studies would be of translational relevance to human infertility disorders.

DESCRIPTION (provided by applicant): Endocrine disrupting compounds (EDCs) are hormonally active, synthetic or natural chemicals that interfere with normal functioning of the endocrine system. Exposure to EDCs continues to be a significant and contentious public health issue. Because of the universal and crucial role estradiol plays in reproduction and other biologic processes, estrogenic pollutants in the environment are of particular concern. Given that sex steroids play a crucial role in organ differentiation during development, it is reasonable to expect in utero exposure to exogenous estrogen mimics may alter the developmental trajectory of the fetus. Our preliminary studies using sheep as a model revealed that prenatal exposure to the plasticizer bisphenol A (BPA), an environmentally pervasive estrogenic EDC, at levels approaching that found in human maternal blood and amniotic fluids, resulted in low birth weight female offspring. In this proposal, we will test the following hypotheses: Poor pregnancy outcomes and low birthweight offspring in humans are related to increased exposure to BPA. Experimentally, treatment of pregnant sheep with BPA, at levels approaching those found in human maternal and fetal circulation, would reduce fetal IGF bioavailability and culminate in fetal growth retardation and low birth weight offspring. Even more importantly, the programming effects of BPA on fetal developmental trajectory are transgenerationally transferable. Three Specific Aims will test these hypotheses. In Specific Aim 1 we will measure BPA concentrations in maternal and cord blood of US women and correlate it with pregnancy outcome and birthweight of offspring. In Specific Aim 2, we will determine if BPA, at levels found in human maternal circulation, reduces fetal IGF bioavailability and culminates in intrauterine growth restriction and low birthweight offspring. In Specific Aim3, we will determine if detrimental effects of BPA on fetal growth trajectory are transgenerationally transferable. The research proposed here has important implications for reducing poor pregnancy outcomes by identifying potentially modifiable risk factors for IUGR and low birth weight. Environmental exposures are good candidates for modifiable risk factors as they can be effectively regulated at the personal, behavioral, as well as the regulatory levels. PUBLIC HEALTH RELEVANCE: Endocrine disrupting compounds (EDC) are hormonally active, synthetic or natural chemicals that interfere with normal functioning of the endocrine system. Exposure to EDCs continues to be a significant and contentious public health issue. This proposal will determine 1) to what extent human fetuses are exposed to BPA and if a relationship exists between level of BPA exposure and pregnancy outcomes, 2) if exposure of pregnant sheep to BPA at levels seen in human circulation will lead to low birthweight offspring by altering growth factor availability, and 3) if the effects of BPA on fetal development is carried across subsequent generations.

The objective of the Sheep Core is to provide a mechanism whereby the services of common personnel are shared by all projects of this Program Project Grant, enabling cost-effective breeding and animal maintenance programs of exceptionally high standards. The underlying rationale for this Core is simple. A staff of well-qualified individuals can meet the needs of multiple research projects more effectively and economically than if each project were to meet its needs separately. This is especially important given that a major function of this core will be to generate sheep that have been programmed prenatally by exposure to gonadal steroids at known stages of pregnancy. This requires extensive experience and expertise of managing fertility, breeding, and early postnatal care in this experimental species. The Specific Aims of the Sheep Core are as follows. Specific Aim 1. To operate a breeding program to generate sheep those are prenatally treated with gonadal steroids at specific fetal ages and vehicle-treated controls for use by the three research projects that make up this Program Project Grant. Specific Aim 2. To coordinate effective and economical usage of sheep by three interrelated research projects. Specific Aim 3. To provide a uniform and high standard of animal maintenance and veterinary care. Specific Aim 4. To provide a technical knowledge base for reproductive research in sheep and train personnel in proper handling of animals. Specific Aim 5. To determine the metabolic status of the experimental animals required for meeting the needs of all 3 projects. By achieving these aims, the Sheep Core will greatly enhance the productivity of all projects in the Program Project Grant.

The objective of the Administrative Core is to provide comprehensive administrative support to the Program Project grant "Prenatal Programming of Reproductive Health and Disease" consistent with NICHD POI Research" guidelines. University of Michigan policy, and applicable State and Federal regulations. The Administrative Core will: 1) foster and coordinate communication amongst members of the Program Project, between projects and other research programs. Centers and Institutes in the University;2) maintain communication with and writing reports to granting agency;3) aid in sustaining scientific momentum, maintaining the quality of research performed, and ensure timely publications of findings;4) ensure quality control;5) organize the Steering Committee meetings;6) plan and arrange the annual External Committee meeting;and 7) maintain minutes of the Steering and the External Committee meetings. The Administrative Core will manage the Program Project budget and consult with steering committee as needed to assure that Program Project and Core budgets are efficiently utilized and scientific progress is maintained.

DESCRIPTION (provided by applicant): Endocrine disrupting compounds (EDC) are hormonally active, synthetic or natural chemicals that interfere with normal functioning of the endocrine system, most notably the reproductive endocrine axis. Concern about EDC has mainly been fueled by studies that point to likely effects of EDC exposure on humans including dramatic increases in estrogen sensitive cancers, decline in human sperm quality and quantity, a notable rise in endometriosis, and early puberty in women. Because of the universal and crucial role estradiol plays in reproduction and other biologic processes, estrogenic pollutants in the environment are of particular concern. Given that sex steroids play a crucial role in organ differentiation during development, it is reasonable to expect in utero exposure to exogenous steroid mimics may alter the developmental trajectory of the fetus culminating in adult reproductive dysfunction. Our preliminary studies using sheep as a model revealed that prenatal exposure to the plasticizer bisphenol A (BPA), an environmentally relevant EDC, at levels approaching that found in human maternal blood and amniotic fluids, resulted in low birth weight female offspring, early postnatal hypergonadotropism, and cycle defects manifested as severe dampening of the LH surge. In this proposal, we will test the hypothesis that prenatal exposure to BPA, an environmental estrogen mimic, at levels similar to what human fetuses are exposed to, will disrupt adult reproductive function by disrupting the mechanisms controlling postnatal neuroendocrine feedback controls of GnRH/LH secretion and ovarian sensitivity to gonadotropins. Further, prenatal exposure to BPA will exacerbate postnatal reproductive susceptibility to steroid exposure and culminate in reproductive failure. Three Specific Aims will test these hypotheses. In Specific Aim 1, we will determine if unconjugated BPA in the maternal circulation reaches the fetus and at levels seen by the human fetus disrupt adult reproductive function. In Specific Aim 2, we will determine if prenatal BPA effect is mediated at the neuroendocrine or ovarian level and involves disruption of the mechanisms controlling postnatal neuroendocrine feedback controls of GnRH/LH secretion and/or ovarian sensitivity to gonadotropins. In Specific Aim 3, we will determine if prenatal exposure to BPA exacerbates reproductive susceptibility to postnatal estradiol exposure culminating in reproductive failure. The proposal targets key elements of strategic plans that emanated from workshops convened by the NICHD in 2000-01 by focusing on three targeted areas: fetal antecedents of disease, reproductive health for the 21st century, and developmental biology. The findings will be relevant to research on fetal origin of infertility and the threat estrogenic environmental disruptors at current exposure levels pose to human health.

DESCRIPTION (provided by applicant): The postdoctoral Pediatric Endocrinology Training Program (PETP) at the University of Michigan is designed to recruit and provide high quality research training in one of two major tracks, Basic Science or Clinical Investigation &Epidemiology to qualified candidates. The theme of the Program is developmental programming of endocrine disorders. The program is organized to complement the established training program in clinical pediatric endocrinology for M.D.s, and for basic scientist pursuing a career in endocrine research, by providing 2 years of intensive postdoctoral training using closely-mentored research training program individually structured for each trainee. The program is actively supported by 12 faculty from 9 Departments at 4 Schools (Medicine, LS&A, Public Health and Kinesiology), all of whom have extensive research and mentoring experience. The Program also includes an extensive didactic component tailored to each trainee's educational background and research interests. Trainees in the Basic Sciences complete a 3- month course designed to expose them to a variety of state-of-the-art techniques, as well as to skills in performing hypothesis-driven, controlled research studies. Trainees in the Clinical Investigation &Epidemiology choose between Outcomes &Epidemiology or Clinical Research. They complete courses leading to either a Master of Public Health in Biostatistics &Epidemiology or a Master of Science in Clinical Research Design &Statistical Analysis. In addition to the formal curriculum, trainees attend weekly clinical and research seminars. Mentored research is supported the Medical School resources including the Centers of Integrative Genomics, Proteomics, Organogenesis and,Diabetes and the Michigan Institute for Clinical and Health Research. To optimize each trainee's potential for development of a successful academic career, trainees are required to submit abstracts to national meetings, submit a first-authored research manuscript, and apply for individual grant funding. Expansion of our Program will help alleviate the critical shortage of successful Pediatric Endocrinologists and Pediatric Physician-Scientists. RELEVANCE (See instructions): Biomedical research faces great challenges nationwide. Academic centers need to provide an environment that allows an integrated vision of research, which in turn will deepen our understanding of biology, stimulate interdisciplinary research teams, and reshape clinical research to accelerate medical discovery and improve health. Research training is integral to the future biomedical advancement. The present proposal will train the medical doctors (MDs) and scientist (PhDs) to study the origins of hormonal disorders in children. The overarching goal of the training program is to improve children's health, prevent development and/or ameliorate severity of diseases thereby improving the health ofthe United States population.

The objectives ofthe Data Management and Modeling Core (DMMC) are to provide integrated biostatistics, bioinformatics, and database management support for all Projects ofthe U-M NIEHS/EPA Children's Environmental Health and Disease Prevention Center (CEHC). The DMMC will promote integration of knowledge and understanding of common statistical, bioinformatics, and database issues across the U-M CEHC, and accelerate the research goals ofthe projects by not only fully engaging in all aspects ofthe U-M CEHC, but also employing appropriate and advanced study design and data analysis methodologies that can enhance the types of research questions addressed by study data. The primary aims ofthe core include (i) providing statistical and bioinformatics support for all projects, including study design, conduct of statistical analyses, integration with biological information and interpretation of research findings, (ii) supporting data management, data quality controls, and integration of databases for conduct of the projects, and (iii) participating in dissemination of research findings, including writing of manuscripts and presentations, and data sharing and dissemination strategies. The DMMC personnel have a great wealth of knowledge and experience of analyzing longitudinal data and genetic/genomic data, including mixed-effects models, GEE models, joint modeling of longitudinal and survival data, robust mixed-effects models, modern variable selection techniques (e.g. group-lasso), novel methods of handling missing data and high-throughput data analysis and biomarker network analysis for interpretation of metabolomic data. All these are critical and beneficial to the study aims in all projects. To study mixtures of exposures, an innovative approach based on the multi-index regression model will be applied to identify important pollutants and pollutant mixtures that may be associated with somatic grov\/th, sexual maturation, and metabolomic outcomes.

DESCRIPTION (provided by applicant) The overall goal of the Administrative Core (AC) is to provide oversight, coordination and integration of Center activities. The specific aims are to: 1) Coordinate and integrate the scientific aims of the Projects and Cores, track and evaluate progress and outputs, and ensure successful completion of all Center aims; 2) Manage resources to assure the needs and priorities of every Project and Cores are met; 3) Act as coordinating center by convening meetings of Project, Core, and subcontract investigators, organize External Advisory Committee, ensure timely translation of research findings, prepare Center-wide reports, and interface with NIEHS and EPA project officers; 4) Evaluate the progress and success of the career, development plan, with specific focus on the career development of Faculty Development Investigator; 5) Ensure quality control of all stages of research namely study design, establishment of standard operating procedures and study protocols, secure sample and data transfer and management, data analysis, and accurate interpretation and translation of research findings to academic, government, healthcare and community stakeholders. The AC will operate under the direction of the Core co-Leaders, Consultant, and Pediatric Health Specialist in collaboration with Leaders of each Project and Core. The AC will ensure coordination of Center activities, manage resources across Projects and Cores, identify the most efficient use of infrastructure and communication and resolve needs as they arise, in order to increase the efficiency and productive output of the Center. AC Leaders will interact with the External Advisory Board to ensure the merit and value of all UM-CEHC elements to accomplish the overall Center aims. The Center Manager will prioritize coordination with the Community Outreach and Translation Core to optimize outreach and research translation to community stakeholders. Finally, the AC will oversee career development and training of New Investigators, particularly the UM-CEHC Co-investigator designated as the Faculty Development Investigator. Career development and training activities will draw upon extensive institutional resources and will be fully integrated with the Center's Research Projects and Cores.

Epidemiological and animal studies now firmly establish that environmental exposures during eariy embryonic development play a critical role in disease susceptibility in later-life. Moreover, such exposures during gestation have been directly linked with subsequent disease formation through epigenetic mechanisms. Little research, however, has considered the combined effects of perinatal and peripubertal , exposures on life course metabolic syndrome risk and reproductive development. Utilizing an established mouse model of perinatal exposures and focusing on bisphenol A (BPA), phthalates, and lead (Pb) as representative toxicants, we focus on the influence of perinatal and peripubertal exposures on offspring metabolic status and epigenetic gene regulation. Specifically, we will investigate whether perinatal exposure to BPA/phthalates/Pb mixture results in epigenetic alterations and disrupts life course physiologic status, events that are subject to modification by diet and continued peripubertal exposure. First, capitalizing on our mouse models of physiologically relevant maternal toxicant levels, we will assess whether pregnancy and postnatal high-fat diet modifies the effects of perinatal BPA or Pb exposure on life course physiological parameters. Second, we will use sophisticated mouse phenotyping to examine the relationship of perinatal and peripubertal exposure to chemical mixtures on metabolic and reproductive status and identify key epigenetic labile genes important for metabolic homeostasis and hormonal regulation. Finally, animal models are well-poised to elucidate complex relationships among exposures, epigenetic tissue specificity, and timedependent epigenetic drift. Thus, we will conduct unbiased lineage specific whole methylome, transcriptome, and histone mark analysis at multiple time points. Results of these comprehensive studies will elucidate issues of tissue specificity and drift with age, inherent complexities in conducting epigenetic epidemiological studies. Knowledge from this project is crucial for deciphering the role of epigenetic programming by early exposures in the pathogenesis of diseases, including metabolic syndrome and reproductive success, and for the development of novel epigenetic-based diagnostic and therapeutic strategies for human diseases.

The developmental origins hypothesis relates early exposures to endocrine disrupting chemicals (EDCs) to the development of chronic diseases, including metabolic syndrome, a condition affecting up to 25% of US adults and 30% of obese adolescents. Limited research in humans has considered the mechanisms by which exposures to EDCs mixtures interact with diet to alter maternal and child metabolic homeostasis, nor considered whether subsequent exposures during adolescence exacerbate risk of metabolic syndrome. Pilot human and animal data from our Formative CEHC: 'Perinatal exposures, epigenetics, child obesity and sexual maturation (P20 ESDI 8171/ RD834800, Pl: Peterson) suggest that the disruptive effects of representative maternal EDCs on metabolic and epigenetic markers may differ across sensitive periods of child development. Drawing on unparalleled institutional resources including the UM NIEHS Center of Excellence Epigenetics Laboratory (P30 ES017885) and the Michigan Nutrition and Obesity Research Center (MNORC, P30 DK089503), this project will test the hypothesis that EDC mixtures (BPA, phthalates, lead, cadmium) via epigenetic mechanisms induce oxidative stress (tyrosine oxidation products), disrupt metabolic homeostasis (free fatty acids, amino acids, Acyl-carnitine) and lead to changes in gene transcription and metabolic function. We further hypothesize that dietary macro- and micronutrient intake and dietary patterns during pregnancy and adolescence will modify the impact of EDC mixtures on these outcomes. Study participants include the Michigan Mother-Infant Pairs (MMIP) cohort (n=80) (extension of R01 ES017005, PI: Padmanabhan) and 400 children followed from pregnancy to 8-15 years of age through our 18-yr Early Life Exposures in Mexico to ENvironmental Toxicants (ELEMENT) collaboration with Mexico's Instituto Nacional de Salud Publica (INSP). Findings will: 1) provide proof of concept that EDC mixtures perturb metabolic homeostasis, 2) clarify the role of diet in amplifying or negating such effects, 3) illustrate the epigenetic and transcriptional changes involved and 4) inform the design of future interventions to modify metabolic consequences of EDC exposures both in utero and during the pubertal transition.

The goal ofthe Community Outreach and Translation Core (COTC) is to enhance the multiple, diverse stakeholders' understanding ofthe role that environmental exposures have on children's health and the potential points of prevention and intervention. To achieve this goal, the COTC aims to: (1) Develop and evaluate innovative, state-wide translation networks and activities connecting cutting-edge academic research findings to public health practice; (2) Increase the awareness, understanding, and translation of the most recent scientific findings in children's environmental health into practice among families, community members, and community public health practitioners; (3) Translate children's environmental health research for use by advocacy organizations and policy makers at the local, state, national, and international level in order to advance policies which protect children's health. This Core an integral part of the proposed overall Center, designed to offer input into the Center's activities and to benefit from cutting-edge research findings, integration ofthe COTC within the UM-CEHC will accelerate the typical timeline of research to translation and seeks to create dialogue from the outset on research questions that are inherently relevant to children's clinical and public health practitioners and policy makers. Directed by the University of Michigan Office of Public Health Practice, COTC leadership brings more than 25 years of experience in the translation of research into practice and extensive networks of diverse stakeholders frorn across the state of Michigan. In order to fully engage these stakeholders and provide materials and resources that are the greatest public health priorities; or the issues of greatest need as perceived by community members, a community-based participatory research approach will be taken to all COTC activities. A Community Advisory Board will be established to ensure and enhance the two-way interaction between the UM-CEHC and community stakeholders. The COTC will accomplish these aims with an efficient use of funds by leveraging extensive institutional resources for community engagement, such as the P30 NIEHS Core Center's Community Outreach and Education Core, and others.

DESCRIPTION (provided by applicant): Endocrine disrupting chemicals (EDC) are pollutants that disturb normal functioning of the endocrine system. Exposure to such pollutants during pre-natal development can have detrimental effects on the developing fetus because the system is highly plastic and responsive during critical periods. There has been much concern recently about the action of these pollutants on fetuses in utero. One such compound that is prevalent in the environment is bisphenol A (BPA). It is used to make polycarbonate bottles, including baby bottles and linings of cans, so exposure is widespread. Studies in rodents have implicated prenatal BPA exposure in a variety of health problems. We propose to use sheep as a model to examine the effects of prenatal BPA on complex behaviors. Similar work with prenatal testosterone exposure has demonstrated that sheep are an excellent model for such studies. Studies at our facility using sheep as a model for prenatal BPA exposure indicate that exposure to BPA leads to reproductive disruptions. However, no work has yet been done to examine the lifespan development of complex behaviors in a long-lived social species, such as ourselves. The goal of this study is to investigate the effects of prenatal BPA exposure and adolescent BPA exposure on complex behaviors in the sheep model across development. These behaviors will be correlated with physiological endpoints including steroid hormone levels, stress reactivity and body weight. Additionally, we will be examining steroid receptor number and distribution in areas of the brain associated with motivation and consummation of mating behavior. We hypothesize that prenatal exposure to BPA, at levels similar to the exposure of human fetuses, will disrupt a variety of complex behaviors including play, aggressive, motivational and sex behaviors over the lifespan of the animal. A second period of vulnerability is adolescence, so we will also examine the effects of BPA exposure during adolescence. Specific Aims 1 and 2 will examine complex behaviors throughout the lifespan of both male and female exposed to either prenatal, adolescent or prenatal and adolescent BPA compared to controls. Specific Aim 3 will examine the effect of BPA exposure during these vulnerable periods on steroid receptor number and distribution in areas of the brain associated with motivation and consummation of mating behavior. Results from this study are important for characterizing the behavioral and developmental effects of prenatal exposure to the endocrine disruptor BPA. This is an important issue in society, since many products that mothers and young children are exposed to contain BPA.

There is great concern regarding the potential developmental and reproductive effects resulting from environmental exposure to endocrine disrupting compounds (EDCs). Recent population trends showing shifts in the age of puberty onset and progression have contributed to this concern, as has evidence for interrelationships between EDC exposures, overweight and obesity, and eadier onset of puberty. Early and late puberty onset are associated with social and psychological challenges and may indicate risk of current or future endocrine-related disorders. EDCs, such as phthalates and BPA, have been hypothesized to be associated with earlier puberty, while animal and human studies suggest exposure to lead and cadmium may be associated with delayed growth and puberty onset via endocrine-mediated pathways. There are a lack of longitudinal studies that have considered exposure to these agents, individually and as mixtures, and at multiple sensitive stages of development, in relation to physical growth and sexual maturation. The proposed study will address these gaps by expanding on pilot work ofthe University of Michigan (UM) Formative P20 Children's Center to undertake a more robust (N = 400) prospective assessment ofthe relationship between exposure to a select mixture of EDCs (phthalates, BPA, lead, and cadmium) and the tempo of physical growth and timing of sexual maturation in a long-standing longitudinal cohort study in Mexico. Existing data on life stage lead exposure and anthropometry (height, weight, BMI) 6-month intervals between birth and 5 years of age will be utilized, along with newly collected data. In the proposed study, children in the ongoing cohort will be re-recruited for two clinic visits (18 months apart) between the ages of 8 and 15 where Tanner stages (along with testicular volume among boys), anthropometry, and skjn fold thickness will be assessed by a clinician, a blood sample will be collected for analysis of lead and reproductive and thyroid hormones, and a urine sample will be collected for analysis of phthalates, BPA and cadmium. These same exposure measures will additionally be analyzed from samples collected at all 3 trimesters of pregnancy when the children were in utero. Four years of repeated self-reported Tanner stage

The overall goal ofthe Administrative Core (AC) is to'provide oversight, coordination and integration of Center activities. The Specific Aims are to: (1) Coordinate and integrate the scientific aims ofthe Projects and Cores, track and evaluate progress and outputs, and ensure successful completion of all Center aims; (2) Manage resources to assure the needs and priorities of every Project and Cores are met; (3) Act as coordinating center by convening meetings of Project, Core, and subcontract investigators, organize External Advisory Committee, ensure timely translation of research findings, prepare Center-wide reports, and interface with NIEHS and EPA project officers; (4) Evaluate the progress and success ofthe career, development plan, with specific focus on the career development of Faculty Development Investigator; (5) Ensure quality control of all stages of research namely study design, establishment of standard operating procedures and study protocols, secure sample and data transfer and management, data analysis, and accurate interpretation and translation of research findings to academic, government, healthcare and community stakeholders. The AC will operate under the direction of the Core Co-Leaders, Consultant, and Pediatric Health Specialist in collaboration with Leaders of each Project and Core. The AC will ensure coordination of Center activities, manage resources across Projects and Cores, identify the most efficient use of infrastructure and communication and resolve needs as they arise, in order to increase the efficiency and productive output ofthe Center. AC Leaders will interact with the External Advisory Board to ensure the merit and value of all UM-CEHC elements to accomplish the overall Center aims. The Center Manager will prioritize coordination with the Community Outreach and Translation Core to optimize outreach and research translation to community stakeholders. Finally, the AC will oversee career development and training of New Investigators, particularly the UM-CEHC Co-lnvestigator designated as the Faculty Development Investigator. Career development and training activities will draw upon extensive institutional resources and will be fully integrated with the Center's Research Projects and Cores.

Endocrine-disrupting chemicals (EDCs) can have adverse impacts on children's physical growth and tempo of maturation and have been related to development of adult chronic conditions, e.g., obesity, type 2 diabetes mellitus and cardiovascular disease. Metabolic syndrome, a constellation of these outcomes, affects up to 25% of US adults and 30% of obese adolescents. Research on developmental origins has focused primarily on early life exposures, with less attention on the role exposures to EDC mixtures during the pubertal transition may play in exacerbating metabolic consequences leading to metabolic syndrome. Research on the effects of maternal prenatal diet on developmental origins of chronic disease has not been well integrated with environmental health research, despite growing evidence that nutrition is a modulator of susceptibility to environmental pollutants through effects on absorption, distribution, and/or toxicity. Hypothesized mechanisms for dietary modulation include down-regulation of signaling pathways involved in the inflammatory response, protection against oxidative stress, and restoration of metabolic homeostasis. Because nutrients may serve as agonists or antagonists of toxic effects of environmental chemicals depending on factors such as dose, more studies are necessary to accurately determine the role it plays in altering the effects of environmental toxicants and the future development of disease. This application aims to understand the mechanisms by which exposure to EDC mixtures (BPA, phthalates, lead, cadmium) interact with diet in utero and peripuberty to impact markers of metabolic homeostasis, oxidative stress and risk of metabolic syndrome.. Capitalizing on two existing human cohorts [Early Life Exposures in Mexico to ENvironmental Toxicants (ELEMENT) and the Michigan Mother-Infant Pairs (MMIP)] and the viable yellow agouti mouse model, the focus of this center is to dissect out the impact of EDC mixtures and its interaction with diet on metabolic health. Research findings will shed light on epigenetic and transcriptional changes leading to changes in growth, maturation and metabolic outcomes, and provide an invaluable base for designing future interventions to reduce the impact of pervasive EDCs on children's health. RELEVANCE (See instructions); Exposure to endocrine disrupting chemicals (EDCs) during different periods of child development can impact growth, tempo of maturation and risk of metabolic syndrome, but these effects may be amplified or dampened by dietary intake. Research conducted through this center will foster a better understanding of how chemical and diet interact and inform the design of future interventions to improve children's health.

? DESCRIPTION (provided by applicant): The postdoctoral Pediatric Endocrinology and Diabetes Training Program at the University of Michigan is designed to recruit and provide high quality research training in one of two major tracks, Basic Science or Clinical Investigation & Epidemiology to qualified candidates. Now on its 9th year, the theme of the Program is developmental programming of disorders of glucose metabolism. The program is organized to complement the established training program in clinical pediatric endocrinology for M.D.s, and for basic scientist pursuing a career in endocrine research, by providing 2 years of intensive postdoctoral training using closely-mentored research training program individually structured for each trainee. The program is actively supported by 11 faculty from 7 UM Departments, all with extensive research and mentoring experience within their respective areas of expertise. The Program also includes an extensive didactic component tailored to each trainee's educational background and research interests. Trainees in the Basic Sciences complete a 3-month course designed to expose them to a variety of state-of-the-art techniques, as well as to skills in performing hypothesis-driven, controlled research studies. Trainees in Clinical Investigation & Epidemiology choose between Outcomes & Epidemiology or Clinical Research. They complete courses leading to either a Master of Public Health in Biostatistics & Epidemiology or a Master of Science in Clinical Research Design & Statistical Analysis. In addition to the formal curriculum, trainees attend weekly clinical and research seminars; receive instruction preparing and delivering oral presentations; ethical conduct of research and can choose short certificate leading courses in public policy, or entrepreneurship. Mentored research is supported the Medical School resources including the Centers of Comprehensive Diabetes Research, Nutrition Obesity Research, Organogenesis, Human Growth and Development, Comprehensive Metabolomic Center, Molecular & Behavioral Neuroscience Institute and the Michigan Institute for Clinical and Health Research. To optimize each trainee's potential for development of a successful academic career, trainees are required to submit abstracts to national meetings, submit a first-authored research manuscript, and apply for individual grant funding. Expansion of our Program will help alleviate the critical shortage of successful Pediatric Endocrinologists and Pediatric Physician-Scientists.

? DESCRIPTION (provided by applicant): The postdoctoral Pediatric Endocrinology and Diabetes Training Program at the University of Michigan is designed to recruit and provide high quality research training in one of two major tracks, Basic Science or Clinical Investigation & Epidemiology to qualified candidates. Now on its 9th year, the theme of the Program is developmental programming of disorders of glucose metabolism. The program is organized to complement the established training program in clinical pediatric endocrinology for M.D.s, and for basic scientist pursuing a career in endocrine research, by providing 2 years of intensive postdoctoral training using closely-mentored research training program individually structured for each trainee. The program is actively supported by 11 faculty from 7 UM Departments, all with extensive research and mentoring experience within their respective areas of expertise. The Program also includes an extensive didactic component tailored to each trainee's educational background and research interests. Trainees in the Basic Sciences complete a 3-month course designed to expose them to a variety of state-of-the-art techniques, as well as to skills in performing hypothesis-driven, controlled research studies. Trainees in Clinical Investigation & Epidemiology choose between Outcomes & Epidemiology or Clinical Research. They complete courses leading to either a Master of Public Health in Biostatistics & Epidemiology or a Master of Science in Clinical Research Design & Statistical Analysis. In addition to the formal curriculum, trainees attend weekly clinical and research seminars; receive instruction preparing and delivering oral presentations; ethical conduct of research and can choose short certificate leading courses in public policy, or entrepreneurship. Mentored research is supported the Medical School resources including the Centers of Comprehensive Diabetes Research, Nutrition Obesity Research, Organogenesis, Human Growth and Development, Comprehensive Metabolomic Center, Molecular & Behavioral Neuroscience Institute and the Michigan Institute for Clinical and Health Research. To optimize each trainee's potential for development of a successful academic career, trainees are required to submit abstracts to national meetings, submit a first-authored research manuscript, and apply for individual grant funding. Expansion of our Program will help alleviate the critical shortage of successful Pediatric Endocrinologists and Pediatric Physician-Scientists.

PROJECT SUMMARY Inadvertent fetal exposure to excess steroids or steroid mimics poses risks to reproductive and metabolic health in humans. At risk is the offspring whose mother has elevated levels of endogenous or exogenous steroids during pregnancy for a variety of reasons, including disease states and exposure to environmental compounds with steroidogenic activity. Experimental manipulation of the fetal steroid environment provides a powerful tool not only to unravel the mechanisms underlying the development of reproductive dysfunction and infertility, but also to develop intervention strategies to improve reproduction and prevent transmission of undesirable traits to subsequent generations. Our studies using sheep as the animal model demonstrated that prenatal exposure to excess testosterone from days 30-90 of pregnancy leads to reproductive neuroendocrine, ovarian and metabolic perturbations in the female offspring that recapitulate those seen in women with polycystic ovary syndrome (PCOS). These perturbations include oligo-anovulation, multifollicular ovarian morphology, functional hyperandrogenism, and insulin resistance. Furthermore, excess postnatal weight gain exacerbated the severity of such dysfunctions in female sheep prenatally exposed to testosterone excess. With mounting evidence supporting that developmentally-programmed traits are transmitted across multiple generations, elucidating the mechanisms by which reproductive and metabolic dysfunctions are passed on to the next generation in the sheep model of PCOS may help develop intervention strategies to alleviate adverse multigenerational effects and improve the health of subsequent generations. Using the day 60 to 90 gestational testosterone exposure model that allows natural mating (female offspring are not virilized like the 30-90 day exposure model), this proposal tests the novel hypotheses that: 1) prenatal testosterone excess promotes epigenetic, molecular, and functional alterations at multiple levels of the reproductive and metabolic systems that will carry over to subsequent generations, thus contributing to the vertical transmission of disease traits; and 2) lifestyle modifications via dietary intervention will considerably mitigate expression of these adverse events and will protect the second-generation (F2) offspring from inheriting several reproductive and metabolic defects programmed by prenatal testosterone excess and aggravated by increased adiposity. The studies proposed in this application target the developmental origins of adult disease and focus on a large animal model of translational relevance that exhibits a developmental trajectory that parallels that of humans. Because gestational exposure to excess steroids due to maternal disease and/or environmental factors impairs fertility, the findings from these studies will provide crucial biological information for improving reproduction across generations and will be of relevance in meeting the scientific missions of NIH.

Cardiovascular disease (CVD) is one of the leading causes of death worldwide accounting for 32.2% of all deaths in the United States. Compelling epidemiological data in human has shown that adverse in utero environment leads to intrauterine growth restriction that has been associated with increased risk of CVD and left ventricular hypertrophy. Prenatal exposure to excess testosterone (T) has been found to adversely program multiple organ systems in the well-validated sheep model of developmental programming. Prenatal T excess induces IUGR in both sexes in this model and culminates in insulin resistance, increased left ventricular mass and hypertension in the female offspring in adulthood (the impact of prenatal T on cardiometabolic outcomes in the male offspring has not been studied). Considering the sexual dimorphism that exists in CVD mortality and morbidity it is critically important to gain an insight into the role sexual dimorphism plays in left ventricular hypertrophy to enable development of sex-specific prevention strategies. In this regard, the prenatal T model provides a valuable resource to determine to what extent excess sex steroid exposure via disease states (Polycystic ovary syndrome, Congenital adrenal hyperplasia, or environmental chemicals with steroidogenic potential during fetal development programs adverse cardiac tissue remodeling resulting in increased mortality from CVD in adulthood. Our preliminary data provide evidence that prenatal T excess leads to pathological left ventricular remodeling in adult female offspring thus allowing us to probe underlying mechanisms and sex-specific functional outcomes. The objective of this application is to identify the mechanism(s) by which in utero exposure to excess T programs adverse cardiac remodeling and susceptibility to cardiac disease during adult life and to discern sex differences that might exist in this programming. We hypothesize that prenatal T excess will lead to adverse left ventricular structural and functional changes over the lifespan and these changes will be driven by epigenetic modifications of pathways involved in maintaining left ventricular morphology and function leading to increased susceptibility to insult later in life. The proposed studies will provide insight into the 1) mechanisms by which excess T in-utero leads to adverse left ventricular remodeling, 2) long term cardiac functional and structural consequences of prenatal T excess and 3) sex differences in prenatal cardiac programming. Knowledge gained through these studies will help identify strategies targeted toward treatment for LVH and heart failure and of translational relevance.

Abstract Global challenges to human health include a well-documented decline in human fertility and a dramatic increase in the prevalence of obesity related metabolic disorders. There is now compelling evidence that these health challenges are associated with environmental chemicals that can influence endocrine activity. However, this premise has evolved from studies with acute single chemical exposures in altricial, genetically restricted, animal models. The proposed study will address the central hypothesis that exposure to a real-life mixture of environmental chemicals, prior to and throughout pregnancy, results in transgenerational epigenetic effects in offspring that affect metabolism and reproduction. The proposal will use a unique, real-life chemical exposure and a translationally relevant animal model whereby sheep are exposed to biosolids. Humans are exposed to a wide mixture of environmental chemicals, at typically low concentrations, throughout their lifespan. Grazing pregnant sheep on pastures treated with biosolids generated from human sewage provides a precocial model with a developmental trajectory similar to humans. This provides a novel approach to examine the effects of environmental chemical mixtures to which we are all exposed. The specific aims are: (1) to characterise lifetime changes in metabolic function of male and female offspring spanning three generations (i.e. F1, F2 and F3) following F0 exposure to biosolids treated pasture; (2) to assess changes in reproductive potential of male and female offspring across three generations (i.e. F1, F2 and F3) following F0 exposure to biosolids pasture; (3) to determine epigenetic changes, and their functional relationships, to transcriptional networks governing transgenerational effects of maternal biosolids exposure on offspring metabolism and fertility. This comprehensive analysis will provide proof-of-concept data on the effects of real-life chemical exposure in a relevant experimental paradigm. This will significantly enhance our ability to predict and inform policy development with the aim of ameliorating detrimental effects of environmental-chemical exposure and to improve metabolic and reproductive health.

Abstract Maternal obesity during pregnancy increases the risk of hypertension, gestational diabetes and abnormalities in fetal growth with higher infant birth weight and BMI in both early and late childhood. Extensive literature provides evidence for epigenetic programming of the developing fetus in response to the maternal metabolic milieu, but there is minimal direct evidence that changes in the fetal environment can alter the epigenome. The proposed study will test the hypothesis that significant weight loss prior to conception will improve the intrauterine metabolic environment as reflected in the maternal metabolome and inflammation-related proteome and result in changes in methylation patterns in cord blood leukocytes. To test this hypothesis, three Specific Aims are proposed. Aim 1, will examine the metabolic intrauterine environment in 300 obese women (BMI>30 kg/m2 ? 45 kg/m2) who will be randomized to either Very Low Energy Diet (VLED) targeting a >15% body weight loss or Standard of Care (SOC) interventions. Along with 120 lean women (LEAN) serving as comparators, VLED and SOC women will undergo extensive prepregnancy clinical and physiological phenotyping and blood collections. Obese women will undergo additional phenotyping after weight loss and all women will have additional testing at each trimester. We expect 87 offspring in each group. Fetal growth will be assessed by ultrasound and offspring birth weight (Ponderal index), adiposity (Pea Pod Air Displacement Plethysmography). In Aim 2, plasma metabolomics and (hybrid targeted/untargeted and lipidomics) and inflammatory markers will be used to assess intervention associated changes in VLED and SOC women and compared to VLED women. To assess the intrauterine environment, metabolomic profiles and inflammatory proteome will be measured in the first trimester and at term in all mothers and in fetal cord blood. Multivariate computational models will assess the association of maternal and neonate metabolome and inflammatory markers to fetal growth and newborn weight and adiposity. In Aim 3, DNA methylation patterns and RNA-seq will be obtained from fetal cord blood lymphocytes of all offspring. Differences in methylation patterns between VLED, SOC and LEAN will be assessed and changes in mRNA levels will be determined to assess the effect of methylation on gene expression. Multivariate analysis of methylation patterns will be related to the metabolome and to fetal growth and birth outcomes. Using sparse multivariate factor analysis regression model (smFARM) and other statistical approaches, we will determine how the maternal metabolome and proteome is associated with cord blood DNA methylation and investigate whether fetal growth or birth weight and other outcomes are mediated by specific metabolites. The results of these studies will provide the first prospective assessment of the benefit of preconception weight loss on the intrauterine environment and molecular changes in the newborn and will provide a potential pathway from maternal intrauterine environment and programmed changes in weight and metabolic status in offspring.