Relationship between Caspase Activity and Apoptotic Markers in Human Sperm in Response to Hydrogen Peroxide and Progesterone

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1 Journal of Reproduction and Development, Vol. 55, No. 6, 2009, Full Paper Relationship between Caspase Activity and Apoptotic Markers in Human Sperm in Response to Hydrogen Peroxide and Progesterone Graciela M. LOZANO 1), Ignacio BEJARANO 2), Javier ESPINO 2), David GONZÁLEZ 2), Águeda ORTIZ 1), Juan F. GARCÍA 2), Ana B. RODRÍGUEZ 1) and José A. PARIENTE 1) 1) Extremadura Center of Human Assisted Reproduction, Infantile Hospital, Badajoz and 2) Department of Physiology, University of Extremadura, Badajoz, Spain Abstract. Apoptosis plays an essential role in normal spermatogenesis, but deregulations of this biological process, which is closely associated with male infertility, have been found. Whereas calcium homeostasis is a key regulator of cell survival, sustained elevation of intracellular calcium plays a role in apoptosis. The aim of this research was to determine the role of two different calcium mobilizing agents, hydrogen peroxide (H 2O 2) and the physiological agonist progesterone, on the apoptosis process of human ejaculated spermatozoa. Translocation of membrane phosphatidylserine was examined with an annexin V binding assay, DNA damage was detected by terminal deoxynucleotidyl transferase-mediated dutp nick-end labelling (TUNEL assay) and caspase-3 activity was assessed using a fluorometric assay. After incubation of spermatozoa for 1 h with either 10 μm H 2O 2 or 20 μm of progesterone, there was a significant increase in both caspase-3 activity and the percentage of annexin V-positive cells. Similarly, the TUNEL results were significantly higher 1 h after incubation with either 10 μm H 2O 2 or 20 μm of progesterone. In fact, progesterone-treated cells showed a three-fold increase (from 17.6 to 52.9%) of TUNEL-positive cells compared to untreated cells, while H 2O 2-treated cells exhibited a two-fold increase (from 17.6 to 37.9%). In sum, our results suggest that spermatozoa treated with calcium mobilizing agents, such as H 2O 2 and progesterone, seem to undergo an apoptosis process that is dependent on caspase-3 activation. Key words: Apoptosis, Caspases, Phosphatidylserine exposure, Spermatozoa, TUNEL (J. Reprod. Dev. 55: , 2009) poptosis is a distinctive form of cell death characterized by a series of morphological and biochemical changes that plays a critical role in many physiological processes such as development, tissue homeostasis, inflammatory responses and disease [1]. It also plays an essential role in gamete maturation and embryogenesis, contributing to appropriate formation of various organs and structures. In fact, many studies in animal models have demonstrated that apoptosis is the underlying mechanism of germ cell death during normal spermatogenesis [2], which has also been reported in humans [3]. On the other hand, deregulations of this biological process, which is closely associated with male infertility, have been found, and both men with azoospermia and men with severe oligozoospermia have an increased frequency of apoptotic germ cells in their testicular tissues compared with patients having normal spermatogenesis [2, 4, 5]. In addition, a certain degree of spontaneous apoptosis and relatively high rates of apoptosis have been detected using an in situ end-labelling (TUNEL) assay in testicular biopsies of infertile patients with testicular insufficiency [6, 7]. Similarly, disturbance of cell membrane symmetry with translocation of phospatidylserine residues to the outer layer of the plasma membrane has been observed in germ cells from patients with complete spermiogenesis failure [8]. Subsequently, some studies have attempted to link programmed Accepted for publication: July 21, 2009 Published online in J-STAGE: September 7, 2009 Correspondence: AB Rodríguez ( moratino@unex.es) cell death in spermatozoa to conventional seminal parameters [9 11]. In fact, a few studies concerning apoptosis in ejaculated spermatozoa have been reported in the recent years. Thus, Gorczyca et al. were among the first researchers to suggest that DNA strand breaks in abnormal spermatozoa are analogous to apoptosis in somatic cells [12]. This apoptotic feature is called DNA fragmentation, which usually indicates the presence of a late apoptotic nucleus caused by endonuclease activation [13]. In this sense, many studies have reported DNA strand breaks as important evidence for apoptosis in spermatozoa [14 17]. Furthermore, the translocation of phosphatidylserine residues to the outer layer of the plasma membrane has also been detected as an early apoptotic event in sperm cells from infertile men [15]. Likewise, morphological evidence has been provided by the existence of characteristic ultraestructural alterations in apoptosis in ejaculated sperm cells [18]. Therefore, it seems that the typical features of apoptosis in somatic cells are also indicative of apoptosis in fresh ejaculated spermatozoa [12, 15]. Aditionally, another mechanism that has been extensively studied in previous years is the oxidative stress, which is caused by overproduction of intracellular reactive oxygen species (ROS), and its relationship with sperm apoptosis and male infertility [19, 20]. In this sense, it has been demonstated that high ROS levels cause lipid peroxidation of the sperm plasma membrane, resulting in alteration of sperm function and fertilizing capacity [21]. Moreover, ROS are also known to attack DNA, inducing strand breaks and other oxidative-based damage in human spermatozoa [22, 23]. Similarly, DNA fragmentation in ejaculated spermatozoa (assessed

2 616 LOZANO et al. by TUNEL assay) has been correlated with oxidative stress (endogenous generation or exogenous stimulus) and with impaired sperm functional competence, including poor fertilization rates using in vitro fertilization [23 26]. Finally, it is well-known that one of the earliest and most consistently observed features of apoptosis is the activation of a series of cytosolic cysteine proteases called caspases [27], which cleave multiple protein substrates en masse, leading to loss of cellular structure and function and ultimately resulting in cell death [28]. In particular, caspase-3, caspase-8 and caspase-9 play relevant roles in apoptosis: caspase-9 in the mitochondrial pathway, caspase-8 in Fas/CD95 pathway, and caspase-3 an executioner caspase activated by multiple pathways more downstream [29]. On the other hand, it has been reported that calcium ion, a key regulator of cell survival, also plays a important role in cell death [30] since sustained elevation of intracellular calcium produces a calcium overload in mitochondria and may subsequently induce apoptosis by both stimulating release of apoptosis-promoting factors from the mitochondrial intermembrane space into the cytoplasm and impairing mitochondrial function [31]. Interestingly, it has been very recently reported that the agonist progesterone [32] and H 2O 2 induced an increase in the cytosolic free-calcium concentration ([Ca 2+ ] c) by depletion of intracellular calcium stores and mitochondrial apoptosis in human spermatozoa, which was strongly dependent on caspase-3 and caspase-9 activation [33]. The aim of this study was to determine the role of two different calcium mobilizing agents, hydrogen peroxide (H 2O 2) and the physiological agonist progesterone, on the apoptosis process of human ejaculated spermatozoa as well as to analyze the relationship beetwen caspase activity with both early (phosphatidylserine externalization) and late (DNA fragmentation assessed by TUNEL assay) apoptotic markers in spermatozoa from infertile patients. Materials and Methods Reagents Progesterone, H 2O 2, CHAPS, N-acetyl-Asp-Glu-Val-Asp-7- amido-4-ethylcoumarin (AC-DEVD-AMC), dithiothreitol (DTT) and RPMI-1640 medium were obtained from Sigma (Spain). N- acetyl-leu-glu-his-asp-7-amido-4-fluoromethylketone (z- LEHD-FMK) was obtained from Bachem (Germany). An in situ cell death detection kit, fluorescein, was obtained from Boehringer Mannheim (Indianapolis, IN, USA). Subjects Human semen was obtained from infertile patients at the Extremadura Centre of Assisted Reproduction (Badajoz, Spain), as approved by a local committee, the institutional review board of the University of Extremadura and the ethics committee of Infantile Hospital (Badajoz, Spain) in accordance with the Declaration of Helsinki. Semen was obtained from forty-five men (20 50 years old) being counseled for infertility and who were undergoing evaluation at our andrology laboratory. The patients suffered from primary infertility of at least 1 year in duration. Each subject was ascertained to be in good health by means of their medical histories and a clinical examination including routine laboratory test and screening. The subjects were all non smokers, were not using any medication and abstained from alcohol. Informed consent was obtained from all the participants. Sperm preparation Samples were collected by masturbation after 4 5 days of sexual abstinence and allowed to liquefy for 30 min at room temperature. Routine seminal parameters were evaluated according to the World Health Organization criteria [34]. The variables taken into consideration were ejaculated volume; spermatozoa concentration > cells/ml, in order to be able to perform multiple assays in the same sample (patients with total concentrations of motile sperm < cells/ml in a liquefied sample were excluded from the study); total spermatozoa number > cells; motility (grades a+b, where a and b indicate rapid and slow progressively motile spermatozoa, respectively), which was analized using a computer-assisted semen analysis (CASA) system; round cells (ratio of spermatozoa to leukocytes > 100:1); vitality; and morphology based on strict criteria (normal morphology > 15%) (see Table 1). Semen was washed twice in RPMI medium (250 g, 10 min), the supernatant was discarded and the pellet was resuspended in Na-HEPES solution containing 140 mm NaCl, 4.7 mm KCl, 1.1 mm CaCl 2, 10 mm glucose and 10 mm HEPES, (ph adjusted to 7.4 with NaOH). The samples were divided in two fractions. The first fraction of sperm was assayed for caspase activity after incubation with 10 μm H 2O 2 and 20 μm of progesterone for 60 min, and the second one Table 1. Characteristics of seminal parameters in fresh ejaculates from the studied infertile patients according to the World Health Organization criteria Parameters Patients values (mean ± SEM) WHO criteria (range) Age ± 6.15 Volume 2.93 ± 1.70 Concentration (cell/ml) ± Motilitiy A (%) ± Motility B (%) ± Motility A+B (%) ± Round cells (%) 1.51 ± Vitality (%) ± Normal morphology range (%) ±

3 APOPTOTIC ACTIVITY IN HUMAN SPERMATOZOA 617 was evaluated using a TUNEL assay after the same treatments. These doses and incubation time were chosen because it has been previously demonstrated by our research group that they are able to induce calcium release from intracellular stores [32, 33] and apoptosis, which is due to mitochondria being subjected to an excessive calcium load [33]. Caspase activity assay To determinate caspase activity, stimulated and resting cells were sonicated, and cell lysates were incubated with 2 ml of substrate solution (20 mm HEPES, ph 7.4; 2 mm EDTA, 0.1% CHAPS, 5 mm DTT and 8.25 μm of caspase substrate) for 1 h at 37 C, as described in elsewhere [33]. Substrate cleavage was measured by using a fluorescence spectrophotometer with an excitation wavelength of 360 nm and an emission wavelength of 460 nm. Preliminary experiments showed that caspase-3 substrate clearing was not detected in the presence of an inhibitor of caspase-3, DEVD-CMK. The activity of caspase-3 was calculated from the cleavage of its respective specific fluoregenic substrate, AC- DEVD-AMC. The data were calculated as fluorescence units per milligrams of protein and were presented as percentages above the control (untreated samples). Determination of phosphatidylserine externalization The phosphatidylserine externalization of resting and stimulated cells was determined according to a procedure published elsewhere [35]. Briefly, cells were stimulated in HEPES-buffered saline, and samples of cell suspensions (500 μl) were transferred to 500 μl of ice-cold 1% (wt/vol) glutaraldehyde in phosphate-buffered saline for 10 min. Cells were then incubated for 10 minutes with annexin V-fluorescein isothiocyanate conjugate (0.6 μ/ml) in phosphatebuffered saline that was supplemented with 0.5% (w/v) bovine serum albumin and 2 mm CaCl 2. After incubation, the cells were collected by centrifugation for 60 sec at 10,000 g and were resuspended in phosphate-buffered saline. Cell staining was measured by using a Hitachi spectrofluorimeter. Samples were excited at 488 nm, and emission was recorded at 516 nm. The data were calculated as fluorescence per milligram protein and presented as percentages above the control (untreated samples). TUNEL assay DNA damage can be detected by TdT (terminal deoxynucleotidyl transferase)-mediated dutp nick-end labelling (TUNEL) assay, as previously described [36]. Thus, DNA fragmentation was measured using an in situ cell death detection kit, fluorescein, using fluorescein-dutp as a label according to the manufacturer s instructions. Semen samples were washed, the supernatant was then discarded and the cell pellet was finally resuspended in Na-HEPES solution. Each sample was divided into three fractions. One fraction, which contained untreated spermatozoa, was considered as the control slide, and the other two fractions, which were treated with 10 μm H 2O 2 and 20 μm of progesterone for 60 min, respectively, were considered the treated slides. The three slides were air-dried for 24 h. Afterwards, the airdried slides were washed in PBS. Cells were then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated in the dark at 37 C for 1 hour in TUNEL reaction mixture containing 50 μl of the mixture of terminal deoxynucleotidyl transferase and dutp. At least 100 cells were randomly analyzed per slide in five fields. Spermatozoa were first identified in the field using phase contrast microscopy and then analyzed using fluorescence microscopy. Each cell was categorized as apoptotic (intense green nuclear fluorescence) or normal (no fluorescence). Statistical analysis Data were expressed as means ± SEM of the number of determinations. Analysis of statistical significance was performed by using the Student s t-test. One-way analysis of variance followed by Tukey s test was used for multiple comparisons. P<0.05 was considered a statistically significant difference. Results H 2O 2- and progesterone-induced DNA damage It has been previously reported that the reactive oxygen species H 2O 2 and the physiological agonist progesterone induce calcium release from intracellular stores [32, 33] in human spermatozoa, which is related to the apoptotic process. The TUNEL assay is a well-established method for detection of DNA cleavage, a relatively late apoptotic marker [36]. In addition, this technique has been widely used to determine DNA damage in human sperm [37, 38]. As shown in Figs. 1A and 1B, treatment of spermatozoa from infertile men with both 10 μm H 2O 2 and 20 μm of progesterone for 60 min leads to DNA fragmentation, which could be observed as intense green fluorescence in the nuclear region, indicating that spermatozoa might be undergoing the late stages of apoptosis. Furthermore, we found that a relatively high proportion of untreated spermatozoa (control cells) had DNA damage (17.6 ± 1.4%; Fig. 2). However, when control cells were exposed to treatment with 10 μm H 2O 2, a significantly higher percentage of TUNEL-positive cells was found after 60 min of incubation (37.9 ± 2.5% above the untreated cells; P<0.05), which represents a twofold increase above the control (see Fig. 2). Similarly, when control cells were stimulated with 20 μm of progesterone, a three-fold increase of TUNEL-positive cells took place after 60 min of incubation (from 17.6 ± 1.4 to 52.9 ± 2.4%; P<0.05) (see Fig. 2). Interestingly, the percentage of TUNEL-positive cells induced by progesterone was significantly greater (P<0.05) than those induced by H 2O 2. Phosphatidylserine externalization and caspase-3 activation in human spermatozoa To further investigate the relationship of DNA fragmentation with the mechanism of apoptosis, we checked the plasma membrane translocation of phosphatidylserine residues, which reflects a relatively early apoptotic stage. As shown in Fig. 3, treatment of spermatozoa with 10 μm H 2O 2 for 60 min induced a significant increase in phosphatidylserine externalization (22.4 ± 4.2% above the control cells; P<0.05). Moreover, when spermatozoa from

4 618 LOZANO et al. A Fig. 2. H 2O 2 and progesterone (PROG) induce DNA fragmentation in human spermatozoa. Cells were stimulated for 60 min with 10 μm H 2O 2 or 20 μm progesterone, and then DNA fragmentation (assessed by TUNEL assay) was determined as described in Materials and Methods. Values are presented as means ± SEM of seven independent experiments and expressed as percentages of TUNEL-positive cells. *P<0.05 compared to control values. P<0.05. B Fig. 1. TUNEL assay results observed under fluorescence microscopy. Panels A and B show TUNEL-positive cells after stimulation with 10 μm H 2O 2 or 20 μm progesterone (PROG) for 60 min, respectively. DNA fragmentation assessed by TUNEL assay was determined as described in Materials and Methods. A spermatozoon with DNA fragmentation shows intense green fluorescence in the nuclear region, whereas normal cells showed no green fluorescence. Fig. 3. Annexin-V binding assay results after the treatments with H 2O 2 and progesterone (PROG). Cells were stimulated for 60 minutes with 10 μm H 2O 2 or 20 μm progesterone, and then phosphatidylserine (PS) exposure was determined as described in Materials and Methods. Values are presented as means ± SEM of five to eight separate experiments and expressed as percentages above the control (untreated samples). *P<0.05 compared to control values. P<0.05. infertile patients were treated with 20 μm of progesterone for 60 min, a statistically significant increase was found in the percentage of annexin V-positive cells (12.9 ± 3.7% above the control cells; P<0.05; see Fig. 3). Although the percentage of annexin V-positive cells was significantly higher (P<0.05) in the H 2O 2-treated spermatozoa than in the prgesterone-treated spermatozoa, the results of annexin V staining demonstrated a similar proportion of cells depicting membrane phospholipid externalization in both spermatozoa subsets. Likewise, to examine the effect of H 2O 2 on caspase-3 activation, human spermatozoa were treated with 10 μm H 2O 2 for 60 min. As shown in Fig. 4, the treatment produced significant activation of caspase-3 (20.0 ± 5.3% above the control cells; P<0.05). Similarly, when we incubated the cells with 20 μm of progesterone for 60 min, caspase-3 activation was also significantly increased (33.5 ± 7.2% above the control cells; P<0.05; Fig. 4). In this case, caspase- 3 activation was significantly higher (P<0.05) in progesteronetreated spermatozoa than in H 2O 2-treated spermatozoa. It is worth noting that the spontaneous (without adding any apoptotic inducer) phosphatidylserine externalization and casapase-3 activation after 60 min were 98.3 ± 4.1 and 93.5 ± 7.2% compared with the phosphatidylserine and caspase-3 activity at the beginning of incubation (time=0; data not shown). Additionally, it appears that there was a positive correlation between late apototic markers

5 APOPTOTIC ACTIVITY IN HUMAN SPERMATOZOA 619 Fig. 4. Caspase activity induced by H 2O 2 and progesterone (PROG). Cells were stimulated for 60 min with 10 μm H 2O 2 or 20 μm progesterone, and then caspase-3 activity was estimated as described in Materials and Methods. Values are presented as means ± SEM of seven independent experiments and expressed as percentages above the control (untreated samples). *P<0.05 compared with to their respective control. P<0.05. (DNA fragmentation) and early apoptotic markers (phosphatidylserine exposure and caspase-3 activity) in all cases. Taken together, these findings clearly suggest that spermatozoa treated with calcium mobilizing agents, such as H 2O 2 and the physiological agonist progesterone, seem to be undergoing an apoptosis process. In other words, agents that are able to produce alterations in calcium homeostasis or simply sustain increases in [Ca 2+ ] c may be one of the mechanisms that lead to an apoptotic process in human spermatozoa. Discussion Apoptosis is an important process involved in normal spermatogenesis [39]. However, deregulations of this biological process involve abnormalities in the production of male gametes and male infertility. In recent years, much attention has been given to the role of apoptosis in ejaculated sperm. In fact, a previous report has shown that ejaculated sperm exhibit several apoptosis-related proteins, express caspase catalytic activity that could be elevated by some somatic cell apoptosis stimuli and display some somatic cell apoptosis markers in both fertile donors and infertile patients [40]. Moreover, it has been recently displayed that H 2O 2 and the physiological agonist progesterone increase the cytosolic free-calcium concentration due to calcium release from intracellular stores in human spermatozoa, which leads to the apoptotic process owing to calcium overloading in mitochondria [33]. In the present study, we mainly analyzed the changes that H 2O 2 or progesterone caused in mature, ejaculated sperm cells from infertile men at the membrane and nuclear levels, and we then checked the relationship between such apoptosis markers and caspase-3 activity. Thus, treatment of ejaculated sperm with either H 2O 2 or progesterone for 60 min resulted in increased DNA fragmentation (as depicted by TUNEL), confirming the results of Lopes et al. [25] and Wang et al. [41], but in contrast to those presented by Hughes et al. [42]. Therefore, some of these spermatozoa might be undergoing late stages of apoptosis. Nevertheless, even detection of fragmented DNA using a TUNEL assay may fail to discriminate among apoptosis, necrosis and autolytic cell death [43]. Our findings are indeed consistent with those obtained by Ramos and Wetzels [37], although these authors only found a significant increase of TUNEL positive cells in samples that were incubated for 24 h after damage induction with H 2O 2. The differences can be explained by the fact that the other researchers used samples from normal donors, which are probably more resistant to DNA damage than spermatozoa from infertile men. Furthermore, various levels of H 2O 2 were used in the studies. Interestingly, we have previously reported that approximately 17% of freshly ejaculated spermatozoa (untreated cells) from infertile patients show DNA fragmentation. In this sense, previous reports have demonstrated DNA fragmentation in ejaculated spermatozoa from infertile men. Sun et al. communicated that, in motile sperm cells obtained after swin-up separation from infertile men, the percentage of spermatozoa with fragmented DNA assessed by TUNEL was < 4% in the majority of the samples [24]. Moreover, Oosterhuis et al. concluded that 20% of spermatozoa showed DNA fragmentation using TUNEL in ejaculated sperm from infertile men [17]. Similarly, Barroso et al. found that nuclear damage (TUNEL assay) was present in approximately 11% of the sperm cells from infertile patients [15]. The differences in the proportion of sperm with DNA damage among all these studies may be due to the sperm samples analyzed (donors versus different patient populations), the various sperm separation methods and the different methods used to detect DNA fragmentation. On the other hand, spermatozoa have high levels of polyunsaturated fatty acids in their membranes, which are responsible for fluidity and, therefore, the motility of the sperm. The presence of free radicals causes changes in the distribution of the phosphatidylserine of the membrane, which can be measured with the Annexin V-binding assay [44]. In this way, we have established that treatment with either H 2O 2 or progesterone for 60 min also induced staining with annexin V in ejaculated sperm from infertile patients, confirming the results obtained in previous studies [37, 40]. Additionally, we previously demonstrated that translocation of phosphatidylserine induced by H 2O 2 or progesterone is an event dependent on caspase activation because H 2O 2- and progesteroneinduced phosphatidylserine exposure was abolished by pretreatment with z-lehd-fmk, a specific caspase-9 inhibitor [33]. Moreover, other investigators have shown evidence that phosphatidylserine exposure in human sperm is characteristic of apoptosis [45]. Therefore, it seems that some of these spermatozoa might be undergoing the early stages of apoptosis. Conversely, we cannot rule out the possibility that phosphatidylserine exposure is involved in capacitation of human spermatozoa and plays a physiological role in sperm, being necessary for acquisition of fertilizing ability. Caspase-dependent apoptosis is a well-characterized and ubiquitous mechanism in eukaryotes for removing senescent, defective or unnecessary somatic cells, but the roles for caspases and apoptosis in ejaculated sperm are still in question. Nonetheless, we substantiate herein that both H 2O 2 and progesterone provoke caspase-3 activation, as assessed by a fluorometric assay. In this sense, Weng

6 620 LOZANO et al. et al. showed unequivocal evidence for the presence of inactive and active caspases (including caspase-3) in ejaculated human sperm from both infertile patients and fertile donors by three distinctly different methods, including an in vitro fluorometric assay, immunoblot analysis and immunocytochemistry [11]. In spite of the fact that our findings are consistent with those obtained in previous reports showing caspase-3 activation in response to H 2O 2 and progesterone by fluorometric assay and immunoblotting [33, 40], there is still some doubts concerning the presence of inactive and active caspases in human sperm samples. In fact, de Vries et al. reported that both forms of caspase-3 are absent in mature sperm from fertile donors [46]. However, it seems that caspases (including caspase-3) are present in a restricted site for apoptosis (cytoplasmatic droplets) in spermatids and immature spermatozoa [47, 48], which contribute to poor sperm quality in samples obtained from infertile men. Furthermore, we previously found that H 2O 2- and progesteroneevoked caspase-3 activation is dependent on caspase-9 activation, as proved by pretreatment with a specific caspase-9 inhibitor, z- LEHD-FMK [33]. Curiously, we observed that treatments with both H 2O 2 and progesterone induced relatively low levels of both caspase enzyme activity and phosphatidylserine exposure compared with the untreated cells, which is consistent with the results reported in previous studies [40]. This might be because the caspase activity and membrane damage were already elevated in the infertile patients ejaculates and/or perhaps due to the fact that such sperm contain more defective machinery, making them less susceptible to the induction. In summary, our findings demonstrate that two calcium mobilizing agents, H 2O 2 and the physiological agonist progesterone, stimulate two well-known somatic cell caspase-dependent events, annexin V-binding to phosphatisylserine on the outer leaflet of the membrane, a relatively early apoptotic event, and DNA fragmentation, a relatively late apoptotic event. Additionally, our results show that either H 2O 2 or progesterone also increase caspase-3 enzyme activity. However, the roles of caspase-dependent apoptosis mechanisms in post-ejaculated human sperm clearly require further analysis. Acknowledgements This work was supported by MEC-DGI grant BFU , Merck Farma y Química Laboratories and Foundation Salud 2000 (Spain). References 1. Bellamy CO, Malcomson RD, Harrison DJ, Wyllie AH. Cell death in health and disease: the biology and regulation of apoptosis. Semin Cancer Biol 1995; 6: Kierszenbaum AL. Apoptosis during spermatogenesis: the thrill of being alive. 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