John H. Richburg - US grants

In this research project, activation of an apoptotic signal transduction process in germ cell is proposed as a ~final common pathway~ of germ cell death in the testis after toxicant-induced testicular Sertoli cell injury. To test this idea, the Sertoli cell toxicant, mono- (2-ethylhexyl) phthalate (MEHP), will be used to stimulate germ cell apoptosis in young (28 day old) rat testis. Apoptosis of germ cells in the testis occurs in rats routinely as a physiologic mechanism to limit the clonal expansion of germ cells in the testis. In preliminary experiments, exposure of young rats to MEHP resulted in an unexpected initial (3 h) inhibition of germ cell apoptosis followed later by a stimulation of germ cell apoptosis (12 h). These findings indicate that MEHP exposure alters the regulation of germ cell apoptosis in the testis. In addition, the expression of three components of the Fas-mediated pathway in testis: Fas receptor (Fas), Fas ligand (FasL) and FAP-1, was induced after MEHP exposure. The Fas-mediated pathway is a well characterized apoptotic signal transduction system where Fas acts as a receptor protein to trigger an intrinsic suicide program in the cell. These fundamental observations have led to the development of the following working hypothesis: MEHP disrupts Sertoli cell-germ cell interactions resulting in a stimulation of the Fas-mediated signal transduction process between these cells altering the physiologic mechanism of Sertoli cell-directed germ cell apoptosis. This hypothesis will be tested by specific aims designed 1) to characterize MEHP-induced alterations in the expression of components of the Fas-mediated pathway in testis and determine the cell-specific localization of the Fas-associated protein components and, 2) to delineate the role of Fas-mediated apoptosis in MEHP- induced germ cell loss by inhibiting the expression of FasL both in vitro and in vivo. Recent evidence suggests that environmental exposures to toxicants play a role in male infertility. However, despite the association of exposure to environmental agents and infertility, little is known of the mechanisms by which these agents cause decreased numbers of sperm. Phthalates, the model toxicants used in this project, are found widespread in the environment due to their use as plasticizers in food packaging and biomedical devices. The investigation of phthalate-induced testicular injury in the rat will provide clues into the mechanisms of both physiologic and toxicant-induced germ cell death in the testis and give insight into mechanisms of infertility resulting from environmental toxicant exposures.

DESCRIPTION (provided by applicant) The purpose of this training grant is to provide short-term research training in environmental toxicology carcinogenesis for minority undergraduate students. By providing these minority students with an intensive, highly mentored research experience the investigators hope to encourage them to pursue careers in biomedical science. The program is conducted at two geographically close sites within the University of Texas (UT) system, which have multiple, established training and research collaborations. These interactions are fostered by the joint NIEHS-sponsored Center for Research on Environmental Disease and a shared T32 training grant that supports graduate students on both campuses. The state of Texas has a Hispanic population that is more than 30% of the total population and an African American population that is approximately 12% of the total population. The UT system has a strong historical tradition of enrolling minorities in their undergraduate professional program; and one of the training sites, the College of Pharmacy, has the highest percentage (13 to 20% over the last 5 years) of undergraduate Hispanics enrolled at UT Austin. Unfortunately, very few Hispanics or African American students continue for an advanced degree in science, medicine or allied health. The investigators' program recruits from a rich source of highly trained minority students at UT and other colleges and universities, and then assists students in continuing into post-graduate training in biomedical sciences, including toxicology and carcinogenesis. The students are selected for the program on the basis of grades, letters of recommendations, and their motivation to consider a career in the environmental health sciences. The students spend approximately 12 weeks working in a laboratory of their choosing; the faculty mentor and student will work closely on a research project in which the student will learn experimental design, research methodology and interpretation of experimental data. The organized program includes a variety of educational activities such as weekly research seminars presented by post-doctoral fellows, field trips to academic institutions and a biotech company, and a final mini-symposium at which students present their research results. The investigators believe their training program offers the students an excellent opportunity to learn first-hand the experiences and excitement of cutting edge research in toxicology and carcinogenesis and to encourage them to consider science careers. Faculty have an excellent history of collaboration and sharing of research resources, such as advanced instrumentation, that foster multi- disciplinary research. Thus, (i) the maturity of their graduate program, (ii) the institutional commitment to the educational mission of the toxicology program via the establishment of the Center for Molecular and Cellular Toxicology, and (iii) in combination with the recent renewal of their NIEHS supported Center grant, all combine to provide an environment ideal for an increase in the number of NIEHS supported minority undergraduate training positions.

DESCRIPTION (provided by applicant) The investigators are requesting the continuation of a training grant in support of a predoctoral program promoting excellence in the toxicological sciences, focusing on molecular mechanisms of environmental chemical induced toxicity and disease. The Program is located at two geographically close sites within the University of Texas (UT) system, and which have multiple, well established training and research collaborations. These interactions are fostered by a joint NIEHS sponsored Center for Research on Environmental Disease (CRED) and a shared T35 training grant that supports summer research training for undergraduate minority students on both campuses. An important aspect of this program is the graduation of trainees who are actively recruited by colleagues from around the nation. This training program is particularly effective at attracting underrepresented groups into the discipline of Toxicology (women and minorities). Although each trainee selects from a wide variety of ongoing research programs, and works primarily in the laboratory of one of the training faculty, the training program promotes and supports collaborative research. The research programs of the training faculty include: 1) mechanisms of experimental and human carcinogenesis (Conti); 2) mechanisms and regulation of protein kinases (Dalby); 3) molecular mechanisms of multi-stage skin carcinogenesis (DiGiovanni); 4) role of inflammation in chemical carcinogenesis (Fischer); 5) hormonal carcinogenesis (Fuchs-Young); 6) E2F transcription factors (Johnson); 7) free radical biochemistry and molecular mechanisms of apoptosis (Kehrer); 8) role of vitamin E in signaling pathways leading to cell death (Kline and Sanders); 9) metabolism, chemical-induced nephro-carcinogenicity, prostaglandin mediated cytoprotection and proteomics (Lau); 10) molecular interactions of carcinogens with chromatin (MacLeod); 11) molecular mechanisms of oncotic and apoptotic cell death in response to reactive oxygen species (ROS)-induced DNA damage, ROS-induced changes in chromatin structure and function (Monks); 12) male reproductive toxicology and germ cellapoptosis (Richburg); 13) mechanisms of apoptosis in epithelial tumorigenesis (Tang); 14) mechanisms of DNA damage recognition by DNA repair and recombination pathways (Vasquez); and, 15) role of genetic alterations in tumor development (Walker). Predoctoral trainees are evaluated for admission into the training program on the basis of GPA, GRE, letters of recommendation previous research experience, and interviews. Progress is monitored throughout the year by participation, each semester, in seminars, course work, research progress in the laboratory, and an annual report prepared by the trainees describing their progress. Faculty have an excellent history of collaboration and sharing of research resources, such as advanced instrumentation, that foster multidisciplinary research. Thus, (i) the maturity of the graduate program, (ii) the clear demand for the graduates which exceeds the investigators' supply, (iii) the institutional commitment to the educational mission of the toxicology program via the establishment of the Center for Molecular and Cellular Toxicology, (iv) in combination with the established NIEHS Center (CRED), all combine to provide an environment ideal for an increase in the number of NIEHS supported predoctoral positions. The investigators therefore believe this is the optimum time to expand the training grant.

[unreadable] DESCRIPTION (provided by applicant): The chemotherapeutic drug, cisplatin, is highly efficient at removing tumorigenic cells from the body. However, it is not selective and results in loss of normal germ cells from the testes. It has been historically believed that loss of stem spermatogonia during chemotherapeutic treatment accounts for long-term infertility and/or delayed recovery of spermatogenesis. We recently showed, in mice, that a large proportion of stem spermatogonia survive clinic-like repeated cisplatin exposure, without an ensuing recovery in spermatogenesis. This indicates damage to other somatic cells within the testis. Histopathology of testes from cisplatin-treated mice revealed large Sertoli cell vacuoles, apical sloughing, shedding of cellular material into the lumen and loss of germ cells by apoptosis, which are all manifestations of Sertoli cell injury. In this research proposal, we will first test the primary hypothesis that cisplatin directly injures Sertoli cells and creates a testicular microenvironment unsuitable for the development and differentiation of resident germ cells during the recovery period after chemotherapeutic treatment. Cisplatin treated W/W-v mice will be used as recipients of normal stem cell transplants to test if testicular microenvironment is indeed altered as a result of cisplatin exposure. These mice lack endogenous germ cells due to a mutation in c-kit receptor, but have normal Sertoli cells and stem cell factor signaling, which creates a unique system to study normal Sertoli cells, without the interfering presence of germ cells. [unreadable] In the second part of this proposal, we will focus on elucidating the mechanism(s) of the injury to Sertoli cells. Cisplatin has been shown to enter and exit target cells by utilizing transporters that have evolved for the management of copper homeostasis. Our preliminary in vitro evaluation of cisplatin-treated TM4 Sertoli cells revealed the downregulation of the main copper efflux transporter ATP7B (to levels below our detection limits). No alteration in the levels of the main influx transporter, Ctrl, was observed. We propose that cisplatin disrupts the expression of ATP7B in Sertoli cells, resulting in increased accumulation of cisplatin (and copper) in a Wilson's disease-like fashion resulting in exacerbation of the injury to this cell. We will test this hypothesis by measuring intracellular levels of platinum and copper in testes of mice exposed to cisplatin and correlate their levels with alterations in secretion of Sertoli cell specific factors. Additional experiments will employ mice that show a 50% reduction in copper (cisplatin) uptake, due to a mutation in Ctrl, to demonstrate that the sensitivity of Sertoli cell injury correlates with cisplatin uptake and excretion from the cell. The long-term goal of this research is to understand the mechanism(s) of cisplatin-induced Sertoli cell injury and thus spermatogenic injury. Data obtained during the course of this project will provide insights that will allow for the development of strategies for prevention and/or reversal of cisplatin-induced Sertoli cell injury and the reestablishment of functional spermatogenesis. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the cellular and molecular signals instigated in the testis that lead to germ cell apoptosis as a result of environmental toxicant-induced Sertoli cell injury. Since analogous decreases in Sertoli cell support and germ cell apoptosis occur during early postnatal testis development, revealing the mechanisms by which germ cell apoptosis is regulated will enhance our understanding of both the physiological importance of this process during distinctive periods of testicular development as well as to provide insights into cellular targets of environmental toxicants and possible mechanisms of infertility. We have previously revealed the participation of FasL and Fas, two members of the tumor necrosis factor (TNF) superfamily of proteins, in triggering apoptosis of specific germ cell sub- types after exposure to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of the widely dispersed environmental agent DEHP. Here we propose to use this established Sertoli cell injury model to decipher the mechanism(s) responsible for conferring the unique sensitivity of spermatocytes/early round spermatids to undergo apoptosis after MEHP exposure. Our intriguing preliminary findings show that a soluble form of TNF-? (sTNF-?) is elicited by germ cells, via the action of distinct metalloproteinase (MP) enzymes, after MEHP-treatment and that sTNF-? may act upon TNFR1 on Sertoli cells to stimulate an increase in their expression of FasL. Our preliminary findings further indicate that Sertoli cells regulate the activity of MPs via their secretion of TIMP (tissue inhibitor of metalloproteinase) proteins into the adluminal space. Finally, the sensitivity of germ cells to FasL-mediated apoptosis is largely dependent on the ubiquitination, and subsequent degradation, of the anti-apoptotic protein c-FLIP. Our preliminary data suggest that the E3 ligase, Itch, ubiquitinates c-FLIP in germ cells secondary to MEHP- induced Sertoli cell injury. Taken together, these findings led to the development of the central hypothesis of this research proposal that decreases in Sertoli cell-derived TIMP proteins into the adluminal space, and the consequent activation of MPs, is a critical key event that instigates ensuing changes in specific germ cell sub-types that accounts for their sensitivity to undergo apoptosis during periods of reduced Sertoli cell supportive capacity. The first specific aim is designed to delineate the MP and TIMP family members in the testes and assess their role in the regulation of germ cell apoptosis. In the second aim, the functional participation of sTNF-? in FasL-mediated germ cell apoptosis will be challenged. In the last aim, experiments are focused on revealing the mechanisms regulating the levels of the c-FLIP protein and its role in altering the sensitivity of germ cell sub-types to undergo FasL-triggered apoptosis.

PROJECT SUMMARY/ABSTRACT The goal of this project is to decipher the functional role of testicular macrophages and other leukocytes in the pubertal testis after exposure to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP). Although these cells have traditionally been known for their phagocytic and antigen presentation functions, there is a growing body of evidence that these cells are critical for normal development, homeostasis and repair/regeneration of tissues after toxicant injury. We have previously described a robust infiltration of CD11b+ immunoreactive cells, representing macrophages, neutrophils, monocytes, natural killer and dendritic cells, into the testicular interstitial space in pubertal aged rats (PND 28) after MEHP (700 mg/kg, p.o.) treatment. The most pronounced infiltration of these cells occurs in pubertal rats, with lesser amounts in adult rats. Further, C57BL/6J mice at both ages do not have a significant infiltration due to MEHP exposure. The species and age-dependent sensitivity of MEHP- induced testicular injury is well recognized, although the mechanisms that account for these differences remain unresolved. A detailed evaluation of the cells that lie adjacent to the basement membrane of the seminiferous tubules revealed a specific testicular macrophage sub-population, the peritubular macrophages (ptM?s), that is increased in number and in specific regional localization along the periphery of the seminiferous tubules for a sustained period (>2 weeks) after MEHP exposure. The ptM? subtype has gained attention in the field of testis biology because they are predicted to play a critical role in the maintenance of the microenvironment supporting the spermatogonial niche. These findings have led to the hypothesis that MEHP-induced Sertoli cell injury incites an increase in the number and localization of distinct testicular macrophage subtypes as a mechanism to facilitate the efficient repair and recovery of spermatogenesis. To test this hypothesis, the first specific aim will characterize and determine the localization of macrophages subtypes in the pubertal testis in response to a range of MEHP doses (from a low of 10 mg/kg with increased dose intervals of 100, 250, 500 or 700 mg/kg). In the second aim, the cellular mechanisms that induce an infiltration of leukocytes into the testis will be defined. In the final aim, the extent that testicular macrophage subtypes are involved in mediating the recovery of spermatogenesis after a sub-chronic low dose MEHP treatment regimen will be determined as a strategy to more accurately translate the findings of this research project to human relevant exposures. Insights gained from this work will be valuable for predicting individuals susceptible to toxicant-induced infertility and the prevention of reproductive health risks associated with this ubiquitous environmental toxicant. Assessing the individual susceptibility to interactions of environmental toxicants with the immune system is consistent with goals of the NIEHS 2018-2023 strategic plan.