Stimulation of Steroidogenesis in Immature Rat Leydig Cells Evoked by Interleukin-1 Is Potentiated by Growth Hormone and Insulin-Like Growth
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内分泌学杂志 2005年第1期
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@kbh.ki.se.
Abstract
The cytokine IL-1 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.
Introduction
IL-1, 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-1?, 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-1?, 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-1? 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 3?-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-3,17?-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-3,17?-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).
Materials and Methods
DMEM-Ham’s nutrient mixture F-12, MEM, Hanks’ Balanced Salts Solution without Ca2+ or Mg2+, and penicillin and streptomycin were purchased from Invitrogen Life Technologies, Inc. (Paisley, UK); BSA (fraction V), Percoll, hCG, dibutryl cAMP, HEPES, and collagenase type I were obtained from Sigma-Aldrich Corp. (St. Louis, MO); rat recombinant IL-1 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 3?-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.
Results
Leydig cell proliferation
A possible trivial explanation for the findings described here would be an induction of Leydig cell proliferation in response to GH and/or IGF-I or IGF-II. We therefore examined the possible mitogenic action of GH, IGF-I, and IGF-II on Leydig cell cultures. Alone or in combination with IL-1, 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).
FIG. 1. Stimulation of IL-1-induced Leydig cell steroidogenesis by GH. Leydig cells were cultured for 24 h in the absence (control) or presence of an optimal concentration of rat IL-1 (1 ng/ml) together with increasing concentrations of rat GH (0.1–100 ng/ml). After 24 h of incubation, the T and DHT released into the culture medium were quantified by RIA and ELISA, respectively. The results represent the mean ± SD of five independent experiments, each performed in triplicate. Letters above values indicate significant differences. Difference between IL-1 and control: DHT, a, P < 0.001; T, a, P < 0.001. Cells stimulated by the combination of IL-1 and different doses of GH (0.1–100 ng/ml) in comparison with IL-1 (1 ng/ml) alone: b, P < 0.05; c, P < 0.001. Dose effects were analyzed using a one-way repeated measures ANOVA. Tukey’s post hoc test was performed to make pairwise comparisons among the concentrations. Difference between doses: DHT: GH (0.1 ng/ml) vs. GH (1 ng/ml), d, P < 0.001; GH (1 ng/ml) vs. GH (100 ng/ml), d, P < 0.001; T: GH (0.1 ng/ml) vs. GH (1 ng/ml), b*, P < 0.01; GH (1 ng/ml) vs. GH (10 ng/ml), d, P < 0.001. Linear trend, P < 0.001; quadratic trend, P < 0.001.
TABLE 1. Influence of GH, IGF-I, and IGF-II alone (0.1–100 ng/ml) on androgen production by immature Leydig cells in vitro
Cotreatment of IL-1 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).
FIG. 2. Stimulation of IL-1-induced Leydig cell steroidogenesis by IGF-I and IGF-II. Leydig cells were cultured for 24 h in the absence (control) or presence of an optimal concentration of IL-1 (1 ng/ml) together with increasing concentrations (0.1–100 ng/ml) of rat IGF-I (A) or IGF-II (B). The T and DHT released into the culture medium were quantified by RIA and ELISA, respectively. The results represent the mean ± SD of five independent experiments, each performed in triplicate. A, Letters above values indicate differences between IL-1 and control: DHT, a, P < 0.033; T, c, P < 0.001. Cells stimulated by the combination of IL-1 and various doses of IGF-I (0.1–100 ng/ml) compared with cells stimulated by IL-1 (1 ng/ml) alone in the case of DHT or T: b, P < 0.05; c, P < 0.001. Dose effects were analyzed using a one-way repeated measures ANOVA. Tukey’s post hoc test was performed to make pairwise comparisons among the concentrations. Difference between doses: DHT: IGF-I (0.1 ng/ml) vs. IGF-I (1 ng/ml), a*, P < 0.05; IGF-I (1 ng/ml) vs. IGF-I (10 ng/ml), c*, P < 0.001; IGF-I (10 ng/ml) vs. IGF-I (100 ng/ml), d, P < 0.001; T: IGF-I (0.1 ng/ml) vs. IGF-I (1 ng/ml), c*, P < 0.001; IGF-I (1 ng/ml) vs. IGF-I (10 ng/ml), d, P < 0.001. B, Difference between IL-1 and control: DHT: a, P < 0.05; T: c, P < 0.001. Data from cells stimulated by the combination of IL-1 and IGF-II (0.1–100 ng/ml) in comparison with cells stimulated with IL-1 (1 ng/ml) alone: c, P < 0.001. Difference between doses; DHT: IGF-II (0.1 ng/ml) vs. IGF-II (1 ng/ml), c*, P < 0.001; IGF-II (1 ng/ml) vs. IGF-II (10 ng/ml), c*, P < 0.001; IGF-II (10 ng/ml) vs. IGF-II (100 ng/ml), d, P < 0.001; T: IGF-II (0.1 ng/ml) vs. IGF-II (1 ng/ml), c*, P < 0.001; IGF-II (1 ng/ml) vs. IGF-II (10 ng/ml), d, P < 0.001; IGF-II (10 ng/ml) vs. IGF-II (100 ng/ml), d, P < 0.001. IGF-I: linear trend, P < 0.001; quadratic trend, P < 0.001; cubic trend, P < 0.001. IGF-II: linear trend, P < 0.001; quadratic trend, P < 0.001.
Control experiments were performed to investigate the possibility that IL-1 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).
FIG. 3. Influence of GH and/or IL-1 on the secretion of IGF-I by cultured Leydig cells. The IGF-I secreted into the medium was extracted with acidic ethanol and quantified with an RIA procedure. Cells were incubated for 24 h (A) or for various time periods (B) in the absence (control) or presence of IL-1 (1 ng/ml) and/or GH (10 ng/ml). The values shown are the mean ± SD of three independent experiments, each performed in triplicate. Letters in the graph indicate significant differences. A, Analysis of different treatments groups: IL-1 vs. control: a, P < 0.036; GH vs. control: b, P < 0.011; IL-1 plus GH vs. IL-1: c, P < 0.027; IL-1 plus GH vs. control: d, P < 0.001. B, Data were analyzed using a two-way ANOVA with repeated measures on two factors, hormone and time. Simple effects tests were performed to analyze the interaction effect. The hormone x time interaction (change over time between GH plus IL-1 and GH) was significant (P < 0.002). Tests for simple effects showed that there was a significant difference between GH plus IL-1 and GH at 24 h: a, P < 0.001. The main effect of time was highly significant (P < 0.001). Difference between GH plus IL-1 and GH: with regard to the linear trend, P < 0.01; quadratic trend, P < 0.004.
RT-PCR analysis detected a basal level of expression of IGF-I mRNA in unstimulated control cultures of Leydig cells. In the presence of GH alone and IL-1 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).
FIG. 4. The influence of GH and/or IL-1 on the level of IGF-I mRNA in cultured rat Leydig cells. RT-PCR analysis of the level of IGF-I mRNA in Leydig cells from 40-d-old-rats was conducted as described in Materials and Methods. Total RNA was isolated from 2 x 106 cells incubated for 24 h in the absence (control) or presence of IL-1 (1 ng/ml) and/or GH (10 ng/ml). A, Representative RT-PCR pattern is shown. B, Quantitative mean ± SD from five independent experiments are presented. These data were normalized to the level of mRNA from the housekeeping gene S27a. GH vs. control: a, P < 0.027; GH plus IL-1 vs. control: b, P < 0.004.
Effect of inhibition of IGF-IR on steroidogenesis by Leydig cells
To evaluate the role played by IGF-IR in our system, PPP, a specific inhibitor of the IGF-IR tyrosine kinase, was employed. PPP prevented the potentiation of IL-1-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).
FIG. 5. The IGF-IR inhibitor PPP abolishes the stimulatory effect of GH and IGF-I on androgen production by Leydig cells. Cells were cultured for 24 h in the absence (control) or presence of optimal concentrations of IL-1 (1 ng/ml) and/or GH (10 ng/ml) or IGF-I (10 ng/ml). PPP (1 μM) was added to the culture medium alone or in combination with these other factors. T production was quantified by an RIA procedure. DHT was measured by ELISA. The results presented are the mean ± SD, of the values of four independent experiments. For DHT: GH vs. GH plus PPP, a, P < 0.05; GH plus IL-1 vs. GH, IL-1, plus PPP, c, P < 0.021. For IGF-I: IGF-I vs. IGF-I plus PPP, b, P < 0.05; IGF-I plus IL-1 vs. IGF-I, IL-1, plus PPP, d, P < 0.018. For T: GH plus IL-1 vs. GH, IL-1, plus PPP, c, P < 0.039; IGF-I plus IL-1 vs. IGF-I, IL-1, plus PPP, d, P < 0.020.
Secretion of IGFBPs
Because IGF-I activity is regulated by IGFBPs, we examined the secretion of these binding proteins by untreated and treated Leydig cells employing Western ligand blotting with radioactive IGF-I as a probe. The ligand blot displayed a doublet band with an apparent molecular mass of 42–45 kDa and another band at 30 kDa, corresponding to IGFBP-3 and IGFBP-2, respectively, as demonstrated by the use of specific antiserum (Fig. 5A).
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).
Discussion
From the present data we conclude that immature rat Leydig cells increase their rate of steroidogenesis upon stimulation with GH, IGF-I or IGF-II provided that the cells are exposed simultaneously to IL-1. 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, 3?-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 5?1 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.
FIG. 6. Effects of IL-1 and/or GH and hCG on the secretion of IGFBP-3 and IGFBP-2 by rat Leydig cells in culture. The cells were plated and incubated for 24 h in the absence or presence of IL-1 (1 ng/ml) and/or an optimal concentration of GH (10 ng/ml), after which Western ligand blotting (WB) was performed (for additional details, see Materials and Methods). The effect of hCG alone (10 ng/m) was also investigated. WB bands obtained in three independent experiments were quantified by phosphorimage (Fuji) scanning, and the intensity of each band was expressed as a percentage of the control intensity. A, Representative experiment. B, Mean ± SD for three independent experiments. In the case of IGFBP-3: IL-1 alone vs. control, a, P < 0.001; GH vs. control, b, P < 0.001. For IGFBP-2: GH plus IL-1 vs. control, c, P < 0.032; hCG alone vs. control, d, P < 0.029.
Despite high circulating concentrations and a phylogenetically well preserved testicular expression (64), the physiological role of IGF-II remains a mystery. Thus, the function, if any, of IGF-II in the regulation of Leydig cell steroidogenesis is unknown. However, the present results show clearly that such a function is possible. They also strengthen the IGF-I data, because IGF-II is supposed to act via binding to the IGF-IR.
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.
References
Dinarello CA, Savage N 1989 Interleukin-1 and its receptor. Crit Rev Immunol 9:1–20
di Giovine FS, Duff GW 1990 Interleukin 1: the first interleukin. Immunol Today 11:13–20
Granholm T, S?der O 1991 Constitutive production of lymphocyte activating factors by normal tissues in the adult rat. J Cell Biochem 46:143–151
Parvinen M, S?der O, Mali P, Fr?ysa B, Ritzén EM 1991 In vitro stimulation of stage-specific deoxyribonucleic acid synthesis in rat seminiferous tubule segments by interleukin-1. Endocrinology 129:1614–1620
P?ll?nen P, S?der O, Parvinen M 1989 Interleukin-1 stimulation of spermatogonial proliferation in vivo. Reprod Fertil Dev 1:85–87
Calkins JH, Sigel MM, Nankin HR, Lin T 1988 Interleukin-1 inhibits Leydig cells steroidogenesis in primary culture. Endocrinology 123:1605–1610
Hales DB 2002 Testicular macrophage modulation of Leydig cell steroidogenesis. J Reprod Immunol 57:3–18
Svechnikov K, Sultana T, S?der O 2001 Age-dependent stimulation of Leydig cell steroidogenesis by interleukin-1 isoforms. Mol Cell Endocrinol 182:193–201
Verhoeven G, Cailleau J, Van Damme J, Billiau A 1988 Interleukin IL-1 stimulates steroidogenesis in cultured rat Leydig cells. Mol Cell Endocrinol 57:51–60
Svechnikov K, DM Stocco, S?der O 2003 Interleukin-1 stimulates steroidogenic acute regulatory protein expression via p 38 MAP kinase in immature Leydig cells. J Mol Endocrinol 30:59–67
Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 63:193–213
Jonsson CK, Zetterstrom RH, Holst M, Parvinen M, S?der O 1999 Constitutive expression of interleukin-1 messenger ribonucleic acid in rat Sertoli cells is dependent upon interaction with germ cells. Endocrinology 140:3755–3761
Gerard N, Syed V, Jegou B 1992 Lipopolysaccharide, latex beads and residual bodies are potent activators of Sertoli cell interleukin-1 production. Biochem Biophys Res Commun 185:154–161
Wahab-Wahlgren A, Holst M, Ayele D, Sultana T, Parvinen M, Gustafsson K, Granholm T, S?der O 2000 Constitutive production of interleukin-1 mRNA and protein in the developing rat testis. Int J Androl 23:360–365
Sultana T, Svechnikov K, Weber G, S?der O 2000 Molecular cloning and expression of a functionally different alternative splice variant of prointerleukin-1 from the rat testis. Endocrinology 141:4413–4418
Gustafsson K, Sultana T, Zetterstrom CK, Setchell BP, Siddiqui A, Weber G, S?der O 2002 Production and secretion of interleukin-1 proteins by rat testis. Biochem Biophys Res Commun 297:492–497
Jonsson CK, Setchell BP, Martinelle N, Svechnikov K, S?der O 2001 Endotoxin-induced interleukin 1 expression in testicular macrophages is accompanied by downregulation of the constitutive expression in Sertoli cells. Cytokine 14:283–288
Bergh A, S?der O 1990 Interleukin-1?, but not interleukin-1, induces acute inflammation-like changes in the testicular microcirculation of adult rats. J Reprod Immunol 17:155–165
Carter-Su C, Schwartz J, Smit LS 1996 Molecular mechanism of growth hormone action. Annu Rev Physiol 58:187–207
Childs GV 2000 Growth hormone cells as co-gonadotropes: partners in the regulation of the reproductive system. Trends Endocrinol Metab 11:168–175
Hull KL, Harvey S 2000 Growth hormone: roles in male reproduction. Endocrine 13:243–250
Arsennijevic Y 1989 Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor delays sexual maturation in the male rat. Endocrinology 124:3050–3059
Laron Z, Klinger B 1994 Laron syndrome: clinical features, molecular pathology and treatment. Horm Res 42:198–202
Bartlett JM, Charlton HM, Robinson IC, Nieschlag E 1990 Pubertal development and testicular function in the male growth hormone-deficient rat. J Endocrinol 126:193–201
Gravance CG, Breier BH, Vickers MH, Casey PJ 1997 Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf (dw/dw) rats. Anim Reprod Sci 49:71–76
Zipf WB, Payne AH, Kelch RP 1978 Prolactin, growth hormone, and luteinizing hormone in the maintenance of testicular luteinizing hormone receptors. Endocrinology 103:595–600
Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244
Kansaki M, Morris P 1999 Growth hormone regulates steroidogenic acute regulatory protein expression and steroidogenesis in Leydig cell progenitors. Endocrinology 140:1681–1686
Kansaki M, Morris P 1998 Lactogenic hormone-inducible phosphorylation and -activated site-binding activities of Stat5b in primary rat Leydig cells and MA-10 mouse Leydig tumor cells. Endocrinology 139:1872–1882
Chandrashekar V, Bartke A 1993 Induction of endogenous insulin-like growth factor-I secretion alters the hypothalamic-pituitary-testicular function in growth hormone-deficient adult dwarf mice. Biol Reprod 48:544–551
Lin T, Wang D, Hu J, Stocco DM 1998 Upregulation of human chorionic gonadotropin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat Leydig cells. Endocrine 8:73–81
Hull KL, Harvey S 2000 Growth hormone: a reproductive endocrine-paracrine regulator? Rev Reprod 5:175–182
Koike S, Noumura T 1995 Immunohistochemical localization of insulin-like growth factor-II in the perinatal rat gonad. Growth Regul 5:185–189
Chubb C, Ewing LL 1981 Steroid secretion by sexually immature rat and rabbit testes perfused in vitro. Endocrinology 109:1999–2003
Girnita A, Girnita L, del Prete F, Bartolazzi A, Larsson O, Axelson M 2004 Cyclolignans as inhibitors of the insulin-like growth factor-I receptor and malignant cell growth. Cancer Res 64:236–242
Klinefelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36:769–783
Ge RS, Shan LX, Hardy MP 1996 Pubertal development of Leydig cells. In: Payne AH, Hardy MP, Russell LD, eds. The Leydig cell. Vienna, IL: Cache River Press; 159–174
Khan SA, Khan SJ, Dorrington JH 1992 Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro. Endocrinology 131:1853–1857
Bang P, Eriksson U, Sara V, Wivall IL, Hall K 1991 Comparison of acid ethanol extraction and acid gel filtration prior to IGF-I and IGF-II radioimmunoassays: improvement of determinations in acid ethanol extracts by the use of truncated IGF-I as radioligand. Acta Endocrinol (Copenh) 124:620–629
Chomczynski P, Sacchi S 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Casella SJ, Smith EP, van Wyk JJ, Joseph DR, Hynes MA, Hoyt EC, Lund PK 1987 Isolation of rat testis cDNAs encoding an insulin-like growth factor I precursor. DNA 6:325–330
Chan Y, Susuki K, Wool IG 1995 The carboxyl extensions of two rat ubiquitin fusion proteins are ribosomal proteins S27a and L40. Biochem Biophys Res Commun 215:682–690
Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-growth factor binding protein using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138–143
Tapanainen PJ, Bang P, Wilson K, Unterman TG, Vreman HJ, Rosenfeld RG 1994 Maternal hypoxia as a model for intrauterine growth retardation: effects on insulin-like growth factors and their binding proteins. Pediatr Res 36:152–158
Lamson G, Giudice LC, Cohen P, Liu F, Gargosky S, Muller HL, Oh Y, Wilson KF, Hintz RL, Rosenfeld RG 1993 Proteolysis of IGFBP-3 may be a common regulatory mechanism of IGF action in vivo. Growth Regul 3:91–95
Haider SG, Passia D, Overmeyer G 1986 Studies on the fetal and postnatal development of rat Leydig cells employing 3?-hydroxysteroid dehydrogenase activity. Acta Histochem 32:197–202
Kuhn-Velten N, Bos D, Schermer R, Staib W 1987 Age dependence of the rat Leydig cell and Sertoli cell function. Development of the peripheral testosterone level and its relation to mitochondrial and microsomal cytochrome P450 and to androgen binding protein. Acta Endocrinol (Copenh) 115:275–281
Lin T, Vinson N, Haskett J, Murono EP 1987 Induction of 3?-hydroxysteroid dehydrogenase activity by insulin-like growth factor-I in primary cultures of purified Leydig cells. Adv Exp Biol Med 219:603–607
Benahmed M, Morera AM, Chauvin MC, de Peretti E 1987 Somatomedin-C/insulin-like growth factor-1 as a possible intratesticular regulator of Leydig cell activity. Mol Cell Endocrinol 50:69–77
Gelber SJ, Hardy MP, Mendis-Handagama SMLC, Casella SJ 1992 Effects of insulin like growth factor-1 on androgen production by highly purified pubertal and adult Leydig cells. J Androl 13:125–130
Wang G, Hardy MP 2004 Development of Leydig cells in the insulin-like growth factor-I (IGF-I) knockout mouse: effects of IGF-I replacement and gonadotropic stimulation. Biol Reprod 70:632–639
Wang GM, O’Shaughnessy PJ, Chubb C, Robaire B, Hardy MP 2003 Effects of insulin-like growth factor-I on steroidogenic enzyme expression levels in mouse Leydig cells. Endocrinology 144:5058–5064
Cohen PE, Pollard JW 1998 Normal sexual function in male mice lacking a functional type I interleukin-1 (IL-1) receptor. Endocrinology 139:815–818
Lin T, Wang D, Hu J, Stocco DM 1998 Upregulation of human chorionic gonadotropin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat Leydig cells. Endocrine 8:73–78
Chatelain P, Sanchez P, Saez J 1991 Growth hormone and insulin-like growth factor I treatment increase testicular luteinizing hormone receptors and steroidogenic responsiveness of growth hormone deficient dwarf mice. Endocrinology 128:1857–1862
Keene DE, Suescun MO, Bostwick MG, Chandrashekar V, Bartke A, Kopchick JJ 2002 Puberty is delayed in male growth-hormone receptor gene-disrupted mice. J Androl 23:661–668
Chandrashekar V, Bartke A, Awoniyi CA, Tsai-Morris CH, Dufau ML, Russel LI, Kopchick JJ 2001 Testicular function in GH receptor gene disrupted mice. Endocrinology 142:3443–3450
Zhang FP, El-Hafnawy T, Huhtaniemi I, El-Hafnawy T 1998 Regulation of luteinizing hormone receptor gene expression by insulin growth factor-I in an immortalized murine Leydig tumor cell line (BLT-1). Biol Reprod 59:1116–1123
Dombrowicz D, Sente B, Reiter E, Closset J, Hennen G 1996 Pituitary control of proliferation and differentiation of Leydig cells and their putative precursors in immature hypophysectomized rat testis. J Androl 17:639–650
LeRoith D, Werner H, Beitner-Johnson D, Roberts Jr CT 1995 Molecular and cellular aspects of the insulin growth factor-I receptor. Endocr Rev 16:142–163
Bicsak TA, Shimonaka M, Malkowski M, Ling N 1990 Insulin-growth factor binding protein-3 inhibition of granulosa cell function effect on cyclic 3'5'-monophosphate deoxyribonucleic acid synthesis and comparison of effect of IGF-I antibody. Endocrinology 126:2184–2189
Wang D, Nagpai ML, Lin T, Shimasaki S, Ling N 1994 Insulin-like growth factor-binding protein-2: the effect of human chorionic gonadotropin on its gene regulation and protein secretion and its biological effects in rat Leydig cells. Mol Endocrinol 8:69–76
Kelley KM, Oh Y, Gargosky SE, Gucev Z, Matsumoto T, Hwa V, Ng L, Simpson DM, Rosenfeld RG 1996 Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics. Int J Biochem Cell Biol 28:619–637
Loffing-Cueni D, Schmid AC, Reinecke M 1999 Molecular cloning and tissue expression of the insulin-like growth factor II prohormone in the bony fish Cottus scorpius. Gen Comp Endocrinol 113:32–37[CrossRef](Eugenia Colón, Konstantin)
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@kbh.ki.se.
Abstract
The cytokine IL-1 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.
Introduction
IL-1, 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-1?, 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-1?, 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-1? 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 3?-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-3,17?-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-3,17?-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).
Materials and Methods
DMEM-Ham’s nutrient mixture F-12, MEM, Hanks’ Balanced Salts Solution without Ca2+ or Mg2+, and penicillin and streptomycin were purchased from Invitrogen Life Technologies, Inc. (Paisley, UK); BSA (fraction V), Percoll, hCG, dibutryl cAMP, HEPES, and collagenase type I were obtained from Sigma-Aldrich Corp. (St. Louis, MO); rat recombinant IL-1 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 3?-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.
Results
Leydig cell proliferation
A possible trivial explanation for the findings described here would be an induction of Leydig cell proliferation in response to GH and/or IGF-I or IGF-II. We therefore examined the possible mitogenic action of GH, IGF-I, and IGF-II on Leydig cell cultures. Alone or in combination with IL-1, 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).
FIG. 1. Stimulation of IL-1-induced Leydig cell steroidogenesis by GH. Leydig cells were cultured for 24 h in the absence (control) or presence of an optimal concentration of rat IL-1 (1 ng/ml) together with increasing concentrations of rat GH (0.1–100 ng/ml). After 24 h of incubation, the T and DHT released into the culture medium were quantified by RIA and ELISA, respectively. The results represent the mean ± SD of five independent experiments, each performed in triplicate. Letters above values indicate significant differences. Difference between IL-1 and control: DHT, a, P < 0.001; T, a, P < 0.001. Cells stimulated by the combination of IL-1 and different doses of GH (0.1–100 ng/ml) in comparison with IL-1 (1 ng/ml) alone: b, P < 0.05; c, P < 0.001. Dose effects were analyzed using a one-way repeated measures ANOVA. Tukey’s post hoc test was performed to make pairwise comparisons among the concentrations. Difference between doses: DHT: GH (0.1 ng/ml) vs. GH (1 ng/ml), d, P < 0.001; GH (1 ng/ml) vs. GH (100 ng/ml), d, P < 0.001; T: GH (0.1 ng/ml) vs. GH (1 ng/ml), b*, P < 0.01; GH (1 ng/ml) vs. GH (10 ng/ml), d, P < 0.001. Linear trend, P < 0.001; quadratic trend, P < 0.001.
TABLE 1. Influence of GH, IGF-I, and IGF-II alone (0.1–100 ng/ml) on androgen production by immature Leydig cells in vitro
Cotreatment of IL-1 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).
FIG. 2. Stimulation of IL-1-induced Leydig cell steroidogenesis by IGF-I and IGF-II. Leydig cells were cultured for 24 h in the absence (control) or presence of an optimal concentration of IL-1 (1 ng/ml) together with increasing concentrations (0.1–100 ng/ml) of rat IGF-I (A) or IGF-II (B). The T and DHT released into the culture medium were quantified by RIA and ELISA, respectively. The results represent the mean ± SD of five independent experiments, each performed in triplicate. A, Letters above values indicate differences between IL-1 and control: DHT, a, P < 0.033; T, c, P < 0.001. Cells stimulated by the combination of IL-1 and various doses of IGF-I (0.1–100 ng/ml) compared with cells stimulated by IL-1 (1 ng/ml) alone in the case of DHT or T: b, P < 0.05; c, P < 0.001. Dose effects were analyzed using a one-way repeated measures ANOVA. Tukey’s post hoc test was performed to make pairwise comparisons among the concentrations. Difference between doses: DHT: IGF-I (0.1 ng/ml) vs. IGF-I (1 ng/ml), a*, P < 0.05; IGF-I (1 ng/ml) vs. IGF-I (10 ng/ml), c*, P < 0.001; IGF-I (10 ng/ml) vs. IGF-I (100 ng/ml), d, P < 0.001; T: IGF-I (0.1 ng/ml) vs. IGF-I (1 ng/ml), c*, P < 0.001; IGF-I (1 ng/ml) vs. IGF-I (10 ng/ml), d, P < 0.001. B, Difference between IL-1 and control: DHT: a, P < 0.05; T: c, P < 0.001. Data from cells stimulated by the combination of IL-1 and IGF-II (0.1–100 ng/ml) in comparison with cells stimulated with IL-1 (1 ng/ml) alone: c, P < 0.001. Difference between doses; DHT: IGF-II (0.1 ng/ml) vs. IGF-II (1 ng/ml), c*, P < 0.001; IGF-II (1 ng/ml) vs. IGF-II (10 ng/ml), c*, P < 0.001; IGF-II (10 ng/ml) vs. IGF-II (100 ng/ml), d, P < 0.001; T: IGF-II (0.1 ng/ml) vs. IGF-II (1 ng/ml), c*, P < 0.001; IGF-II (1 ng/ml) vs. IGF-II (10 ng/ml), d, P < 0.001; IGF-II (10 ng/ml) vs. IGF-II (100 ng/ml), d, P < 0.001. IGF-I: linear trend, P < 0.001; quadratic trend, P < 0.001; cubic trend, P < 0.001. IGF-II: linear trend, P < 0.001; quadratic trend, P < 0.001.
Control experiments were performed to investigate the possibility that IL-1 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).
FIG. 3. Influence of GH and/or IL-1 on the secretion of IGF-I by cultured Leydig cells. The IGF-I secreted into the medium was extracted with acidic ethanol and quantified with an RIA procedure. Cells were incubated for 24 h (A) or for various time periods (B) in the absence (control) or presence of IL-1 (1 ng/ml) and/or GH (10 ng/ml). The values shown are the mean ± SD of three independent experiments, each performed in triplicate. Letters in the graph indicate significant differences. A, Analysis of different treatments groups: IL-1 vs. control: a, P < 0.036; GH vs. control: b, P < 0.011; IL-1 plus GH vs. IL-1: c, P < 0.027; IL-1 plus GH vs. control: d, P < 0.001. B, Data were analyzed using a two-way ANOVA with repeated measures on two factors, hormone and time. Simple effects tests were performed to analyze the interaction effect. The hormone x time interaction (change over time between GH plus IL-1 and GH) was significant (P < 0.002). Tests for simple effects showed that there was a significant difference between GH plus IL-1 and GH at 24 h: a, P < 0.001. The main effect of time was highly significant (P < 0.001). Difference between GH plus IL-1 and GH: with regard to the linear trend, P < 0.01; quadratic trend, P < 0.004.
RT-PCR analysis detected a basal level of expression of IGF-I mRNA in unstimulated control cultures of Leydig cells. In the presence of GH alone and IL-1 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).
FIG. 4. The influence of GH and/or IL-1 on the level of IGF-I mRNA in cultured rat Leydig cells. RT-PCR analysis of the level of IGF-I mRNA in Leydig cells from 40-d-old-rats was conducted as described in Materials and Methods. Total RNA was isolated from 2 x 106 cells incubated for 24 h in the absence (control) or presence of IL-1 (1 ng/ml) and/or GH (10 ng/ml). A, Representative RT-PCR pattern is shown. B, Quantitative mean ± SD from five independent experiments are presented. These data were normalized to the level of mRNA from the housekeeping gene S27a. GH vs. control: a, P < 0.027; GH plus IL-1 vs. control: b, P < 0.004.
Effect of inhibition of IGF-IR on steroidogenesis by Leydig cells
To evaluate the role played by IGF-IR in our system, PPP, a specific inhibitor of the IGF-IR tyrosine kinase, was employed. PPP prevented the potentiation of IL-1-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).
FIG. 5. The IGF-IR inhibitor PPP abolishes the stimulatory effect of GH and IGF-I on androgen production by Leydig cells. Cells were cultured for 24 h in the absence (control) or presence of optimal concentrations of IL-1 (1 ng/ml) and/or GH (10 ng/ml) or IGF-I (10 ng/ml). PPP (1 μM) was added to the culture medium alone or in combination with these other factors. T production was quantified by an RIA procedure. DHT was measured by ELISA. The results presented are the mean ± SD, of the values of four independent experiments. For DHT: GH vs. GH plus PPP, a, P < 0.05; GH plus IL-1 vs. GH, IL-1, plus PPP, c, P < 0.021. For IGF-I: IGF-I vs. IGF-I plus PPP, b, P < 0.05; IGF-I plus IL-1 vs. IGF-I, IL-1, plus PPP, d, P < 0.018. For T: GH plus IL-1 vs. GH, IL-1, plus PPP, c, P < 0.039; IGF-I plus IL-1 vs. IGF-I, IL-1, plus PPP, d, P < 0.020.
Secretion of IGFBPs
Because IGF-I activity is regulated by IGFBPs, we examined the secretion of these binding proteins by untreated and treated Leydig cells employing Western ligand blotting with radioactive IGF-I as a probe. The ligand blot displayed a doublet band with an apparent molecular mass of 42–45 kDa and another band at 30 kDa, corresponding to IGFBP-3 and IGFBP-2, respectively, as demonstrated by the use of specific antiserum (Fig. 5A).
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).
Discussion
From the present data we conclude that immature rat Leydig cells increase their rate of steroidogenesis upon stimulation with GH, IGF-I or IGF-II provided that the cells are exposed simultaneously to IL-1. 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, 3?-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 5?1 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.
FIG. 6. Effects of IL-1 and/or GH and hCG on the secretion of IGFBP-3 and IGFBP-2 by rat Leydig cells in culture. The cells were plated and incubated for 24 h in the absence or presence of IL-1 (1 ng/ml) and/or an optimal concentration of GH (10 ng/ml), after which Western ligand blotting (WB) was performed (for additional details, see Materials and Methods). The effect of hCG alone (10 ng/m) was also investigated. WB bands obtained in three independent experiments were quantified by phosphorimage (Fuji) scanning, and the intensity of each band was expressed as a percentage of the control intensity. A, Representative experiment. B, Mean ± SD for three independent experiments. In the case of IGFBP-3: IL-1 alone vs. control, a, P < 0.001; GH vs. control, b, P < 0.001. For IGFBP-2: GH plus IL-1 vs. control, c, P < 0.032; hCG alone vs. control, d, P < 0.029.
Despite high circulating concentrations and a phylogenetically well preserved testicular expression (64), the physiological role of IGF-II remains a mystery. Thus, the function, if any, of IGF-II in the regulation of Leydig cell steroidogenesis is unknown. However, the present results show clearly that such a function is possible. They also strengthen the IGF-I data, because IGF-II is supposed to act via binding to the IGF-IR.
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.
References
Dinarello CA, Savage N 1989 Interleukin-1 and its receptor. Crit Rev Immunol 9:1–20
di Giovine FS, Duff GW 1990 Interleukin 1: the first interleukin. Immunol Today 11:13–20
Granholm T, S?der O 1991 Constitutive production of lymphocyte activating factors by normal tissues in the adult rat. J Cell Biochem 46:143–151
Parvinen M, S?der O, Mali P, Fr?ysa B, Ritzén EM 1991 In vitro stimulation of stage-specific deoxyribonucleic acid synthesis in rat seminiferous tubule segments by interleukin-1. Endocrinology 129:1614–1620
P?ll?nen P, S?der O, Parvinen M 1989 Interleukin-1 stimulation of spermatogonial proliferation in vivo. Reprod Fertil Dev 1:85–87
Calkins JH, Sigel MM, Nankin HR, Lin T 1988 Interleukin-1 inhibits Leydig cells steroidogenesis in primary culture. Endocrinology 123:1605–1610
Hales DB 2002 Testicular macrophage modulation of Leydig cell steroidogenesis. J Reprod Immunol 57:3–18
Svechnikov K, Sultana T, S?der O 2001 Age-dependent stimulation of Leydig cell steroidogenesis by interleukin-1 isoforms. Mol Cell Endocrinol 182:193–201
Verhoeven G, Cailleau J, Van Damme J, Billiau A 1988 Interleukin IL-1 stimulates steroidogenesis in cultured rat Leydig cells. Mol Cell Endocrinol 57:51–60
Svechnikov K, DM Stocco, S?der O 2003 Interleukin-1 stimulates steroidogenic acute regulatory protein expression via p 38 MAP kinase in immature Leydig cells. J Mol Endocrinol 30:59–67
Stocco DM 2001 StAR protein and the regulation of steroid hormone biosynthesis. Annu Rev Physiol 63:193–213
Jonsson CK, Zetterstrom RH, Holst M, Parvinen M, S?der O 1999 Constitutive expression of interleukin-1 messenger ribonucleic acid in rat Sertoli cells is dependent upon interaction with germ cells. Endocrinology 140:3755–3761
Gerard N, Syed V, Jegou B 1992 Lipopolysaccharide, latex beads and residual bodies are potent activators of Sertoli cell interleukin-1 production. Biochem Biophys Res Commun 185:154–161
Wahab-Wahlgren A, Holst M, Ayele D, Sultana T, Parvinen M, Gustafsson K, Granholm T, S?der O 2000 Constitutive production of interleukin-1 mRNA and protein in the developing rat testis. Int J Androl 23:360–365
Sultana T, Svechnikov K, Weber G, S?der O 2000 Molecular cloning and expression of a functionally different alternative splice variant of prointerleukin-1 from the rat testis. Endocrinology 141:4413–4418
Gustafsson K, Sultana T, Zetterstrom CK, Setchell BP, Siddiqui A, Weber G, S?der O 2002 Production and secretion of interleukin-1 proteins by rat testis. Biochem Biophys Res Commun 297:492–497
Jonsson CK, Setchell BP, Martinelle N, Svechnikov K, S?der O 2001 Endotoxin-induced interleukin 1 expression in testicular macrophages is accompanied by downregulation of the constitutive expression in Sertoli cells. Cytokine 14:283–288
Bergh A, S?der O 1990 Interleukin-1?, but not interleukin-1, induces acute inflammation-like changes in the testicular microcirculation of adult rats. J Reprod Immunol 17:155–165
Carter-Su C, Schwartz J, Smit LS 1996 Molecular mechanism of growth hormone action. Annu Rev Physiol 58:187–207
Childs GV 2000 Growth hormone cells as co-gonadotropes: partners in the regulation of the reproductive system. Trends Endocrinol Metab 11:168–175
Hull KL, Harvey S 2000 Growth hormone: roles in male reproduction. Endocrine 13:243–250
Arsennijevic Y 1989 Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor delays sexual maturation in the male rat. Endocrinology 124:3050–3059
Laron Z, Klinger B 1994 Laron syndrome: clinical features, molecular pathology and treatment. Horm Res 42:198–202
Bartlett JM, Charlton HM, Robinson IC, Nieschlag E 1990 Pubertal development and testicular function in the male growth hormone-deficient rat. J Endocrinol 126:193–201
Gravance CG, Breier BH, Vickers MH, Casey PJ 1997 Impaired sperm characteristics in postpubertal growth-hormone-deficient dwarf (dw/dw) rats. Anim Reprod Sci 49:71–76
Zipf WB, Payne AH, Kelch RP 1978 Prolactin, growth hormone, and luteinizing hormone in the maintenance of testicular luteinizing hormone receptors. Endocrinology 103:595–600
Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244
Kansaki M, Morris P 1999 Growth hormone regulates steroidogenic acute regulatory protein expression and steroidogenesis in Leydig cell progenitors. Endocrinology 140:1681–1686
Kansaki M, Morris P 1998 Lactogenic hormone-inducible phosphorylation and -activated site-binding activities of Stat5b in primary rat Leydig cells and MA-10 mouse Leydig tumor cells. Endocrinology 139:1872–1882
Chandrashekar V, Bartke A 1993 Induction of endogenous insulin-like growth factor-I secretion alters the hypothalamic-pituitary-testicular function in growth hormone-deficient adult dwarf mice. Biol Reprod 48:544–551
Lin T, Wang D, Hu J, Stocco DM 1998 Upregulation of human chorionic gonadotropin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat Leydig cells. Endocrine 8:73–81
Hull KL, Harvey S 2000 Growth hormone: a reproductive endocrine-paracrine regulator? Rev Reprod 5:175–182
Koike S, Noumura T 1995 Immunohistochemical localization of insulin-like growth factor-II in the perinatal rat gonad. Growth Regul 5:185–189
Chubb C, Ewing LL 1981 Steroid secretion by sexually immature rat and rabbit testes perfused in vitro. Endocrinology 109:1999–2003
Girnita A, Girnita L, del Prete F, Bartolazzi A, Larsson O, Axelson M 2004 Cyclolignans as inhibitors of the insulin-like growth factor-I receptor and malignant cell growth. Cancer Res 64:236–242
Klinefelter GR, Hall PF, Ewing LL 1987 Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36:769–783
Ge RS, Shan LX, Hardy MP 1996 Pubertal development of Leydig cells. In: Payne AH, Hardy MP, Russell LD, eds. The Leydig cell. Vienna, IL: Cache River Press; 159–174
Khan SA, Khan SJ, Dorrington JH 1992 Interleukin-1 stimulates deoxyribonucleic acid synthesis in immature rat Leydig cells in vitro. Endocrinology 131:1853–1857
Bang P, Eriksson U, Sara V, Wivall IL, Hall K 1991 Comparison of acid ethanol extraction and acid gel filtration prior to IGF-I and IGF-II radioimmunoassays: improvement of determinations in acid ethanol extracts by the use of truncated IGF-I as radioligand. Acta Endocrinol (Copenh) 124:620–629
Chomczynski P, Sacchi S 1987 Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Casella SJ, Smith EP, van Wyk JJ, Joseph DR, Hynes MA, Hoyt EC, Lund PK 1987 Isolation of rat testis cDNAs encoding an insulin-like growth factor I precursor. DNA 6:325–330
Chan Y, Susuki K, Wool IG 1995 The carboxyl extensions of two rat ubiquitin fusion proteins are ribosomal proteins S27a and L40. Biochem Biophys Res Commun 215:682–690
Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-growth factor binding protein using Western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138–143
Tapanainen PJ, Bang P, Wilson K, Unterman TG, Vreman HJ, Rosenfeld RG 1994 Maternal hypoxia as a model for intrauterine growth retardation: effects on insulin-like growth factors and their binding proteins. Pediatr Res 36:152–158
Lamson G, Giudice LC, Cohen P, Liu F, Gargosky S, Muller HL, Oh Y, Wilson KF, Hintz RL, Rosenfeld RG 1993 Proteolysis of IGFBP-3 may be a common regulatory mechanism of IGF action in vivo. Growth Regul 3:91–95
Haider SG, Passia D, Overmeyer G 1986 Studies on the fetal and postnatal development of rat Leydig cells employing 3?-hydroxysteroid dehydrogenase activity. Acta Histochem 32:197–202
Kuhn-Velten N, Bos D, Schermer R, Staib W 1987 Age dependence of the rat Leydig cell and Sertoli cell function. Development of the peripheral testosterone level and its relation to mitochondrial and microsomal cytochrome P450 and to androgen binding protein. Acta Endocrinol (Copenh) 115:275–281
Lin T, Vinson N, Haskett J, Murono EP 1987 Induction of 3?-hydroxysteroid dehydrogenase activity by insulin-like growth factor-I in primary cultures of purified Leydig cells. Adv Exp Biol Med 219:603–607
Benahmed M, Morera AM, Chauvin MC, de Peretti E 1987 Somatomedin-C/insulin-like growth factor-1 as a possible intratesticular regulator of Leydig cell activity. Mol Cell Endocrinol 50:69–77
Gelber SJ, Hardy MP, Mendis-Handagama SMLC, Casella SJ 1992 Effects of insulin like growth factor-1 on androgen production by highly purified pubertal and adult Leydig cells. J Androl 13:125–130
Wang G, Hardy MP 2004 Development of Leydig cells in the insulin-like growth factor-I (IGF-I) knockout mouse: effects of IGF-I replacement and gonadotropic stimulation. Biol Reprod 70:632–639
Wang GM, O’Shaughnessy PJ, Chubb C, Robaire B, Hardy MP 2003 Effects of insulin-like growth factor-I on steroidogenic enzyme expression levels in mouse Leydig cells. Endocrinology 144:5058–5064
Cohen PE, Pollard JW 1998 Normal sexual function in male mice lacking a functional type I interleukin-1 (IL-1) receptor. Endocrinology 139:815–818
Lin T, Wang D, Hu J, Stocco DM 1998 Upregulation of human chorionic gonadotropin-induced steroidogenic acute regulatory protein by insulin-like growth factor-I in rat Leydig cells. Endocrine 8:73–78
Chatelain P, Sanchez P, Saez J 1991 Growth hormone and insulin-like growth factor I treatment increase testicular luteinizing hormone receptors and steroidogenic responsiveness of growth hormone deficient dwarf mice. Endocrinology 128:1857–1862
Keene DE, Suescun MO, Bostwick MG, Chandrashekar V, Bartke A, Kopchick JJ 2002 Puberty is delayed in male growth-hormone receptor gene-disrupted mice. J Androl 23:661–668
Chandrashekar V, Bartke A, Awoniyi CA, Tsai-Morris CH, Dufau ML, Russel LI, Kopchick JJ 2001 Testicular function in GH receptor gene disrupted mice. Endocrinology 142:3443–3450
Zhang FP, El-Hafnawy T, Huhtaniemi I, El-Hafnawy T 1998 Regulation of luteinizing hormone receptor gene expression by insulin growth factor-I in an immortalized murine Leydig tumor cell line (BLT-1). Biol Reprod 59:1116–1123
Dombrowicz D, Sente B, Reiter E, Closset J, Hennen G 1996 Pituitary control of proliferation and differentiation of Leydig cells and their putative precursors in immature hypophysectomized rat testis. J Androl 17:639–650
LeRoith D, Werner H, Beitner-Johnson D, Roberts Jr CT 1995 Molecular and cellular aspects of the insulin growth factor-I receptor. Endocr Rev 16:142–163
Bicsak TA, Shimonaka M, Malkowski M, Ling N 1990 Insulin-growth factor binding protein-3 inhibition of granulosa cell function effect on cyclic 3'5'-monophosphate deoxyribonucleic acid synthesis and comparison of effect of IGF-I antibody. Endocrinology 126:2184–2189
Wang D, Nagpai ML, Lin T, Shimasaki S, Ling N 1994 Insulin-like growth factor-binding protein-2: the effect of human chorionic gonadotropin on its gene regulation and protein secretion and its biological effects in rat Leydig cells. Mol Endocrinol 8:69–76
Kelley KM, Oh Y, Gargosky SE, Gucev Z, Matsumoto T, Hwa V, Ng L, Simpson DM, Rosenfeld RG 1996 Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics. Int J Biochem Cell Biol 28:619–637
Loffing-Cueni D, Schmid AC, Reinecke M 1999 Molecular cloning and tissue expression of the insulin-like growth factor II prohormone in the bony fish Cottus scorpius. Gen Comp Endocrinol 113:32–37[CrossRef](Eugenia Colón, Konstantin)