Identification, Proliferation, and Differentiation of Adult Leydig Stem Cells

Abstract

Leydig cells, the testosterone-producing cells of the adult testis, rarely turn over. However, their elimination with ethane dimethanesulfonate (EDS) is followed by the appearance of new, fully functional adult Leydig cells. The cells that give rise to the new Leydig cells have not been well characterized, and little is known about the mechanism by which they are regulated. We isolated cells expressing platelet-derived growth factor receptor-α, but not 3β-hydroxysteroid dehydrogenase (3β-HSD^neg) from the testes of EDS-treated adult rats. Depending on conditions, these cells proliferated indefinitely or differentiated and produced testosterone. To localize these cells and to determine the effect of the testicular environment on their function, the seminiferous tubules and testicular interstitium were physically separated and cultured. During the first 72 h in culture, 3β-HSD^neg cells on the tubule surfaces underwent divisions. Some of these cells later expressed 3β-HSD and produced testosterone. Removal of the newly formed 3β-HSD^pos cells from the tubule surfaces with EDS, followed by further culture of the stripped tubules, resulted in the reappearance of testosterone-producing cells. These results, taken together, suggest that the precursors for newly formed Leydig cells are stem cells, with many if not all situated on the surfaces of the seminiferous tubules. Although normally quiescent, the stem cells are capable of self-renewal and differentiation. The development of the tubule culture system should provide a valuable in vitro approach to assess the role(s) of niche components on the function of adult Leydig stem cells despite their residing in a complex mammalian tissue.

Leydig cells are the testosterone-producing cells of the adult testis. Once formed, these cells rarely die or divide. However, after the depletion of the adult Leydig cells by injecting rats with the alkylating agent ethane dimethanesulfonate (EDS), a new generation of Leydig cells is formed (1–6). It has been suggested in some studies that the cells that give rise to the new Leydig cells reside on the outer surface of the seminiferous tubules, whereas others have suggested that they are associated with blood vessels (4–9). Wherever their location, the cells have not been well characterized, and we know little about how they are regulated.

Clues as to the nature of the Leydig cell precursors in the adult testis may be gleaned from studies of the development of the adult population of Leydig cells. In both the human and rat, testosterone production gradually increases from the peripubertal period through the adult, coincident with the development of the adult Leydig cells (10–12). There now is strong evidence that the adult cells ultimately arise from stem cells. Thus, Ge et al. (13) isolated cells from the testes of postnatal d 7 rats, which, depending on culture conditions, were able to divide without differentiating or to differentiate and ultimately produce testosterone. Additionally, the cells were found able to differentiate in vivo after their transplantation into the testis. These observations defined the cells as stem Leydig cells.

Based in part on these studies, we hypothesized that there are stem Leydig cells in the adult testis as well and that these cells are capable of giving rise to a new generation of functional Leydig cells in the adult testis after the original population is depleted. Studies of diverse systems have shown that stem cell self-renewal and differentiation are regulated by extracellular cues from their local environment or their niche (14) and perhaps also from intrinsic signals (15). In mammals, the anatomic complexity of most tissues makes it difficult to identify the stem cells and/or to characterize the stem cell niche. In the case of the testis, it is possible to physically separate the seminiferous tubular and interstitial compartments. This made it feasible to develop a novel approach by which to identify the testicular compartment(s) in which the putative adult stem Leydig cells are localized and to address the influence of the physical location of the cells on their function.

Herein we provide evidence that the precursor cells in the adult testis that are capable of giving rise to new populations of adult Leydig cells indeed are stem cells. We show further that both the proliferation and differentiation of the stem Leydig cells occur under the apparent influence of the seminiferous tubules with which they are associated.

Materials and Methods

Chemicals

Rat epidermal growth factor (EGF), leukemia inhibitory factor (LIF), platelet-derived growth factor BB (PDGF-BB), IGF-I, fetal bovine serum (FBS), anti-β-actin, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were from Sigma (St. Louis, MO). The Click-iT EDU (5-ethynyl-2′-deoxyuridine) kit and M-199 media were from Invitrogen (Carlsbad, CA). Collagenase-D and dispase II were from Roche Applied Biosciences (Indianapolis, IN). BSA (fraction V) was from MP Biochemicals (Solon, OH). [1,2,6,7,16,17-³H(N)]-testosterone (115.3 Ci/mmol) was from PerkinElmer Life Sciences, Inc. (Boston, MA). Testosterone antibody was from ICN Biochemicals (Costa Mesa, CA). Steroidogenic acute regulatory protein (StAR) antibody was from Affinity BioRegents, Inc. (Golden, CO). Cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) antibody was from Chemicon International (Temecula, CA). PDGF receptor-α (PDGFRα) antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Bovine LH (USDA-bLH-B-6) was provided by the U.S. Department of Agriculture Animal Hormone Program (Beltsville, MD). Testosterone was from Steraloids (Newport, RI). Fluorescent secondary antibodies (fluorescein antirabbit IgG; fluorescein antimouse IgG) were from Vector Laboratories (Burlingame, CA).

Animals

Young adult (3–4 months old) male Brown Norway rats were obtained through the National Institute on Aging, supplied by Harlan Sprague Dawley Inc. (Indianapolis, IN). The rats were housed in the animal facilities of the Johns Hopkins School of Public Health, under conditions of controlled light (14 h light, 10 h dark) and temperature (22 C) and with free access to rat chow and water. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, according to protocols approved by the Johns Hopkins Animal Care and Use Committee.

Isolation of Leydig cell precursor cells

To eliminate the adult Leydig cells from the testes, rats received a single, ip injection of EDS (75 mg/kg body weight). Rats were killed by decapitation 4 d after EDS, by which time the adult Leydig cell population had been eliminated. To isolate putative stem/precursor cells, decapsulated testes were dispersed with 0.25 mg/ml collagenase-D and 0.16 U/ml dispase II in Medium 199 (Life Technologies, Grand Island, NY). Cells were resuspended in 55% isotonic Percoll and centrifuged at 22,000 × g for 1 h. Precursor cells were collected from the Percoll gradient between densities of 1.045 and 1.090 g/ml and further purified through a BSA gradient (0–10% BSA, 120 g, 10 min). The purified cells were plated on PDGFRα antibody-coated plates to select for PDGFRα^pos cells (13). The isolated cells were washed, dispersed with trypsin, and plated on fibronectin-coated plates in expansion media (EM), which consisted of DMEM:F12, sodium bicarbonate (1.2 mg/ml), BSA fraction V (1 mg/ml), gentamycin (12 μg/ml), and penicillin/streptomycin (100 U/ml and 100 μg/ml), supplemented with 2% FBS, 1 μm dexamethasone, 1 ng/ml LIF, 10 ng/ml PDGF-BB, and 5 ng/ml EGF.

Cell growth

The growth of cells cultured in EM was determined by the following formula: Td = (t2-t1)[log (2)/log (q2/q1)], where Td is doubling time, t1 is the initial time at which cells were seeded into flasks, t2 is the time at which cells were at least 70% confluent, q1 is number of cells initially seeded, and q2 is total number of recovered cells (16). Cumulative population doublings were obtained by determining the number of doublings between t1 and t2 (16). A minimum of three independent isolations were conducted.

Differentiation of precursor cells

Isolated precursor cells were cultured for up to 35 d in differentiation-inducing media (DIM) consisting of 2% FBS, 70 ng/ml IGF-I, 1 ng/ml LH, 1 nm T₃, 10 ng/ml PDGF-BB, and Sigma insulin/transferrin/selenium cell culture supplement solution (5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite). Media were collected and replaced every 3–5 d. Collected media were analyzed by RIA to determine testosterone concentration. For these analyses, there were at least three replicates for each of three isolations.

Immunofluorescence

Isolated cells were split onto chamber slides and incubated overnight. Slides were rinsed with PBS and fixed in 10% neutral buffered formalin for 20 min. After the slides were washed three times with PBS, the appropriate serum block was added for 1 h. Then primary antibody was added for 1 h (CYP11A1) or overnight (PDGFRα) at 1:200 dilution. For PDGFRα, rabbit IgG was used as a negative control. For CYP11A1, freshly isolated primary adult Leydig cells were used as a positive control and rabbit IgG was used as a negative control. CYP11A1 signal was amplified using biotin. Fluorescent secondary antibodies (fluorescein antirabbit IgG; fluorescein antimouse IgG, 1:1000) were used after the primary antibody.

Isolation and culture of seminiferous tubules and interstitium

The adult Leydig cells were eliminated with EDS, as above. Four days later, testes were decapsulated and placed in Medium 199. Seminiferous tubules and interstitium were mechanically separated under a dissection microscope using fine forceps (17, 18). The separated fractions were incubated in M199 medium supplemented with 0.1% BSA, 15 mm HEPES, 2.2 mg/ml sodium bicarbonate, penicillin/streptomycin (100 U/ml and 100 μg/ml), and insulin/transferrin/selenium in a humidified atmosphere of 5% CO₂ in air at 34 C. After 48 h, the tubules and interstitium were separated into 24-well Costar culture plates with ultralow attachment surface (Corning, NY) for longer-term culture. Each well contained interstitium or tubules (multiple fragments of 2 in. total length) cultured in M199 medium containing 1% FBS with or without LH (10 ng/ml) for up to 13 wk. For the first week, the medium was changed every 1–2 d. Thereafter the medium was changed every 3–4 d. After completion of the given experiments, the total ATP content of the tissue was quantified in each well to assess cell survival. Culture media were frozen for subsequent assay of testosterone, and tissue was frozen for Western blots or fixed for morphology or immunohistochemical staining.

For some experiments, tubules were first cultured with LH for 49 d so as to restore testosterone-producing Leydig cells to the tubule surfaces, and the tubules then were treated with EDS (75 μg/ml) to eliminate the newly differentiated Leydig cells. Twenty-four hours later, the tubules were washed three times and new media were added. The tubules were cultured further for another 6 wk (up to 13 wk total) to examine whether another round of Leydig cells would be regenerated.

3β-Hydroxysteroid dehydrogenase (3β-HSD) activity

To examine 3β-HSD enzyme activity, a previously published protocol was used (19) with minor modifications. Briefly, cells or tubules were first washed in PBS and then dried at room temperature for 20 min. Staining solution (0.4 mm 5β-androstan-3β-ol-17-one steroid substrate, 1 mg/ml nicotinamide adenine dinucleotide, and 0.2 mg/ml tetranitro blue tetrazolium) was added to slides for 40 min and then removed by two successive washes in PBS. The slides were then placed in 10% formalin for 30 min. Mouse Leydig tumor cells (MA-10 cells) were used as a positive control. Negative controls were run without the substrate 5β-androstan-3β-ol-17-one steroid.

Labeling cell proliferation with Click-iT EdU

Cell divisions on the surface of the tubules were monitored with the Click-iT EdU imaging kit from Invitrogen. The seminiferous tubules were labeled with EdU (10 μm) for 2 or 24 h and examined immediately or 4 wk later. Labeled nuclei (green) were examined by fluorescence microscopy with excitation/ emission at 495/519 nm.

Western blot analysis

Tissues or cells were lysed with Tris-HCl buffer (100 mm Tris; 0.1% Triton X-100; 50 mm dithiothreitol; 1× Sigma protease inhibitor cocktail, pH 6.8). After sonication and centrifugation (18,000 × g, 10 min), the supernatant was mixed with 3× sodium dodecyl sulfate loading buffer (New England BioLab, Ipswich, MA). Equal amounts of total protein (about 30 μg) from each sample were separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. After incubation with primary antibody (1:400) and horseradish peroxidase-conjugated secondary antibody (1:5000), the signals were detected by the enhanced chemiluminescence Western blot kit from Pierce (Rockford, IL). The bound antibodies on the membranes were stripped by Restore Western blot stripping buffer (Pierce), and the membranes were reprobed with new antibodies in the following sequence: StAR, CYP11A1, and GAPDH. The Western blots were repeated three times, using samples from three independent experiments.

Statistical analyses

Data are expressed as the mean ± sem. Group means were evaluated by one-way ANOVA. If group differences were revealed by ANOVA (P < 0.05), differences between individual groups were determined with the Student-Neuman-Kuels test, using SigmaStat software (Systat Software Inc., Richmond, CA). Values were considered significant at P < 0.05.

Results

Isolation of putative Leydig cell stem cells from the adult testis

Adult rats were administered a single injection of EDS (75 mg/kg body weight). Four days later, when the Leydig cells had been eliminated, testicular cells purified by Percoll and BSA gradient centrifugation were immunoselected for their expression of PDGFRα protein (Fig. 1A). On average, about 200,000 cells were isolated per testis compared with the 25 million Leydig cells in the adult testis (20). The immunoselected cells did not express the Leydig lineage marker CYP11A1 (Fig. 1B). When cultured in EM, which contained dexamethasone, LIF, PDGF-BB, and EGF, the isolated cells showed linear growth characteristics for at least 1.5 yr, undergoing about 325 population doublings during that time period (Fig. 1C) and remaining CYP11A1 negative (CYP11A1^neg) (Fig. 1D). The cells did not produce testosterone (Fig. 1E). However, when the cells were cultured in DIM containing LH (1 ng/ml) along with IGF-I and PDGF-BB, in time they stained for CYP11A1 (Fig. 1D) and produced testosterone (Fig. 1E).

Fig. 1.

Characterization and differentiation of isolated putative stem Leydig cells (SLC). A, Putative SLC isolated from adult rat testes. The isolated cells expressed PDGFRα protein (green). Rabbit IgG served as a negative control (−control). B, Western blots of CYP11A1 expression by isolated SLC and adult Leydig cells (ALC). C, Growth characteristics of SLC cultured in EM. D, CYP11A1 expression (green) by cells cultured in EM vs. DIM. Freshly isolated ALC were used as a positive control (+control); the higher magnification insert shows that the CYP11A1 staining is cytoplasmic. Rabbit IgG was used as a negative control (−control). Cell nuclei were stained with 4′,6′-diamino-2-phenylindole. E, Testosterone production by SLC cultured in EM and DIM.

Differentiation in vitro of steroidogenically active cells by seminiferous tubules

We wanted to determine the testicular location of the precursor cells that give rise to new generations of testosterone-producing Leydig cells in the adult testis and to examine the effect of the anatomic location (niche) on their proliferation and differentiation. Seminiferous tubules were mechanically separated from the interstitial compartment of the testis 4 d after rats received EDS, by which time the Leydig cells had been eliminated (Fig. 2A). The freshly isolated seminiferous tubules had no apparent interstitial cell contamination (Fig. 2A; Supplemental Fig. 1, A and B, published on The Endocrine Society's Journals Online web site at http://endo.endojournals.org). The vasculature of the freshly isolated interstitial compartment remained interconnected and there was no cross contamination of seminiferous tubule remnants (Supplemental Fig. 1, C–E). There were no 3β-HSD^pos cells associated with the tubules (Fig. 2B) or the interstitium. The tubules and interstitium were cultured separately in medium containing LH. In time, cells appeared on the surfaces of the cultured tubules that stained for 3β-HSD (Fig. 2, C and D) but not in the cultured interstitial compartment. Consistent with this, two key proteins involved in steroidogenesis, StAR and CYP11A1, were detected by Western blot in tubules cultured with LH but not the interstitial fraction (Fig. 2E).

Fig. 2.

Differentiation of cells associated with cultured seminiferous tubules. A, Seminiferous tubule physically separated from the interstitial compartment 4 d after rats received EDS. B, Freshly isolated seminiferous tubule stained for 3β-HSD (purple). C and D, Seminiferous tubules stained for 3β-HSD after culture with LH for 7 wk. 3β-HSD^pos cells appeared on surface of the tubules (arrow). E, Western blots of CYP11A1 and StAR expression by freshly collected (0 d) interstitial fraction (IF) and seminiferous tubules (T) and by IF and T cultured for 28 d without LH (C) or with LH. Purified Leydig cells (LC) served as positive controls, and GAPDH served as the loading control. F, Testosterone production by the interstitial (IF) and seminiferous tubule (T) fractions when cultured with or without LH.

We then assessed the relative abilities of the cultured tubule and interstitial fractions to produce testosterone. To this end, testosterone was measured in the medium of seminiferous tubules and interstitium cultured with LH (Fig. 2F). At 1 wk of culture, no testosterone was produced by either the tubules or interstitium. By 14 d, testosterone began to appear in the medium of the tubules cultured with LH but not in the medium of tubules cultured in the absence of LH or of the interstitial fraction whether or not cultured in the presence of LH. Testosterone produced by the tubules continued to increase with further culture, whereas no testosterone was detected in the interstitial fraction. These results showed clearly that the tubule fraction contains precursor cells for the production of testosterone-producing Leydig cells.

Are the precursor cells stem cells?

We reasoned that if the seminiferous tubule-associated precursor cells that give rise to testosterone-producing Leydig cells were stem cells, these cells should be capable of proliferation/self-renewal as well as differentiation and should not be depleted, even when repeatedly stimulated to differentiate. Spindle-shaped cells were seen on the outer surfaces of the freshly isolated seminiferous tubules (Fig. 3A). Using the Click-iT EdU cell proliferation assay (Invitrogen), spindle-shaped, EdU-labeled cells were observed on the surfaces of the tubules during the first 72 h in culture (Fig. 3, B and C). Four weeks later, some of the cells labeled during the first 72 h remained labeled with EdU (Fig. 3D) and some among the EdU-labeled cells also stained for 3β-HSD (Fig. 3E). The latter indicates that at least some of the dividing cells at the beginning of the culture subsequently entered into the Leydig cell lineage.

Fig. 3.

Proliferation and differentiation of putative stem Leydig cells (SLC). A, Semithin section of freshly isolated tubule. Spindle-shaped cells were seen on the surfaces of the tubules (arrows). B and C, Click-it EdU labeling of dividing cells (Invitrogen). During the first 72 h in culture, spindle-shaped, actively dividing cells (green) cells were seen on the tubule surfaces (arrows). D and E, 3β-HSD staining of tubules 4 wk after Click-it EdU pulse labeling. Most of the 3βHSD^pos cells (cells with blue dots in the cytoplasm; black arrows show examples) were not labeled with EdU; but some EdU-labeled cells (white arrow, Fig. 3C) also stained for 3β-HSD (Fig. 3E).

If the cells on the surface of the tubules were stem cells, inducing differentiation should not result in their depletion. To test this, tubules from EDS-treated rats first were cultured with LH for 7 wk, which resulted in newly formed 3β-HSD^pos cells on the tubule surfaces (Fig. 4A). Treatment of these tubules with EDS in vitro resulted in the removal of the newly formed cells (Fig. 4B). Further culture of the stripped tubules with LH for an additional 6 wk resulted in the appearance of another round of 3β-HSD^pos cells (Fig. 4C) that also expressed CYP11A1 (Fig. 4D). The ability of the tubules to produce testosterone after treatment with EDS was consistent with the disappearance and then reappearance of 3β-HSD^pos cells. Testosterone production by cultured tubules was lost abruptly when the tubules were treated with EDS, which removed the newly formed Leydig cells and then restored with the further culture of the tubules to produce a new round of adult Leydig cells (Fig. 4E).

Fig. 4.

Effect of depletion of newly formed 3β-HSD^pos cells on the ability of stem Leydig cell to regenerate another round of 3β-HSD^pos cells on the tubule surface. A, Newly generated 3β-HSD^pos cells on the surfaces of tubules that had been cultured in vitro with LH for 7 wk. B, Tubules after elimination of the newly formed 3β-HSD^pos cells by EDS treatment of the tubules in vitro. C, Appearance of another round of 3β-HSD^pos cells after culturing the stripped tubules with LH for 6 wk. D, CYP11A1 expression after EDS treatment of the tubules. E, Testosterone production by tubules cultured for 100 d with LH and by tubules cultured for 7 wk and then treated with EDS to remove the newly formed Leydig cells and then continued in culture.

Discussion

A number of studies have shown that the elimination of the adult Leydig cells from the rat testis in response to EDS administration is followed by the reappearance of a new population of testosterone-producing adult Leydig cells (1–9). The nature of the precursor cells that give rise to the new Leydig cells, how the precursor cells are regulated, and where in the testis they are located had not been established. There has been speculation that the precursor cells might be stem cells but an equally feasible alternative is that they are quiescent progenitor cells (i.e. cells that already have entered the Leydig cell lineage). With regard to their location, previous studies have suggested that they are associated with the seminiferous tubules and/or with blood vessels (4–9). Our major objectives in the present study were to determine whether, in fact, there are stem cells in the interstitial compartment of the adult testis that are capable of giving rise to new Leydig cells, where these cells are located, and how they are regulated.

We reasoned that if, in fact, there were Leydig stem cells in the adult testis, it should be possible to isolate and identify undifferentiated cells with the ability to proliferate in vitro for extended periods of time without expressing Leydig cell lineage markers and also the ability to differentiate into cells capable of producing testosterone. Using methods previously used to isolate stem Leydig cells from the postnatal d 7 rat testis (13), cells were isolated from Leydig cell-depleted adult testes that expressed PDGFRα but not the steroidogenic proteins 3β-HSD or CYP11A1. Depending on culture conditions, the isolated cells proliferated in vitro for well over a year without expressing Leydig cell lineage markers or differentiated and ultimately produced testosterone. As indicated, these are among the characteristics expected of stem cells.

There are suggestions in the literature that steroidogenic factor 1, Hedgehog, Notch signaling molecules, nestin, and/or PDGFRα might be expressed by stem Leydig cells (8, 21–24). Unfortunately, none of these molecules has been established as identifiers of the stem Leydig cells. Indeed, it has proven difficult to definitively identify stem cells in most complex mammalian tissues. With this caveat in mind, we set about to determine where in the complex interstitial compartment of the adult testis the putative stem Leydig cells are located, that is, whether the cells are associated with the seminiferous tubules, other elements (such as blood vessels) of the interstitial compartment (8, 9), or both. To do so, we first physically separated the seminiferous tubules from the interstitium. Careful examination of the tubules and interstitium by light and electron microscopy revealed no apparent cross-contamination between the two. During the first 72 h of culture, there was proliferation of 3β-HSD^neg cells on the surfaces of the tubules. The observation that some of these dividing cells ultimately became 3β-HSD^pos indicates that the progeny of at least some of the undifferentiated dividing cells differentiated into Leydig cells. Initially, there were no cells with characteristics of steroidogenic cells that were associated with either the tubules or the interstitium. A month after culture with growth factors and LH, however, there were 3β-HSD^pos cells on the surfaces of the seminiferous tubules; and moreover, the tubules produced testosterone in vitro. In contrast, the interstitial compartment did not develop 3β-HSD^pos cells or produce testosterone when cultured for comparable periods of time. The proliferation and differentiation of cells on the surface of the tubules suggested that the cells were likely to be stem cells.

We reasoned that if the precursor cells indeed were stem cells, it should be possible to repeatedly induce their proliferation and differentiation without their depletion. To test this, tubules from the testis of EDS-treated adult rats were cultured so as to produce new Leydig cells on their surfaces, and subsequently the tubules were incubated with EDS to remove the newly formed Leydig cells. We found that new 3β-HSD^pos cells reappeared on the stripped tubule surfaces and that the newly developed cells produced testosterone. These results indicate that the differentiation of the putative stem Leydig cells to steroidogenic cells does not result in their depletion and provides further evidence that the precursor cells are stem cells.

Although we cannot be certain that the cells on the surface of the tubules shown to be capable of differentiating into Leydig cells are identical to the stem cells isolated from whole testes, a number of observations suggest this to be the case. First, depending on culture conditions, both the isolated cells and the cells on the tubule surfaces were found to proliferate or to differentiate into Leydig cells, whereas cells associated with the interstitium did not differentiate into Leydig cells. Second, treatment of the isolated tubules with collagenase and dispase, which was used in the procedure to isolate stem Leydig cells from whole testes, depleted the ability of tubules to generate Leydig cells (our unpublished results). Third, as was true of the isolated cells, the cells on the surface of the seminiferous tubules do not express 3β-HSD or LH receptor but do express PDGFRα (Ref. 24–26 and our observations).

As yet, we know little about how the stem Leydig cells are regulated either during the neonatal period or in the adult. The observation that in the adult testis, functional Leydig cells formed in association with tubules suggests that either cells and/or factors associated with the tubules or perhaps the three-dimensional structure of the tubules in some way regulate the proliferation and differentiation of the stem cells. The fact that functional Leydig cells are able to differentiate in the absence of interstitium suggests that macrophages and cells associated with blood vessels in the interstitial compartment (vascular smooth muscle cells, pericytes) may not be critical for the development of new Leydig cells, as suggested in some previous studies (8, 27, 28). However, due to the limitation of our culture method, we cannot rule out the possibility that there might be some contamination of the tubule fraction by interstitial cells which, in turn, might contribute to Leydig cell regeneration. Also, we recognize that a failure to generate Leydig cells from the interstitium in vitro does not exclude the possibility that interstitial components might contribute to Leydig cells regeneration in vivo.

In summary, the results of our studies indicate that there are stem cells in the interstitial compartment of the adult testis which, depending on culture conditions, have the ability to proliferate for months in vitro without differentiating or to differentiate to become functional Leydig cells. There is good evidence that many, and perhaps all, of the stem cells are located on the surface of the seminiferous tubules. Removal of newly differentiated, testosterone-producing Leydig cells from the tubule surfaces did not result in the depletion of the precursor cells. Rather, new Leydig cells were able to form repeatedly. These results, taken together, provide strong evidence that the cells that give rise to new Leydig cells in the adult testis indeed are stem cells and suggest that their proliferation and/or differentiation are regulated by factors associated with the seminiferous tubules or perhaps by the substrate provided by the tubules. Finally, the new tubule culture system that we describe should provide a valuable in vitro approach to address critical questions about the role(s) of niche components on the proliferation and differentiation of adult stem cells in a complex mammalian tissue.