Human Reproduction Vol.22, No.2 pp. 380388, 2007 doi:10.1093/humrep/del399Advance Access publication November 8, 2006.

380 The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: journals.permissions@oxfordjournals.org

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.Levy1,51Laboratoire de Biologie de la ReproductionGIMAP, Hpital Nord, Saint-Etienne, 2INSERM U418INRA UMR1245, Hpital Debrousse, Lyon, 3Centre Commun de Cytomtrie en Flux, Universit Jean Monnet, Saint-Etienne Cedex 2 and 4Laboratoire de Biologie de la ReproductionEA 975, Facult de Mdecine, Clermont-Ferrand, France5To whom correspondence should be addressed at: Laboratoire de Biologie de la Reproduction, Hpital Nord, 42055 Saint-Etienne, France. E-mail: rachel.levy@chu-st-etienne.fr

BACKGROUND: Cryopreservation/thawing of bovine spermatozoa induces a reduction in cell viability and is possi-bly 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 ofapoptosis during the cryopreservation process. We also studied the presence and/or activation in bovine sperm cellsof specific proteins involved in somatic cell apoptosis by western blot and fluorimetry. RESULTS: A decrease of themitochondrial 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 completeprocess of cryopreservation/thawing. The presence of the pro-apoptotic factor Bax, a protein that facilitates the for-mation of mitochondrial pores, was observed in bovine spermatozoa, but the anti-apoptotic factor Bcl-2 was notdetectable. 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, involvedin the mitochondrial pathway, was detected in bovine spermatozoa but not caspase-3 and -8. CONCLUSIONS: Theearly features of apoptosis appear as ordered events during the cryopreservation/thawing process of bovine spermcells. Bovine spermatozoa contain the machinery necessary to proceed to apoptosis involving especially the mitochon-drial pathway.

Key words: bovine spermatozoa/cryopreservation/apoptosis/mitochondria/caspases

IntroductionCryopreservation and/or thawing is known to induce manychanges in mammalian spermatozoa (Medeiros et al., 2002;Martin et al., 2004). Diminished motility and membrane altera-tions are two of the main deleterious effects of cryopreserva-tion. Cryopreservation definitely affects sperm viability.

Apoptosis represents a universal and tightly regulated physi-ological process of cell death (Kerr et al., 1972; Wyllie et al.,1980). Apoptosis is a complex phenomenon that can bedivided into three phases: induction, execution and degrada-tion. Mitochondria are known to play a central role during theexecution phase (Green and Reed, 1998). Following inductionof apoptosis, mitochondrial pores are opened, resulting in adecrease of the mitochondrial membrane potential (Ym). TheBcl-2 protein family members are potent regulators of apopto-sis that can influence the permeability of the outer mitochon-drial membrane (Reed, 1997b). Whereas the family memberBax promotes apoptosis, Bcl-2 inhibits it. Opening mitochon-drial pores leads to the release of pro-apoptotic factors from themitochondria into the cytoplasm, like cytochrome c and the

flavoprotein apoptosis-inducing factor (AIF) (Ravagnan et al.,2002). Cytochrome c normally functions in energy productionbut, upon its release from mitochondria, participates in the acti-vation of caspase-9 (Reed, 1997a). Caspases (cysteine pro-teases with aspartate specificity) are synthesized as inactivezymogens (pro-caspases) and are activated by cleavage duringthe cascade of ordered events of apoptosis (Cohen, 1997). Initi-ators caspase-8 and -9 activate the effector caspase-3. Duringthe degrading phase, caspase-3 and/or AIF can induce changesat both the cell surface and in the nucleus (Cohen, 1997; Susinet al., 1999).

We demonstrated previously that cryopreservation of spermcells induces some of the main apoptotic features: Ym dissi-pation, caspase activation and membrane permeabilityincrease; this phenomenon, which we named apoptosis-like,is supposed to involve at least some of the mechanisms thatoccur in the execution phase and the early degrading phase ofapoptosis in somatic cells, without the nuclear manifestationsof apoptosis (Martin et al., 2004). As cryopreserved bull semenis extensively used in breeding industry, a better understanding

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of the deleterious effects of cryopreservation on bovine sper-matozoa is necessary. Data concerning caspase activation inbovine sperm throughout the cryopreservation process arelacking. Moreover, the possible implication of the pro- andanti-apoptotic factors Bax and Bcl-2 and of AIF has not beenestablished.

We therefore studied specific apoptotic parameters through-out the main steps of the cryopreservation/thawing process(dilution, equilibration and freezing/thawing) of bovine sper-matozoa and studied the expression of additional markersbefore and after cryopreservation/thawing. In a first step, allthe techniques were validated both on the human myeloid leu-kaemia cell line U937, because induction and visualization ofapoptosis in this cell line are easy and well documented (Naitoet al., 1997; Widmann et al., 1998; Shrivastava et al., 2000;Sordet et al., 2001), and on the MadinDardy bovine kidney(MDBK) cell line to assess their efficiency in the bovine spe-cies (Cristina et al., 2001; He et al., 2001; Jordan et al., 2002;Yazici et al., 2004; Li and Elsasser, 2005).

Materials and methodsChemicals and antibodiesThe Vybrant Apoptosis Assay Kit and 3, 3-dihexylocarbocyanineiodide [DiOC6(3)] were purchased from Molecular Probes (Montluon,France), propidium iodide (PI), etoposide (VP-16), Peanut Agglutininconjugated with fluorescein isothiocyanate (FITC) (PNA-FITC) fromSigma (Saint Quentin Fallavier, France), the CaspACE FITC-VAD-FMK In Situ Marker from Promega (Charbonnires-les-Bains,France), Roswell Park Memorial Institute (RPMI) 1640 medium fromEurobio (Les Ulis, France) and Biociphos Medium from IMV Tech-nologies (LAigle, 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 ( 21) were purchased from Tebu-Bio (Le Perray enYvelines, France) and the antibodies against AIF from Sigma. Mousemonoclonal IgG antibodies against Bcl-2 (C-2), cytochrome c (7H8)and secondary anti-goat IgG from donkey were from Tebu-Bio; anti-rabbit IgG from goat and anti-mouse IgG from rabbit were fromSigma.

Semen collection and cryopreservationA total of 26 healthy bulls (Charolais) were used in this study. Semenwas collected with an artificial vagina. The volume of each ejaculatewas measured and sperm cell concentration assessed under lightmicroscopy. After collection, the ejaculate was divided into two aliq-uots. Less than 2 h after collection, the first aliquot was analysed byflow cytometry and/or proteins were extracted for fluorimetry and/orwestern blot analysis. Immediately after collection, the second aliquotwas diluted to 100 106 sperm/ml in Biociphos Medium pre-warmedat 37C. The semen was equilibrated at 4C for 3 h and frozen in liq-uid nitrogen vapour for 10 min before plunging into liquid nitrogen.The cryopreservation method was adapted from previously describedprotocols (Vishwanath and Shannon, 2000; van Wagtendonk-deLeeuw et al., 2000; Anzar et al., 2002). Before the various analyses,cryopreserved samples were thawed at 37C for 1 min.

Culture and apoptosis induction in U937 and MDBK cellsThe different apoptosis tests developed by flow cytometry were previ-ously validated on U937 cells (Martin et al., 2004). In the present

study, U937 and MDBK cells were used to check for the efficiency ofthe tests assessing caspase activities and to verify that the different anti-bodies used in western blotting experiments cross-reacted with thebovine proteins. U937 and MDBK cells were grown in RPMI 1640 sup-plemented with 10% heat-inactivated neonatal calf serum. The mediumincluded penicillin and streptomycin. Cells were maintained at 37C ina water-saturated atmosphere of 95% air and 5% CO2. Apoptosis wasinduced after 6 h of incubation in 25 M VP-16 for U937 cells and 100M VP-16 for MDBK cells. Before analysis, cells were centrifuged for5 min at 1000 g and were washed twice with pre-warmed (37C)phosphate-buffered saline (PBS) 1 to eliminate culture medium.

Flow cytometry analysisCell viability was determined by studying the permeability to PI(Martin et al., 2004). PI (6 M) was added to each tube at room tem-perature, and flow cytometry analysis was conducted within 10 min.When PI was used with other fluorochromes, PI was added at the endof the incubation with the other probe.

Accumulation of the cationic lipophilic fluorochromes DiOC6(3) inthe inner membrane of mitochondria enables detection of mitochon-drial membrane potential variations (Castedo et al., 2002). About 1 106cells were diluted in 1 ml of PBS. DiOC6(3) was added up to a finalconcentration of 90 nM (Castedo et al., 2002; Martin et al., 2004).The tubes were gently mixed and incubated for 15 min at roomtemperature.

The CaspACE FITC-VAD-FMK In Situ Marker was used to detectactive caspases. The structure of the cell-permeable caspase inhibitorpeptide VAD-FMK (Val-Ala-Asp-Fluoromethylketone) conjugated toFITC allows delivery of the inhibitor into the cell where it binds toactivated caspases, serving as an in situ marker for apoptosis (Duvalet al., 2002; Martin et al., 2004). About 0.5 106 cells were diluted in0.5 ml of PBS. One microlitre of FITC-VAD-FMK (5 mM) wasadded. The tubes were gently mixed and incubated for 20 min at roomtemperature in the dark. Then, the cells were washed twice with PBSand the pellets were re-suspended in 500 l of PBS.

The Vybrant Apoptosis Assay Kit was used to detect changes inplasma 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 M) was added. The tubes were gen-tly mixed and incubated for 20 min at room temperature.

PNA-FITC was used to detect spermatozoa with a reacted acro-some (Idziorek et al., 1995). About 1 106 cells were diluted in 1 mlof PBS. Ten microlitres of PNA-FITC (1 g/ml) was added. The tubeswere gently mixed and incubated for 20 min at room temperature.

Flow cytometry analysis was performed using the fluorescence-activated cell sorting (FACS) Vantage SE cell sorter (BD Biosciences,San Jose, CA, USA). Fluorochromes were excited with the 488-nmline of the Enterprise laser (Coherent, San Jose, USA). Green and redfluorescence signals were detected using FL1 and FL3 detectors,respectively, through BP 530/30 nm and BP 695/40 nm filters. Alldata were analysed with Cell Quest Pro 3.1 software (BD Bio-sciences). For DiOC6(3)/PI, caspase inhibitor/PI, Yo-Pro-1/PI andPNA-FITC/PI, 10 000 events were analysed. FL1 and FL3 fluores-cence signals were recorded after logarithmic amplification.

MicroscopyBefore examination under a conventional microscope (LeicaMicrosystems, Wetzlar, Germany), all samples were washed twicewith PBS. Green and red fluorescence signals were respectivelydetected using L5 (BP 440520 nm) and N 2-1 (BP 515560 nm) fil-ters. Images were captured by a CollSnapfx camera (Roper Scientific,Evry, France) using Meta Imaging 4.6.6. software (Universal Imaging,Downingtown, USA).

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Sodium dodecyl sulphatepolyacrylamide gel electrophoresis and immunoblottingCell pellets were washed twice with PBS, and cell lysates were pre-pared in lysis buffer. This composed of protease inhibitor cocktail(Roche), 50 mM TrisHCl (pH 7.5), 150 mM NaCl, 10 mM glycero-phosphate, 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 con-tent measured by the Dc protein assay kit (Bio-Rad, Marnes-la-Coquette, France). Fifty micrograms of total lysate protein was loadedand separated by sodium dodecyl sulphatepolyacrylamide gel elec-trophoresis (SDSPAGE) using stacking and resolving gels with 4and 12% of acrylamide, respectively. Prestained proteins with a broadrange were used as molecular weight markers (BioRad). Then, pro-teins were transferred to polyvinylidene difluoride (PVDF) mem-branes (BioRad).

After overnight incubation at 4C 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 incu-bated for 90 min at room temperature with appropriate peroxydase-conjugated antibodies: anti-mouse IgG (1:5000), anti-rabbit IgG(1:1000) or anti-goat IgG (1:8000). After washing with 0.05% Tweenin PBS, blots were revealed with enhanced chemiluminescence (ECL)western blotting detection reagents (Amersham, Orsay, France) andtransferred on X-ray film. To detect small amount of the cleaved frag-ments of activated caspases (p10 for caspase-9, p20 for caspase-8 andp20, p17 and p11 for caspase-3), we lengthened exposure durations ascompared to caspase detection.

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

Preparation of samples for lipid analysis and high-performance thin-layer chromatographyFresh or frozen thawed spermatozoa were washed twice with PBS toremove seminal plasma, freezing medium and lysed cells. Sperm cho-lesterol and phospholipids were extracted by the method of Folch et al.(1957) adapted for spermatozoa. About 0.2 ml of sperm cell suspen-sion was mixed with 8 ml of chloroformmethanol (2:1, v/v). Distilledwater (1.5 ml) was added and the mixture was allowed to stand at roomtemperature for 1 h before centrifugation at 500 g for 10 min. Theupper layer was reextracted in 8 ml of chloroformmethanolwater

(86:16:1, v/v) and centrifuged at 500 g for 10 min, after which thechloroform extracts were pooled. Finally, the organic layers wereevaporated under vacuum. The dried material was dissolved in a ade-quate volume of chloroformmethanol (2:1, v/v). Total phospholipidswere 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) plateswere used after prewashing with chloroformmethanol (1:1, v/v) fol-lowed by heating at 110C for 10 min. Lipid extracts were appliedunder a flow of nitrogen on the HPTLC plate using Linomat IV(CAMAG, Muttenz, Switzerland) and separated by using the follow-ing sequential development system: development either to half finaldistance in methyl acetatechloroformn-propanolmethanol0.25%KCl in water (25:25:25:10:9, v/v) (Vitiello and Zanetta, 1978) or toimprove the separation of the cardiolipin chloroformacetonemethanolacetic acidwater (6:8:2:2:1, v/v) (Grizard et al., 2000)followed by full development in hexanediethyletheracetic acid(80:20:2, v/v) to resolve the non-polar lipids. The quantification wasperformed after staining [10% CuSO4 w/v in 8% H3PO4 (v/v)] andcharring at 160C. For quantification, lipids were run under the sameconditions. The plates were scanned and quantification was performedusing density measurements with standard lipids and SIGMA scan prosoftware (SPSS Inc, Chicago, USA).

Sperm motilityFresh and cryopreserved spermatozoa were diluted to 25 106spermatozoa/ml with PBS and observed under a light microscope onslides warmed at 37C. Three fields per sample were evaluated with atotal of 100 observed spermatozoa. For each sample, the percentageof motile spermatozoa was determined.

Statistical methodsStatistical analyses were performed using the Statistica 6.0 program(StatSoft, Tulsa, USA). Population means for fresh and cryopreservedspermatozoa were compared by t-test for dependent samples. Valuesare presented as mean standard error of the mean (SEM) and wereconsidered statistically significant when P < 0.05. Analysis of vari-ance (ANOVA) with subsequent post hoc Fisher tests were applied totest for potential differences between cryopreservation steps or differ-ent times of incubation in PBS after cryopreservation/thawing.

ResultsChanges in various apoptotic markers during the cryopreservation processThe percentage of living spermatozoa (assessed by their PI per-meability) was affected by cryopreservation, but no variationin the proportion of dead sperm cells was observed either afterdilution in the medium used for cryopreservation (Biociphos)or after 4 h of incubation at 4C 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 normalYm showed normal mean green fluorescence intensity and(iii) living cells with low Ym, characteristic of apoptotic phe-nomena, showed low mean fluorescence intensity (Figure 1A).The mean values of the apoptotic Ymlow/PI sperm cell popu-lation during the cryopreservation process are summarized inTable I. Cryopreservation induced a significant increase in theproportion of bovine sperm cells with low Ym (P < 0.001).Before cryopreservation, 4.5 0.6% of cells in the ejaculate

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showed mitochondria with low Ym, whereas after cryopreserva-tion, this proportion dramatically increased to 54.4 2.2%. Impor-tantly, an increase of the Ymlow/PI sperm cell population wasalready observed after 5 min of incubation in the cryopreservationmedium Biociphos (P < 0.001). After cryopreservation/thawing,

this population decreased for the benefit of PI+ cells (both P 40% of the spermato-zoa were still motile whereas 10% exhibited a normal Ym.This result could be explained by the fact that most of theenergy required for sperm motility is generated by glycolysisrather than by oxidative phosphorylation (Miki et al., 2004).

A consequence of the Ym decrease may be the release ofpro-apoptotic factors from the mitochondria to the cytoplasm(Ravagnan et al., 2002). Hence, some proteins implicated inthe mitochondrial pathway were studied next. The pro-apoptoticfactor Bax was detected in bovine spermatozoa but not theanti-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 spermatozoaappear highly sensitive to the cryopreservation process, exhib-iting a rapid and important Ym decrease already after dilutionin the cryopreservation medium (Reed, 1997b).

Furthermore, cytochrome c, which is involved in caspase-9activation (Reed, 1997a), was detected in bovine spermatozoain higher levels than in U937 or MDBK cells. This result couldbe explained by the fact that spermatozoa require high quantitiesof energy for movement and contain high number of mitochondria(Turner, 2003). Lastly, AIF, which acts in a caspase-independentmanner, was also detected in bovine spermatozoa. Takingthese results together, it is tempting to suggest that the decreaseof Ym after sperm dilution led to the release of both AIF andcytochrome c and that the latter induced an activation of cas-pases in equilibrated sperm preparations.

Experiments were therefore conducted to specify whichcaspases might be implicated in the apoptosis-like processinduced by cryopreservation. The validity of the antibodies andof the substrates was first assessed in the control cell linesU937 and MDBK. It is interesting to note that the anti-caspase-8and -3 antibodies recognized additional proteins of lowermolecular weight than the human pro-enzyme in MDBK cells.It could be a non-specific staining or cleaved intermediates ofpro-caspase-8 and -3 (Cohen, 1997). Neither activated caspase-8,involved in the membrane pathway, nor caspase-3, an effectorcaspase, was detected by fluorimetry or western blot in bovinespermatozoa. It would be of interest to investigate the activityof alternative caspases such as the effector caspase-7 and thecaspase-10 implicated in the membrane pathway (Slee et al.,1999). By contrast, pro-caspase-9 and activated caspase-9 (p10fragment), involved in the mitochondrial pathway, wereobserved in bovine spermatozoa and higher levels of activatedcaspase-9 could be detected after cryopreservation, supportingthe implication of the mitochondrial pathway (Zou et al.,1999). The fact that in human spermatozoa caspase-3 and -8were also activated by cryopreservation (Paasch et al., 2004a,b,2005) highlights the species specificity of this process.

Peter and Linde-Forsberg (2003) found that blocking caspaseactivity with the anti-caspase agent zVAD-fmk had no effecton post-thaw motility or cell viability in canine spermatozoa.These authors hypothesized that either the caspase inhibitor orits concentration was not adapted. It would be of interest to testa panel of caspase inhibitors on cryopreserved/thawed bovinesperm cells.

In this study, the total phospholipid and cholesterol contentsof bull spermatozoa we found were similar to previous reports(Parks et al., 1987). In addition, we confirmed that PC, PE andSM are the major phospholipids present in bull spermatozoa(Hinkovska-Galcheva and Srivastava, 1993). Too small quanti-ties of PS were recovered in sperm membranes to be analysed.This latter result should explain, at least partly, why we previ-ously observed only small populations of bovine sperm cellsreacting with annexin V (Martin et al., 2004). Cryopreserva-tion induced an increase in the proportion of CL, a major phos-pholipid of the mitochondrial membrane (Hoch, 1992).Moreover, cryopreservation resulted in a decrease in the mem-brane-rigidifying phospholipids PE and SM and in a relativeincrease in the membrane-fluidizing phospholipid PC (White,1993 and present results). Some spermatozoa can be lysed dur-ing the freezing thawing process (Anzar et al., 2002). Hence, itcan be hypothesized that cells having more PC and less PE andSM would resist cryopreservation more easily. Exchange ofphospholipids between the cryopreservation medium and thespermatozoa membrane could also explain the variations ofspermatozoa phospholipid contents during cryopreservation(Cookson et al., 1984). Taking into consideration all theseresults, it is not unexpected that cryopreservation induced anincrease of the permeability of bovine spermatozoa plasmamembrane.

A consequence of the increase of permeability of spermato-zoa membrane could be an early cell death or a premature acro-somal reaction (Medeiros et al., 2002). This hypothesis is inline with our results showing an increase of the proportion ofdead spermatozoa and of cells with a reacted acrosome aftercryopreservation/thawing. Capacitation is characterized byvarious membrane changes. In boar spermatozoa, cooling to5C induces a capacitation-like process that is not analogous totrue capacitation (Green and Watson, 2001). It would be ofinterest to investigate if early steps of cryopreservation inducea similar phenomenon in bovine spermatozoa.

Apoptosis and plasma membrane alterations can be inducedby reactive oxygen species (Carmody and Cotter, 2001). Theincorporation of anti-oxidant in cryopreservation mediummight be a way of investigation to improve bovine sperm cryo-preservation efficiency.

In summary, we have confirmed that cryopreservationinduces the occurrence of some apoptotic features in bovinespermatozoa. More importantly, these apoptotic characteristicsappeared as ordered events during the cryopreservation proc-ess, as a decrease of the Ym could be observed immediatelyafter dilution in the cryopreservation medium, caspase activa-tion after equilibration and changes in membrane permeabilityafter the complete freezing/thawing process. The Ym decreasemight be facilitated by the fact that bovine spermatozoa con-tain the pro-apoptotic factor Bax and only small amounts, if

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any, of the anti-apoptotic factor Bcl-2. The presence of highlevels of cytochrome c and AIF together with caspase-9strongly suggests the importance of the mitochondrial pathwayin this apoptosis-like phenomenon.

AcknowledgementsWe are grateful to R. Touraine (Laboratoire de Gntique Molculaire,Saint-Etienne, France) for his support to N. Laroche (INSERME0366, Saint-Etienne) for his help in the microscopy study and toT. Bourlet (GIMAP, Saint-Etienne) for providing MDBK cells.

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Submitted on April 11, 2006; resubmitted on June 7, 2006; accepted onJune 30, 2006

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