Barry Zirkin - US grants

The objectives of the proposed research are to investigate the factors involved in the packing and regulation of cell nuclei during mammalian spermatogenesis and in the formation and functioning of the male pronucleus during fertilization. Proposed experiments are designed: (1) to determine the factors involved in condensing sperm chromatin during spermiogenesis; (2) to determine the effect on spermatid template activity of changes in the protein content of spermatid nuclei; and (3) to determine the factors involved in sperm chromatin decondensation and male pronuclear formation during fertilization. The results of the proposed autoradiographic, ultrastructural and electrophoretic studies of spermatogenesis are expected to provide information on the functions of one or more of the several testis-specific proteins known to replace the histones during mammalian spermiogenesis. By employing newly developed egg microinjection methods for studies of early events of fertilization, the roles of disulfide bond reduction and proteolysis in the decondensation of the fertilization, the roles of disulfide bond reduction and proteolysis in the decondensation of the fertilizing sperm nucleus and the formation of the male pronucleus will be investigated. The results obtained from these latter studies should contribute to an understanding of critical events of early fertilization.

We propose to examine the quantitative response of specific spermatogenic cells to defined changes in testosterone concentration within the seminiferous tubules of the adult rat. Two specific aims are proposed. The first is to determine the effect on germ cell numbers per testis of clamping intratubular testosterone concentrations at levels below those normally present in the tubules of control rats. To this end, experiments are proposed that are designed to show: 1) whether the production of advanced spermatids by the adult rat testis can be maintained quantitatively at intratubular testosterone concentrations that are clamped below the concentrations found in the tubules of control rats; 2) Whether the same minimum intratubular testosterone concentration is required to maintain normal numbers of advanced spermatids per testis in rats with, and deprived of, endogenous FSH; and 3) whether and when specific spermatogenic cell types change in number per testis in response to a series of defined step reductions in intratubular testosterone concentration. The second specific aim is to determine the quantitative effect of reduced intratubular testosterone concentrations on the number of specific germ cells per testis at particular stages of the cycle of the seminiferous epithelium. To this end, rats whose testes have been synchronized at particular stages of the cycle of the seminiferous epithelium will be used to show: 1) whether there are significant differences in intratubular testosterone concentrations at different stages of the cycle; 2) whether the maintenance of normal numbers of specific spermatogenic cell types at different stages requires different intratubular testosterone concentrations; 3) whether there are stage-specific changes in the number of specific germ cells in response to changes in their intratubular testosterone environments; and 4) whether Leydig cell structure/function and androgen receptor concentrations are related to intratubular testosterone concentrations at different stages of the cycle. The results of these studies should substantially increase our understanding of the quantitative response of specific spermatogenic cells to changes in the hormonal milieu to which they are exposed. This information should increase our understanding of how spermatogenesis is regulated.

The effect of aging on the male reproductive tract is receiving increasing attention for several reasons. Among these are: 1) the population for the United States is aging rapidly, and thus there is increased interest in geriatric medicine; 2) a number of diseases of aged men (e.g. benign prostatic hyperplasia) are dependent on testicular hormones; 3) there is evidence that some drugs commonly used in elderly men (e.g. Gemfibrozil, a blood cholesterol-lowering drug) cause Leydig cell neoplasia; 4) there is evidence for statistically significant correlation between diminished testosterone production and diminished libido and potency in aged menl; 5) the pharmacologic effects of a number of drugs have been shown to change with aging; and 6) diseases of endocrine origin such as osteoporosis are likely to become major problems in geriatric male. In this program project application, we will determine whether age- related changes in the rat male reproductive tract result exclusively from changes in somatic cells extrinsic to the testis, ro whether there also are deleterious age-associated changes intrinsic to Leydig, Sertoli and germ cells. We also will examine how age affects androgen control of biologic functions associated with the prostate gland. To accomplish these objectives, we present five highly integrated research projects. In Project 1, Dr. Ewing will examine the effect of aging on the structure and function of the Leydig cell the cell within the testis that produces testosterone. Dr.Zirkin, in Project 2, will examine the possible consequences of age-associated changes in Leydig cells by studying the morphologic response of proliferating, meiotic and differentiating ells of the seminiferous tubules of aging rats to changes in intratesticular testosterone concentration. In Project 3, Dr. Wrights will examine to effect of aging and of changes in testicular testosterone on Sertoli cells and testicular stem cells. Dr. McCarrey, in Project 4, will examine age-associated changes in spermatogenic cell gene expression, and so will focus on the consequences of aging on spermatogenic cell function. Finally, Dr. Brown, in Project 5, will examine the effect of aging o androgen control of the rat prostate, a sex accessory organ that is exquisitely sensitive to androgen changes.

The Leydig cell produces testosterone in mammals. Testosterone is required for the development of the male reproductive tract, for embryonic and neonatal "imprinting" of testosterone responsive structures such as the hypothalamus, prostate and liver, for potency and libido in the adult make, and for testicular, epididymal and accessory sex organ function in the adult. Yet, little is known regarding the factors regulating development of and sustaining steroidogenic function in Leydig cells. Therefore, we propose studies in the neonatal rat designed to: first, identify transitional cells intermediate between primitive mesenchymal precursor cells and the adult Leydig cell; second, describe unequivocal morphologic and biochemical markers for such intermediate transitional cells; third, study the regulation of this Leydig cell developmental process both in situ and in vitro. In the adult rat, we propose studies designed to: first, elucidate the "trophic" effect of LH on Leydig cell division, number and intratesticular mitogenic activity and transforming growth factor beta content; and second, test the effect of LH on Leydig cell peroxisome structure and function.

Age-related changes in the male reproductive tract are of importance for a number of reasons, including: 1) the population of the U.S. is aging rapidly; 2) a number of diseases of aged men, such as benign prostatic hyperplasia and prostatic cancer, depend upon testicular hormones; 3) other endocrine diseases, such as osteoporosis, are likely to become major problems in geriatric males; 4) aga-related changes in drug and xenobiotic effects have been shown; and 5) couples are waiting until later in life to have children. The male reproductive tract offers many advantages for studies of age-related phenomena, including: 1) experimental manipulation of the tests/epididymides is unlikely to cause death or morbidity; 2) much is known about the regulation of the male tract; and 3) the male reproductive system is an excellent model for generalized aging because it is possible to study, at once, the influence of age on cells that have different proliferative capacities and are at different stages of differentiation. Thus, there are cells that are proliferating rapidly by mitosis (spermatogonia, prostatic epithelia cells), undergoing meiosis (spermatocytes), permanently postreplicative (Sertoli cells), slowly turning over (Leydig cells), differentiating (spermatids), terminally differentiated (spermatozoa), and exquisitely sensitive to hormones and to changes in testicular function (epididymal and prostatic epithelia cells). In the rat, as in the human, striking changes occur with aging, including reduced secretion of testosterone, diminished germ cell production, reduced numbers of testicular and epididymal sperm, atrophy of the seminiferous epithelium, and increased sensitivity of the prostate gland to androgens. The major objectives of this program project application are to elucidate the critical structural and functional changes associated with aging of Leydig (Drs. Zirkin and Wright, Project 1), prostatic (Dr. Brown, Project 2), Sertoli/germ (Drs. Zirkin and Wright, Project 1), spermatozoal (Dr. Robaire, Project 3) and epididymal (Dr. Robaire, Project 3) cells, and to determine the mechanism by which such changes occur.

The overall objectives of the Morphology Core have not changed: to facilitate research in reproductive biology by providing investigators the ability to combine morphologic with endocrinologic, cellular and/or molecular approaches; and importantly, to encourage collaboration among associates with common interests in the reproductive sciences who represent several divisions of the University (School of Medicine, School of Hygiene and Public Health, and School of Arts and Sciences). The Core has evolved to offer expertise in immunocytochemistry, immunofluorescence, analytical methods for the quantification of signal density, stereological methods for the quantifiation of cells and cell ultrastructure and in situ hybridization. PCR-based in situ hybridization was also considered seriously, and is now underway. With the realization that it would not be possible to offer this wide variety of services without increased staff, and, moreover, that it often is in the best interests of investigators to perform their own studies independently, the training of associates and their staffs in morphological methods has become an increasingly central function of the Lab. As most investigators cannot afford the expensive equipment that morphological studies require, the Core also provides investigators the ability to use the equipment and space as needed, without charge, and continues to provide expert advice on the design and conduct of experiments, and on the interpretation of results.

The maximal capacity of the testes of otherwise healthy Brown Norway rats to produce testosterone declines significantly with age. The long-term objectives of this project are to understand the intracellular and extracellular mechanisms that underlie this decline, and its consequences. In Part I, we will determine the effect of age on the intracellular transport of cholesterol to and into the mitochondria, and on its conversion to pregnenolone. The similarity of aged Leydig cells to LH-deprived adult cells suggests the possibility of age-related restricted access of LH. In Part II, we will compare LH levels in the interstitial compartment of young and old rat testes, and the ability of exogenously administered LH to enter the interstitial compartment. We will determine whether LH will stimulate steroidogenesis by old Leydig cells in vivo and vitro. Using reciprocal testicular transplants, we also will determine whether the steroidogenic abilities of Leydig cells from old rats can be restored when transplanted into young rats. In Part III, we will test the hypothesis that after the removal of old Leydig cells with EDS, ~new~ Leydig cells repopulate the old tests. We will compare the cells that repopulate young and old testes post-EDS with respect to cell structure, LH responsiveness, cholesterol transport proteins, and steroidogenic enzymes. We will determine when after their appearance the new Leydig cells become steroidogenically hypofunctional in order to understand whether extrinsic factors produced by aged animals cause Leydig cells to become steroidogenically hypofunctional, versus the possibility that this occurs independently of such factors. In Part IV, we will test the hypothesis that chronically active steroidogenesis in some way cause age-related reduced steroidogenesis, perhaps by producing oxygen free radicals. We will determine whether the chronic suppression of Leydig cells steroidogenesis will prevent reduced steroidogenesis. In the same experiments, we will determine whether the suppression of endogenous steroidogenesis will suppress age-related germ cell loss.

Adult mammalian spermatogenesis is highly dependent upon testosterone. Androgen receptors are localized to Sertoli cells, making it likely that the effects of testosterone are mediated via the Sertoli cell. However, remarkably little is known about how testosterone functions in vivo. Reduced intratesticular testosterone can lead to increased germ cell apoptosis, suggesting that testosterone may serve a cell survival factor. The mechanism(s) by which testosterone protects germ cells from apoptotic death has received very little attention. The Fas system, including Sertoli cell Fas ligand, and Fas, expressed by germ cells, and germ cell Fas, has been implicated in testicular germ cell apoptosis. The Bcl-2 family of proteins plays a central role in the regulation of apoptotic cell death in a variety of cell types. Some among these proteins, including Bcl-2 and Bcl-xlong, inhibit cell death, while others, including Bax and Bcl-xshort, promoter apoptosis. As yet, we do not know which of these proteins is present in particular germ cells, nor how they respond to testosterone. A central hypothesis of this grant is that testosterone promotes the survival of germ cells via its effects on the Sertoli cell/germ cell Fas system, and on the Bcl-2 family of proteins involved in cell survival/cell death. We will first determine the extent to which binding of testosterone by ABP explains the high intratesticular testosterone concentration is required to maintain spermatogenesis in the adult rat. We then will test the concept that testosterone, acting through the Sertoli cell, influences the Fas system and, through this system, cell survival/cell death proteins of the Bcl-2 family, thereby influencing whether or not internucleosomal cleavage and subsequent apoptotic death of germ cells occurs. The results of these studies, taken together with the results of studies proposed by Dr. Jarow in Project I, should elucidate common principles involved in regulating spermatogenesis in rats and men, and at long last should provide concrete information on mechanism(s) by which testosterone acts in vivo to regulate spermatogenesis.

This application proposes four integrated projects centered on male infertility and its biological basis. Project I will examine the role of inadequate intratesticular testosterone concentration in human male infertility. The hypothesis that reduced intratesticular testosterone concentration is responsible for some conditions of infertility will be tested directly in infertile men. Project II will examine the mechanism(s) by which testosterone protects germ cells from apoptotic death. A central hypothesis is that testosterone promotes the survival of germ cells via its effects on the Sertoli cell/germ cell Fas system, and through the Bcl- 2 family of cell survival/cell death proteins of germ cells. The results of Projects I and II should elucidate common principles involved in regulating spermatogenesis in rats and men. Project II focuses on how testosterone functions to regulate spermatogenesis through the androgen receptor (AR). The hypothesis will be tested that stage-specific regulation of AR gene expression in Sertoli cells is affected via the differential expression of endogenous transcription factors that bind to the AR promoter and enhance or repress transcription of the AR gene. The hypothesis that the AR is able to recruit endogenous co-regulatory proteins in Sertoli cells, and that these protein-protein interactions activate or repress AR transcriptional activity of genes required during spermatogenesis also will be examined. Thus Project III also provides a link between intratesticular testosterone (Projects I and II) and stage- specific gene expression (Project IV). Project IV examines the interactions between germ cells and Sertoli cells, critical for how spermatogenesis is coordinated and regulated. It will examine the function and regulation of stage-specific gene expression of analyses of Cyclic Protein-2, the pro-enzyme form of the cysteine protease Cathepsin L. It will be determined whether cis-acting elements in the CP-2/Cathepsin L gene and trans-acting factors in Sertoli cells interact in a stage- specific manner; and whether these regions are responsible for stage- specific transcription of the CP-2/Cathepsin L gene.

DESCRIPTION (provided by applicant): The maximal capacity of Brown Norway rat Leydig cells to produce testosterone decreases significantly with age. Our major objective is to elucidate the molecular mechanisms that are responsible. A central hypothesis of this application is that reactive oxygen species (ROS), produced by the Leydig cells themselves, play an important role in age-related reductions in Leydig cell testosterone production. Three specific aims are proposed. The first is to determine whether age-related reductions in Leydig cell testosterone production are reversed by administering LH directly to the testes of old rats, or by encapsulating old cells and implanting them into young rats. These studies will test the hypothesis that factors outside the Leydig cells might be responsible for the reduced ability of old Leydig cells to produce testosterone. In the second specific aim, we will determine whether the increases in Leydig cell reactive oxygen production that occur as these cells age are from the mitochondrial transport chain, the P450 reactions of steroidogenesis, or both; and whether there are age-related changes in mRNA, protein, and/or activity levels of the major enzymatic scavengers of reactive oxygen species in Leydig cells - SOD, glutathione peroxidase and catalase. The third aim is to examine the effects of manipulating reactive oxygen load on Leydig cell steroidogenesis during aging, based on the hypothesis that reactive oxygen, whether derived from the electron transport chain, steroidogenesis, or both, plays an important role in the reduced testosterone produced by aging Leydig cells. We will test this hypothesis by examining the consequences of manipulating oxidative stress load in vivo on Leydig cell function, including: the effects of vitamin E supplementation and deprivation; the effects of age on the acute response of Leydig cells to depletion of its major non-enzymatic antioxidant, glutathione; and the effects of caloric restriction. Together, these studies will provide new insights into how Leydig cells cope with stressors that are present or increase during aging, and will shed light on the underlying molecular basis for functional changes in Leydig cells that occur during aging.

DESCRIPTION (provided by applicant): This proposal requests support for trainee attendance at the 2002 Annual Meeting of the American Society of Andrology (ASA) in Seattle, Washington, April 24-27, 2002, and for selected scientific sessions of the meeting. The American Society of Andrology (ASA), a scientific society of approximately 750 members, is a unique partnership of basic scientists and clinicians. Its primary missions are to advance and promote basic knowledge of the male reproductive tract and clinical andrology by fostering interdisciplinary collaboration and communication, and to attract young investigators to the field. An important means by which ASA fulfills its mission is by holding an annual scientific meeting, which typically attracts 250-350 attendees. The participation of trainees (predoctoral students, medical students, postdoctoral fellows, medical fellows) is essential to maintaining and instilling new vigor into andrology. There is ample opportunity for active participation in the 2002 meeting by trainees in the meeting's three oral sessions and two poster sessions. If this application is successful, trainee travel awards will be based on the dual criteria of scientific merit and need. We also request funding to help defray the costs of travel and honorarium for the speakers of a State-of-the-Art Lecture entitled "Hormone replacement therapy in the male" (Lisa Tenover) and for two symposia, entitled: "Do gene defects impact male reproduction?" (Eva Eicher, Juliana Imperato-McGinley, Stephanie Seminara) and "Are there estrogen effects on the testis?" (Ken Korach, Richard Sharpe, and Fred vom Saal).

DESCRIPTION (provided by applicant): It is well established for both rat and man that the total testosterone concentration within the testes is far higher than that in serum. We know for the that intratesticular testosterone can be reduced by 50-60% without effect on spermatogenesis, but that the required testosterone concentration is still 10-fold greater than serum testosterone concentration. This kind of information, if available for the human, could prove invaluable for understanding and treating at least subsets of men with infertility. Unfortunately, we know little about the androgen content of intratesticular fluid within the human testis or the relationship between intratesticular androgens and human spermatogenesis. Our recent studies of the human have demonstrated that, as in the rat, there is a gradient between the concentration of testosterone in serum and within the testis; intratesticular testosterone levels were found to be 100-fold higher than normal serum testosterone levels. We do not yet know how much of the testosterone within the human testis is required either to maintain or restore quantitatively normal spermatogenesis because, as yet, experimental studies comparable to those performed in the rat have not been feasible for the human. Moreover, we know little about the concentration of bioavailable androgens within the testes of any mammal, and therefore virtually nothing about the relationship between bioavailable androgen concentration and spermatogenesis in rat or man. The major objectives of this project are to identify and quantify the androgen content of the human testis, to assess the bioavailability of intratesticular androgens, to experimentally determine the relationship between bioavailable intratesticular androgen concentration and spermatogenesis, and to examine the effect of testosterone replacement modalities on intratesticular bioavailable androgen concentration and on spermatogenesis in subfertile men.

DESCRIPTION (provided by applicant): The focus of this application is on the hormonal and paracrine regulation of spermatogenesis in humans and rodents. Project I (Barry Zirkin and Jonathan Jarow) addresses the biological mechanisms that explain why only some men who receive hormonal contraception become azoospermic. Naturally occurring ethnic differences in the response to testosterone (T)-based contraception will be studied to address this biological issue. We will compare intratesticular concentrations of bioactive androgen in Asian and Caucasian men before and after they receive hormone-based contraceptive doses of T, and will determine the quantitative relationship between hCG [human chorionic gonadotropin]-induced intratesticular bioactive androgens and spermatogenesis. These studies should provide important new information needed to develop an effective hormone-based male contraceptive, as well as provide substantial new insight into the physiological mechanisms through which T regulates spermatogenesis in men. The biological effects of androgens on spermatogenesis are mediated via the androgen receptor (AR) in Sertoli cells. The primary objective of Project II (Terry Brown) is to elucidate the molecular mechanisms that drive expression of the AR gene in Sertoli cells and to identify the specific transcription factors and their cis-acting regulatory elements that control the pubertal maturation dependent and adult stage-specific transcription of the AR gene. Project III (William Wright) focuses on paracrine regulation of spermatogenesis by examining the in vivo function of Sertoli cell growth factors, GDNF, FGF-2 and IGF-1, which have been implicated as involved in the control of stem spermatogonial replication and differentiation. Its central hypothesis is that differentiated germ cells, via feedback effects, regulate the expression by Sertoli cells of growth factors that, in turn, regulate the differentiation and replication of stem spermatogonia. Finally, we propose a Pilot Project (Paul Miller) that is based on our understanding that germ cell DNA is subject to damage and may be an important factor in male infertility. A rapid, simple analytical method to assess the level of oxidative DNA damage in germ cells will be developed using beacon aptamers, oligonucleotides that have the ability to bind ligands in a highly specific manner. These biosensors will be designed to measure oxidized nucleosides, 8-oxodeoxyadenosine and 8-oxodeoxyguanosine, in the DNA of sperm from infertile men.

ABSTRACT-PROJECT 1 Principal Investigator: Barry R. Zirkin The administration of testosterone (T) can reversibly suppress spermatogenesis in men. However, although azoospermia is achieved in a majority of men, significant numbers typically maintain high enough sperm production to pose a risk of initiating a pregnancy. A significantly higher prevalence of azoospermia has been noted in Asians than in Caucasians. The failure of individuals or groups of men to become azoospermic diminishes the promise of hormone-based contraception. The long-term goal of the proposed research is to determine the biological mechanisms that explain why some men become azoospermic while others do not. We will take advantage of the naturally occurring ethnic differences, namely the response of Asians and Caucasians, to T-based contraception to address this biological issue. Three specific aims are proposed. In the first, we hypothesjze that differences in T metabolites (e.g. lower DHT levels) in Asian than in Caucasian men might help explain the increased responsiveness of Asians to T-based contraception. To address this hypothesis, we first will quantify T and DHT present within the testes of untreated fertile Asian and Caucasian men, using a highly sensitive liquid chromatography tandem mass spectroscopy method (LC/MS/MS) to do so. A second objective will be to compare the total intratesticular bioactive androgen concentrations in Asian and Caucasian men, using a highly sensitive assay of androgen bioactivity based upon stimulation of androgen receptor transcriptional activity in mammalian cells. The central hypothesis to be tested is that a greater percentage of androgen is bound and therefore not bioavailable in Asian than in Caucasian men. If true, this would suggest that lesser reduction of T and DHT would be required to achieve azoospermia in Asian than in Caucasian men. In the second aim, we will determine whether there are differences in the bioactive, bioavailable pool of androgens within the testes'of Asians and Caucasians following the men receiving a contraceptive dose of T. The central hypothesis that we will test is that in response to a contraceptive T dose, the Caucasian men; will have greater amounts of intratesticular bioactive androgens than Asian men. In the third aim, we will test the hypothesis that at specific total intratesticular T concentrations, there will be less bioavailable, bioactive androgen in the testes of Asian than Caucasian men. The results of these studies should be invaluable to the development of a safe and effect hormonebased male contraceptive. Moreover, comparing two groups with different sensitivities to a contraceptive dose of T should provide significant insight into the physiological mechanisms in men through which T regulates spermatogenesis.

The Administrative Core will be centered in the Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Johns Hopkins University School of Public Health. The Administrative Core will have four major responsibilities: 1) to assure that there is consistent communication among the scientists of the Center; 2) to attract interested faculty, students and fellows at Johns Hopkins University to participate in Center activities, as appropriate: 3) to maintain frequent contact with the Center's Advisory Board (Drs. Mary Ann Handel, Robert Braun and Arthur Burnett), both informal and formal, so as to keep the Board informed of Center activities and research; and 4) to foster communication between Centers. To accomplish its objectives, the Administrative Core will: i. Organize weekly meetings among Center investigators to discuss research objectives and data. ii. Organize an annual meeting with Advisory Board members, and otherwise maintain contact through email, phone, teleconferencing. iii. Encourage research collaboration within the Hopkins Center and between Centers. iv. Sponsor a monthly series of seminars in Reproductive Biology with funds pledged by the Department of Biochemistry and Molecular Biology, Department of Urology, and Department of Gynecology and Obstetrics. v. Organize the endowed annual Larry L. Ewing Lectureship. vi. Co-Sponsor the Mid-Atlantic Reproductive Biology meeting, which is attended by faculty, students and fellows from Johns Hopkins University, Georgetown University, University of Virginia and University of Pennsylvania. vii. Co-sponsor the Maryland-Hopkins Reproductive Biology retreat, which is attended by faculty, students and fellows from Johns Hopkins University and the University of Maryland at Baltimore. viii. Encourage participation in Center activities by students and fellows of the Training Program in the Reproductive Sciences. The preceptors of the program include Drs. Greg Ball (School of Arts and Sciences), Terry Brown, Janice Evans, Jonathan Jarow, Erika Matunis (School of Medicine), Geraldine Seydoux (School of Medicine), William Wright and Barry Zirkin. Drs. Brown, Jarow, Miller, Wright and Zirkin, co-investigators of the Center application, will invite Drs. Ball, Evans, Matunis and Seydoux and their students and fellows to participate in the annual meeting of the Center, the monthly seminar series in Reproductive Biology, the Larry L. Ewing Lectureship, and the Mid-Atlantic Reproductive Biology meeting.

The administration of testosterone (T) can reversibly suppress spermatogenesis in men. However, although azoospermia is achieved in a majority of men, significant numbers typically maintain high enough sperm production to pose a risk of initiating a pregnancy. A significantly higher prevalence of azoospermia has been noted in Asians than in Caucasians. The failure of individuals or groups of men to become azoospermic diminishes the promise of hormone-based contraception. The long-term goal of the proposed research is to determine the biological mechanisms that explain why some men become azoospermic while others do not. We will take advantage of the naturally occurring ethnic differences, namely the response of Asians and Caucasians, to T-based contraception to address this biological issue. Three specific aims are proposed. In the first, we hypothesjze that differences in T metabolites (e.g. lower DHT levels) in Asian than in Caucasian men might help explain the increased responsiveness of Asians to T-based contraception. To address this hypothesis, we first will quantify T and DHT present within the testes of untreated fertile Asian and Caucasian men, using a highly sensitive liquid chromatography tandem mass spectroscopy method (LC/MS/MS) to do so. A second objective will be to compare the total intratesticular bioactive androgen concentrations in Asian and Caucasian men, using a highly sensitive assay of androgen bioactivity based upon stimulation of androgen receptor transcriptional activity in mammalian cells. The central hypothesis to be tested is that a greater percentage of androgen is bound and therefore not bioavailable in Asian than in Caucasian men. If true, this would suggest that lesser reduction of T and DHT would be required to achieve azoospermia in Asian than in Caucasian men. In the second aim, we will determine whether there are differences in the bioactive, bioavailable pool of androgens within the testes'of Asians and Caucasians following the men receiving a contraceptive dose of T. The central hypothesis that we will test is that in response to a contraceptive T dose, the Caucasian men;will have greater amounts of intratesticular bioactive androgens than Asian men. In the third aim, we will test the hypothesis that at specific total intratesticular T concentrations, there will be less bioavailable, bioactive androgen in the testes of Asian than Caucasian men. The results of these studies should be invaluable to the development of a safe and effect hormonebased male contraceptive. Moreover, comparing two groups with different sensitivities to a contraceptive dose of T should provide significant insight into the physiological mechanisms in men through which T regulates spermatogenesis.

? DESCRIPTION (provided by applicant): Reduced serum testosterone (T), or hypogonadism, is estimated to affect about 5 million American men, including both aging and young. Low serum T has been linked to mood changes, worsening cognition, fatigue, depression, decreased lean body mass, reduced bone mineral density, increased visceral fat, and decreased libido. In men with low serum T that results from reduced luteinizing hormone (LH), treatment with hCG will stimulate Leydig cell T formation and thus ameliorate symptoms. Typically, however, decline in serum T is not due to reduced LH, but rather (or in addition) to reduced Leydig cell T production resulting from a primary testicular problem. Administration of hCG to these men rarely elevates serum T. Exogenous T can be administered to raise serum T levels in these men and thus treat symptoms. However, based on a number of alarming recent studies, the FDA issued a report warning that men who take exogenous T-boosting products may face increased risk of stroke and heart attack. Additional recent evidence suggests that exogenous T also might promote prostate tumorigenesis. Moreover, it is well established that administered T can have suppressive effects on LH, resulting in lower Leydig cell T production and therefore in reduced intratesticular T and sperm numbers. This makes exogenous T administration inappropriate for men who wish to father children. In this application, we propose to explore a novel method by which to increase serum as well as intratesticular T levels by restoring the ability of Leydig cell themselves to produce young levels of T. Our approach is based on current understanding of the molecular mechanisms involved in Leydig cell T formation. Translocator protein (18-kDa TSPO) is a ubiquitous mitochondrial protein that is expressed at very high levels in Leydig cells. It is clear from in vitro studies of cell lines and primary cells that TSPO drug ligands are able t induce the translocation of cholesterol from the outer to the inner mitochondrial membrane in steroidogenic cells, and thereby stimulate steroid formation. We propose to undertake proof-of-principle, preclinical studies designed to pharmacologically increase cholesterol transfer into the Leydig cell mitochondria in conditions of hypogonadism, and in this way stimulate T production. We will test the hypothesis that drug ligand-induced activation of TSPO will restore Leydig cell T production to higher levels in hypogonadal Brown Norway rats, elevate serum and intratesticular T levels, and maintain spermatogenesis. The effect of treatment on adrenal and brain steroid synthesis, and on prostate and cardiac outcomes, will be assessed, as will overall toxicity. If proven successful and safe, the potential impact of this approach to elevate T could be paradigm shifting for the clinical treatment of hypogonadism in aging men and in all hypogonadal men who desire children.