William Wright - US grants

Spermatogenesis occurs in a highly organized tissue, the seminiferous epithelium which contains proliferating germ cells and a static population of somatic cells. The germ cells exist in defined cellular associations and are intimately associated with the nongerminal, Sertoli cells. There is substantial evidence that Sertoli cells are important components in the regulation of spermatogenesis. Furthermore, the morphology and physiology of the Sertoli cells change in consort with the development of their neighboring germ cells. It seems likely that the germ and Sertoli cells interact with each other and that this interaction is important in the regulation of spermatogenesis. This cellular interaction may be facilitated by the secretion and binding of specific regulatory proteins. Consequently, it is proposed to examine a newly discovered protein, Cyclic Protein-2, which is secreted maximally by seminiferous tubules at States VI-VIIa, b of the cycle of the seminiferous epithelium. In particular, it is proposed: first, to isolate this protein and raise a monospecific antiserum; second, to use this antiserum to identify the cellular orgin of CP-2; third, ot identify the cellular target of CP-2 and biochemically characterize the binding of CP-2 to that cell and finally; to examine changes in the secretion and binding of this protein with testicular maturation and with progression of the epithelial cycle in the mature animal.

the seminiferous epithelium, the site of spermatogenesis, is populated by somatic Sertoli cells and developing spermatogenic cells. There is evidence that these cells interact extensively and it has been proposed that these interactions are essential for spermatogenesis. Recently, we described a new glycoprotein, Cyclic Protein-2 (CP-2), which is secreted by Sertoli cells in response to specific germ cells. Cyclic Protein-2 is the principal in vitro secretory product of Sertoli cells in Stage VI seminiferous tubules while it is secreted in minimal amounts at Stage XII. This protein is also secreted in vivo as it can be purified in microgram amounts from the seminiferous tubule fluid of a single male rat. In this application, we proposed the first thorough analysis of the composition, function, cellular distribution and biosynthesis of a protein which is secreted in a stage-dependent manner by Sertoli cells. We propose to characterize CP-2 both immunochemically and biochemically. We will identify, clone and sequence a cDNA for CP-2. Comparison of this sequence with that of proteins of known function may indicate the role of CP-2 in the seminiferous epithelium. Finally, we will examine the cellular distribution and biosynthesis of CP-2 at different stages of the epithelial cycle and we will measure the Sertoli cell's mRNA content for this protein at these different stages.

Spermatogenesis and sperm maturation result in dramatic changes in the plasma membrane of the developing male gamete. These changes include the acquisition of cell surface molecules required for fertilization. In the mouse, one of these cell surface molecules is the enzyme UDP-galactosyltransferase (GT). GT is a unique molecule in the reproductive tract as it may exist in three different forms: as a plasma membrane component, as a constituent of the golgi membrane and as a secretory product of the somatic cells in the seminiferous and epididymal epithelia. It has not been established whether a biochemically distinct form of GT resides in each compartment. Neither has it been established how the biosynthesis of these molecules is regulated. However, as the structure and function of the male reproductive tract are hormonally regulated, both the synthesis and the distribution of GT among the three compartments may be influenced by hormones. Recently, we have developed two reagents with which to carefully examine the source, biochemistry and regulation of germ cell surface GT. These reagents are (1) a polyclonal, monospecific antiserum against bovine GT which specifically binds GT on the mouse sperm plasma membrane, and (2) a cloned cDNA for murine GT mRNA. Using these specific reagents, we propose the following experiments: First, we will establish which germ cells have a cell surface GT and whether they synthesize the protein and insert it into their plasma membranes. We will also explore the alternative possibility that germ cell surface GT is an extrinsic membrane component which is derived from secretions of the Sertoli cell and the epididymal epithelium. Second, we will compare the biochemical structures of galactosyltransferase recovered from the plasma membrane, from the golgi membrane, and from the pool of proteins secreted by Sertoli cells and the epididymal epithelium. Additionally, we will determine whether each form of GT is encoded by a distinct GT mRNA. Third, we will compare the hormonal regulation of germ cell surface GT, golgi membrane GT and secreted GT. Completion of these experiments will result in the first thorough analysis of the distribution, source, biochemistry and regulation of a germ cell membrane protein required for fertilization.

the seminiferous epithelium, the site of spermatogenesis, is populated by somatic Sertoli cells and developing spermatogenic cells. There is evidence that these cells interact extensively and it has been proposed that these interactions are essential for spermatogenesis. Recently, we described a new glycoprotein, Cyclic Protein-2 (CP-2), which is secreted by Sertoli cells in response to specific germ cells. Cyclic Protein-2 is the principal in vitro secretory product of Sertoli cells in Stage VI seminiferous tubules while it is secreted in minimal amounts at Stage XII. This protein is also secreted in vivo as it can be purified in microgram amounts from the seminiferous tubule fluid of a single male rat. In this application, we proposed the first thorough analysis of the composition, function, cellular distribution and biosynthesis of a protein which is secreted in a stage-dependent manner by Sertoli cells. We propose to characterize CP-2 both immunochemically and biochemically. We will identify, clone and sequence a cDNA for CP-2. Comparison of this sequence with that of proteins of known function may indicate the role of CP-2 in the seminiferous epithelium. Finally, we will examine the cellular distribution and biosynthesis of CP-2 at different stages of the epithelial cycle and we will measure the Sertoli cell's mRNA content for this protein at these different stages.

Spermatogenesis and sperm maturation result in dramatic changes in the plasma membrane of the developing male gamete. These changes include the acquisition of cell surface molecules required for fertilization. In the mouse, one of these cell surface molecules is the enzyme UDP-galactosyltransferase (GT). GT is a unique molecule in the reproductive tract as it may exist in three different forms: as a plasma membrane component, as a constituent of the golgi membrane and as a secretory product of the somatic cells in the seminiferous and epididymal epithelia. It has not been established whether a biochemically distinct form of GT resides in each compartment. Neither has it been established how the biosynthesis of these molecules is regulated. However, as the structure and function of the male reproductive tract are hormonally regulated, both the synthesis and the distribution of GT among the three compartments may be influenced by hormones. Recently, we have developed two reagents with which to carefully examine the source, biochemistry and regulation of germ cell surface GT. These reagents are (1) a polyclonal, monospecific antiserum against bovine GT which specifically binds GT on the mouse sperm plasma membrane, and (2) a cloned cDNA for murine GT mRNA. Using these specific reagents, we propose the following experiments: First, we will establish which germ cells have a cell surface GT and whether they synthesize the protein and insert it into their plasma membranes. We will also explore the alternative possibility that germ cell surface GT is an extrinsic membrane component which is derived from secretions of the Sertoli cell and the epididymal epithelium. Second, we will compare the biochemical structures of galactosyltransferase recovered from the plasma membrane, from the golgi membrane, and from the pool of proteins secreted by Sertoli cells and the epididymal epithelium. Additionally, we will determine whether each form of GT is encoded by a distinct GT mRNA. Third, we will compare the hormonal regulation of germ cell surface GT, golgi membrane GT and secreted GT. Completion of these experiments will result in the first thorough analysis of the distribution, source, biochemistry and regulation of a germ cell membrane protein required for fertilization.

This is a Small Grants for Exploratory Research (SGER) effort. The work is a pilot experiment to determine the feasibility of using a virtual reality environment with a scanning tunneling microscope (STM) for real-time viewing and manipulation of atoms and molecules. Recent advances make it possible to link a STM to force feedback and head-mounted display system for new types of scientific instruments. These instruments will give the user the subjective experience of presence at the atomic scale on a physical surface. Scanning can be at video rates for real-time visualization. Force feedback provides the capability for tactile exploration of the contours of the electronic environment presented at the atomic, molecular and substrate surfaces. Atoms and molecular fragments can be manipulated interactively with the probe. The same probe can cut molecular bonds and bring fragments together for reactions. The products of resulting reactions can be explored to determine their morphology and other characteristics. The STM collaborators are Dr. R. Stanley William and graduate students from the Chemistry Department of UCLA.

9511215 William Wright One of the goals of neuroscience is to understand what happens in our brains when we learn. A powerful approach to finding answers to this question is provided by studying the brains of early animals and asking how much of what happens is similar to processing in early animals and how much occurs only in higher animals. The work of this group of neuroscientists examines mechanisms of learning in marine mollusks. It charts changes in specific cellular mechanisms of learning in a specific behavior in several species of these animals. By monitoring in parallel correlated changes in learning capabilities, this study will provide more precise insights into the roles of these cellular mechanisms in learning. Besides the knowledge of whether and how mechanisms of learning change across species, this work will also give insights into learning mechanisms in higher animals.

9632069 Wright What happens in our brains when we learn? How much of what happens is due to processes that first appeared in the brains of ancient ancestors, and how much is due to more recently evolved mechanisms? The work proposed here examines, for the first time, the evolution of mechanisms of learning. Through neurophysiological and behavioral studies of present-day species with known relationships to each other, we propose to chart the evolution of specific cellular mechanisms of learning. This approach will 1) address the question of whether and how mechanisms of learning change across evolution, and 2) provide new insights into learning mechanisms of modern species.

Abstract ATM-9810474 Leavitt, Steven W. University of Arizona Title: Tree-Ring Formation, Isotopes, and El Nino in Ponderosa Pine of the Southwest Climate conditions and variability over several centuries in the American Southwest have been investigated via standard dendroclimatological research during the past few decades. Analysis of reconstructed data and modern instrumental data indicates this region's climate appears particularly impacted by El Nino teleconnections and the vagaries of the annual southwestern summer monsoon. New tools in the form of stable isotopes have recently become a viable option for probing present and past climate by means of analysis of tree rings and precipitation. This award supports a study designed to establish the linkage between tree-ring isotopic composition and environment (including climate). The primary focus of the proposed research will be to advance the numerical tree-ring growth process model through the addition of isotopic subroutines. The fundamental goal of the process model development is to take inputs of climate and isotopic composition of atmosphere and soil (water), and accurately generate the detailed patterns of isotopic variation (and cell size) for individual wood cells within rings. The isotopic experimentation and model development will go hand-in-hand to better understand relationships of isotopic composition and climate. With escalating use of tree-ring isotopes in climate research, especially at the subannual level, this model should provide important guidance to understanding the isotopic results.

There is considerable evidence that in normal, mature testes, developing male gametes interact with surrounding Sertoli cells and that these interactions are essential for spermatogenesis. In rats, one result of these interactions is the expression by Sertoli cells of specific genes at particular stages of the cycle of the seminiferous epithelium. It is proposed that these genes' products regulate the development of germ cells present in those stages or other important testis functions. To investigate stage-specific gene expression by Sertoli cells, we have focused on Cyclic Protein-2, the pro-enzyme form of the cysteine protease, Cathepsin L. This protein is secreted by Sertoli cells within stage V-VIII tubules. Recent and published results propose three different in vivo functions for this protein in rat testes: [1] Degradation of residual bodies that have been engulfed by Sertoli cells, preventing the release of the germ cell-specific antigens into the general physiological environment. [2] Cleavage of adhesive functions between compacted spermatids and Sertoli cells in stage VI tubules, facilitating the movement of spermatids towards the lumen of the tubule. [3] Stimulation of testosterone secretion by Leydig cells. Experiments in the first specific aim test, in vivo, these proposed functions. The second specific aim examines whether the stage-specific expression of CP-2/Cathepsin L mRNA by Sertoli cells also occurs in humans, rabbits, boar and bulls. Such expression in other species would indicate that our proposed studies in rodents are applicable to those species and in the future might provide insight into causes of human idiopathic fertility. This application also proposes to define the molecular mechanisms which regulate the stage- specific transcription of the gene encoding CP-2/Cathepsin L. We have previously demonstrated that with progression of the stages of the cycle, germ cells sequentially trigger the repression, derepression (or loss of repression), stimulation and re-repression of transcription of this gene. Such regulation by germ cells may be mediated by an enhancer and by repressor and derepressor regions that we have identified in the CP- 2/Cathepsin L gene. We, therefore, propose to identify the cis-acting elements in these regions which bind specific transacting factors in a stage-specific manner. We also propose to determine, by studying mice carrying specific CP-2/Cathepsin L-beta-galactosidase transgenes, whether these regions are responsible for stage-specific gene transcription. Results from these studies will begin to elucidate the molecular basis for a stage-specific germ cell-Sertoli cell interaction.

The tight binding of a spermatozoan to the zona pellucida surrounding an egg is a critical event in fertilization. Thus, the identification of the molecules responsible for this binding is of fundamental importance. It is generally agreed that in the mouse, receptors on the sperm plasma membrane bind to the nonreducing termini of specific O-linked glycans on zona pellucida glycoprotein-3 (ZP3). Current debate centers on the identity of the receptor(s) on sperm and the structure(s) of the glycans they bind. To gain insight into the structures of the ligands bound by sperm, we analyzed the activities of a series of small glycans of known structure in a competitive sperm-zona binding assay. Results showed that a beta- Gal-capped but not a beta-C1cNAc-capped trisaccharide was a low activity competitive inhibitor (ED50=42muM). Addition of a nonreducing terminal alpha3-Gal to the beta-Gal-capped glycan increased activity 8-fold. However, additional of an alpha3-fucose residue to either the beta-Gal- or the alpha-Gal-capped trisaccharide produced a highly effective competitive inhibitor (ED50 approximately 0.45muM for both glycans). Lastly, mixing experiments with pairs of competitive inhibitors demonstrated additive effects when saturating does of fucosylated, Gal-capped glycans and a beta-Gal-capped glycan were added to sperm and eggs. These results lead to the hypotheses that fucosylated, Gal-capped glycans have high affinity for a receptor on sperm, that beta-Gal-capped glycans have a lower affinity for a receptor and that these two classes of glycans bind different receptors on the sperm surface. To test these hypotheses, we propose to synthesize and radiolabel two neoglycoproteins, and use these probes to directly determine whether they bind separate receptors. We will also examine how changes in a glycan's structure affects its affinity for these receptors. We next propose to test the function of these receptors by examining whether their expression is affected by capacitation, whether they mediate the acrosome reaction or are required for fertilization. We will then identify the glycan receptors on the sperm surface by cross linking the neoglcoproteins to their respective receptors and examining the cross-linked protein. Finally, using antibodies, glycosyltransferases, glycosidases and lectins with defined specificities for glycan structures, we propose to determine whether glycans with specific fucosylated, Gal-capped and/or beta-Gal-capped termini are present on ZP3. Completion of these experiments will provide substantial insight into the structures and functions of the molecules on murine sperm and the zona pellucida that mediate critical events in fertilization.

The n-body problem - i.e. simulating the motion of many particles that attract or repel each other - is a classic one with many applications. N-body algorithms are the computational means of solving this problem. The many users of these algorithms include biophysiologists and biochemists studying biological phenomena, pharmaceutical researchers dealing with drug structures and interactions, astrophysicists studying the structure of the cosmos, and engineering researchers studying hydrodynamics. The algorithms are also interesting in their own right to computer scientists. This project will develop an innovative approach to n-body algorithms called self-scheduling n-body algorithms. This family of algorithms promises not only reduced computational complexity, but also a straightforward implementation.

Technically, the research will use a multiple time step method where each pairwise interaction is evaluated using a dynamic schedule that attempts to equalize the error of each interaction, drastically reducing the computational cost. The fundamental new idea is to equalize the impulses for all interactions, rather than equalizing the time steps for all interactions (which is too conservative in most cases). Mathematically, the constant time step t is traded for a constant impulse I, defined as Fij tij, where Fij is the force between particles i and j and tij becomes the time step used to re-evaluate Fij. This leads to an expected execution complexity of O(n4/3) per simulation step. Algorithmic improvements that rely on the first and second derivatives of force reduce the per step computational complexity to O(n log n) and O(n), respectively. The project will fully explore these algorithms, analyze their error bounds and computational complexity, implement prototype versions, and explore additional topics (such as efficient parallelization and cache-efficient memory layouts) as time permits.

Lay abstract Wright IBN-0131743

What happens in our brains when we learn? How do evolutionary changes in mechanisms underlying one form of learning impact upon the evolution of other forms of learning? In previous research this investigator used neurophysiological and behavioral studies of several present-day species (related to the popular models species, Aplysia californica) with known relationships to each other to establish that fundamental mechanisms of sensitization, a simple form of learning, have changed across evolution. In this research the consequences of evolution will be examined with respect to a more complex form of learning, classical conditioning. The hypothesis to be examined is whether loss or reduction in the simple mechanisms of sensitization reduces the persistence of the more complex neural changes underlying classical conditioning. This study will lead to a deeper understanding of the neural mechanisms of learning and memory.

[unreadable] DESCRIPTION (provided by applicant): Interactions between developing male gametes and adjacent Sertoli cells are essential for spermatogenesis. Sertoli cells express genes encoding cell adhesion molecules, growth factors, transport proteins, proteases and protease inhibitors required by male germ cells. Germ cells regulate expression by Sertoli cells of many of these genes. Consequently, expression of these genes changes as the adjacent germ progress through the stages of the cycle of the seminiferous epithelium. This application examines the regulation and biological consequences of stage-specific gene expression by Sertoli cells from the perspective of the cathepsin L gene. Transcription of this gene increases more than 10-fold as adjacent germ cells progress from stage I to stages VI and VII and then decreases to undetectable levels when germ cells reach stage X. This cycle of gene expression is a response to germ cells which causes sequential repression, stimulation and re-repression of transcription of the cathepsin L gene. We have identified 2 domains in the cathepsin L gene that potentially respond to these signals: [1] An upstream repressor domain responds to inhibitory signals from germ cells [2] A 120 bp activation domain is stimulated upon the culmination of testis maturation, which is characterized by the completion of the first wave of spermatogenesis. These domains are functional in vivo; a 3kb fragment of the cathepsin L gene confers both Sertoli cell-specific and stagespecific expression of a reporter in transgenic mice. This application also proposes to examine the function of cathepsin L in the seminiferous epithelium. Those experiments are prompted by our observation that mice which express catalytically inactive cathepsin L exhibit an increased incidence of seminiferous tubule atrophy and produce 30% fewer spermatids in nonatrophic tubules than are produced by control mice. Building on all of these data, this proposal asks four questions which are our specific aims: 1 What are the specific cis-acting elements in the activation domain and what are their functions? 2. What is the identity of the transcription factors that bind to the maturation domain? Is their expression stage-specific amd maturation-dependent? 3. What cis-acting elements and which spermatogenic cells repress cathepsin L promoter activity in Sertoli cells? 4. What is the function of cathepsin L in the seminiferous epithelium? [unreadable] [unreadable] [unreadable]

This award will enable a five-year project using the science of dendrochronology to examine the relationship between the Asian monsoon and the large-scale coupled processes that drive much of its variability. The researchers will examine these interactions on inter-annual to centennial time scales and anticipate that this project will yield information about how the monsoon system has varied over the past several centuries. Specifically, they will investigate the relationship of the Asian Monsoon to the large-scale coupled processes that drive much of its variability; namely air temperatures and sea surface temperatures (SST).

The project is motivated by a series of research question such as: "How have the processes responsible for driving much of the observed Asian monsoon climate dynamics varied and interacted on inter-annual to centennial time scales?" and "How have Asian surface air temperatures, Indian Ocean SSTs, and Tropical Pacific SSTs covaried and interacted over the past several centuries?"

The Asian monsoon climate system plays a significant role in large-scale climate variability over much of the globe. Due to its considerable importance to global climate and implications for the world's population, a greater understanding of this system, with the ultimate goal being improved prediction on annual to decadal and longer time scales is warranted.

This research will help the science community understand climate variability in a densely populated region of the world. On the practical side, improved knowledge of climate variability could be used to improve long-term agricultural planning in the region by buffering agrarian societies against the effects of monsoon variability. The award will support post-doctoral, graduate, and undergraduate students at Columbia University and the Lamont Doherty Earth Observatory and help build scientific capacity, infrastructure, and professional partnerships among Asian colleagues.

ABSTRACT PI WRIGHT # IOS-0721800

William G. Wright
IOS-0721800
Predation-induced sensitization in Aplysia californica

Understanding how we learn and remember, a fundamental goal of basic research, has been greatly enriched by the study of behavioral sensitization in the marine snail Aplysia californica. Sensitization refers to the observation that a noxious stimulus may result in an increased likelihood of performing defensive behaviors. Four decades of experiments in Aplysia have unveiled a vast landscape of knowledge about sensitization, ranging from behavioral to molecular levels. Earlier research by the PI and colleagues established an evolutionary history of neural mechanisms of sensitization in related sea hares, but knowledge of the ecological basis of this evolution is completely lacking. The PI and his undergraduate colleagues will explore the hypothesis that sensitization benefits a survivor of a predatory attack by decreasing its risk of subsequent attack. Their pilot experiments demonstrated that attacks by two different co-occurring predators cause sensitization with distinct anatomical and temporal differences, suggesting specific anti-predator strategies. This research will further explore these learning differences, will analyze their neuronal basis using conventional nerve cell recording techniques, and will identify other ecologically relevant behavioral changes, (e.g., locomotion, movement toward light) following sub-lethal predator attack. This research will thus provide a uniquely integrative ecological picture of sensitization in Aplysia.
The PI has extensive experience with all proposed methods, including coordination of a group of undergraduates to execute them. This proposal represents a rare opportunity for 15-20 undergraduates (across 3 years) to do full-time summer research on a question with fully publishable answers. The students will perform experiments, analyze data, present results at national meetings, and publish in peer-reviewed journals. Field observations and experiments by additional students in an upper level Marine Biology (Biol440) course will be directly integrated into this research.

The continuous production of male gametes requires the precise regulation of the differentiation and replication of stem spermatogonia. These critical processes occur in a niche that is a dynamic environment created in part by Sertoli cells, the somatic cells with which all spermatogenic cells interact. As yet, the in vivo mechanisms by which Sertoli cells regulate stem spermatogonial differentiation and replication have not been elucidated. Neither is it understood how those regulatory functions of Sertoli cells are themselves controlled. The central hypothesis of this application is that differentiated spermatogenic cells control the expression by Sertoli cells of growth factors that regulate the differentiation and replication of stem spermatogonia. We propose that by regulating expression of those growth factors, differentiated spermatogenic cells control the functioning of the stem spermatogonia niche. Using mice as a model, this proposal focuses on three growth factors that been shown to stimulate the growth of stem spermatogonia either in vivo or in vitro. These growth factors are: glial cell line derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2) and insulin like growth factor 1 (IGF-1). Specific Aim I tests the hypothesis that in vivo the stage-specific expression of GDNF by Sertoli cells regulates replication and differentiation of stem spermatogonia and that FGF-2 and IGF-1 act in consort with GDNF in the regulation of these processes. Specific Aim II tests the hypothesis that Type A1 spermatogonia or more advanced spermatogenic cells regulate expression of GDNF, FGF-2 and IGF-1 by Sertoli cells. To begin to define the molecular basis of this regulation, we also propose an unbiased search for transcription factors and components of signal transduction pathways that mediate the effects of spermatogenic cells on GDNF expression by Sertoli cells. Specific Aim III will test the hypothesis that differentiated spermatogenic cells by their feedback on Sertoli cells control stem spermatogonia renewal and differentiation. Stage-specific gene expression by Sertoli cells is a well-established phenomenon in many species, including humans and GDNF, IGF-1 and FGF-2 are all expressed in human testes. Therefore, results from our proposed experiments hold promise for understanding the biological basis of the infertility of some men.

[unreadable] DESCRIPTION (provided by applicant): Spermatogenesis occurs in a unique environment that is created by the somatic Sertoli cells. Not only do these cells bind to the developing male germ cells and spatially organize them within the seminiferous epithelium, they also secrete a large number of paracrine factors, carrier proteins, proteases and protease inhibitors. While it is assumed that these secretory products affect germ cell development, it is remarkable that to date no Sertoli cell secretory product has been proven to be required for production of fertile male gametes. This application tests the hypothesis that the secretion by Sertoli cells of the protein, Doppel, is absolutely required for development of fertile sperm. The rationale for this hypothesis comes from four observations. First, published data from two laboratories demonstrate that male mice with a knockout of the gene encoding Doppel, Prnd, are infertile because their sperm are incapable of undergoing the acrosome reaction. Second, the testis expresses substantially higher concentrations of Prnd mRNA than any other organ. Third, most of the Doppel in the testis is localized to Sertoli cells. Fourth, recent data from our laboratory indicate that Sertoli cells express very high levels of Prnd mRNA while spermatids, spermatocytes and spermatogonia do not express detectable levels of this transcript. It should be noted, however, that Prnd mRNA is also expressed in the caput epididymis of the mouse, albeit at much lower levels than in the testis. Thus, while all data identify Sertoli cells as the most likely source of the Doppel that is required for male fertility, one cannot a priori exclude a role for Doppel produced by the epididymis. The goal of this R03 application is to specifically evaluate the importance to fertility of the secretion of Doppel by Sertoli cells. We propose to generate transgenic mice that carry a floxed Prnd allele and to breed these mice to Amh-Cre mice, which express Cre recombinase specifically in Sertoli cells. The Sertoli cells of their progeny will have a conditional knockout of the Prnd gene. We also propose to breed mice with the floxed Prnd allele to EIIa-Cre mice, which express Cre recombinase as preimplantation embryos. [unreadable] Public Health Relevance: All cells of their progeny will have a conditional knockout of the Prnd gene. We will then compare the effects on fertility of the specific loss of Doppel secretion by Sertoli cells to the effects of the loss of Doppel secretion by all cells. By making this comparison, we will directly test the hypothesis that the secretory product of Sertoli cells, Doppel, is required for development of fertile sperm. Sertoli cells are the somatic cells that support and nourish developing male gametes. These cells bind to spermatogenic cells, spatially organize them within the seminiferous epithelium and secrete a large repertoire of products that are assumed to be required for male fertility. However, despite the obvious intimate relationship between Sertoli cells and spermatogenic cells, the crucial mechanisms by which Sertoli cells support spermatogenesis are mostly unknown. This application will determine if the Sertoli cell secretory protein, Doppel, is essential for male fertility. We propose to generate mice with a conditional knockout in Sertoli cells of the gene that encodes Doppel and to determine if loss of Doppel secretion by Sertoli cells renders male mice infertile. Completion of these proposed experiments hold promise for providing the first direct evidence that a Sertoli cell product is required for male fertility. Such evidence may open new avenues to the study of male fertility and, thus, to the treatment of the large population of infertile men that live in the United States. [unreadable] [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY (See instructions): An essential requirement for sustaining male fertility is maintaining an adequate number of stem spermatogonia, the foundation of spennatogenesis. To achieve this, when the stem cells divide, some progeny must remain stem spermatogonia while other progeny differentiate. It is obvious that the correct balance between self-renewing replication and differentiation of stem spermatogonia is crucial to male fertility, and there is indirect evidence that GDNF plays an important role in maintaining this balance in the normal mature testis. However, we known almost nothing about the in vivo regulation of this balance in the mature organ, of the specific function of GDNF in the adult testis, or if physiological changes in GDNF expression significantly affect the replication or differentiation of the stem cells. To address these critical issues, we have developed a unique mouse model that allows GDNF signaling to the stem spermatogonia to be specifically and reversibly inhibited in vivo by an ATP antagonist. Using this model, we have generated the first direct evidence that GDNF is required for maintaining the stem spermatogonial pool in a normal mature testis. Additionally, we have shown that when inhibition of GDNF signaling is reversed, the stem cells begin to rebuild the stem cell pool. Importantly, our data demonstrate that some stem spermatogonia are lost when GDNF signaling is inhibited for as little as 2 days, while other stem cells survive for up to 11 days. This suggests that factors intrinsic or extrinsic to the stem cells modulate the response to GDNF. Using this new mouse model we propose in Specific Aim 1 to define the mechanisms'responsible for the loss of stem spermatogonia upon inhibition of GDNF signaling and to detenmine why some cells are lost rapidly while others more slowly. Building on these results, we also propose to collaborate with Project 1 to determine if GDNF mRNA expression and/or stem spermatogonial numbers are significantly reduced in some oligospermic men. Specific Aim 2 studies the capacity of stem spermatogonia to repopulate the testis once their numbers are partially depleted. In doing so, we will identify the extent to which a defined number of stem spermatogonia can restore this pool and the biological processes responsible for this restoration. Additionally, in collaboration with Project 1 we will study oligospermic men who are receiving endocrine therapy to increase} sperm production in order to determine if in men who have a positive response to therapy, there is bath an increase in GDNF expression and/or an increase in numbers of stem spermatogonia. To jour knowledge, these specific aims constitute the first detailed in vivo analysis of theresponse of stem sjaermatogonia or any other adult stem cell to the inhibition and then restoration of signaling by an essbntial growth factor. We anticipate that the results from these studies will make an important contribution to the evaluation and eventually the treatment of infertile men.

DESCRIPTION (provided by applicant): This R13 conference grant application requests funds to support the XXIInd North American Testis Workshop that will be held from April 13 to April 16, 2013 in San Antonio, Texas. Since 1972, the North American Testis Workshop has been the premiere international forum for bringing basic and clinician-scientists together to present and discuss the most recent findings on the regulation and function of the fertile and infertile testis The first goal of this meeting is to promote solutions for two major medical and public health problems: the high incidence of male infertility in the developed world and the lack of a safe and reversible male contraceptive. Another important goal of this meeting is to foster the development of future leaders in this field, especially if they currently are trainees, or if they re women or underrepresented minorities. The theme of the XXIInd North American Testis Workshop is, The Foundations of Male Fertility. The program includes three major lectures: the keynote address, the benchmark lecture, and the European Molecular Biology Organization Young Investigator Lecture. There also will be 18 invited talks, which are grouped in five sessions. Session 1 is: Regulation and Restoration of Fertility in Men; Session 2 is: Genome Integrity; Session 3 is: Regulation of Gene Expression; Session 4 is: Somatic Cells; and Session 5 is: Stem Cells and Their Niche. In addition to the invited talks, each session will end with two short talks from abstracts that are submitted by young investigators (trainees and Assistant Professors who have been in rank for no more than 2 years.) The meeting also includes two poster sessions, and the posters will remain on display throughout the meeting. The keynote address begins the meeting and sets its tone. Dr. David Zarkower, Professor of Cell Biology and Genetics at the University of Minnesota, will present this lecture. Dr. Zarkower is one of the world's leading experts in the molecular and genetic control of sex determination. His research focuses on the function of DMRT1 and other DM-domain genes in sex determination of metazoans. As the key event in testis determination is the formation of Sertoli cells, his talk provides a foundation for Session 4, Somatic Cells. Additionally, recent evidence indicates that mutations in the DMRT1 gene cause testicular cancer, a subject that is addressed in Session 2. The benchmark lecture highlights major advances in our understanding of testis regulation that are achieved through use of state-of-the-art technologies. Dr. Michael Griswold, Regents Professor, Washington State University, will present a genome-wide perspective on the regulation of spermatogenesis. Dr. Griswold pioneered the use of microarrays in the study of spermatogenesis and is now applying deep sequencing for a more detailed view of the changes in gene expression during male germ cell development. His talk will provide a foundation for Session 3. Dr. Antoine Peters of the Friedrich Miescher Institute will present The European Molecular Biology Organization Young Investigator Lecture. This lecture will address the epigenetic regulation of germ cell development, a topic that is also addressed in Session 3. A notable aspect of the program is the topic of Session 1, Regulation and Restoration of Fertility in Men. The Program Committee decided to begin with this topic in order to stimulate discussion throughout the meeting on the translation of basic studies in testis regulation and function to the treatment of infertile men and the development of male contraceptives. Presentations in the subsequent four sessions complement those in Session 1. In this application, we request funds to partially defray the cost of attendance of the 10 young investigators whose abstracts will be selected for short platform talks. The rest of the funds will defray part of the housing costs for the 20 distinguished scientists who have been invited to speak at this meeting.

The past decade has witnessed numerous developments in technology that can be applied to rehabilitation practice. The biennial International Conference for Virtual Rehabilitation (ICVR) evolved from the desire and need for rehabilitation practitioners to play an integral role in the development and evaluation of new technologies, which incorporate virtual reality (VR) technology. One goal of the conference is to assist engineers who develop the technology to recognize the value they could derive by consulting with rehabilitation professionals in order to make their machine-user interfaces more efficient, user friendly, and effective for specific disabilities. Another goal is to advance translation efforts that bridge the gap between knowledge generation from research and knowledge uptake in clinical practice so that rehabilitation professionals become more comfortable with technology and recognize how to incorporate it into their individualized interactions with patients. ICVR 2013, the 9th conference in the series, will be collocated with the 8th annual Saffran conference entitled "Virtual Reality Technology and Rehabilitation of Speech and Language Disorders," which will focus on virtual reality applications being developed for rehabilitation of aphasia. The joint ICVR-Saffron conference is a 4-day event which will take place August 26-29, 2013, in Philadelphia. A combined attendance of approximately 150-200 at the two conferences is expected from clinical practitioners, scientists, faculty and students with interest in the many disciplines ICVR has historically embraced, such as motor rehabilitation with virtual technology, brain-computer interfaces, rehabilitation robotics, haptic interfaces, novel applications of game consoles, psychological and environmental rehabilitation in virtual reality, cognitive rehabilitation in virtual reality, tele-rehabilitation, balance and gait rehabilitation in virtual reality, regulatory and educational efforts to promote virtual rehabilitation, and sociological, demographic and legal aspects of virtual rehabilitation. More information about the ICVR conference is available online at http://virtual-rehab.org/2013.

This is funding to promote student involvement and educational initiatives in these conferences. Aside from the usual reduced student registration fees and stipends to assist with hotel and travel costs, four novel events are planned for ICVR 2013, three of which are specifically designed with student and post-doc advancement in mind. First, there will be two moderated poster sessions with one session dedicated specifically to student presenters. Posters will be grouped by topic area with teams of two senior investigators assigned to all posters within a topic area, who will facilitate discussion following a brief presentation by the investigator. Coffee breaks and dessert will be provided during poster sessions to encourage attendance. Second, lunch on Day 3 will be lengthened and held in the room with the commercial exhibitors, who will be encouraged to provide hands-on experiences with their technologies. Third, on Day 4 an oral platform session by graduate students and post-docs will give them the opportunity to present their research; student presentations will be selected by members of the organizing committee based on a written recommendation by the student's mentor and a one-page abstract. (these abstracts will be uploaded as part of the conference proceedings). Lastly, Day 4 will conclude with a panel-led discussion where clinicians, scientists, and technology developers can generate ideas about future promotion of technology transfer into the clinical environment; one student and one post-doc will be invited to serve on this panel. The net result of these innovations is that ample opportunities will exist for students and young investigators to interact with the keynote speakers, established scientists and clinicians throughout the conference. The organizers expect these initiatives will encourage more students to attend, both by reducing financial burden and by rewarding meritorious scientific achievements.

Broader Impacts: Raising awareness for science and technology students of the need for their skills in the interdisciplinary field of technology-oriented rehabilitation science is a necessary step to promoting interest in the field. Informing students at the earliest possible stages of their education about the opportunities that will be available to them later will help remove one major barrier to interdisciplinary collaboration, which is student readiness to engage in this type of research. This conference will create a forum whereby students and experts alike can gain interdisciplinary knowledge and develop a common language. Not only will this conference increase the opportunity to form interdisciplinary partnerships, but it will also help bridge any generational gap that exists as we transition from the traditional compartmentalized modes of research to new cross-disciplinary modes. The conference organizers will take special steps to encourage the participation of racial/ethnic minorities and persons with disabilities.

? DESCRIPTION (provided by applicant): This R13 conference grant application requests funds to support the XXIIIrd North American Testis Workshop that will be held from April 15 to April 18, 2015 in Salt Lake City, Utah. Since 1972, the North American Testis Workshop has been the premiere international forum for bringing basic and clinician-scientists together to present and discuss the most recent findings on the regulation and function of the fertile and infertile testis The first goal of this meeting is to promote solutions for two major medical and public health problems: the high incidence of male infertility in the developed world and the lack of a safe and reversible male contraceptive. Another important goal of this meeting is to foster the development of future leaders in this field, especially if they currently are trainees, or if they re women or underrepresented minorities. The theme of the XXIIIrd North American Testis Workshop is, Health Sperm-Healthy Children. The program includes four major lectures: the keynote address, and three benchmark lectures from international speakers. There also will be 17 invited talks, which are grouped in six sessions. Session 1 is: Male Fertility Across Generations - Setting the Stage; Session 2 is: Healthy Stem Cells, Healthy Aging; Session 3 is: Regulation of Gene Expression from Progenitor Cells through Meiosis; Session 4 is: Determining and Perturbing Testicular Function; Session 5 is: Somatic Cells; and Session 6 is: Human Fertility, Infertility and the Next Generation. In addition to the invited talks, each session will end with to short talks from abstracts that are submitted by young investigators (trainees and Assistant Professors who have been in rank for no more than 2 years.) The meeting also includes two poster sessions, and the posters will remain on display throughout the meeting. The keynote address begins the meeting and sets its tone. Dr. Amander Clark, from the Department of Molecular, Cell and Developmental Biology at the University of California in Los Angeles will present this lecture. Dr. Clark is an engaging, motivating lecturer and one of the world's leading experts in the study of human primordial germ cells. Abnormal development of the early germ cell lineage can result in infertility, germ cell tumor formation or result in birth defects in the offspring. Her talk provides a foundation for many of the talks throughout the meeting especially those on germ cells and their unique genetic and epigenetic programs. Furthermore, the keynote address links well with the final session of the meeting on human fertility. The first benchmark lecture highlights major advances in our understanding of primate spermatogonial stem cells and their clinical applications. Dr. Stefan Schlatt, Professor at the University of Muenster, Germany, is internationally recognized for his research using of the non-human primate model in the study of spermatogenesis and spermatogonia. His talk will provide a strong foundation for Session 2. Professor Sarah Robertson of the University of Adelaide in Australia will present the second benchmark lecture addressing how factors in seminal fluid can interact with the maternal uterine environment to influence the metabolic phenotype of the offspring, a dramatic example of paternally-mediated intergenerational effects, a topic that is also addressed in Session 1. The third benchmark lecture will be delivered by Professor Lee Smith from the MRC Centre for Reproductive Health at the University of Edinburgh. Professor Smith's group specializes in identifying genetic and hormonal control signals that support male fertility and steroid hormone (testosterone) production. His topic links not only to basic topics in sessions 2 and 5 but also focuses on the important question of how androgen production is controlled in the testis and how this influences male fertility. In this application, we request funds to partially defray the cst of attendance of the 10 young investigators whose abstracts will be selected for short platform talks. The rest of the funds will defray part of the housing costs for the 21 distinguished scientists who have been invited to speak at this meeting.

This award will fund the purchase of a scanning micro X-Ray Fluorescence (Micro-XRF) spectrometer for dendrochemical and other research. The research it enables is significant because it permits scientists to reconstruct multiple aspects of past environments and environmental change over time. Many species of trees add a visibly distinct ring of wood each year. The width of the annual ring may vary substantially from year to year, and these patterns of variation may be sufficiently distinctive to allow wood from a given site to be dated by comparison to a master chronology for the region. Dendrochronology was first developed at the University of Arizona in the 1930's. Once dated, the rings may be used to reconstruct variation in the factors controlling ring width. These include annual rainfall, annual temperature (at high altitude), the frequency of forest fires, and in which region the tree grew. A relatively new science is dendrochemistry - the study of variation in chemical composition from ring to ring within a tree. This award will allow researchers at the University of Arizona to undertake novel dendrochemical research on the enormous archive of cores from modern and ancient wood in the University of Arizona's Laboratory of Tree Ring Research (LTRR). The instrument will also be a valuable tool for LTRR'S outreach work with visiting high school science classes.

The instrument will be installed in LTRR. A priority will be the search for evidence in dated tree rings of former volcanic eruptions and major forest fires, both of which result in spikes of certain chemical elements that are then taken up by growing trees. Since volcanic eruptions often spread ash (microtephra) over a very large area, they offer a potentially valuable means of correlating archaeological and paleoenvironmental sequences across a large region. Dendrochemical analysis by micro-XRF potentially offers a quick and efficient means of detecting and dating these past eruptions. An estimated 30-40,000 wood samples in LTRR cannot currently be assigned a cutting date because the outer ring is indistinct. Pilot research has however shown distinctive chemical changes at the heartwood/sapwood junction in several species, and also in the penultimate ring. Micro-XRF will be used to scan currently undated cores to look for these signals, from which cutting dates can be derived. This research can potentially make available many more dated samples for archaeological and paleoenvironmental interpretation.

Abstract Drug addiction is a prominent medical and social problem with no effective treatments. The malfunction of the nucleus accumbens (NAc) has been identified to critically contribute to the development of addiction and related behaviors. To understand the drug-induced functional alterations in the NAc contributing to addiction, extensive efforts have been spent investigating NAc medium spiny neurons (MSNs), which constitute over 90% of NAc neurons and directly mediate the output of the NAc. In contrast fast-spiking interneurons (FSIs) have largely been ignored. While FSIs only constitute a very small portion of NAc neurons (<1%), our preliminary results show that FSIs within the NAc provide robust inhibition to MSNs in response to specific excitatory inputs, gating MSN activity and in turn shaping the functional output of the NAc. This regulation allows FSIs the capacity to greatly influence behaviors mediated by the NAc, including those associated with addiction. The objective of this application is to determine the role of FSIs in mediating addiction-related behaviors, using a clinically relevant rodent model of cue-induced cocaine seeking. Our preliminary studies have found that upon exposure to cocaine-associated cues following abstinence from cocaine self- administration, FSIs display a transient increase in activity while MSNs are simultaneously suppressed. This timing-contingent cue-induced activity pattern and other results lead us to hypothesize!that the local FSI-MSN connection dictates the output of NAc by controlling the activation of MSNs to promote cue-induced cocaine seeking after abstinence. We will test this hypothesis by pursuing two aims. Our Aim 1 will thoroughly characterize the cellular and circuit properties of FSI-mediated regulation of MSNs within the NAc to understand the mechanistic and functional dynamics of this circuit in gating the functional output of the NAc to influence behavior. Our Aim 2 will directly test the hypothesis that cue-induced FSI activity gates MSNs within the NA to promote cue-induced cocaine seeking after withdrawal from cocaine self-administration. The expected outcomes will provide a new conceptual basis in understanding how information is integrated and computed within the NAc to shape addiction-related behaviors by introducing FSIs as critical components within the NAc circuit. This information may provide new avenues for the development of novel therapeutic strategies that target this small neural population for the treatment of addiction. As such, this proposal is highly relevant to the mission of NIDA and NIH.