Production of 25-hydroxycholesterol by testicular macrophages and its effects on Leydig cells.

Testicular macrophages secrete 25-hydroxycholesterol, which can be converted to testosterone by neighboring Leydig cells. The purposes of the present studies were to determine the mode of production of this oxysterol and its long-term effects on Leydig cells. Because oxysterols are produced both enzymatically and by auto-oxidation, we first determined if testicular macrophages possess cholesterol 25-hydroxylase mRNA and/or if macrophage-secreted products oxidize cholesterol extracellularly. Rat testicular macrophages had 25-hydroxylase mRNA and converted 14C-cholesterol to 14C-25-hydroxycholesterol; however, radiolabeled cholesterol was not converted to 25-hydroxycholesterol when incubated with medium previously exposed to testicular macrophages. Exposure of Leydig cells to 10 microg/ml of 25-hydroxycholesterol, a dose within the range known to result in high basal production of testosterone when tested from 1 to 6 h, completely abolished LH responsiveness after 2 days of treatment. Because 25-hydroxycholesterol is toxic to many cell types at 1-5 microg/ml, we also studied its influence on Leydig cells during 4 days in culture using a wide range of doses. Leydig cells were highly resistant to the cytotoxic effects of 25-hydroxycholesterol, with no cells dying at 10 microg/ml and only 50% of cells affected at 100 microg/ml after 2 days of treatment. Similar conditions resulted in 100% death of a control lymphocyte cell line. These results demonstrate that 1) testicular macrophages have mRNA for cholesterol 25-hydroxylase and can convert cholesterol into 25-hydroxycholesterol, 2) macrophage-conditioned medium is not capable of auto-oxidation of cholesterol, 3) Leydig cells are highly resistant to the cytotoxic influences of 25-hydroxycholesterol, and 4) long-term treatment with high doses of 25-hydroxycholesterol results in loss of LH responsiveness. These results support the concept that testicular macrophages enzymatically produce 25-hydroxycholesterol that not only is metabolized to testosterone by Leydig cells when present at putative physiological levels but also may exert inhibitory influences on Leydig cells when present for extended periods at very high concentrations that may occur under pathological conditions.

Specificity of a new lipid mediator produced by testicular and peritoneal macrophages on steroidogenesis.

Macrophage-derived factor (MDF) is a lipophilic factor produced by rat testicular and peritoneal macrophages that maximally stimulates testosterone production by rat Leydig cells through a steroidogenic acute regulatory protein independent mechanism. The purpose of the present study was to determine whether MDF is also produced by human macrophages, and/or if it acts on human steroidogenic cells. We also studied the tissue-specific functions of MDF by determining if it also acts on steroidogenic cells of the ovary and adrenal glands and, if so, does it require new protein synthesis. It was found that MDF was produced by human peritoneal macrophages, and was capable of stimulating human steroidogenic cells. In terms of tissue specificity, it was found that primary cultures of rat adrenocortical cells respond to MDF with increased secretion of aldosterone and corticosterone, as did rat granulosa cells by producing progesterone. MDF acted in the presence of cycloheximide, indicating that it does not require new protein synthesis. These results indicate that MDF may have significant therapeutic potential and provide a basis for future studies concerning its physiological role in humans. These results further suggest that MDF is not only involved in paracrine regulation of Leydig cells, but also has the potential for the local regulation of steroidogenesis in both granulosa and adrenal cortical cells.

Identification of the lipophilic factor produced by macrophages that stimulates steroidogenesis.

Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns. A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol. Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol. When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected. Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity. Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions. Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.

Short-term leptin infusion does not affect circulating levels of LH, testosterone or cortisol in food-restricted pubertal male rhesus macaques.

OBJECTIVES: Although the adipocyte protein leptin has been implicated in the control of reproductive function in rodents, its role in primate reproductive physiology is poorly understood. Because primates in puberty show nighttime LH secretion and there is considerable evidence that the fertile state requires adequate nutrition, we reasoned that animals on the verge of reproductive competence would respond to leptin infusions by secreting LH. Food restriction reduces circulating leptin levels and slows or stops the GnRH pulse generator. Therefore, we examined the endocrine effects of leptin infusions in food-restricted male pubertal primates during the night when they normally secrete LH. In addition, we investigated the effect of leptin on in vitro testosterone production by Leydig cells. SUBJECTS: Four pubertal male rhesus macaques (Macaca mulatta), 4-5.5 kg in weight (2.5-4-year-old) were examined in this study. Leydig cells from adult male rats were to investigate in vitro effects of leptin. DESIGN: To document that animals had entered puberty, blood samples were collected from each of the four animals at 15-minute intervals for 15 h both during the day and at night. Since at this age animals secrete LH mainly at night, blood samples were collected at 15-minute intervals from each of the four animals on two separate occasions for 15 h between 1500 and 0600h. During the experiment, animals were feeding from 0800 to 0830h, cages were completely cleaned of food at 0900h and the afternoon meal was not given to individual animals on the day they were studied. One of the studies served as the control (food restricted group) and during the other, 2 mg (n = 4) or 0.3 mg (n = 3) of recombinant human leptin was administered intravenously during 2000-0100h (food restricted plus leptin group). Blood samples (1 ml) were collected through the indwelling catheter and immediately transferred from the plastic syringe into chilled glass tubes containing 10 microl 14% EDTA. The samples were centrifuged at 5-h intervals and the plasma withdrawn and stored frozen at - 20 degrees C in polypropylene vials until assays were performed. MEASUREMENTS: Bioactive LH was determined and testosterone, cortisol and leptin were measured by radioimmunoassay. RESULTS: During daytime experiments in these animals, LH pulses were sometimes observed late in the day and generally continued for 12-15 h. Food-restricted pubertal males showed delayed or absent LH pulses. Short-term leptin administration to food-restricted male rhesus macaques had no effect on LH, testosterone, or cortisol levels either during or after the infusion. Leptin also had no direct effect on basal or LH-stimulated testosterone production in Leydig cells. CONCLUSIONS: Our data support the notion that leptin is not the missing signal for the acute suppression of reproductive hormones secretion in food-restricted primates.

Regulation of Leydig cells through a steroidogenic acute regulatory protein-independent pathway by a lipophilic factor from macrophages.

The purpose of this investigation was to study the mechanism of action of a macrophage-derived factor that stimulates steroid production by Leydig cells. This factor increased testosterone production within 30 min, and reached a half-maximal response by 6-8 h. At a maximal dose, it stimulated testosterone production 20-fold at 24 h. Its efficacy was consistently higher than that achieved with a maximal dose of human chorionic gonadotropin (hCG). However, Leydig cells treated with a maximal dose of both the macrophage-derived factor and hCG secreted the same amount of testosterone as when given a maximal dose of only the macrophage-derived factor. The macrophage-derived factor did not require new protein synthesis to stimulate testosterone production, nor did it alter the amount of steroidogenic acute regulatory protein (StAR). While the macrophage-derived factor required an active cholesterol side-chain cleavage complex system, it did not alter the capacity of this enzyme complex. Finally, the macrophage-derived factor was unable to stimulate the production of progesterone by isolated mitochondria. In summary, the macrophage-derived factor is a highly active, acute regulator of steroidogenesis that acts through a high capacity StAR-independent pathway.

FSH does not directly influence testicular macrophages.

We have previously demonstrated that conditioned medium from testicular macrophages stimulates testosterone production by Leydig cells. It was also reported that conditioned medium from macrophages treated with follicle-stimulating hormone (FSH) had an even greater amount of Leydig cell-stimulating activity than medium from untreated macrophages, indicating that this factor is under the regulation of FSH. However, most other laboratories have been unable to reproduce this effect of FSH. We have recently purified and partially characterized the stimulatory factor from macrophage-conditioned medium that stimulates Leydig cells. The purpose of the present investigation was to reinvestigate the effect of FSH by determining whether it regulates the production of this purified factor and by determining whether macrophages have mRNA for the FSH receptor. Testicular macrophages were isolated from adult rats and incubated 24 hours with human recombinant FSH (20 units/ml), ovine FSH (200 ng/ml), fetal bovine serum (2%), or dibutyryl cyclic adenosine monophosphate (1 mM). The macrophage-derived factor (MDF) was then purified from conditioned medium of the various treatment groups and added to Leydig cells. The concentration of testosterone in the Leydig cell medium was then measured after 16 hours. It was found that serum significantly stimulated production of the MDF. However, FSH had no effect on production of the MDF in the presence or absence of serum. Dibutyryl cyclic adenosine monophosphate exerted a slight inhibitory effect on production of the macrophage-derived factor. Most importantly, testicular macrophages did not express detectable levels of FSH receptor mRNA, either in vivo or in vitro, when evaluated using either in situ hybridization or northern analysis, under identical conditions that clearly demonstrated FSH receptor mRNA in Sertoli cells. We conclude that testicular macrophages are not a direct target for FSH.

Ethane dimethane sulfonate and NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine inhibit steroidogenic acute regulatory (StAR) protein expression in MA-10 Leydig cells and rat Sertoli cells.

Apoptosis inhibits steroid biosynthesis, but it is not clear how the Steroidogenic Acute Regulatory (StAR) protein, is affected. To characterize StAR expression during apoptosis, mouse MA-10 Leydig tumor cells were treated with ethane dimethane sulfonate (EDS), an inducer of apoptosis, and the metal ion chelator NNN'N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), an inducer of cell death. Both chemicals induced cell death and similarly inhibited dbcAMP-stimulated steroidogenesis and accumulation of the 30 kDa form of StAR. Utilizing the dye JC-1, it was found that TPEN and EDS also impaired the mitochondrial electrochemical potential (delta psi). In Sertoli cells, which also express StAR, EDS induced cell death and attenuated StAR expression. We conclude 1) steroidogenesis and accumulation of mature StAR protein are inhibited as a consequence of the induction of apoptosis; 2) reduced levels of StAR may be partially attributed to inhibition of import because of the loss of delta psi; 3) loss of steroidogenesis is probably due to loss of StAR synthesis and disruption of delta psi.

Effects of clodronate-containing liposomes on testicular macrophages and Leydig cells in vitro.

We undertook the present studies to determine if clodronate-containing liposomes have direct effects on Leydig cells. Macrophages and Leydig cells were isolated and maintained separately in culture. Following treatment with clodronate-containing liposomes, macrophages were killed in a dose-response fashion over a range of 5-200 microliters liposomes. By comparison, a 500 microliters dose was required to kill Leydig cells, but this was not dependent upon clodronate since liposomes containing buffer elicited an identical response. The concentration of testosterone in medium from Leydig cells treated with clodronate-containing liposomes was significantly reduced compared with untreated cells. However, we subsequently found that liposomes can adsorb testosterone. Therefore, testosterone production was determined at various times following removal of liposomes from Leydig cells, thereby circumventing this complication. It was found that testosterone production was not altered by liposomes under these conditions. Finally, free clodronate had no effect on testosterone production, even at doses representing the amount present within the 500 microliters dose of liposomes. In summary, clodronate-containing liposomes killed testicular macrophages at a far smaller dose than required to kill Leydig cells. Most importantly, neither liposomes no free clodronate had a direct effect on testosterone production. Thus, clodronate-containing liposomes represent a valuable tool to study Leydig cell-macrophage interactions.

Purification and characterization of a lipophilic factor from testicular macrophages that stimulates testosterone production by Leydig cells.

Testicular macrophages have been shown to secrete a factor that stimulates testosterone production by Leydig cells. The purpose of this investigation was to purify and characterize this factor. Medium was collected from 24- to 48-hour cultures of testicular macrophages isolated from adult rats. This medium induced a sevenfold increase in testosterone production by cultured Leydig cells. When the medium was extracted with ether, all biological activity was found in the organic phase, indicating that the factor was lipophilic. The ether extract was then fractionated on a C18 reversed-phase high-performance liquid chromatography (HPLC) column, using a gradient of acidified methanol as the mobile phase. Leydig cell-stimulating activity eluted at approximately 11 minutes. Standards of testosterone, dihydroepiandrosterone (DHEA), pregnenolone, progesterone, dihydrotestosterone (DHT), and prostaglandin E2 (PGE2) all had elution times of between 5 and 6 minutes, under identical column conditions. The biological activity of the HPLC-purified fraction was partly resistant to boiling but was completely abolished by Dextran-coated charcoal treatment. Biological activity of testicular macrophage-conditioned medium was not abolished following chymotrypsin treatment, indicating that this molecule was not a hydrophilic peptide. It was found that the factor obtained by reversed-phase HPLC could be further purified by normal-phase HPLC. The results of this investigation demonstrate that the testicular macrophage-derived factor that stimulates testosterone production by Leydig cells can be purified by organic extraction and HPLC, and that it is a highly potent chymotrypsin-resistant heat-stable lipophilic factor.

Corticotropin-releasing hormone stimulates steroidogenesis in mouse Leydig cells.

The actions of corticotropin-releasing hormone (CRH) on steroidogenesis in enriched preparations of mouse and rat Leydig cells were investigated. Primary cultures of purified Leydig cells as well as a Leydig tumor cell line were used in these studies. CRH had a stimulatory effect on steroid production in both isolated preparations of mouse Leydig cells (80-90% Leydig cells) and MA-10 cells (a mouse Leydig tumor cell line). In primary cultures of mouse Leydig cells, CRH was effective over a range of 1 nM-100 nM, while MA-10 cells were responsive over a wider range (10 nM-100 microM). When a submaximal dosage of CRH was given together with a maximal dosage of hCG, steroid production was stimulated even more highly in MA-10 cells. However, when primary cultures of mouse Leydig cells were treated with CRH and hCG, no similar response was observed. In addition, a CRH antagonist, alpha-helical CRH9-41, reversed the CRH stimulatory effect on steroidogenesis in both mouse Leydig cells and MA-10 cells. The accumulation of intracellular cAMP after CRH treatment was dose-responsive to CRH in both cell types, a finding similar to the results described above for steroid production. CRH had no effect on steroidogenesis in rat Leydig cells (60-80% Leydig cells) in the present study. These results indicate that mouse Leydig cells respond to CRH through specific receptors with increased production of cAMP and steroids.

Physiological relevance of tumor necrosis factor in mediating macrophage-Leydig cell interactions.

Previously, we reported that testicular macrophages constitutively release tumor necrosis factor (TNF) in vitro and are unresponsive to bacterial endotoxins [lipopolysaccharides (LPS)]. These properties are not typical of other tissue macrophages. The goals of the present study were, therefore, to establish 1) if testicular macrophages also release TNF in vivo, and 2) if secretion of TNF in vitro is influenced by the isolation procedure. In vivo TNF production was assessed by assaying testicular interstitial fluid for TNF. Using the L929 cytotoxicity assay for TNF, we found that interstitial fluid contained a cytotoxic factor(s), but this bioactivity was not due to either authentic TNF or a TNF-like molecule acting through the TNF receptor. This was established by showing that 1) antibodies to TNF alpha and -beta could not neutralize interstitial fluid cytotoxicity; 2) interstitial fluid was cytotoxic to TNF-resistant L929 cells; and 3) there was no detectable TNF immunoreactivity in interstitial fluid, as measured by enzyme-linked immunosorbent assay. Therefore, we evaluated whether the release of TNF in vitro was induced by the isolation procedure, particularly by collagenase, which is used to free interstitial cells. Testicular macrophages obtained without the use of collagenase (agitation of testes in buffer) did not release TNF, but responded to the TNF-releasing effect of LPS. Exposure of peritoneal macrophages to collagenase resulted in constitutive TNF release in vitro and lack of responsiveness to LPS. There was no evidence that a non-TNF cytotoxic factor was released in the conditioned medium by any macrophage preparation. Taken together, our findings show that testicular macrophages do not constitutively release TNF, and collagenase has a significant activating effect on macrophages. Testicular macrophages will, however, release TNF when exposed to LPS, indicating that TNF could be a paracrine regulator of testicular steroidogenesis under pathological conditions.

Regulation of the macrophage population in postnatal rat testis.

Testicular macrophages increase in concentration during postnatal development in rats. This process may be under hormonal control since administration of hCG stimulates a similar increase to occur precociously. The purpose of the present studies was to determine how the macrophage population is regulated during normal postnatal development and in response to exogenous hCG. We first determined that testicular macrophages proliferate in situ during development and that hCG administration results in an increase in proliferation when given to 10-day-old rats. We next evaluated whether hCG might exert its effects through enhanced secretion of testosterone from Leydig cells. We found that testosterone could not induce a precocious increment in the macrophage concentration when it was administered to newborn pups for 10 days. Finally, the normal increase in macrophage concentration that occurs prior to puberty could not be blocked by treatment with the antiandrogen Casodex. The results are consistent with the hypothesis that the macrophage population expands by proliferation, perhaps under gonadotropin control. In addition, neither the precocial expansion that occurs in response to hCG nor the normal expansion that occurs before puberty is mediated by testosterone.

Secretion of tumor necrosis factor alpha by testicular macrophages.

While it has been shown that culture medium from testicular macrophages can influence testosterone production when added to Leydig cells, the identity of the factor(s) responsible for this activity remains unknown. Since tumor necrosis factor alpha (TNF alpha) has been shown to be capable of influencing testosterone production by Leydig cells, a series of studies was conducted to determine if testicular macrophages produce TNF alpha. It was found that testicular macrophages from adult rats produce a factor that is capable of lysing L929 cells, which are used as a traditional bioassay for TNF alpha. The TNF alpha activity in the macrophage-conditioned medium could be neutralized by the addition of anti-murine TNF alpha but not with the addition of preimmune IgG. While lipopolysaccharide (LPS) slightly increased the release of TNF alpha, neither follicle-stimulating hormone (FSH) nor testosterone had a similar effect. It was not determined if the isolation procedure had artificially 'activated' the macrophages. Medium from cultured Sertoli cells, Leydig cells and peritubular cells did not contain TNF alpha activity. These studies are consistent with the hypothesis that the paracrine interaction between testicular macrophages and Leydig cells is mediated in part by TNF alpha.

Development of cytoplasmic digitations between Leydig cells and testicular macrophages of the rat.

Testicular macrophages and Leydig cells from adult animals are known to be functionally coupled. For example, secreted products from macrophages stimulate testosterone secretion by Leydig cells. In adult rat testes, structural coupling also exists between these cells. This coupling consists of cytoplasmic projections from Leydig cells located within cytoplasmic invaginations of macrophages. Although macrophages are known to exist in the testis in immature animals, it is not known when these digitations develop. The purpose of the present study was to determine whether the time of their development coincides with known maturational events that occur in Leydig cells, particularly during the peripubertal period. Testes from rats at 20, 30 and 40-days-of-age as well as testes from mature rats weighing more than 500 gm were prepared for ultrastructural analysis. It was found that digitations form between 20 and 30-days-of-age. These structures varied from simple tubular projections to complicated branched structures, suggesting that digitations are more than simple invaginations of microvilli into coated vesicles as previously described. Subplasmalemmal linear densities were also observed within macrophages juxtaposed to Leydig cells. Collagen was commonly observed between macrophages and Leydig cells in animals 20 days old. These studies demonstrate that although macrophages are present in the testis in maximal numbers at 20 days-of-age, they do not form junctions with Leydig cells until day 30. This is just prior to the major increase in secretory activity of rat Leydig cells that occurs during puberty.

Human chorionic gonadotropin increases the concentration of macrophages in neonatal rat testis.

The purpose of these studies was to determine whether treatment of newborn rats with exogenous FSH or hCG would alter the concentration or size of testicular macrophages. Animals were injected once daily with various doses of FSH, hCG, or vehicle for 8-10 days beginning the day following birth. After immunohistochemical labeling of the macrophages with a monoclonal antibody specific for rat macrophages, the concentration and size of macrophages were determined by use of a point-counting method. Body weight, testis weight, and serum levels of testosterone and FSH were also measured. It was found that hCG significantly increased the concentration of macrophages within the interstitium but did not affect the size of the cells. Both testicular weight and serum testosterone concentrations increased after hCG treatment. Although FSH increased the weight of the testis, neither the size nor concentration of macrophages was altered. These results raise the possibility that the number of macrophages within the interstitial compartment of the normally maturing rat testis is under the control of LH.

Changes in the concentration and size of testicular macrophages during development.

Structural and functional interactions exist between Leydig cells and testicular macrophages of adult rats. Since the function of Leydig cells changes during critical periods of development and postnatal maturation, it is possible that macrophages are in part involved in regulating this process. As a first step towards gaining an understanding of the development of this paracrine phenomenon, I have undertaken a series of studies designed to determine when macrophages first become identifiable in the fetal tests and to determine whether the concentration or size of macrophages changes during important stages of testicular maturation. Macrophages were identified immunohistochemically in frozen sections of testis from rats at various prenatal and postnatal ages using commercially available monoclonal antibodies to proteins specific to rat macrophages. It was found that macrophages positive for these antigens were found only within the interstitial compartment and were commonly associated with clusters of presumptive Leydig cells that were negative for these antigens. Macrophages were first identifiable in the testis at Day 19 of fetal development. The number of macrophages/unit area of interstitium increased 15-fold between Day 20 of gestation and Day 47 postpartum. The cross-sectional area of the macrophages increased 1.7-fold between Days 13 and 47 postpartum. These results demonstrate that the number and size of testicular macrophages changes with age, suggesting a role for these cells during important times of testicular development and maturation.

Comparison of cellular and secreted proteins of macrophages from the testis and peritoneum on two-dimensional polyacrylamide gels: evidence of tissue specific function.

It has been shown that the testis contains a population of cells with many characteristics typical of macrophages of other tissues. However, these macrophages are unique in that they secrete a product(s) that is responsible for stimulating testosterone secretion by Leydig cells while peritoneal macrophages have no similar effect. The purpose of the present study was to compare the pattern of cellular and secreted proteins of rat testicular macrophages to those of peritoneal macrophages using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Cellular and secreted proteins of cultured macrophages from the testis and peritoneum were labeled with 35S-methionine and 35S-cystine and then analyzed by 2D-PAGE using a computer-assisted image analysis system. It was found that the patterns of both cellular and secreted proteins of testicular macrophages were vastly different than those of peritoneal macrophages. One striking difference was the presence of a family of proteins in the 47 kd range, with a range of pI from 5.7-6.3 that represented approximately 57% of the total protein secreted by testicular macrophages. Proteins of similar molecular weight and pI represented only approximately 15% of the total peritoneal macrophage secreted proteins. Many other differences were observed in less abundant cellular and secreted proteins. These studies strongly support the concept that macrophages are functionally heterogeneous and secrete a group of proteins characteristic to the histological location of these cells.

Leydig cells do not have Fc receptors.

It has been reported that Leydig cells have Fc receptors, which traditionally have been considered markers specific for macrophages and polymorphonuclear leukocytes. The purpose of this investigation was to study further this phenomenon and also to determine if Leydig cells and macrophages could be separated from each other either by density gradient centrifugation using Percoll or by differential detachment with trypsin treatment of cultures of crude interstitial preparations. Interstitial cells were obtained by collagenase digestion of rat testis and established in culture. These cultures were reacted for 3 beta-steroid dehydrogenase and Fc receptor and viewed with phase contrast microscopy. No individual cells were positive for both steroid dehydrogenase activity and Fc receptors. The order in which the cells were stained for these two markers did not influence the results. Trypsin treatment of these crude interstitial cultures removed over 90% of the Leydig cells and approximately 20% of the macrophages. Macrophages were located in the same portion of Percoll gradients as the less dense (Population I) Leydig cells, while Leydig cells found in the dense area of the gradient (Population II) were not contaminated with macrophages. These studies indicate that Leydig cells do not have Fc receptors and that a subpopulation of Leydig cells can be isolated free of macrophages using density gradient centrifugation.