Caspase-dependent and -independent DNA fragmentation in Sertoli and germ cells from men with primary testicular failure: relationship with histological diagnosis

Jan Tesarik1,2,5, Filippo Ubaldi3, Laura Rienzi3, Francisco Martinez4, Marcello Iacobelli3, Carmen Mendoza1,4 and Ermanno Greco3

1 MAR&Gen (Molecular Assisted Reproduction & Genetics), Gracia 36, 18002 Granada, Spain, 2 Laboratoire d’Eylau, 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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Germ cell elimination and sperm DNA fragmentation in men with primary testiculopathies involve apoptosis-related processes whose mechanisms are poorly understood. This study examines the participation of typical (caspase-dependent) and atypical (caspase-independent) pathways in these processes. METHODS: Caspase activity and DNA fragmentation were evaluated in Sertoli and germ cells from 63 men with non-obstructive azoospermia and with different histological diagnoses who were undergoing testicular biopsy for an assisted reproduction attempt. In eight of these men, phosphatidylserine externalization was also examined. RESULTS: The percentage of Sertoli cells showing caspase activity and DNA fragmentation was low and uniform in all diagnoses. In germ cells that remained tightly associated with Sertoli cells despite vigorous mechanical treatment, the incidence of both caspase activity and DNA fragmentation was high, particularly in men with maturation arrest. In Sertoli cell-free germ cells, high incidence of DNA fragmentation contrasted with low incidence of caspase activity and phosphatidylserine externalization. CONCLUSIONS: In men with primary testicular failure, apoptosis of Sertoli cells is insignificant. Some germ cells undergo caspase-dependent apoptosis, show phosphatidylserine externalization and are tightly associated with Sertoli cells. Other germ cells show caspase-independent DNA fragmentation, do not externalize phosphatidylserine and lack a tight association with Sertoli cells.

Key words: apoptosis/caspase activity/DNA fragmentation/germ cells/Sertoli cells


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Programmed cell death (apoptosis) occurs in the seminiferous epithelium of the developing mammalian testis and is required to reduce germ cells to a number that can be effectively supported by the existing population of Sertoli cells (Rodriguez et al., 1997Go; Blanco-Rodriguez, 1998Go; Tres and Kierszenbaum, 1999Go; Kierszenbaum, 2001Go). However, the existing knowledge of the apoptotic mechanisms operating in the adult human testis with normal or deranged spermatogenesis is confusing. In fact, DNA fragmentation, which is one of the essential endpoints of apoptosis, is a frequent finding in germ cells from men with maturation arrest (Tesarik et al., 1998aGo; Jurisicova et al., 1999Go) and from mice with targeted disruption of the PP1c{gamma} gene, representing an animal model for this human pathology (Jurisicova et al., 1999Go).

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., 1998Go). In fact, in vitro apoptosis of human male germ cells can be prevented by caspase inhibition (Pentikainen et al., 1999Go). 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., 2002Go), 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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
This study involves 63 men, with normal, 46,XY karyotype and with non-obstructive azoospermia, in whom testicular biopsy was performed in an attempt to obtain sperm or immature germ cells for assisted reproduction. The patients had given their informed consent for the use of part of the tissues obtained by testicular biopsy for this study.

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 Bouin’s 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 (1979Go) 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 Sertoli–germ 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., 2002Go). 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., 2002Go).

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 manufacturer’s 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., 1998aGo). 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 {chi}2 and Kruskal–Wallis tests.


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 Materials and methods
 Results
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 References
 
General observations on Sertoli–germ cell associations in treated samples
In spite of vigorous mechanical disintegration of seminiferous tubules at the beginning of sample processing, only a small proportion of the germ cells dissociated themselves from Sertoli cells and floated free in the medium, either as isolated cells or forming clusters. On the other hand, many germ cells remained intimately associated with Sertoli cells (Figure 1). Caspase activity and DNA fragmentation was analysed separately for Sertoli cells, Sertoli cell-associated germ cells and Sertoli cell-free germ cells.



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Figure 1. Germ cells (GC) remaining tightly associated with a Sertoli cell despite vigorous mechanical disintegration of the seminiferous tubules. SCN = Sertoli cell nucleus. Bar = 10 µm.

 
Caspase activity
Caspase activity was absent in most Sertoli cells, irrespective of histological diagnosis (Table I). When present, it produced only a weak, diffuse or finely granular FITC-VAD-FMK signal. In contrast, strong caspase activity was present in the Sertoli cell-associated germ cells (Figure 2). The percentage of Sertoli cell-associated germ cells with caspase activity was high in samples from men with meiotic and post-meiotic maturation arrest but low in samples from men with hypospermatogenesis (Table I). In this latter diagnosis, preparations of intact segments of seminiferous tubules showed a characteristic labelling distribution with portions showing heavy labelling alternating with those with scarce caspase activity (Figure 3). This pattern contrasted with all the other histological diagnoses in which seminiferous tubules were either uniformly labelled (meiotic or post-meiotic maturation arrest) or uniformly unlabelled (SCO) all along their length.


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Table I. Quantitative analysis of FITC-VAD-FMK binding of Sertoli and germ cells from azoospermic men with different histological diagnoses
 


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Figure 2. Caspase activity in Sertoli cell-associated germ cells. (A) Fluorescence micrograph visualizing the binding of pan-caspase marker FITC-VAD-FMK. (B) Phase contrast. Arrow indicates a Sertoli cell nucleus. Bar = 40 µm.

 


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Figure 3. Caspase activity in a whole-tubule preparation from a man with hypospermatogenesis showing alternating segments with high and low activities. (A) Fluorescence micrograph visualizing the binding of pan-caspase marker FITC-VAD-FMK. (B) Phase contrast. Bar = 100 µm.

 
Unlike the Sertoli cell-associated germ cells, the percentage of Sertoli cell-free germ cells showing caspase activity was very low, especially in samples from men with meiotic arrest and hypospermatogenesis (Table I). As compared with these two diagnoses, the percentage of Sertoli cell-free germ cells with caspase activity was higher in samples from men with post-meiotic maturation arrest, but even in these samples it was markedly lower as compared to Sertoli cell-associated germ cells (Table I). In caspase-positive Sertoli cell-free post-meiotic germ cells, the caspase activity was typically located in a restricted cytoplasmic region adjacent to the cell nucleus (Figure 4A and B).



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Figure 4. (A and B) Caspase activity in a round spermatid from a man with post-meiotic maturation arrest. (A) Fluorescence micrograph visualizing the binding of pan-caspase marker FITC-VAD-FMK. (B) Phase contrast. Arrow indicates the spermatid nucleus. Bar = 7 µm. (C and D) Detection of fragmented DNA in an aggregate of vesicles and granules in the cytoplasm of a Sertoli cell. This extranuclear fragmented DNA is likely to have originated from an apoptotic germ cell previously engulfed by the healthy Sertoli cell whose own nucleus is TUNEL negative. (C) Fluorescence micrograph visualizing TUNEL reaction. (D) Phase contrast. Arrow indicates a Sertoli cell nucleus. Bar = 25 µm.

 
DNA fragmentation
Similar to caspase activity, nuclei with fragmented DNA were found relatively rarely in Sertoli cell nuclei from all men involved in this study, irrespective of histological diagnosis (Table II). Interestingly, TUNEL-positive reaction was occasionally detected outside Sertoli cell nuclei, in aggregates of small vesicles and granules within Sertoli cell cytoplasm, while the nucleus of the Sertoli cell itself was TUNEL-negative (Figure 4C and D). This apparently cytoplasmic TUNEL staining is likely to correspond to phagosomes containing fragmented DNA originating from nuclei of apoptotic germ cells previously engulfed by healthy Sertoli cells.


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Table II. Quantitative analysis of TUNEL labelling of Sertoli and germ cell nuclei from azoospermic men with different histological diagnoses
 
The occurrence of DNA fragmentation in nuclei of germ cells depended on histological diagnosis. The percentage of TUNEL-positive nuclei was high in both Sertoli cell-associated and Sertoli cell-free germ cells from men with the histological diagnoses of meiotic and post-meiotic maturation arrest, but it was lower in samples from men with hypospermatogenesis (Table II).

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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Table III. Simultaneous visualization of TUNEL and annexin V staining in Sertoli and germ cells
 


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Figure 5. (A and B) Simultaneous visualization of DNA fragmentation (TUNEL reaction, red fluorescence) and phosphatidylserine externalization (annexin V binding, green fluorescence). Images for both fluorochromes (TRITC and FITC) are merged into a single picture. (A) Primary spermatocyte (green arrow) showing simultaneously both DNA fragmentation and phosphatidylserine externalization, contrasting with a round spermatid (long red arrow) and three late elongated spermatids (short red arrows) that only display signs of DNA fragmentation without phosphatidylserine externalization. Bar = 10 µm. (B) Late elongated spermatid showing DNA fragmentation without phosphatidylserine externalization (red arrow) and three round spermatids devoid of any labelling (green arrows). Bar = 10 µm.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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In contrast to our previous study in which acute apoptosis was induced in Sertoli cells detached from basement membrane of the seminiferous tubules and incubated in vitro in the absence of testosterone (Tesarik et al., 2002Go), the present study has shown that in vivo caspase activation and DNA fragmentation in human adult Sertoli cells are rare phenomena. The proportion of Sertoli cells with active caspases and fragmented DNA was low in all azoospermic men involved in this study, was not related to histological diagnosis and was similar to that previously reported in men with normal spermatogenesis (Tesarik et al., 2002Go). It is well known that artificial separation from the basement membrane induces apoptosis in Sertoli cells in vitro and that this effect can be partly prevented by FSH, testosterone and some other survival factors (Dirami et al., 1995Go; Tesarik et al., 2002Go). This study shows that, contrary to in vitro conditions, Sertoli cell apoptosis is not a dominant phenomenon under in vivo conditions, even in cases of severe primary testiculopathies leading to non-obstructive azoospermia. In fact, caspase activity was abundant in preparations of intact segments of seminiferous tubules from men with meiotic and post-meiotic maturation arrest, inconspicuous in tubules from men with SCO and mosaic-like in tubules from men with hypospermatogenesis.

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., 2002Go). 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., 2002Go), 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., 2000Go). By analogy with other systems in which caspase-independent apoptosis was observed (Carmody and Cotter, 2000Go; Kim et al., 2000Go; Krishnamurthy et al., 2000Go), 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., 1999Go). 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., 2002Go). 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., 2003Go) and post-meiotic differentiation (Aslam and Fishel, 1998Go; Tesarik et al., 1998bGo,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., 2003Go). 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., 1993Go; Aravindan et al., 1997Go; Sakkas et al., 1999Go; Barroso et al., 2000Go; Gandini et al., 2000Go). 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.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aravindan GR, Bjordahl J, Jost LK and Evenson DP (1997) Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Exp Cell Res 236,231–237.[CrossRef][ISI][Medline]

Aslam I and Fishel S (1998) Short-term in-vitro culture and cryopreservation of spermatogenic cells used for human in-vitro conception. Hum Reprod 13,634–638.[Abstract]

Barroso G, Morshedi M and Oehninger S (2000) Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 15,1338–1344.[Abstract/Free Full Text]

Blanco-Rodriguez J (1998) A matter of death and life: the significance of germ cell death during spermatogenesis. Int J Androl 21,236–248.[CrossRef][ISI][Medline]

Carmody RJ and Cotter TJ (2000) Oxidative stress induces caspase-independent retinal apoptosis in vitro. Cell Death Differ 7,282–291.[CrossRef][ISI][Medline]

Dirami G, Ravindranath N, Kleinman HK and Dym K (1995) Evidence that basement membrane prevents apoptosis of Sertoli cells in vitro in the absence of known regulators of Sertoli cell function. Endocrinology 136,4439–4447.[Abstract]

Erkkila K, Pentikainen V, Wikstrom M, Parvinen M and Dunkel L (1999) Partial oxygen pressure and mitochondrial permeability transition affect germ cell apoptosis in the human testis. J Clin Endocrinol Metab 84,4253–4259.[Abstract/Free Full Text]

Francavilla S, D’Abrizio P, Rucci N, Silvano G, Properzi G, Straface E, Cordeschi G, Necozione S, Gnessi L, Arizzi M et al (2000) Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab 85,2692–2700.[Abstract/Free Full Text]

Francavilla S, D’Abrizio P, Cordeschi G, Pelliccione F, Necozione S, Ulisse S, Properzi G, Francavilla F (2002) Fas expression correlates with human germ cell degeneration in meiotic and post-meiotic arrest of spermatogenesis. Mol Hum Reprod 8,213–220.[Abstract/Free Full Text]

Frungieri MB, Calandra RS, Lustig L, Meineke V, Kohn FM, Vogt HJ and Mayerhofer A (2002) Number, distribution pattern, and identification of macrophages in the testes of infertile men. Fertil Steril 78,298–306.[CrossRef][ISI][Medline]

Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G, Verlengia C, Pondero F and Lenzi A (2000) Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 15,830–839.[Abstract/Free Full Text]

Gorczyca W, Traganos F, Jesionowska H and Darzynkiewicz Z (1993) Presence of DNA strand breaks and increased sensitivity of DNA in situ to denaturation in abnormal human sperm cells: analogy to apoptosis in somatic cells. Exp Cell Res 207,202–205.[CrossRef][ISI][Medline]

Jurisicova A, Lopes S, Meriano J, Oppedisano L, Casper RF and Varmuza S (1999) DNA damage in round spermatids of mice with targeted disruption of the Pp1c{gamma} gene and in testicular biopsies of patients with non-obstructive azoospermia. Mol Hum Reprod 5,323–330.[Abstract/Free Full Text]

Kierszenbaum AL (2001) Apoptosis during spermatogenesis: the thrill of being alive. Mol Reprod Dev 58, 1–3.[CrossRef][ISI][Medline]

Kim DK, Cho ES and Um HD (2000) Caspase-dependent and -independent events in apoptosis induced by hydrogen peroxide. Exp Cell Res 257,82–88.[CrossRef][ISI][Medline]

Krishnamurthy PK, Mays JL, Bijur GN and Johnson GV (2000) Transient oxidative stress in SH-SY5Y human neuroblastoma cells results in caspase dependent and independent cell death and tau proteolysis. J Neurosci Res 61,515–523.[CrossRef][ISI][Medline]

Levin HS (1979) Testicular biopsy in the study of male infertility: its current usefulness, histologic techniques, and prospects for the future. Hum Pathol 10,569–584.[ISI][Medline]

Pentikainen V, Erkkila K and Dunkel L (1999) Fas regulates germ cell apoptosis in the human testis in vitro. Am J Physiol 276,E310–E316.[ISI][Medline]

Rodriguez I, Ody C, Araki K, Garcia I and Vassalli P (1997) An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. EMBO J 16,2262–2270.[Abstract/Free Full Text]

Sakkas D, Mariethoz E and St John JC (1999) Abnormal sperm parameters in humans are indicative of an abortive apoptotic mechanism linked to the Fas-mediated pathway. Exp Cell Res 15,350–355.

Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH and Peter ME (1998) Two CD95 (APO-1/Fas) signalling pathways. EMBO J 17,1675–1678.[Abstract/Free Full Text]

Tanaka A, Nagayoshi M, Awata S, Mawatari Y, Tanaka I and Kusunoki H (2003) Completion of meiosis in human primary spermatocytes through in vitro coculture with Vero cells. Fertil Steril 79,795–801.[CrossRef][ISI][Medline]

Tesarik J, Greco E, Cohen-Bacrie P and Mendoza C (1998a) Germ cell apoptosis in men with complete and incomplete spermiogenesis failure. Mol Hum Reprod 4,757–762.[Abstract]

Tesarik J, Greco E, Rienzi L, Ubaldi F, Guido M, Cohen-Bacrie P and Mendoza C (1998b) Differentiation of spermatogenic cells during in-vitro culture of testicular biopsy samples from patients with obstructive azoospermia: effect of recombinant follicle stimulating hormone. Hum Reprod 13,2772–2781.[Abstract/Free Full Text]

Tesarik J, Guido M, Mendoza C and Greco E (1998c) Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J Clin Endocrinol Metab 83,4467–4473.[Abstract/Free Full Text]

Tesarik J, Martinez F, Rienzi L, Iacobelli M, Ubaldi F, Mendoza C and Greco E (2002) In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium. Hum Reprod 17,1811–1819.[Abstract/Free Full Text]

Tres LL and Kierszenbaum AL (1999) Cell death patterns of the rat spermatogonial cell progeny induced by Sertoli cell geometric changes and Fas (CD95) agonist. Dev Dyn 214,361–371.[CrossRef][ISI][Medline]

Submitted on March 31, 2003; resubmitted on September 15, 2003; accepted on October 20, 2003.