Interleukin-1 Superfamily Genes Expression in Normal Or Impaired Human Spermatogenesis

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 fraction or tissue Number of samples divided into samples homogenates pathological subgroups

Azoospermia (age 21–40 years) 33 (45) MA 20 SCOS 7 O/A 6

Testicular tumors (age 18–37 years) 12 (15) Carcinoma embryonale, 7 Seminoma 5

Normal gonad 21 (25) G (heterogenous suspension) 8 Purified gametogenic cells (RS) 4 I9 Peripheral blood lymphocytes 10

Abbreviations: G, gametogenic cells; I, interstitial cells; MA, maturation arrest; O/A, obstructive azoospermia; RT-PCR, reverse transcription- polymerase chain reaction; SCOS, Sertoli Cell Only Syndrome.

Genes and Immunity Interleukin-1 superfamily genes expression N Rozwadowska et al 102 Table 2 Primer sequences

Gene Primer name Sequences Size of the product

b-Act QACTf 50-CATgTACgTTgCTATCCAggC-30a 250 QACTr 50-CTCCTTAATgTCACgCACgAT-30a IL-1a qIL-1a 50-gAATgACgCCCTCAATCAAAgT-30a 186 qIL-1b 50-TCATCTTgggCAgTCACATACA-30a IL-1b IL-1C (f) 50-AAACAgATgAAgTgCTCCTTCCAgg-30 388 IL-1D (r) 50-TGGAgAACACCACTTgTTgCTCCA-30 IL-1RA IL-1RAF 50-AATCCAgCAAgATgCAAgCC-30 406 IL-1RAR 50-ACgCCTTCgTCAggCATATT-30 IL-1R1 IL-1R1F 50-gTggAgTCAAAgATAggCTC-30 298 IL-1R1R 50-AgCACTgggTCATCTTCATC-30 IL-1R2 IL-1 R2F 50-ATgACTCTgCTAggACggTC-30 439 IL-1R2R 50-TATTgCTggCCTTCATgggC-30 IL-1RAcP IL-1RAcPF 50-AACCAggAgAggAgCTACTCATTCCC-30 445 IL-1RAcPR 50-gAATCACCACTAgCAggACTgTgg C-30 ICE qICEf 50-CTCAggCTCAgAAgggAAT-30a 220 qICEr 50-CgCTgTACCCCAgATTTTgT-30a IL-18 qIL-18f 50-gCTgCTgAACCAgTAgAAgAC-30a 186 qIL-18r 50-CCgATTTCCTTggTCAATgAAgA-30a IL-18Ra qIL-18Rf 50-AgTgTgCCTAgTgACTgTgTg-30a 146 qIL-18Rr 50-CATTTTCAggTCggCATTCTTTT-30a IL-18Rb RAPr 50-CTAAAATCATCTTgACACAACAggC-30 140 Ex10 50-ATCCTTAATTCgCTCTCCTgCAAC-30

aPrimer sequences obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/).

3.50E+05 * * 1.20E+04 I l 3.00E+05 G 1.00E+04 G RS RS 2.50E+05 PBL 8.00E+03 * 6.00E+03 2.00E+05 4.00E+03 1.50E+05 * 2.00E+03 * 1.00E+05 0.00E+00

Relative expression level (number of (number level Relative expression mRNA copies in relation to beta actin) ICE IL-18 5.00E+04

Relative expression level (number of (number level Relative expression mRNA copies in relation to beta actin) Figure 2 Expression of ICE and IL-18 gene in isolated cell fractions: interstitial cells (I), gametogenic cells (G), purified gametogenic cells 0.00E+00 IL-1 IL- IL-1RA IL-1R1 IL-1R2 IL- (RS). *Po0.05. beta 1alpha 1RAcP Figure 1 Expression of IL-1 gene family in isolated cell fractions: interstitial cells (I), gametogenic cells (G), purified gametogenic cells changed in parallel to the biopsies histopathology (RS), peripheral blood mononuclear leucocytes (PBL). *Po0.05. (Figure 3a–c). For example, the analysis of mRNA quantity in tissue homogenates from the testis with maturation arrest spermatocyte round spermatids cell fraction where (MA), Sertoli cell only syndrome (SCOS) and obstructive such expression almost completely faded (Figure 2). A azoospermia showed an inversely related balance be- pattern similar to that of ICE expression in the case of tween IL-1a and IL-1RA (Figure 3e). The oligobiopsies IL-1RII receptor (Figure 1). We also found that IL-18 from SCOS patients showed exceptionally high expres- expression was most eminent in the RS cell fraction than sion for IL-1a in proportion to IL-1RA mRNA. The in other testicular cell types (Figure 2). Gene expression maturation block of spermatogenesis (azoospermia) was from isolated testicular cells and mononuclear leucocytes connected in more equal proportions between both these (control) was corroborated (Figures 1 and 2). genes. When a spermatogenesis was close to normal (obstructive azoospermia), the expression pattern resem- bled the physiological status of testis (prevalence of Testicular pathology IL-1RA mRNA over IL-1a). We next created a quotient

Quantitative specific mRNA (obstructive azoospermia) value (QIL-1/RA) for the potentially predictive diagnostic studies on tissue homogenates from testis with normal or tool. The Kruskal–Wallis test showed significant differ- impaired spermatogenesis or samples subjected to tumor ences between QIL-1/RA measured in pathological (MA overgrowth yielded interesting data. We found that a (Po0.003) and SCOS (Po0.0003)) than in physiological profile of IL-1 receptor antagonist gene expression was conditions (OA) (Figure 3f and Table 3).

Genes and Immunity Interleukin-1 superfamily genes expression N Rozwadowska et al 103 d *** a 250000 ** Series1 200000

150000

100000

50000 b

Relative expression level (number of (number level Relative expression mRNA copies in relation to beta actin) A.O M.A. SCOS 221756.7 42804.44 2752.574

e 100 *** IL-1alpha 90 IL-1RA ** 80 * 70 c 60 50 40 30 20 % of normalized expression 10 0 123

f 100 *** Table 3 ** Mean QIL-1/IL-1RA OA 0.45±0.1 MA 7.8±5.8 SCOS 12.9±6.4 10 IL-1/RA Q 1 01234

0.1 Figure 3 IL-1a/IL-1RA genes expression in tissue biopsies obtained from azoospermic patients. (a) Histological examination of obstructive azoospermia (OA): normal spermatogenesis. (b) Histological examination of MA: severe impairment of spermatocytogenesis – no spermatid visible. (c) SCOS: there is no germ cell in the seminiferous tubules. (d) Expression of IL-1RA gene in investigated samples. (e) The inversely related proportion between IL-1a and IL-1RA expression in tissue homogenates from testis with obstructive azoospermia (OA), MA and SCOS – depiction. (f) Quotient value of IL-1a and IL-1RA expression. *Po0.05, **Po0.005, ***Po0.0005.

The oligobiopsies from testicular neoplastic tissues bioactive IL-1 was not connected, however, with any generally showed high expression of IL-1RA and IL-1RII signs of local inflammation. It seems that the role of the genes (Figure 4a). We also found relatively high amounts IL-1 gene system in testis and its paracrine influence of caspase-1 mRNA in neoplastic tissue samples is still far from our understanding. In our study, we (Figure 4b). have shown the intense expression of IL-1b gene in the interstitial cells (Figure 1), earlier observed in our previous work.16 Numerous studies, reported an inhibi- tory role of IL-1 on testosterone production, whereas in Discussion another scenario this cytokine appeared as a growth While using the real-time PCR technique we have shown factor for immature Leydig cells.17 It was also shown that that the IL-1 genes system was fully expressed in human Leydig cells express both Fas and FasL without markers testis but the profile of this expression depended on the of visible apoptosis whereas the other groups demon- different cell fractions studied or certain defined testi- strated that FasL induced production and secretion of cular pathologies. IL-1b.18,19 Therefore it cannot be ruled out that FasL The constitutive expression of IL-1 molecules within presented on Leydig cells can be a potent activator of the murine and/or human testis has been postulated IL-1b gene. Immune cells have a strong impact on testi- since the early 1990s.14,15 Production and secretion of cular function; above especially the testicular macro-

Genes and Immunity Interleukin-1 superfamily genes expression N Rozwadowska et al 104 a Gene expression profiling of reproductive cells and *** tissues, and the molecular mechanisms underlying the *** 1.20E+05 male infertility or testicular tumor growth have been *** intensively pursued in recent years.27 IL-1 is a pluripo- 1.00E+05 tent cytokine which is not only involved in immunolo- 8.00E+04 gical response and tumor growth but is also critical for spermatogenesis, namely for regulation of cell prolifera- 6.00E+04 28 ** tion and apoptosis. Expression of IL-1 gene family 4.00E+04 * members in obstructive azoospermia (OA), owing to the 2.00E+04 ongoing spermatogenesis, served as a reference standard for non-obstructive azoospermia samples (spermato- 0.00E+00 Relative expression level (number of (number level Relative expression mRNA copies in relation to beta actin) genic arrest) and testicular tumor evaluation. The profile IL-1 beta IL-1alpha IL-1RA IL-1RI IL-1RII IL-1RAcP of gene expression in OA samples was distinctly b ** different from the profiles found in MA and SCOS. 1.20E+04 ** According to our data, the expression of IL-1RA * deteriorated in conjunction with severity of spermatoge- 1.00E+04 netic impairment. MA samples showed intermediate 8.00E+03 levels of IL-1RA (Figure 3d). Based on these results, we have proposed the determination of the quotient value ICE 6.00E+03 (QIL-1/RA), which appeared to illustrate the studied gene expression proportion in MA and SCOS, thus enabling 4.00E+03 a molecular classification of spermatogenetic disorder 2.00E+03 (Figure 3f). The low levels of IL-1RA and the relatively high Relative expression level (number of (number level Relative expression mRNA copies in relation to beta actin) 0.00E+00 transcription of IL-1a in SCOS testicular tissue samples TT A.O. M.A SCOS could be explained in several ways. The decreased Figure 4 (a) IL-1 gene family expression in testicular tumor level of IL-1RA could come from the intratubular biopsies. *Po0.05, **Po0.005, ***Po0.0005. (b) ICE gene expression compartment, where the lack of germ cells could be a in testicular tumor biopsies compared to normal (OA), impaired reason for the lost balance in expression between these spermatogenesis (MA) and SCOS. *Po0.05, **Po0.005. two IL-1 family members. On the other hand, one could not rule out that the observed tendency originated from other than only the intratubular cell compartment. For example, it could also depend on the expression in phages have been suggested to participate in providing Leydig cells or testicular immune cells. The latter ones the appropriate microenvironment for spermatogenesis were found in large numbers in biopsies from infertile utilizing a variety of cytokines and growth factors.20 patients not only in the interstitium but also in the Our study also demonstrated that the normal tubular wall and lumen.29 One could not rule out that human male gonad can be a rich source not only for IL-1a/IL-1Ra imbalance in SCOS patients originated IL-1 mRNA (a and b) but also for the receptor antagonist from Sertoli cells because IL-1a mRNA was found mainly (IL-1RA) (Figure 1) – the potent immunosuppressive in Sertoli cells.11 The RNA obtained from SCOS biopsies protein that could be involved in immune privileged could then be enriched with mRNA derived from status the testis besides the high amounts of IL-1b Sertoli cells. transcripts.21 The role for the IL-1 gene family in tumorigenesis has The experiments originating from the group of Soder also been intensively studied, and Voronov et al.9 have and co-workers indicated that the so-called testicular reported that IL-1 is required for in vivo angiogenesis and IL-1 was identical (in rats) with IL-1a and further invasion of tumor cells. The high level of this cytokine analysis revealed its origin mostly to the intratubular occurring in normal conditions in the male gonad could compartment of the testis particularly connected to the then be a predisposing factor for the early development Sertoli cells.22 Our previous studies of the human of testicular cancer. High expression of IL-1 family genes gonad confirmed that the purified gametogenic cells (IL-1, IL-1RA and IL-1RII), present in a tumor micro- can express the high levels of IL-1a.23 Haugen et al.24 environment, could also participate in the secretion of additionally reported constitutive IL-1a expression in numerous pro-tumorigenic proteins, similarly to breast rat germ cells. Recent studies have focused on the role cancer cells contributing to tumor cell proliferation and of IL-1a in seminiferous epithelium as a growth factor invasion.30 (i.e. factor stimulating dividing spermatogonia). The Caspase-1 plays an important role in the apoptotic N-terminal piece of proIL-1a contains the nuclear locali- pathway of many cell types. In our experiments, the zing sequence (NLS) and is actively translocated to mRNA for this gene was (relatively) highly expressed the nucleus. Muscle cell stably transfected with either in testicular tumor samples. Overexpression of this IL-1a-(1–271) or IL-1a-(113–271) expression plasmids enzyme was previously found in numerous cancers but proliferated rapidly when compared to the non-trans- the postulated function of ICE was moderately con- fected cells and displayed a distinct morphology.25 nected only to certain malignancies.31–33 On the other Regardless of all the reports concerning the postulated hand, high ICE expression augmented the apoptotic role for IL-1a in mice, the lack of functional signaling response of prostate tumor cells to irradiation. Surpris- receptor or cytokine itself did not impair their normal ingly, it also correlated with proliferation markers such fertility and good testosterone levels.26 as cyclin-D expression.34

Genes and Immunity Interleukin-1 superfamily genes expression N Rozwadowska et al 105 Here, we report that all investigated human testis was carried out according to the standard histological samples are the source of IL-18 mRNA. The role of this staining (PAS-H). cytokine in human testicular physiology is unknown but Soder’s group results suggest its host-protecting and Tissue biopsies growth-promoting function in the rat testis.35 The origin Testicular samples were obtained from patients with of IL-18 in rat seminiferous tubules was rather restricted azoospermia (n ¼ 45) who underwent testicular oligo- to spermatocytes and spermatids.35 This phenomenon biopsies as part of their infertility diagnosis. Histopatho- seems to be observed (at least at mRNA level) also in logical analysis determined the classification of obtained human testis. tissue samples into – obstructive azoospermia (OA)and We have herein shown that the IL-1 gene system is non-obstructive azoospermia (NOA). OA tissue samples expressed throughout the male gonad and the profile were histologically normal whereas NOA biopsies of its expression can be a promising predictive marker showed a different degree of spermatogenetic impair- of spermatogenetic impairment. Spermatogenesis is a ment (SCOS – Sertoli cell only syndrome, MA – matu- complex process that is regulated by a variety of endo- ration arrest). Surgical specimens of a total 15 samples crine and auto/paracrine factors. Under pathological with testicular germ cell tumors were additionally conditions, such as testicular infection, inflammation, investigated. The tissue sections were stained with MA or tumor development, the levels of cytokines in hematoxilin/eosin for histopathological evaluation. All the male gonad can be severely affected, disrupting the tissue samples were subjected to rapid freezing in cooled sophisticated and precisely regulated gonadal home- iso-pentane and then stored in liquid nitrogen until use. ostasis. Apart from the molecular predictive factor that can be calculated on the basis of IL-1a/IL-1RA propor- Peripheral blood leukocytes isolation tion, the next question is of even greater importance. Peripheral blood samples were obtained from healthy, Can the altered proportion between these genes be male volunteers (n ¼ 10) and mononuclear leuko- reversed, and would it serve as a potentional cure for at cytes were separated on Histopaque (Sigma) gradient least some types of azoospermia? This question needs to (1.077 g/ml). be answered immediately, especially as a good medical cure for the male factor of infertility is not known. RNA extraction and cDNA synthesis Total RNA from testicular cell fractions and leukocytes was isolated as described previously following the 37 Materials and methods method of Chomczynski and Sacchi. Tissue specimens were homogenized in LN2 and total RNA was isolated The Local Ethical Committee (University Medical School using Trizol Reagent (Invitrogen, Paisley, UK). Quality of Poznan) has approved the protocols with application and quantity of isolated nucleic acids were next of human samples that were necessary to accomplish the evaluated. Target RNA (3 mg) was reversely transcribed planned project. with Super Script (Invitrogen) reverse transcriptase enzyme and oligo(dT)15 primer.

Human testicular cell preparation: fractionation and Real-time PCR purification To evaluate the level of IL-1 gene family expression, real- Human testicular tissue samples (n ¼ 10) were obtained time PCR with SYBR Green dye was applied. The iCycler from males who had undergone orchidectomy due to (BioRad, Herules, CA, USA) equipment for reaction the prostatic cancer treatment. The presence of mature monitoring was used. spermatozoa obtained from testicular tissue was accu- The PCR standards for each investigated gene con- rately checked to confirm ongoing spermatogenesis. sisted of known number of purified PCR products Principal cell fractions representing two main functional prepared as described previously.38 Briefly, the product compartments of the male gonad were obtained using was purified by using gel electrophoresis and agarose the methodology previously described by Janitz et al.36 gel nebulization with subsequent filtration (Millipore, Briefly, enzymatic digestion was performed for 15 min Billerica, MA, USA). The calculation of the number of 1 at 33 C with 0.1% collagenase type I (Sigma, St Louis, cDNA copies/ml was based on OD260/280 measurements MO, USA) dissolved in Hank’s balanced salt solution according to the following formula: (HBSS) supplemented with 1 mg/ml DNase I, in order to 6:023Â1023ÂCÂOD separate all the interstitial cell types. After first digestion, cDNA copies=ml ¼ 260 seminiferous tubules were allowed to settle down, cut MWt into 2 mm fragments and subjected to the second where C ¼ 5 Â 10À5 g/ml and MWt ¼ molecular weight of enzymatic treatment. After 15 min incubation, the super- PCR product. natant containing cells and tissue debris was filtered Then, the serial dilutions of standard were prepared through an 80 mm mesh. Almost completely dissociated (102–106 copies/ml) and stored in 201C until use. The intratubular cell aggregates and crude fractions of primer sequences, together with amplicon size for cDNA interstitial cells were next overlayered on discontinuous amplification, were listed in Table 1 (including b-actin Percoll gradient (23, 45, 68 and 90%). Pure testicular sequence as standard reference). cell fractions (intratubular and interstitial ones) were The SYBR Green real-time PCR reaction was per- collected from the formed interphases. Cells from an formed in 25 ml final volume using 96-well plates. The intratubular fraction were subjected to further purifica- reaction mixture contained 12.5 ml SyberGreenSuperMix tion applying a continuous Percoll gradient. Morpho- (Biorad), 10 pM of each primer (forward and reverse) logical identification fixed in 2.5% glutaraldehyde cells and 0.5 ml cDNA or determined copy number of PCR

Genes and Immunity Interleukin-1 superfamily genes expression N Rozwadowska et al 106 standard (102–106). All samples were run in duplicate. 12 Sultana T, Svechnikov KV, Gustafsson K, Wahlgren A, Tham The thermal protocol was as follows: 1 min 951C, follo- E, Weber G et al. Molecular identity, expression and functional wed by 50 cycles (20 s at 951C – denaturation, 20 s at 601C analysis of interleukin-1alpha and its isoforms in rat testis. – annealing and 20 s at 721C – elongation – when the Asian J Androl 2004; 6: 149–153. signal was acquired). A melting curve of PCR products 13 Svechnikov KV, Sultana T, Soder O. Age-dependent stimula- (60–961C) was performed to ensure the absence of tion of Leydig cell steroidogenesis by interleukin-1 isoforms. artifacts. To calculate normalized gene expression levels, Mol Cell Endocrinol 2001; 182: 193–201. b-actin gene expression was applied. Each sample was 14 Soder O, Sultana T, Jonsson C, Wahlgren A, Petersen C, Holst M. The interleukin-1 system in the testis. Andrologia 2000; 32: b normalized on the basis of its -actin content according 52–55. to the formula 15 Soder O, Syed V, Callard GV, Toppari J, Pollanen P, Parvinen Inv:geneÂ105 M et al. Production and secretion of an interleukin-1-like factor Ninv:gene ¼ is stage-dependent and correlates with spermatogonial DNA b-actin synthesis in the rat seminiferous epithelium. Int J Androl 1991;

where, Ninv.gene is the normalized expression of investi- 14: 223–231. gated gene, Inv.gene the number of cDNA copies for 16 Janitz M, Fiszer D, Lukaszyk A, Skorupski W, Kurpisz M. investigated gene in test sample and b-actin the number Analysis of mRNA expression for interleukin-1 genes on of cDNA copies for b-actin. human testicular cells. Immunol Lett 1995; 48: 139–143. Samples were subjected to further analysis when the 17 Svechnikov K, Stocco DM, Soder O. Interleukin-1alpha stimulates steroidogenic acute regulatory protein expression b 5 -actin expression level was greater than 10 copies per via p38 MAP kinase in immature rat Leydig cells. JMol sample. Endocrinol 2003; 30: 59–67. 18 Francavilla S, D’Abrizio P, Rucci N, Silvano G, Properzi G, Statistical analysis Straface E et al. Fas and Fas ligand expression in fetal and Statistical analysis was performed using the Kruskal– adult human testis with normal or deranged spermatogenesis. Wallis test for non-parametric analysis of variances in J Clin Endocrinol Metab 2000; 85: 2692–2700. 19 Miwa K, Asano M, Horai R, Iwakura Y, Nagata S, Suda T. order to compare among the test values obtained in Caspase 1-independent IL-1beta release and inflammation different analyzed groups of samples. induced by the apoptosis inducer Fas ligand. Nat Med 1998; 4: 1287–1292. 20 Hedger MP. Macrophages and the immune responsiveness of the testis. J Reprod Immunol 2002; 57: 19–34. Acknowledgements 21 Fiszer D, Rozwadowska N, Lukaszyk A, Slomski R, Kurpisz This work was funded by Committee for Scientific M. Quantitative mRNA analysis of IL-1 gene system in human 50 Research of Poland. testis. Am J Reprod Immunol 2003; : 389–398. 22 Gustafsson K, Sultana T, Zetterstrom CK, Setchell BP, Siddiqui A, Weber G. Production and secretion of interleukin-1alpha proteins by rat testis. Biochem Biophys Res Commun 2002; 297: References 492–497. 23 Rozwadowska N, Fiszer D, Kurpisz M. Interleukin-1 system 1 Holstein AF, Schulze W, Davidoff M. Understanding sperma- in testis – quantitative analysis. Expression of immuno- togenesis is a prerequisite for treatment. Reprod Biol Endocrinol modulatory genes in male gonad. Adv Exp Med Biol 2001; 2003; 1: 107–123. 495: 177–180. 2 Fiszer D, Ulbrecht M, Fernandez N, Johnson JP, Weiss EH, 24 Haugen TB, Landmark BF, Josefsen GM, Hansson V, Hogset Kurpisz M. Analysis of HLA class Ib gene expression in male A. The mature form of interleukin-1 alpha is constitutively gametogenic cells. Eur J Immunol 1997; 27: 1691–1695. expressed in immature male germ cells from rat. Mol Cell 3 Hedger MP, Meinhardt A. Cytokines and the immune- Endocrinol 1994; 105: R19–R23. testicular axis. J Reprod Immunol 2003; 58: 1–26. 25 Stevenson FT, Turck J, Locksley RM, Lovett DH. The N- 4 Dinarello CA. IL-1b. In: Oppenheim JJ, Feldman M (eds). Cyto- terminal propiece of interleukin 1 alpha is a transform- kine Reference. Academic Press: San Diego, 2001, pp 351–374. ing nuclear oncoprotein. Proc Natl Acad Sci USA 1997; 94: 5 Dinarello CA. IL-1a. In: Oppenheim JJ, Feldman M (eds). Cyto- 508–513. kine Reference. Academic Press: San Diego, 2001, pp 307–318. 26 Fantuzzi G. Lessons from interleukin-deficient mice: the 6 O’Neill LAJ, Dower SK. IL-1 receptor family. In: Oppenheim interleukin-1 system. Acta Physiol Scand 2001; 173: 5–9. JJ, Feldman M (eds). Cytokine Reference. Academic Press: San 27 Song GJ, Lee H, Park Y, Lee HJ, Lee YS, Seo JT et al. Expression Diego, 2001, pp 1565–1585. pattern of germ cell-specific genes in the testis of patients 7 Wilson KP, Black JA, Thomson JA, Kim EE, Griffith JP, Navia with nonobstructive azoospermia: usefulness as a molecular MA et al. Structure and mechanism of interleukin-1 beta marker to predict the presence of testicular sperm. Fertil Steril converting enzyme. Nature 1994; 370: 270–275. 2000; 73: 1104–1108. 8 Pollock AS, Turck J, Lovett DH. The prodomain of interleukin 28 Rozwadowska N, Fiszer D, Kurpisz M. Function of the 1alpha interacts with elements of the RNA processing interleukin-1 gene system in immunomodulation, apoptosis apparatus and induces apoptosis in malignant cells. FASEB J and proliferation in the male gonad. Post Hig Med Dosw 2003; 17: 203–213. (Online) 2005; 59: 56–67. 9 Voronov E, Shouval DS, Krelin Y, Cagnano E, Benharroch D, 29 Hussein MR, Abou-Deif ES, Bedaiwy MA, Said TM. Iwakura Y et al. IL-1 is required for tumor invasiveness and MustafaPhenotypic characterization of the immune and angiogenesis. Proc Natl Acad Sci USA 2003; 100: 2645–2650. mast cell infiltrates in the human testis shows normal 10 Pollanen P, Soder O, Parvinen M. Interleukin-1 alpha and abnormal spermatogenesis. Fertil Steril 2005; 83: stimulation of spermatogonial proliferation in vivo. Reprod 1447–1453. Fertil Dev 1989; 1: 85–87. 30 Pantschenko AG, Pushkar I, Anderson KH, Wang Y, Miller LJ, 11 Petersen C, Boitani C, Froysa B, Soder O. Interleukin-1 is a Kurtzman SH et al. The interleukin-1 family of cytokines and potent growth factor for immature rat sertoli cells. Mol Cell receptors in human breast cancer: implications for tumor Endocrinol 2002; 186: 37–47. progression. Int J Oncol 2003; 23: 269–284.

Genes and Immunity Interleukin-1 superfamily genes expression N Rozwadowska et al 107 31 Yang YM, Ramadani M, Huang YT. Overexpression of 35 Strand ML, Wahlgren A, Svechnikov K, Zetterstrom C, Setchell Caspase-1 in adenocarcinoma of pancreas and chronic BP, Soder O. Interleukin-18 is expressed in rat testis and may pancreatitis. World J Gastroenterol 2003; 9: 2828–2831. promote germ cell growth. Mol Cell Endocrinol 2005; 240: 64–73. 32 Ricote M, Garcia-Tunon I, Bethencourt FR, Fraile B, Paniagua 36 Janitz M, Fiszer D, Michalczak-Janitz K, Lukaszyk A, Fernandez R, Royuela M. Interleukin-1 (IL-1alpha and IL-1beta) and its N, Skorupski W et al. Analysis of mRNA for class I HLA receptors (IL-1RI, IL-1RII, and IL-1Ra) in prostate carcinoma. on human gametogenic cells. Mol Reprod Dev 1994; 38: 231–237. Cancer 2004; 100: 1388–1396. 37 Chomczynski P, Sacchi N. Single-step method of RNA 33 Tsai WC, Tsai ST, Ko JY, Jin YT, Li C, Huang W et al. The isolation by acid guanidinium thiocyanate-phenol-chloroform mRNA profile of genes in betel quid chewing oral cancer extraction. Anal Biochem 1987; 162: 156–159. patients. Oral Oncol 2004; 40: 418–426. 38 Yin JL, Shackel NA, Zekry A, McGuinness PH, Richards C, 34 Ramadani M, Gansauge F, Schlosser S, Yang Y, Beger HG, Putten KV et al. Real-time reverse transcriptase-polymerase Gansauge S. Overexpression of caspase-1 in pancreatic chain reaction (RT-PCR) for measurement of cytokine and disorders: implications for a function besides apoptosis. growth factor mRNA expression with fluorogenic probes or J Gastrointest Surg 2001; 5: 352–358. SYBR Green I. Immunol Cell Biol 2001; 79: 213–221.

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