Molecular Human Reproduction, Vol. 10, No. 2, pp. 123-128, 2004
© European Society of Human Reproduction and Embryology 2004
Characterization of tumour necrosis factor-
-related apoptosis-inducing ligand and its receptors in the adult human testis
1Institut National de la Santé et de la Recherche Médicale, INSERM U-407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Médecine Lyon-Sud, BP 12, F-69921 Oullins Cedex, 2Centre de Médecine et de Biologie de la Reproduction and 3Service d’Anatomo-Pathologie, Centre Hospitalier de Poissy, 10 rue du Champs Gaillard, BP 3082, 4Service d’Urologie and 5Service d’Anatomo-Pathologie, Centre Hospitalier Lyon-Sud, 69921 Oullins Cedex, France
6 To whom correspondence should be addressed. e-mail: gratarol{at}lsgrisn1.univ-lyon1.fr
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ABSTRACT |
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Tumour necrosis factor-
-related apoptosis-inducing ligand (TRAIL) is a member of the tumour necrosis factor- (TNF-) family of cytokines which is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated by immunohistochemistry in adult human testes. In addition, TRAIL and its receptors were studied in terms of protein and mRNA using western blot analysis and RT–PCR respectively. TRAIL and its receptors were immunodetected according to the different testicular cell types: TRAIL, DR5/TRAIL-R2 and DcR2/TRAIL-R4 were localized in Leydig cells, DR4/TRAIL-R1 was seen in peritubular and Sertoli cells whereas ligand and all receptors were detected in germ cells. Proteins and mRNA corresponding to TRAIL and its receptors were also identified in adult human testes. In conclusion, TRAIL and its receptors DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1/TRAIL-R3 and DcR2/TRAIL-R4 are expressed in the human testis, and are predominantly localized in different germ cell types.
Key words:
Key words: apoptosis/human testis/spermatogenesis/TNF--related apoptosis-inducing ligand
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Introduction |
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During spermatogenesis, the process of germ cell proliferation and differentiation of stem spermatogonia into mature sperm, a considerable number of germ cells, especially during the first spermatogenetic wave, die by apoptosis (Allan et al., 1992; Bartke, 1995; Billig et al., 1995). It is suggested that germ cell death during spermatogenesis is a mechanism to adjust the number of germ cells to the supporting capacity of Sertoli cells. In addition to the physiological germ cell apoptosis that occurs continuously throughout life (Bartke, 1995; Billig et al., 1995), testicular apoptosis of germ cells may be induced by a variety of conditions such as gonadotrophin (Sinha Hikim et al., 1995; Woolveridge et al., 1999) or growth factor (Yoshinaga et al., 1991) withdrawal, heat (Miraglia et al., 1993) or exposure to toxins (Richburg et al., 1996; Lee et al., 1999), ischaemia–reperfusion (Lysiak et al., 2000, 2001) and irradiation or treatment with chemotherapeutic compounds (Meistrich et al., 1993). Elevated germ cell apoptosis was also found to be involved in infertility in adult human males (Lin et al., 1997).
To date, three apoptosis-related systems have been characterized in the testis: the tumour suppressor protein p53 (Hasegawa et al., 1998; Yin et al., 1998), the Bcl-2 family members (Krajewski et al., 1994a,b; Knudson et al., 1995; Rodriguez et al., 1997), and the Fas system (French et al., 1996; Lee et al., 1997; Sugihara et al., 1997; Woolveridge et al., 1999). The Fas system belongs to the tumour necrosis factor- (TNF-) gene superfamily (Ashkenazi et al., 1998). Members of the TNF- ligand and receptor superfamily regulate many biological processes, including cell growth, differentiation, activation and apoptosis, and are mainly involved in the maintenance and function of the immune system (Smith et al., 1994). Three of the ligands, Fas ligand, TNF- and TRAIL (TNF--related apoptosis-inducing ligand), which are type II transmembrane proteins, exhibit potent cytotoxic activity, inducing apoptosis of susceptible cells (Wiley et al., 1995; Pitti et al., 1996; Nagata, 1997). These cytotoxic ligands bind to and aggregate type I transmembrane receptors with cytoplasmic ‘death domains’, Fas receptor, TNF- receptor 1 (TNFR1) and TRAIL receptors which ultimately activate a protease cascade leading to apoptosis. TRAIL also designated as APO-2 ligand has been identified recently (Wiley et al., 1995; Pitti et al., 1996). It is widely expressed in normal cells and is highly homologous to FasL. Currently, five TRAIL receptors belonging to the TNF- receptor superfamily have been identified. Two of them, DR4/TRAIL-R1 (Pan et al., 1997a; Schneider et al., 1997a) and DR5/TRAIL-R2 (Pan et al., 1997b; MacFarlane et al., 1997) contain a cytoplasmic death domain and transmit an apoptotic signal in response to TRAIL. Two other cellular TRAIL receptors, DcR1/TRAIL-R3 (Degli-Esposti et al., 1997a; Sheridan et al., 1997), a glycosylphosphatidylinositol (GPI)-linked protein without an intracellular domain, and DcR2/TRAIL-R4 (Master et al., 1997; Pan et al., 1998), containing a truncated death domain, have been identified. DcR2 binds TRAIL without activation of the apoptotic machinery and seems to antagonize the death domain containing TRAIL receptors. Finally, osteoprotegerin, a regulator of osteoclastogenesis, was reported to be a soluble receptor for TRAIL (Emery et al., 1998). The Fas system has been implicated as a major key regulator of germ cell apoptosis in the testes of the adult rat and in the human (French et al., 1996; Lee et al., 1997; Pentikäinen et al., 1999; Francavilla et al., 2000). By engaging TNFR1, TNF- activates several transduction pathways leading to regulation of the testicular expression of several genes (Besset et al., 1996; Mauduit et al., 1996; Grataroli et al., 2000); however, its role in the regulation of spermatogenesis and apoptosis is unclear. A recent study has reported that TNF- down-regulates the Fas ligand and inhibits germ cell apoptosis in the human testis (Pentikäinen et al., 1999). Recently we have reported that TRAIL and its receptors are expressed in the rat testis during normal development, and that TRAIL protein is present in the different germ cell types whereas its receptors were predominantly detected in post-meiotic germ cells (Grataroli et al., 2002). The present study reports the immunolocalization of TRAIL and its receptors and their identification in terms of protein and mRNA in human testes.
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Human tissues
Tissue sections were obtained from biopsies of five patients (34–50 years) with obstructive azoospermia (Centre de Médecine et de Biologie de la Reproduction de Poissy, France). Biopsies were performed for a testicular sperm extraction and an ICSI. Only tissues showing a normal histological structure and preserved spermatogenesis, this confirming the obstructive aetiology of azoospermia, were used in immunolocalization studies. Informed consent for participation was obtained and the project was approved by Saint Germain research and ethics committee.
We used five human testicular tissues for RT–PCR and five for western blotting. The five human testicular tissues used for RT–PCR and two for western blotting resulted from orchidectomy of patients (70–75 years) who were being treated for prostate cancer without any hormonal therapy (Pr. M.Devonec, Service d’Urologie du Centre Hospitalier Lyon-Sud, Pierre-Bénite, France) and displaying a testis histology with a spermatogenesis (Pr. F.Berger, service d’Anatomo-Pathologie du Centre Hospitalier Lyon-Sud, Pierre-Bénite, France). Informed consent was obtained from all patients. In addition, testes obtained from three deceased men (65–79 years) who were part of a body donation programme (Saint Pères University, Paris) and displaying a normal testis histology with a preserved spermatogenesis were used for western blot analysis. In each case, after collection, a piece of each testis was immediately frozen in liquid nitrogen and stored at –80°C until use.
Materials
Anti-TRAIL is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 25–281 mapping at the carboxy terminus of human TRAIL, (Santa Cruz Biotechnology, USA). Anti-DR4 is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1–130 mapping at the amino terminus of human DR4 (Santa Cruz Biotechnology). Anti-DR5 and anti-DcR1 are affinity-purified goat polyclonal antibodies raised against a peptide mapping at the amino terminus of human DR5 and DcR1 respectively (Santa Cruz Biotechnology). The anti-DcR2 is an affinity-purified rabbit polyclonal antibody raised against a peptide in an intramolecular region of human DcR2 (StressGen Biotechnologies Corp, Canada). Secondary antibody horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG and the chemiluminescence western blotting detection kit were obtained from Covalab (Lyon, France). Non-immune rabbit or goat sera and secondary antibody HRP-conjugated donkey anti-goat IgG were purchased from Santa Cruz Biotechnology.
The same antibodies were used for the immunohistochemical approach and western blot analysis. All antibodies in this study have been previously used in investigation of TRAIL/TRAIL receptors proteins in the rat testis (Grataroli et al., 2002).
Immunohistochemistry
Human testis biopsies were fixed for 24 h in Bouin’s fluid. Paraffin-embedded tissues were sectioned at 5 µm and the sections were mounted on positively charged glass slides (Superfrost plus; Menzel-Glaser, Germany). The sections were deparaffinized, hydrated, treated for 20 min at 93–98°C in citric buffer at pH 6, rinsed in deionized water (2x5 min), washed (2x5 min) in phosphate-buffered saline with 0.1% Tween 20 (PBST), then incubated overnight at 4°C with the primary antibody diluted (TRAIL: 1/100; DR4: 1/250; DR5: 1/300; DcR1: 1/500; DcR2: 1/200) in antibody diluent (Dako Corp., France). After incubation with the primary antibody, the sections were rinsed, washed (2x5 min) in PBST, then incubated for 30 min at 37°C in the presence of the secondary antibody. Secondary antibodies, rabbit or goat immunoglobulins, were attached to a peroxidase-conjugated polymer backbone or biotinylated (respectively, in Envision+ kit and LSAB+ kit; Dako). After incubation with the secondary antibody, the sections were rinsed, washed (2x5 min) in PBST, incubated for 10 min at room temperature with AEC (3-amino-9-ethylcarbazole; Dako) or sequentially with streptavidin conjugated to HRP and with DAB (3,3'-diaminobenzidine) which generated a red colour or a brown colour respectively at the site of peroxidase activity. Sections were then rinsed and washed (2x5 min) in deionized water and the nuclei were counterstained with Mayer’s haematoxylin (Dako). Finally, the sections were mounted in Faramount (Dako). In negative control slides, the primary antibody was either replaced by the antibody diluent or by normal serum control.
Western blot analysis
Whole testicular protein extracts were prepared by direct addition of 5 volumes of cold lysis buffer to the samples and mechanical homogenization of tissue. The lysis buffer consisted of 50 mmol/l Tris (pH 7.4), 250 mmol/l NaCl, 5 mmol/l EDTA, 50 mmol/l NaF, and was supplemented immediately prior to use with a cocktail of protease inhibitors [4-(2-aminoethyl)-benzenesulphonyl fluoride, pepstatine A, transepoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), bestatin, leupeptin and aprotinin] (Sigma, France) and sodium orthovanadate 1.10–3 mmol/l. Whole HeLa cell lysate used as positive control was obtained from Santa Cruz Biotechnology. The protein concentration of the tissue lysates was determined using a colorimetric Bradford method. Protein samples were resolved by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted onto a nitrocellulose membrane. The membrane was blocked by soaking in PBS–0.05% Tween 20 (PBST), 5% non-fat dried milk for 1 h and then incubated with diluted primary antibody (TRAIL, DR4, DR5, DcR1 or DcR2, dilution 1:1000) overnight at 4°C. Membranes were then washed three times (15 min) with PBST, incubated with diluted secondary antibody, HRP-conjugated goat anti-IgG rabbit (dilution 1:2000) (Covalab, France) or donkey anti-IgG goat (dilution 1:3000) (Santa Cruz Biotechnology) for 1 h and washed three times with PBST. Bound antibodies were detected using the chemiluminescence western blotting detection kit according to the manufacturer’s recommendations and Kodak Biomax films.
Isolation of RNA and RT–PCR
Total RNA were prepared using TRIzol, a monophasic solution of phenol and guanidine isothiocyanate (Life Technologies, France). This reagent is an improvement over the single-step RNA isolation method developed by Chomczynski (1993). The amount of RNA was estimated by spectrophotometry at 260 nm. Human HeLa cell total RNA, used as positive control, was obtained from Clontech Laboratories, USA.
cDNA were obtained from reverse transcription (RT) of 5 µg total RNA using random hexanucleotides as primers (50 µmol/l) in the presence of dNTP (250 µmol/l) (Gibco BRL), dithiothreitol (10 µmol/l), and Moloney murine leukaemia virus (10 IU/µl) for 1 h at 37°C. cDNA (reverse transcription mixtures, see Table I) were amplified by PCR (GenAmp PCR system 9700; Perkin–Elmer) with Taq DNA polymerase (Promega) (0.05 IU/µl), dNTP (250 µmol/l) [-33P]dATP (0.75µCi), and specific oligonucleotide primers (10 µmol/l) designed with the BLAST Search Results program from human sequence and inside separate exons to avoid any bias due to residual genomic contamination (Life Technology, France). PCR amplification was performed by first heating the mixture at 94°C for 5 min, followed by x cycles (see Table I) at 94°C for 30 s, Tm°C (melting temperature °C, see Table I) for 30 s, 72°C for 45 s, then 72°C for 7 min. PCR products were analysed on an 8% polyacrylamide gel. Dried gels were exposed to Biomax MR films (Eastman Kodak Co., USA) for 2 days at room temperature.
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Primers used in this study (Table I) have been previously used in identification of the TRAIL/TRAIL receptor mRNA in rat testes (Grataroli et al., 2002); they were generated with the BLAST Search Results program, except for DR4 (Vignati et al., 2002) and DR5 (Saleh et al., 2001). The nucleotide sequence of the PCR products, from HeLa cell mRNA extract, was confirmed by sequence analysis. Sequencing reactions were prepared using the Big dye terminator chemistry (PE Applied Biosystems, USA) and analysed on the ABI Prism 310 Genetic Analyser.
Data analysis
The different experiments were performed in duplicate or in triplicate and repeated at least three times with five independent tissue preparations. A representative experiment of each series is presented.
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Results |
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Immunolocalization of TRAIL ligand and its receptors in the adult human testis
Immunolocalization of TRAIL ligand and its receptors in the adult human testis was investigated by immunohistochemistry and with antibodies used in western blot analysis.
TRAIL ligand was detected in Leydig cells and in seminiferous tubules (Figure 1a1); TRAIL protein was seen abundantly in spermatocytes (Figure 1a2), in round and elongated spermatids (Figure 1a3), depending on the stage of the seminiferous epithelium cycle.
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In the adult human testis, DR4 receptor immunostaining was faintly detected in some myoid cells and strongly in Sertoli cells (Figure 1b1 and 1b2). Spermatocytes and round and elongated spermatids exhibited staining according to the stages of the seminiferous epithelium cycle (Figure 1b3).
In the human testis, leydig cells were weakly stained for DR5 receptor (Figure 1c1 and 1c2) whereas staining was strongest in spermatocytes and in round and elongated spermatids (Figure 1c3).
The two decoy receptors DcR1 (Figure 1d1, 1d2 and 1d3) and DcR2 (Figures 1e1, 1e2 and 1e3) were immunostained in round and elongated spermatids whereas DcR2 was also faintly detected in Leydig cells (Figure 1e1 and 1e2) and in spermatocytes (1e3).
TRAIL and receptor proteins in the adult human testis
The presence of TRAIL ligand and its receptor proteins in the adult human testis was investigated by using western blot analysis. Total proteins from adult human testes were analysed together with a whole HeLa cell lysate used as positive control. The data in Figure 2 show that protein bands corresponding to the mol. wt of DR4 receptor (
60 kDa), DR5 receptor (60 kDa) and DcR2 decoy receptor (36 kDa) were immunodetected in adult human testis as in HeLa cells. A protein band according to the mol. wt of TRAIL ligand (35 kDa) was present in both the adult human testis and in HeLa cells. As previously reported in the rat gonad (Grataroli et al., 2002), DcR1 protein from adult human testis migrated on SDS–PAGE with an apparent mol. wt (70 kDa) higher than the theoretical Mr value (30 kDa), suggesting the presence of extensive post-translational modifications and/or unusual structural motifs in DcR1 protein as in 293T cells (Schneider et al., 1997b). The polyclonal antibodies we used also detected a protein band with a mol. wt >100 kDa (anti-TRAIL and DR5), and one protein band with mol. wt <30 kDa (anti-TRAIL) (data not shown). These observations may be due to high proteinaceous material with unusual migration or degradation products respectively. The presence of monomeric, dimeric or trimeric forms may also be evoked for these protein bands.
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Identification of TRAIL and its receptor mRNA in the adult human testis
The expression of mRNA corresponding to TRAIL and its four receptors in adult human testes was investigated by using a RT–PCR approach. HeLa cell mRNA were used as positive controls. Figure 3 shows that a major transcript is detected at the expected value, 391 bp in length for TRAIL, 218 and 189 bp for DR4 and DR5 death receptors respectively, and 431 and 583 bp for DcR1 and DcR2 decoy receptors respectively.
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Discussion |
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The present study reports that TRAIL and its receptors including DR4, DR5, DcR1 and DcR2 are expressed in the adult human testis. Post-meiotic germ cells immunoexpressed both TRAIL and its four receptors and Leydig cells expressed TRAIL, DR5 and DcR2; these results were globally consistent with those obtained previously in the adult rat testis (Grataroli et al., 2002). In contrast with the rat testis, DR4 was also immunodetected in myoid and Sertoli cells in the human testis. The results obtained were consistent with those related to the identification of proteins and mRNA corresponding to TRAIL and its receptors in the human testis.
TRAIL rapidly induces apoptosis in a wide variety of tumour cell lines by interaction with the two death domain-containing receptors TRAIL-R1/DR4 (Pan et al., 1997a; Schneider et al., 1997a), TRAIL-R2/DR5 (MacFarlane et al., 1997; Pan et al., 1997b). Although DR4, DR5 and TRAIL mRNA are expressed in many tissues, most normal cells are resistant to apoptosis induction by this ligand (Ashkenazi et al., 1999; Walczak et al., 1999). As several normal tissues express DcR1 and/or DcR2, it was suggested that these receptors may contribute to the physiological resistance of certain cells to TRAIL. By contrast, several tumour cell lines express DR4 and DR5, but little or no DcR1 and DcR2 protein, suggesting that cancer cells may be more sensitive to the apoptotic signal of TRAIL. DcR1 and DcR2 were thus considered to inhibit TRAIL induced apoptosis (Degli-Esposti et al., 1997a; Master et al., 1997; Sheridan et al., 1997; Pan et al., 1998) either by acting as decoy receptors or by providing inhibitory signals such as activation of the transcription factor NF-
B (Degli-Esposti et al., 1997b) which is known to regulate several inhibitors of apoptosis. However, the death receptors DR4 and DR5 are also known to be able to activate NF-B upon ligation (Pan et al., 1997a,b; Schneider et al., 1997a). Despite several investigations, the mechanisms of apoptosis initiation by TRAIL remain unclear.
We find a co-localization of TRAIL ligand and TRAIL receptors in human testis germ cells. Recently we have reported the co-expression of TRAIL/TRAIL receptors in germ cells of the rat testis (Grataroli et al., 2002). Previous results have reported the co-expression of Fas L/Fas receptor in the same cell type (Weller et al., 1998) or TRAIL/TRAIL receptors in the same tissue (Bretz et al., 1999), and the ligand message is mostly envisioned in a physio-pathological context. Although the exact role of TRAIL ligand and its receptors in the apoptosis or in physio-pathological context in the testis is yet unknown, one could suggest the following possibilities. (i) Like the Fas system, TRAIL/TRAIL receptors will be one paracrine (Weller et al., 1998) or autocrine signalling system, by which cells expressing TRAIL ligand can initiate killing of TRAIL receptor-expressing germ cells. (ii) In the adult human testis, TRAIL and its receptor proteins are detected in post-meiotic germ cells; the possibility exists that TRAIL/TRAIL receptors are involved in post-meiotic germ cell apoptosis following, for example, hormonal (androgen) withdrawal. Such possibilities are currently under investigation in our laboratory. (iii) TRAIL may play a role unrelated to apoptosis probably related to signal transduction. In summary we report here the presence of TRAIL and its receptors in terms of protein and mRNA in the adult human testis. This is, to our knowledge, the first report of this system in human testicular tissue.
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Acknowledgements |
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This work was supported in part by INSERM and by Ligue contre le Cancer: Comité de l’Ardèche (R.G.). We thank Mr David Thoiron for English revision.
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Submitted on October 8, 2003; accepted on October 20, 2003.
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