| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
interstitial cells, Leydig cells, puberty, testis, testosterone
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Regulation of Leydig cells during these phases of differentiation involves a variety of growth factors and hormones including LH, androgens, thyroid hormones, estradiol, insulinlike growth factor I, transforming growth factors 
and ß, müllerian inhibiting substance, and peptides derived from Sertoli cells [7–11]. Early studies demonstrated that macrophages are present in the interstitium [12], can be isolated and maintained in culture [13], and influence testosterone production by Leydig cells in vitro [14]. When macrophages are experimentally removed from the testis, Leydig cells produce less testosterone, further indicating that macrophages play a role in Leydig cell function [15–17]. Similarly, in colony stimulating factor-1 mutant animals that have very few macrophages, Leydig cells do not develop the typical morphology of adult Leydig cells and also produce less testosterone [18, 19]. We have found that macrophages from adult animals produce a 25-hydroxylase that converts cholesterol to 25-hydroxycholesterol, an oxysterol that can be efficiently converted to testosterone by adjacent Leydig cells [20–24]. Because 25-hydroxycholesterol has also been shown to be involved in the differentiation of other cell types including keratinocytes [25] and hepatocytes [26], we hypothesized that the deficiencies in Leydig cell development in macrophage-depleted animals is in part due to the absence of 25-hydroxycholesterol. However, in order for 25-hydroxycholesterol to be involved in Leydig cell maturation, it must be produced during the postnatal period. Therefore, the first aim of the present study was to determine if cultured macrophages from animals at various postnatal ages produce 25-hydroxycholesterol and mRNA for the enzyme responsible for its production (25-hydroxylase mRNA). Our second goal was to determine if chronic exposure to 25-hydroxycholesterol induces maturational changes in cultured immature Leydig cells using basal and LH-stimulated testosterone production and 3ß-hydroxysteroid dehydrogenase histochemistry as end points.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Male rats (Charles River Laboratories, Inc., Wilmington, MA) were handled in accordance with protocols approved by the Animal Use and Care Committee of Texas Tech University Health Sciences Center. Dulbecco modified Eagle/Ham F12 medium (DME/F12), BSA (fraction V), penicillin, streptomycin, collagenase (Type I), and other routine chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Percoll was obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). 25-Hydroxycholesterol (cholest-5-ene-3ß,25-diol) was obtained from Steraloids (Newport, RI). Ovine LH (biological preparation) was from the National Pituitary Program of the National Institute of Arthritis Metabolic and Digestive Diseases. The 100- and 35-mm diameter dishes were obtained from Becton Dickinson Labware (Franklin Lakes, NJ), and the 96-well plates were from Corning Glass Works (Corning, NY). HPLC grade organic solvents were purchased from Fisher Scientific (Fair Lawn, NJ). HPLC columns (C18, Microsorb MV, 10 nm, 4.6 x 25 cm) were from Varian Chromatography Systems (Walnut Creek, CA). The gas chromatography column (DB-5 fused silica, 30 m x 0.25 mm internal diameter, 0.25 µm film thickness) was from J & W Scientific (Rancho Cordova, CA). The Light Cycler and FastStart DNA Master SYBR Green I reagent kit and glycogen were from Roche Molecular Biochemicals (Indianapolis, IN). Primers for reverse transcription-polymerase chain reaction (RT-PCR) were from Midland Certified Reagent Company (Midland, TX). Plasmid containing the 25-hydroxylase cDNA was generously provided by Dr. David W. Russell (University of Texas Southwestern Medical Center, Dallas, TX). Molecular biology grade agarose and TRIzol Reagent were from Life Technologies (Grand Island, NY). RNase-free DNase I was from Amersham Pharmacia Biotech (Umea, Sweden). The testosterone RIA kit was from Diagnostic Systems Laboratories (Webster, TX).
Cell Isolation and Culture
Testicular macrophages Testicular macrophages from adult and 40-day-old rats were isolated as previously described [20]. Animals were killed with CO2, and the testes were removed and perfused through the gonadal veins (deep to the tunica albuginea) with collagenase (100 U/ml in culture medium containing 0.1% BSA) with a 30-gauge needle. The testes were then decapsulated and digested in collagenase in a shaking water bath at 120 cycles per minute at 37°C. Undigested seminiferous tubules were removed by unit gravity sedimentation, and the interstitial cells were recovered from the supernatant by centrifugation (350 x g, 6 min). Interstitial cells were suspended in medium and plated into culture dishes (described subsequently) for 7–12 min to allow rapidly adhering cells (primarily macrophages) to attach. Nonadherent cells were then removed by vigorous washing with medium. Macrophages from immature animals (10 and 20 days of age) were obtained using the adult isolation procedure except that testes were digested in collagenase for 18 min in a shaking water bath at 60 cycles per minute, and DNase (1 mg/ml) was added drop-wise to reduce clumping at the end of the digestion.
Cells from adult and 40-day-old animals were plated into 100-mm dishes in 6 ml medium, and cells from 10- and 20-day-old animals were plated into 35-mm dishes in 1 ml serum-free medium. In a separate single experiment, cells were assessed for purity using Fc receptor binding [13]. Cultures from the 10-, 20-, and 40-day-old animals were 71%, 93%, and 85% positive for Fc receptor, respectively. Testicular macrophages from adult animals were approximately 95% positive for Fc receptor. Purity and yield depend on a balance between plating time and the force used to wash the unattached cells from the cultures. The majority of the contaminating cells were Leydig cells, with a few cells having the morphology of peritubular cells.
Leydig cell isolation Leydig cells were retrieved from the interstitial cells that were washed from the macrophage cultures described previously. These cells were collected by centrifugation (350 x g, 6 min, 4°C) and then centrifuged through Percoll gradients as previously described [20]. Fetal Leydig cells (10 days of age), immature Leydig cells (40 days of age), and adult Leydig cells (250–500 g body weight) were collected from fractions with an approximate density range of 1.07 g/ml and greater (up to the red blood cell band). Progenitor Leydig cells (20 days of age) were isolated from fractions at an approximate density range of 1.064–1.070 g/ml. The percentage of the total population that was 3ß-hydroxysteroid dehydrogenase-positive at the end of the experiment was as follows: 20-day-old, 82%; 40-day-old, 83%; and adult, 88%. The 10-day-old preparations were negative for 3ß-hydroxysteroid dehydrogenase. Leydig cells were plated into 96-well plates in 100 µl of serum-free culture medium (0.5–1 x 105 cells per well) and maintained in culture as previously described [20].
Quantitative Measurement of Steady-State Levels of 25-Hydroxylase mRNA Using Real-Time RT-PCR
After 26 h of culture, total RNA was isolated from testicular macrophages and Leydig cells (as a control for cellular specificity) using TRIzol Reagent plus glycogen (250 µg/ml). Complementary DNAs were generated using avian myelobastosis virus reverse transcriptase and oligo(dT)12–18 primers at 48°C for 45 min. Complementary DNAs were amplified in a Roche LightCycler using the FastStart DNA Master SYBR Green I reagent kit. Cycle conditions were 95°C for 30 sec and 40 cycles of 95°C for 0 sec, 58°C for 5 sec, and 72°C for 8 sec. Primer sequences were: 5'-GCGACCCAATACATGAGCTT-3' and 5'-CAAAGGGCACAAGTCTGTGA-3' spanning bases 507–691 of the mouse 25-hydroxylase (GenBank accession No. AF059213). Controls included omission of the reverse transcriptase or template, which were consistently found to be negative. Copy number was determined by comparing the linear portion of the DNA accumulation curves generated by the samples to those curves generated by serially diluted standards (plasmid carrying the 25-hydroxylase cDNA). Products were also analyzed on agarose minigels stained with ethidium bromide. We have previously sequenced this product and found it to be 25-hydroxylase [24]. RT-PCR was used since both 25-hydroxylase mRNA and protein were undetectable using approximately 3 x 106 testicular macrophages by Northern analysis and Western analysis, respectively [23].
Detection of 25-Hydroxycholesterol
Culture medium from testicular macrophages was extracted with 3 volumes of ether, and the organic phase was blown to dryness with nitrogen and dissolved in 180 µl of methanol. Extracts were analyzed by HPLC using a C18 column with methanol as the mobile phase at a flow rate of 1 ml/min. The HPLC isolates were analyzed on a Hewlett Packard 5890 Series II gas chromatograph (GC) (Palo Alto, CA) in the splitless mode on a DB-5 fused-silica column using a temperature program of 170–280°C developed over 7 min. Isolates were analyzed with a Hewlett Packard 5970 mass spectrometer and compared with a reference preparation of 25-hydroxycholesterol.
Effects of 25-Hydroxycholesterol on Basal and LH-Stimulated Testosterone Production by Leydig Cells
After 1 h of culture, the medium was removed, and fresh medium (100 µl) containing 0, 0.5, 5, 10, or 50 µg/ml 25-hydroxycholesterol was added to Leydig cells. After 24 h, the concentration of testosterone in the medium was determined by RIA with a standard curve prepared in culture medium. The cells were washed 3 times with fresh medium, allowing 10-min rests between washes for 25-hydroxycholesterol to diffuse out of the cells. After the final wash, 50 µl of medium was added to the cultures with or without a maximal dose of LH (1 ng/ml). After 6 h, the concentration of testosterone in the medium was determined by RIA. The cells were then stained for 3ß-hydroxysteroid dehydrogenase activity as previously described using etiocholane as the substrate [27]. Substrate was omitted from control dishes to establish specificity. Staining intensity was graded from + to ++++ by visual inspection using an inverted light microscope.
Statistics
All experiments were repeated at least 3 times using a separate cell isolation for each experiment with exceptions as noted. Replicates (3 to 6) were used for each group within an experiment. ANOVA followed by the Fisher probable least-squares difference test was used to assess statistical differences between group means. Groups were considered statistically different at P 
0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Macrophages from animals at all ages had a very similar morphologic appearance and attached rapidly to the culture dish. The major difference was that the yield of cells from immature animals was far less than that from adult animals, as would be expected given the number of macrophages per testis during the early postnatal period [6, 25, 26]. This was overcome by using more animals per culture. The approximate yield of macrophages per rat at the various ages was 4 x 103 cells at 10 days of age; 1 x 104 cells at 20 days of age; 1.6 x 105 cells at 40 days of age; and 1.5 x 106 cells at adult age.
Steady-State Levels of 25-Hydroxylase mRNA
25-Hydroxylase transcripts were present in macrophages from rats at all ages, with the highest number in cells present in 10-day-old animals (Table 1). Agarose gel analysis of the PCR products, as well as the melting curve analysis from the RT-PCR, demonstrated that a single product was formed at the appropriate size (184 base pairs). No amplification was observed when input RNA or the reverse transcriptase was omitted. No product was formed when RNA from Leydig cells was used.
|
Identification of 25-Hydroxycholesterol in Medium from Testicular Macrophages
25-Hydroxycholesterol was present in media from testicular macrophages isolated from rats at 10, 20, and 40 days of age as well as from adult animals. The elution time for 25-hydroxycholesterol from cells and the reference standard was 4.9 min by HPLC and 17.1 min by GC. The ion spectrum obtained by mass spectrometry for cellularly derived 25-hydroxycholesterol and the reference preparation were identical to those previously reported [21].
Responsiveness of Leydig Cells to 25-Hydroxycholesterol
Treatment of Leydig cells with 25-hydroxycholesterol for 24 h resulted in elevation of levels of testosterone in the medium in a dose-dependent fashion at all ages (Fig. 1). The amount of testosterone produced was dependent on the state of cell differentiation, as expected. Within the adult population, the amount of testosterone per 106 Leydig cells increased under basal conditions as follows: progenitor, 1.0 ng/ml; immature, 21.5 ng/ml; and adult, 645 ng/ml. In response to the highest dose of 25-hydroxycholesterol (50 µg/ml), the following increases in testosterone production was observed: progenitor, 13-fold; immature, 19-fold; and adult, 27-fold. The amount of testosterone produced by fetal Leydig cells was 15.1 ng/ml per 106 cells under basal conditions, with a 23-fold increase in response to the highest dose of 25-hydroxycholesterol (Fig. 1).
|
Effects of 25-Hydroxycholesterol on Leydig Cell Differentiation
Pretreatment of Leydig cells at all ages for 24 h with 25-hydroxycholesterol dose-dependently elevated basal testosterone production during a subsequent 6-h period in the absence of 25-hydroxycholesterol (Fig. 2). Conversely, the amount of testosterone produced by 25-hydroxycholesterol-pretreated Leydig cells in the presence of LH was reduced in Leydig cells at all ages (Fig. 2). When considered as a percentage of control values, the ability to respond to LH was markedly reduced by 25-hydroxycholesterol in a dose-dependent manner.
|
Whereas untreated cultures of progenitor and immature Leydig cells were unstained (except for an occasional cluster of cells that was lightly stained), cells treated for 24 h with 25-hydroxycholesterol (50 µg/ml) were clearly positive (1+) for 3ß-hydroxysteroid dehydrogenase (Fig. 3). We found that 3ß-hydroxysteroid dehydrogenase-positive cells from 20-day-old animals were also present in more dense regions of the Percoll gradients (1.07 g/ml and greater). This population of cells was affected by 25-hydroxycholesterol in a manner similar to that of the traditionally defined progenitor cells (1.064–1.07 g/ml) presented previously. Changes in 3ß-hydroxysteroid dehydrogenase activity were not observed after 25-hydroxycholesterol treatment in fetal or adult Leydig cells.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Steady-state levels of 25-hydroxylase were highest in macrophages of 10-day-old animals, indicating that this may be an important developmental time to focus on the regulation of this enzyme, as well as the production of 25-hydroxycholesterol and its effects on undifferentiated Leydig cells. It is yet to be determined if this increase in 25-hydroxylase message is due to a change in the rate of transcription or message stability, or if these levels result in increased production of 25-hydroxycholesterol. Because the macrophage preparations were not 100% pure, it is possible that contaminating cells were involved in this age-related response. It should be noted that Leydig cells, which are the major contaminating cell type of the testicular macrophage cultures, were found to be negative for 25-hydroxylase mRNA. We have previously found that primary cultures of peritoneal macrophages also express 25-hydroxylase and produce 25-hydroxycholesterol, strongly supporting the conclusion that this oxysterol is a product of macrophages [24].
We also found that 25-hydroxycholesterol has significant effects on the maturation of Leydig cells. First, basal testosterone production was increased in Leydig cells at all ages after pretreatment with 25-hydroxycholesterol. We wanted to test the effects of 25-hydroxycholesterol that were not associated with its known role as a substrate for cholesterol side chain cleavage enzyme. Therefore, we treated Leydig cells for 24 h and then washed the Leydig cells with three medium changes over the next 30 min in the absence of 25-hydroxycholesterol. The amount of testosterone made during the 6-h period after removal of 25-hydroxycholesterol was then measured. Although it can not be completely ruled out that small amounts of 25-hydroxycholesterol remained within Leydig cells or bound to the culture dish and thereby served as a precursor for testosterone, it seems likely that most of the observed effect was due to a direct effect of 25-hydroxycholesterol on one or more steps in the steroidogenic pathway.
Potentially the most important finding was that 25-hydroxycholesterol increased 3ß-hydroxysteroid dehydrogenase activity in progenitor and immature Leydig cells. We do not believe that this is due to residual 25-hydroxycholesterol within Leydig cells serving as a substrate for 3ß-hydroxysteroid dehydrogenase since the histochemical reaction was conducted with the substrate at a saturating concentration. Thus, any residual 25-hydroxycholesterol would represent only a small amount of the total concentration of substrate. It is not known if this effect was at the level of transcription and/or if other steps in the steroidogenic pathway were also influenced. Although 3ß-hydroxysteroid dehydrogenase is distal to the molecular and enzymatic rate-limiting steps, this phenomenon may in part explain why testosterone production is reduced in animals deficient in testicular macrophages [15–19]. It is curious that macrophage-derived 25-hydroxycholesterol has this effect on the adult population of Leydig cells at ages when the ratio of macrophages to Leydig cells is low (21 days old, 1:10.6; 40 days old, 1:6.7) [31].
As we previously found in adult animals [23], the LH-responsiveness of fetal, progenitor, and immature Leydig cells was dose-dependently inhibited by chronic treatment with 25-hydroxycholesterol. If this phenomenon also occurs in vivo, it may serve as a dampening mechanism preventing overstimulation by LH. This inhibitory effect would most likely occur only with high concentrations of 25-hydroxycholesterol (micrograms per milliliter), whereas lower concentrations may act more simply by serving as a substrate for steroidogenesis. Although the concentration of 25-hydroxycholesterol in the interstitial fluid that directly surrounds Leydig cells is unknown, it seems that the most significant role of 25-hydroxycholesterol is as a substrate for steroidogenesis, rather than inhibiting LH-responsiveness, since animals lacking macrophages have reduced levels of testosterone [15–19]. Because Leydig cells and macrophages are commonly found in direct contact in vivo [12, 32], even low levels of 25-hydroxycholesterol would have direct access to Leydig cells. These postulates are supported by the finding that 25-hydroxycholesterol has been shown to be present in the testis of normal rats [24]. Additional studies concerning the regulation of 25-hydroxycholesterol production and its rate of turnover in interstitial fluid are needed.
It has been shown that 25-hydroxycholesterol and other oxysterols increase steroidogenic factor 1-mediated steroidogenic acute regulatory (StAR) gene transactivation in CV-1 cells [33]. Since StAR is the rate-limiting step involved in controlling access of cholesterol to the side chain cleavage system [34], it seems that 25-hydroxycholesterol should have stimulated LH-driven steroidogenesis. The simplest explanation for this apparent discrepancy is that there are multiple sites of action of oxysterols in the steroidogenic pathway, some inhibitory and some stimulatory. It has been shown that approximately 15% of the MA-10 Leydig tumor cell's capacity to produce progesterone is not mediated by StAR [35], indicating that separate regulatory mechanisms exist for basal and LH-mediated steroidogenesis. We are currently conducting studies to determine the mechanisms responsible for this paradoxical influence.
In conclusion, the results of the present study support the hypothesis that 25-hydroxycholesterol from testicular macrophages is involved in the differentiation of Leydig cells that occurs in the adult population of cells during postnatal maturation of the testis.
|
FOOTNOTES |
|---|
1 This work was supported by a grant from the NIH (HD34708) to J.C.H. 
2 Correspondence: James C. Hutson, Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, 3601 4th St., Lubbock, TX 79430. FAX: 806 743 2990; jim.hutson{at}ttmc.ttuhsc.edu
Accepted: November 28, 2001.
Received: August 24, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. C. Hutson Physiologic Interactions Between Macrophages and Leydig Cells Experimental Biology and Medicine, January 1, 2006; 231(1): 1 - 7. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Morales, P. Santana, R. Diaz, C. Tabraue, G. Gallardo, F. L. Blanco, I. Hernandez, L. F. Fanjul, and C. M. Ruiz de Galarreta Intratesticular Delivery of Tumor Necrosis Factor-{alpha} and Ceramide Directly Abrogates Steroidogenic Acute Regulatory Protein Expression and Leydig Cell Steroidogenesis in Adult Rats Endocrinology, November 1, 2003; 144(11): 4763 - 4772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Lukyanenko, J.-J. Chen, and J. C. Hutson Testosterone Regulates 25-Hydroxycholesterol Production in Testicular Macrophages Biol Reprod, November 1, 2002; 67(5): 1435 - 1438. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
