1 MAR&Gen (Molecular Assisted Reproduction & Genetics), Gracia 36, 18002 Granada, Spain, 2 Laboratoire dEylau, 55 Rue Saint-Didier, 75116 Paris, France, 3 Center of Reproductive Medicine, European Hospital, Via Portuense 700, 00149 Rome, Italy and 4 Department of Biochemistry and Molecular Biology, University of Granada Faculty of Sciences, Campus Universitario Fuentenueva, 18071 Granada, Spain
5 To whom correspondence should be addressed at: MAR&Gen, Gracia 36, 18002 Granada, Spain. e-mail: cmendoza{at}ugr.es
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Abstract |
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Key words: apoptosis/caspase activity/DNA fragmentation/germ cells/Sertoli cells
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Introduction |
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In most cell types, the apoptotic machinery responsible for DNA fragmentation involves a family of intracellular cystein proteases (caspases) some of which, termed signalling caspases, are activated by upstream signalling molecules and activate, in turn, executioner caspases that are directly engaged in dismantling vital cellular components (Scaffidi et al., 1998). In fact, in vitro apoptosis of human male germ cells can be prevented by caspase inhibition (Pentikainen et al., 1999
). On the other hand, caspase activity could not be detected in human adult germ cells obtained from men with normal spermatogenesis and cultured in vitro under conditions that led to massive DNA fragmentation (Tesarik et al., 2002
), suggesting the implication of an alternative, caspase-independent mechanism. No information is currently available about in vivo caspase activity in the seminiferous epithelium of men with primary testicular failure.
This study was undertaken to analyse the relationship between caspase activity and DNA fragmentation in Sertoli and germ cells from azoospermic men with different histopathological diagnoses.
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Materials and methods |
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Bilateral, multiple-site testicular biopsy was performed under general anaesthesia. Part of the biopsied tissue was immediately fixed and processed for histological examination. The rest of the tissue was partly disintegrated by stretching between two sterile microscope slides to obtain segments of seminiferous tubules. Approximately half of this tissue was used for the assisted reproduction attempt. The remaining tissue was divided into two parts that were processed for the detection of caspase activity and DNA fragmentation respectively.
Histological diagnosis
Specimens used for histological diagnosis were fixed in Bouins solution, embedded in paraffin and subjected to standard qualitative histological examination of haematoxylin- and eosin-stained sections. At least 100 seminiferous tubules were examined for each patient. The biopsies were classified according to Levin (1979) as Sertoli cell-only syndrome (SCO), early maturation arrest (here called meiotic arrest), late maturation arrest (here called post-meiotic arrest) and hypospermatogenesis. All cases showing meiotic and post-meiotic maturation arrest were characterized by complete arrest of spermatogenesis at the primary spermatocyte and round spermatid stage respectively.
In situ labelling of active caspases
Pieces of testicular tissue, partly disintegrated by previous stretching between two microscope slides, were further disintegrated mechanically by repeated aspiration into a 1 ml tuberculin syringe. This manipulation resulted in complete disintegration of seminiferous tubules to a suspension of free-floating Sertoli and germ cells as well as Sertoligerm cell clusters of different sizes. This suspension was incubated, at 30°C for 20 min, with 10 µmol/l of the fluorochrome-tagged in situ marker of active caspases FITC-VAD-FMK (CaspACETM, USA), washed from unbound probe and examined in the native state in a fluorescence microscope (Nikon, Japan) as described previously (Tesarik et al., 2002). Some intact segments of seminiferous tubules were also incubated with FITC-VAD-FMK without the final complete disintegration to evaluate the distribution of caspase activity along the tubules. Control incubations were performed, under the same conditions, with specimens preincubated (30°C, 30 min) with 50 µmol/l of Z-VAD-FMK, a non-fluorescent analogue of FITC-VAD-FMK (Tesarik et al., 2002
).
Evaluation of DNA fragmentation
After thorough mechanical disintegration, as described in the previous section, specimens were fixed with 5% glutaraldehyde in 0.05 mol/l sodium cacodylate buffer (pH 7.4) and processed for terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) using In Situ Cell Death Detection Kit with fluorescein isothiocyanate (FITC)-labelled dUTP (Roche, Italy) according to the manufacturers instructions. Nuclei with fragmented DNA (FITC-labelled) were identified in a fluorescence microscope. Dead cells, detected by supravital staining with propidium iodide (included in the commercial kit), were excluded from the analysis.
Positive and negative controls were performed by incubating specimens for 24 h at 37°C in Gamete medium (Vitrolife, Sweden) without hormone supplementation before processing for TUNEL and by omitting terminal deoxyribonucleotidyl transferase from the reaction mixture respectively.
Double staining for DNA fragmentation and phosphatidylserine externalization
This experiment was performed with only eight out of the 63 patients included in this study. All of these patients showed histological diagnosis of hypospermatogenesis. Disintegrated testicular tissue samples were incubated with FITC-labelled annexin V (Annexin-V-FLUOS; Boehringer Mannheim, Germany) as previously described (Tesarik et al., 1998a). At the end of incubation the cells were pelleted by centrifugation (200 g, 10 min), smeared on microscope slides, fixed with 5% glutaraldehyde in 0.05 mol/l sodium cacodylate buffer (pH 7.4) and processed for TUNEL using an in situ cell death detection kit containing tetramethylrhodamine isothiocyanate (TRITC)-labelled dUTP and 4',6-diamidino-2-phenylindole (DAPI) as supravital stain (Roche). This combination allowed simultaneous visualization of DNA fragmentation (red fluorescence) and externalized phosphatidylserine (classical apoptotic pathway, green fluorescence) while excluding dead cells (blue fluorescence).
Quantitative evaluation and statistical analysis
Two hundred cells were evaluated for each patient and for each type of analysis. Cells to be evaluated were chosen by viewing randomly selected microscope fields (10) for each specimen.
Percentages of cells showing caspase activity and DNA fragmentation were calculated for each patient, and data for patients with different histological diagnoses were compared by 2 and KruskalWallis tests.
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Results |
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Relationship between DNA fragmentation and phosphatidylserine externalization
All TUNEL-positive Sertoli cells showed at the same time phosphatidylserine externalization as detected by simultaneous annexin V staining (Table III). In contrast, many germ cells with fragmented DNA failed to display externalized phosphatidylserine (Figure 5). Most of Sertoli cell-associated germ cells with fragmented DNA showed at the same time phosphatidylserine externalization, which contrasted with the absence of phosphatidylserine externalization in most Sertoli cell-free germ cells with fragmented DNA (Table III).
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Discussion |
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The present data have shown that caspase activation and DNA fragmentation are frequent phenomena in germ cells from men with non-obstructive azoospermia, especially in cases of meiotic and post-meiotic maturation arrest. The incidence of caspase activation and DNA fragmentation is somewhat lower in samples from patients with hypospermatogenesis, in which some germ cells achieve the late elongated spermatid stage. Interestingly, in patients with any of these three histological diagnoses, two distinct populations of germ cells were observed. One of them consisted of germ cells that remained firmly attached to Sertoli cells, despite vigorous mechanical disintegration of the seminiferous tubules. These cells were characterized by a high incidence of both caspase activation and DNA fragmentation. The similarity of the percentages of caspase-positive and TUNEL-positive cells in this population suggests that both phenomena occur simultaneously in the same cells, although double-labelling experiments were not performed in this study.
The other germ cell population detached from Sertoli cells as a result of the same mechanical manipulation. When obtained from men with histological diagnosis of meiotic or post-meiotic maturation arrest, these germ cells showed a high incidence of DNA fragmentation, but few of them contained caspase activity. In men with histological diagnosis of hypospermatogenesis, the incidence of both DNA fragmentation and caspase activation was low in the Sertoli cell-free germ cell subpopulation.
These observations suggest the co-existence of two distinct mechanisms of germ cell demise, one caspase dependent and the other caspase independent. The tight association of caspase-positive germ cells with Sertoli cells suggests an active role of Sertoli cells in the activation of caspase-dependent apoptosis of germ cells and in the subsequent disposal of the apoptotic cells. It has been shown previously that the seminiferous tubules from men with maturation arrest show an increased number of apoptotic germ cells expressing Fas, and these cells appeared to be at least partly removed through phagocytosis by Sertoli cells (Francavilla et al., 2002). It is possible that the caspase-positive germ cells that remained tightly associated with Sertoli cells even after vigorous mechanical treatment employed in the present study were in fact cells engulfed by Sertoli cells. The detection of fragmented DNA in aggregates of vesicles and granules in Sertoli cell cytoplasm, probably representing remnants of engulfed cells, supports this interpretation, although an unambiguous distinction between phagocytosis and a simple tight contact cannot be made with the methodology used. Anyway, the results of this study show that apoptosis of a fraction of germ cells in the seminiferous tubules of men with primary testicular failure proceeds through a classical pathway including caspase activation, probably initiated through Fas, and that these cells remain in a tight contact with Sertoli cells until their complete disintegration.
On the other hand, another population of germ cells displays DNA fragmentation without presenting detectable signs of caspase activity, and these cells are found either free of Sertoli cells or only loosely associated with them. Thus, this non-classical, caspase-independent apoptosis, similar to that developing during in vitro incubation of explanted human Sertoli-germ cell clusters in media devoid of FSH (Tesarik et al., 2002), appears to be a consequence of the loss of Sertoli cell support rather than a process actively initiated and controlled by Sertoli cells. It is not known whether the germ cells undergoing this type of apoptosis also express Fas, but even so, it might not be accessible to activation without proximity of Sertoli cells which are the only known cells bearing Fas ligand within the human seminiferous tubules (Francavilla et al., 2000
). By analogy with other systems in which caspase-independent apoptosis was observed (Carmody and Cotter, 2000
; Kim et al., 2000
; Krishnamurthy et al., 2000
), DNA fragmentation in these germ cells may be induced by oxidative stress after deprivation of protective factors provided by Sertoli cells. In fact, in vitro apoptosis of human germ cells is attenuated by lowering oxygen pressure from 21 to <10% (Erkkila et al., 1999
). Germ cells undergoing this type of demise are likely to be released into the lumen of the seminiferous tubules or eliminated by macrophages. A significant increase in the number of CD68-positive macrophages and a shift of these cells from the interstitium to the tubules have been described in cases of maturation arrest as compared with normal spermatogenesis (Frungieri et al., 2002
). It appears that this type of germ cell disposal does not involve phosphatidylserine externalization, since externalized phosphatidylserine was not detected on most Sertoli cell-free meiotic germ cells with fragmented DNA observed in this study. It remains to be elucidated whether some germ cells fall off Sertoli cells as part of a random process and then, in the absence of Sertoli cell interaction, proceed down the caspase-independent pathway. Alternatively, caspase-independent demise may concern a special subpopulation of germ cells fundamentally different from other germ cells and characterized by a propensity to become dislodged. The present data showing that Sertoli cell-detached germ cells with DNA fragmentation not only lack caspase activity but also fail to undergo phosphatidylserine externalization are in favour of the latter possibility.
These observations open several questions about the actual biological efficacy of apoptosis in the diseased human seminiferous tubules as a barrier against developmental progression of damaged germ cells. Those cells that are undergoing caspase-dependent apoptosis under the tight control by Sertoli cells are likely to be efficiently excluded from further development and removed from the seminiferous tubules. In contrast, the caspase-independent apoptosis, occurring without a direct control of Sertoli cells, may allow cells with fragmented DNA to escape phagocytosis and to progress up to the late stages of spermatogenesis. In vitro studies have shown that isolated human germ cells can proceed through meiosis (Tanaka et al., 2003) and post-meiotic differentiation (Aslam and Fishel, 1998
; Tesarik et al., 1998b
,c) faster than Sertoli cell-associated germ cells under in vivo conditions, and it was hypothesized that this acceleration could be related to the loss of Sertoli cell control (Tanaka et al., 2003
). Thus, it cannot be excluded that similar events occur in vivo, leading to uncontrolled differentiation of detached germ cells and the formation of late spermatids and sperm from Sertoli cell-detached germ cells carrying DNA damage, which would otherwise be removed by Sertoli cell-controlled apoptosis. In fact, it has been observed that, in some men, many sperm with fragmented DNA escape internal testicular quality controls and subsequently appear in the ejaculate (Gorczyca et al., 1993
; Aravindan et al., 1997
; Sakkas et al., 1999
; Barroso et al., 2000
; Gandini et al., 2000
). The presence of sperm with fragmented DNA in the ejaculate may thus not be a consequence of deranged control of apoptosis in the individuals concerned but rather a sequela of reduced viability or disturbed function of Sertoli cells which are leaving a fraction of germ cells on their own without being capable of actively destroying and removing these surplus cells. This hypothetical pathogenetic mechanism, however, still remains to be substantiated by in vivo experimental studies.
Concerning the safety of ICSI and other assisted reproduction techniques using immature male germ cells for fertilization, the possible consequences of male-derived DNA damage for embryonic and fetal development also remain to be evaluated.
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Submitted on March 31, 2003; resubmitted on September 15, 2003; accepted on October 20, 2003.