Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

Guillaume Martin1,2, Odile Sabido3, Philippe Durand2 and Rachel Levy1,4

1 Laboratoire de Biologie de la Reproduction, GIMAP, Hôpital Nord, 42055 Saint-Etienne, 2 INSERM U418 – INRA UMR1245, Hôpital Debrousse, 29 rue Sur Bouvier, 69322 Lyon, 3 Centre Commun de Cytométrie en Flux, Université Jean Monnet, 15 rue Ambroise Paré, 42023 Saint-Etienne Cedex 2, France

4 To whom correspondence should be addressed. E-mail: rachel.levy{at}chu-st-etienne.fr


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp–fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.

Key words: acrosome reaction/apoptosis/calcium ionophore A23187/capacitation/phosphatidylserine exposure


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Successful fertilization requires a sperm plasma membrane with normal integrity and function (Flesch and Gadella, 2000Go). The numerous functions of the membrane are related to cell metabolism, for maintaining sperm motility, capacitation, acrosome reaction and sperm–oocyte interaction (Cross and Hanks, 1991Go).

The early phase of disruption of the integrity of the membrane is characterized by the loss of phospholipid asymmetry due to translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane (Vermes et al., 1995Go). This translocation of PS is one of the earliest features of cells undergoing apoptosis (Martin et al., 1995Go). Annexin V is a 35–36 kDa protein that binds very selectively to PS in a calcium-dependent manner. Annexin V binding enables the identification of cells with altered membrane integrity at an earlier stage than staining with supravital stains (Vermes et al., 1995Go).

Sperm showing evidence of PS exposure were found in human semen at higher levels in infertile men than in sperm donors (Barroso et al., 2000Go; Schuffner et al., 2002Go; Shen et al., 2002Go; Paasch et al., 2003Go). However, correlation between PS exposure and semen quality is still much debated (Duru et al., 2000Go; Oosterhuis et al., 2000Go; Ricci et al., 2002Go). Nevertheless, annexin V staining is more frequent in sperm recovered in the low motility fractions (Weng et al., 2002Go). Hence, the most efficient technique to recover non-apoptotic sperm seems to be the swim-up (Lachaud et al., 2004Go).

Normal spermatogenesis depends on the efficiency of apoptosis. Approximately 25–75% of germ cells degenerate in the adult mammalian testis. Apoptosis has been clearly observed in the rat testis, affecting spermatogonia, spermatocytes and spermatids (Lue et al., 1999Go). Thus, the basal apoptotic cell population in the ejaculated sperm might result from abortive apoptosis of sperm that escaped the elimination mechanism operating during spermatogenesis (Sakkas et al., 1999Go, 2004Go). Since externalized PS is expected to be involved in the recognition of apoptotic cells by phagocytes, ejaculated sperm exhibiting PS externalization may represent apoptotic sperm cells that escaped phagocytosis during spermatogenesis (Tesarik et al., 1998Go).

Cryopreservation–thawing (Glander and Schaller, 1999Go; Duru et al., 2001a,bGo), incubation in BM1 medium (Kotwicka et al., 2002Go), incubation in human tubal fluid with/without human serum albumin (Schuffner et al., 2002Go), incubation with H2O2 (Ramos and Wetzels, 2001Go) and spermicidal potency of oxovanadium IV complexes (D’Cruz et al., 1999Go) can induce a significant increase in the number of cells positively stained with annexin V. However, the significance of induced PS exposure is not fully understood; according to the studied model, it might reflect a deterioration of membrane functions (Glander and Schaller, 1999Go), an apoptotic process (D’Cruz et al., 1999Go), and/or changes of the plasma membrane occurring during capacitation (Kotwicka et al., 2002Go).

One of the key processes in human fertilization is the acrosomal reaction, usually triggered in sperm upon their binding to the zona pellucida of the oocyte. The acrosomal reaction involves fusion between the plasma membrane and the underlying outer acrosomal membrane, as a result of which the acrosomal content is released, including a variety of hydrolytic enzymes that enable penetration of the sperm into the oocyte (Wassarman, 1987Go). Capacitated cells that have the capacity to undergo the acrosomal reaction can be induced to do so, not only by the zona pellucida (Morales et al., 1994Go), but also by the pharmacological agent calcium ionophore A23187 (Tao et al., 1993Go; Jaiswal et al., 1999Go).

The objective of the present study was to examine the effects of A23187 on capacitated human sperm. We therefore compared annexin V binding with other probes related to apoptosis, plasma membrane scrambling and acrosomal reaction with and without A23187 exposure.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sperm preparation, capacitation and induction of the acrosome reaction
Human sperm samples were obtained by masturbation after 3 days of sexual abstinence. They were allowed to liquefy for 30 min at room temperature. Men displayed semen parameters according to the World Health Organization (1999)Go criteria. Samples with detectable leukocytes in the semen were excluded from the study. Motile sperm were then isolated using a discontinuous Percoll gradient (Sigma, Saint Quentin Fallavier, France) of 90, 70 and 50%. Following centrifugation for 25 min at 300 g, the 90% Percoll fraction was recovered and the sperm were washed twice with BM1 medium (Eurobio, les Ullis, France). The supernatants were discarded, the final pellets were re-suspended in BM1 medium and the sperm concentration was adjusted to 2x107 cells/ml. After selection, motility was >90% in all the samples.

Sperm suspensions were incubated in BM1 medium under 5% CO2 in air at 37°C for 24 h for capacitation (Kotwicka et al., 2002Go). BM1 medium contains both calcium and bicarbonate that are necessary for capacitation induction. For induction of the acrosomal reaction, cells were then diluted to 106 in phosphate-buffered saline (PBS) and incubated for different periods with A23187 (Sigma, Saint Quentin Fallavier, France) 10 µmol/l final (Tao et al., 1993Go; Jaiswal et al., 1999Go). In order to check whether changes could be attributed to A23187, human sperm were also incubated in PBS only for various periods of time.

Annexin V–fluoroscein isothiocyanate (FITC)/propidium iodide (PI) assay
The Annexin-V–FLUOS Staining Kit (Roche, Meylan, France) was used to detect PS translocation from the inner to the outer leaflet of the plasma membrane. Following the instructions of the manufacturer, for each assay, 106 cells were washed, then diluted in 100 µl of annexin V buffer. Five microlitres of annexin V–FITC were added to the sample. The tubes were incubated for 15 min at room temperature in the dark; 900 µl of binding buffer and 6 µmol/l PI were then added to each tube. Flow cytometry or fluorescence microscopy analysis was conducted within 10 min.

DiOC6(3)/PI assay
DiOC6(3) (Molecular Probes, Montluçon, France) was used to detect mitochondrial membrane potential ({Delta}{Psi}m). A total of 10 6 cells were diluted in 1 ml of PBS. DiOC6(3) was added up to a final concentration of 90 nmol/l (Castedo et al., 2002Go). The tubes were then gently mixed and incubated for 15 min at room temperature and 6 µmol/l PI were added to each tube. Flow cytometry analysis was conducted within 10 min. The test was validated using carbonyl cyanide m-chlorophenylhydrazone (FCCP), an uncoupler of mitochondrial oxidative phosphorylation, which makes the inner mitochondrial membrane permeable to protons and induces dissipation of {Delta}{Psi}m. In this control, before adding DiOC6(3), 4 µl of 50 mmol/l FCCP were added and the cells were incubated for 30 min at room temperature.

Caspase inhibitor/PI assay
The CaspACE FITC-VAD-FMK In Situ Marker (Promega, Charbonnières-les-Bains, France) was used to detect activated 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., 2002Go). A total of 0.5x106 cells was diluted in 0.5 ml of PBS; 1 µl of FITC-VAD-FMK (5 mmol/l) was added. The tubes were gently mixed and incubated for 20 min at room temperature in the dark. The cells were then washed twice with PBS and the pellets were re-suspended in 500 µl of PBS; 6 µmol/l PI were added to each tube. Flow cytometry analysis was conducted within 10 min.

Yo-Pro-1/PI assay
The Vybrant Apoptosis Assay Kit (Molecular Probes, Montluçon, France) was used to detect changes in plasma membrane permeability to Yo-Pro-1 related to apoptosis (Idziorek et al., 1995Go; Martin et al., 2004Go). A total of 106 cells were diluted in 1 ml of PBS; 1 µl of Yo-Pro-1 (100 µmol/l) was added. The tubes were gently mixed and incubated for 20 min at room temperature and 6 µmol/l PI were added to each tube. Flow cytometry analysis was conducted within 10 min.

TUNEL assay
The polymerization of labelled nucleotides in DNA breaks gave information on DNA fragmentation (Li and Darzynkiewicz, 1995Go; Martin et al., 2004Go). The DNA fragmentation was assessed using the Apo BrdU Kit from Phoenix Flow Systems (San Diego, CA, USA). Sperm were diluted to 5x106 cells/ml, centrifuged at 1000 g for 5 min, fixed, and permeabilized in 70% ethanol at –20°C for >12 h. After two washes with 1 ml of PBS, the elongation reaction was performed by incubating the sperm in 50 µl of labelling solution containing the TdT enzyme and dUTP, for 1 h at 37°C. 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 labelling reaction, the highly dUTP-specific fluorescein-PRB1 antibody was incubated with sperm for 30 min at room temperature in the dark. Before flow cytometry analysis, the sperm were washed twice with PBS, labelled with 150 µmol/l PI, and filtrated. Positive controls were prepared as described above, but with an additional treatment with 10 IU DNAse I (from Qbiogene, Illkirch, France), for 1 h at 37°C before the elongation reaction.

M540
Merocyanine 540 (molecular probes, Montluçon, France) was used to detect plasma membrane scrambling (Schlegel et al., 1986Go; Rathi et al., 2001Go; Muratori et al., 2004Go). For each assay, 106 cells were diluted in 1 ml of PBS and 3 µl of M540 from a stock solution of 0.5 mg/ml in DMSO were added. The tubes were incubated for 20 min at room temperature. Flow cytometry analysis was conducted within 10 min.

CD46-PE assay
The monoclonal anti-human CD46-PE antibody (CD46-PE; Ancell, Bayport, MN, USA) that recognizes an antigen on the inner acrosomal membrane was used to detect complete acrosomal reaction (Tao et al., 1993Go; Jaiswal et al., 1999Go). For each assay, 106 cells were washed, then diluted in 50 µl of PBS; 1 µl of the antibody was added to the sample. The tubes were incubated for 1 h at room temperature. The cells were then washed and diluted in 1 ml of PBS.

Flow cytometry analysis
For each experimental set of double staining [annexin V-FITC/PI, DiOC6(3)/PI, FITC-VAD-FMK/PI, Yo-Pro-1/PI, annexin V-FITC/CD46-PE and annexin V-FITC/M540] and triple staining (annexin V-FITC/M540/PI and annexin V-FITC/CD46-PE/PI), different sperm suspensions were prepared for instrumental setting and data analysis: (i) by omitting all fluorochromes (non-specific fluorescence sample); (ii) by adding only one fluorochrome (samples for compensation). Analysis was performed using the FACS Vantage SE cell-sorter (BD Biosciences, San José, CA, USA). Fluorochromes were excited with the 488 nm line of the Enterprise laser (Coherent, San José, CA, USA). Fluorescence was detected using FL1 [for annexin V-FITC, DiOC6(3), FITC-VAD-FMK, Yo-Pro-1], FL2 (for M540 and CD46-PE) and FL3 (for PI) detectors respectively through BP 530/30 nm, BP 575/26 nm and BP 695/40 nm filters. All data were analysed with Cell Quest Pro 3.1 software (BD Biosciences). Except for TUNEL assay, for all cytometric analysis, 10 000 events were analysed. FL1, FL2 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/s. Cell doublets and debris were excluded using an FL3-A versus FL3-w gate. Analysis of DNA fragmentation was performed using an FL3-A versus FL1-H cytogram. FL1 and FL3 fluorescence signals were respectively recorded before logarithmic and linear amplification. For triple stainings, cells were first gated as living (PI) and dead (PI+) on FL3 channel. Then cells on each gate were studied on FL1 versus FL2 cytograms.

Fluorescence microscopy
Before examination under a DMRB microscope (Leica Microsystems, Wetzlar, Germany), all samples were washed twice with PBS. Green and red fluorescences 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, USA).

Statistical analysis
Values are presented as mean ± SEM. Population means for non-incubated sperm and sperm incubated with A23187 were compared by t-test for dependent samples, and were considered statistically significant when P < 0.05. ANOVA was applied to test potential differences between times of incubation with A23187. Statistical analyses were performed using the Statistica 6.0 program (StatSoft, Tulsa, OK, USA).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Effect of A23187 on PS externalization
PS externalization was studied using the annexin V probe. Typical cytograms of cells labelled with annexin V–FITC/PI are shown in Figure 1. Three different patterns could be observed: (i) necrotic cells, labelled with PI, were found in the top quadrant; (ii) living cells without PS exposure were found in the lower left quadrant; and (iii) living cells with PS exposure were found in the lower right quadrant. After capacitation, only 7.3 ± 3.2% cells of the ejaculate showed PS exposure (Table I). However, A23187 induced a significant (P < 0.001) increase of the proportion of cells exhibiting exposure of PS: after 1 h of treatment, this proportion reached 47.5 ± 5.6%. The increase of the living annexin V+ cell population was accompanied by a decrease of the living annexin V cell population from 76.8 ± 4.0% to 16.6 ± 4.1% after 1 h of incubation with A23187. Sperm cells incubated for 1 h in PBS presented minor signs of cell death and PS exposure compared to cells incubated in PBS with A23187 (Figure 1). Hence, the observed phenomenon can be clearly attributed to A23187. During the first hour of incubation, the proportion of annexin V+/PI sperm cells was multiplied by ~6, whereas the proportion of dead cells (PI+) increased 2-fold. An additional 30 min incubation did not induce further increase of the annexin V+/PI population. However, at 2 and 3 h of incubation, the proportion of annexin V+/PI cells decreased sharply for the benefit of PI+ sperm (Table II).



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Figure 1. Flow cytometry study of phosphatidylserine (PS) externalization. Typical cytograms of annexin V-FITC/PI of human sperm incubated under capacitating conditions (24 h in BM1) and not treated (–A23187) or incubated with 10 µmol/l A23187 for 1 h (+A23187). (a) Dead cells are PI+; (b) living cells without PS exposure are annexin V-FITC/PI; and (c) living cells with PS exposure are annexin V-FITC+/PI. All these cytograms are representative of three independent assays.

 

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Table I. Sperm cell populations analysed with annexin V-FITC/PI assay after incubation under capacitating conditions (24 h with BM1) and different times of incubation with 10 µmol/l A23187

 

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Table II. Sperm cell populations analysed with DiOC6(3)/PI, FITC-VAD-FMK/PI, Yo-Pro-1/PI and annexin V-FITC/PI assays after incubation under capacitating conditions (24 h with BM1) and different times of incubation with 10 µmol/l A23187

 

Under fluorescence microscopy, all cells stained by annexin V exhibited PS exposure in the post-acrosomal region. However, additional PS exposure was also detected on the rest of the head and/or on the middle piece of the sperm (Figure 2).



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Figure 2. Localization of annexin V-FITC fluorescence in human sperm incubated under capacitating conditions (24 h in BM1) and treated with 10 µmol/l A23187 for 1 h. Arrows show cells with phosphatidylserine (PS) externalization: localization in (a) post-acrosomal region and middle piece; (b) whole head and middle piece; or (c) whole head only. Scale bar = 10 µm.

 

Effect of A23187 on apoptotic markers
PS exposure is often associated with the early stages of apoptosis. The accumulation of the cationic lipophilic fluorochromes DiOC6(3) in the inner membrane of mitochondria enables detection of {Delta}{Psi}mvariations (Castedo et al., 2002Go). Using DiOC6(3)/PI, three cell patterns were detected: (i) necrotic cells were labelled with PI; (ii) living cells with normal {Delta}{Psi}m showed ‘normal mean’ green fluorescence intensity; and (iii) living cells with low {Delta}{Psi}m, characteristic of the apoptotic phenomenon, showed low mean fluorescence intensity (data not shown). The apoptotic DiOC6(3)low/PI cell population, representing only 8.5 ± 2.2% before A23187 induction, did not change significantly, even after 3 h of incubation in the presence of A23187 (Table II).

During apoptosis, the decrease in {Delta}{Psi}m results from the opening of membrane pores located in the mitochondrial membrane. The consequence is the translocation and activation of the various pro-apoptotic factors (Duval et al., 2002Go). Our study focused on the activation of the proteins of the caspase family. Using FITC-VAD-FMK/PI, three cell patterns were detected: (i) necrotic cells, labelled with PI; (ii) living cells without activated caspase; and (iii) apoptotic cells (i.e. living cells containing activated caspases) (data not shown). The apoptotic FITC-VAD-FMK+/PI cell population, representing 5.4 ± 1.2% before A23187 induction, did not increase, even after 3 h of incubation with A23187 (Table II).

During the degradation phase of apoptosis, the cytoplasmic membrane becomes slightly permeable. Apoptotic cells become permeable to Yo-Pro-1 green fluorochrome while remaining impermeable to PI. Thus, use of combined Yo-Pro-1 and PI dyes provides a sensitive indicator for apoptosis (Idziorek et al., 1995Go). Using Yo-Pro-1/PI, three patterns of cells were detected: (i) necrotic cells, labelled with PI; (ii) living cells, with low permeability membrane; and (iii) apoptotic cells (i.e. living cells with modified membrane) (data not shown). The apoptotic Yo-Pro-1+/PI cell population represented only 5.4 ± 1.0% before A23187 induction, and did not increase significantly after A23187 incubation (Table II).

In order to check whether the changes could be attributed to A23187, human sperm were incubated in PBS only for various periods of time (Table III). It can be seen that even after 3 h of incubation no effect on apoptosis markers ({Delta}{Psi}m, caspase activity, membrane permeability, PS exposure) nor on viability could be observed.


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Table III. Sperm cell populations analysed with DiOC6(3)/PI, FITC-VAD-FMK/PI, Yo-Pro-1/PI and annexin V-FITC/PI assays after incubation under capacitating conditions (24 h with BM1) and different times of incubation in PBS without A23187

 

Another manifestation of the degradation phase of apoptosis is the increase in DNA fragmentation (Li and Darzynkiewicz, 1995Go). Before incubation with A23187, the percentage of sperm with fragmented DNA represented 14.8 ± 4.8%. Actually, as shown in Table IV, a 4 h incubation with A23187 induced high cell death but no significant variation of the proportion of cells with fragmented DNA. It can be noted that permeability to PI following incubation in the presence of A23187 may vary between different experiments (compare Tables II and IV). However, in every case, A23187 resulted in a significant increase in cell death.


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Table IV. DNA fragmentation and necrosis in sperm analysed with TUNEL assay and PI after incubation under capacitating conditions (24 h with BM1) and with or without a 4 h treatment with 10 µmol/l A23187

 

Effect of A23187 on cell membrane scrambling and acrosomal reaction
A 1 h incubation of sperm in the presence of A23187 induced a significant increase in the percentage of cells with membrane scrambling (from 18.1 ± 4.0 to 57.3 ± 8.3%, P < 0.001) and with acrosomal reaction (from 10.1 ± 2.2 to 23.0 ± 3.9%, P < 0.01) detected by M540 and CD46-PE respectively (Table V). As expected, the immunolocalization revealed that CD46-PE staining was restricted to the acrosomal region of the sperm head (data not shown).


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Table V. Summary of the various populations of cells exhibiting membrane scrambling (M540+) and of cells with a reacted acrosome (CD46-PE+) after incubation under capacitating conditions (24 h with BM1) and with or without a 1 h treatment with 10 µmol/l A23187

 

In order to look for a relationship between PS exposure and plasma membrane scrambling or acrosome reaction, we performed double staining of sperm. Typical cytograms of cells labelled with annexin V-FITC and M540 are shown in Figures 3 and 4. Three patterns of cells were mainly detected: (i) cells exhibiting cell membrane scrambling with PS exposure were found in the upper right quadrant; (ii) cells exhibiting cell membrane scrambling without PS exposure were found in the upper left quadrant; and (iii) cells without PS exposure and membrane scrambling were found in the lower left quadrant. The cell population exhibiting PS exposure without any membrane scrambling was weak (<1%) and would rather represent background. The fact that annexin V+ cells were mostly M540+ (Figure 3) suggests that cell scrambling and PS exposure are linked. However, cells with membrane scrambling did not always present PS exposure. Indeed, after incubation with A23187, the proportion of M540/annexin V sperm cells decreased for the benefit of both M540+/annexin V+ and M540+/annexin V populations. As shown in Figure 4, adding PI for triple staining assay revealed that living cells (PI, population I) were mostly annexin V–/M540 (population c) without A23187 and that some sperm became M540+/annexin V+ or M540+/annexin V (populations b and a respectively) with A23187. PI+ cells (population II) were mostly M540+/annexin V+ or M540+/annexin V and the proportions in the various populations remained roughly stable despite A23187 induction of acrosomal reaction.



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Figure 3. Flow cytometry study of cell membrane scrambling and phosphatidylserine (PS) externalization. Typical cytograms of M540/annexin V-FITC in human sperm incubated under capacitating conditions (24 h in BM1) and for 1 h in the absence (upper panel) or presence (lower panel) of 10 µmol/l A23187. (a and b) Cells with cell membrane scrambling are M540+; (b and d) cells with PS externalization are annexin V-FITC+. The percentages of the different populations are indicated. All these cytograms are representative of three independent assays.

 


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Figure 4. Flow cytometry study of cell membrane scrambling and phosphatidylserine (PS) externalization in living or dead sperm. Typical triple staining M540/annexin V-FITC/PI in human sperm incubated under capacitating conditions (24 h in BM1) and for 1 h in the absence (–A23187) or presence (+A23187) of 10 µmol/l A23187. Cytograms of M540/annexin V-FITC are shown for PI (I) and PI+ (II) cells. (a and b) Cells with cell membrane scrambling are M540+; (b and d) cells with PS externalization are annexin V-FITC+. The percentages of the different populations are indicated. All these cytograms are representative of three independent assays.

 

Typical cytograms of cells labelled with both annexin V-FITC and CD46-PE are shown in Figures 5 and 6. Three patterns of cells were mainly detected: (i) cells with a reacted acrosome and PS exposure were found in the upper right quadrant, (ii) unreacted cells with PS exposure were found in the lower right quadrant; and (iii) unreacted cells without PS exposure were found in the lower left quadrant. The cell population exhibiting a reacted acrosome without any PS exposure was weak (<1%) and would rather represent background. The fact that CD46-PE+ cells were mostly annexin V+ (Figure 5) suggests that acrosome reaction and PS exposure are linked. However, the acrosome of cells with PS exposure was not always reacted. Incubation with A23187 induced a decrease of the proportion of CD46/annexin V sperm cells for the benefit of annexin V+/CD46PE+ and annexin V+/CD46-PE populations. As shown in Figure 6, adding PI for triple staining assay revealed that living cells (PI, population I) were mostly annexin V/CD46 (population c) without A23187 and that some sperm became annexin V+/CD46-PE (population d) with A23187. Only few living and CD46+ cells could be observed. PI+ cells (population II) were mostly annexin V+/CD46+ or annexin V+/CD46-PE (populations b and d respectively) and the proportions in the various populations remained roughly stable despite A23187 induction of acrosome reaction.



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Figure 5. Flow cytometry study of acrosomal reaction and phosphatidylserine (PS) externalization. Typical cytograms of CD46-PE/annexin V-FITC in human sperm incubated under capacitating conditions (24 h in BM1) and for 1 h in the absence (upper panel) or presence (lower panel) of 10 µmol/l A23187. (a and b) Cells with a reacted acrosome are CD46-PE+; (b and d) cells with PS externalization are annexin V-FITC+. The percentages of the different populations are indicated. All these cytograms are representative of three independent assays.

 


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Figure 6. Flow cytometry study of acrosomal reaction and phosphatidylserine (PS) externalization in living or dead sperm. Typical triple staining CD46-PE/annexin V-FITC/PI in human sperm incubated under capacitating conditions (24 h in BM1) and for 1 h in the absence (–A23187) or presence (+A23187) of 10 µmol/l A23187. Cytograms of CD46-PE/annexin V-FITC are shown for PI (I) and PI+ (II) cells. (a and b) Cells with a reacted acrosome are CD46-PE+; (b and d) cells with PS externalization are annexin V-FITC+. The percentages of the different populations are indicated. All these cytograms are representative of three independent assays.

 


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Being alive and having the capacity to undergo the acrosome reaction are requirements for a spermatozoon to fertilize an oocyte. These functions are highly dependent on the efficiency of the sperm cells to control and modify the quality and the integrity of their plasma membrane. In human sperm, PS exposure to the outer leaflet of the plasma membrane has been observed (Barroso et al., 2000Go; Duru et al., 2000Go; Oosterhuis et al., 2000Go; Ricci et al., 2002Go; Schuffner et al., 2002Go; Shen et al., 2002Go; Weng et al., 2002Go; Paasch et al., 2003Go; Lachaud et al., 2004Go) and induced (D’Cruz et al., 1999Go; Glander and Schaller,1999Go Duru et al., 2001aGo,bGo; Ramos and Wetzels, 2001Go; Kotwicka et al., 2002Go; Schuffner et al., 2002Go). However, the significance of PS externalization is not fully understood. Here, we demonstrate that incubating capacitated sperm with the calcium ionophore A23187 induces high PS exposure in the living cell population which cannot be related solely to apoptosis.

Before incubation with A23187
Conflicting results have been reported concerning the significance of the induction of PS exposure under capacitating conditions regarding the species and the various agents studied (Gadella and Harrison, 2002Go; Kotwicka et al., 2002Go; De Vries et al., 2003Go; Avalos-Rodriguez et al., 2004Go; Muratori et al., 2004Go). In the present work, we observed that <10% of capacitated sperm were alive and presented PS exposure (annexin V+/PI), a low {Delta}{Psi}m, [DiOC3(6)low/PI], activated caspases (FITC-VAD-FMK+/PI) or a high permeability to Yo-Pro-1 (Yo-Pro-1+/PI). We also observed ~15% DNA-fragmented sperm. According to Sakkas et al. (1999)Go, these populations could result from ‘abortive apoptosis’ of male germ cells that escaped the physiological elimination mechanism occurring during spermatogenesis. 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 (Grasl-Kraupp et al., 1995Go). Histone/protamine transition defect is a mechanism that might explain the observed basal DNA fragmentation rate. Indeed, during spermiogenesis, histones are progressively replaced by protamines. The result of this exchange is a high DNA compaction in order to protect sperm DNA (Kierszenbaum, 2001Go). Sakkas et al. (2002)Go have shown that incomplete replacement of histones by protamines could be partly responsible for enhanced sperm DNA sensitivity to fragmentation.

Using the M540 probe, ~20% of human capacitated sperm showed cell membrane scrambling. In sperm from different species, like stallion (Rathi et al., 2001Go) and boar (Green and Watson, 2001Go), the cell membrane scrambling detected by M540 has been considered a manifestation of capacitation. However, the relatively low percentage of M540 that we observed after capacitation suggested that M540 is not able to specifically detect capacitation-related membrane modifications in human sperm. In support of this hypothesis, a recent study indicates that neither annexin V nor M540 is able to detect membrane modifications under conditions leading to capacitation of human sperm (Muratori et al., 2004Go). Interestingly, the cell membrane scrambling detected with M540 probe could also detect the cell membrane scrambling related to apoptosis and, as a consequence, bind to the highly disordered membranes of apoptotic cells (Laakko et al., 2002Go). After incubation in the capacitating medium, we observed that sperm cells with PS exposure were also M540+. As a consequence, sperm having PS exposure after incubation in capacitating medium might represent true apoptotic cells.

After incubation with A23187
Our experiment clearly showed an increase of PS externalization to the outer leaflet of the plasma membrane in living human sperm after 1 h of incubation with the calcium ionophore A23187. However, the proportion of ‘apoptotic’ sperm cells with low {Delta}{Psi}m, activated caspases, high membrane permeability and DNA fragmentation, did not show any significant increase after A23187 treatment. Furthermore, preliminary western blot analysis did not reveal any detectable active caspase-3 on the same samples (data not shown). Thus, the induced PS exposure could not be related simply to apoptosis. In line with this, De Vries et al. (2003) observed that PS exposure in human sperm was associated neither with caspase activation nor mitochondria degeneration nor DNA fragmentation. It is of interest to observe that a longer incubation with A23187 increased the permeability to PI, without any change of the different apoptotic features. Therefore, under our experimental conditions, the PS scrambling response appears to be independent of apoptosis. The phospholipid scramblase has been reported to cause cell surface exposure of PS by enhancing the bidirectional transbilayer movement of phospholipid in the plasma membranes during sperm capacitation (Gadella and Harrison, 2000Go; De Vries et al., 2003). In red blood cells, it has been shown that scrambling can be induced through a rise in intracellular Ca2+, which activates the scramblase and inhibits the flippase (Bevers et al., 1998Go). It would be of interest to investigate whether A23187 (thus Ca2+) acts through the same pathways in sperm and in red blood cells.

Flow cytometric analysis conducted by Gadella and Harrison (2002)Go revealed that PS exposure in boar sperm was closely related to a decrease in lipid packing order as detected by enhanced binding of M540. This relationship could be explained by the fact that PS is an important contributor to membrane scrambling (Waczulikova et al., 2002Go). Indeed, all the cells presenting PS exposure were also M540+. Phosphatidylcholine content and/or transmembrane potential, that may also affect M540 binding (Waczulikova et al., 2002Go), would explain that we could observe M540+ cells without PS exposure. Thus, PS exposure induced after incubation with A23187 might probe cell membrane scrambling. In the absence of A23187, most of the cells in the small population of M540+ cells were also PI+. By contrast, when A23187 was present in the incubation medium, a rather large population of living and M540+ appeared. Some of these living M540+ cells were also labelled with annexin V. Hence, PS scrambling could not be attributed only to necrosis and might have a role in the acrosome reaction induced by A23187.

The acrosome reaction, a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome, is a key feature during mammalian fertilization (Ramalho-Santos et al., 2002Go). As we observed, incubation of capacitated sperm cells with A23187 is known to induce an increase of the acrosome reaction (Tao et al., 1993Go; Jaiswal et al., 1999Go). Our experiments showed evidence of a link between the acrosome reaction and PS exposure: both phenomena could be induced by the same drug and acrosome-reacted sperm cells usually exhibited PS exposure too. However, the immunolocalization revealed that CD46-PE staining was restricted to the acrosomal region, whereas annexin V staining could also be observed in the post-acrosomal region and in the intermediate piece of the spermatozoon. Moreover, not all the sperm cells with PS exposure presented a reacted acrosome. De Vries et al. (2003) have shown that under their capacitating conditions PS scrambling cells are acrosome intact. Moreover, we have observed that PS+ cells could also be acrosome intact. It may well be that PS exposure occurred before the acrosome reaction and that those PS scrambling cells acrosome-reacted after addition of A23187. As a consequence, if a link exists between PS exposure and acrosome reaction, their contribution in fertilization, in the case of a role of PS externalization, might be different.

We observed that A23187 induced a significant increase of the proportion of dead cells and that most of the CD46+ cells were also PI+. However, after acrosome reaction, the sperm nucleus and cytoplasm will still be covered with an intact membrane. Hence, it might be hypothesized that A23187 produces two different actions on sperm cells: induction of cell death (PI+ cells) and acrosome reaction (evidenced by CD46 labelling) which may not be related.

Once the sperm has crossed the zona pellucida, the gametes fuse. The molecular mechanism of sperm–oocyte fusion remains to be elucidated (Kaji and Kudo, 2004Go). However, for the sperm, this process takes place through a specific membrane domain in the head, the equatorial segment (Yanagimachi and Noda, 1970Go; Bedford et al., 1979Go), where we observed the A23187-induced PS exposure. Phagocytosis of most of apoptotic somatic cells requires that the dying cells lose phospholipid asymmetry, thus exposing PS on the external leaflet of their plasma membrane (Fadok et al., 2001Go; Hoffmann et al., 2001Go). Preventing loss of phospholipid asymmetry or blocking exposed PS with annexin V appears to abrogate uptake of the cell corpse (Fadok et al., 2001Go). Recently, it was demonstrated that common key features of apoptosis (caspase-3) can be implicated in non-apoptotic function such as erythroïd maturation (Carlile et al., 2004Go). It would be of interest to investigate the role of PS exposure at the sperm cell membrane in the sperm–oocyte interaction, considering a possible analogy with the recognition of apoptotic PS-exhibiting cells by specific receptor on the phagocytes. Phosphatidylserine receptor (PSR), the only specific receptor molecule described in direct interaction between PS and the phagocyte (Moreira and Barcinski, 2004Go), and the class B scavenger receptor type I (SR-BI) responsible for the PS-mediated phagocytosis of apoptotic spermatogenic cells by Sertoli cells (Kawasaki et al., 2002Go) have not been studied in oocyte, to the best of our knowledge. However, PS redistribution and the ATP binding cassette transporter ABC1, observed on phagocytes (Moreira and Barcinski, 2004Go), have been described in mouse and human oocytes (Van Blerkom and Davis, 1998Go) and in Xenopus laevis oocytes (Becq et al., 1997Go) respectively.

PS exposure was induced by A23187, which is an artificial way to induce the acrosome reaction. Moreover, inductions and assays were assessed at room temperature in absence of bicarbonate or calcium. The consequence could be the reversal of the capacitation process. Results obtained after capacitation and acrosome reaction under our experimental conditions might be underestimated. Therefore, a close comparison of A23187-induced PS exposure and IVF-induced PS exposure cannot be made.

In conclusion, in our in vitro conditions, few capacitated human sperm exhibited PS exposure. These cells might have escaped the apoptotic phenomenon occurring during spermatogenesis. However, A23187 induced a significant increase in the proportion of living cells exhibiting PS exposure. This PS exposure seemed to be independent of apoptosis and would rather be linked to early signs of necrosis and/or to the acrosome reaction. Further studies on the model of acrosome reaction induced by A23187 are now needed to determine the possible role of PS exposure during fertilization.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are grateful to R.Touraine (Laboratoire de Génétique Moléculaire, Saint-Etienne, France) for his support and to N.Laroche (INSERM E0366, Saint-Etienne) for his help in the microscopy study.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 24, 2005; resubmitted on July 11, 2005; accepted on July 12, 2005.





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