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Articles |
Production in Murine Sertoli Cells by Interleukin-11
a Department of Obstetrics and Gynecology, Soroka University Medical Center and Department of Microbiology and Immunology and Department of Pathology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
b Christian-Albrechts-University of Kiel Medical School, Kiel, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, -1ß, FSH, and lipopolysaccharide (LPS) in the regulation of IL-1 and -1ß production by Sertoli cells under in vitro conditions. Sertoli cell cultures from immature mice produced constitutively basal levels of intracellular IL-1. Stimulation of Sertoli cell cultures with LPS (5 µg/ml) resulted in a maximal production of intracellular IL-1 2 h after the stimulation. Thereafter, these levels decreased but remained significantly higher within 24 h after stimulation than those in control cultures. The effect of LPS on IL-1 production was dose dependent. FSH did not show any effect on intracellular IL-1 production by Sertoli cells. IL-1 could not be detected in supernatants of unstimulated or stimulated Sertoli cell cultures. Sertoli cell cultures stimulated with recombinant IL-1 induced optimal intracellular levels of IL-1 within 2 h of stimulation. These levels remained high 24 h after stimulation. However, stimulation of Sertoli cell cultures with IL-1ß induced a peak of IL-1 production 8 h after stimulation. These levels decreased 24 h after the stimulation but were still found to be significantly higher than those in control cultures. The addition of IL-1 receptor antagonist (IL-1ra) to Sertoli cell cultures did not significantly alter their capacity to produce IL-1. However, the stimulatory effects of recombinant IL-1 on IL-1 production by Sertoli cell cultures were reversed by the concomitant addition of recombinant IL-1ra. No immunoreactive IL-1ß could be detected in lysates or conditioned media of immature murine Sertoli cells under any of the stimulatory conditions outlined.
Our results may suggest the involvement of physiological (IL-1) and pathophysiological factors (LPS) in the regulation of spermatogenesis and spermiogenesis processes and male fertility.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-1ß, the former acting mainly on an intracellular level, the latter almost exclusively on an extracellular level [1]. IL-1 is known to be produced predominantly by tissue macrophages and monocytes in response to lipopolysaccharide (LPS), IL-1, and other cytokines [2]. A unique feature of the IL-1 system is the naturally occurring IL-1 receptor antagonist (IL-1ra). Its genetic structure is homologous with the IL-1 and IL-1ß genes to a certain degree [3, 4]. It binds to the same receptors without transmitting any signal; thus it inherits an equally important role in the regulation of IL-1 hormonal action [5, 6].
It has been shown that IL-1 is present in lysates of testicular tissue [7, 8]. Further investigations have identified several sources of IL-1 in the testis. In the interstitium, Leydig cells [9, 10] and testicular macrophages [11] were found to produce and secrete IL-1, preferably the ß form. In contrast, Sertoli cells have been shown to produce and secrete exclusively the form of IL-1 [10, 12–14]. Recently we have demonstrated that IL-1-like activity is present in conditioned media of mature human sperm cells [15, 16]. Infection-stimulatory agents such as lipopolysaccharides (LPS) as well as gonadotropins or steroid hormones have been shown to alter IL-1 levels in interstitial as well as tubular cells [10, 13], thus suggesting that IL-1 is participating in physiological and infection-regulatory autocrine and paracrine functions. While most investigations have focused on IL-1 responses detected in cell-conditioned media [10, 13, 17, 18], little is known about intracellular responses of IL-1.
Recently, we have demonstrated the presence of IL-1ra in germ cells and Sertoli cells. It was shown that Sertoli cells from prepubertal mice constitutively produce high amounts of IL-1ra, which can be enhanced within 4–8 h by LPS, FSH, or IL-1 [19].
Various investigations have indicated that IL-1 interferes in the regulation of spermatogenesis and spermiogenesis [20–27]. IL-1 receptors are identified, characterized, and localized in the mouse testis [20]. IL-1 or IL-1-containing media have been found to alter testosterone release of Leydig cells [21–24] and to have impact on Sertoli cell activity parameters such as transferrin or the aromatase activity [25, 26]. A correlation between IL-1 levels and the meiotic DNA synthesis of spermatogonia has been indicated [27].
In the present study, we examined the capacity of Sertoli cells isolated from prepubertal mice to produce IL-1 under physiological and pathological conditions. Specifically, we examined the effect of LPS, IL-1, IL-1ß, and IL-1ra, as well as FSH, in regulating IL-1 and -1ß production and secretion by Sertoli cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagenase (359 U/mg) and hyaluronidase (295 U/mg) were obtained from Sigma Chemical Co. (St. Louis, MO). Eagle's Minimum Essential Medium (MEM), penicillin, streptomycin, and fetal calf serum were purchased from Beit Haemek Biological Industries (Beit-Haemek, Israel). BSA was purchased from ICN Biomedicals (Aurora, OH). The Ares Serono group (Geneva, Switzerland) kindly provided recombinant human FSH (Gonal-F). Recombinant human IL-1 (92.5 U/ng) and IL-1ß (280 U/ng) were obtained from Genzyme (Cambridge, MA). Recombinant human IL-1ra was purchased from R&D Systems (Minneapolis, MN). LPS was obtained from Difco Laboratories (Detroit, MI). All other chemicals (analytical grade) were purchased from commercial sources.
Isolation and Culture of Murine Sertoli Cells
The investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Highly purified Sertoli cells were isolated from 15-day-old Balb/c mice (Harlan Laboratories, Jerusalem, Israel) using a modification and combination of the methods described by Toebusch et al. [28] and Skinner and Fritz [29] as follows. Twenty testes were decapsulated and mechanically digested by multiple aspirations through pipette tips (8 aspirations through 2-mm followed by 10 aspirations through 1-mm diameter opening) into a 50-ml syringe after addition of 20 ml MEM. Mechanical digestion was continued until tubules were completely dissociated. Thereafter the tubules were allowed to settle at unit gravity and washed three times with PBS. Supernatants containing the interstitial cells were discarded. The tubules were transferred to a 50-ml culture flask and subjected to collagenase treatment (8 mg/20 ml PBS, 25 min). All enzymatic digestions were carried out in a shaking water bath (120 cycles/min at 37°C). The resulting cell clusters and tubule fragments were washed two times with PBS (centrifugation at 100 x g for 4 min), followed by three needle aspirations into a 18-gauge x 1.5 needle syringe to break up the remaining tubule fragments. Finally the cell clusters were subjected to hyaluronidase digestion (20 mg/20 ml PBS, 30 min); thereafter they were filtered through sterile surgical gauze and washed three times with MEM (centrifugation at 100 x g for 5 min).
The resulting, almost completely dissociated Sertoli cells and germ cells were counted under phase-contrast microscopy in a Neubauer (Nalge Nunc International, Naperville, IL) counting chamber. Cells were seeded in MEM containing streptomycin (100 mg/L), penicillin (105 IU/L), L-glutamine, and 5% fetal calf serum in 96-well plates at a density of 1.3 x 105 Sertoli cells per well (3.3–3.4 x 105 Sertoli cells/cm2). Cells were also seeded on Permanox 4-chamber culture slides (Nalge Nunc, Naperville, IL) at lower densities.
After 48 h of incubation (36°C, 5% CO2), most of the remaining germ cells were removed by hypotonic shock treatment with 10% MEM in distilled water for 2.5 min, unless otherwise indicated, and washed three times with MEM, the plates being vigorously shaken in a horizontal direction. The culture was continued for another 2 days in the presence of 0.5% BSA. At the end of the 4-day preincubation period, cells were incubated for various times with MEM containing 0.5% BSA in the absence or presence of FSH, LPS, or various cytokines as indicated. At the end of incubation, supernatants were collected and stored at -20°C. Cells were washed and fresh medium was added to each well. Cells were lysed by three cycles of rapid freezing/thawing. Culture slides were fixed with methanol (absolute, 10 min, -20°C) and air dried until subjected to procedures to evaluate the purity and composition.
Evaluation of Viability, Purity, and Composition
The viability was evaluated at the end of the incubation period using trypan blue.
To examine the purity of the preparations, fixed and air-dried culture slides were stained by Mayer's hematoxylin (1 min) and eosin (1.5 min) or subjected to immunocytochemical staining using neurofilament-200 antibodies (Sigma, Rehovot, Israel) in 1:1000 final dilution. Sertoli cells were identified by their distinctive morphology [30].
The presence of peritubular cells was examined according to the method used by Oonk et al. [31]. Part of the final cell pellet was fixed in a mixture of ethanol and acetic acid (3:1, v:v) and air dried on microscope slides. After addition of a drop of acetic acid (45%, v:v), the slides were examined under phase-contrast microscopy. Contamination of Leydig cells was assessed at the time of isolation by phase-contrast microscopy of the final Sertoli cell-enriched fraction. Leydig cells presented the characteristic illumination described by Schumacher et al. [32].
Isolation and Culture of Murine Splenic Leukocytes
Murine splenic leukocytes were obtained by mechanical digestion of mouse spleens. Cells were filtered, washed two times with MEM, and subsequently seeded at a density of 5 x 106/ml in 6-well plates. After 24 h of stimulation (MEM in the presence of 5 µg/ml LPS), supernatants were collected and stored at -20°C until used in IL-1 assays as positive control.
Murine IL-1 and IL-1ß ELISA
Immunoreactive murine IL-1 and IL-1ß of Sertoli cell culture supernatants and lysates were quantified using specific ELISAs (IL-1: Serotec, Oxford, UK, and Biodesign International, Kennebunk, ME; IL-1ß: BioSource International, Camarillo, CA). According to the manufacturers, no cross-reactivity was measured between IL-1ß, -2, -3, -4,-5, -6, -7, -8, -10, -11, -13, -15, murine IL-1ß, granulocyte-macrophage colony-stimulating factor, insulin-like growth factor-1, interferon- and -
, tumor necrosis factor- or-ß, vascular endothelium-derived growth factor, and IL-1 ELISA. Also no cross-reactivity was measured between murine IL-1 and IL-1ß ELISA. Sensitivity of IL-1 and -1ß ELISAs was 4 and 15 pg/ml, respectively.
Murine IL-1ra ELISA
Immunoreactive murine IL-1ra of Sertoli cell culture supernatants and lysates was quantified using a specific ELISA (R&D Systems). No cross-reactivity was measured between murine IL-1, -1ß, human IL-1, -1ß, and murine IL-1ra ELISA. The sensitivity of IL-1ra ELISA was 120 pg/ml.
Statistical Procedure
Results are expressed as means ± SD. Each experiment was performed independently at least twice. ANOVA and Student's t-test were used to calculate P values. Significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The viability of Sertoli cell cultures at the end of incubation periods was always > 95%. After 4-day preincubation periods including hypotonic shock treatment, contamination of germ cells and macrophages was less than 0.5% and 0.1%, respectively. The presence of peritubular cells, as evaluated by the method of Oonk et al. [31], was always less than 0.5% at the time of isolation. At the same time, no contaminating Leydig cells were observed in the Sertoli cell-enriched fraction under phase-contrast microscopy.
Production of IL-1/ß by Sertoli Cells
Immature murine Sertoli cells are capable of producing intracellular immunoreactive IL-1 under basal culture conditions (3.5–9.5 pg/106 cells). Exposure of Sertoli cell cultures to LPS (5 µg/ml) resulted in enhanced production of intracellular IL-1 after 30 min of stimulation (Fig. 1). Maximal intracellular IL-1 levels were observed 2 h after stimulation with LPS (70 pg/106 Sertoli cells). Thereafter, these levels were decreased but remained significantly higher for 24 h after stimulation compared to those of unstimulated controls (20–28 pg/106 cells, P < 0.001) (Fig. 1). The effect of LPS on IL-1 production by Sertoli cells was dose dependent (Fig. 2). Significant induction was observed in response to concentrations of LPS as low as 0.625 µg/ml (24 h after stimulation; P < 0.001).
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Various doses of FSH (0.5–50 U/ml) did not show any alternating effect on intracellular IL-1 levels in Sertoli cells compared to controls at any observed time after stimulation (0.5–24 h) (data not shown).
No immunoreactive IL-1 could be observed in supernatants of unstimulated or stimulated Sertoli cell cultures.
IL-1 Activated Sertoli Cell IL-1 Production
Sertoli cell cultures subjected to recombinant IL-1 (50 U/ml) or -1ß (50 U/ml) responded with a significant increase of intracellular IL-1 production compared to that in control cultures (P < 0.01) (Fig. 3). Both IL-1 and-1ß induced elevated intracellular levels of IL-1 within 2 h of stimulation of Sertoli cell cultures. IL-1 production by IL-1-stimulated Sertoli cells increased continuously, with observed maximal levels 2–24 h after stimulation, whereas IL-1ß induced a peak of IL-1 production by Sertoli cells 8 h after stimulation. These levels decreased 24 h after the stimulation but were significantly higher than the levels in control cultures (P < 0.001) (Fig. 3). Intracellular IL-1 production by Sertoli cells, in response to IL-1, was shown to be dose dependent (Fig. 4).
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The addition of IL-1ra to immature Sertoli cells did not significantly alter their capacity to produce IL-1 (Fig. 3).
IL-1ra Reversed Stimulatory Effects of IL-1 on IL-1 Production by Sertoli Cells
The stimulatory effects of recombinant IL-1 (50 U/ml) on IL-1 production by Sertoli cells were dose-dependently reversed by the concomitant addition of recombinant IL-1ra (Fig. 5). In the presence of 5000 ng/ml IL-1ra, intracellular IL-1 levels in IL-1-stimulated Sertoli cells were comparable to levels in controls (Fig. 5).
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No immunoreactive IL-1ß could be detected in lysates or conditioned media of immature murine Sertoli cells under any of the stimulatory conditions used. Supernatants collected from splenic leukocyte cultures stimulated for 24 h without or with LPS (5 µg/ml) were used as positive control.
Time-Course Kinetic Effect of LPS on Immunoreactive Intracellular IL-1 and IL-1ra Production by Immature Murine Sertoli Cells
The levels of IL-1 and IL-1ra were examined in lysates of Sertoli cell cultures stimulated by LPS (5 µg/ml) for various time periods (0.5–24 h). As depicted in Figure 6, maximal levels of intracellular IL-1 production by Sertoli cells were examined after 2 h of stimulation and thereafter were decreased to basal levels. After 2 h of Sertoli cell stimulation, the levels of intracellular IL-1ra were basal; they reached maximal levels after 7–9 h of stimulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by immature Sertoli cells can be stimulated by recombinant IL-1 and IL-1ß. Furthermore, our data reveal that Sertoli cells are capable of reacting in specific time and concentration patterns of IL-1 production to different stimuli. LPS induces optimal levels of intracellular IL-1 production in Sertoli cells shortly (2 h) after it has been added. IL-1 and -1ß show their maximal effect much later (8 h) compared to LPS. The data suggest that LPS and IL-1 inherit different roles in the scenery of autocrine IL-1 signals in Sertoli cells. The fact that LPS induces IL-1 production by Sertoli cells differently from IL-1 and -1ß (Fig. 1 and Fig. 3, respectively) may indicate that LPS acts through additional/different mechanisms than through intracellular IL-1 or -1ß. It seems that IL-1 and IL-1ß differently induce the production of IL-1 by Sertoli cells. This may suggest different regulatory effects of the autocrine and paracrine IL-1 (IL-1 and IL-1ß, respectively) on Sertoli cell functions. The fact that we could not find detectable IL-1ß production by Sertoli cell cultures may also indicate the possible paracrine effects of IL-1ß produced by other testicular cells such as Leydig cells and macrophages on Sertoli cell functions.
IL-1 is primarily an intracellular regulator that is not secreted by most cells producing it [1, 2]. Mature Sertoli cells are capable of producing and secreting IL-1 [33]. It is known that during the process of sexual maturation, Sertoli cells isolated from rat testes increase the amount of secreted IL-1 [33]. While Sertoli cells isolated from 45-day-old rats secrete twice as much as similar cultures prepared from 35-day-old animals, there is no detectable amount of IL-1 present in Sertoli cell-conditioned media from prepubertal animals (20 day old) [33]. Our experiments using the murine system confirm the findings by Jegou et al. [33] that immature Sertoli cells produce but do not secrete IL-1. Therefore we suspect possible sites of action of Sertoli cell IL-1 in the cell itself as an autocrine regulator. It is known that intracellular IL-1 affects Sertoli cell functions like transferrin secretion and possibly cell growth and proliferation [25, 26, 34].
IL-1 can be presented on membranes after undergoing cleavage by membrane proteases called calpains [2]. Thus, the possibility that Sertoli cell-produced IL-1 acts on neighboring germ cells has to be taken into account.
Cudicini et al. [10] have indicated that IL-1 production and secretion by mature human Sertoli cells can be induced by FSH when cell cultures have been treated by hypotonic shock. However, our data obtained from use of immature murine Sertoli cells does not show any evidence to support these findings. Therefore it remains to be investigated whether sensitivity to FSH concerning IL-1 production by Sertoli cells may be under the regulating process of sexual maturation.
We have previously reported that immature murine Sertoli cells are capable of producing but not secreting IL-1ra [19]. It was of interest now to assemble data of IL-1 and IL-1ra produced by the same cells in order to estimate the final activity of IL-1. Therefore, we compared intracellular IL-1ra levels to intracellular levels of IL-1 under LPS stimulatory conditions. As presented here, intracellular amounts of IL-1 show a peak after 2 h of Sertoli cell stimulation by LPS. However, the intracellular IL-1ra production by LPS-stimulated Sertoli cells reached its maximum 7 and 9 h after stimulation [19]. By that time intracellular IL-1 levels had already dropped to about one quarter of the initially observed peak. The ratio of intracellular IL-1ra to IL-1 at that time mark was estimated to be about 8 to 10 (Fig. 6). As the present study has demonstrated, IL-1ra has the capacity to completely reverse the effects of IL-1 on Sertoli cells. Lin et al. [35] have performed a similar series of experiments on the effects of recombinant IL-1ra on IL-1ß-inhibited Leydig cell steroidogenesis, indicating that the ratio IL-1ra/IL-1ß of 8 to 10 could be enough to reverse 80% of the IL-1 action [35].
The present study indicates a possible role of IL-1 and IL-1ra in the autocrine regulation of Sertoli cell functions. IL-1 and IL-1ra have to be seen in relation to each other and their specific time patterns of expression to evaluate the final IL-1 action. It has been indicated that physiological and pathological factors and conditions affect the levels of IL-1 and IL-1ra in Sertoli cells. Thus, they may be involved in the regulation of spermatogenesis and spermiogenesis processes and male fertility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant (No. 4467) from the Ministry of Health, Jerusalem, Israel. During the course of this work D.Z. was the recipient of a Deutscher Akademischer Austauschdienst (DAAD) scholarship. 
2 Correspondence. FAX: 7 6400932; huleihel{at}bgumail.bgu.ac.il
Accepted: December 15, 1999.
Received: August 24, 1999.
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REFERENCES |
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messenger RNA expression in primary cultures of Leydig cells. Endocrinology 1991; 129:2862–2866. synthesis in rat testis. Mol Cell Endocrinol 1991; 82:R13–16. stimulate expression of mRNA for IL-1 and IL-1 receptor type I in cultured rat Sertoli cells. Mol Cell Endocrinol 1998; 137:139–144.[CrossRef][Medline] (IL-1) release, which triggers IL-6 production by an autocrine mechanism, through the lipoxygenase pathway. Endocrinology 1995; 136:3070–3078.[Abstract] and ß on Leydig cell function. Biochem Biophys Res Commun 1990; 167:548–553.[CrossRef][Medline] as a potent inhibitor of gonadotropin action in porcine Leydig cells: sites of action. Biol Reprod 1992; 46:1119–1126.[Abstract]-subunit mRNA expression and inhibin secretion are enhanced by FSH but not by testosterone. J Endocrinol 1989; 122:757–762.This article has been cited by other articles:
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  M. Ganaiem, M. AbuElhija, E. Lunenfeld, N. Cherniy, N. Weisze, S. B.-S. Itach, H. Breitbart, R. Apte, and M. Huleihel Effect of Interleukin-1 Receptor Antagonist Gene Deletion on Male Mouse Fertility Endocrinology, January 1, 2009; 150(1): 295 - 303. [Abstract] [Full Text] [PDF]
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