Hum. Reprod. Advance Access published November 8, 2006

Human Reproduction Page 1 of 9

doi:10.1093/humrep/del399

Kinetics of occurrence of some features of apoptosis during the cryopreservation process of bovine spermatozoa G.Martin1,2, N.Cagnon1, O.Sabido3, B.Sion4, G.Grizard4, P.Durand2 and R.Levy1,5 1 Laboratoire de Biologie de la Reproduction—GIMAP, Hôpital Nord, Saint-Etienne, 2INSERM U418—INRA UMR1245, Hôpital Debrousse, Lyon, 3Centre Commun de Cytométrie en Flux, Université Jean Monnet, Saint-Etienne Cedex 2 and 4Laboratoire de Biologie de la Reproduction—EA 975, Faculté de Médecine, Clermont-Ferrand, France 5

To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, Hôpital Nord, 42055 Saint-Etienne, France. E-mail: [email protected]

BACKGROUND: Cryopreservation/thawing of bovine spermatozoa induces a reduction in cell viability and is possibly associated with a form of programmed cell death that we previously named ‘apoptosis-like phenomenon’. METHODS: In this study, we specified, by flow cytometry, the moment of appearance of some characteristics of apoptosis during the cryopreservation process. We also studied the presence and/or activation in bovine sperm cells of specific proteins involved in somatic cell apoptosis by western blot and fluorimetry. RESULTS: A decrease of the mitochondrial membrane potential (DYm) was detectable 5 min after sperm dilution in the cryopreservation medium, caspase activation after 3 h of equilibration and an increase in plasma membrane permeability after the complete process of cryopreservation/thawing. The presence of the pro-apoptotic factor Bax, a protein that facilitates the formation of mitochondrial pores, was observed in bovine spermatozoa, but the anti-apoptotic factor Bcl-2 was not detectable. Moreover, it was observed that bovine spermatozoa contain cytochrome c and apoptosis-inducing factor (AIF), two proteins usually released from the mitochondria during the apoptotic process. Activated caspase-9, involved in the mitochondrial pathway, was detected in bovine spermatozoa but not caspase-3 and -8. CONCLUSIONS: The early features of apoptosis appear as ordered events during the cryopreservation/thawing process of bovine sperm cells. Bovine spermatozoa contain the machinery necessary to proceed to apoptosis involving especially the mitochondrial pathway. Key words: bovine spermatozoa/cryopreservation/apoptosis/mitochondria/caspases

Introduction Cryopreservation and/or thawing is known to induce many changes in mammalian spermatozoa (Medeiros et al., 2002; Martin et al., 2004). Diminished motility and membrane alterations are two of the main deleterious effects of cryopreservation. Cryopreservation definitely affects sperm viability. Apoptosis represents a universal and tightly regulated physiological process of cell death (Kerr et al., 1972; Wyllie et al., 1980). Apoptosis is a complex phenomenon that can be divided into three phases: induction, execution and degradation. Mitochondria are known to play a central role during the execution phase (Green and Reed, 1998). Following induction of apoptosis, mitochondrial pores are opened, resulting in a decrease of the mitochondrial membrane potential (ΔYm). The Bcl-2 protein family members are potent regulators of apoptosis that can influence the permeability of the outer mitochondrial membrane (Reed, 1997b). Whereas the family member Bax promotes apoptosis, Bcl-2 inhibits it. Opening mitochondrial pores leads to the release of pro-apoptotic factors from the mitochondria into the cytoplasm, like cytochrome c and the

flavoprotein apoptosis-inducing factor (AIF) (Ravagnan et al., 2002). Cytochrome c normally functions in energy production but, upon its release from mitochondria, participates in the activation of caspase-9 (Reed, 1997a). Caspases (cysteine proteases with aspartate specificity) are synthesized as inactive zymogens (pro-caspases) and are activated by cleavage during the cascade of ordered events of apoptosis (Cohen, 1997). Initiators caspase-8 and -9 activate the effector caspase-3. During the degrading phase, caspase-3 and/or AIF can induce changes at both the cell surface and in the nucleus (Cohen, 1997; Susin et al., 1999). We demonstrated previously that cryopreservation of sperm cells induces some of the main apoptotic features: ΔYm dissipation, caspase activation and membrane permeability increase; this phenomenon, which we named ‘apoptosis-like’, is supposed to involve at least some of the mechanisms that occur in the execution phase and the early degrading phase of apoptosis in somatic cells, without the nuclear manifestations of apoptosis (Martin et al., 2004). As cryopreserved bull semen is extensively used in breeding industry, a better understanding

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of the deleterious effects of cryopreservation on bovine spermatozoa is necessary. Data concerning caspase activation in bovine sperm throughout the cryopreservation process are lacking. Moreover, the possible implication of the pro- and anti-apoptotic factors Bax and Bcl-2 and of AIF has not been established. We therefore studied specific apoptotic parameters throughout the main steps of the cryopreservation/thawing process (dilution, equilibration and freezing/thawing) of bovine spermatozoa and studied the expression of additional markers before and after cryopreservation/thawing. In a first step, all the techniques were validated both on the human myeloid leukaemia cell line U937, because induction and visualization of apoptosis in this cell line are easy and well documented (Naito et al., 1997; Widmann et al., 1998; Shrivastava et al., 2000; Sordet et al., 2001), and on the Madin–Dardy bovine kidney (MDBK) cell line to assess their efficiency in the bovine species (Cristina et al., 2001; He et al., 2001; Jordan et al., 2002; Yazici et al., 2004; Li and Elsasser, 2005). Materials and methods Chemicals and antibodies The Vybrant Apoptosis Assay Kit and 3, 3′-dihexylocarbocyanine iodide [DiOC6(3)] were purchased from Molecular Probes (Montluçon, France), propidium iodide (PI), etoposide (VP-16), Peanut Agglutinin conjugated with fluorescein isothiocyanate (FITC) (PNA-FITC) from Sigma (Saint Quentin Fallavier, France), the CaspACE FITCVAD-FMK In Situ Marker from Promega (Charbonnières-les-Bains, France), Roswell Park Memorial Institute (RPMI) 1640 medium from Eurobio (Les Ulis, France) and Biociphos Medium from IMV Technologies (L’Aigle, France). For western blot analysis, rabbit polyclonal immunoglobulin G (IgG) antibodies against caspase-3 (H-277), caspase-8 (H-134), caspase-9 (H-83) and Bax (Δ 21) were purchased from Tebu-Bio (Le Perray en Yvelines, France) and the antibodies against AIF from Sigma. Mouse monoclonal IgG antibodies against Bcl-2 (C-2), cytochrome c (7H8) and secondary anti-goat IgG from donkey were from Tebu-Bio; antirabbit IgG from goat and anti-mouse IgG from rabbit were from Sigma. Semen collection and cryopreservation A total of 26 healthy bulls (Charolais) were used in this study. Semen was collected with an artificial vagina. The volume of each ejaculate was measured and sperm cell concentration assessed under light microscopy. After collection, the ejaculate was divided into two aliquots. Less than 2 h after collection, the first aliquot was analysed by flow cytometry and/or proteins were extracted for fluorimetry and/or western blot analysis. Immediately after collection, the second aliquot was diluted to 100 × 106 sperm/ml in Biociphos Medium pre-warmed at 37°C. The semen was equilibrated at 4°C for 3 h and frozen in liquid nitrogen vapour for 10 min before plunging into liquid nitrogen. The cryopreservation method was adapted from previously described protocols (Vishwanath and Shannon, 2000; van Wagtendonk-de Leeuw et al., 2000; Anzar et al., 2002). Before the various analyses, cryopreserved samples were thawed at 37°C for 1 min. Culture and apoptosis induction in U937 and MDBK cells The different apoptosis tests developed by flow cytometry were previously validated on U937 cells (Martin et al., 2004). In the present

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study, U937 and MDBK cells were used to check for the efficiency of the tests assessing caspase activities and to verify that the different antibodies used in western blotting experiments cross-reacted with the bovine proteins. U937 and MDBK cells were grown in RPMI 1640 supplemented with 10% heat-inactivated neonatal calf serum. The medium included penicillin and streptomycin. Cells were maintained at 37°C in a water-saturated atmosphere of 95% air and 5% CO2. Apoptosis was induced after 6 h of incubation in 25 μM VP-16 for U937 cells and 100 μM VP-16 for MDBK cells. Before analysis, cells were centrifuged for 5 min at 1000 × g and were washed twice with pre-warmed (37°C) phosphate-buffered saline (PBS) 1× to eliminate culture medium. Flow cytometry analysis Cell viability was determined by studying the permeability to PI (Martin et al., 2004). PI (6 μM) was added to each tube at room temperature, and flow cytometry analysis was conducted within 10 min. When PI was used with other fluorochromes, PI was added at the end of the incubation with the other probe. Accumulation of the cationic lipophilic fluorochromes DiOC6(3) in the inner membrane of mitochondria enables detection of mitochondrial membrane potential variations (Castedo et al., 2002). About 1 × 106 cells were diluted in 1 ml of PBS. DiOC6(3) was added up to a final concentration of 90 nM (Castedo et al., 2002; Martin et al., 2004). The tubes were gently mixed and incubated for 15 min at room temperature. The CaspACE FITC-VAD-FMK In Situ Marker was used to detect active caspases. The structure of the cell-permeable caspase inhibitor peptide VAD-FMK (Val-Ala-Asp-Fluoromethylketone) conjugated to FITC allows delivery of the inhibitor into the cell where it binds to activated caspases, serving as an in situ marker for apoptosis (Duval et al., 2002; Martin et al., 2004). About 0.5 × 106 cells were diluted in 0.5 ml of PBS. One microlitre of FITC-VAD-FMK (5 mM) was added. The tubes were gently mixed and incubated for 20 min at room temperature in the dark. Then, the cells were washed twice with PBS and the pellets were re-suspended in 500 μl of PBS. The Vybrant Apoptosis Assay Kit was used to detect changes in plasma membrane permeability to Yo-Pro-1 (Idziorek et al., 1995; Martin et al., 2004). About 1 × 106 cells were diluted in 1 ml of PBS. One microlitre of Yo-Pro-1 (100 μM) was added. The tubes were gently mixed and incubated for 20 min at room temperature. PNA-FITC was used to detect spermatozoa with a reacted acrosome (Idziorek et al., 1995). About 1 × 106 cells were diluted in 1 ml of PBS. Ten microlitres of PNA-FITC (1 g/ml) was added. The tubes were gently mixed and incubated for 20 min at room temperature. Flow cytometry analysis was performed using the fluorescenceactivated cell sorting (FACS) Vantage SE cell sorter (BD Biosciences, San Jose, CA, USA). Fluorochromes were excited with the 488-nm line of the Enterprise laser (Coherent, San Jose, USA). Green and red fluorescence signals were detected using FL1 and FL3 detectors, respectively, through BP 530/30 nm and BP 695/40 nm filters. All data were analysed with Cell Quest Pro 3.1 software (BD Biosciences). For DiOC6(3)/PI, caspase inhibitor/PI, Yo-Pro-1/PI and PNA-FITC/PI, 10 000 events were analysed. FL1 and FL3 fluorescence signals were recorded after logarithmic amplification. Microscopy Before examination under a conventional microscope (Leica Microsystems, Wetzlar, Germany), all samples were washed twice with PBS. Green and red fluorescence signals were respectively detected using L5 (BP 440–520 nm) and N 2-1 (BP 515–560 nm) filters. Images were captured by a CollSnapfx camera (Roper Scientific, Evry, France) using Meta Imaging 4.6.6. software (Universal Imaging, Downingtown, USA).

Apoptotic features in cryopreserved bull sperm

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotting Cell pellets were washed twice with PBS, and cell lysates were prepared in lysis buffer. This composed of protease inhibitor cocktail (Roche), 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mM glycerophosphate, 2 mM EDTA, 2 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM Na3VO4, 1 mM NaF and 1% Nonidet P-40. After centrifugation, the supernatant was collected and protein content measured by the Dc protein assay kit (Bio-Rad, Marnes-laCoquette, France). Fifty micrograms of total lysate protein was loaded and separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) using stacking and resolving gels with 4 and 12% of acrylamide, respectively. Prestained proteins with a broad range were used as molecular weight markers (BioRad). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (BioRad). After overnight incubation at 4°C in saturation buffer (8% milk, 0.05% Tween in PBS), blots were incubated with anti-AIF (1:2000), anti-cytochrome c (1:1000), anti-caspase-3 (1:300), anti-caspase-8 (1:200), anti-caspase-9 (1:200), anti-Bax (1:1000) or anti-Bcl-2 (1:200) for 90 min at room temperature. Then, the blots were incubated for 90 min at room temperature with appropriate peroxydaseconjugated antibodies: anti-mouse IgG (1:5000), anti-rabbit IgG (1:1000) or anti-goat IgG (1:8000). After washing with 0.05% Tween in PBS, blots were revealed with enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham, Orsay, France) and transferred on X-ray film. To detect small amount of the cleaved fragments of activated caspases (p10 for caspase-9, p20 for caspase-8 and p20, p17 and p11 for caspase-3), we lengthened exposure durations as compared to caspase detection.

(86:16:1, v/v) and centrifuged at 500 × g for 10 min, after which the chloroform extracts were pooled. Finally, the organic layers were evaporated under vacuum. The dried material was dissolved in a adequate volume of chloroform–methanol (2:1, v/v). Total phospholipids were determined by measuring the amount of inorganic phosphorus (Bartlett, 1959) but using HNO3 as an oxidant instead of H202. High-performance thin-layer chromatography (HPTLC) plates were used after prewashing with chloroform–methanol (1:1, v/v) followed by heating at 110°C for 10 min. Lipid extracts were applied under a flow of nitrogen on the HPTLC plate using Linomat IV (CAMAG, Muttenz, Switzerland) and separated by using the following sequential development system: development either to half final distance in methyl acetate–chloroform–n-propanol–methanol–0.25% KCl in water (25:25:25:10:9, v/v) (Vitiello and Zanetta, 1978) or to improve the separation of the cardiolipin chloroform–acetone– methanol–acetic acid–water (6:8:2:2:1, v/v) (Grizard et al., 2000) followed by full development in hexane–diethylether–acetic acid (80:20:2, v/v) to resolve the non-polar lipids. The quantification was performed after staining [10% CuSO4 w/v in 8% H3PO4 (v/v)] and charring at 160°C. For quantification, lipids were run under the same conditions. The plates were scanned and quantification was performed using density measurements with standard lipids and SIGMA scan pro software (SPSS Inc, Chicago, USA).

Caspase activity analysis Cell pellets were washed twice with PBS, and cell lysates were prepared in lysis buffer: 50 mM Hepes, 100 mM NaCl, 0.1% 3-[(3Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 10 mM dithiothreitol (DTT), 1 mM EDTA and 10% glycerol. Cell lysates were cryopreserved in liquid nitrogen and thawed at 37°C for three times and centrifuged at 16 000 × g for 10 min at 4°C. Caspase-3 and -8 activities were measured with the fluorogenic peptide substrates Acetyl-Asp-Glu-Val-Asp-7-Amino-4-methyl coumarin (Ac-DEVD-AMC) and Z-Ile-Glu-Thr-Asp-7-Amino-4-trifluoromethyl coumarin (Z-IETDAFC), respectively (Kanaoka et al., 1982; Karahashi and Amano, 2000). Twenty microlitres of supernatant was incubated with 50 μM Ac-DEVD-AMC or Z-IETD-AFC in lysis buffer at 37°C. The change in fluorescence (excitation at 360/400 nm and emission at 355/460 nm for AMC and excitation at 400/505 nm and emission at 390/510 nm for AFC) was monitored for 60 min of incubation with a Fluoroskan Ascent (Labsystems). Protein concentrations in cell lysates were measured by the Dc protein assay kit. The final results were expressed as picomoles of AFC or AMC release per microgram of protein.

Statistical methods Statistical analyses were performed using the Statistica 6.0 program (StatSoft, Tulsa, USA). Population means for fresh and cryopreserved spermatozoa were compared by t-test for dependent samples. Values are presented as mean ± standard error of the mean (SEM) and were considered statistically significant when P < 0.05. Analysis of variance (ANOVA) with subsequent post hoc Fisher tests were applied to test for potential differences between cryopreservation steps or different times of incubation in PBS after cryopreservation/thawing.

Preparation of samples for lipid analysis and high-performance thin-layer chromatography Fresh or frozen thawed spermatozoa were washed twice with PBS to remove seminal plasma, freezing medium and lysed cells. Sperm cholesterol and phospholipids were extracted by the method of Folch et al. (1957) adapted for spermatozoa. About 0.2 ml of sperm cell suspension was mixed with 8 ml of chloroform–methanol (2:1, v/v). Distilled water (1.5 ml) was added and the mixture was allowed to stand at room temperature for 1 h before centrifugation at 500 × g for 10 min. The upper layer was reextracted in 8 ml of chloroform–methanol–water

Sperm motility Fresh and cryopreserved spermatozoa were diluted to ∼25 × 106 spermatozoa/ml with PBS and observed under a light microscope on slides warmed at 37°C. Three fields per sample were evaluated with a total of ∼100 observed spermatozoa. For each sample, the percentage of motile spermatozoa was determined.

Results Changes in various apoptotic markers during the cryopreservation process The percentage of living spermatozoa (assessed by their PI permeability) was affected by cryopreservation, but no variation in the proportion of dead sperm cells was observed either after dilution in the medium used for cryopreservation (Biociphos) or after 4 h of incubation at 4°C in this medium (equilibration) (Table I). Using DiOC6(3)/PI, three cell patterns were detected: (i) necrotic cells were labelled with PI, (ii) living cells with normal ΔYm showed ‘normal’ mean green fluorescence intensity and (iii) living cells with low ΔYm, characteristic of apoptotic phenomena, showed low mean fluorescence intensity (Figure 1A). The mean values of the apoptotic ΔYmlow/PI– sperm cell population during the cryopreservation process are summarized in Table I. Cryopreservation induced a significant increase in the proportion of bovine sperm cells with low ΔYm (P < 0.001). Before cryopreservation, 4.5 ± 0.6% of cells in the ejaculate 3

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Table I. Changes in the various populations of sperm cells analysed by flow cytometry during the different steps of the cryopreservation process Test

Population

Fresh

Diluted

Equilibrated

Cryopreserved

Analysis of variance (ANOVA)

DiOC6(3)/propidium iodide (PI) (n = 11)

ΔYmnormal/PI– ΔYmlow/PI– PI+ FITC-VAD-FMK–/PI– FITC-VAD-FMK+/PI– PI+ Yo-Pro-1–/PI– Yo-Pro-1+/PI– PI+

67.8 ± 3.5a 4.5 ± 0.6a 26.7 ± 2.6a 65.1 ± 3.9a 2.6 ± 0.6a 32.3 ± 3.8a 70.8 ± 4.5a 1.9 ± 0.4a 27.3 ± 4.4a

2.5 ± 1.1b 73.1 ± 3.8b 24.4 ± 3.8a 68.6 ± 4.7a 3.4 ± 0.6a 28.0 ± 4.6a 73.8 ± 4.3a 3.4 ± 2.5a 22.8 ± 3.8a

3.0 ± 1.0b 66.9 ± 2.3b 20.1 ± 1.9a 66.1 ± 5.0a 7.3 ± 1.5b 26.6 ± 4.4a 75.9 ± 2.9a 2.5 ± 0.3a 21.6 ± 2.7a

1.5 ± 0.6b 54.4 ± 2.2c 44.1 ± 2.0b 43.2 ± 4.4b 10.2 ± 1.0c 46.6 ± 4.1b 45.0 ± 3.6b 9.3 ± 1.5b 45.7 ± 3.0b

*** *** *** ** *** ** *** *** ***

FITC-VAD-FMK/PI (n = 9) Yo-Pro-1/PI (n = 10)

DiOC6(3), 3, 3′-dihexylocarbocyanine iodide; FITC, fluorescein isothiocyanate; VAD-FMK, Val-Ala-Asp-fluoromethylketone. Means ± SEM are indicated. ANOVA results are specified for each parameter: **P < 0.01; ***P < 0.001. Different letters indicate that means between two steps are significantly different.

A 10

FL3

B

Fresh

a

103

103

102

2 FL3 10

101

101

100

c 100 101

104

b 102 103 FL1

10

103

101

b c 102 103 104 FL1

100 101

Cryopreserved

104

104

103

103

2 FL3 10

2 FL3 10

2 FL3 10

101

101

101

100

100

100

100 101

102 103 FL1

PI

104

DiOC6(3)

100 101

102 103 FL1

104

b

100

103

PI

a

2 FL3 10

100 101

Cryopreserved

Fresh

104

a

0

104

C

Fresh

104

4

PI

FITC-VAD-FMK

c

102 103 104 FL1

Cryopreserved

100 101

102 103 104 FL1

Yo-Pro-1

Figure 1. Flow cytometry study of apoptosis in fresh and cryopreserved bovine spermatozoa. Typical cytograms of 3, 3′-dihexylocarbocyanine iodide [DiOC6(3)]/propidium iodide (PI) (A), fluorescein isothiocyanate (FITC)-VAD-FMK/PI (B) and Yo-Pro-1/PI (C). (a) Dead cells are PI+, (b) living non-apoptotic cells are DiOC6(3)normal/PI–, FITC-VAD-FMK–/PI– and Yo-Pro-1–/PI–, (c) living apoptotic cells are DiOC6(3)low/PI–, FITC-VAD-FMK+/PI– and Yo-Pro+1/PI–.

showed mitochondria with low ΔYm, whereas after cryopreservation, this proportion dramatically increased to 54.4 ± 2.2%. Importantly, an increase of the ΔYmlow/PI– sperm cell population was already observed after 5 min of incubation in the cryopreservation medium Biociphos (P < 0.001). After cryopreservation/thawing,

this population decreased for the benefit of PI+ cells (both P < 0.001). It is interesting to note that this ΔYm decrease was partially reversible (Table II). When cryopreserved/thawed sperm cells were incubated for 2 h with PBS, a small but significant increase of the proportion of spermatozoa with a normal ΔYm

Table II. Changes in the various populations of sperm cells analysed by DiOC6(3)/propidium iodide (PI) after cryopreservation/thawing and dilution in phosphatebuffered saline (PBS) Test

Population

0h

2h

6h

Analysis of variance (ANOVA)

DiOC6(3)/PI (n = 10)

ΔYmnormal/PI– ΔYmlow/PI– PI+

6.5 ± 0.6a 55.5 ± 2.3 38.0 ± 2.3

13.5 ± 1.7b 48.3 ± 3.1 38.2 ± 4.0

15.1 ± 2.8b 47.2 ± 3.9 37.7 ± 3.4

** NS NS

DiOC6(3), 3, 3′-dihexylocarbocyanine iodide; NS, not significant. Means ± SEM are indicated. ANOVA results are specified for each parameter: **P < 0.01. Different letters indicate that the means between two incubation times are significantly different.

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Apoptotic features in cryopreserved bull sperm

Presence of proteins involved in the mitochondrial pathway of apoptosis To determine the possible causes and consequences of the ΔYm decrease, we studied the presence of two proteins implicated in the regulation of mitochondrial permeability (Bax and Bcl-2) and two proteins usually released from mitochondria during apoptosis (cytochrome c and AIF). The relevance of each antibody used in western blot was first validated on the human somatic cell line U937. The second somatic cell line, MDBK, was used to test the cross-reactivity of the antibodies with the bovine proteins. Using light microscopy, we noted that VP-16 induced changes in MDBK shape from healthy (adherent, with

bright cytoplasm) to apoptotic cells (less adherent, with a condensed cytoplasm) (Figure 2). The anti-apoptotic Bcl-2, pro-apoptotic Bax, Cytochrome c and AIF proteins were observed in U937 and MDBK cells (Figure 3). However, in fresh as in cryopreserved/thawed bovine spermatozoa, the presence of Bcl-2 could not be detected, whereas a small amount of the pro-apoptotic factor Bax could be observed. It is interesting to note that the antibodies raised against Bax recognized another protein of higher molecular weight than Bax in bovine cells (MDBK and spermatozoa). Cytochrome c and AIF were detected in bovine

MDBK

Figure 2. Optical microscopy of Madin–Dardy bovine kidney (MDBK) cells without (A) and with (B) induction of apoptosis by 100 μM etoposide (VP-16). Scale bar = 50 μM.

U937

was observed (P < 0.01). However, a longer incubation of 6 h did not lead to further changes of this population. Nevertheless, as expected, the proportion of motile spermatozoa was lower after cryopreservation/thawing (43.3 ± 1.4%) than in fresh samples (75.5 ± 0.5%) (n = 20; P < 0.001). During apoptosis, the decrease in ΔYm results from the opening of membrane pores of the mitochondrial membrane. The consequence is the translocation of various pro-apoptotic factors leading to caspase activation (Ravagnan et al., 2002). Using FITC-VAD-FMK/PI, three cell patterns were detected: (i) necrotic cells, labelled with PI, (ii) living cells without active caspase and (iii) apoptotic cells (i.e. living cells containing active caspases) (Figure 1B). The mean values of the apoptotic FITC-VAD-FMK+/PI– sperm cell population during the cryopreservation process are summarized in Table I. Cryopreservation of bovine spermatozoa induced a significant increase in the proportion of cells with active caspases (P < 0.001). About 2.6 ± 0.6% of the fresh PI– cells of the ejaculate showed active caspases, whereas after cryopreservation this proportion reached 10.2 ± 1.0%. Contrary to the increase of ΔYmlow/PI– sperm cell population, no significant increase of caspase activation was observed within 5 min after dilution in the cryopreservation medium. However, a significant increase of the FITC-VADFMK+/PI– population was detected in equilibrated sperm cells, after 3 h of incubation with Biociphos medium at 4°C (P < 0.01). The protease activity of the apoptotic factors contributes to the degradation phase of apoptosis, when the cytoplasmic membrane becomes slightly permeable. Apoptotic cells are permeable to Yo-Pro-1 green fluorochrome and impermeable to PI. Thus, use of combined Yo-Pro-1 and PI dyes provides a sensitive indicator for apoptosis (Idziorek et al., 1995). Using Yo-Pro-1/PI, three patterns of cell were clearly detected: (i) necrotic cells, labelled with PI, (ii) living cells, with low membrane permeability, and (iii) apoptotic cells (i.e. living cells with modified membrane) (Figure 1C). The mean values of the apoptotic Yo-Pro-1+/PI– cell population during the cryopreservation process are summarized in Table I. Cryopreservation of bovine spermatozoa induced a significant increase in the proportion of cells exhibiting high permeability to Yo-Pro-1 (P < 0.001): only 1.9 ± 0.4% of the fresh cells of the ejaculate showed permeability to Yo-Pro-1, whereas after cryopreservation this proportion reached 9.3 ± 1.5%. Plasma membrane permeability to Yo-Pro-1 was affected neither by dilution in Biociphos medium nor by a 4-h equilibration period in this medium.

Bovine spermatozoa

F

C Bcl-2 (26 kDa)

Bax (23 kDa)

Cytochrome c (12 kDa)

AIF (57 kDa) Figure 3. Western blot analysis of different proteins involved in apoptosis. U937 and Madin–Dardy bovine kidney (MDBK) cells, fresh (F) and cryopreserved (C) bovine spermatozoa were studied. All western blots are representative of three independent assays.

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G.Martin et al.

spermatozoa (Figure 3), and bovine spermatozoa appear to contain more cytochrome c than the somatic cells U937 and MDBK.

U937

-

+

Bovine MDBK spermatozoa

-

+

F

C Pro-Caspase-9 (47 kDa)

Caspase activities The activities of caspase-3 and -8 studied by the fluorogenic peptide substrates Ac-DEVD-AMC and Z-IETD-AFC, respectively, are presented in Figure 4. These caspase activities were first validated in U937 and in MDBK by using the apoptosis inducer VP-16, but both activities were lower in MDBK than in U937. Moreover, for fresh as well as for cryopreserved bovine spermatozoa, the activities of caspase-3 and -8 were very low (40% of the spermatozoa were still motile whereas ∼10% exhibited a normal ΔYm. This result could be explained by the fact that most of the energy required for sperm motility is generated by glycolysis rather than by oxidative phosphorylation (Miki et al., 2004). A consequence of the ΔYm decrease may be the release of pro-apoptotic factors from the mitochondria to the cytoplasm (Ravagnan et al., 2002). Hence, some proteins implicated in the mitochondrial pathway were studied next. The pro-apoptotic factor Bax was detected in bovine spermatozoa but not the anti-apoptotic factor Bcl-2. The low level, if any, of Bcl-2, which is known to protect mitochondria from pore formation, would explain that the mitochondria of bovine spermatozoa appear highly sensitive to the cryopreservation process, exhibiting a rapid and important ΔYm decrease already after dilution in the cryopreservation medium (Reed, 1997b). Furthermore, cytochrome c, which is involved in caspase-9 activation (Reed, 1997a), was detected in bovine spermatozoa in higher levels than in U937 or MDBK cells. This result could be explained by the fact that spermatozoa require high quantities of energy for movement and contain high number of mitochondria (Turner, 2003). Lastly, AIF, which acts in a caspase-independent manner, was also detected in bovine spermatozoa. Taking these results together, it is tempting to suggest that the decrease of ΔYm after sperm dilution led to the release of both AIF and cytochrome c and that the latter induced an activation of caspases in equilibrated sperm preparations. Experiments were therefore conducted to specify which caspases might be implicated in the apoptosis-like process induced by cryopreservation. The validity of the antibodies and of the substrates was first assessed in the control cell lines U937 and MDBK. It is interesting to note that the anti-caspase-8 and -3 antibodies recognized additional proteins of lower molecular weight than the human pro-enzyme in MDBK cells. It could be a non-specific staining or cleaved intermediates of pro-caspase-8 and -3 (Cohen, 1997). Neither activated caspase-8, involved in the membrane pathway, nor caspase-3, an effector caspase, was detected by fluorimetry or western blot in bovine spermatozoa. It would be of interest to investigate the activity of alternative caspases such as the effector caspase-7 and the caspase-10 implicated in the membrane pathway (Slee et al., 1999). By contrast, pro-caspase-9 and activated caspase-9 (p10 fragment), involved in the mitochondrial pathway, were observed in bovine spermatozoa and higher levels of activated caspase-9 could be detected after cryopreservation, supporting the implication of the mitochondrial pathway (Zou et al., 1999). The fact that in human spermatozoa caspase-3 and -8 were also activated by cryopreservation (Paasch et al., 2004a,b, 2005) highlights the species specificity of this process. 8

Peter and Linde-Forsberg (2003) found that blocking caspase activity with the anti-caspase agent zVAD-fmk had no effect on post-thaw motility or cell viability in canine spermatozoa. These authors hypothesized that either the caspase inhibitor or its concentration was not adapted. It would be of interest to test a panel of caspase inhibitors on cryopreserved/thawed bovine sperm cells. In this study, the total phospholipid and cholesterol contents of bull spermatozoa we found were similar to previous reports (Parks et al., 1987). In addition, we confirmed that PC, PE and SM are the major phospholipids present in bull spermatozoa (Hinkovska-Galcheva and Srivastava, 1993). Too small quantities of PS were recovered in sperm membranes to be analysed. This latter result should explain, at least partly, why we previously observed only small populations of bovine sperm cells reacting with annexin V (Martin et al., 2004). Cryopreservation induced an increase in the proportion of CL, a major phospholipid of the mitochondrial membrane (Hoch, 1992). Moreover, cryopreservation resulted in a decrease in the membrane-rigidifying phospholipids PE and SM and in a relative increase in the membrane-fluidizing phospholipid PC (White, 1993 and present results). Some spermatozoa can be lysed during the freezing thawing process (Anzar et al., 2002). Hence, it can be hypothesized that cells having more PC and less PE and SM would resist cryopreservation more easily. Exchange of phospholipids between the cryopreservation medium and the spermatozoa membrane could also explain the variations of spermatozoa phospholipid contents during cryopreservation (Cookson et al., 1984). Taking into consideration all these results, it is not unexpected that cryopreservation induced an increase of the permeability of bovine spermatozoa plasma membrane. A consequence of the increase of permeability of spermatozoa membrane could be an early cell death or a premature acrosomal reaction (Medeiros et al., 2002). This hypothesis is in line with our results showing an increase of the proportion of dead spermatozoa and of cells with a reacted acrosome after cryopreservation/thawing. Capacitation is characterized by various membrane changes. In boar spermatozoa, cooling to 5°C induces a capacitation-like process that is not analogous to true capacitation (Green and Watson, 2001). It would be of interest to investigate if early steps of cryopreservation induce a similar phenomenon in bovine spermatozoa. Apoptosis and plasma membrane alterations can be induced by reactive oxygen species (Carmody and Cotter, 2001). The incorporation of anti-oxidant in cryopreservation medium might be a way of investigation to improve bovine sperm cryopreservation efficiency. In summary, we have confirmed that cryopreservation induces the occurrence of some apoptotic features in bovine spermatozoa. More importantly, these apoptotic characteristics appeared as ordered events during the cryopreservation process, as a decrease of the ΔYm could be observed immediately after dilution in the cryopreservation medium, caspase activation after equilibration and changes in membrane permeability after the complete freezing/thawing process. The ΔYm decrease might be facilitated by the fact that bovine spermatozoa contain the pro-apoptotic factor Bax and only small amounts, if

Apoptotic features in cryopreserved bull sperm

any, of the anti-apoptotic factor Bcl-2. The presence of high levels of cytochrome c and AIF together with caspase-9 strongly suggests the importance of the mitochondrial pathway in this ‘apoptosis-like phenomenon’. Acknowledgements We are grateful to R. Touraine (Laboratoire de Génétique Moléculaire, Saint-Etienne, France) for his support to N. Laroche (INSERM E0366, Saint-Etienne) for his help in the microscopy study and to T. Bourlet (GIMAP, Saint-Etienne) for providing MDBK cells.

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