:: Asian Journal of Andrology ::

Molecular identity, expression and functional analysis of interleukin-1a and its isoforms in rat testis

T. Sultana^{1, 2, 3}, K. V. Svechnikov¹, K. Gustafsson⁴, A. Wahlgren¹, E. Tham², G. Weber², O. Söder¹

¹Pediatric Endocrinology Unit, Department of Woman and Child Health, ²Centre for Molecular Medicine and Tumor Biochemistry, Karolinska Institute and Hospital, S-17176 Stockholm, Sweden
³Department of Biological and Biomedical Sciences, Faculty of Health Sciences, Aga Khan University, Karachi- 74800, Pakistan
⁴Department of Chemistry and Biomedical Sciences, University of Kalmar, SE-391 82 Kalmar, Sweden

Asian J Androl 2004 Jun; 6: 149-153

Keywords: IL-1; spermatogenesis; testis; Sertoli cells; Leydig cells; calpain

Abstract

Interleukin-1 (IL-1) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL-1 in testicular physiology. Bioactive IL-1 isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in a thymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1 are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL-1 is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis. Pathophysiologically, testicular IL-1a may contribute to testicular relapse of acute lymphocytic leukemia in boys.

1 Introduction

Normal testicular function requires both the controlled secretion of LH, FSH and testosterone during fetal and postnatal life [1, 2], and a well-organised intratesticular network of local regulators. The exquisitely timed and highly rationalized expression of these regulators initiates and maintains spermatogenesis [3-6].

More than 100 local factors considered important for the testis have been described to be involved in the regulation of Sertoli and Leydig cell function and these factors enable the co-ordination of spermatogenesis and steroidogenesis [5, 7]. Such factors include growth factors and cytokines, like insulin like growth factor-1 (IGF-1) and interleukin-1 (IL-1).

IL-1 was first discovered as a pro-inflammatory cytokine and was later found also to be constitutively produced by Sertoli cells in the absence of local inflammation. This indicate that IL-1 has non-inflammatory paracrine function(s) at this site [8, 9]. In the rat testis, we have found that IL-1, but not IL-1 exerts pro-inflammatory activity after local injection [8]. The presence of bioactive IL-1 in the interstitial fluid in biologically relevant concentration raises the question, why the testis is not an inflamed tissue. Our laboratory has been actively involved in studying the role of IL-1 in testicular function.

The present review is focused on the role of IL-1 and its isoforms in testicular physiology. A putative functional relevance of these isoforms might be extrapolated to a role in disorders of spermatogenesis and steroidogenesis in humans leading to infertility.

2 Interleukin-1

IL-1 belongs to a family consisting of two agonist proteins IL-1 and IL-1, one antagonist IL-1 receptor antagonist (IL-1ra) and two receptors: type I and type II IL-1 receptors, of which the former is the signalling receptor. IL-1 and b are both synthesized as precursor proteins of 33 kDa (proIL-1 and -) [10]. In activated macrophages, proIL-1s are processed to 17 kDa mature forms that exert most of the biological functions. Both mature forms of IL-1 and are potent enhancers of immune responses and also potent inducers of acute phase responses and inflammation.

In our studies we have shown that an IL-1 like protein was age- and stage-dependently expressed by Sertoli cells and was secreted bidirectionally into both the interstitial and intratubular compartments of the rat testis [9, 11]. Testicular IL-1 (tIL-1) was suggested to serve as a paracrine regulator in the testis. It was also shown that IL-1 was mitogenic to certain germ cell types [12] and acted on Leydig cells to regulate steroidogenesis [13, 14].

2.1 Molecular identity of tIL-1

Physicochemical and molecular characterisation of isolated and purified IL-1 from rat testis showed that it is most probably the same IL-1 protein secreted by activated macrophages. This conclusion was made by the use of specific blockers of the bioactivity such as, IL-1ra, anti-IL-1 antibodies and soluble type I IL-1 receptor [15].

The study also confirmed the existence of micro-heterogeneity of tIL-1 proteins as reported previously [16, 17]. Chromatographic studies revealed three bioactive IL-1 protein species with apparent molecular sizes of 45, 31 and 17 kDa, respectively, and two charged species with isoelectric points (pI) of 5.7 and 6.0. Testicular IL-1 was found to share charge properties with macrophage IL-1, but not IL-1, as no charged bioactive species of pI 7 (pI for IL-1) was found at chromato-focusing [15].

The molecular heterogeneity also existed at the transcriptional level as two transcripts of 941 and 767 bp were found [18]. The 941 bp transcript corresponded to rat macrophage-derived proIL-1 and encoded a protein of 32 kDa. The smaller transcript represented a splice variant that lacked an internal 174 bp that corresponded to exon 5 of the mouse and human IL-1 gene. The resulting protein product of 24 kDa lacked the calpain cleavage site (Figure 1). Calpain plays an important role in the production of the active 17 kDa IL-1 cytokine. The remaining 16 kDa propeptide has a nuclear localisation signal and has been reported to act as an oncogenic protein by some investigators [19].

Figure 1. Genomic organization and processing of rat IL-1. The genomic sequence shows the presence of responsive elements in the promoter region (black arrows) including glucocorticoid-responsive elements (GRE), activator protein-1 (AP1) and nuclear factor kappa B (NFkB). A transcript of 941 bp and a splice variant of 767 bp are produced corresponding to 32 kDa (32proIL-1) and 24 kDa (24proIL-1) proteins, respectively. The 32proIL-1 protein is processed by calpain to produce mature 17 kDa IL-1 and a 16 kDa pro-peptide. 24proIL-1 lacks the calpain cleavage site and remains uncleaved.

The microheterogeneity of testicular IL-1, in addition to alternative splicing, may also be due to differential post-translational modifications. Such modifications may increase the plasticity of the system by activation of different signal transduction pathways and may also direct the protein to different locations. As reported in some studies, post-translational modifications may influence membrane targeting of the isoforms [20]. It has been reported that proIL-1 is glycosylated, myristyl-acylated and phosphorylated in its N terminal region [21-23].

2.2 Expression and secretion of IL-1 isoforms in rat testis

Studies using in situ hybridisation to localize the transcripts in normal rat testis showed that the two isoforms of IL-1 (32proIL-1 and 24proIL-1) had a similar localisation in the periphery of the seminiferous tubules and more specifically in Sertoli cells [18, 24]. These studies indicated that expression of IL-1 depends on interaction with germ cells. Furthermore some studies showed that there is a rapid increase in the expression of IL-1 and its receptor in cultured Sertoli cells upon exposure to residual bodies [25].

Protein expression analysis of IL-1 isoforms revealed that tissues other than the testis express the classical precursor form of IL-1 and mature 17 kDa IL-1, whereas the testis showed three distinct molecular protein species with apparent molecular sizes of 45, 24 and 19 kDa [15]. A similar expression pattern was observed in mouse Sertoli cell line (MSC-1) with a constitutive exclusive localisation to the nucleus (Figure 2). This again confirms a Sertoli cell origin of tIL-1.

Figure 2. Constitutive expression of IL-1 isoforms by murine Sertoli cell line (MSC-1).
(A) Immunolocalisation of endogenously produced IL-1 isoforms in MSC-1 cells using antibodies against 32proIL-1. The cells were cultured on cover slips for 24 hr and analysed by immunofluorescent staining (left photo). The expression was dominant in the nucleus. Right photo shows nuclear staining by DAPI.
(B) IL-1a protein isoforms produced in MSC-1 cells. Western blot analysis of IL-1 proteins extracted from MSC-1 cells and recombinant 32 kDa, 24 kDa and 17 kDa IL-1 expressed in COS-1 cells. Detection was performed with chicken antibodies raised against rat recombinant 32proIL-1. Lane 1 (24) represents recombinant 24 kDa proIL-1 protein. Lane 2 (MSC-1) shows protein extract from MSC-1 cells. Lane 3 (32+17) represents a mixture of COS-1 cell expressed 32proIL-1 and 17 kDa mature IL-1. kDa shows molecular sizes markers.

All tIL-1 isoforms were found to be bidirectionally secreted by the producing Sertoli cells into the interstitial and intratubular testis compartments. The mechanism of extracellular release is unclear but IL-1 has been reported to utilize a non-Golgi route of secretion [10]. Alternative mechanisms of protein secretion have been shown in other studies for IL-1, FGF and HMGB1 [26-28]. These proteins have also been reported to be produced by the testis.

2.3 Functional analysis of tIL-1a isoforms

2.3.1 Thymocyte proliferation

IL-1 has the ability to induce the proliferation of thymocytes. Therefore, a murine thymocyte proliferation assay [29] was utilised to analyse the activity of IL-1 isoforms. The bioactivity of these isoforms was also compared with mature 17 kDa IL-1 and IL-1. A clear biopotency difference between these isoforms was found in an order of 17 kDa IL-1 > 17 kDa IL-1 >32proIL-1 > 24proIL-1 [18].

The lymphocyte activating properties of tIL-1 may have pathophysiological relevance, as the testis is a frequent site of relapse in boys suffering from acute lymphoblastic leukaemia [30].

2.3.2 Leydig cell steroidogenesis

Studies have indicated that IL-1 inhibits hCG-induced Leydig cell steroidogenesis in rats [13]. We found an age-dependent difference in the ability of IL-1 to regulate basal testosterone production. In Leydig cells from 80-day-old rats IL-1 showed an inhibitory, while cells from 40-day-old rats displayed a stimulating effect on testosterone production in response to IL-1 [31]. The age-dependent IL-1 responsiveness may be due to the level of IL-1 receptor expression as IL-1RI was expressed in relatively high amounts in Leydig cells from 40-day-old rats, whereas the inhibitory decoy receptor IL-1RII was more predominant in Leydig cells from 80-day-old rats. An interesting observation was that 24proIL-1 showed no effect on basal steroid production in Leydig cells from 80-day-old rats.

Testosterone production induced by hCG was inhibited by IL-1 isoforms in both 40- and 80-day-old rats. 24proIL-1 had no effect on Leydig cells from 80-day-old rats but showed a weak but significant stimulatory effect in cells from 40-day-old rats. The site of inhibition was suggested to be P450c17 as indicated by other studies [32]. The stimulatory effect of IL-1 occurs mainly through protein kinase A and the calcium signalling pathway [31]. The tested IL-1 isoforms displayed the same biopotency difference in the steroidogenesis assay as in the thymocyte proliferation system.

2.4 Regulation of 32proIL-1 processing

Due to their shared substrate specificity, calpain I and II act as key regulators in processing of IL-1 and cleave 32proIL-1 to produce mature 17 kDa IL-1 and the 16 kDa propeptide [33]. Both calpain I and II were expressed in the adult rat testis, where Sertoli cells, Leydig cells and peritubular cells were the main expressing cells [33]. There was no change in developmental expression of calpain II, while calpain I showed some increase in 20- and 25-day-old rats. This increase correlated to a parallel temporal increase in IL-1 expression and an important regulatory period of spermatogenesis. At day 20 post-partum a peak of apoptosis was observed particularly in pachytene spermatocytes [34]. It could therefore be speculated that high expression of calpain at day 20 and 25 may contribute to germ cell apoptosis as supported by other studies [35].

Testosterone depletion studies using EDS showed decreased calpain I expression but no effect on calpain II. Although not fully confirmed, this indicates that calpain expression may be regulated by testosterone [36].

2.5 Inflammation and IL-1

It is well established that endotoxin, a potent proin-flammatory activator, induces expression of IL-1 in most tissues. Under such conditions, testicular macrophages were found to express IL-1, whereas Sertoli cells showed a down-regulation of the constitutively expressed IL-1. This may serve as a mechanism to protect germ cells from potentially harmful effects of increased IL-1 [37]. Concomitantly, the expression of calpain I and II proteins was decreased stage-dependently in response to inflammatory stimuli. There was almost no change in stage VII, a site known to have virtually no IL-1 expression [11]. In contrast, whole testis extracts showed decreased calpain I but not calpain II expression together with a decreased calpain I enzyme activity after endotoxin treatment. In contrast, the liver displayed increased IL-1 and calpain I and II expression. We have proposed that both calpain I and II may contribute to regulation of IL-1 in the testis.

3 Conclusion

The present review focuses on the molecular heterogeneity of testicular IL-1 and has presented some of the molecular and functional aspects of IL-1 in testicular physiology. Several important aspects could be the targets of future studies. One such issue is the expression of the 45 kDa IL-1 isoform in testis and its potential function. Another important point is to incorporate IL-1 and its isoforms in an integrative perspective with the hormones and other factors in the testis. Most local factors act together in concert and each factor may be only one word of the whole book of signals that the cellular machinery recognizes to produce a biological response. Nevertheless, we believe that it is still important to investigate and describe the functions of individual factors in detail.

Acknowledgments

The author's own work cited was supported by grants from the Swedish Research Council (8282), the Children Cancer Foundation, the Cancer Foundation, Frimurare Barnhuset in Stockholm, H.R.H. Crown Princess Lovisa Society of Pediatric Health Care, the Society for Child Care and the Karolinska Institute.

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Correspondence to: Taranum Sultana, Ph.D., Department of Biological and Biomedical Sciences, Faculty of Health Sciences, Aga Khan University, Stadium Road, P.O. Box 3500, Karachi- 74800, Pakistan.
Tel: +92-21-4859 4567, Fax: +92-21-493 4294
Email: taranum.sultana@aku.edu
Received 2003-07-24 Accepted 2004-03-22