James Hutson - US grants

We have shown that macrophages isolated from the testis respond to FSH while macrophages isolated from the peritoneal cavity do not. FSH stimulated protein and lactate secretion in a dose-dependent manner in vitro and in vivo. Unlike the effects of FSH Sertoli cells, the accumulation of intracellular protein by macrophages was unaffected by FSH. LH, insulin, testosterone, or estradiol had no similar effects. Macrophages obtained from the peritoneal cavity were not responsive to FSH. Most recently, we have obtained evidence that secreted products of macrophages influence testosterone secretion by cultured Leydig cells. Since it is not known if these two phenomena are causally related, the experiments of this proposal will be undertaken in two phases to evaluate this possibility. First, we will determine the molecular weights and isoelectric points of secreted proteins using two-dimensional polyacrylamide gel electrophoresis. These studies will allow us to compare the pattern of proteins secreted by macrophages with and without FSH treatment and from macrophages from various tissues. These studies will allow us to determine if the secreted proteins of testicular macrophages are different from those secreted by macrophages from other tissues and also provide information concerning the ability of FSH to increase the appearance of new proteins. We will also determine if these proteins have similar biological activities (lysozyme and collagenase activity) as those proteins known to be secreted by macrophages from other tissues. Finally, we will test the validity of our preliminary studies which indicated that macrophages are capable of influencing testosterone secretion by Leydig cells. The general chemical nature (heat stability, molecular weight, solubility in organic solvents and activity after charcoal stripping) of the putative secretory product(s) that macrophages produce that are responsible for influencing testosterone secretion in Leydig cells will be determined. We will also determine if FSH stimulates the secretion of this factor(s) and if the factor(s) is(are) capable of potentiating the effects of LH on Leydig cells.

Sertoli cells; testis; cell cell interaction; all trans retinol; cyclic AMP; insulin; hormone regulation /control mechanism; androgens; aging; testosterone; cell type; follicle stimulating hormone; uridine; tissue /cell culture; phase contrast microscopy;

Introduction: Macrophages reside within the interstitial compartment of the testis and form junction complexes with Leydig cells in vivo. We have previously shown that testicular macrophages produce a factor that stimulates testosterone secretion by Leydig cells while peritoneal macrophages have no effect. The purpose of the studies of this application is to investigate the development and regulation of this paracrine phenomenon. Hypothesis: Monocytes from bone marrow migrate to the testis at an important time of development and a subpopulation of these cells are then influenced by local testicular factors in interact with Leydig cells. Specific Aims: 1. determine if the initial appearance of macrophages in the testis or the development of junctions between Leydig cells and macrophages occurs during an important time in testicular development, 2. determine if factors from the local testicular environment are involved in the differentiation or regulation of macrophages. Methods: We will determine the stage of development that macrophages first appear in the testis using light microscopic immunocytochemical methods. To determine when junctional complexes form between the macrophages and Leydig cells we will study the testes from animals at various stages of development or postnatal maturation using electron microscopic techniques. We will employ retrovirus mediated gene-transfer studies to determine if stem cells in the bone marrow produce testicular macrophages. To determine if local testicular factors are capable of regulating or inducing the testis- specific phenotype, we will treat macrophages from the testis, blood (monocytes), peritoneum, lung and ovary with factors form testicular extracts, interstitial fluid or culture medium of various testicular cell types and then assay for testis-specific functions. Significance: These studies will contribute to an understanding of the regulation and differentiation of the interaction between macrophages and Leydig cells. Since these multidisciplinary studies are broad-based and employ both in vivo and in vitro methods, important information concerning the physiological relevance of testicular macrophages will be gained.

It has long been established that in the absence of LH, as occurs in hypophysectomized animals, Leydig cell development and function are drastically altered. However, it has recently been shown that even when LH is present, Leydig cells produce far less testosterone when macrophages are depleted from the testis. Similarly, when macrophages are depleted from the testis of immature animals, Leydig cells fail to develop properly. Thus testicular macrophages, like LH, are essential for normal function of Leydig cells. We have partially purified and characterized a lipophilic factor secreted by macrophages that stimulates testosterone production by Leydig cells. We hypothesize that this factor mediates some of the actions that macrophages exert on Leydig cells that have been described in these macrophage-depletion studies, including a role in Leydig cell maturation, stimulation of testosterone production, and involvement in maintaining LH- responsiveness. Thus this factor has the potential to be of great physiological significance. In Aim 1 we propose to use mass spectroscopy, nuclear magnetic resonance, and other physico-chemical approaches to identify the chemical structure of this factor. A CoPI and a collaborator have been added to the proposal as recommended who have expertise in this area. In Aim 2, we will continue studies concerning the mechanism of action of the factor. We have found that the factor elicits a greater response than does LH/hCG, does not require new synthesis of StAR, does not act directly on mitochondria, is not itself a substrate for steroidogenesis, and exerts its influence proximal to cholesterol sidechain cleavage, all indicating that its mode of action is very different than that of LH. We now propose to determine if the factor activates protein kinase A and/or C, causes changes in calcium metabolism, and/or changes in intracellular levels of cAMP. In Aim 3, we will determine if the factor is produced by testicular macrophages from immature animals. If so, we will then test its ability to induce maturation of postnatal Leydig cells. Finally, we will determine if the factor can restore Leydig cell function (testosterone production and ability to respond to LH) in macrophage- depleted animals. These gain-of-function studies will determine the physiological significance of this factor, and if positive results are obtained, new opportunities to treat infertility and/or create new male contraceptives may emerge, since testicular macrophages are present in high numbers in humans as they are in rodents.