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Reproduction (2005) 130 615-626
DOI: 10.1530/rep.1.00561
Copyright © 2005 Society for Reproduction and Fertility
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RESEARCH

Localization of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction

Anke Kurz1,2, Dagmar Viertel3, Andreas Herrmann1 and Karin Müller2

1 Humboldt-Universität zu Berlin, Institut für Biologie, Invalidenstrasse 42, D-10099 Berlin, Germany, 2 Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V., Bernauer Allee 10, D-16321 Bernau, OT Schönow, Germany, 3 Institut für Zoo- und Wildtierforschung, Alfred-Kowalke-Strasse 17, D-10315 Berlin, Germany

Correspondence should be addressed to K Müller; Email: k.mueller{at}ifn-schoenow.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
One of the essential properties of mammalian, including sperm, plasma membranes is a stable transversal lipid asymmetry with the aminophospholipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), typically in the inner, cytoplasmic leaflet. The maintenance of this nonrandom lipid distribution is important for the homeostasis of the cell. To clarify the relevance of lipid asymmetry to sperm function, we have studied the localization of PS in boar sperm cell membranes. By using labeled annexin V as a marker for PS and propidium iodide (PI) as a stain for nonviable cells in conjunction with different methods (flow cytometry, fluorescence and electron microscopy), we have assessed the surface exposure of PS in viable cells during sperm genesis, that is, before and during capacitation as well as after acrosome reaction. An approach was set up to address also the presence of PS in the outer acrosome membrane. The results show that PS is localized in the cytoplasmic leaflet of the plasma membrane as well as on the outer acrosome membrane. Our results further indicate the cytoplasmic localization of PS in the postacrosomal region. During capacitation and acrosome reaction of spermatozoa, PS does not become exposed on the outer surface of the viable cells. Only in a subpopulation of PI-positive sperm cells does PS became accessible upon capacitation. The stable cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane, is assumed to be essential for a proper genesis of sperm cells during capacitation and acrosome reaction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
During capacitation, several molecular and cellular processes prime mammalian sperm cells for fertilization. A visible characteristic of capacitating sperm cells is their hyperactivated motility. Capacitation affects membrane properties such as ion permeability, surface charge, lectin binding, and protein phosphorylation. Removal of sperm-associated proteins and cholesterol triggers basic structural membrane modifications in many species that lead to the acrosome reaction. Those modifications include changes of lateral lipid diffusion, redistribution of glycolipids and proteins, and the development of protein-free areas in the acrosomal region (for review, see Yanagimachi 1989, Töpfer-Petersen et al. 1996, Flesch & Gadella 2000, Visconti et al. 2002). The molecular mechanisms which mediate membrane fusion during acrosomal exocytosis and the subsequent fertilization of the oocyte are still unknown.

Fresh viable ejaculated ram (Müller et al. 1994), bull (Nolan et al. 1995) and boar (Gadella et al. 1999) spermatozoa have the same characteristic asymmetric phospholipid distribution between both halves of the plasma membrane as other eukaryotic cells. An aminophospholipid translocase activity is responsible for the rapid ATP-dependent inward directed translocation of the aminophospholipids phosphatidylserine (PS) and phosphatidy-lethanolamine (PE) and, thus, for their enrichment on the cytoplasmic leaflet. Recently, Wang et al.(2004) described an aminophospholipid translocase in mouse sperm cells that is exclusively expressed in the acrosomal region and shares 62% similarity with the P-type ATPase FIC1, which has been suggested to act as an aminophospholipid translocase in hepatocytes (Ujhazy et al. 2001). Investigating the transverse lipid distribution during sperm genesis, Gadella & Harrison (2002) observed an exposure of PS and PE in the apical head region of a boar sperm subpopulation under in vitro capacitation conditions. The authors suggested the transversal redistribution of lipids with the surface exposure of PS and PE as a prerequisite for acrosomal exocytosis. A capacitation-related scrambling of lipids has already been hypothesized by Nolan & Hammerstedt (1997). On the other hand, Muratori et al.(2004) demonstrated for human spermatozoa that exposure of PS is characteristic of dead cells or cells with early membrane degeneration. Annexin V as a marker for PS (Tait & Gibson 1994) could not detect capacitation-related membrane modifications. Wang et al.(2004) reported that mice with disrupted sperm FIC1 genes partly develop sperm cells which expose PS on their surface, have a restricted acrosome-reacting capacity, and are less fertile in vivo and in vitro than the wild type.

To address this controversy about the relevance of breakdown of lipid asymmetry to sperm function, we have undertaken a comprehensive study on the PS localization in boar sperm cell membranes. Employing fluorescent FITC-annexin V and propidium iodide (PI) as a stain for nonviable cells, we studied the surface exposure of PS in viable cells before and during capacitation as well as after acrosome reaction. In parallel, cell morphology was characterized, and the accessibility of the outer acrosome membrane was probed by the fluorescent peanut agglutinin (Alexa 549-PNA) (Fazeli et al. 1997, Flesch et al. 1998). By this approach and also taking advantage of the artificial removal of the sperm plasma membrane by nitrogen cavitation, we were able to assess selectively the exposure of PS in the plasma and in the outer acrosome membrane. In addition, with biotin-annexin V, we studied PS localization by electron microscopy.

An important new finding of our study is that PS is localized on the outer acrosome membrane in boar sperm cells. The opposing cytoplasmic leaflets of the outer acrosome membrane and of the sperm plasma membrane probably harbor PS. We confirmed our and others’ previous finding (Müller et al. 1994, Nolan et al. 1995, Gadella et al. 1999) that PS is exclusively localized in the cytoplasmic leaflet of the plasma membrane in viable fresh sperm cells. The exposure of PS may serve as an early signal for the elimination of impaired or dead cells, as was shown for apoptotic spermatogenic cells in the male genital tract (Shiratsuchi et al. 1997). Indeed, PS exposure was typical of PI-labeled spermatozoa. During capacitation and acrosome reaction, PS did not become exposed on the surface of the PI-negative boar sperm cells. The cytoplasmic localization of PS in the plasma membrane, as well as in the outer acrosome membrane of viable sperm cells, might be essential for the genesis of distinct internal structures during capacitation, by priming the sperm cell for acrosomal exocytosis, and for the maintenance of internal postacrosomal structures after the acrosome reaction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Chemicals
FITC- and biotin-conjugated annexin V was purchased from VPS Diagnostics (Hoeven, The Netherlands); PI, Alexa 549-conjugated with peanut agglutinin (Alexa 549-PNA), Fluo3-acetoxymethylester (Fluo3) and Pluronic F127 from MoBiTec GmbH (Göttingen, Germany). The goat antibiotin antibody (Aurion, 10 nm colloidal gold) was obtained from BIOTREND (Köln, Germany); paraformaldehyde, glutaraldehyde and LR White resin from AGAR Scientific (Stansted, UK); EPON 812, lead citrate, osmium tetroxide and uranyl acetate from Serva (Heidelberg, Germany). FITC-conjugated Pisum sativum agglutinin (FITC-PSA), BSA, CaCl2, glucose, KCl, NaHCO3, NaH2PO4, sodium lactate, MgCl2, HEPES, heparin (H3149), the Ca2+-ionophore A23187 [GenBank] , DMF and DMSO were obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany), and sodium pyruvate (L0473) and Dulbecco’s buffered salt solution (L1823) from Biochrom GmbH (Berlin, Germany).

Sperm cell preparation, capacitation and induction of acrosome reaction
Semen from Pietrain, Landrace or Duroc boars was collected and diluted with Beltsville thawing solution (BTS; Minitüb GmbH, Tiefenbach, Germany) at boar stations of the Land Brandenburg. Tubes of liquid-preserved boar semen were transported and stored at 16 °C until use within 48 h.

For capacitation in vitro, sperm cells were washed twice (6 min, 600 g) in basic Tyrode’s capacitation medium (TALP: 100 mmol NaCl/l, 20 mmol sodium lactate/l, 20 mmol NaHCO3/l, 10 mmol HEPES/l, 6 mmol glucose/l, 2.7 mmol KCl/l, 2 mmol CaCl2/l, 1 mmol sodium pyruvate/l, 0.5 mmol MgCl2/l, 0.4 mmol NaH2PO4/l, 0.6% BSA, 300 mOsmol, pH 7.4) that was adapted from Cordova et al.(2001). Before use, TALP was saturated with CO2 in a cell-culture incubator at 38.5 °C (preincubated). After washing, sperm cells were diluted to a final concentration of about 5 x 107 cells/ml in preincubated TALP and supplemented with 1.18 USP/ml heparin and 2 mmol CaCl2/l (final concentrations). Samples were then incubated for 1 h in a thermoblock at 38.5 °C. To minimize the loss of CO2 all incubations and labeling procedures were performed in closed tubes. For some experiments, capacitation was extended to 2 h and performed in a cell-culture incubator with a 5% CO2 atmosphere. The acrosome reaction was induced after 1 h capacitation by addition of the Ca2+-ionophore A23187 [GenBank] at a final concentration of 3 µmol/l, and cells were observed after 10- or 30-min incubation at 38.5 °C as indicated.

Nitrogen cavitation of sperm cells
A nitrogen cavitation under appropriate conditions specifically leads to a loss of the cellular membrane around the acrosomal region in most of the sperm cells while leaving the acrosome intact (Peterson et al. 1980, Althouse et al. 1995). By the method of Peterson et al.(1980), about 109 washed, capacitated or acrosome-reacted sperm cells were resuspended in 15 ml preincubated TALP containing 15 µl protease inhibitor cocktail from a stock (500 units/ml, P8340; Sigma-Aldrich Chemie GmbH, Tauf-kirchen, Germany). Cavitation was performed in a Parr-bomb (Parr Instrument Company, Moline, IL, USA) at 650 psi (45 bar) for 10 min on ice. After slow decompression, the cavitate was collected at 4 °C, and subsequently stained and labeled for microscopic evaluation.

Propidium iodide (PI) staining
We applied the DNA-stain PI to discriminate between viable and dead sperm cells, usually defined as PI impermeable and permeable respectively. The PI concentration per cell and volume was made as low as possible to minimize the cytotoxic effect of the dye itself, but still allow a stable viable/dead discrimination in nonfixed, in vitro capacitated boar sperm cells. Sperm cells were stained at 38.5 °C during the last 5 min of labeling with FITC-annexin V or fluorescent lectin. The PI concentration of 6 µmol/l corresponds to 2 nmol PI per 106 sperm cells. The stained cells were then transferred to microscopic slides or, immediately before flow cytometry, diluted 1:8 in measuring tubes with preincubated TALP at 38 °C to about 4 x 105 cells/ml. The resulting PI concentration in the measuring tube was 0.75 µmol/l. As shown in Fig. 1Go, a cytotoxic effect of PI is negligible under these conditions, since the proportion of PI-positive sperm cells did not increase over at least 30 min. About 1 nmol PI would be sufficient to saturate the DNA of 106 sperm cells with about 5 nmol bp (base pairs), assuming one molecule of PI per 4–5 bp (instructions of the provider), a DNA-content of 3 µg per 106 sperm cells (Anand et al. 1967) and a mean molecular mass of 600 g/mol bp (about 5 nmol bp per 106 sperm cells).



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  		Figure 1 Flow cytometric analysis of capacitated boar sperm cells after staining with propidium iodide (PI). Capacitated cells (3 x 106/ml) were stained for up to 30 min at 38.5 °C, applying concentrations of 6 or 15 µmol/l PI, and subsequently transferred to measuring tubes with preincubated TALP at 38.5 °C. The dilution was 1:8, and the resulting volume concentrations of PI during measurement were 0.75 µmol/l (open circles) and 1.88 µmol/l PI (closed circles) respectively. Cells were measured immediately after transfer to the measuring tube. For each time point, mean percentage of PI-positive (PI+) sperm cells ± S.D. of six experiments is given.

 
Annexin V staining
To detect surface exposure of endogenous PS, sperm samples were treated with FITC-labeled annexin V. A volume of 1 µl FITC-annexin V from a stock (0.25 mg/ml) was diluted in a final volume of 235 µl preincubated TALP containing 2 mmol CaCl2/l. Subsequently, an aliquot (15 µl) of sperm suspension was added and incubated for 10 min at 38.5 °C in closed tubes in the dark. After cavitation, cells were labeled for 10 min at 4 °C.

Peanut agglutinin (PNA) staining
To discriminate between plasma membrane-intact, plasma membrane-defect, acrosome-intact and acrosome-defect cells, sperm samples were treated with Alexa 549-labeled PNA, which has been described as a marker for the outer acrosome membrane (Fazeli et al. 1997). A volume of 2.5 µl Alexa 549-PNA from a stock (0.5 mg/ml) was diluted together with FITC-annexin V (as described above) in a final volume of 235 µl preincubated TALP. An aliquot (15 µl) of sperm suspension was added and incubated for 10 min at 38.5 °C in closed tubes in the dark. After cavitation, cells were labeled for 10 min at 4 °C.

Fluo3 loading
To characterize their intracellular free Ca2+ content, sperm cells were loaded with Fluo3 according to Wiesner et al.(2001). A 20% stock solution of Pluronic F127 was prepared in DMSO. An amount of 1 mg Fluo3 was dissolved in 200 µl DMF. A volume of 0.6 ml liquid-preserved boar semen was added to Fluo3 and Pluronic F127 prediluted in 1 ml Dulbecco’s buffered salt solution to give final concentrations of 4 µmol/l and 0.01% respectively. After 30 min incubation at 38.5 °C in the dark, an aliquot was diluted to about 3 x 106 cells/ml and stained for 5 min with PI before flow cytometry. The remaining Fluo3-loaded sperm suspension was divided and diluted 1:2 with preincubated TALP for capacitation or Dulbecco’s buffered salt solution as noncapacitating control. After two washings (6 min, 500 g) a final concentration of about 5 x 107 cells/ml was prepared in the respective media and supplemented with 1.18 USP/ml heparin and 2 mmol CaCl2/l (final concentrations) for capacitation, or with 2 mmol CaCl2/l (final concentration) for control. Both samples were incubated for 1 h in a thermoblock at 38.5 °C. Before flow cytometric measurement, aliquots (15 µl) were diluted with 235 µl of the respective medium and stained for 5 min with PI.

Flow cytometry
FITC-annexin V- and PI-labeled cell suspensions were gently diluted in a measuring cuvette with 1750 µl preincubated TALP containing 2 mmol CaCl2/ml, and measured in a flow cytometer (Partec GmbH, Münster, Germany) equipped with a 400 mW argon laser (Ex 488 nm), a 515–560 band-pass for FITC, and a 620 nm long-pass filter for PI. The system was triggered on the forward light scatter (FSC), and 10 000 cells per sample were characterized for their fluorescence at a flow rate of about 100–150 cells per second. Fluo3- and PI-labeled cell suspensions were diluted in a measuring cuvette with 1750 µl preincubated TALP or Dulbecco’s buffered salt solution, each containing 2 mmol CaCl2/ml, and measured in the same acquisition mode as for FITC and PI fluorescence.

Fluorescence microscopy
Labeled sperm cells were observed on wet slides with an inverse standard microscope (Olympus IX81, Hamburg, Germany) equipped with appropriate standard fluorescence facilities for green (NIBA) and red (NG) dyes at a magnification of x1000. To visualize the nonstained cells, phase-contrast (or DIC) optics were used. For each filter set, digital pictures were recorded by a Spot RT camera (Diagnostic Instruments, Sterling Heights, MI, USA) and overlayed by MetaVue Software (Universal Imaging Corp., Las Vegas, NV, USA). Cells labeled with FITC-annexin V and Alexa 549-PNA were classified by their morphology and the binding of the respective marker (FITC-annexin V and/or PNA) (Fig. 2Go). For every treatment, three independent samples with 200 cells each were evaluated for their morphology and fluorescence.



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  		Figure 2 Scheme of characteristic fluorescence patterns in the acrosomal head region of sperm cells after FITC-annexin V (green) and Alexa 549-PNA (red) binding observed microscopically. Category A: plasma membrane and normal apical ridge with pronounced optical density (NAR) are present – no staining; categories B–D: plasma membrane and NAR are present or only a smooth acrosome is present without overlaying plasma membrane – staining as indicated only for annexin V (category B), only for PNA (category C) or for both markers simultaneously (category D); categories E and F: plasma membrane or plasma and acrosome membranes are visibly expanding or detaching – staining as indicated only for PNA (category E) or both markers simultaneously (category F); category G: plasma membrane and acrosome are lost – only remaining membrane shreds are stained (exceptional staining patterns are described in the appropriate figures).

 
Electron microscopy
Freshly cavitated or capacitated sperm samples were fixed in 2.5% (w/v) glutaraldehyde, washed in PBS (pH 7.2) and fixed again in 2% osmium tetroxide. Dehydration was performed in ethanol, and samples were embedded in EPON 812 before preparation of ultrathin sections and staining with uranyl acetate and lead citrate. For detection of PS, sperm cells were exposed for 10 min to 50 µl biotin-annexin V per 108 cells (final concentration 1 mg/ml) and fixed for 30 min with Immuno-Karnovski (containing 2 mmol CaCl2/l) in TALP. After centrifugation (5 min, 1200 g), the sedimented sperm cells were fixed for 1 h in fresh Immuno-Karnovski with CaCl2. Cells were washed with PBS (pH 7.2), dehydrated in ethanol and embedded in LR White resin. Ultrathin sections were incubated for 3 min in a drop of PBS and exposed for 30 min at room temperature to a 1:5 dilution of goat antibiotin antibody coupled to 10 nm colloidal gold. Controls were prepared without annexin V (-biotin) incubation. Ultrathin sections stained as above were examined with a TEM Zeiss 902A electron microscope (80 kV; Oberkochen, Germany).

Statistical analysis
Results are expressed as mean ± S.D. with the referred number of samples or experiments. For the flow cyto-metric data, differences were analyzed by Student’s paired t-test with the indicated P value. Pearson product moment correlation was performed with the data in Fig. 7Go, using SigmaStat software (Version 2.03, SPSS Inc.).



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  		Figure 7 The percentage of FITC-annexin V-labeled and propidium iodide-positive (PI + ) sperm cells in capacitated boar semen samples measured by flow cytometry. Cells were washed and subsequently capacitated for 1 h in preincubated TALP in closed tubes in a thermo-block at 38.5 °C (closed circles) or for 2 h in a CO2 cell-culture incubator (open circles), as described in Materials and Methods.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
PS is localized on the cytoplasmic site of plasma membrane and on the outer acrosome membrane of viable liquid-preserved sperm cells
Liquid-preserved semen samples from 10 boars were washed at room temperature in BTS or capacitation medium (see Materials and Methods); subsequently, binding of FITC-annexin V was analyzed by flow cytometry. Immediately after washing in BTS, 82 ± 4% of the whole cell population were viable and not labeled by FITC-annexin V. Only 3.3 ± 4.2% of the sperm cells were annexin V positive, and most of those cells (2.6 ± 3.6%) were also stained with PI. The total amount of dead cells (PI positive) was 17 ± 4%. Similar results were obtained for TALP-washed sperm cells (Table 1Go; see also Fig. 6aGo): 5 ± 3% of the cells were annexin V positive, and 4 ± 2% were additionally stained by PI. The total fraction of dead cells after washing in capacitation medium was 12 ± 5%. Thus, viable sperm cells immediately after washing in BTS or TALP did not expose PS on their surface. Appearance of PS on the cell surface was detected for only some of the PI-positive cells.


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  		Table 1 Development of FITC-annexin V and propidium iodide (PI) staining in boar sperm cells in the course of capacitation and acrosome reaction
 


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  		Figure 6 Flow cytometric analysis of boar sperm cells after staining with FITC-annexin V and propidium iodide (PI). Two-parameter dot plots are shown with the FITC-annexin V fluorescence (green) and PI fluorescence (red) after gating sperm cells in the scatter plot. Liquid-preserved cells were washed (a) and capacitated for 1 h in TALP at 38.5 °C, as described in Materials and Methods. The staining pattern after capacitation is shown for five different boars (b–f).

 
To support these findings, we studied sperm cells on wet slides by fluorescence microscopy. In analogy to flow cytometric investigations, we used PI as dead cell marker and binding of FITC-annexin V as a marker for PS. In the samples investigated, we never observed FITC-annexin V fluorescence for PI-negative sperm cells. Even most of the PI-positive sperm cells did not show FITC-annexin V binding (images not shown).

To assess the intracellular organization of PS, particularly its presence in the outer acrosome membrane, nitrogen cavitation of liquid-preserved boar sperm cells was performed immediately after washing in TALP. This treatment is known to remove specifically the sperm head plasma membrane covering the acrosome (Peterson et al. 1980, Flesch et al. 1998). We simultaneously incubated the cells with FITC-annexin V and fluorescently (Alexa 549) labeled peanut agglutinin (PNA). PNA has been described as a marker for the outer acrosome membrane. Therefore, labeling of cells with PNA indicates an access to the outer acrosome membrane caused by a defect or a removed plasma membrane. Before cavitation, about 1% of those control cells were solely labeled by FITC-annexin V (Fig. 2Go, category B), 5% of cells were solely stained with PNA (category C) and up to 10% of the cells were stained with both FITC-annexin V and PNA (category D, images not shown). Thus, FITC-annexin V binds only to a very small fraction of liquid-preserved cells with a membrane defect detected by PNA.

Upon nitrogen cavitation, we observed, by phase or interference contrast optics, that the apical ridge had lost its sharp contrast. The loss of the plasma membrane reduced the optical density in that region (Fig. 3aGo, categories B, C and D according to Fig. 2Go). In many cases, the acrosome appeared to be expanded after cavitation (Fig. 3aGo, categories E and F). Upon nitrogen cavitation of liquid-preserved boar sperm cells, 80 ± 20% of the cells were FITC-annexin V positive. Importantly, those cells always simultaneously bound PNA, reflecting the accessibility of the outer acrosome membrane (Fig. 3aGo, categories D and F). Therefore, we conclude, that the outer acrosome membrane contains PS that became accessible to FITC-annexin V after nitrogen cavitation. Cavitation also increased the fraction of cells with a completely lost acrosome to 14 ± 13% (Fig. 3aGo, category G). These egg-cup-like sperm heads showed no fluorescence, and only loosely attached membrane fragments were stained by annexin V and/or PNA.



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  		Figure 3 Characteristic fluorescence patterns of FITC-annexin V and Alexa 549-PNA in differentially treated boar sperm cells, as indicated in the figure. Categories A–G correspond to the scheme in Fig. 2Go. Data represent mean percentages of cells in the corresponding category ± S.D. of three experiments with 200 counted cells each. Images were taken as described in Materials and Methods (x1000). Morphology was evaluated under differential interference or phase-contrast optics (left); green and red fluorescence images were superimposed (right) by MetaVue Software (Universal Imaging Corporation).

 
Electron microscopic images confirmed that cavitation of fresh cells removed the cell membrane above the acrosome, leaving most acrosomes intact but slightly swollen. In accordance with binding of FITC-annexin V, gold-labeled antibodies against biotin-annexin V, which were added before fixation, stained the outer site of the acrosome (Fig. 4aGo). This confirms the presence of PS on the outer acrosome membrane.



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  		Figure 4 Electron microscopic images of uncapacitated but cavitated sperm cells (a) and capacitated sperm cells (b–e). Samples a and b were treated with biotin-annexin V before fixation, and a gold-labeled goat antibiotin antibody was used. Nitrogen cavitation, capacitation and sample preparation were performed as described in Materials and Methods. The bar represents 500 nm. Inserts enlarge characteristic details of the respective image.

 
PS does not become surface exposed in viable sperm cells during capacitation
To validate the capacitation state of boar sperm cells, we applied the Ca2+-sensitive dye Fluo3. Besides a hyperactivated motility, we observed in PI-negative (viable), capacitated sperm cells a gradual increase in the intracellular free Ca2+-content with time (Fig. 5Go). At 15, 30 and 60 min after washing in TALP, the fraction of viable sperm cells with a high Ca2+ content was significantly enlarged compared with control cells, which had been washed and incubated in a noncapacitating medium (P < 0.05). About 68% of the viable sperm cells were counted as ‘high Ca2+ cells’ after 60-min capacitation. In controls, we found only 9% of the viable cells with high Ca2+ content. The presence of Fluo3 and Pluronic F127 during capacitation severely affected the viability of sperm cells. We observed a decrease of the amount of PI-negative sperm cells to 30 ± 13% after 60 min capacitation compared with 62 ± 8% PI-negative cells in the absence of Fluo3 and Pluronic F127 (see FITC-annexin V-labeling experiments, Table 1Go).



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  		Figure 5 Changes in intracellular free Ca2+ content measured flow cytometrically with Fluo3 in combination with PI, as described in Materials and Methods. The percentages of viable (PI negative) cells with low (open symbols and bar) and high (closed symbols and bar) Ca2+ content are shown after loading with the dye in Beltsville thawing solution (BTS) (bar) and subsequent washing and incubation in noncapacitating (circles) and capacitating medium (squares).
Means ± S.D. of five different boar semen samples are given.

 
The percentage of FITC-annexin V-positive sperm cells increased continuously for 60 min after washing in capacitation medium (Table 1Go). Capacitation for 1 h caused a significant increase in the number of FITC-annexin V-stained but also of PI-positive (nonviable) cells compared with the samples immediately after washing in TALP or BTS. Again, most of the FITC-annexin V-stained cells were PI positive. Only 6 ± 5% of the whole sperm population bound exclusively FITC-annexin V, whereas a large proportion of the cells (56 ± 11%) was found to be viable and labeled by neither FITC-annexin V nor PI. Repeated observation of samples by fluorescence microscopy confirmed that PS was not surface exposed in PI-negative sperm cells during 1-h capacitation (images not shown).

Figure 6aGo shows characteristic flow cytometric patterns for a sperm sample immediately after washing in TALP, and Fig. 6b–fGo shows five samples after 1-h capacitation. The FITC-annexin V/PI-staining pattern of semen varied between animals. In total, sperm cells of 33 boars were measured after 1 h capacitation. Figure 7Go shows the fractions of FITC-annexin V- and PI-stained cells for each sample. Again, the variability between the samples is evident. The fractions of PI-positive and annexin V-positive sperm cells were closely correlated (Pearson correlation, P = 0.005). Thus, PS exposure correlates with the fraction of nonviable, capacitated sperm cells.

We extended capacitation up to 2 h under 5% CO2 atmosphere in a cell-culture incubator to ensure the supply of CO2. Under those conditions, a significant increase of the fraction of dead sperm cells (from 29 ± 12% after 1 h to 36 ± 14%; P = 0.034) was accompanied by a moderate enhancement of FITC-annexin V-labeled cells (from 19 ± 13% after 1 h to 25 ± 8%). FITC-annexin V binding to viable sperm cells was similarly rare for both treatments (8 ± 5% and 9 ± 6% respectively). All boars from which samples were taken provided fertile semen, as deduced from artificial insemination (unpublished data).

Almost all (97%) (Fig. 3bGo, categories A–D) of the capacitated sperm cells were morphologically intact, as deduced from phase or differential interference contrast microscopy. About half of the cells (49 ± 9%) (Fig. 3bGo, category A) showed neither FITC-annexin V nor PNA binding. About 26 ± 3% of the sperm cells showed FITC-annexin V binding to heads (Fig. 3bGo, categories B, D and F). This number agrees well with the total fraction of FITC-annexin V-positive cells detected by flow cytometry after capacitation. PNA binding indicates membrane injuries in 10 ± 2% of the cells (category D), and this was not observed for the remaining FITC-annexin V-positive cells (16 ± 4%) (category B). However, costaining experiments with PI indicated that cells with an obvious exposure of endogenous PS on the cell surface were dead. Electron microscopy confirmed limited labeling of capacitated sperm cells by annexin V. Gold-labeled antibodies against biotin-annexin V were found occasionally on sites of membrane bending, as shown in Fig. 4bGo. Moreover, loosely bound or detaching membranes showed labeling by gold particles (not shown).

Acrosome-reacted sperm cells do not expose PS
Contact with the zona pellucida triggers the acrosome reaction of capacitated sperm cells in vivo. A crucial step in the signal cascade that leads to the fusion of cell and acrosome membrane, and finally the release of acrosomal contents, is a sharp rise in intracellular sperm Ca2+ concentration. It has been shown that the Ca2+-ionophore A23187 [GenBank] induces the acrosome reaction in boar sperm cells (Aguas & Pinto Da Silva 1989). In combination with PI, the application of FITC-conjugated Pisum sativum lectin (FITC-PSA), which selectively binds to glycoprotein structures of the acrosomal contents, allows the determination of viable, acrosome-reacted sperm cells (Mattioli et al. 1996). When capacitated cells after 1 h were treated with the Ca2+-ionophore for 30 min, flow cytometry revealed an increase of viable, acrosome-reacted cells (PI negative, PSA positive) from 8 ± 4% to 42 ± 14% (data not shown). Hardly any FITC-annexin V binding was visible in viable or nonviable, acrosome-reacted sperm cells (Table 1Go).

Flow cytometry data were strongly supported by fluorescence microscopy observations. In acrosome-reacted sperm cells, FITC-annexin V binding was rare and restricted to membrane shreds on cells of category G (Fig. 3dGo). About 58 ± 19% of the cells passed the acrosome reaction within 30 min after addition of the Ca2+-ionophore (Fig. 3dGo, category G). No staining of those egg-cup-like cells was observed, in agreement with the loss of the plasma and outer acrosome membranes. About 27 ± 14% of sperm cells showed detaching membranes. These cells were stained only by PNA (category E).

Interaction between the plasma membrane and outer acrosome membrane during capacitation
A remarkable proportion of capacitated sperm cells (22 ± 4%) bound PNA, but not FITC-annexin V (Fig. 3bGo, category C). This was the case in only about 5% of sperm cells immediately after washing (as stated above). The absence of FITC-annexin V binding suggests that the plasma membrane was intact and FITC-annexin V had access neither to PS of the plasma membrane nor to PS of the outer acrosome membrane. Moreover, phase/interference contrast microscopy supports a morphologically intact appearance of the plasma membrane, whereas binding of PNA demonstrates its access to the outer acrosome membrane via plasma membrane injuries. Since the molecular mass of annexin V (about 35 kDa) is much smaller than that of PNA (about 110 kDa), we can exclude steric hindrance of the annexin V entry through those plasma membrane injuries. Similar results were obtained with annexin V bearing Alexa 549 as fluorescent moiety in combination with FITC-PNA, and independently of the order of label application (images not shown).

To clarify this unexpected labeling pattern of capacitated sperm cells, electron microscopic studies were done, revealing different morphologic structures for the head membranes of capacitating sperm cells. Besides cells with an intact head structure, we often observed strong membrane bending with patches of close apposition between the plasma and the outer acrosome membrane (Fig. 4cGo). Furthermore, we found areas of hole-like structures of the plasma membrane with tight contact to the outer acrosome membrane (Fig. 4dGo). We surmise that PNA has access to the outer acrosome membrane through those hole-like structures. However, the absence of FITC-annexin V binding indicates that PS is not exposed in that region. Premature acrosome reactions with vesicles obviously originating from both the plasma and outer acrosome membrane were also observed (Fig. 4eGo). These results indicate an interaction between the plasma membrane and the acrosome during capacitation not accompanied by exposure of PS. Evidence of an interaction came from the observation that after cavitation of capacitated cells, we typically did not observe a removal of the plasma membrane leaving the outer acrosome membrane intact (categories C and D). Essentially, only cells belonging to categories B, E, F and G were found (Fig. 3cGo).

Cytoplasmic PS pattern in the postacrosomal region
FITC-annexin V did not bind to the postacrosomal region of freshly TALP-washed boar sperm cells. However, this region was labeled by FITC-annexin V for 15 ± 8% of 1 h capacitated boar sperm cells. Like the PS exposure in the acrosomal region, the spontaneous exposure of PS in the postacrosomal part of the plasma membrane occurred only in nonviable (PI positive), capacitated sperm cells. When capacitated sperm cells were cavitated, FITC-annexin V binding to the postacrosomal region was observed in 63 ± 12% cells. (Notably, similar results were found after solely cooling capacitated cells to 4 °C.) The postacrosomal binding pattern varies somewhat and can be described principally as (i) a smaller, sharp or (ii) a broader, stained ring, both mostly in combination with (iii) a stained head–tail connection (Fig. 3cGo). For washed and immediately cavitated samples, a postacrosomal FITC-annexin V binding pattern was found for only 21 ± 13% of the cells. Presumably, capacitation induced postacrosomal membrane changes that lead to a higher fragility and finally to the perturbation or removal of the postacrosomal plasma membrane by cavitation treatment.

The postacrosomal region of acrosome-reacted boar sperm samples was labeled by FITC-annexin V in 6 ± 10% of cells. Only after nitrogen cavitation of those sperm samples was FITC-annexin V binding to the postacrosomal region observed for the whole cell population (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
One of the essential properties of mammalian plasma membranes is the maintenance of a transversal lipid asymmetry with the majority of glycosphingolipids and PC typically in the outer, exoplasmic leaflet, and the aminophospholipids PS and PE in the inner, cytoplasmic leaflet (for review, see Zachowski 1993, Devaux & Zachowski 1994). The asymmetric lipid distribution across the plasma membrane is regulated by the concerted action of specific membrane proteins controlling lipid movement across the bilayer, in addition to passively occurring transmembrane movement of lipids (for review, see Pomorski et al. 2004). For example, the aminophospholipids PS and PE are rapidly transported from the exoplasmic to the cytoplasmic leaflet by an active, ATP-dependent and protein-mediated process maintaining lipid asymmetry (Seigneuret & Devaux 1984, Daleke & Lyles 2000, Wang et al. 2004).

Maintenance of this nonrandom lipid distribution is important for diverse cellular functions. Any change in this distribution generally has a physiologic consequence. For example, exposure of PS at the surface of activated blood or endothelial cells serves to promote blood coagulation or to signal the removal of injured and apoptotic cells by the reticuloendothelial system (Zwaal & Schroit 1997). For intra- and intercellular fusion processes, the localization of PS at the fusion-competent membrane faces is also considered to play an essential role (for review, see Herrmann et al. 1991, Williamson & Schlegel 1994, Devaux 2000).

Freshly ejaculated sperm cells of different species are characterized by such a stable phospholipid asymmetry based on the activity of an aminophospholipid translocase (Müller et al. 1994, Nolan et al. 1995, Gadella et al. 1999). Very recently, Wang et al.(2004) reported an aminophospholipid translocase that is exclusively expressed in the acrosomal region of mouse sperm cells. The activity of this protein is thought to ensure the localization of PS almost exclusively to the cytoplasmic leaflet of the sperm plasma membrane. Therefore, we observed a very rare binding of FITC-annexin V to liquid-preserved, washed boar sperm cells, which remained alive under these storage conditions for several days. This binding was confined to a small population of nonviable cells identified by PI staining.

So far, nothing is known about the transversal organization of phospholipids, in particular PS, in the acrosome membrane of (fresh) sperm cells. By applying nitrogen cavitation to liquid-preserved sperm samples, we were able to assess the intracellular organization of PS. Nitrogen cavitation caused destruction and removal of the plasma membrane in the acrosomal region, uncovering the outer acrosome membrane. As indicated by PNA labeling, the acrosome and its outer membrane were mostly preserved by this approach. Costaining of FITC-annexin V with PNA clearly showed that the outer acrosome membrane harbors PS, probably on the cytoplasmic leaflet. The localization of PS on the outer acrosomal face and on fragments of detaching membranes could be confirmed by electron microscopy. These data indicate that the cytoplasmic leaflets of the two facing membranes –the acrosome and the plasma membrane – contain the aminophospholipid PS. Taking into account the specific properties of the PS head group, a cytoplasmic orientation of PS might be of significance for the interaction and/or fusion between the plasma and acrosome membrane during capacitation and acrosome reaction (see below). At the present state of investigation, we do not know whether an aminophospholipid translocase activity realizes a cytoplasmic localization of PS in the outer acrosome membrane. However, there are strong indications that such a transport activity is also present in subcellular membranes of mammalian cells as in the late Golgi apparatus (Holthuis et al. 2003). Notably, the sperm acrosome has been suggested to originate from the Golgi apparatus during cell genesis (Abou-Haila & Tulsiani 2000).

During capacitation of sperm cells, a large fraction of cells remained viable (PI negative). The majority of these cells did not expose PS as judged from the absence of FITC-annexin V binding. Therefore, the transverse phospholipid asymmetry in the plasma membrane was maintained in most sperm cells that remained alive under capacitation conditions, suggesting that the aminophospholipid translocase was still active. Indeed, with use of the fluorescent phosphoplipid analog NBD-PS, which is a substrate of the aminophospholipid translocase, only a slight decrease of the rate of the inward transport of the lipid analog was observed in viable capacitated boar sperm cells: the final asymmetric distribution was not affected (Kurz & Müller, unpublished results). This observation is in accordance with previous findings, using the same approach, of Gadella & Harrison (2000) on boar sperm cells. Those studies also showed that the translocase activity in PI-stained sperm cells is abolished, and thus cannot prevent redistribution of NBD-PS to the exoplasmic leaflet by passive flop (Müller et al. 1999). However, passive phospholipid flop is slow, a fact which may also explain why not all dead sperm cells were labeled by FITC-annexin V. The latter observation has also been made for uncapacitated and capacitated mouse sperm cells (Wang et al. 2004).

The increasing fraction of FITC-annexin V-binding boar sperm cells during capacitation correlated with the amount of PI-positive spermatozoa. In contrast, the number of viable cells exposing PS was not significantly increased. This is in accordance with the results of Muratori et al.(2004), who did not detect an increased annexin V binding in viable human sperm cells upon capacitation. In mice, a small population of viable PS-exposing sperm cells during capacitation was detected (Wang et al. 2004). In contrast, Gadella & Harrison (2002) found a much more frequent binding of FITC-annexin V to capacitated boar sperm cells that were scored as viable. We have no satisfying explanation of this discrepancy. While only 15% of the PI-stained, uncapacitated (liquid-preserved) boar sperm cells exposed PS, PS was accessible in about 45% of the nonviable sperm cells after 1 h capacitation. This comparatively fast PS exposure upon capacitation could be a process different from cellular death during sperm storage. Although we were able in all of our experiments to define clearly a viable sperm cell population, the fraction of capacitated, PS-exposing cells may represent a scrambling and very fragile subpopulation that did not survive the experimental procedures, particularly of microscopy, and therefore was counted ‘falsely’ as dead. From electron microscopic images, we occasionally observed membrane shedding and biotin-annexin V-labeling on membrane pullouts involving probably both the plasma and outer acrosome membrane (Fig. 4bGo). Those alterations of membrane structure might be a consequence of lipid scrambling. Lipid scrambling and membrane shedding in a certain fraction of cells could represent an alternative to or complementary mechanism of the classical zona pellucida-induced acrosome reaction in order to release acrosomal contents.

As already discussed, for the major population of viable fresh and capacitated boar sperm cells, we observed a localization of endogenous PS on the cytoplasmic leaflet of the sperm plasma membrane and on the opposite acrosome membrane. However, surprisingly, for a significant fraction of viable capacitated sperm cells, we found accessible PNA-binding sites on the acrosome membrane, although PS was not accessible to FITC-annexin V. Electron microscopic images of capacitated sperm samples revealed connective areas between the plasma and outer acrosome membrane. The strong interaction between both membranes is supported by experiments which showed that, upon cavitation of capacitating cells, the integrity of those putative connective structures was only partly disrupted. Thus, capacitated sperm cells labeled solely by PNA, but not by annexin V, could represent an intermediate stage during acrosome reaction. In this context, we found it an interesting observation that a large fraction of viable sperm cells from mice with disrupted aminophospholipid transporter gene did not expose PS on their surface, even after capacitation (Wang et al. (2004). Based on electron microscopic studies, Peterson et al.(1987) have already suggested that the outer acrosomal membrane at the apical rim of the boar sperm head might be rich in acidic phospholipids that could provide the sites for initiating the acrosome reaction. PS belongs to the fusogenic lipids whose structure promotes the creation of fluid and fusogenic membrane areas, and whose negative charge promotes the Ca2+-mediated close relation of membranes (for comprehensive review, see Herrmann et al. 1991). Indeed, exocytotic events in chromaffin or neurosecretory cells require anionic phospholipids selectively located at the interacting (cytoplasmic) faces of vesicle and plasma membranes (Buckland et al. 1978, Westhead 1987, Zachowski et al. 1989, Devaux & Zachowski 1994, De Haro et al. 2004). Moreover, proteins such as synexin in chromaffin cells or SNARE proteins in neurosecretory cells, which – modulated by Ca2+ – participate in exocytotic membrane approach and fusion, were shown to need preferentially acidic phospholipids at the cytoplasmic site of the complementary membranes for binding (Scott et al. 1985, Pollard et al. 1988, Quetglas et al. 2002, De Haro et al. 2004). However, further studies are warranted to understand the role of PS in priming sperm cells for fertilization.

Upon completion of the acrosome reaction (triggered by Ca2+-ionophore treatment), we observed binding of neither FITC-annexin V nor PNA. This suggests that the releases of the plasma membrane and the outer acrosome membrane are closely related and supports the view of a strong interaction between both membranes, including their merging in the course of acrosome reaction. Indeed, the clots of vesicles observed after acrosome reaction showed staining by PNA and/or annexin V (images not shown). Therefore, a fusion-related reorganization/scrambling of phospholipids could have occurred.

While PS was not exposed on the outer surface of the majority of acrosome-reacted sperm cells, this lipid was present in distinct internal postacrosomal structures, in the intracellular postacrosomal region and in the connective piece between head and tail. A binding of annexin V to that region was possible only when the cells were treated with nitrogen cavitation. The same pattern of PS was found for almost all acrosome-reacted sperm cells after cavitation or cooling to 4 °C. An interesting hypothesis is that PS contributes to the stabilization of the postacrosomal region during the subsequent fertilization process. Previous studies have already shown the specific structure of this region. Yagi and Paranko (1995) detected cyto-skeletal actin in the postacrosomal segment of epididymal bull semen that was not extractable by incubation with Triton X-100. Castellani-Ceresa et al.(1992) found F-actin also after acrosome reaction in the respective postacrosomal cytoplasmic regions. Studies on SNARE proteins have shown that these proteins are confined to the equatorial region after acrosome reaction of several mammalian sperm cells (Ramalho-Santos et al. 2000). The cooperative interaction of endogenous PS with proteins and/or cyto-skeletal elements remains to be proven in future studies.

Our study demonstrates that viable boar spermatozoa, whether freshly washed after liquid preservation, capacitated or acrosome reacted, do not expose PS on their outer membrane surface. Surface-exposed PS could serve as a signal for the removal of dead sperm cells in the epididymis or in the first parts of the female genital tract. Upon capacitation, a subpopulation of PS-exposing cells, mainly stained by PI, appears and may represent a highly fragile fraction of sperm cells with scrambled plasma membrane lipids. Their relevance to fertilization remains to be studied. The functional role of the high activity of the aminophospholipid translocase in fresh and capacitated cells (Gadella & Harrison 2000, Kurz & Müller, unpublished results) may be (at least) two-fold. First, it is important to maintain the exclusive localization of PS in the cytoplasmic leaflet in the sperm plasma membrane, thus, preventing the removal of viable cells from the genital tract. Secondly, the accumulation of PS on the cytoplasmic leaflet ensures optimal conditions for the exocytotic fusion process of the acrosome reaction and for postacrosomal stabilization.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We gratefully acknowledge the advice from Gudrun Wibbelt (Institute for Zoo Biology and Wildlife Research, Berlin, Germany) on electron microscopy as well as the support in routine sperm evaluation by Anita Retzlaff and Karen Reguszynski (Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V., Schönow). This work was supported by a grant from the Deutsche Forschungsgemeinschaft (A H and K M).


   Footnotes
 
Received 8 November 2004
First decision 19 January 2005
Revised manuscript received 30 June 2005
Accepted 14 July 2005


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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