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Biology of Reproduction 66, 1707-1715 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Expression of Tumor Necrosis Factor-{alpha}-Related Apoptosis-Inducing Ligand and Its Receptors in Rat Testis During Development

Renée Grataroli1,a, David Vindrieuxa, Alain Gougeona, and Mohamed Benahmeda

a Institut National de la Santé et de la Recherche Médicale, INSERM U-407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Médecine Lyon-Sud, F-69921 Oullins Cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-{alpha} family of cytokines that 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 in the rat testis during development. TRAIL and its receptors were immunolocalized to the different testicular cell types. TRAIL and its receptors were also identified in the rat testis in terms of protein and mRNA. Our immunohistochemical studies indicate that TRAIL, DR5/TRAIL-R2, and DcR2-TRAIL-R4 are detected in Leydig cells, whereas ligand and all receptors are localized in germ cells. TRAIL was permanently immunodetected in germ cells from the fetal stage to adulthood, whereas its receptors were immunolocalized exclusively in postmeiotic germ cells. The expression of TRAIL and receptor mRNAs was consistent with the immunodetection of TRAIL and receptor proteins. Indeed, TRAIL ligand mRNA was also identified in the rat testis from the fetal stage to adulthood. The mRNAs of the death receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2, were weakly detected during the perinatal period and increased from the pubertal stage to adulthood. The mRNAs of the decoy receptors, DcR1 and DcR2, were present in the rat testis at all ages studied, but the DcR2/TRAIL-R4 mRNa level was higher from the pubertal period to adulthood. Together, the present findings demonstrate that 1) TRAIL and its receptors are expressed in the testis during normal development, and 2) TRAIL protein is present in the different germ cell types, whereas its receptors were predominantly detected in the postmeiotic germ cells.

apoptosis, cytokines, male reproductive tract, polypeptide receptors, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a dynamic and complex process of male germ cell proliferation and differentiation from stem spermatogonia to spermatozoa [1]. In the mammalian testis, germ cells clonally expand, but this expansion could be excessive, and requires mechanisms to match the number of germ cells with the supporting capacity of Sertoli cells. The occurrence of spontaneous germ cell selective apoptosis in the mammalian testis has been recognized for many years [2–4]. Testicular germ cell apoptosis occurs normally and continuously throughout life [2, 4]. A large number of spermatocytes undergo apoptosis in the testis of 4-wk-old rats, and spermatogonia have been reported as the main cell type undergoing apoptosis in the adult rat [3, 4]. In addition, massive testicular germ cell loss is known to result from toxicant exposure [5, 6], depletion of growth factors [7], alterations of hormonal (testosterone or FSH) support [8, 9], heat exposure [10], and radiation or treatment with chemotherapeutic compounds [11]. In both spontaneous and injury-associated germ cell death, apoptosis appears to be the major pathway of cell death [12, 13].

To date, three apoptosis-related systems have been characterized in the testis: the tumor suppressor protein p53, the Bcl-2 family members, and the Fas system. Some results indicate that p53 plays a role in activation of germ cell death, because mice deficient in p53 develop normally and are fertile, but they exhibit a decreased or delayed onset of germ cell apoptosis after radiation exposure [14] or experimental cryptorchidism [15]. In rodent testes, the expression of Bax, Bad, Bcl-xl, and Bcl-2 has been demonstrated [16–19]. Studies using knockout and transgenic mice suggest that members of the Bcl-2 family are important regulators of apoptosis in the testis. Bax-knockout mice are infertile as a result of the accumulation of premeiotic germ cells and an absence of mature haploid sperm [18], and adult transgenic mice expressing high levels of Bcl-xl or Bcl-2 show a highly abnormal spermatogenesis accompanied by infertility [19].

An alternative and often more rapid form of programmed cell death is mediated by the "death ligands and death receptors pathway" [20], including Fas/Fas ligand, tumor necrosis factor-{alpha}/tumor necrosis factor-{alpha} receptor 1 (TNF/TNFR1) and receptors for tumor necrosis factor-{alpha}-related apoptosis-inducing ligand/tumor necrosis factor-{alpha}-related apoptosis-inducing ligand receptors (TRAIL/TRAIL receptors). In the testis, the Fas system has been identified as one paracrine signaling system by which Sertoli cells, expressing Fas-L, can initiate killing of Fas-expressing germ cells [21, 22]. However, it has been reported that Fas-L is also expressed in the germ cells and that Fas is expressed in Sertoli cells [23, 24]. The role of the Fas system has been assessed in various injury models by inducing a massive germ cell death [6, 24]. Fas and TNF-{alpha} systems belong to the tumor necrosis factor-{alpha} gene superfamily [20]. The presence of the TNF/TNFR1 system was characterized in the testis. By engaging TNFR1, TNF-{alpha} activates several transduction pathways leading to a regulation of the testicular expression of several genes, including inhibin, aromatase, insulin-like growth factor binding protein 3, and lactate dehydrogenase A [25–30]. Unlike Fas-L, however, TNF-{alpha} rarely triggers apoptosis unless protein synthesis is blocked, which suggests the preexistence of cellular factors that can suppress the apoptotic stimulus generated by TNF-{alpha}.

TRAIL, also designated as APO-2 ligand [31, 32], is another member of the tumor necrosis factor family, and is capable of inducing apoptosis in several cell lines. TRAIL is widely expressed in normal cells and is highly homologous to Fas-L. Currently, five TRAIL receptors belonging to the TNF-{alpha} receptor superfamily have been identified. Two of them, TRAIL-R1 (DR4) [33, 34] and TRAIL-R2 (DR5) [35, 36] contain a cytoplasmic death domain and transmit an apoptotic signal in response to TRAIL. Two other cellular TRAIL receptors, TRAIL-R3 (DcR1) [37, 38], a glycosylphosphatidylinositol (GPI)-linked protein without an intracellular domain; and TRAIL-R4 (DcR2) [39, 40], which contains 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 [41].

Although Fas/Fas ligand and TNF/TNFR1 systems have been identified in the testis during normal and pathological development, testicular TRAIL and its receptors expression in the gonad remains to be investigated. The present study aimed to 1) immunolocalize TRAIL and its receptors to the different cell types of the rat testis during its normal development and 2) identify TRAIL and its receptor mRNAs and proteins in the gonad.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Sprague-Dawley rats were purchased from IFFA CREDDO (Lyon, France). Virgin females were individually housed in controlled conditions of lighting (12L:12D), temperature (20°C–24°C), and humidity (40%–70%), and were given free access to water and feed. Females were mated on a one-to-one basis with male animals of the same strain and the same supplier. Day 0 of gestation was assigned when a vaginal plug was observed. At the appropriate times, rats were killed by asphyxiation in CO2. Five to nine rats of each age during fetal to postnatal development were killed for each tissue preparation.

All studies on animals were approved by the INSERM Animal Care Committee.

Materials

The 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 (sc-7877; Santa Cruz Biotechnology, Santa Cruz, CA). The anti-DR4 is a rabbit polyclonal antibody raised against a recombinant protein corresponding to amino acids 1–130, which map at the amino terminus of human DR4 (sc-7863; Santa Cruz Biotechnology). Anti-DR5 and anti-DcR1 are affinity-purified goat polyclonal antibodies raised against a peptide that maps at the amino terminus of human DR5 and DcR1, respectively (sc-7192 and sc-7193; Santa Cruz Biotechnology). The anti-DcR2 is an affinity-purified rabbit polyclonal antibody raised against a peptide in an intracellular region of human DcR2 (AAP-371 from StressGen Biotechnologies Corp, Victoria, BC, Canada). According to the manufacturers, anti-TRAIL, anti-DR4, and anti-DcR2 cross-react with rat proteins. DcR1 control peptide, nonimmune rabbit or goat serums, and secondary antibody horseradish peroxidase conjugate, and donkey anti-goat immunoglobulin (Ig) G were purchased from Santa Cruz Biotechnology. Secondary antibody horseradish peroxidase conjugated goat anti-rabbit IgG and the chemiluminescence Western blotting detection kit were obtained from Covalab (Lyon, France).

The same antibodies were used for the immunohistochemical approach and Western blot analysis.

Immunohistochemistry

The freshly dissected testes were fixed for 24 h in Bouin fluid. Paraffin-embedded testes were sectioned at 5 µm, and were mounted on positively charged glass slides (superfrost plus; Menzel-Glaser, Braunscheig, Germany). The sections were deparaffinized, hydrated, treated for 20 min at 93–98°C in citric buffer at pH 6, rinsed twice for 5 min each in osmosed water, washed twice for 5 min in PBS with 0.1% Tween 20, then incubated overnight at 4°C with the primary antibody dilution (TRAIL, 1:100; DR4, 1:500; DR5, 1:250; DcR1, 1:1500; and DcR2, 1:250) in antibody diluent (DAKO Corp, Trappes, France). After incubation with the primary antibody, the sections were rinsed, washed twice for 5 min each in PBS-Tween 20, 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 an Envision+ kit and an LSAB+ kit; both from DAKO). After incubation with the secondary antibody, the sections were rinsed, washed twice for 5 min in PBS-Tween 20, incubated for 10 min at room temperature with 3-amino-9-ethylcarbazole (AEC; DAKO) or sequentially with streptavidin conjugated to horseradish peroxidase and with 3,3'-diaminobenzidine (DAB), which generated a red color or a brown color, respectively, at the site of peroxidase activity. The sections were then rinsed again, washed twice for 5 min in osmosed water, and then the nuclei were counterstained with Mayer hematoxylin (DAKO). Finally, 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.

Isolation of RNA and Semiquantitative Reverse Transcriptase-Polymerase Chain Reaction

Total RNAs were prepared using TRIzol, a monophasic solution of phenol and guanidine isothiocyanate (Life Technologies, Eragny, France). This reagent is an improvement over the single-step RNA isolation method developed by Chomczynski and Sacchi [42]. 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, Palo Alto, CA (catalog number 64021-1).

Complementary DNAs were obtained from reverse transcription (RT) of 3 µg of total RNA using random hexanucleotides as primers (50 µM) in the presence of dNTPs (250 µM; Gibco BRL, Cergy Pontoise, France), dithiothreitol (10 µM), and Moloney murine leukemia virus (10 U/µl) for 1 h at 37°C. Complementary DNAs (X µl RT mixture; Table 1) were amplified by polymerase chain reaction (PCR; GenAmp PCR system 9700, Perkin-Elmer, Courtaboeuf, France) with Taq DNA polymerase (Promega, Charbonnière, France; 0.05 U/µl), dNTP (250 µM), [{alpha}-33P]dATP (0.75 µCi), and specific oligonucleotide primers (10 µM) designed from a human sequence and inside separate exons to avoid any bias due to residual genomic contamination (Life Technologies). PCR amplification was performed by first heating the mixture at 94°C for 5 min, followed by X cycles (see Table 1) at 94°C for 30 sec, Tm°C (melting temperature, °C) for 30 sec, 72°C for 45 sec, then 72°C for 7 min. Complementary DNAs were simultaneously amplified in the same conditions with specific or with ß-actin oligonucleotide primers in separate tubes. PCR products were analyzed on an 8% polyacrylamide gel. Dried gels were exposed to Biomax MR films (Eastman Kodak Company, Rochester, NY) for 2 days at room temperature. Intensities of autoradiographic bands were estimated by densitometric scanning using the BioImage (Cheshire, U.K.) scanner. The data were expressed as TRAIL (or DR4, DR5, DcR1, Dcr2)/ß-actin mRNA ratio.


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  		TABLE 1. PCR amplification

Western Blot Analysis

Whole testis protein extracts were prepared by direct addition of 5 volumes of cold lysis buffer to the samples. Lysis buffer consisted of 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, and 50 mM NaF, and was supplemented immediately before use with a cocktail of protease inhibitors (Sigma, Isle d'Abeau, France). Whole Hela cell lysate (sc2200) 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% SDS-PAGE and electroblotted onto a nitrocellulose membrane. The membrane was blocked by soaking in PBS, 0.05% Tween 20, and 5% nonfat dried milk for 1 h; incubated with diluted primary antibody (dilution 1:1000) overnight at 4°C; washed three times (15 min each) with PBS and 0.05% Tween 20; (PBST) incubated with diluted secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG; dilution 1:2000; Covalab, Lyon, France) or donkey anti-goat IgG (dilution 1:3000; Santa Cruz Biotechnology) for 1 h, and washed three times with PBST. Bound antibodies were detected using the chemiluminescence Western blot detection kit according to the manufacturer's recommendations and Kodak Biomax films (Eastman Kodak).

Data Analysis

Unless mentioned in the figure legends, the different experiments were performed in triplicate and were repeated at least three times with independent tissue preparations. A representative experiment of each series is presented.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunolocalization of TRAIL Ligand During Rat Testis Development

TRAIL ligand was detected in the testis at all different ages (Fig. 1). At the fetal period (19 days postcoitum; Fig. 1a) and perinatal period (3 days, Fig. 1b), a strong immunostaining was observed in both gonocytes and fetal Leydig cells. At the prepubertal period (15 days, Fig. 1c) Leydig cells, spermatogonia, and young spermatocytes showed an intense TRAIL staining. At the pubertal period (21 days, Fig. 1d), TRAIL ligand was present in Leydig cells, in spermatogonia, and spermatocytes. At the young adult period (30 days, Fig. 1e), TRAIL staining was slightly observed in spermatogonia but it was most abundantly detected in Leydig cells and in round spermatids. At adulthood (>60 days, Fig. 1f), TRAIL ligand was still present in the interstitial Leydig cells, and in the seminiferous tubules, staining for TRAIL was most present in round and elongated spermatids, depending on the stage of the seminiferous epithelium cycle.



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  		FIG. 1. Developmental immunoexpression of TRAIL ligand in the rat testis. Testis sections were incubated with antibody raised against TRAIL (dilution 1:100). a) Postcoital Day 19 testis, b) Postnatal Day 3, c) Postnatal Day 15 testis, d) Postnatal Day 21 testis, e) Postnatal Day 30 testis, f) Postnatal Day 60 testis, (f, lower right corner) Postnatal Day 60 testis, negative control obtained with buffer instead primary antibody. Magnification x480 (a, b, c, d), x240 (e, f), and x80 (f, lower right corner)

Immunolocalization of TRAIL Receptors During Testis Development

At the perinatal and prebubertal periods, we detected no TRAIL receptors in the rat testis.

DR4 receptor immunostaining was not detected in 30-day-old rat testis (Fig. 2a). It was observed in 60-day-old rat testis, in elongated spermatids, and in residual bodies (Fig. 2b).



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  		FIG. 2. Developmental immunoexpression of DR4 and DR5 receptors in rat testis. Testis sections were incubated with antibody raised against DR4 (dilution 1:500), or with antibody raised against DR5 (dilution 1:250). DR4: a) Postnatal Day 30 testis, b) Postnatal Day 60 testis, and (b, lower right corner) Postnatal Day 60 testis negative control obtained with buffer instead of primary antibody. DR5: c) Postnatal Day 15 testis, d) Postnatal Day 21 testis, e) Postnatal Day 30 testis, f) Postnatal Day 60 testis, and f, lower right corner) Postnatal Day 60 testis negative control obtained with buffer instead primary antibody. Magnification x240 (a, c, d, e), x120 (b, f), x80 (b, lower right corner), and x40 (f, lower right corner)

DR5 receptor immunostaining was not present in the testis at the prepubertal period (Fig. 2c). At the pubertal period (Fig. 2d), Leydig cells and spermatocytes were weakly stained for DR5 receptor. In a 30-day-old rat (Fig. 2e), DR5 receptor was immunodetected in Leydig cells and in round spermatids. In adults (Fig. 2f), DR5 staining was preponderant in Leydig cells and in round and elongated spermatids, according to the stages of the seminiferous epithelium cycle.

DcR1 receptor was not immunodetected before the pubertal period (Fig. 3a). It was weakly immunoexpressed in round spermatids in 30-day-old rat testis (Fig. 3b). Round and elongated germ cells from the adult testis (>60 days) exhibited a strong staining pattern, according to the stages of the seminiferous epithelium cycle (Fig. 3c).



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  		FIG. 3. Developmental immunoexpression of DcR1 and DcR2 receptors in rat testis. Testis sections were incubated with antibody raised against DcR1 (dilution 1:1500), or with antibody raised against DcR2 (dilution 1:250). DcR1: a) Postnatal Day 21 testis, b) Postnatal Day 30 testis, c) Postnatal Day 60 testis, and c, lower right corner) Postnatal Day 60 testis negative control obtained with buffer instead primary antibody. DcR2: d) Postnatal Day 21 testis, e) Postnatal Day 30 testis, f) Postnatal 60 testis and f, lower right corner), Postnatal Day 60 testis negative control obtained with buffer instead primary antibody. Magnification x240 (a, b, d, e), x120 (c, f), x80 (c and e, lower right corner)

DcR2 staining began to be detected at the pubertal period, Leydig cells were faintly stained (Fig. 3d), whereas primary spermatocytes in various stages of degeneration were intensely stained. In 30-day-old rat testis (Fig. 3e), Leydig cells, spermatocytes, and round spermatids were strongly stained. In the adult, DcR2 immunostaining was preponderant in Leydig cells and in round and elongated spermatids (Fig. 3f).

TRAIL and Receptors Proteins in the Adult Testis

The presence of TRAIL ligand and its receptors in the male gonad was investigated by using Western blot analysis and antibodies that were used in immunohistochemistry studies. Total proteins from adult rat testis were analyzed together with a whole Hela cell lysate used as positive control (33–40). The data in Figure 4a show that proteic bands corresponding to theoretical molecular weight of DR4 receptor (about 60 kDa), DR5 receptor (about 60 kDa), and DcR2 decoy receptor (about 36 kDa) were immunodetected in adult rat testis as in Hela cells. A proteic band according to a theoretical molecular weight of TRAIL ligand (about 35 kDa) was present in both the adult rat testis and in Hela cells (the same result was obtained with brain extract; data not shown). The DcR1 decoy receptor was detected as a doublet of about 55 and 70 kDa in the adult testis, but as a single band of 70 kDa in Hela cells. The identity of the two bands detected in the testis was assessed by coincubation of the antibody, anti-DcR1 alone, and with the corresponding peptide, which resulted in a slight decrease of a 70-kDa band in Hela cells and testis extract, and in the disappearance of 55-kDa band in testis (Fig. 4b).



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  		FIG. 4. Identification of TRAIL ligand and receptor proteins in adult rat testis. Sixty micrograms of proteins from adult rat testis (T) (>60 days) were used to perform Western blot analysis, and 50 µg of whole Hela cell lysate (H) were used as positive control. Individual membranes were incubated with primary antibody a) against TRAIL (dilution 1: 800), DR4 (dilution 1:1000), DR5 (dilution 1:1000) DcR2 (dilution 1:1000) or b) with antibody against DcR1 (dilution 1:1000) or with antibody against DcR1 plus corresponding peptide

In all cases, the polyclonal antibodies we used also detected a proteic band with a molecular weight larger than 100 kDa, and for some of them, one or two proteic bands with molecular weights less than 30 kDa (anti-TRAIL, DR4, and DcR2; data not shown). These observations may be due to high proteic material with unusual migration or degradation products, respectively. The presence of monomeric, dimeric, or trimeric forms may be also evoked for these proteic bands.

Identification of TRAIL and Its Receptors mRNAs in the Testis During Postnatal Development

The expression of mRNAs corresponding to TRAIL and its four receptors in adult rat testis was investigated by using a RT-PCR approach. Hela cell mRNAs were used as positive controls. Figure 5 shows that a major transcript is detected at an expected value, 391 base pairs (bps) in length for TRAIL; 677 bp and 606 bp for DR4 and DR5 receptors, respectively; and 431 bp and 583 bp for DcR1 and DcR2 decoy receptors, respectively.



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  		FIG. 5. Identification of TRAIL ligand and receptor mRNAs in adult rat testis. Complementary DNAs obtained from RT of total RNAs from adult rat testis (T) and total RNAs of Hela cells (H) used as positive control were amplified by PCR with corresponding primers (Table 1, and Materials and Methods)

For the developmental study in postnatal rat testis (Fig. 6), ß-actin was used as the standard (see Materials and Methods). The data in Figure 6a show that TRAIL mRNA was detected in the rat testis at all the different ages studied. Whereas death receptors DR4 (Fig. 6b) and DR5 (Fig. 6c) mRNAs were weakly detected at 3 and 15 days, their levels increased from the pubertal period to adulthood. Messenger RNAs for decoy receptors, DcR1 (Fig. 6d) and DcR2 (Fig. 6e), were evidenced at all ages studied, but DcR2 mRNA levels were increased from the pubertal period (21 days) to adulthood (>60 days).



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  		FIG. 6. Developmental expression of TRAIL ligand and receptor mRNAs in postnatal rat testis. RNAs from testes of rats at Postnatal Days 3–60 were analyzed by semiquantitative RT-PCR for the evaluation of TRAIL and its mRNA receptor levels. Hela cells were used as positive control (data not shown) and ß-actin as a standard. In the upper panel, representative autoradiographs are shown, whereas in the lower panel, the mean of 3 triplicate determinations of the ligand or receptor/ß-actin ratio is presented. a) TRAIL-ligand, b) DR4-receptor, c) DR5-receptor, d) DcR1-receptor, and e) DcR2-receptor


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present paper, we have shown that TRAIL and its receptors, including DR4, DR5, DcR1, and DcR2, were expressed in the testis during development. Although Leydig cells immunoexpressed TRAIL, DR5, and DcR2, germ cells expressed both TRAIL and its four receptors. During development, TRAIL was permanently immunoexpressed in germ cells and Leydig cells from fetal stages to adulthood, whereas its receptors were immunodetected exclusively in postmeiotic cells.

These results, which are related to the immunoexpression of TRAIL and its receptors, were globally consistent with those obtained with the analysis of their mRNAs and proteins in the testis, except for DcR1. Indeed, under denaturing and reducing conditions, DcR1 protein from adult testis migrated as two protein bands with an apparent molecular weight that was higher than the theoretical Mr value (about 30 kDa), suggesting the presence of extensive posttranslational modifications, or unusual structural motifs (or both) in the testicular DcR1 protein. However, this unusual migration of the receptor DcR1 on SDS-PAGE gels as the different forms of DcR1 have been also previously reported in 293T cells [43].

There are at least two possible explanations for this unusual migration. First is the presence in the protein of five repeat units of 15 amino acids, rich in Thr, Ala, Pro, and Glu residues (TAPE repeats migrate approximately four times slower than predicted), which are located in the extracellular domain, close to the membrane interaction site. Second, the cDNA sequence of DcR1 predicts several sites for posttranslational modifications, including signal peptide cleavage, N-and O-glycosylation and GPI addition. Furthermore, these observations may well explain the apparent discrepancy between the immunodetection of DcR1 protein from mRNAs. Indeed, DcR1 protein was present at Postnatal Day 30, whereas DcR1 mRNAs were present at all the different periods of testis development. Moreover, and because mRNA expression is more sensitive than immunodetection, the presence of DcR1 on Sertoli cells cannot be excluded, because the 3-day-old rat testis contains mainly Sertoli cells and only few germ cells.

Systematic screenings of adult human tissues by Northern blot analysis has shown that mRNA receptors are expressed in most human tissues, including the testis [33, 35, 38, 40]. TRAIL induces apoptosis by interacting with two death domain-containing receptors, TRAIL-R1/DR4 [33, 34], and TRAIL-R2/DR5 [35, 36]. Both of these receptors are type I transmembrane proteins, and their mRNAs were found to be widely expressed in normal tissues. Normal tissues are believed to be protected from apoptosis by the two decoy receptors, TRAIL-R3/DcR1 [13, 14] and TRAIL-R4/DcR2 [15, 16], which do not signal apoptosis induction. TRAIL-R3 and TRAIL-R4 were thus considered to inhibit TRAIL-induced apoptosis either by acting as decoy receptors or by providing inhibitory signals such as activation of the transcription factor, NF-{kappa}B [44], which is known to regulate several inhibitors of apoptosis. However, the death receptors, TRAIL-R1 and TRAIL-R2, are also known to be able to activate NF-{kappa}B upon ligation [33–35].

One intracellular C-terminal portion of the death receptors, termed the "death domain," is a protein-protein interaction module that is responsible for the transduction of cytotoxic death signals. The receptor death domain binds to death domains in downstream molecules such as TRADD (for TNFR1-associated death domain), FADD (for Fas-associated death domain), TRAF2 (for TNF-R associated factor 2), RIP (for receptor interacting protein), and RAIDD (for RIP-associated Ich1/CED3 homologous protein with a death domain), which act as adapter molecules that interact with the ICE-like caspases, which are the effectors of the death program [20]. Despite several investigations, the mechanisms of apoptosis initiation by TRAIL remain unclear. It is unknown whether DR4 and DR5 may assemble a signaling complex that reassembles that of TNFR1 with the adapters FADD, TRADD, and RIP rather than of Fas (with FADD as a unique adapter only). Although some studies have, alternatively, implicated the physiological adapters FADD, TRADD, and RIP, other studies did not. Also, there are conflicting reports about the involvement of endogenous caspase-8 or caspase-10 [20, 45]. Although DR4 and DR5 transcripts and TRAIL mRNA are expressed in many tissues, most normal cells are resistant to apoptosis induction by this ligand [46, 47]. Because several normal tissues express DcR1, DcR2, or both, it was suggested that these receptors may contribute to the physiological resistance of certain cells to TRAIL. By contrast, several tumor 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.

We have reported the presence of TRAIL/TRAIL receptors in germs cells. Previous results have reported the coexpression of Fas L/Fas receptor in the same cell type [48] or TRAIL/TRAIL receptors in the same tissue [49], and the ligand message is mostly envisioned in a physiopathological context. Our present results show that TRAIL ligand is present in the testis from fetal to adult ages in Leydig cells and in germ cells, whereas by contrast, the TRAIL-receptor proteins are detected only in postmeiotic germ cells. The immunodetection of TRAIL but not DR4 and DR5 receptors in fetal and in immature (premeiotic) testicular germ cells presents different possibilities. First, the TRAIL receptors are present as ligands from the fetus to adulthood but are not detectable by the immunohistochemical approach used here. Second, TRAIL ligand can bind to one or more receptors that have not yet been identified. Third, TRAIL might play a role unrelated to apoptosis, and therefore its action is not mediated by the two TRAIL receptors. Although the precise role of TRAIL ligand and its receptors in apoptosis in the testis is not yet known, one could suggest some possibilities: 1) as a Fas system, it will be one paracrine or autocrine signaling system by which cells expressing TRAIL ligand can initiate killing of TRAIL receptor-expressing germ cells; 2) because TRAIL protein is present in all the germ cell types, whereas its receptor proteins are exclusively detected in postmeiotic germ cells, one could speculate that spermatogonial apoptosis (for example, following chemotherapy or irradiation) probably does not involve TRAIL and its receptors. By contrast, in postmeiotic germ cell apoptosis following, for example, hormonal (androgen) withdrawal, the possibility exists that TRAIL and its receptors are at play. Such possibilities are currently being investigated in our laboratory.


   FOOTNOTES
 
First decision: 19 July 2001.

1 Correspondence: R. Grataroli, INSERM U-407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Medecine Lyon Sud, B.P. 12, 165 Chemin du Grand Revoyet, F-69921 Oullins Cedex, France. FAX: 33 4 78 86 31 16; gratarol{at}lsgrisn1.univ-lyon1.fr Back

Accepted: December 26, 2001.

Received: June 20, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Griswold MD, Morales C, Sylvester SR. Molecular biology of the Sertoli cell. Oxf Rev Reprod Biol 1998; 10:124-161
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