Interleukin-1 Superfamily Genes Expression in Normal Or Impaired Human Spermatogenesis
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Genes and Immunity (2007) 8, 100–107 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene ORIGINAL ARTICLE Interleukin-1 superfamily genes expression in normal or impaired human spermatogenesis N Rozwadowska1, D Fiszer1, P Jedrzejczak2, W Kosicki3 and M Kurpisz1 1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; 2Department of Infertility and Reproductive Endocrinology, Poznan University of Medical Sciences, Poznan, Poland and 3Department of Urology, District Regional Hospital of Poznan, Poznan, Poland Interleukin-1 (IL-1) is a pleiotropic cytokine that may play a role in contributing to the specific immune environment of mammalian testis and in regulating cell differentiation. We have determined the transcription activity of the IL-1 gene family (using real-time polymerase chain reaction (PCR)) in two main functional testicular compartments (interstitial and intratubular ones), and in tissue homogenates obtained from patients with fertility disorders (spermatogenic arrest and testicular tumors). We observed the prominent expression of gene coding for IL-1 receptor antagonist (IL-1RA) in a purified fraction of gametogenic cells (normal gonad). Caspase-1 (ICE – IL-1b-converting enzyme) was highly expressed (on mRNA level) in interstitial compartments as well in testicular tumors (immune enhancement?). In addition we found, that the activity of IL-1RA gene decreased along spermatogenic alteration in an inversely related manner with IL-1a (from normal gonad through spermatogenic arrest to Sertoli cell only syndrome). Therefore, the quotient value of IL-1a/IL-1RA could potentially serve as the diagnostic molecular probe for spermatogenesis assessment. The precise level of mRNA for IL-1–IL-18 cytokines and their receptors, and specifically of the receptor antagonist in immune privileged gonad, could be one of the main factors responsible for maintaining testicular homeostasis, thus enabling generation of the mature spermatozoa. Genes and Immunity (2007) 8, 100–107. doi:10.1038/sj.gene.6364356; published online 11 January 2007 Keywords: interleukin-1 family; spermatogenesis; immune privilege; real-time PCR; cytokines Introduction logical reactions, and its biological activities are precisely regulated. The IL-1 family comprises several ligands Spermatogenesis is a dynamic process containing mitotic (two agonists – IL-1a and IL-1b, and receptor antagonist cell divisions (spermatogonial stem cells), meiosis (sper- – IL-1RA), and surface or soluble receptors: IL-1RI, IL- matocytes) and mature sperm formation (spermio- 1RII and IL-RAcP. IL-1 signaling is based on a ligand genesis). Homeostasis in the seminiferous epithelium binding to IL-1RI and, subsequently, IL-1RAcP coupling requires a permanent balance between cell proliferation to the complex. This results in signal transduction and apoptosis. The altered equilibrium between these through the intracellular protein cascade.4–6 The IL-18 two processes could lead to testicular pathologies such as is closely related to the IL-1 superfamily, including fertility disorder or cancerogenesis.1 Testicular home- its receptor structure (IL-18Ra and IL-18Rb) and signal ostasis requires a unique immune status within the transduction pathway. Both proIL-1b and proIL-18 male gonad. The immune system provides protection require caspase-1 (ICE – IL-1b-converting enzyme) for for immunogenic male germ cells and simultaneously their cleavage into the active molecules. Caspase-1 is the permits normal inflammatory response against invading cysteine protease, homologous to ced-3 gene (Caenohab- pathogens.2 Proinflammatory cytokines must be tightly ditis elegans) – the central component of apoptotic regulated in order to prevent autoimmune orchitis. The machinery.7 Both IL-1b and IL-1a are processed and cytokine network in the testis also works as the cell released during apoptosis but only the alpha form is a growth and differentiation promoting system that helps potent inducer of malignant cell death.8 to coordinate multifactorial interaction during steroido- Besides being the best-known key regulator of inflam- and spermatogenesis.3 matory reaction, the IL-1 system modulates the expres- Interleukin-1 (IL-1) family members are known to be sion of genes involved in cell survival, proliferation and involved in host response to inflammatory or immuno- angiogenesis. Several studies demonstrated in vivo and in vitro ability of IL-1 to regulate a variety of tumor cell function. IL-1 is also the major cytokine involved in Correspondence: Professor M Kurpisz, Polish Academy of Sciences, carcinogenesis, angiogenesis and metastasis.9 Institute of Human Genetics, ul. Strzeszynska 32, 60-479 Poznan, The male gonad is an abundant source of bioactive Poland. E-mail: [email protected] IL-1. Numerous studies have shown that IL-1a is Received 23 January 2006; revised and accepted 10 October 2006; produced mainly by seminiferous epithelium, where it published online 11 January 2007 has been reported as a potent growth factor for immature Interleukin-1 superfamily genes expression N Rozwadowska et al 101 Sertoli cells and also for spermatogonia.10,11 Further oligobiopsies obtained from azoospermic as well as studies have revealed at least three bioactive isoforms cancer patients. Peripheral blood mononuclear leuco- of IL-1a in the mammalian testis.12 On the other hand, cytes isolated from healthy volunteers served as systemic IL-1b is mainly expressed in interstitial tissue, where it controls. was shown to affect steroidogenesis. However, in several Evaluation of the expression of the listed genes was studies where the effect of IL-1b in the interstitium achieved by real-time quantitative PCR kinetics using was investigated, some discrepancies were noted. SybrGreen I chemistry. Verification of the accuracy of Prevailing observations suggest that IL-1 inhibits both performed amplifications was carried out by analyzing basal and LH/hCG-stimulated testosterone production, the product melting curve and by using classical agarose and stimulates immature Lydig cell proliferation in gel electrophoresis. All the quantification results ob- in vitro culture.13 tained were subsequently normalized according to the In this study, we examined, by using a real-time PCR, b-actin gene reference. Thus, among the collected the expression profile of the IL-1 gene superfamily in samples (n ¼ 95), only 75 were included for further normal and impaired spermatogenesis, including testi- analysis on the basis of b-actin gene expression (4105 cular malignancies, to define whether cytokine gene copies/per sample was required). expression levels differ in various pathophysiological Expression of all the genes under study was observed conditions and to establish possible correlations. both in tissue samples or in isolated cell fractions; however, the pattern of expression was different in physiological and pathological testis. Observations indi- Results cated that IL-18 and its receptors were expressed on a Testicular cells and tissues lower level than the IL-1 gene members. Also, there was During two-step collagenase digestion with mechanical a tendency to overall weaker expression in the tissue agitation, heterogeneous suspensions of testicular cells of gonads with impaired spermatogenesis in comparison were obtained. Further purification process, that was to normal or neoplastic testis. based on Percoll discontinuous gradient centrifugation technique, allowed cell fractions of satisfying purity to be Normal spermatogenesis obtained. Histological staining confirmed the identity The real-time RT–PCR (Q-RT–PCR) analysis of cDNA and homogeneity of obtained cell fractions, that is, samples obtained from testicular cell fractions revealed 99–100% of Leydig cells in the interstitial fraction (I) that there were two distinct expression patterns of and 98% of gametogenic cells in the intratubular one (G). IL-1 family genes in testicular compartments under The purified gametogenic cell fraction contained mainly study. A high expression of IL-1a gene was noted in spermatocytes and round spermatids (RS). the intratubular cell fraction (especially in the purified Table 1 summarizes the histopathological classification gametogenic cell suspensions) as compared with the and number of collected cell/tissue samples. extratubular compartment. One could concomitantly observe the intense expression of IL-1b in the Leydig Real-time PCR cell fraction (interstitium), where a far less intense Both isolation methods yielded comparable RNA sam- expression of IL-1a gene was found (Figure 1). Testis ples in quality and amount. Samples in were validated appeared as a rich source of IL-1RA mRNA in both and subjected to quantification assays when the b-actin testicular compartments, although we observed a ten- gene expression was over 105 copies in the investigated dency toward higher expression of this gene in the sample. intratubular compartment, especially in a purified We have examined the following IL-1 superfamily gametogenic cells fraction. We also observed a tendency gene members: IL-1a, IL-1b, IL-1RA, IL-1RI, IL-1RII, for the deteriorating transcription of caspase-1 gene IL-18, IL-18Ra and IL-18Rb (primers listed in Table 2) beginning from the interstitium through the hetero- in interstitial and intratubular cell fractions or in tissue geneous gametogenic cell suspensions as well as in the Table 1 Analyzed samples (total collected samples in parentheses) by real-time RT-PCR Material Number of Cell