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Is Potentiated by Growth Hormone and Insulin-Like Growth Factors
Department of Woman and Child Health, Pediatric Endocrinology Unit, Astrid Lindgren Children’s Hospital, Karolinska Institute and University Hospital, S-171 76 Stockholm, Sweden
Address all correspondence and requests for reprints to: Dr. Olle Soder, Department of Woman and Child Health, Pediatric Endocrinology Unit, Q2:08, Karolinska Institute and Hospital, Astrid Lindgren Children’s Hospital, S-171 76 Stockholm, Sweden. E-mail: olle.soder{at}kbh.ki.se.
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is produced constitutively by the intact testis, but its function in this organ remains largely unknown. In this study we examined cooperation between IL-1 and GH and IGFs with regard to stimulation of steroidogenesis by Leydig cells from 40-d-old rats in vitro. IL-1 alone stimulated testosterone (T) and dihydrotestosterone (DHT) production. GH, IGF-I, or IGF-II alone was without effect on T production, but they were found to elevate DHT release, albeit without an obvious dose-response effect. Costimulation with IL-1 and GH or with IL-1 and IGF-I or IGF-II elevated the rate of steroidogenesis (both T and DHT) above that observed with IL-1 alone. GH was found to increase the level of IGF-I in the cultured Leydig cells, an effect that was potentiated by IL-1. The costimulatory effect of GH on steroidogenesis was abolished by treatment with picropodophyllin, a specific inhibitor of the IGF-I receptor, indicating that the action of GH is mediated via IGF-I. Moreover, cells costimulated with IL-1 and GH exhibited a marked decrease in the level of intact IGF-binding protein-3 in the culture medium due to the induction of proteolytic activity toward this binding protein. In contrast, secretion of IGF-binding protein-2 was increased by such costimulation. These findings suggest that the stimulation of steroidogenesis in Leydig cells evoked by GH and IGFs requires cooperation with IL-1. This cooperation may play an important role in connection with postnatal Leydig cell maturation and steroidogenesis. |
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, A PROINFLAMMATORY cytokine, was originally shown to be synthesized and released by activated mononuclear phagocytes (1). This cytokine has been found to be produced even in tissues such as the skin and testis in the absence of inflammation, indicating that it plays a noninflammatory, paracrine role at such sites (2, 3). Although the precise function of IL-1 in the testis is not clearly understood, findings suggest that this signal is involved in the paracrine regulation of spermatogenesis (4, 5). IL-1 has been demonstrated to exert both inhibitory (6, 7) and stimulatory (8, 9, 10) effects on steroidogenesis by Leydig cells. Recent studies have revealed that the effect exerted by IL-1 in this context depends on the stage of maturation of the Leydig cells (8) and that it is mediated by stimulation of the steroidogenic acute regulatory protein (StAR) (10, 11). Stimulation of the expression of StAR in immature Leydig cells by IL-1 is dose- and time-dependent (10). The proposed sources of constitutive production of IL-1 in the testis are the Sertoli cells (12, 13). The testicular level of this cytokine varies with age and stage of development, and in the case of the rat testis, IL-1 mRNA and protein are detectable from 20–25 d after birth (14). Several isoforms of IL-1 are present in rat testis and are secreted into both interstitial and intratubular compartments (15, 16). IL-1ß, the more abundant proinflammatory isoform of IL-1, synthesized by activated macrophages (1) and is not produced constitutively in the testis, but its expression in this organ is readily induced by proinflammatory stimuli such as endotoxin (7, 17). The findings that IL-1, in contrast to IL-1ß, does not evoke an inflammatory response in the testis after local injection (18), and that it is at least 10 times more potent than IL-1ß to stimulate Leydig cell steroidogenesis in vitro (15) indicate differential functions of these two isoforms of IL-1 in the testis. GH, a 22-kDa protein secreted by the anterior pituitary, is required for longitudinal growth and certain aspects of global metabolism (19). There are also indications that GH plays a role in gonadal steroidogenesis and gametogenesis (20), exerting endocrine action either directly at gonadal sites or indirectly via IGF-I (21). Thus, male rats immunized against GHRH exhibit delays in both testicular growth and differentiation of germ cells (22). In male subjects, congenital GH deficiency may result in a delay in the onset of puberty (23) and has also been associated with decreased sperm counts and motility and reduced testicular size (24, 25). Furthermore, GH evokes an increase in the number of precursor mesenchymal cells in the testis of immature, hypophysectomized male rats (26). In addition, this hormone influences Leydig cell steroidogenesis, increasing the expression of several genes that code for steroidogenic enzymes, including 3ß-hydroxysteroid dehydrogenase (27, 28). GH also increases the levels of mRNA encoding StAR in progenitor, immature, and adult rat Leydig cells, indicating an effect on the differentiation of this cell type (28, 29).
GH is known to regulate the secretion of IGF-I by Leydig cells (30), thereby enhancing steroidogenesis in these same cells via up-regulation of human chorionic gonadotropin (hCG)-induced expression of StAR (31, 32). Little information concerning the regulation of IGF-II production by the testis and possible interactions between this factor and cytokines is presently available. Some studies suggest that IGF-II participates in the regression of fetal and/or the proliferation of adult Leydig cells around the time of birth as well as in the initial stage of gonadal differentiation (33).
The pattern of steroidogenesis in Leydig cells varies in parallel with their postnatal maturation. The major androgen produced by 30-d-old rat testes is 5-androstane-3,17ß-diol (34). At this age, the activity of 5-reductase increases, converting steroids with a 3-oxo-
structure to the corresponding 5-reduced metabolite. In the case of testosterone (T), this leads to formation of dihydrotestosterone (DHT), which is one substrate for the formation of 5-androstane-3,17ß-diol.
Regulation of the onset and maintenance of steroidogenesis during puberty is governed primarily by LH. Once this process has been initiated, other hormones and factors also modulate steroidogenesis, and identification and characterization of the roles played by such factors in the testis are of central importance. In the present study we examined the cooperation between paracrine IL-1 and the GH-IGF axis in connection with Leydig cells steroidogenesis in vitro. In particular, we investigated the manner in which such cooperation might modulate local expression of IGF-I and production of the IGF-binding proteins (IGF-BPs).
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was purchased from R&D Systems (Abingdon, UK); recombinant rat GH, IGF-I, and IGF-II and rabbit antirat IGF-I antiserum were obtained from GroPep Ltd. (Adelaide, Australia); 125I was obtained from NEN Life Science Products (Boston, MA); and Sepharose-coupled secondary antibodies (sheep antirabbit IgG) was purchased from BD Biosciences (Uppsala, Sweden). The cyclolignan 7R,7'R,8R,8'R-7-hydroxy 3',4',5'-trimethoxy-5-methylenedioxy-2,7'cyclolignan-9,9'-oxide (PPP) (35) was a gift from Drs. Olle Larsson and Magnus Axelson (Karolinska Hospital, Stockholm, Sweden).
Isolation and culture of Leydig cells
Immature Leydig cells were isolated from 40-d-old male Sprague Dawley rats (B&K Laboratories, Sollentuna, Sweden) (36). These animals were provided with a standard pellet diet and water ad libitum. All experiments were conducted in accordance with institutional guidelines and approved in advance by the northern Stockholm ethics committee, for animal experimentation. Leydig cells were prepared by subjecting rat testes to collagenase treatment as described previously (8). Briefly, decapsulated testes were incubated with collagenase (0.25 mg/ml) for 20 min at 37 C, and the suspension thus obtained was filtered through nylon gauze (70 µm pore size; BD Biosciences, Piscataway, NJ). A crude mixture of interstitial cells was collected by centrifugation of the resulting filtrate at 300 x gav for 8 min, then washed twice in MEM containing 0.1% (wt/vol) BSA. To obtain purified Leydig cells, 3 ml of this crude cell suspension were loaded on top of a discontinuous Percoll gradient (20%, 40%, 60%, and 90% Percoll in Hanks’ Balanced Salts Solution), then centrifuged at 800 x gav for 20 min. Leydig cells enriched in these fractions were finally collected by centrifugation through a 90% Percoll at 20,000 x gav for 30 min at 4 C. After such density centrifugation, Leydig cells are recovered in Percoll fractions with a density greater than 1.068 g/ml. According to Ge et al. (37), we designated the Leydig cells isolated in this manner from 40-d-old rats as immature. Cell viability, assessed on the basis of trypan blue exclusion, was greater than 90%. The cell preparation consisted of 87–90% of Leydig cells, as determined by histochemical staining for 3ß-hydroxysteroid dehydrogenase. For purposes of culture, the purified Leydig cells were first washed twice with DMEM-F-12, then resuspended in DMEM-F-12 supplemented with 15 mM HEPES (pH 7.4), BSA (1 mg/ml), glutamine (365 mg/ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml). Subsequently, 1.5 x104 cells/ml in a total volume of 200 µl were plated into each well of 96-well plates (Falcon, BD Biosciences, Franklin Lakes, NJ) and incubated for 24 h. At this point, the culture medium was replaced by fresh medium with or without IL-1 (1 ng/ml). This concentration is optimal for stimulation of steroidogenesis by immature Leydig cells steroidogenesis (10), toward which IL-1 alone exhibits the same efficacy as hCG (8).
In addition to IL-1, GH, IGF-I, or IGF-II was included in the medium at various concentrations, and the cells were incubated for another 24 h. In the case of incubations continued for 48 h, fresh culture medium containing IL-1 (1 ng/ml) or the appropriate hormones was added after 24 h. In all cases, four independent experiments were performed.
In additional experiments, the cells (2 x 106) in a total volume of 2 ml were plated into each of 12 wells (Falcon) and incubated for 24 h. Thereafter the original culture medium was replaced with fresh medium with or without IL-1 (1 ng/ml) and/or an optimal concentration of GH (10 ng/ml), and incubation was continued for another 24 h. Finally, the culture medium was collected and stored at –80 C for later determination of IGF-I by RIA and of IGF-I binding proteins by Western ligand blotting and Western immunoblotting. Three independent experiments were carried out.
Analysis of Leydig cell proliferation in vitro
IL-1 has been shown previously to stimulate the proliferation of Leydig cells from 10- to 20-d-old rats in culture, but did not affect the proliferation of Leydig cells from older animals (38), including 40-d-old rats employed in the present study (8). To exclude that the observed effects of GH, IGF-I, and IGF-II were due to increased Leydig cell proliferation in vitro, cells were cultured on 96-well plates, labeled with [3H]thymidine (1 µCi/well; Amersham Biosciences, Little Chalfont, UK) during the final 4 h of incubation, and subsequently harvested (4, 5). Incorporation of radioactivity (counts per minute) was measured in a scintillation spectrometer (Beckman Coulter, Fullerton, CA). In each of two independent experiments, three or four cultures were characterized in this manner.
The number of Leydig cells present in a given culture was estimated by the addition of the supravital precursor dye thiazolyl blue (0.05 mg/well; 3-[4,5-dimethlthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich Corp.) before addition of the factor to be tested and after 44 h of incubation. After 4 h of labeling with this dye, the cells were lysed with 10% sodium dodecyl sulfate in 0.01 M HCl (90 µl/well), and the lysate thus obtained was additionally incubated at 37 C for an additional 24 h. Finally, the absorbance at 570 nm was determined using an ELIZA reader (Anthos Labtec Instruments, Salzberg, Austria) and was recorded as absorbance units. In each of two independent experiments, three or four cultures subjected to each treatment were examined in this manner.
Hormone analyses
Media collected from the cultures described above were stored at –20 C before assaying T and DHT employing a Coat-a-Count RIA kit for T (Diagnostic Products Corp., Los Angeles, CA) and an ELISA procedure for DHT (catalog no. 1940, Diagnostic International, San Antonio, TX), according to the manufacturer’s instructions. No steroid extraction procedure was employed due to the small volumes of analyzed medium. The reported cross-reactivity of the T RIA for DHT was 3.3%, and that for T with the DHT ELISA was 8%.
Media were also collected at different time points for determination of the IGF-I secreted. First, this IGF-I was removed from its binding proteins by acid-ethanol extraction (39). For this purpose, the sample of medium was mixed with acetic acid to obtain a final concentration of 1 M and with ethanol (1:4), incubated for 1 h at 20 C, then centrifuged. The supernatants thus obtained were lyophilized, and the residue was dissolved in a volume of PBS, pH 7.4, corresponding to 50% of the original volume.
Subsequently, the level of IGF-I in these extracts was determined employing a RIA procedure developed in our own laboratory and involving rabbit antirat IGF-I, recombinant rat IGF-I as the standard, and 125I-labeled rat IGF-I, (iodinated using the chloramine-T) as the radioligand. One hundred microliters of a standard sample or extract were incubated with 100 µl [125I]IGF-I (
10,000 cpm) and 100 µl rabbit antirat IGF-I (final dilution, 1:60,000) in PBS containing 0.5% BSA for 18 h at 4 C. Sheep antirabbit immunoglobulin G (IgG) antibodies coupled to Sepharose beads (500 µl) were then added, and this mixture was incubated for 30 min at room temperature. After adding 500 µl H2O, bound and free radioligand was subsequently separated by centrifugation at 3000 x gav for 15 min at 4 C. Finally, the supernatants were discarded, and the content of 125I in the pellets counted in a 
-counter for 1 min (39).
Recovery of IGF-I (1.5–10 ng/ml) added to control and test media as an internal standard on the second day of culture was 100 ± 10% (mean ± SD of six determinations), and the lower limit of detection limit was 0.31 ng/ml. The extent of the cross-reactivity with recombinant human IGF-I was shown to be less than 0.5%, and no cross-reactivity with recombinant human and rat IGF-II could be detected.
RT-PCR analysis of IGF-I mRNA
After incubation, 2 x 106 control or treated cells were collected with a rubber policeman and homogenized for 30 sec using a rotor-stator. Total RNA was then extracted from these homogenates employing the TRIzol reagent (Invitrogen Life Technologies, Inc.) and a modification of the guanidine isothiocyanate/phenol-chloroform extraction procedure described by Chomczynski and Sacchi (40). The RNA concentration in these extracts was determined on the basis of the absorbance at 260 nm (measure with a model U-2000 spectrophotometer, Hitachi, Hialeah, FL), and these RNA samples were subsequently stored at –80 C.
RT was carried out employing a cDNA kit (First-Strand cDNA Synthesis Kit, Amersham Biosciences), according to the manufacturer’s instructions. cDNA was synthesized from total RNA using the Superscript cDNA Kit (Invitrogen Life Technologies, Inc.). Primer pairs specific for the cDNA corresponding to rat IGF-I and the ribosomal protein S27a (used as internal standard) were designed and were ordered from Genset Oligos (Paris, France).
The primers for rat IGF-I were designed on the basis of published amino acid sequence of this protein using the Primer 3.cgivo.2c program (www-genome.wl.mit.edu). The sequence of the forward primer, located in exon 3, was 5'-CAATTCGTGTGTGGACCAAG-3'; that of the reverse primer located in exon 4 was 5'-GACTTTGTAGGCTTCAGCGG-3'. With the use of these forward and reverse primers, the predicted size of the PCR product was 164 bp (41).
The primers for S27a were designed on the basis of the published amino acid sequence of the rat protein (42). The sequence of the forward primer was 5'-CCAGGATAAGGAAGGAATTCCTCCTG-3'; that of the reverse primer was 5'-CCAGCACCACATTCATCAGAAGG-3'. The use of these primers produced a PCR product containing 296 bp, as reported previously (42).
PCR was performed using the Gene Amp PCR 2400 system (PerkinElmer, Palo Alto, CA) in a total reaction volume of 50 µl containing 15 µM primers, 0.2 mM deoxy-NTP, 15 mM MgCl2, and 3.5 U Expand High Fidelity/ml (Roche Diagnostics Scandinavia, Bromma, Sweden). The conditions employed for the amplification were as follows: 5 min at 96 C, followed by 30–35 cycles of denaturation at 96 C for 30 sec, annealing at 52–58 C for 30 sec, and extension at 72 C for 1 min or, in the final round, for 5 min. A control PCR without added cDNA revealed no contamination from the components of the reaction mixture. Rat liver was used as a positive control. S27a was used to normalize the RT-PCR data for the IGF-I mRNA. The primer sets for S27a were added to separate tubes and run in parallel.
The PCR products amplified from each cDNA were separated by electrophoresis on 2% agarose gels and visualized by staining with ethidium bromide. The intensities of the bands thus obtained were analyzed using the EDAS 120 Kodak Gel documentation system (Eastman Kodak Co., Rochester, NY), and the level of IGF-I mRNA was standardized to that of S27a, which was assumed to remain constant.
Inhibition of IGF-IR
In attempt to elucidate the role of IGF signaling via the IGF-I receptor (IGF-IR) in our system, we employed cyclolignan PPP as a specific inhibitor of the IGF-IR tyrosine kinase (35). Cells (100 µl of 1.5 x104 cells/ml) were cultured for 24 h, as described above, then incubated in the presence of IL-1 (1 ng/ml), PPP (1.0 µM), GH (10 ng/ml), or IL-1 (1 ng/ml) and IGF-I (10 ng/ml) for another 24 h, after which the culture medium was collected for determination of T. In this case, PPP was added to the medium 30 min before the addition of hormone, and three independent experiments were performed.
Determination of IGFBPs
Media from primary cultures of control Leydig cells and cells stimulated with IL-1 and/or GH for 24 h were collected to analyze secretion of IGFBPs. IGFBPs were quantitated by Western ligand blotting as originally described by Hossenlopp et al. (43), with minor modifications. Briefly, normal rat serum (5 µl), recombinant human IGFBP-1 and -2 (40 ng of each), or the medium from Leydig cells (100 µl) was diluted with nonreducing sodium dodecyl sulfate sample buffer for separation by SDS-PAGE (12% gels). The resulting bands were electroblotted onto nitrocellulose filters (0.45 µm pore size) in a Hoefer Semi-Dry Transphor unit at 200 mA (Amersham Biosciences). Thereafter filters were treated sequentially with 0.1% Tween 20 in Tris-buffered saline (TBS-T) and 1% BSA in TBS-T, then probed with 125I-labeled IGF-I in TBS-T containing 1% BSA (2 x 106 cpm/50 ml). Subsequently, the filters were washed with TBS-T, dried, and subjected to phosphorimage analysis (Fujifilm BAS-1000 System, Fuji, Tokyo, Japan). The density of each of the phosphorimage bands was analyzed and the average band density associated with each treatment, expressed as the fold increase compared with the corresponding control values. Three independent experiments of this kind were carried out.
IGFBP-2 and -3 were quantitated by Western immunoblotting using the enhanced chemiluminescence detection technique. In brief, normal rat serum (5 µl) or the residue from lyophilized culture medium (200 µl) was diluted with nonreducing sodium dodecyl sulfate sample buffer for separation by SDS-PAGE (12% gels) and subsequent electroblotting as described above for the ligand blots. The filters were then treated sequentially with TBS-T and 3% nonfat dry milk in TBS-T, after which they were probed with antirat IGFBP-2/-HEC antibody (44) and antirat IGFBP-3 antibody (GroPep Ltd.) at a 1:1000 dilution in TBS-T containing 3% nonfat dry milk overnight at 4 C. Thereafter, the filters were washed with TBS-T and incubated with horseradish peroxidase-conjugated donkey antirabbit IgG (no. 9340, Amersham Biosciences; diluted 1:5000 in TBS-T) for 90 min at room temperature, followed by washing with TBS-T and water. Finally, the filters were exposed to enhanced chemiluminescence reagents (Amersham Biosciences) for 1 min at 20 C, then to x-ray film for 1–40 min at 20 C. In this manner the levels of IGFBP-3 and IGFBP-2 after 24 or 48 h of incubation were determined in triplicate in three independent experiments.
Determination of proteolytic activity toward IGFBP-3
Proteolytic activity toward IGFBP-3 was monitored on the basis of the degradation of glycosylated human recombinant IGFBP-3. To this end, medium (30 µl) from control and treated cells was incubated with 50 ng IGFBP-3 in a total volume of 10 µl 50 mM HEPES (pH 7.5), 3 mM CaCl, and 0.1% BSA for 5 h at 37 C. This reaction was terminated by the addition of 30 µl sodium dodecyl sulfate sample buffer, and the resulting mixture was separated by SDS-PAGE and electroblotted as described above for the ligand blots. Thereafter, the filters were treated sequentially with TBS-T and 3% nonfat dry milk in TBS-T, after which they were probed with antihuman IGFBP-3 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) at 1:1000 dilution in TBS-T containing 3% nonfat dry milk overnight at 4 C. After washing, the filters were incubated with horseradish peroxidase-conjugated donkey antirabbit IgG and subjected to enhanced chemiluminescence analysis as described above (45).
Statistical analysis
All results are presented as the mean ± SD. In the case of DHT and T production, data were analyzed using one-way repeated measures ANOVA. Tukey’s post hoc test was performed for pairwise comparisons of different concentrations. Additional analyses were performed to investigate linear, quadratic, and cubic trend components of the data. In the case of IGF-I secretion, data were analyzed by two-way ANOVA with repeated measures on two factors. Simple effects tests were performed to analyze any interaction effects. Additional analyses were performed to investigate linear and quadratic trend components of the data. For data on mRNA expression and IGF-BP levels, one-way ANOVA and Tukey’s post hoc test were performed for pairwise comparisons. Differences were regarded as significant at P < 0.05.
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, neither GH, IGF-I, nor IGF-II had any significant effect on DNA synthesis (assessed by incorporation of tritiated thymidine) or the number of viable cells (based on thiazolyl blue staining; data not shown). In all cases the level of DNA synthesis in these primary cultures was very low (<500 cpm), indicating that spontaneous proliferation of the Leydig cells from 40-d-old rats was very limited.
Effects of IL-1 and GH or IGF-I/IGF-II on androgen production by immature rat Leydig cells
At the optimal concentration of 1 ng/ml, IL-1 alone significantly increased T and DHT production by 40-d-old Leydig cells (4.3-fold increase in T compared with untreated cells, P < 0.001; 2.86-fold increase in DHT compared with untreated cells, P < 0.001; n = 5; Fig. 1
). In contrast, GH alone exerted no stimulatory effect on T production at any of the concentrations tested (Table 1). However, in the case of DHT, GH alone induced a 4.27-fold increase compared with untreated cells (P < 0.001), although no obvious dose-response effect of GH on DHT release was found (Table 1). Coadministration of IL-1 and the two highest concentrations of GH (10–100 ng/ml) significantly stimulated production of DHT and T compared with IL-1 alone (Fig. 1). Neither IGF-I nor IGF-II alone had any effect on T production by immature Leydig cells, but showed a stimulatory effect on DHT production at all concentrations tested, albeit without any obvious dose-response effect (Table 1).
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and IGF-I (1, 10, and 100 ng/ml) resulted in a significant increase in T and DHT production compared with IL-1 alone (Fig. 2A). The same pattern of responsiveness was observed with rat IGF-II in combination with IL-1 (Fig. 2B). IGF-II (1, 10, and 100 ng/ml) in combination with IL-1 increased T and DHT production compared with the increases induced by IL-1 alone (Fig. 2B).
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could stimulate secretion of GH from cultured Leydig cells or, the reverse action, that GH could stimulate release of IL-1. No secreted GH or IL-1 was detected under such conditions in 40 times concentrated Leydig cell-conditioned culture medium as judged by Western immunoblots (data not shown).
Regulation of the production of IGF-I in rat Leydig cells by IL-1 and GH
The secretion of IGF-I (assayed using a specific RIA for the rat protein) by control and stimulated 40-d-old Leydig cells into the culture medium is documented in Fig. 3A. The most potent increase in IGF-I release (4.9-fold increase compared with control; P < 0.001) was observed when the cells were costimulated with GH and IL-1. GH alone (10 ng/ml) produced a 3.5-fold increase compared with control (P < 0.011), and IL-1 alone produced a 2.3-fold increase over the control (P < 0.036). The combined stimulatory effect of GH and IL-1 on IGF-I release was found to be dependent on time (Fig. 3B).
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and GH together, the level of IGF-I mRNA relative to the housekeeping gene S27a was elevated compared with the control cells (vs. GH, P < 0.027; vs. GH plus IL-1, P < 0.004). A small increase was induced by IL-1 alone, but it did not reach statistical significance (Fig. 4).
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-induced steroidogenesis by GH and also decreased the direct stimulation by IGF-I, affecting both T and DHT production. In contrast, PPP alone was without effect, and the stimulatory action of IL-1 alone on both T and DHT release was not affected by PPP, supporting the specificity of this inhibitor for the IGF-IR (Fig. 5).
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).
Stimulation with IL-1 (1 ng/ml) resulted in an increased secretion of IGFBP-3 compared with either untreated control cells or cells exposed to a combination of IL-1 and GH. GH alone also increased the secretion of this binding protein compared with the control situation or the incubation with both factors (Fig. 5). Surprisingly, exposure to both GH and IL-1 did not lead to any significant change in IGFBP-3 secretion compared with untreated cells. Also, hCG at 10 ng/ml, a concentration resulting in maximal stimulation of testosterone production in the present system, had no effect on IGFBP-3 levels (Fig. 5). Untreated immature Leydig cells also secreted IGFBP-2 and this secretion was elevated by co-stimulation with GH and IL-1. GH or IL-1 alone had no effect on IGFBP-2 levels, whereas hCG (10 ng/ml) inhibited the release of IGFBP-2 into the culture medium (Fig. 5).
A time-course study revealed that the increase in IGFBP-2 secretion induced by IL-1 and GH together after 24 h of incubation had disappeared 24 h later. In contrast, the effects on IGFBP-3 secretion were the same at all time points examined (i.e. after 3, 6, 12, and 24 h of incubation; data not shown).
The low level of IGFBP-3 after coincubation with GH and IL-1 may be explained by an increased degradation of this binding protein. To examine this possibility, we analyzed the proteolytic activity toward IGFBP-3 in the Leydig cell culture medium. Medium from all cell cultures catalyzed degradation of IGFBP-3 with the concomitant appearance of fragments, suggesting that Leydig cells produce an IGFBP-3 protease. A proteolytic fragment with an apparent molecular mass of 30 kDa was detected in the cultures treated with IL-1 or GH alone as well as in control cultures (data not shown).
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. The effect was mediated via IGF-IR and was found to be accompanied by changes in the levels of IGFBPs. These results demonstrate a potentially important and hitherto unknown paracrine role of IL-1 in the testis.
Previous studies have revealed that IL-1 is expressed constitutively in the rat testis after 25 d of postnatal life, suggesting a role in the maturation of this organ (14). This expression of IL-1 is localized exclusively to Sertoli cells (12, 15) and shows a temporal relation to the postnatal maturation and differentiation of Leydig cells in rats. We reported recently the expression of the StAR protein by immature rat Leydig cells, i.e. a protein involved in the maturation of these same cells is induced by IL-1 (10).
During this same maturational period, i.e. 28–56 d of postnatal life, steroidogenesis undergoes maturational changes, and the activities of cytochrome P450scc, 3ß-hydroxysteroid dehydrogenase, and cytochrome P450c17 enzymes increase (34, 46, 47, 48). In rats and wild-type mice, levels of 5-reductase activity are highest during the midpubertal period. Under normal conditions, a midpubertal rise in 5-reductase activity could enhance androgen action before the development of full steroidogenic potential in Leydig cells. A changing balance in the expression of T biosynthetic and metabolizing enzyme activities undoubtedly serves to regulate testicular T levels maintained by the Leydig cell (37, 49, 50, 51) Some studies in the IGF-I-null mutant showed decreased P450scc and P450c17 mRNA levels and were associated with disproportionately higher 5-reductase expression. This accounts for the extremely low T levels in adult IGF-I-null testes and also supports the idea that the absence of IGF-I stimulation results in disproportionate androgen-metabolizing enzyme expression in these animals (37, 50, 51). Several studies have concluded that stimulation of Leydig progenitor cells by IGF-I evokes enhanced expression of steroidogenic enzymes and enhanced steroid production, suggesting that this growth factor promotes the maturation of immature Leydig cells into mature type adult Leydig cells (28, 29, 48, 49, 50, 51, 52).
IL-1 alone has been found to stimulate steroidogenesis in Leydig cells from 40-d-old rats dose-dependently (8). Our present observations indicate that GH, IGF-I, and IGF-II do not alone influence T production at this age, but that costimulation with IL-1 does elevate this production to a level exceeding that reached by an optimal concentration of IL-1 alone. In contrast, we found a markedly increased production of DHT in the presence of GH and both IGFs alone, although this effect was not dose dependent. However, in the presence of IL-1, these growth factors induced a marked stimulation of DHT production. From these findings we conclude that IL-1 sensitizes Leydig cells to the stimulatory action of GH, IGF-I, and IGF-II, although we cannot yet explain the exact mechanism(s) underlying this phenomenon. However, the different effects on T and DHT release indicate multiple actions of IL-1, including a direct modulation of 5-reductase activity. Conversion of T to DHT generally occurs in the androgenic target tissues, although 5-reductase activity is also expressed in the testis. There is a peak of 5-reductase activity in the Leydig cells around puberty, with a marked decline in activity as the animal reaches adulthood. Interestingly, IL-1 appears to decrease the ratio of DHT to T, consistent with a lower 5-reductase activity and a more mature androgen pattern. In contrast, adding increasing concentrations of GH, IGF-I, or IGF-II in combination with IL-1 appears to reverse the IL-1 effect and return the ratio of DHT to T to approximately that found for untreated control cells. This finding is consistent with an increased activity of 5-reductase and Leydig cells with a less mature androgen pattern. Similarly, GH, IGF-I, and IGF-II treatments alone appear to increase the activity of 5-reductase.
Determination of the rate of DNA synthesis and cell numbers in the Leydig cell cultures showed that the effect was not due to a mitogenic action of the tested substances. Such a mitogenic role in Leydig cells has previously been ascribed to IL-1, although this reported effect was limited to cells derived from 10- to 20-d-old rats, with no such action on Leydig cells from older donors (38).
A finding that challenges the physiological relevance of our present observations is that IL-1R type I-deleted mice seem to have a normal testicular phenotype, including Leydig cell steroidogenesis (53). However, a great number of paracrine growth factors and cytokines are operative simultaneously in the testis, and it may well be that IL-1 is not the only signal exerting the observed effect. The referred mediators are known to have pleiotropic, overlapping, and compensatory actions in many systems, and similar mechanisms are likely to also be active in the testis.
Previous investigations have also shown that IGF-I plays an important autocrine/paracrine role in the regulation of Leydig cell function (49, 50, 51, 52, 53, 54, 55). Snell dwarf mice, which are characterized by a low level of GH and prolactin secretion, exhibit delayed puberty and a low serum level of T (56). In line with this finding, GH receptor gene-disrupted mice show a decreased adult Leydig cell volume per testis (57) and delayed puberty (56). Moreover, IGF-I appears to up-regulate the level of mRNA coding for the LH receptor (58), although this mechanism may not contribute to the effects of IGF-I observed here.
Thus, our results suggest that local IGF-I production by Leydig cells in the testis is stimulated by GH after sensitization by IL-1. The role known to be played by IGF-I in the regulation of testicular function and androgen production (49, 50, 54, 55, 58) leads to the conclusion that the elevation in T and DHT production evoked by GH in combination with IL-1 is an indirect effect. Furthermore, this finding with our present model system confirms previous observations that IL-1, GH, and IGF-I elevate the level of StAR protein expressed by Leydig cells (data not shown), a phenomenon that may also be of significance for steroidogenesis and maturation (10, 20, 28). The exact role of the StAR protein in this context remains to be explored.
Cells other than Leydig cells, e.g. macrophages, also appear to be present in the primary cell cultures examined here, because the Leydig cell preparations employed were only 87–90% pure. Although we judge it unlikely (59), one cannot totally exclude that contaminating macrophages influenced by GH may affect the present results either directly or indirectly via an interaction with Leydig cells.
IGF-I mRNA is expressed in rat Leydig cells as well as in interstitial cells in the prepubertal mouse testis, and the IGF-IR is expressed by Leydig cells in both rat and mouse testis (56). Binding of IGF-I to its receptor is known to stimulate tyrosine kinase activity, leading to autophosphorylation of IGF-IR together with phosphorylation of tyrosine residues in several cellular proteins (60). PPP, which efficiently inhibits phosphorylation of IGF-IR, was shown to reverse the effect of GH on IL-1-induced steroidogenesis, indicating that this action of GH is mediated indirectly via increased IGF-I production and action. The effect of IL-1 alone was not influenced by PPP and is thus independent of IGF-IR function in our system.
When Leydig cells were incubated with a combination of GH and IL-1, a decrease in the level of intact IGFBP-3 and a transiently increased level of IGFBP-2, in addition to the increase in IGF-I production were observed in the culture medium. IGFBP-3 has been found to inhibit the stimulation of steroidogenesis by IGF-I as well as to inhibit cAMP synthesis by granulosa cells in response to IGF-I (61). GH alone was found to increase the level of IGFBP-3 in the culture medium, whereas in the case of IL-1-sensitized cells, stimulation by GH results in proteolysis of IGFBP-3.
We suggest that in cells treated with both IL-1 and GH, elevated IGF-I action is facilitated by proteolysis of IGFBP-3, resulting in the stimulation of steroidogenesis, whereas IGFBP-3 blocks the action of elevated IGF-I levels in cells treated with GH alone.
IGFBP-2 may have a complex role in the regulation of IGF-I bioavailability and function. It is obvious that a decreased level of IGFBP-2, shown previously (62) and here (Fig. 6) to be induced by hCG, may result in higher levels of free and active IGF-I. However, it is also possible that the up-regulated levels IGFBP-2 stimulated by the combination of IL-1 and GH (Fig. 6) act in synergy with the elevated production of IGF-I by Leydig cells, in analogy to observations made on other cell systems (63). The tripeptide motif, RGD, present in IGFBP-2 is a recognition site for several cell adhesion molecules, including 5ß1 integrin, which is involved in the association of IGFBP-2 with the cell surface. This RGD sequence and binding to the cell membrane appear to be of importance for potentiation of the effects of IGF-I by IGFBP-2 (63). The questions of whether the elevated level of IGFBP-2 in culture medium from Leydig cells treated with both IL-1 and GH is accompanied by enhanced association of this protein with the cell membrane and thereby potentiated stimulation of steroidogenesis by IGF-I or, in contrast, exerts an opposite modulatory action by binding and inactivation of IGF-I await additional investigation.
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In conclusion, this investigation provides the first evidence that IL-1 with GH and IGF-I interact to stimulate androgen production by immature Leydig cells at a time point corresponding to puberty in humans. Although the effects of IL-1 isoforms on steroidogenesis and testicular function remain controversial (7, 8, 9, 10, 15, 18), the findings described here suggest an important physiological role for constitutively produced IL-1 in the postnatal differentiation of Leydig cells as well as, perhaps, in the onset of puberty in boys.
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Footnotes |
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First Published Online October 14, 2004
Abbreviations: DHT, Dihydrotestosterone; hCG, human chorionic gonadotropin; Ig, immunoglobulin; IGFBP, IGF-binding protein; IGF-IR, IGF-I receptor; PPP, 7R,7'R,8R,8'R-7-hydroxy 3',4',5'-trimethoxy-5-methylenedioxy-2,7'cyclolignan-9,9'-oxide; StAR, steroidogenic acute regulatory protein; T, testosterone; TBS-T, Tween 20 in Tris-buffered saline.
Received April 15, 2004.
Accepted for publication October 8, 2004.
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