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Biology of Reproduction 64, 790-796 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Production of 25-Hydroxycholesterol by Testicular Macrophages and Its Effects on Leydig Cells1

Yevgenia O. Lukyanenkoa, Jau-Jiin Chena, and James C. Hutson2,a

a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

ABSTRACT

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 µg/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 µg/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 µg/ml and only 50% of cells affected at 100 µg/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.

interstitial cells, Leydig cells, male reproductive tract, steroid hormones, testosterone

INTRODUCTION

Macrophages are primarily involved in phagocytosis of foreign material and in secretion of a variety of compounds that signal neighboring cells [1]. The best-known signaling compounds are those that target lymphocytes, but others influence a wide variety of cell types outside the immune system [2]. Among the earliest examples of nonimmune macrophage function was the finding that testicular macrophages produce a factor that stimulates the production of testosterone by neighboring Leydig cells [3]. The factor responsible for this activity, which was previously called macrophage-derived factor, has recently been purified from testicular macrophage culture medium [4] and identified as 25-hydroxycholesterol [5]. Historically, this oxysterol has been used at saturating doses to assess the capacity of the cholesterol side-chain cleavage system without addressing its potential as a physiologically relevant compound in the testis [6]. However, it has long been known to be a naturally occurring sterol in liver [7], and a cholesterol 25-hydroxylase has recently been cloned and sequenced, with high message expression in the lung [8]. 25-Hydroxycholesterol can be produced both by 25-hydroxylase [8], and by auto-oxidation by way of various oxygen species [9]. Whereas testicular macrophages produce superoxide anion [10] and, presumably, other oxygen species, whether they produce 25-hydroxycholesterol by way of these oxygen radicals or convert cholesterol to 25-hydroxycholesterol enzymatically by 25-hydroxylase is not known. To begin to understand how the production of 25-hydroxycholesterol is regulated, we first determined if testicular macrophages have mRNA for 25-hydroxylase and/or can convert radiolabeled cholesterol to 25-hydroxycholesterol. We also determined if testicular macrophages produce sufficient amounts of compounds to oxidize cholesterol (i.e., autooxidation).

In contrast to the beneficial influences of 25-hydroxycholesterol on steroidogenesis mentioned above, it is also highly toxic to a wide variety of cells, including lymphocytes [11], smooth muscle cells [12], hepatomas [13], neurons [14], and fibroblasts [15]. Because we have shown that testicular macrophages have the potential to secrete large amounts of this oxysterol [5], our second phase of study addressed the viability and LH responsiveness of Leydig cells following chronic exposure to various doses of 25-hydroxycholesterol during 2–5 days in culture.

MATERIALS AND METHODS

Materials

Adult male (250–350 g) Harlan Sprague-Dawley (Minneapolis, MN) rats were handled in accordance with protocols approved by the Animal Use and Care Committee of Texas Tech University Health Sciences Center. Dulbecco's modified Eagle/Ham F12 medium (DME/F12), BSA (Fraction V), newborn calf serum (NCS), penicillin, streptomycin, collagenase (type I), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and other routine chemicals were obtained from Sigma (St. Louis, MO). 25-Hydroxycholesterol and cholesterol were obtained from Steraloids (Newport, RI), and 14C-cholesterol (51 mCi/mmol) was obtained from New England Nuclear (Boston, MA). Ovine LH (biological preparation) was from the National Pituitary Program of the NIDDK. The 100-mm diameter culture dishes (catalog no. 3001) and 35-mm diameter dishes (catalog no. 3005) were obtained from Becton Dickinson Labware (Franklin Lakes, NJ), and the 96-well-plates (catalog no. 25860) were from Corning Glass Works (Corning, NY). High-performance liquid chromatography (HPLC)-grade organic solvents were purchased from Fisher Scientific (Fair Lawn, NJ). The HPLC column (C18, Microsorb MV, 100 Å, 4.6 mm x 25 cm) was from Varian Chromatography Systems (Walnut Creek, CA). Primers for reverse transcription-polymerase chain reaction (RT-PCR) were from Midland Certified Reagent Company (Midland, TX). Qiaex II Gel Extraction Kit was purchased from Qiagen (Valencia, CA). Molecular biology-grade agarose and TRIzol Reagent were from Life Technologies (Grand Island, NY). The DNase I was from Amersham Pharmacia Biotech (Umea, Sweden), and the BW 5147.5.2 cells were from the American Type Culture Collection (Manassas, VA). Testosterone RIA kit was from Diagnostic Systems Laboratories (Webster, TX).

Cell Culture

From collagenase dispersions of adult rat testis, macrophages were isolated by differential attachment procedures, and Leydig cells were isolated using Percoll density gradients as previously described [4, 5]. Cells were maintained in DME/F12 plus 0.1% w/v BSA, 100 µg/ml of streptomycin, and 100 U/ml of penicillin (culture medium) at 34°C in an atmosphere of 95% air and 5% v/v CO2. Rat peritoneal macrophages were obtained from peritoneal rinses using culture medium as previously described [4, 5]. The BW 5147.5.2 cells, a lymphocyte cell line, were maintained in culture medium containing 10% heat-inactivated NCS.

RT-PCR for 25-Hydroxylase

Testicular macrophages were plated into 100-mm Petri dishes, at approximately 2.8 x 106 cells/dish, in 7 ml of culture medium and maintained in culture for 2 h. Total RNA was isolated using TRIzol Reagent, and a single-tube procedure for RT-PCR was performed using Access RT-PCR as recommended by the manufacturer with the following modifications to the cycling program: 48°C for 45 min, 94°C for 2 min, 30 cycles at 94°C for 0.5 min, 56°C for 1 min, 68°C for 2 min, and a final extension for 7 min at 68°C. Controls included omission of the reverse transcriptase or pretreatment of the RNA with RNase-free DNase I (10 U for 1 h at 37°C). The following primers were designed based on the mouse 25-hydroxylase sequence (GenBank accession no. AF059213): 5'-GCGACCCAATACATGAGCTT and 5'-TGGAGTCAATGACACTGGGA (from base 507 [from start site of translation] to base 1058). A second set of partially nested primers were 5'-GCGACCCAATACATGAGCTT and 5'-CAAAGGGCACAAGTCTGTGA (bases 507–691). Products were electrophoresed on 2% w/v agarose mini-gels in Tris-borate-EDTA for 3 h at 50 V and stained with ethidium bromide (0.5 µg/ml). The 551-base pair (bp) product was purified using the Qiaex II Gel Extraction Kit as recommended by the manufacturer and amplified using the nested primer. The 184-bp product from that reaction was also electrophoresed and extracted as described above, then sequenced by the Texas Tech University Biotechnology Center using both primers. The RT-PCR was used because both 25-hydroxylase mRNA and protein were undetectable using approximately 3 x 106 testicular macrophages by Northern and Western analysis, respectively. (Plasmid and antibody were generously provided by Dr. David Russell, UT Southwestern Medical School, Dallas, TX.)

Conversion of 14C-Cholesterol to 14C-25-Hydroxycholesterol by Testicular Macrophages

Testicular macrophages were plated into 35-mm dishes in 1 ml of medium (~200 000 cells/dish). After 2 h, 0.08 µCi 14C-cholesterol in 2 µl of ethanol was added, and the cells were maintained in culture for an additional 19 h in medium containing 0.2% w/v BSA. The medium was removed, centrifuged at 350 x g for 5 min, and extracted with two volumes of water-saturated, HPLC-grade ether. The organic phase was evaporated under a gentle stream of nitrogen, dissolved in 200 µl of methanol, and chromatographed on a C18 HPLC column using methanol as the mobile phase at 1 ml/min. Fractions (50 drops, ~0.5 ml each) were collected and assessed for radioactivity, and 14C-25-hydroxycholesterol was identified by comparison of its elution time to that of a reference preparation of 25-hydroxycholesterol (ultraviolet A206nm).

Autooxidation of 14C-Cholesterol by Macrophage-Conditioned Medium

Testicular (0.9 x 106) and peritoneal (17.4 x 106) macrophages were plated separately into 100-mm dishes in 6 ml of medium plus 0.2% w/v BSA. After 24 h, the medium was removed and centrifuged at 350 x g for 5 min. The 14C-cholesterol (0.16 µCi in 4 µl of ethanol) was then added to the supernatant, and the medium was incubated for an additional 24 h at 34°C in an atmosphere of 5% CO2/95% v/v air. The medium was then extracted with ether and 14C-25-hydroxycholesterol measured as described above.

Effects of 25-Hydroxycholesterol on Viability of Leydig Cells and Lymphocytes

25-Hydroxycholesterol (dissolved in ethanol) was added at various concentrations to Leydig cells and BW 5147.5.2 lymphocytes in 96-well plates. Leydig cells were plated at approximately 40 000 cells/well, and lymphocytes were plated at approximately 1000 cells/well. Control cells received only ethanol at the same volume. After 2 and 4 days, viability was assessed using 0.5 mg/ml of MTT. Living cells with active mitochondrial dehydrogenases become blue when incubated with MTT for 3 h [16]. The percentage of viable cells per well was then estimated using an inverted phase-contrast microscope fitted with a gridded eyepiece calibrated to a stage micrometer. Using a 20x objective, the grid area represented 62 500 µm2. The number of grid areas sampled was sufficient to produce SEMs generally less than 10% of the mean.

Effects of 25-Hydroxycholesterol on LH Responsiveness of Leydig Cells

Leydig cell function was also assessed by testing their ability to produce testosterone in response to a 6-h exposure to 0.001–1.0 ng/ml of LH following 1–5 days of preincubation with 25-hydroxycholesterol. (25-Hydroxycholesterol was washed from the cells before adding the LH, thereby excluding its influence as a substrate of side-chain cleavage.) Leydig cells were cultured in 96-well plates in 100 µl of culture medium at approximately 40 000 cells/well. The medium was then assayed for testosterone by RIA [4, 5]. Purity of the Leydig cell preparation was determined by 3ß-hydroxysteroid dehydrogenase histochemistry at the time of plating [17].

RESULTS

RT-PCR of 25-Hydroxylase

As predicted from the mouse 25-hydroxylase sequence, 184- and 551-bp products were generated using RT-PCR of total RNA from rat testicular macrophages (Fig. 1A). No product was formed when the reverse transcriptase was omitted. Treatment of total RNA with RNase-free DNase did not reduce formation of PCR products. When the 551-bp product was purified by gel electrophoresis and used as the template for nested primers, a 184-bp product was obtained with greater than 90% sequence similarity to mouse 25-hydroxylase (Fig. 1B).



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  		FIG. 1. RT-PCR analysis of total RNA from rat testicular macrophages for 25-hydroxylase. Using a primer set based on the mouse sequence, a 551-bp product was obtained as predicted (A, illustrated in the lane to the left of the 100-bp ladder). Using one of these primers and a second primer designed to a 20-bp sequence within the 551-bp fragment, a 184-bp product was obtained, again as predicted from the mouse sequence (A, illustrated in the lane to the right of the 100-bp ladder). When the 551-bp product was purified from the gel and used as a template for the second set of nested primers, the predicted 184-bp product was again obtained (B). The 184-bp band was then purified, sequenced, and found to be highly homologous to the same region of the mouse 25-hydroxylase (>90%)

Conversion of 14C-Cholesterol to 14C-25-Hydroxycholesterol by Testicular Macrophages

Testicular macrophages converted 14C-cholesterol to 14C-25-hydroxycholesterol (Fig. 2). No 25-hydroxycholesterol was detected in the radiolabeled cholesterol before incubation with macrophages. Approximately 1.2% of the radiolabeled cholesterol was converted to 25-hydroxycholesterol, demonstrating that sufficient substrate was available during this 19-h period. The identity of the compound eluting in fractions 6 and 7 of Figure 2 is unknown.



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  		FIG. 2. Testicular macrophages converted 14C-cholesterol to 14C-25-hydroxycholesterol. Macrophages were incubated for 19 h in the presence of 14C-cholesterol, and then the medium was extracted and analyzed for labeled 25-hydroxycholesterol on a C18 HPLC column. 25-Hydroxycholesterol eluted in fraction 10, representing approximately 1.2% of the counts in cholesterol (fractions 29–32). Reference preparations of 25-hydroxycholesterol and cholesterol also eluted in fractions 10 and 29–32, respectively (data not illustrated). The compound eluting in fractions 6 and 7 is unidentified. This representative experiment was repeated three times with similar results

Autooxidation of 14C-Cholesterol by Macrophage-Conditioned Medium

In four separate experiments (three using medium from peritoneal macrophages and one using medium from testicular macrophages), the fractions eluting at the time of 25-hydroxycholesterol did not contain significant levels of 25-hydroxycholesterol compared to background (data not illustrated).

Effects of 25-Hydroxycholesterol on Viability of Leydig Cells and Lymphocytes

Lymphocytes were killed by 25-hydroxycholesterol in a dose- and time-dependent fashion, with approximately 70% of the cells dying by 2 days of treatment at 5 µg/ml (Fig. 3A). At 10 µg/ml, all cells were killed by 2 days. Leydig cells, however, required 50–100 µg/ml of 25-hydroxycholesterol to attain a similar level of cytotoxicity—and even then, only on Day 4 (Fig. 3B). At the time of plating, approximately 90% of the Leydig cells were positive for 3ß-hydroxysteroid dehydrogenase activity. Testosterone production increased in response to 25-hydroxycholesterol during the first 2 days in culture. No further accumulation was observed from Day 2 to Day 4 of culture at the higher concentrations of 25-hydroxycholesterol (Fig. 3C).



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  		FIG. 3. Leydig cells were highly resistant to the cytotoxic effects of 25-hydroxycholesterol. A) Lymphocyte viability was drastically affected at 5 µg/ml, with cells being completely killed at 10 µg/ml, following 2 and 4 days of exposure (mean and SEM of values from three culture wells). B) 25-Hydroxycholesterol had no effect on Leydig cells at 10 µg/ml, a dose that completely killed lymphocytes (compare to A). Even at 100 µg/ml, nearly 50% of the Leydig cells (compared to control) remained alive (mean and SEM of three experiments). C) Accumulation of testosterone following 2 and 4 days of exposure to 25-hydroxycholesterol (mean and SEM of three experiments). Most of the testosterone likely was produced during the first 1–2 days, particularly at the higher moderately toxic doses, because the amounts of testosterone produced at these higher doses were not different between 2 and 4 days. Even though these high doses were toxic after extended times in culture (see B), they caused significant production of testosterone during the acute phase of treatment (mean and SEM of experiments in B)

Effects of 25-Hydroxycholesterol on LH Responsiveness of Leydig Cells

Leydig cells are well known to become unresponsive to LH and to die with time in culture. 25-Hydroxycholesterol at 0.5 µg/ml had no effect on this decline in LH responsiveness when tested from 1–5 days (Fig. 4). However, when Leydig cells were exposed to higher doses of 25-hydroxycholesterol for 2 days and then treated with LH for 6 h in the absence of 25-hydroxycholesterol, LH responsiveness declined at 5 µg/ml and was completely lost at 10 µg/ml (Fig. 5). Compare these results to those in Figure 3B, demonstrating that 10 µg/ml of 25-hydroxycholesterol had no effect on Leydig cell viability. Similar results were observed after 4 days in culture (data not illustrated).



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  		FIG. 4. Pretreatment of Leydig cells with a low dose of 25-hydroxycholesterol (0.5 µg/ml) for 1, 2, 3, 4, or 5 days had no influence on their ability to produce testosterone under basal conditions or in response to LH in a subsequent 6-h period (in the absence of 25-hydroxycholesterol). 25-Hydroxycholesterol also had no influence on the expected decline in Leydig cell responsiveness to LH with time in culture (mean and SEM of three experiments)



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  		FIG. 5. Treatment of Leydig cells for 2 days with high doses of 25-hydroxycholesterol dramatically reduced their ability to respond to LH. Note that at 10 µg/ml, LH responsiveness was completely eliminated, even though Leydig cell viability was not influenced (compare to Fig. 3B; mean and SEM of three experiments)

DISCUSSION

The 25-hydroxycholesterol produced by testicular macrophages under the conditions used in the present studies likely arose by enzymatic conversion of cholesterol. This speculation is supported by the findings that mRNA for 25-hydroxylase was present in testicular macrophages, that conditioned medium from macrophages was unable to convert cholesterol to 25-hydroxycholesterol, and that authentic 25-hydroxycholesterol was produced from radiolabeled cholesterol by cultured macrophages. Testicular macrophages secrete superoxide anion [10], but it appears that this or other potential oxygen radicals are either present at levels too low to oxidize cholesterol and/or are insufficiently stable under the conditions of the present studies. It is important to note that only 25-hydroxycholesterol was detected in medium from macrophages incubated with 14C-labeled cholesterol (Fig. 2). If autooxidation of cholesterol had occurred, it is predicted that many different oxysterol species would have been produced. Whether the cholesterol used for conversion to 25-hydroxycholesterol arises by de novo synthesis or is attained through lipoprotein receptor-mediated mechanisms in testicular macrophages has yet to be determined.

25-Hydroxycholesterol is toxic to a wide variety of cell types at 1–5 µg/ml [14, 15, 18–21], as confirmed in the present studies using a lymphocyte cell line (Fig. 3). This may explain, in part, the extremely low number of lymphocytes in the interstitium of the testis [22, 23]. That Leydig cells survive in very high concentrations of 25-hydroxycholesterol indicates that this oxysterol possibly represents a significant proportion of the naturally occurring sterol pool used as substrate for side-chain cleavage without its negative cytotoxic effects. This speculation is supported by the fact that Leydig cells are located in a loose connective tissue compartment of the testis that contains an unusually high number of macrophages [24] and was previously shown to produce large amounts of 25-hydroxycholesterol [5]. The physiological significance of 25-hydroxycholesterol is further supported by the findings that animals deficient in macrophages produce less testosterone than intact controls [25–28]. It has not been conclusively established that 25-hydroxycholesterol is the factor that mediates this interaction, but we have shown that this oxysterol is present in the testis [29] and stimulates testosterone production when administered to the testis of animals depleted of macrophages [30]. 25-Hydroxycholesterol also possibly has clinical relevance, because it is produced by human macrophages as well [29].

That long-term treatment with very high levels of 25-hydroxycholesterol (50–100 µg/ml) was cytotoxic to Leydig cells may have resulted from the known inhibitory effects of this oxysterol on cholesterol synthesis [31]. However, when considering the effects of lower levels of 25-hydroxycholesterol (5–10 µg/ml) on LH responsiveness, it is more difficult to speculate on a mechanism of action, because the precise source of the cholesterol used for testosterone production is unclear and varies among species [32–34].

If testicular macrophages were secreting 25-hydroxycholesterol in vivo at a rate sufficient to mimic the high levels used in the present study, LH responsiveness would be blocked, and at the highest levels tested, Leydig cells would be killed. Clearly, however, Leydig cells are responsive to LH in vivo and, therefore, must be normally bathed in a lower concentration of 25-hydroxycholesterol than that used in the present in vitro experiments. Even so, under pathological conditions, high levels of 25-hydroxycholesterol may be produced and, thereby, inhibit LH responsiveness. Such conditions may explain, in part, why some laboratories have found inhibition of steroidogenesis with macrophage-conditioned medium and others a stimulatory response [2, 35–38]. Further studies are needed to determine the precise role of this oxysterol under in vivo conditions, but it seems that its role may be purely stimulatory under physiological conditions given the results of the macrophage-depletion studies [25–28].

Paradoxically, Leydig cells treated with 100 µg/ml of 25-hydroxycholesterol had a high rate of both cytotoxicity and steroid production compared to Leydig cells treated with lower concentrations of this oxysterol. This is speculated to result from a temporal difference in these two mechanisms, with rapid conversion of 25-hydroxycholesterol early in the experiment followed by cytotoxicity toward the end of the treatment period. However, additional studies are needed to rule out the possibility that Leydig cells remaining viable in the presence of 100 µg/ml of 25-hydroxycholesterol exhibit unusually high levels of sterol conversion.

The gift of 25-hydroxycholesterol to Leydig cells is analogous to the production of androstenedione by theca interna cells that is subsequently metabolized to estradiol by adjacent granulosa cells of the ovary [39]. Even with steroids such as testosterone, which are destined for more distant endocrine targets, conversion to more pertinent hormones such as dihydrotestosterone and estradiol occurs within the target cells [40, 41]. Thus, the transfer of sterols from one cell to another for metabolism to a more relevant compound appears to be a recurring theme in lipid-mediated, cell-signaling systems.

The number of macrophages increases during postnatal maturation of the testis, as does the number of Leydig cells [24, 42–44]. Whereas testosterone secretion has been shown to increase dramatically between Days 40 and 60, the ratio of macrophages to Leydig cells does not change concomitantly [44]. Whether the rate of secretion of 25-hydroxycholesterol by testicular macrophages or the ability of Leydig cells to utilize this oxysterol changes during this important time of puberty has yet to be determined.

In summary, the present findings indicate that testicular macrophages 1) possess 25-hydroxylase mRNA, 2) do not release sufficient levels of long-lived compounds that auto-oxidize cholesterol to 25-hydroxycholesterol, and 3) convert radiolabeled cholesterol to authentic 25-hydroxycholesterol. All these findings are consistent with the hypothesis that macrophages produce 25-hydroxycholesterol enzymatically. 25-Hydroxycholesterol stimulates testosterone production under acute conditions at a wide range of doses, but we found that chronic treatment of Leydig cells with high doses of 25-hydroxycholesterol completely blocks LH responsiveness. Finally, we found that Leydig cells are far more resistant than other cell types to the cytotoxic effects of 25-hydroxycholesterol, indicating an adaptive response to the unique environment within the interstitial compartment, where they are in direct contact with oxysterol-producing macrophages.

FOOTNOTES

First decision: 28 September 2000.

1 Supported by a grant from the National Institutes of Health (HD34708) to J.C.H. Back

2 Correspondence. FAX: 806 743 2990; jim.hutson{at}ttmc.ttuhsc.edu Back

Accepted: October 11, 2000.

Received: August 24, 2000.

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