BIOLOGY OF REPRODUCTION 71, 28–37 (2004) Published online before print 18 February 2004. DOI 10.1095/biolreprod.103.024281

Cryopreservation Induces an Apoptosis-Like Mechanism in Bull Sperm Guillaume Martin,2,3 Odile Sabido,4 Philippe Durand,3 and Rachel Levy1,2 Laboratoire de Biologie de la Reproduction—GIMAP,2 Hoˆpital Nord, 42055 Saint-Etienne, France INSERM U418—INRA UMR1245,3 Hoˆpital Debrousse, 69322 Lyon, France Centre Commun de Cytome´trie en Flux,4 Universite´ Jean Monnet, 42023 Saint-Etienne Cedex 2, France ABSTRACT

Opening of mitochondrial pores leads to the release of proapoptotic factors from the mitochondria [4]. In the cytoplasmic compartment, the proapoptotic factors—for example, different proteases related to the caspases family (cysteine proteases with aspartate specificity)—are subsequently activated, leading to the degradation phase. During this phase, changes at both the cell surface and the nucleus occur. Phosphatidylserine (PS), ordinarily sequestered in the plasma membrane inner leaflet, appears in the outer leaflet, where it triggers noninflammatory phagocytic recognition of the apoptotic cell [5]. In the apoptotic cells, internucleosomal cleavage of DNA by specific endonucleases produces ;180-base pair DNA fragments [2]. Normal spermatogenesis depends on the efficiency of apoptosis. Approximately 25–75% of germ cells degenerate and die in the adult mammal testis. Spontaneous apoptosis has been clearly observed in rat testis seminiferous epithelium, affecting spermatogonia, spermatocytes, and spermatids [6]. Using electron microscopy, ejaculated spermatozoa have been shown to exhibit certain characteristics of apoptotic somatic cells: DNA fragmentation and chromatin condensation, lobulation of the acrosome membrane, and mitochondrial distention [7, 8]. Furthermore, apoptotic spermatids and apoptotic corpses phagocytosed by macrophages can be observed in ejaculated sperm [7]. In 1993, Gorczyca et al. [9] first observed spermatozoa with DNA fragmentation, analogous to apoptosis in somatic cells. They hypothesized that this phenomenon would inactivate abnormal ejaculated spermatozoa, which could be dangerous. Failure to remove these defective germ cells efficiently during spermatogenesis would result in high numbers of abnormal sperm cells in the semen and consequent low fertility. The pathway of such putative apoptosis in sperm cells is the subject of controversy. Weil et al. [10], using in situ immunofluorescence, defended the hypothesis of a caspaseindependent pathway on the basis of the observation that less than 8% of dead cells expressed caspase-3. In contrast, Weng et al. [11] concluded in favor of caspase-dependent apoptosis after studying caspase enzymatic activity by fluorometry and immunoblot assay. Few studies have focused on the effects of cryopreservation on apoptotic manifestations in sperm cells. They have shown that cryopreservation is associated with induction of membrane PS translocation in human [12–14], boar [15], and bull [16, 17] sperm cells. This membrane modification is not correlated with DNA fragmentation in human [13] or in bull [16] sperm or with free-radical production in human sperm [12]. We therefore compared the expression of various apoptosis markers before and after cryopreservation-thawing. As a single assay is not sufficient to assess apoptosis, var-

Cryopreservation induces many changes in sperm cells, including membrane disorders and cell death. We tested the hypothesis that apoptosis, a form of programmed cell death, can contribute to the fatal effect of cryopreservation on sperm cells. A multiparametric study of apoptosis on bovine sperm is proposed, using flow cytometry, including mitochondrial membrane potential (DCm), caspase activation, membrane permeability, nucleus condensation, DNA fragmentation, and phosphatidylserine (PS) externalization. The relevance of each test was first validated on a human somatic cell line, U937. Cryopreservation and/or thawing induced significant changes in all apoptotic markers in living bull sperm cells except those concerning the nucleus. After cryopreservation, 44.9% 6 17% (vs. 11.3% 6 10.6% before cryopreservation) of sperm cells showed low DCm, 12% 6 6.3% (vs. 2.2% 6 1.0% before) contained active caspases, and 10.8% 6 5.8% (vs. 1.4% 6 1.1% before) exhibited high membrane permeability. However, cryopreservation had no effect on DNA fragmentation (9.1% 6 7.7% before vs. 11.1% 6 5.7% after cryopreservation) or on nucleus condensation (46% 6 12.7% before vs. 43.8% 6 13.1% after). Cryopreservation acts as an apoptotic mechanism inducer in bovine sperm cells, where the earliest but not the latest features of cells undergoing apoptosis occur. We have named this abortive process an apoptosis-like phenomenon.

apoptosis, gamete biology, sperm

INTRODUCTION

Cryopreservation and/or thawing induce many changes in mammalian spermatozoa [1]. Diminished motility and membrane changes, including sperm capacitation or acrosomal reaction, are some of the main forms of damage brought out by cryopreservation. Cryopreservation definitely affects sperm viability. Necrosis and apoptosis are two forms of cell death. Necrosis results from injury and affects large numbers of cells, causing cell swelling and membrane rupture; in contrast, apoptosis is physiologically programmed cell death that affects single cells without any related inflammation in the surrounding tissue [2, 3]. 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. After induction of apoptosis, mitochondrial pores are opened, characterized by decreased mitochondrial membrane potential (DCm). Correspondence. FAX: 33 0 4 77 82 84 61; e-mail: [email protected]

1

Received: 14 October 2003. First decision: 5 November 2003. Accepted: 15 January 2004. Q 2004 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

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ious probes specific to the main apoptotic targets were studied. First, all the techniques were validated on the human myeloid leukemia cell line U937 because induction and visualization of apoptosis in this cell line is easy and abundantly documented [18–21]. We here present our results concerning apoptosis markers, which shed light on the effects of cryopreservation on bovine spermatozoa. This multiparametric study provided evidence that a phenomenon, similar to the apoptosis observed in somatic cells, with the exception of nucleus changes, is induced in ejaculated bovine spermatozoa by cryopreservation and/or thawing. MATERIALS AND METHODS

Chemicals The Live/Dead Sperm Viability Kit, the Vybrant Apoptosis Assay Kit, and 3,39-dihexylocarbocyanine iodide (DiOC6(3)) were purchased from Molecular Probes (Montluc¸on, France); propidium iodide (PI), etoposide (VP-16), and carbonyl cyanide m-chlorophenylhydrazone (FCCP) were from Sigma (Saint Quentin Fallavier, France); the CaspACE FITC-VADFMK In Situ Marker was from Promega (Charbonnie`res-les-Bains, France); the Annexin V-FLUOS Staining Kit was from Roche (Meylan, France); the Apo BrdU Kit was from Phoenix Flow Systems (San Diego, CA); DNase was from Qbiogene (Illkirch, France); RPMI-1640 was from Eurobio (Les Ulis, France); and Biociphos Medium was from IMV Technologies (L’Aigle, France).

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tion of DCm. In this control, before adding DiOC6(3), 4 ml of 50 mM FCCP was added and the cells were incubated for 30 min at room temperature (RT). The tubes were gently mixed and incubated for 15 min at RT and 6 mM PI was added to each tube. Flow cytometry or fluorescence microscopy analysis was conducted within 10 min.

Caspase Inhibitor/PI Assay 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-Phe-Met-Lys) 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 [26]. In 0.5 ml of PBS, 0.5 3 106 cells were diluted. Then 1 ml of FITC-VAD-FMK (5 mM) was added. The tubes were gently mixed and incubated for 20 min at RT in the dark. Then the cells were washed twice with PBS and the pellets were resuspended in 500 ml of PBS. To each tube was added 6 mM PI. Flow cytometry or fluorescence microscopy analysis was conducted within 10 min.

Annexin V-FITC/PI Assay Annexin V-FITC/PI assay was conducted on U937 and human and bovine sperm cells. Human sperm was obtained from normospermic men by masturbation after obtaining informed written consent. The AnnexinV-FLUOS Staining Kit was used to detect the PS translocation from the inner to the outer leaflet of the plasma membrane. Following manufacturer instructions, for each assay, 1 3 106 cells were washed and then diluted in 100 ml of annexin V buffer. Then 5 ml of annexin V-FITC was added to the sample. The tubes were incubated for 15 min at RT in the dark. Then 400 ml of additional binding buffer and PI was added to each tube. Flow cytometry analysis was conducted within 10 min.

Semen Collection and Cryopreservation Procedures relating to the care and use of animals were approved by the French Ministry of Agriculture and Fishing according to the French regulations for animal experimentation (guideline 19/04/1988). Twenty-six healthy bulls (Charolais) were used in this study. Semen was collected with an artificial vagina. The volume of each ejaculate was measured and the sperm cell concentration assessed under light microscopy. After collection, ejaculate was divided into two parts. Less than 2 h after collection, the first part was analyzed with SYBR-14/PI, DiOC6(3)/PI, Annexin VFITC/PI, Yo-Pro-1/PI, and caspase inhibitor/PI or fixed for terminal deoxynucleotidyl transferase (TdT)-mediated nick end labeling (TUNEL) assay. Immediately after collection, the second part was diluted to 100 3 106 sperm/ml in Biociphos Medium prewarmed at 378C. The semen was cooled to 48C over 3 h and frozen in liquid nitrogen vapor for 10 min before being plunged into liquid nitrogen. The cryopreservation method was adapted from previously described protocols [16, 22, 23]. Prior to the various analyses, cryopreserved samples were thawed at 378C for 1 min. Ten thawed ejaculates were diluted to 10 3 106 cells/ml in PBS and either incubated at 378C for 4 h for caspase inhibitor/PI analysis or for 30 h for viability analysis and TUNEL assay.

Culture and Apoptosis Induction in U937 U937 cells were grown in RPM1-1640 supplemented with 10% heatinactivated neonatal calf serum. The medium included penicillin and streptomycin. Cells were maintained at 378C, in a water-saturated atmosphere of 95% air and 5% CO2. Apoptosis was induced after 6 h of incubation in 25 mM VP-16. Before analysis, the U937 samples were centrifuged for 5 min at 6000 rpm and washed twice with prewarmed 378C PBS 13 to eliminate culture medium.

Viability Assay Sperm sample viability was assessed using the Live/Dead Sperm Viability Kit. Briefly, ejaculates were diluted to 1 3 106 sperms/ml in prewarmed 378C PBS. Then samples were incubated at 378C for 20 min with 100 nM SYBR-14 and 12 mM PI before flow cytometric analysis [24].

DiOC6(3)/PI Assay DiOC6(3) was used to detect mitochondrial membrane potential. In 1 ml of PBS, 1 3 106 cells were diluted. DiOC6(3) was added up to a final concentration of 90 nM [25]. The test was validated using FCCP, an uncoupler of mitochondrial oxidative phosphorylation, which makes the inner mitochondrial membrane permeable for protons and induces dissipa-

Yo-Pro-1/PI Assay The Vybrant Apoptosis Assay Kit was used to detect changes in plasma membrane permeability to Yo-Pro-1 [27]. The 1 3 106 cells were diluted in 1 ml of PBS. Then 1 ml of Yo-Pro-1 (100 mM) was added. The tubes were gently mixed and incubated for 20 min at RT and 6 mM PI was added to each tube. Flow cytometry analysis was conducted within 10 min.

TUNEL Assay Nucleus status was studied using the TUNEL assay. The polymerization of labeled nucleotides in DNA breaks gave information on DNA fragmentation [28] and the varying amounts of PI molecules incorporated into each permeabilized cell gave information on the nucleus condensation level [29]. The DNA fragmentation was assessed using the Apo BrdU Kit. Spermatozoa were diluted to a 5 3 106 cells/ml concentration, centrifuged at 1000 3 g for 5 min, fixed, and permeabilized in 70% ethanol at 2208C for more than 12 h. After two washes with 1 ml of PBS, the elongation reaction was performed by incubating the sperm in 50 ml of labeling solution containing the TdT enzyme and dUTP, for 1 h at 378C. For each experimental set, a negative control was prepared by omitting TdT from the reaction mixture. Two subsequent washes were performed to stop the reaction. To perform the labeling reaction, the highly dUTP-specific fluorescein-PRB1 antibody was incubated with sperm for 30 min at RT in the dark. Before flow cytometry analysis, the sperm was washed twice with PBS, labeled with 150 mM PI, and filtered. Positive controls were prepared as described above but with an additional treatment with 10 IU DNase I for 1 h at 378C before the elongation reaction.

Flow Cytometry Analysis Analysis was performed using the FACS Vantage SE cell-sorter (BD Biosciences, San Jose, CA). Fluorochromes were excited with the 488-nm line of the Enterprise laser (Coherent, San Jose, CA). Green and red fluorescence were detected using FL1 and FL3 detectors, respectively, through a bandpass (BP) 530/30 nm and a BP 695/40 nm filter. All data were analyzed with Cell Quest Pro 3.1 software (BD Biosciences). For viability, DiOC6(3)/PI, caspase inhibitor/PI, Yo-Pro-1/PI, and annexin V-FITC/PI, 10 000 events were analyzed. FL1 and FL3 fluorescence signals were recorded after logarithmic amplification. For TUNEL assay, 15 000 events were recorded at a flow rate stabilized at 200–300 cells/sec. Cell doublets and debris were excluded using an FL3-A vs. FL3-w gate. Analysis of DNA fragmentation was performed using an FL3-A vs. FL1H cytogram. FL1 and FL3 fluorescence signals were, respectively, record-

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Statistical Methods Statistical analyses were performed using the Statistica 6.0 program (StatSoft, Tulsa, OK). Population means for fresh and cryopreserved spermatozoa were compared by t-test for dependent samples. Values are presented as mean 6 SD and were considered statistically significant when P , 0.05.

RESULTS

Propidium Iodide Labeling

FIG. 1. Optical microscopy study of U937 without (A) and with (B) induction of apoptosis by 25 mM VP-16. Arrows show cells with a typical apoptotic pattern. Scale bar 5 50 mM.

ed before logarithmic and linear amplification. Percentage DNA fragmentation of normally condensed and decondensed nuclei were quantified through gates drawn on an FL3-A histogram.

Microscopy Before examination under a DMRB microscope (Leica Microsystems, Wetzlar, Germany), all samples were washed twice with PBS. Green and red fluorescence 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, PA).

For each experiment on unfixed cells, the percentage of dead cells (i.e., cells with high permeability to PI) was assessed. Contrary to U937, where 25 mM VP-16 apoptosis induction did not increase PI permeability, cryopreservation induced statistically significant sperm cell death (P , 0.0001). Before cryopreservation, 28% 6 12.7% of the ejaculate cells showed high permeability to PI, whereas after cryopreservation, this proportion increased to 50.8% 6 14.2%. In spite of various precautions (specific medium for freezing, slow cooling), the number of dead cells was multiplied by 2.1% 6 1.1% after cryopreservation. Cell Morphology

Blebbing membranes are morphological modifications characteristic of apoptosis. Using light microscopy, it was noted that VP-16 induced changes in U937 shape from round (healthy) to blebbing cells (apoptotic) (Fig. 1). In

FIG. 2. Flow cytometry study of DCm. A, B) Typical cytograms of DiOC6(3) staining in living cells (PI2). The population with a normal DCm (a) is shown. A) U937 cells without treatment (3), treated with FCCP (50 mM, 30 min) (1) or with VP-16 (25 mM, 6 h) (2). B) Cryopreserved (2) and freshly ejaculated spermatozoa without treatment (3) or treated with FCCP (50 mM, 30 min) (1). C) Means of the various populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P , 0.0001). D) Location of DiOC6(3) fluorescence in fresh spermatozoon using fluorescence microscopy. Scale bar 5 5 mM.

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APOPTOSIS IN CRYOPRESERVED BULL SPERM TABLE 1. Summary of the various populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm.* Test DiOC6(3)/PI FITC-VAD-FMK/PI Yo-Pro-1/PI TUNEL

Population Normal DCm/PI2 Low DCm/PI2 PI1 FITC-FMK-VAD2/PI2 FITC-FMK-VAD1/PI2 PI1 Yo-Pro-11/PI2 Yo-Pro-11/PI2 PI1 DNA fragmentation Normal nucleus condensation

Fresh (%)

Cryopreserved (%)

6 6 6 6 6 6 6 6 6 6 6

4.9 6 3.8† 44.9 6 15.9† 50.2 6 16.4† 34.6 6 8.9† 12 6 6.3† 53.3 6 10.5† 43.8 6 13.2† 10.8 6 5.8† 46.1 6 15.7† 11.1 6 5.7 43.8 6 13.1

62 11.3 26.8 64.2 2.2 33.6 75.7 1.4 22.9 9.1 46

19.3 10.6 19 9.2 1 9.3 10.8 1.1 10.8 7.7 12.7

* Means 6 SD are indicated. † Difference between fresh and cryopreserved samples was significant (P , 0.0001).

contrast, cryopreservation did not induce any change in spermatozoon shape (data not shown). The study then focused on other events in the apoptotic cascade. Determination of Mitochondrial Membrane Potential after Cryopreservation in Living Sperm

The accumulation of the cationic lipophilic fluorochromes DiOC6(3) in the inner membrane of mitochondria enables detection of DCm variations [25]. Using fluorescence microscopy, we found that DiOC6(3) fluorescence was mainly detected in the intermediate piece of the spermatozoa, where sperm mitochondria are located (Fig. 2D). Using DiOC6(3)/PI, three cell patterns were detected: i) necrotic cells were labeled with PI, ii) living cells with

normal DCm showed normal mean green fluorescence intensity, and iii) living cells with low DCm, characteristic of apoptotic phenomena, showed low mean fluorescence intensity. Typical cytograms of living (PI-negative) U937 and bovine sperm cells labeled with DiOC6(3)/PI are shown in Figure 2, A and B, respectively. The DCm decrease induced by FCCP in U937 and in sperm cells confirmed the efficiency of DiOC6(3) labeling (Fig. 2, A and B, respectively). Furthermore, after apoptosis induction in the U937 cell line, the observed increase in the proportion of living cells with low DCm validated the experiment (Fig. 2A). Cryopreservation induced a statistically significant (P , 0.0001) increase in the proportion of bovine sperm cells with low DCm. Before cryopreservation, 11.3% 6 10.6%

FIG. 3. Flow cytometry study of the activation of proteins from the caspase family. Typical cytograms of FITC-VAD-FMK/PI in U937 incubated with 25 mM VP-16 or not (A) and in fresh or cryopreserved ejaculated spermatozoa (B). a) Dead cells are PI1; (b) living nonapoptotic cells are FITC-VADFMK2/PI2; and (c) living apoptotic cells are FITC-VAD-FMK1/PI2. C) Means of the different populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P , 0.0001). D) Location of FITC-VAD-FMK fluorescence in a cryopreserved spermatozoon using fluorescence microscopy. Scale bar 5 5 mM.

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patterns were detected: i) necrotic cells, labeled with PI, were found in the top quadrant; ii) living cells without active caspase were found in the lower left quadrant; and iii) apoptotic cells (i.e., living cells containing active caspases) were found in the lower right quadrant. Induction of apoptosis in the U937 cells resulted in the appearance of living cells containing active caspases (Fig. 3A). Cryopreservation of bovine spermatozoa induced a statistically significant (P , 0.0001) increase in the proportion of cells with active caspases. Of the fresh cells of the ejaculate, 2.2% 6 1% showed active caspases, whereas after cryopreservation, this proportion reached 12% 6 6.3% (Table 1 and Fig. 3C). This proportion was not significantly (P . 0.05) modified after 4 h of incubation in PBS at 378C (7.3% 6 0.7%, n 5 3). Active caspases were found to be mainly detected in the intermediate piece of spermatozoa (Fig. 3D). Most stained spermatozoa had normal morphology without any cytoplasmic droplets (immature sperm cell morphology) or typical characteristics of apoptotic cell (blebbing membranes) (Fig. 3D). Assessment of Membrane Modifications in Living Sperm Cells

FIG. 4. Flow cytometry study of the phosphatidylserine externalization. Typical cytograms of annexin V-FITC/PI in U937 incubated with 25 mM VP-16 or not (A) and in human spermatozoa incubated with 10 mM A23187 or not (B) and in fresh or cryopreserved bull ejaculated spermatozoa (C). a) Dead cells are PI1; (b) living nonapoptotic cells are annexin V-FITC2/PI2; and (c) living apoptotic cells are annexin V-FITC1/PI2. All these cytograms are representative of three independent assays.

cells of the ejaculate showed mitochondria with low DCm, whereas after cryopreservation, this proportion dramatically increased to 44.9% 6 17%. Furthermore, the high DCm population nearly disappeared (4.9% 6 3.8%) (Table 1 and Fig. 3C). Detection of Active Caspases in Sperm

During apoptosis, the decrease in DCm results from the opening of membrane pores located in the mitochondrial membrane. The consequence is the translocation and activation of the various proapoptotic factors. Our study focused on the activation of the caspase family of proteins, one of these main components. Typical cytograms of U937 and bovine sperm cells labeled with FITC-VAD-FMK/PI are shown in Figure 3, A and B, respectively. Using FITC-VAD-FMK/PI, three cell

The protease activity of apoptotic factors contributes to the degradation phase of apoptosis when PS is exposed to the external leaflet of the cytoplasmic membrane. PS externalization was studied using the annexin V probe. Typical cytograms of cells labeled with annexin V-FITC/PI are shown for U937, human sperm, and bovine sperm cells in Figure 4. Three patterns of cell were detected: i) necrotic cells, labeled with PI, were found in the top quadrant; ii) living cells without PS exposure were found in the lower left quadrant; and iii) apoptotic cells with PS exposure were found in the lower right quadrant. This experiment gave clearly different populations in U937 (Fig. 4A) and in human sperm (Fig. 4B) cells, but the green fluorescence intensity of bovine sperm cells was too low to discriminate apoptotic from living cells (Fig. 4C). During the degradation phase, 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 [27]. Typical cytograms of U937 and bovine sperm cells labeled with Yo-Pro-1/PI are shown in Figure 5, A and B, respectively. Using YoPro-1/PI, three patterns of cell were clearly detected: i) necrotic cells, labeled with PI were found in the top quadrant; ii) living cells, with low permeability membranes, were found in the lower left quadrant; and iii) apoptotic cells (i.e., living cells with modified membranes) were found in the lower right quadrant. Induction of apoptosis in the U937 cell line increased the number of living cells exhibiting a high permeability to Yo-Pro-1 (Fig. 5A). Cryopreservation of bovine spermatozoa induced a statistically significant (P , 0.0001) increase in the proportion of cells exhibiting high permeability to Yo-pro-1: only 1.4% 6 1.1% of the fresh cells of the ejaculate showed permeability to Yo-Pro-1, whereas after cryopreservation, this proportion reached 10.8% 6 5.8% (Table 1 and Fig. 5C).

APOPTOSIS IN CRYOPRESERVED BULL SPERM

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FIG. 5. Flow cytometry study of the permeability to Yo-Pro-1 and to PI. Typical cytograms of Yo-Pro-1/PI in U937 incubated with 25 mM VP-16 or not (A) and in fresh or cryopreserved ejaculated spermatozoa (B). a) Dead cells are PI1, (b) living nonapoptotic cells are Yo-Pro-12/PI2, and (c) living apoptotic cells are Yo-Pro-11/PI2. C) Means of the different populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P , 0.0001).

DNA Fragmentation and Nucleus Condensation

Two late manifestations of the degradation phase of apoptosis are DNA fragmentation and nucleus condensation. Typical cytograms of TUNEL assay-labeled bovine sperm cells are shown in Figure 6A. Cells with DNA fragmentation were gated as shown in Figure 6A. Induction of apoptosis in U937 cell line led to DNA fragmentation (data not shown). Moreover, the TUNEL assay was validated using DNase-treated spermatozoa as positive controls (Fig. 6A). Cryopreservation did not induce a statistically significant (P . 0.05) increase in the proportion of DNA-fragmented cells (Table 1 and Fig. 6B). Of the fresh bovine cells, 9.1% 6 7.7% of the ejaculate showed DNA fragmentation and, after cryopreservation, this proportion was 11.1% 6 5.7%. Only 5.9% 6 8.4% of cells survived after 30 h of incubation in PBS at 378C. This massive cell death was not associated with a significant (P . 0.05) increase of the fragmented DNA cells percentage (11.3% 6 3.6%; Fig. 7). Accessibility of PI to the spermatozoon nucleus enabled two populations with differently condensed nuclei to be quantified [29]. Figure 6A shows these cell populations with normally condensed and decondensed chromatin. Of the fresh cells of the bovine ejaculate, 46% 6 12.7% exhibited a normal nucleus condensation, and after cryopreservation, this proportion was 43.8% 6 13.1%. Cryopreservation/thawing or cryopreservation/thawing followed by 30 h of incubation in PBS at 378C did not induce a statistically significant (P . 0.05) decrease in the proportion of cells with a normally condensed nucleus (Table 1, Figs. 6B and 7). We also compared the percentage DNA fragmentation of

cells with normal and with decondensed nuclei. A positive correlation (r 5 0.92) was found for DNA fragmentation in cells with a normally condensed nucleus and in cells with a decondensed nucleus (Fig. 6C). Furthermore, the DNA fragmentation in cells with a normally condensed nucleus was significantly (P , 0.0001) lower than in cells with a decondensed nucleus (17.2% 6 10% vs. 8.3% 6 5.5%) (Fig. 6B). DISCUSSION

It is clear that the cryopreservation and/or thawing process is detrimental to sperm viability. The dead cells with high PI permeability observed may result either from simple necrosis or from necrosis secondary to apoptosis. During conventional freezing, water precipitates as ice and often leads to tissue damage, acts on cytoplasmic structures, or even effects on cytoskeleton or genome-related structures [30]. Ice crystallization induces an unregulated and mechanical death related to necrosis. The present study tested the hypothesis that cryopreservation is associated with the activation of an apoptotic process in spermatozoa. As the existence and origin of apoptosis in ejaculated mature spermatozoa remains controversial, we first validated each test with the human somatic cell line U937. After VP-16 apoptosis induction, changes were observed in all the tested markers along the cascade of events, including i) cell morphology, ii) decreased DCm, iii) caspase activation, iv) increased permeability to Yo-Pro-1, v) PS externalization, and vi) DNA fragmentation. Our results showed that cryopreservation had a dramatic effect on spermatozoon DCm. The synthesis of ATP is under mitochondrial control and dependent on a high DCm.

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FIG. 6. Flow cytometry study of DNA fragmentation and nucleus condensation of bovine spermatozoa. A) Typical cytograms of TUNEL assay in fresh or cryopreserved ejaculated spermatozoa. Negative control was obtained by omitting the TdT enzyme and DNA fragmentation in positive control was obtained by DNase digestion (10 IU, 1 h, 378C). a) Populations with a DNA fragmentation, (b) with a normally condensed nucleus, and (c) with a decondensed nucleus. B) Means of the different populations analyzed from 16 pairs of fresh and cryopreserved ejaculated sperm. Dead (PI1) and viable populations were determined by a Sybr14/PI assay before fixation. Error bars indicate standard deviation. *, Difference between fresh and cryopreserved samples was significant (P , 0.0001). C) Correlation between the DNA fragmentation in normally condensed and in decondensed nuclei. Thirty-two samples were studied (16 fresh and 16 cryopreserved).

A DCm decrease can result in mitochondrial dysfunction, leading to nonrenewal of ATP. Then a lack of energy, as depletion in ATP, can be responsible for the decreased spermatozoon cell motility usually observed after cryopreservation and thawing [1]. During programmed somatic cell death, the release of apoptotic factors located between the outer and inner membrane is another consequence of the loss of mitochondrial integrity [4]. If male germ cells contained these profactors, a mitochondrial disruption induced by cryopreservation would be responsible for the entry of these apoptotic factors into the cytoplasmic compartment, too. Annexin V-FITC failed to discriminate apoptotic from nonapoptotic populations objectively in bovine sperm cells. This form of labeling seems to be inappropriate for precise study of the effects of cryopreservation on PS exposure. This experiment was done three times in correlation with

experiments giving exploitable results on somatic and human germ cells. Moreover, we found similar cytogram profiles using annexin V-FITC (BD Biosciences) (data not shown). These results differ from Anzar et al. [16], where they observed more than 31% of the ejaculate Annexin V1/ PI2 after cryopreservation. Comparing the cytograms, it can be hypothesized that the green fluorescence amplification they applied for annexin V-FITC analysis was higher than ours. As we determined our amplification using somatic and human sperm controls, higher amplification would increase the problem of false positives. Our observation of a weak annexin V1/PI2 population may be supported by the results of Januskauskas et al.[17]. Yo-Pro-1 analysis in sperm cells (which is, to the best of our knowledge, the first in the literature) is inexpensive, quick, and easy to perform and gave interesting information on membrane modifications related to the apoptotic pro-

APOPTOSIS IN CRYOPRESERVED BULL SPERM

FIG. 7. Flow cytometry study of DNA fragmentation and nucleus condensation of bovine spermatozoa. Ten pairs of ejaculated sperm were analyzed immediately after thawing or after 30 h of incubation in PBS at 378C. DNA fragmentation and nucleus condensation was determined using TUNEL assay, and dead (PI1) and viable populations were determined by a Sybr14/PI assay before fixation. Error bars indicate standard deviation. *, Difference between 0 h and 30 h of incubation was significant (P , 0.0001).

cess. This experiment clearly showed an increased permeability of plasma membranes to Yo-Pro-1 in living spermatozoa after cryopreservation. This result is in line with the dramatic effects of cryopreservation usually observed in sperm cell membranes [1]. Furthermore, cryopreservation was shown to induce premature capacitation or acrosomal reaction in sperm cells [31, 32]. Physiologically, these two membrane modifications must occur at the right time and place for successful fertilization [33]. It could be hypothesized that the living and Yo-Pro-1-permeable cell population that we observed corresponded to living and premature reacted cells. A premature acrosomal reaction induced by cryopreservation should be partly responsible for increased cell death. The observed membrane instability could also be a manifestation of capacitation. The effects of cryopreservation on both capacitation and acrosomal reaction, in correlation with apoptotic markers, are currently under investigation. Our preliminary results, using FITCpeanut agglutinin (PNA) (Sigma) and PI, in flow cytometry, confirm that cryopreservation increased the living and acrosomal-reacted cell population (data not shown) [32]. However, it has not been established if cells with a reacted acrosome exhibit an apoptotic-like pattern too and if these two mechanisms might be linked. Before cryopreservation, about 10% DNA-fragmented cells were observed. Detection of DNA fragmentation using the TUNEL assay is not sufficient to discriminate whether fragmentation is internucleosomal and thus apoptosis specific or unregulated, as described for necrosis [34]. Histone/ protamine transition defect or abortive apoptosis are two different hypotheses that could explain the observed basal DNA fragmentation rate. During spermiogenesis, histones are replaced by protamines. The result of this exchange is high DNA compaction to protect the spermatozoon DNA [35]. Sakkas et al. [36] showed that incomplete replacement of histones by protamines could be partly responsible for enhanced sperm DNA sensitivity to fragmentation. The fact that the observed DNA fragmentation rate was higher in sperm cells with less condensed nuclei bears out this hypothesis. The DNA-fragmented cell population in the ejac-

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ulated sperm might also result from abortive apoptosis of spermatozoa that escaped the elimination mechanism occurring during spermatogenesis [37]. The presence of Faslabeled spermatozoa in human ejaculates would seem to point to abortive apoptosis. However, in the present study, cryopreservation had no effect on sperm DNA fragmentation, as previously described in human spermatozoa [13]. In addition, DNA fragmentation was unaffected by the postthawing 30 h of incubation, as previously reported in testicular sperm from fertile men [38]. Anzar et al. [16] found that cryopreservation induces a decrease of the percentage of cells with a DNA fragmentation. This discrepancy in results may be due to differences in sperm cryopreservation protocols: for example, in our experiment, we used cryopreservation medium without egg yolk. Our work demonstrates that normal, living ejaculated spermatozoa contain active caspases. Previously, Weng et al. [11] demonstrated that immature spermatozoa, exhibiting cytoplasmic droplets, contain active caspase-3. Following the hypothesis of Sakkas et al. [37], it is possible that this weak basal population is the result of abortive apoptosis. However, the significant increase in active caspase rates we observed after cryopreservation should not be related to abortive apoptosis. Our experiment, where we found that 12% of the cryopreserved ejaculate was constituted of living spermatozoa containing active caspases, is not in contradiction with Paasch et al. [39] or De Vries et al. [40] results. In the first study, the authors found 20% of cells contained active caspases regardless of whether they were dead or alive. In the second study, following detection of caspase-3 by immunofluorescence, the authors concluded that caspase-3 is only expressed by immature spermatozoa or contaminating cells. The tests we used allowed detection of a wide range of caspases: it is possible that the spermatozoon caspase composition differs from that of other cell types. It would be interesting to use Western blot or enzymatic analysis to specify which caspases are involved under cryopreservation induction [41]. By immunofluorescence, we localized active caspases and mitochondria exclusively in the midpiece area of the spermatozoa. This provides further evidence that the midpiece area of the spermatozoa could be considered as a region specialized for apoptosis in ejaculated spermatozoa. Nevertheless, the role that caspases play in spermatozoa is not yet fully elucidated. Peter et al. [42] found that blocking caspase activation with the anticaspase agent zVAD-fmk had no effect on postthaw acrosomal reaction, mobility, or cell viability in canine spermatozoa. These authors hypothesized that either the caspase inhibitor or its concentration were not adapted. While substrates of cleaved caspase are commonly implicated in apoptosis, a recent review showed that activated caspases can be involved in other essential processes, such as the cell cycle or cell differentiation [43]. To recapitulate, freshly ejaculated bovine sperm exhibits only a few apoptotic characteristics. Abortive apoptosis is one possible explanation for the observed apoptotic base rate. On the contrary, cryopreservation induced the main apoptotic features: i) DCm decrease, ii) caspase activation, and iii) permeability membrane increase. These three different manifestations of programmed cell death can, respectively, be implicated i) in slowing cell motility [1], ii) in cell-cycle regulation and cellular differentiation [43], or iii) in capacitation and acrosomal reactions [31, 32], too. In somatic cells, apoptosis usually leads to final DNA fragmentation, but in our experimental conditions, cryopreser-

36

MARTIN ET AL.

vation and thawing had no effect on sperm nuclei, even after 30 h of incubation after thawing. This phenomenon, that we named apoptosis-like, is supposed to involve at least the same mechanisms as those occurring in the execution phase and the early degrading phase of apoptotic somatic cells. For example, noncleavage of the poly(ADPribose) polymerase would prevent nuclear manifestations of apoptosis [44]. We found that, after cryopreservation, a large majority of living sperm cells showed low DCm; in contrast, few sperm cells presented caspase activation. This result points to the possibility of a caspase-independent apoptosis pathway, where the apoptosis-inducing factor is one of the mitochondrial compounds that can act [45]. A study of appropriate apoptotic markers in the various steps of the sperm freezing-thawing process would be helpful to specify the critical moment of apoptosis-like induction: liquid nitrogen vapor but also sperm dilution or equilibration steps are potential initiators of the apoptosis-like phenomenon. A better understanding of the cellular mechanisms involved in sperm cryopreservation would help improve the preservation of bovine sperm.

15.

16. 17.

18.

19. 20.

21.

ACKNOWLEDGMENTS

22.

We are grateful to R. Touraine (Laboratoire de Ge´ne´tique Mole´culaire, Saint-Etienne, France) for his support and to N. Laroche (INSERM E0366, Saint-Etienne, France) for his help in the microscopy study.

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