Current Specific Research:
1) Peritubular (Myoid) cell-Sertoli cell interactions in the testis via locally produced regulatory proteins and the hormonal and developmental control of these interactions. Analysis from the onset of male and female embryonic sex determination through the adult stage of development.
2) The control of gonadal development and somatic cell differentiation through an examination of cell specific gene/promoter expression (e.g. basic helix-loop-helix transcription factors).
3) Theca cell-granulosa cell interactions in the ovary via locally produced regulatory proteins and the hormonal and developmental control of these interactions. Analysis from the onset of primordial follicle assembly and development, through the Graffian follicle stage of development.
4) Investigation of the epigenetic transgenerational actions of endocrine disruptors on testis development and male and female fertility.
Grant Abstracts
NIH - Testis Development and Male Fertility (click to abstract below)
NIH - Testis Cell Differentiation (click to abstract below)
NIH - Ovary Primordial (click to abstract below)
NIEHS - Epigenetic Transgenerational Actions of Endocrine Disruptors (click to abstract below)
DOD - Epigenetic Transgenerational Origins of Disease (click to abstract below)
NIH - Testis Development and Male Fertility
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CELL-CELL INTERACTIONS AND TESTIS DEVELOPMENT
The proposed research is d esigned to develop a better understanding of the molecular and cellular mechanisms that control testis development. The integration of critical transcriptional events and cell-cell interactions that regulate testis morphogenesis and growth, which directly influences male fertility and sperm production, are of particular interest. Research during the previous grant period demonstrated critical transcription factors appear to regulate the expression of paracrine growth factors to directly influence testis development and male sex determination. THE GENERAL HYPOTHESIS EXAMINED IS THAT CRITICAL TRANSCRIPTIONAL EVENTS DURING GONADAL SEX DETERMINATION REGULATE LOCALLY PRODUCED PARACRINE GROWTH FACTORS THAT ARE ESSENTIAL FOR CELL GROWTH AND DIFFERENTIATION DURING EMBRYONIC AND POSTNATAL TESTIS DEVELOPMENT, AND THAT THIS DIRECTLY INFLUENCES MALE FERTILITY AND SPERM PRODUCTION IN THE ADULT. Abnormal testis development and male infertility may in part be due to inappropriate control of testicular cell differentiation and cell-cell interactions during embryonic development. THE SPECIFIC HYPOTHESIS TESTED IS THAT SUBSEQUENT TO GENETIC MALE SEX DETERMINATION (e.g. SRY) CRITICAL TRANSCRIPTION FACTORS (i.e. SOX9 AND THE BHLH FACTOR TCF21) ARE EXPRESSED IN PRECURSOR SERTOLI CELLS TO PROMOTE THE PRODUCTION OF CRITICAL PARACRINE GROWTH FACTORS (e.g. NT3 AND JAG1) THAT INDUCE TESTIS MORPHOGENESIS (i.e. CORD FORMATION) AND CELL-CELL INTERACTIONS REQUIRED FOR EMBRYONIC TESTIS DEVELOPMENT AND ADULT MALE FERTILITY. Research during the previous grant period indicates that SRY and SOX9 can transcriptionally regulate the Sertoli cell expression of the neurotrophin NT3 which has a critical role in the morphogenesis of the testis (i.e. cord formation). Preliminary studies also indicate that the basic helix-loop-helix (bHLH) family of transcription factors (e.g. TCF21) appear to be important for gonadal development and precursor Sertoli cell differentiation. Further analysis of how these transcriptional events integrate with the growth factor mediated cell-cell interactions to control testis development is the focus of the current proposal and involves the following specific aims: 1. Investigate the role of growth factors on testis morphogenesis and development. 2. Investigate the interactions between SRY/SOX9 and bHLH factors (i.e. TCF21) in the transcriptional regulation of growth factor expression. 3. Investigate the physiological functions of these growth factors during testis development on cell fate and differentiation, as well as testis spermatogenic capacity. The completion of these studies will provide insight into the cell-cell interactions and factors that control embryonic testis growth and function. Abnormal control of these factors and cell-cell interactions during embryonic development are anticipated to result in adult male infertility. Therefore, observations from the current proposal will lead to the design of future preventative or therapeutic treatments of male infertility.
NIH – Testis Cell Differentiation
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SERTOLI CELL DIFFERENTIATION AND TESTIS DEVELOPMENT
The proposed research is designed to investigate the integration of the bHLH and cAMP/CREB transcriptional pathways in the hormone (i.e. FSH) induced control of Sertoli cell differentiation. Emphasis is placed on the transcriptional control of Sertoli cell differentiation during pubertal development. Sertoli cells are the epithelial cells responsible for the onset of embryonic testis development and the maintenance of spermatogenesis in the adult. Previous research has demonstrated that a unique class of transcription factors, basic helix-loop-helix (bHLH), is involved in Sertoli cell differentiation and important for testis development. Preliminary studies have identified a mixture of bHLH factors including scleraxis (Scl), Id regulatory transcription factor (ITF) and inhibitor of differentiation (Id) that regulate Sertoli cell differentiated functions. Interestingly, we have made the recent novel observation that the bHLH proteins (i.e. Scl) interact directly with the cAMP Response Element Binding Protein (CREB) to integrate the HLH transcriptional pathway with the hormone responsive cAMP signal transduction pathway on a transcriptional level. THE HYPOTHESIS TESTED IS THAT bHLH PROTEINS (i.e. Scl) HETERODIMERIZE WITH CREB FACTORS (i.e. ATF4/CREB2) AND THAT A UNIQUE NETWORK OF POSITIVE (i.e. Scl, ITF) AND NEGATIVE (i.e. Id, ATF4) INTERACTING PROTEINS PROMOTES PUBERTAL SERTOLI CELL DIFFERENTIATION AND HORMONE (i.e. FSH) RESPONSIVENESS, AND THAT ABNORMALITIES IN THE INTEGRATION OF THESE TRANSCRIPTIONAL PATHWAYS RESULTS IN MALE INFERTILITY. The proposed research is involved in characterizing the integration of the bHLH and cAMP/CREB transcription pathways required for pubertal Sertoli cell differentiation. Hormones (e.g. FSH) and locally produced paracrine factors promote Sertoli cell differentiation at various stages of testis development. Abnormal testis development and male infertility is anticipated to in part be due to inappropriate transcriptional control of Sertoli cell differentiation involving bHLH and CREB proteins. The experimental approach consists of the following aims: 1) Investigate the role of bHLH and CREB protein interactions in the transcriptional regulation of Sertoli cell differentiation. 2) Investigate interactions between a network of various bHLH and CREB proteins in the control of Sertoli cell differentiation. 3) Investigate the physiological role of specific Sertoli cell bHLH and CREB protein interactions during testis development. The completion of these studies will provide insight into the transcriptional regulation of Sertoli cell differentiation during pubertal testis development. Sertoli cell differentiation is essential for embryonic, pre-pubertal, pubertal and adult testis development. Abnormal Sertoli cell differentiation can cause a number of birth defects and male infertility. Inappropriate expression of Sertoli cell genes results in defects such as sex reversal, ambiguous gonads, and vanishing testis. These conditions will in part be due to abnormal transcriptional regulation of Sertoli cell differentiation. Sertoli cell differentiation is also critical for the maintenance of adult testis function (i.e. spermatogenesis). Abnormal Sertoli cell differentiation and the inappropriate expression of Sertoli cell genes directly effects germ cell development and male fertility. Therefore, observations from the current proposal on transcriptional regulation of Sertoli cell differentiation will provide a better understanding of normal and abnormal testis development and function. The proposed research utilizes a functional genomics approach to investigate the molecular and physiological aspects of Sertoli cell biology and testis development. In addition to insights into FSH actions and Sertoli cell differentiation, the proposed research elucidates the integration of two major transcriptional control pathways. The ability of bHLH and CREB proteins to form heterodimers and regulate each other's signaling adds a level of control and complexity not previously appreciated. It is anticipated this signal integration will allow the cellular differentiation control by bHLH to be integrated more efficiently with the general regulatory control of the cAMP signal transduction pathway. The molecular mechanisms identified in the proposed research in the control of Sertoli cell differentiation will likely be common to many cell types responsive to hormone induced cAMP pathways.
Ovary Primordial
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OVARIAN PRIMORDIAL FOLLICLE DEVELOPMENT
The proposed research is directed toward developing a better understanding of the mechanisms that control primordial follicle development. This process is essential for the establishment and maintenance of female reproduction. Primordial follicle development involves the initial event of primordial follicle assembly and the subsequent event of primordial to primary follicle transition. The process involved in primordial follicle assembly is distinct from that of primordial to primary follicle transition and both will be investigated in the proposed research. The manner by which locally produced ovarian substances and hormones regulate these processes are of particular interest. Emphasis is placed on the elucidation of cell-cell interactions between precursor theca cells, granulosa cells and the oocyte. Previous research and preliminary studies have demonstrated that the control of primordial follicle development and subsequent folliculogenesis appears to be mediated by the local production and action of specific paracrine factors involving theca cells, granulosa cells, and the oocyte. Preliminary studies have shown that steroid hormones (i.e. estrogen and progesterone) play a critical role in the onset of primordial follicle assembly. Preliminary studies also demonstrate that primordial to primary follicle transition requires integrated interactions between the oocyte, granulosa cells and precursor theca cells involving kit ligand (KL), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), keratinocyte growth factor (KGF) and bone morphogenic protein-4 (BMP4). "THE GENERAL HYPOTHESIS TESTED IS THAT HORMONES INFLUENCE THE PARACRINE GROWTH FACTOR MEDIATED INTERACTIONS BETWEEN THE OVARIAN SOMATIC CELLS AND THE OOCYTE TO CONTROL PRIMORDIAL FOLLICLE ASSEMBLY AND DEVELOPMENT". More defined sub-hypotheses are presented for each specific aim below. Interactions between hormones and paracrine growth factors provide an efficient mechanism to control primordial follicle development. It is anticipated that abnormalities in these cellular interactions will influence female fertility, menopause onset and promote pathophysiologies such as premature ovarian failure. The experimental approach consists of the following specific aims: 1) Elucidate the hormonal and paracrine growth factor control of primordial follicle assembly. 2) Elucidate the hormonal and growth factor control of the primordial to primary follicle transition.. 3) Elucidate the physiological importance of specific factors that mediate primordial follicle development. Completion of these specific aims will extend previous observations and provide insight into the role of growth factor mediated cell-cell interactions in regulating primordial follicle development. The critical developmental periods to be examined include the induction of primordial follicle assembly and the primordial to primary follicle transition. It is anticipated that the specific cell-cell interactions will change between these developmental periods. The differential effects of hormones on growth factors and relevant receptors have a critical role in later stages of follicle development, but remain to be determined in primordial follicle development. Observations are anticipated to provide insight into the molecular and cellular control of primordial follicle development. The information obtained will be used to understand the onset of puberty and menopause, as well as provide insight into the potential future design of diagnostic procedures and therapeutic treatments for abnormal ovarian conditions such as premature ovarian failure (POF) and forms of female infertility. An example of a future experiment proposed is to determine if mutation(s) exist in any of the repertoire of growth factors and/or receptors established in the current study as potential causal factors for disease states such as premature ovarian failure.
Testis Toxicology
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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.
DOD
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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.