Current Specific Research:
1) Role of environmentally induced epigenetic transgenerational inheritance in disease etiology and evolutionary biology.
2) Actions of endocrine disruptors and environmental compounds on embryonic gonadal development that cause the epigenetic transgenerational inheritance of adult onset disease.
3) Influence of environmental epigenetics on testis development and Sertoli cell differentiation that promotes adult onset testis disease.
4) Influence of environmental epigenetics on ovarian primordial follicle assembly and development that promotes the transgenerational inheritance of ovarian disease.
NIH - Epigenetic Transgenerational Actions of DDT (abstract below)
NIH - Primordial Follicle Assembly and Disease (abstract below)
NIH - Renewal - Epigenetic Transgenerational Endocrine Disruptor Actions (abstract below)
NIEHS - Epigenetic Transgenerational Actions of Endocrine Disruptors (abstract below)
DOD - Epigenetic Transgenerational Origins of Disease (abstract below)
Epigenetic Transgenerational Actions of DDT
The proposed research is designed to investigate the ability of the environmental toxicant and insecticide DDT to promote the epigenetic transgenerational inheritance of disease (e.g. male infertility). Exposure of F0 generation gestating females to DDT during the period of fetal gonadal sex determination has been shown to promote the transgenerational inheritance of disease such as spermatogenic cell defects and testis disease in the F1 and F3 generation adults. To investigate this phenomena, the molecular events induced by DDT during fetal gonadal sex determination and those associated with the germline epigenetic transmission of disease to adult somatic cells (e.g. Sertoli cells) will be elucidated in the F1 and F3 generations. The hypothesis tested is that exposure (gestating F0 generation female) to the environmental toxicant DDT during fetal (F1 generation) gonadal sex determination alters the cascade of epigenetic and gene expression events in the primordial germ cells (PGC) to cause a permanent epigenetic reprogramming of the germline to promote the epigenetic transgenerational inheritance of disease (e.g. male infertility and obesity). The objective is to elucidate the optimal exposure period, exposure dose and molecular events associated with the direct exposure F1 generation pathology and transgenerational F3 generation disease (e.g. male infertility and obesity). The proposed research involves the following Specific Aims: 1) Investigate the DDT induced transgenerational inheritance of disease: 2) Investigate the germline epigenetic transmission of the DDT induced transgenerational phenotypes. 3) Investigate the transgenerational transmission of germline epimutation information to adult somatic cells (i.e. Sertoli cell and adipocyte) associated with the transgenerational inheritance of disease (i.e. testis disease and obesity). The objectives of the proposed research are to elucidate: 1) optimal exposure and dose parameters: 2) germline epimutation transgenerational transmission; and 3) translation of germline epimutation information to somatic cells associated with transgenerational disease. The testis disease and obesity were selected as model organs and diseases to elucidate how the germline epimutation information is translated to an adult onset disease. Completion of the proposed research will provide insights into the molecular events of environmentally induced epigenetic transgenerational inheritance of disease. The potential that ancestral DDT exposure may have a significant role in the etiology of male infertility and obesity will be investigated.
PRIMORDIAL FOLLICLE ASSEMBLY AND DISEASE
The ovarian primordial follicle pool is one of the most critical elements that directly influences the endocrinology and fertility of the mammalian female. The development of the primordial follicle pool, in the fetus for the human or after birth for the rodent, establishes the reproductive potential of the female to ovulate and generate endocrine hormones such as estrogen and progesterone. The molecular factors that regulate primordial follicle assembly and pool size are starting to be elucidated. Previous research identified a number of growth factors and hormones that control follicle assembly and correlate them to alterations in the ovarian transcriptome. The current proposal extends this molecular information to investigate primordial follicle assembly on a genomic or systems biology level and correlates this with the induction of primordial follicle disease. The hypothesis tested is that a complex and distinct bionetwork of genes exists to regulate primordial follicle assembly and alterations in these gene networks will correlate with the induction of ovarian disease (e.g. primary ovarian insufficiency), and these critical regulatory targets can be used to manipulate the primordial follicle pool size. Previously we have established a rat ovarian culture system to study primordial follicle assembly and assess alterations in the ovarian transcriptome. A gene bionetwork for primordial follicle assembly has recently been identified. This culture system and systems biology approach will be used to further elucidate gene networks and identify targets to manipulate primordial follicle assembly. The following Specific Aims are proposed. 1) Elucidate and validate the molecular control and gene bionetworks involved in primordial follicle assembly. 2) Promote abnormal primordial follicle assembly and pool size to identify alterations in the gene bionetworks involved in primordial follicle disease. 3) Manipulate primordial follicle assembly and pool size. Completion of the proposed research will elucidate the molecular control of primordial follicle assembly, as well as identify and test in vitro pharmacologic targets to manipulate primordial follicle assembly and pool-size. Although follicle assembly will not be a viable in vivo therapy target, investigation of assembly manipulation in vitro is anticipated to provide insights into in vivo therapeutic targets for later stages of follicle development. Observations will identify alterations in gene bionetworks associated with abnormal primordial follicle assembly and disease such as primary ovarian insufficiency (POI).
EPIGENETIC TRANSGENERATIONAL ENDOCRINE DISRUPTOR ACTIONS
The current grant “Epigenetic Transgenerational Endocrine Disruptor Actions” was funded starting 2009. The Specific Aims for this funded grant are listed below. The original specific aims are being pursued to investigate how an endocrine disruptor exposure during embryonic development can promote an epigenetic transgenerational inheritance of disease (1). The transgenerational disease observed include testis and ovary disease, cancer, prostate disease and kidney disease (2). This supplemental application requests funding to help support investigation into the molecular aspects of this phenomenon not previously proposed in the current grant. When the grant was originally proposed and funded, the primary technology to do epigenetic analysis was methylated DNA immunoprecipitation (MeDIP) followed by tiling microarrays (MeDIP-Chip). The genome-wide analysis was approximately $10,000 per assay so was not possible within the budget obtained. Therefore, the grant focused on a promoter MeDIP-Chip analysis. Over the past couple years the advancement of next generation sequencing has allowed MeDIP-Seq to be used. However, a major limitation was a bias to high density CpG sites. Recently we have optimized this technology (MeDIP-Seq), which is less expensive now than MeDIP-Chip. The advancement of this new technology allows several experiments not previously proposed to now be performed in the final year of the current grant that were not previously possible. There are new experiments not proposed in the original grant that are now possible due to the advancement of this new technology. These experiments are outlined below on samples previously collected during the grant period. The supplemental funding will allow the current grant to be expanded and a more thorough molecular analysis of the environmentally induced epigenetic transgenerational inheritance of disease.
EPIGENETIC TRANSGENERATIONAL ENDOCRINE DISRUPTOR ACTIONS
Transgenerational effects of environmental factors, such as pesticides, plastics and fungicides, significantly amplify the impact and health hazards of these compounds. The transgenerational actions of these compounds requires a heritable epigenetic alteration of the germline. This transgenerational epigenetic phenomenon is anticipated to be an important aspect of adult onset disease etiology, and suggests ancestral environmental exposure may influence disease epidemiology. The current proposal is designed to investigate this transgenerational phenomenon and the underlying epigenetic mechanism(s) involved. The model endocrine disruptor utilized will be the fungicide vinclozolin, an anti-androgenic compound, studied in an outbred rodent (i.e. rat) system. Previously, we demonstrated that vinclozolin exposure during embryonic gonadal sex determination promotes an epigenetic reprogramming of the male germline that then induces transgenerational adult onset disease states of male infertility, prostate disease, kidney disease, immune abnormalities and tumor development. The objective of the current proposal is to provide further insights into the molecular, cellular and physiological (i.e. systems biology) actions of this endocrine disruptor on the induction of this transgenerational epigenetic phenomenon. THE OVERALL HYPOTHESIS TO BE TESTED IS THAT TRANSIENT IN UTERO EXPOSURE TO THE ENDOCRINE DISRUPTOR VINCLOZOLIN PROMOTES A PERMANENT REPROGRAMMING OF THE EPIGENOME (I.E. DNA METHYLATION) OF THE MALE GERM LINE THAT THEN, THROUGH ALTERATIONS IN CRITICAL EPIGENETIC REGULATORY MECHANISM(S), TRANSGENERATIONALLY PROMOTES ADULT ONSET DISEASE (E.G. MALE INFERTILITY). Previous studies have shown a transgenerational epigenetic effect on the male germ line (sperm) through alterations in DNA methylation. This epigenetic alteration in the germ line is proposed to subsequently promote transgenerational effects on the epigenomes and transcriptomes of numerous organ systems in the adult. The current proposal is designed to further investigate these transgenerational epigenetic effects on the male germ line to determine the functional relationship with the induction of specific adult onset disease states, and to reveal the underlying epigenetic mechanisms responsible for this phenomenon. The experimental approach to test the above hypothesis consists of the following specific aims: 1) Investigate the transgenerational actions of vinclozolin on the sperm epigenome (DNA methylation) to identify genome-wide epigenetic biomarkers. 2) Characterize the direct and transgenerational effects of vinclozolin exposure on the fetal male germ cell epigenome and transcriptome. 3) Correlate the sperm epigenetic biomarkers with transgenerational adult onset disease phenotypes, and investigate the potential role of the biomarkers in the dysregulation of adult somatic tissue transcriptomes associated with specific disease states. Completion of the proposed research will determine how environmental exposures and compounds may promote adult onset disease in a transgenerational manner. The epigenetic biomarkers associated with the epigenetic reprogramming of the male germ line will be identified. The potential role these biomarkers may have in newly created epigenetic control regions (ECR) to promote transgenerational dysregulation of tissue transcriptomes will be established and provide insight into the etiology of adult onset disease. The potential for epigenetic biomarkers to be used as early stage diagnostic markers for specific adult onset disease states will also be established. The proposed research will more thoroughly investigate this epigenetic transgenerational phenomenon and elucidate the molecular mechanisms involved.
EPIGENETIC ORIGINS OF DISEASE AND IMPACT OF ENVIRONMENTAL TOXICANTS IN IRAQ THEATER OF OPERATIONS
Transgenerational effects of environmental factors (i.e. toxicants), such as pesticides, plastics and environmental compounds, significantly amplify the impact and health hazards of these toxicants. The transgenerational nature of the actions of these compounds requires a permanent epigenetic alteration of the germ-line. The current proposal is designed to investigate the actions of several different mixtures of environmental compounds relevant to the military (e.g. pesticide, herbicide, explosive residue mixtures). A comparison to the actions of the fungicide vinclozolin (i.e. antiandrogen endocrine disruptor used in the fruit industry) will be made as a positive control. A rodent model (i.e. rat) system is used to assess the potential actions of the compounds of interest. Previously, we have demonstrated that the endocrine disruptor vinclozolin exposure during embryonic gonadal sex determination promotes an epigenetic reprogramming of the male germ-line that then induces a transgenerational adult onset disease state of male infertility, prostate disease, kidney disease, immune abnormalities and tumor development. The objective of the proposed research is to determine the actions of several mixtures of environmental compounds relevant to the military on the potential to promote transgenerational disease states and alterations in the epigenome. THE HYPOTHESIS TESTED IS THAT TRANSIENT EXPOSURE AT CRITICAL TIMES DURING DEVELOPMENT TO SPECIFIC MIXTURES OF ENVIRONMENTAL COMPOUNDS WILL PROMOTE AN ALTERATION IN THE EPIGENOME THAT SUBSEQUENTLY PROMOTES ADULT ONSET DISEASE, AND IF THE MALE GERM-LINE IS EPIGENTICALLY MODIFIED THAT TRANSGENERATIONAL DISEASE PHENOTYPES WILL BE INDUCED. Several generations of progeny from exposed F0 gestating females will be collected (F1-F3) to assess effects on adult onset disease, while the F3 generation will be the focus for transgenerational modification in the epigenome. A correlation with epigenetic modification with adult onset disease will be made as proof of concept for the epigenetic transgenerational phenotype and development of epigenetic biomarkers for disease. Once this is established in the rodent model future studies will consider analysis of epigenetic biomarkers in relevant human cohorts. The experimental approach to test the above hypothesis consists of the following specific aims: 1) Investigate the transgenerational effects of environmental compounds on adult onset disease; 2) Investigate the transgenerational effects of environmental compounds on the epigenome; 3) Correlation of the transgenerational disease phenotypes with the epigenome modifications to potentially identify epigenetic biomarkers of exposure and disease. Completion of the proposed research will determine how several mixtures of environmental compounds relevant to the military influence transgenerational adult onset disease in the F1, F2 and F3 generations of exposed gestating females and pubertally exposed animals. The transgenerational effects on the epigenome will also be determined and correlated to identify potential epigenetic biomarkers for disease and exposure. Insights into the role of epigenetics in environment-genome interactions will be provided. The role of epigenetics in adult onset disease etiology and actions of environmental compounds will be elucidated. The biohazard and potential health problems associated with the different environmental compound mixtures will be determined. This information can then be used by the military to assess if problems with exposure exist and the potential epigenetic biomarkers can be used to monitor exposure.