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Gamete Biology |
Institute for Biomembranes,3 Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Graduate School of Animal Health,4 Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
Department of Molecular and Experimental Medicine,5 The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, apoptosis, cyclic adenosine monophosphate, sperm capacitation, sperm maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In analogy to boar sperm cells, we report here, to our knowledge for the first time, on PL asymmetry in human sperm cells. The PKA-dependent exposure of PE and PS in human sperm cells is investigated. Bicarbonate-driven PL scrambling is shown to closely correlate with capacitation markers such as tyrosine phosphorylation of tail proteins and the incidence of capacitation-specific chlortetracyclin (CTC) staining. Importantly, no relation was found between the bicarbonate-stimulated PL scrambling and the presence and/or increase of caspase activity and/or damage to mitochondria or DNA. Therefore, this scrambling response appears to be independent from apoptosis. Also novel is the report on the presence of a 25-kDa isoform of PL scramblase (PLSCR) in human sperm cells. It has been proposed that activation of PLSCR enhances the bidirectional transbilayer movement of PL [14] in membranes without headgroup specificity [15]. Its possible participation in bicarbonate/PKA-mediated PL scrambling in human sperm cells is discussed. Correlation of bicarbonate-induced PL scrambling in the plasma membrane of the sperm head with downstream signaling also is discussed in relation to capacitation-dependent, albumin-mediated cholesterol efflux and tyrosine phosphorylation, both of which are thought to be important in generation of affinity to the zona pellucida and preparation of the acrosome reaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monoclonal antibodies 4D2 and 219.1 were raised against human PLSCR1. The epitope for 4D2 is located within the fragment of Met1–Trp85 [16, 17] and that of 219.1 within Met86–Glu118 [17]. Polyclonal antibody C-term was raised against Glu306–Trp318 of human PLSCR1 [14, 18]. These anti-PLSCR antibodies do not recognize PLSCR3 or PLSCR4. The PLSCR1 was purified from human erythrocytes [19]. Recombinant MBP-PLSCR2 fusion protein [20] was incubated with factor Xa (Promega, Madison, WI; MBP-PLSCR2:factor Xa ratio, 100:1) to cleave of MBP. Monoclonal antibodies against p53 and heat shock protein 70 (Hsp 70) were from BD Transduction Laboratories (Lexington, KY), those against 
-tubulin from Sigma Chemical Company (St. Louis, MO), and those against phosphotyrosine (PY20) from Calbiochem (San Diego, CA). Polyclonal antibodies against active and inactive caspase-3 were from R&D Systems (Minneapolis, MN) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Alkaline phosphatase-conjugated secondary mouse immunoglobulin (Ig) G+IgM, rabbit IgG, and enhanced chemifluorescence (ECF) substrate were purchased from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L), goat anti-rabbit IgG (H+L), and rabbit anti-goat IgG (H+L) were from Molecular Probes (Eugene, OR). Annexin V-FITC was a generous gift from Dr. C.P. Reutlingsperger (Academic Hospital, Maastricht, The Netherlands), and Ro-09-198 streptavidin fluorescein was donated by Dr. M. Umeda (Rinshoken, Tokyo, Japan). The R-phycoerythrin peanut agglutinin (RPE-PNA) was conjugated as described before [6], Mitotracker red, Acridine Orange, Yo-Pro, and propidium iodide (PI) were from Molecular Probes. The TUNEL apoptotic cell death fluorescein detection kit for flow cytometry was from Roche Diagnostics GmbH (Mannheim, Germany). The GPO (pasteurized plasma-protein solution) was obtained from the Central Laboratory of the Red Cross Blood Transmission Services (Amsterdam, The Netherlands). Bovine serum albumin and chlortetracyclin were from Sigma; BSA was defatted as described previously [21].
Sperm Incubations
Human semen was obtained from healthy volunteers by masturbation at the IVF Clinic of the University Medical Centre Utrecht (Utrecht, The Netherlands). Samples were allowed to liquefy at room temperature for 2 h. Semen was diluted to 15 x 106 cells/ml in saline medium containing 137 mM NaCl, 2.5 mM KCl, 10 mM glucose, and 20 mM Hepes (pH 7.4) and washed through isotonic Percoll as described previously [10]. Washed spermatozoa were diluted up to 107 cells/ml in Hepes-buffered Tyrode medium (HBT-control: 120 mM NaCl, 21.7 mM lactate, 20 mM Hepes, 5 mM glucose, 3.1 mM KCl, 1.0 mM pyruvate, 0.4 mM MgSO4, 0.3 mM NaH2PO4, 1 mg/ml of kanamycin, 2 mM CaCl2, 3 mg/ml of BSA, 300 mOsm/kg, pH 7.4). The HBT-control with isotonic addition of 15 mM bicarbonate (final concentration) was further designated as HBT-Bic [9]. For comparative purposes, Percoll-washed human sperm cells were diluted in human tubular fluid (HTF-control: 100 mM NaCl, 20 mM Hepes, 21.4 mM lactate, 2.8 mM glucose, 4.7 mM KCl, 0.2 mM MgSO4, 1.0 mM pyruvate, 0.37 mM KH2PO4, 100 mg/ml kanamycin, 2 mM CaCl2, 10% [v/v] GPO [containing human serum albumin], 300 mOsm/kg, pH 7.4). The HTF-control was also isotonically supplemented with 24 mM bicarbonate (HTF-Bic) [22].
Sperm cells were incubated up to 4 h in control media (HBT-control, HTF-control) at 37°C in air-tight, closed tubes under normal humidified atmosphere or capacitated up to 4 h in HBT-Bic or HTF-Bic at 37°C and in a 5% CO2 humidified atmosphere. Effectors and inhibitors were included in these media as described in Results.
Annexin V Staining
Sperm cells were labeled for 5 min at 37°C under 5% CO2 with 1 µg/ml of annexin V-FITC to detect exposure of PS, 1 µg/ml of RPE-PNA to discriminate acrosome-intact from acrosome-deteriorated cells, and 25 nM PI to discriminate between live and dead cells. This incubation condition was kept during sampling when labeled sperm suspension passed through a FACScalibur flow cytometer equipped with a 100-mW argon laser (Becton Dickinson, San Jose, CA) that was pre-equilibrated at 37°C. Only sperm-specific scatter events were gated for further analysis. Sperm cells with red fluorescence (detected on FL-3 at 620-nm long-pass emission photo multiplier tube [PMT]) were considered to be deteriorated and excluded from analysis [6]. Annexin V-FITC was detected on FL-1 with a 530/30-nm bandpass emission filter. Two dimensional dot-plots were made with FL-1 data expressed on the x-axis and FL-3 data on the y-axis. Computer-generated boundaries were set to define the sperm subpopulation that did or did not expose PS, and 10 000 events corresponding exclusively to live acrosome-intact sperm cells were collected. Labeled sperm suspension was transferred to a microscopical life sample chamber (equilibrated at 37°C and 5% CO2 in humidified atmosphere) and placed on an inverted spectra confocal microscope (Leica TCS-SP, Heidelberg, Germany) with objectives prewarmed at 37°C. Annexin V-FITC labeling was captured in live, acrosome-intact sperm cells using single 488-nm excitation scans that were obtained with a 63x APO-water immersion objective in PMT-1 that selectively recorded emitted photons of a wavelength of 510- to 540-nm (red fluorescence from RPE-PNA and PI were detected in PMT-2 in the emission range of 580–700 nm).
Ro-SA-FITC Staining
The Ro-SA-FITC staining procedure was similar to that described for annexin V-FITC staining; however, 0.5 µg/ml of Ro-SA-FITC was used to detect exposure of PE (instead of annexin V-FITC for PS exposure). The PE exposure was analyzed on live, acrosome-intact sperm cells using flow cytometry and confocal laser-scanning microscopy as described above.
CTC Staining
Chlortetracyclin staining was essentially performed as previously described [23] following the protocol of DasGupta et al. [24]. Briefly, 25 nM PI was used as a counterstain for dead cells instead of ethidium homodimer 1. Stained sperm specimens were placed on an inverted spectral confocal microscope (Leica TCS-SP), and CTC staining was imaged using the 458-nm excitation line of the argon laser. Excitation scans were obtained with a 63x APO-water immersion objective in PMT-1 that selectively recorded emitted photons of a wavelength of 495–535 nm (red fluorescence from PI was detected in PMT-2 using the 568-nm excitation line of the krypton laser, and emitted photons were detected in the emission range of 630–700 nm). After sperm treatment and CTC staining, four slides of each condition were made, and in each slide, 200 cells were counted in 12 randomly selected fields (25 cells/field). Uniform CTC staining over the entire sperm surface was classified as pattern F (uncapacitated sperm), sperm with lower CTC staining at the postacrosomal surface of the head as pattern B (capacitated sperm), and sperm with lower CTC staining in the apical part of the head as pattern AR (acrosome-reacted cells). All cells with red fluorescence (PI positive) were classified as dead cells (see also DasGupta et al. [24]).
Preparation of Human Sperm Protein Samples
Percoll-washed sperm cells were resuspended in solubilization buffer (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM EGTA, 1 mM EDTA, 1% [w/v] SDS, and 1% [v/v] Triton X-100 containing a complete EDTA-free protease-inhibitor cocktail [Roche Diagnostics]) and subjected to sonication (four 5-sec bursts, 15-µm peak-to-peak amplitude; MSE Ultrasone Desintegrator, Soniprep 150; MSE Ltd., Crawley, U.K.). To solubilize membrane proteins, sperm cells were incubated for 14 h at 4°C under continuous rotation. The suspension was centrifuged at 14 000 x g and 4°C for 30 min, after which the resulting supernatant was taken and the protein concentration determined as described previously [25].
Immunoprecipitations
For immunoprecipitations, 250 µg of solubilized human sperm proteins were used. Samples were diluted 10-fold with solubilization buffer without SDS, and antibodies (final concentration, 10 µg/ml) were added and incubated for 14 h at 4°C under continuous rotation. Protein A sepharose CL 4B (Amersham Pharmacia Biotech) was added, and the incubation was extended for at least 4 h. After extensively washing the protein complex with ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4, and 1.5 mM KH2PO4), the pellet was resuspended in 25 µl of Laemmli buffer (66 mM Tris/HCl [pH 6.8], 3% [w/v] SDS, 5% [v/v] glycerol, 2% [v/v] ß-mercaptoethanol, and 0.001% [w/v] bromophenol blue [26]) and boiled for 10 min before separation by SDS-PAGE.
SDS-Gel Electrophoresis and Western Blot Analysis
Proteins were separated by SDS-PAGE using 11% polyacrylamide gels [26]. The proteins were wet blotted onto polyvinylidene difluoride membranes for Western blot analysis. After blocking for 4 h at room temperature with TBSt (20 mM Tris, 0.5 M NaCl, and 0.05% [v/v] Tween-20, pH 7.4) containing 5% (w/v) dry milk and 5% (v/v) normal goat serum, the membranes were incubated with primary and, subsequently, alkaline phosphatase-conjugated secondary antibodies in TBSt (final concentrations, 1 and 0.1–0.2 µg/ml, respectively). After every single incubation step, the membranes were washed four times for 10 min each in TBSt. The Western blots were detected using ECF on a Molecular Dynamics FluorImager STORM 860 (Amersham Pharmacia Biotech) at 450 nm.
Immunofluorescence Microscopy
Incubated sperm cells were fixed in PBS supplemented with 2% (w/v) p-formaldehyde and 0.4% (w/v) polyvinyl pyrrolidone and polyvinyl alcohol for 1 h. Sperm cells (5 x 105) were air-dried on microscope slides and permeabilized in methanol for 1 min. Cells were washed twice in washing buffer (PBS containing 0.05% [v/v] Tween-20) and incubated for 2 h with primary antibodies (final concentration, 10 µg/ml) in antibody buffer (washing buffer containing 1% [w/v] BSA and 5% [v/v] normal goat serum). After washing, the cells were incubated for 2 h with Alexa 488-conjugated secondary antibodies (final concentration, 20 µg/ml) in antibody buffer. After washing, the slides were supplied with two drops of FluorSave Reagent (CalBiochem), and cells were sealed with a coverslip. Sperm cells were imaged using a Leica TCS-SP inverted spectral emission confocal laser-scanning microscope and Leica Confocal Software (488-nm argon laser excitation line, detection of emission wavelength in the range of 510–540 nm) as described above. As a control for the specificity of the primary antibodies, all immunofluorescent-labeling experiments were performed as described above without use of primary antibodies. All these controls showed no detectable fluorescent label (data not shown).
Sperm Sorting for Immunofluorescence Microscopy
Sperm samples were incubated in HTF-Bic for 4 h and stained with Ro-SA-FITC, RPE-PNA, and PI as described above. Sperm suspensions were kept at 37°C in tubes that were pressurized with a humidified atmosphere of 5% CO2 and sorted in a FACS Vantage SE (Becton Dickinson) with optical settings comparable to the FACScalibur settings described above. The flow cytometer was pre-equilibrated at 37°C to avoid thermal stress of the sperm cells during sorting. Three subpopulations of sperm cells were sorted: 1) total population of live sperm cells, 2) live subpopulation of sperm cells that showed no PE exposure, and 3) live subpopulation of sperm cells that showed PE exposure. Next, 15 000 cell events were sorted immediately into 0.2 ml of fixative (4% [w/v] glutaraldehyde in PBS) at room temperature. Smears of this sorted suspension were made on microscopic slides. These slides were processed for immunofluorescence microscopy as described above. In a parallel experiment, approximately 500 000 cells were sorted into 5 ml of fixative (for sorting criteria, see above). The suspension was washed twice in PBS and subsequently permeabilized in 10 ml of methanol, washed two times, and phosphotyrosine or PLSCR immunolabeled as described above. The amount of phosphotyrosine labeling was quantified for each individual cell using the FACScalibur flow cytometer with the 488-nm argon laser for excitation and PMT-1 (510–540 nm) for detection of emitted photons.
Detection of DNA and Mitochondrial Degradation
The DNA and mitochondrial degradation of incubated sperm cells was detected flow cytometrically: 1) Single-stranded DNA was detected by the Acridine Orange assay; 2) DNA fragmentation was measured by extracting sperm nuclei, brief RNase treatment, and quantitative DNA staining with PI; 3) DNA strand breaks were detected using a flow-cytometric TUNEL kit, and 4) depolarization of the inner mitochondrial membrane potential on living and deteriorated sperm was detected by dual-staining with Mitotracker red and Yo-Pro. For experimental details of these procedures, see Gadella and Harrison [7].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm cells incubated for 3 h in HTF without bicarbonate (HTF-control) did not show any aminophospholipid exposure at their surface. These cells were completely nonfluorescent (not depicted in Fig. 1, but in Fig. 2, one can see that the nonexposing cells [NR] only contained <3% of the annexin V-FITC signal compared with scrambling cells [PS+, PE+]). However, in the presence of bicarbonate (HTF-Bic), a variable subpopulation of sperm cells exposed PS (detected with annexin V-FITC [12, 13]) and PE (detected with Ro-SA-FITC [11]) at the apical surface area of the sperm cell (Fig. 1, A and D, respectively). Some of the incubated cells, irrespective of the presence or absence of bicarbonate, showed midpiece labeling and/or cytoplasmic droplet staining (Fig. 1, B and C for annexin V-FITC and E and F for Ro-SA-FITC). Midpiece stained cells were often deteriorated (PI positive) (Fig. 1, B and E), whereas the cytoplasmic droplet-containing cells often were intact but failed to expose PS or PE at the apical plasma membrane in the presence of bicarbonate. Similar results were obtained in HBT-control and HBT-Bic (data not shown).
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Flow-Cytometric Detection of Bicarbonate-Induced Aminophospholipid Exposure in Human Spermatozoa
The proportion of sperm cells that showed bicarbonate-induced exposure of aminophospholipids was tested using triple-staining flow cytometry. Sperm cells were incubated in HTF-control (Fig. 2, A and C) or HTF-Bic (Fig. 2, B and D) for 4 h and then stained with annexin V-FITC to detect PS exposure (Fig. 2, A and B) or with Ro-SA-FITC to detect PE exposure (Fig. 2, C and D). Cells were counterstained red with PI (negative stained cells were considered to be vital or plasma membrane intact) and with RPE-PNA (negative stained cells were considered to be acrosome intact [6]). In the absence of bicarbonate, hardly any cell exposed PE or PS (Fig. 2, A and C; for the amount of scrambling live cells, see Fig. 3). Note also that nonexposing cells (Fig. 2, NR) contained less than 3% of FITC-specific fluorescence compared to PE- and PS-exposing cells (Fig. 2, PE+ and PS+, respectively).
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Bicarbonate-induced aminophospholipid exposure was observed in a considerable subpopulation of intact cells (depicted for HTF-Bic in Fig. 2, B and D; for the amount of scrambling live cells, see Fig. 3). Similar results were obtained when HBT-Bic (Fig. 3) was used previously as porcine sperm capacitation medium [6–9]. Because the detecting probes cannot pass the intact plasma membrane, the PS and PE exposure must have taken place at the plasma membrane. The kinetics of PE and PS exposure differed considerably (Fig. 3). Steady-state labeling for PS was only achieved in 90 min (t1/2 
30 min) (Fig. 3A), whereas maximal subpopulations of spermatozoa exposing PE were detected within 30 min (t1/2 12 min) (Fig. 3B) However, the 4-h incubated sperm samples gave similar relative population sizes of PS- and PE-exposing intact cells (Fig. 3C).
Role of cAMP-Dependent Protein Phosphorylation in the Control of Aminophospholipid Scrambling
The cAMP levels in mammalian spermatozoa rise in response to bicarbonate, which activates a forskolin-insensitive and soluble adenylyl cyclase (sAC) [3]. The second-messenger role of cAMP is generally exerted through its activation of PKA to phosphorylate specific cellular proteins and, thereby, alter their molecular function. Previously, we have shown that this signaling cascade is involved in activating the transbilayer movements of PL analogues and stimulates the exposure of aminophospholipids in porcine sperm cells [6, 7]. In the present study, we investigated the effect of various modulators of cAMP-dependent phosphorylation on the induction of PS and PE exposure in human sperm cells using the annexin V-FITC- and Ro-SA-FITC-binding assays, respectively.
Incubation of sperm cells in HBT-control with the phosphodiesterase-inhibitor papaverine resulted in PE and PS exposure similar to that induced in HBT-Bic (Table 1). The cAMP-analogue 8BrcAMP provoked similar responses, whereas the cGMP-analogue 8BrcGMP did not induce aminophospholipid scrambling (Table 1). The adenylyl cyclase (AC)-activator forskolin did not induce the plasma membrane alterations, even at high concentrations, probably because the sAC involved is sperm specific and forskolin insensitive [3]. On the other hand, in HBT-control, okadaic acid and calyculin (protein phosphatase inhibitors) at low levels of 3-isobutyl-1-methylxanthine (IBMX; <50 µM) induced aminophospholipid exposure (Table 1). Low levels of IBMX on its own did not exert any effect, probably because protein phosphatases are capable of dephosphorylating the PKA substrates and inhibiting these phosphatases clearly shows the PKA dependency of PL scrambling [6, 7, 27]. The above results suggest that bicarbonate was exerting its effect on aminophospholipid exposure by increasing cAMP and, thereby, stimulating PKA to raise protein phosphorylation levels. The stimulation of PKA-dependent protein phosphorylation by 15 mM bicarbonate appears to be maximal, because addition of IBMX and phosphatase inhibitors to HBT-Bic did not further stimulate PL scrambling (Table 1). We also incubated sperm in HBT-Bic in the presence of the PKA-inhibitors H89 [28] and RpcAMPS [29]; both compounds indeed blocked the effect of bicarbonate (Table 1). These data are in line with the effects of these PKA-modulating agents on the stimulation of transbilayer movements of PL in boar sperm [7]. In general, it seems that the bicarbonate/sAC/cAMP/PKA pathway is regulating PL scrambling at the apical area of the sperm head plasma membrane (Table 1).
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Apoptotic Indicators Are Not Associated with Bicarbonate-Induced PL Scrambling
Surface exposure of aminophospholipids can be an early indicator of apoptosis [30]. Two other hallmarks of apoptosis are deterioration of nuclear DNA and mitochondrial degeneration: Cytochrome c that has leaked into the cytosol is a key component of the apoptosome that initiates activation of destructive caspases [31, 32]. In analogy with porcine sperm [7], we tested whether sperm incubation in HTF-Bic affected the DNA integrity (Table 2). Using the flow-cytometric Acridine Orange-labeling assay to detect single-stranded DNA (red fluorescent), we did not observe a significant increase in DNA damage. Similarly, a flow-cytometric TUNEL assay, which detects DNA strand breaks, did not reveal a significant rise of DNA damage in sperm cells. Finally, bicarbonate challenge did not result in increased fractionation of sperm nuclei. In Table 2, data are shown for 0-min, 30-min, and 4-h incubations; DNA damage was not significantly increased (P = 0.32) from the 4-h measurement after 24-h incubation despite the fact that some increase in deteriorated cells was observed. Bicarbonate induced, to some extent, an increase in sperm cells with nonfunctional mitochondria as detected with Mitotracker red (Table 2, 15.6% after 4-h incubation in HTF-Bic compared to 4.9% in HTF-control). However, all Mitotracker red-negative cells also contained a disrupted sperm plasma membrane, which caused positive green staining with Yo-Pro. Therefore, mitochondrial damage is not related to apoptosis but to plasma membrane damage and, thus, to necrosis. By contrast, plasma membranes of apoptotic cells remain intact, followed by extensive blebbing of cytosol-rich membrane vesicles. Apoptotic plasma membrane blebbing has not been observed for human sperm (except for cytoplasmic droplet release).
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Inhibition of caspases with high concentrations of wide-spectrum caspase inhibitors did not result in decreased PL scrambling in human sperm cells after induction with bicarbonate (Table 2). Both zVAD-fmk and BocD-fmk did not significantly reduce PE exposure, indicating that responding sperm cells either do not contain active caspases or are not involved in the PE exposure. Indeed, only minute amounts of active and inactive caspase-3 were detected in human sperm samples on Western blots (data not shown). In line with this, immunofluorescence microscopy of sperm samples revealed the presence of active and inactive caspase-3 (implicated in male germ cell apoptosis [33]), as depicted in Fig. 4. Caspase-3-positive staining appeared to result mainly from the fact that sperm samples include contaminating cells and cytoplasmic droplets that contain both inactive and active caspase-3, with no obvious differences being observed between HTF-control and HTF-Bic-treated sperm samples. By contrast, mature sperm cells contained neither active nor inactive capase-3, irrespective of incubation medium. This suggests that mature sperm cells do not contain caspases and that the bicarbonate-triggered PS/PE-exposure is apoptosis independent.
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Capacitation Parameters Are Strongly Associated with Bicarbonate-Induced PL Scrambling
Bicarbonate is considered to be an important factor inducing sperm capacitation [1]. In the present study, we report on the sAC/cAMP/PKA pathway-dependent induction of PL scrambling in human sperm, but this pathway also is involved in the induction of tyrosine phosphorylation [32–36]. The latter phenomenon has been correlated with the capacitation state of sperm from mouse and other mammalian species [2, 23, 36–39]. Therefore, we investigated whether the bicarbonate treatments used in the present study resulted in increased tyrosine phosphorylation (i.e., induced capacitation). Immunofluorescent labeling of phosphotyrosine residues in sperm cells revealed that in the absence of bicarbonate (HTF-control), only 11% of the sperm cells showed weak fluorescent signal (Fig. 4). However, after 4-h incubations in HTF-Bic, approximately 40% of the sperm cells showed intense fluorescence, especially at the tail, indicating that tyrosine kinase activity has been induced by bicarbonate. Also of interest was the observation that cytoplasmic droplets containing sperm cells did not show any phosphotyrosine labeling (Fig. 4), because such cells also failed to expose aminophospholipids (Fig. 1).
In a separate experiment, we sorted live, bicarbonate-challenged sperm cells with a flow cytometer after their labeling for PE exposure (Fig. 5). Immediately after sorting, the intact sperm cells were fixed and permeabilized with methanol (PE and PS labeling as well as DNA labeling is lost by this step). Permeabilized cells were immunolabeled for phosphotyrosine. The sorted live cell population showed labeling identical to that for the unsorted cells (compare bicarbonate phosphotyrosine labeling pictures of Fig. 4 with the middle of Fig. 5), which indicates that tyrosine phosphorylation had occurred in live sperm cells (in the experiment depicted in Fig. 5, phosphotyrosine labeling was detected in 45.8% of the living cells) and is not a sign of cell death. Intriguingly, all sorted living cells with PE exposure (42.3% of the living sperm cells in Fig. 5) showed clear phosphotyrosine labeling, whereas none of the nonexposing cells showed this clear labeling (Fig. 5). Some of the nonexposing cells showed dull tail labeling and labeling on the equatorial region of the head. Taken together, these experiments indicate that PE exposure is an early sign of capacitation, followed by a later increase of tyrosine phosphorylation (the higher phosphotyrosine-labeling levels were noted only after 90-min incubation in HTF-Bic). Nonscrambling cells appear to be immature and not competent to capacitate as judged by the absence of immunolabel on phosphotyrosine residues in cytoplasmic droplet-containing cells.
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Staining with CTC is the most commonly used technique to assess human sperm capacitation [24]. We tested whether the bicarbonate-induced incidence of PE- and PS-exposing cells correlated with the increased incidence of capacitation-specific CTC staining patterns [23, 24] of live human sperm (Fig. 6). Much variability was found between the eight ejaculates, which enabled the correlation between the parameters to be observed clearly. The proportion of live cells showing PE and PS exposure was closely and positively correlated with the proportion of live cells that showed capacitation-specific CTC staining (P < 0.02, R2 = 0.99 and 0.98 for PE and PS, respectively) (Fig. 6). The incidences of PS or PE exposure are therefore strongly correlated with each other as well as to the incidence capacitation-specific CTC staining.
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Immunolabeling of PLSCR in Human Sperm
Bicarbonate-challenged sperm cells were sorted as described above, and after fixation and permeabilization, we stained these cells for PLSCR using the 4D2 antibody (described previously for immunolocalization studies [16, 40]). Both in the sorted total living cell population as well as in the sorted nonscrambling and scrambling subpopulations of living cells PLSCR was detected (Fig. 5). The PLSCR appears to be localized over the entire sperm surface. Furthermore, no differences in localization pattern were observed between nonexposing and PE-exposing human sperm cells. An identical sorting experiment for PS exposure gave similar results (data not shown). The labeling patterns did not alter from those detected in sperm cells incubated in HTF-control (data not shown). Therefore, it appears that the apical head plasma membrane specificity of PL scrambling is not caused by the local concentration of PLSCR but by a regionalized regulatory mechanism activating the PLSCR pathway at the apical sperm head area only.
Detection of PLSCR in Human Sperm
The antibodies 4D2 [16, 40], C-term [14, 18], and 219.1 [40], raised against human PLSCR1, have been described previously (for epitope mapping, see Fig. 7A). In the present study, we determined the specificity of these antibodies for the PLSCR isoforms using human erythrocyte PLSCR1 and recombinant testis-specific PLSCR2. Of note, these antibodies do not recognize PLSCR3 and PLSCR4. The apparent molecular weights, deduced from the amino acid sequence, of PLSCR1 (35 kDa) and of PLSCR2 (25 kDa) [20] were confirmed by Western blot analysis using these antibodies (Fig. 7B). The antibodies C-term and 219.1 recognize both PLSCR1 and PLSCR2. From Western blot experiments, it became clear that human sperm cells only contained a 25-kDa protein (Fig. 7B) using the C-term and 219.1 antibodies. Extensive staining with the 219.1 antibody also resulted in a detection of a 47-kDa protein, which we attribute to nonspecific antibody binding.
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For immunoprecipitation experiments, the monoclonal antibodies 219.1 and 4D2 were used (Fig. 7C). Although antibody 4D2 was not able to detect the 25-kDa protein in 100 µg of solubilized human sperm proteins (Fig. 7B), it was useful for immunoprecipitation of the human sperm PLSCR. Antibody 4D2 may recognize the native state of PLSCR as present in immunoprecipitation and immunofluorescence microscopy experiments (Figs. 5 and 7C) better than the denatured protein as presented in Western blot experiments (Fig. 7B). The polyclonal antibody C-term did not immunoprecipitate PLSCR (data not shown) and, therefore, was used to visualize immunoprecipitated PLSCR in the subsequent Western blot experiments. In line with the results of the Western blot experiments (Fig. 7B), immunoprecipitation experiments using 4D2 and 219.1 revealed that a 25-kDa protein is present in human sperm cells (Fig. 7C).
Control immunoprecipitation experiments using comparable amounts of monoclonal antibodies against Hsp 70 and p53 were carried out. These experiments showed no proteins on Western blot after visualization with C-term (Fig. 7C), ruling out the idea that the 25-kDa protein band is the light chain of IgG. The C-term antibody recognized the 25-kDa PLSCR in immunoprecipitates of 219.1 and 4D2 but also in immunoprecipitates of -tubulin (Fig. 7C). The presence of the 25-kDa protein on the immunoprecipitate of -tubulin indicates that at least a proportion of PLSCR interacts with -tubulin and, thus, with the cytoskeleton of the human sperm cell. To confirm this finding, a PLSCR immunoprecipitation and subsequent visualization with a -tubulin antibody has to be performed. However, at this moment, we do not have a good combination of antibodies with which to perform this experiment. The -tubulin antibody recognized only the 50- and 35-kDa isoforms of -tubulin on Western blots of human sperm, not the 25-kDa protein (Fig. 7D). Moreover, the C-term antibody did not bind to -tubulin or to subunits of the antibodies 219.1, 4D2, or anti--tubulin on Western blots (data not shown). We derive from these results that human sperm cells contain a 25-kDa form of PLSCR. This could either represent the testis- and germ cell-specific isotype of PLSCR (PLSCR2) or a truncated form of PLSCR1 [20].
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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24 mM), whereas ejaculated semen contains reduced levels (<1 mM). Bicarbonate is known to activate a sAC in a direct manner [3] and promotes production of cAMP. Elevated sperm cAMP levels activate PKA, promoting most of the surface changes mentioned. In the present study, we have investigated whether the bicarbonate/sAC/cAMP/PKA path induces similar surface changes in capacitated human sperm cells. We have chosen to follow the exposure of PE and PS in human sperm after a bicarbonate challenge. The collapse of PL asymmetry is the most rapid cellular response to this challenge, and it can be easily measured and is probably required for further capacitation of sperm [6, 7]. The importance of PKA-mediated capacitation in human sperm is the fact that many IVF clinics add caffeine to the medium to support sperm capacitation by reducing the breakdown of cAMP through inhibiting phosphodiesterases [46, 47]. Both PE and PS remain sequestered to the inner leaflet of the sperm plasma membrane after ejaculation and after washing in bicarbonate-depleted media. However, incubation of washed sperm in bicarbonate-enriched media (15 or 24 mM) resulted in exposure of PE and PS at the apical surface area of intact and mature sperm cells. A very rapid PE exposure was detected in 20–50% of the intact sperm cells. A similar population of sperm cells exposed PS, albeit with slower kinetics. Similar proportions of PE- and PS-exposing cell populations were recorded after approximately 90 min of bicarbonate stimulation. Only well-matured human sperm cells (i.e., without cytoplasmic droplets) showed PL scrambling in response to bicarbonate by activating the sAC/cAMP/PKA-signaling pathway. This signaling cascade increased the bidirectional transverse movement of PL in boar sperm cells [6, 7]. Bicarbonate also induced sperm capacitation in a number of mammalian sperm cells, including rodent [37–39], porcine [2], equine [23], and human [34–36].
Exposure of aminophospholipids at the cell surface is recognized as an early indicator of apoptosis in many cell types, but it is also associated with other cellular events, such as exocytosis and cell adhesion. Bicarbonate induced PL scrambling in approximately 40% of live sperm cells, whereas no effects on DNA or mitochondrial damage were noted in these live cells. It must be noted that longer incubations in HTF-Bic induced, to a small extent, cell death and that those cells also showed mitochondrial deterioration (the latter was not observed in live cells; see also Gadella and Harrison [7]). Furthermore, caspase inhibitors failed to block the PL-scrambling response, and caspase-3, in both its active and inactive form, was not detected in mature sperm, only in cytoplasmic droplets and contaminating somatic cells. We investigated caspase-3 because this enzyme has been implicated in the induction of testicular germ cell apoptosis [33]. Our results are in line with earlier findings that no caspase activity was found in mature sperm but that caspase, c-jun, p53, and p21 were present in a restricted site for apoptosis (cytoplasmic droplets) in spermatids and immature spermatozoa [48, 49]. Furthermore, apoptosis can take place during spermatogenesis and occurs in sperm samples of poor quality in various species [50, 51]. In subfertile cases, higher proportions of PS-exposing and TUNEL-positive cells have been found [52], which corresponds with a high proportion of immature cells. This latter observation supports the abortive apoptosis theory: Apoptosis in mature sperm is initiated during spermatogenesis in which some cells, earmarked for elimination, may escape the removal mechanism and contribute to poor sperm quality [52, 53]. However, in the present study, we selected mature sperm cells by Percoll washing. Bicarbonate-dependent PL scrambling appeared to be independent from apoptosis. In fact, under bicarbonate-free conditions, we failed to induce PL scrambling in these cells by adding Fas ligand or staurosporin/cycloheximide (data not shown). The only reported treatment known to stimulate PS exposure appears to be cryopreservation and, in relation to this, the induction of lipid peroxidation [54–56]. The relation of this temperature-induced membrane damage to apoptosis and/or PL scrambling needs to be elucidated. Another explanation could be that cooling of sperm cells induces an irreversible, lateral phase separation of PL that will be followed by increased permeability to calcium ions, which in turn may be followed by PLSCR activation (for review, see Bevers et al. [15]). This may result in increased populations of PS-exposing (annexin V-binding) sperm cells.
Our observations indicate that mature sperm cells do not contain caspases, do not show bicarbonate/PKA-dependent signs of apoptosis such as fractionation of DNA or mitochondrial inner-membrane depolarization, but do show rapid aminophospholipid exposure. These observations do not contradict those of a recent report concerning the presence of inactive and active caspases in human sperm samples and the incidence of DNA fractionation in subfertile cases [57]. In fact, Weng et al. [57] found that inactive and active caspases (the latter only observed in subfertile cases) are located in the midpiece of a small subpopulation of human sperm cells. The midpiece is quite remote from the apical area of the sperm head, where bicarbonate triggered aminophospholipid exposure. We also detected inactive and active forms of caspase-3 in human sperm samples; however, the finding that both forms of caspase-3 were absent in mature sperm is new. This is relevant because only mature cells showed bicarbonate-stimulated PL scrambling in the apical head area.
Schuffner et al. [58] reported that inclusion of 0.3% or 3% human serum albumin in HTF caused a plateau in PS exposure in sperm cells from human donors after 1-h incubation that lasted for at least 5 h. Different PS-exposure kinetics were observed in the absence of albumin or in sperm cells of patients [58]. We only tested sperm cells from human donors in the presence of high amounts of albumin, and indeed, we detected the same plateau of PS-exposure kinetics in subpopulations of sperm cells. Although we did not test this for human sperm, we did not observe dramatic differences between bicarbonate-stimulated PS and PE exposure in the absence or presence of BSA in the porcine species. Theoretically, PS-exposure delay could depend on the source from which albumin was purified. More likely, the difference between the porcine results [7] and the human results [58] is the use of less advanced techniques to detect cell viability and annexin V binding in the latter study. Immobilization of incubated sperm cells to poly-L-lysin-coated coverslips and examination using a plain epifluorescent microscope [58], especially in the absence of protective macromolecules (e.g., albumin), may well introduce electrostatic membrane stress. Albumin or other protective macromolecules in the buffer probably will protect sperm cells from poly-L-lysin-induced artifacts. Furthermore, Schuffner et al. [58] did not mention where annexin V binding was localized in the absence of albumin and whether this localization was similar to that of annexin V binding in the presence of albumin. As indicated in the present study (see also Gadella and Harrison [7]), direct measurement of sperm under circumstances that optimally mimic IVF conditions may minimize the incidence of such potential artifacts. First, with the use of an inverted rapid-scanning confocal microscope equipped with a cell chamber in which sperm suspensions were analyzed under 0% or 5% CO2 in humidified air at 37°C, we were able to follow sperm cells under incubation conditions. Moreover, using a flow cytometer that was pre-equilibrated to 37°C and sperm tubes that were pressurized by humidified air containing 0% or 5% CO2 (all at 37°C), we were able to objectively and more sensitively quantify the amount of intact sperm cells that exposed aminophospholipids under incubation conditions.
It has also been shown that annexin V binding took place at low (0.4 mM) bicarbonate levels [58]. The response reported is less pronounced when compared to the response we report with 15 or 24 mM bicarbonate. In a previous study, a bicarbonate dose-response curve was made in the range of 0–16 mM in porcine sperm [6]. Significant induction of PL scrambling was detected at 2 mM, with maximum induction at 8 mM. We must note here that these data were obtained with porcine rather than human sperm, and the latter may be more sensitive to bicarbonate stimulation. Furthermore, the transbilayer behavior of PL probes, instead of endogenous PL, was studied. We have mentioned that transbilayer movement of endogenous PL may be more sensitive for bicarbonate [10]. At any rate, in the present study, we did not note significant annexin V binding to sperm cells in media devoid of bicarbonate during the 4-h incubation interval.
Bicarbonate is identified as a key activator of capacitation in sperm of human and other eutherians [2], but it appears to be unrelated to apoptosis. Increased tyrosine phosphorylation also depends on bicarbonate-induced sAC/cAMP/PKA signaling [34, 35] and is modulated by the organization and depletion of cholesterol [59–61]. Bicarbonate-induced sAC/cAMP/PKA signaling itself seems to be involved in cholesterol redistribution and enables albumin-dependent depletion of this sterol [8]. Only sperm cells with the early PL-scrambling response showed a slower reorganization of surface cholesterol, which concentrated in the scrambling area (i.e., the apical plasma membrane of the sperm head) and subsequent albumin-dependent cholesterol depletion was only observed in those cells where cholesterol was concentrated in the scrambling area [8]. Here, we found in human sperm cells that only approximately 10% of the HTF-treated cells (without bicarbonate) showed dull phosphotyrosine labeling, whereas 40% of the HTF-Bic-stimulated cells showed clear phosphotyrosine labeling after 90 min. Interestingly, all PE-exposing cells showed this increase, whereas nonexposing cells did not show phosphotyrosine labeling. Sperm cells with immature appearance and/or cytoplasmic droplets failed to expose PE or PS and also showed no phosphotyrosine labeling. Taken together, this indicates that tyrosine phosphorylation is indeed coupled to the bicarbonate/sAC/cAMP/PKA pathway, either indirectly via cholesterol changes or directly by activation of tyrosine kinase. Increased tyrosine phosphorylation is reported to be instrumental for sperm hypermotility as well as for the generation of high zona pellucida affinity [2, 62]. At present, we are studying whether induction or inhibition of PE or PS exposure is paralleled by an increase or decrease of tyrosine phosphorylation. Most likely, the two phenomena are spatially separated by region-specific PKA modulators such as A kinase-anchoring proteins (AKAP). In this regard, human sperm contain at least one tail-specific AKAP located in the fibrous sheath and linked to tail-specific tyrosine phosphorylation and motility regulation [63], and another known AKAP is thought to play a role in the progesterone-induced acrosome reaction [64]. Therefore, it is tempting to speculate that bicarbonate-induced PKA stimulation is an overall sperm cell phenomenon. In the sperm tail, this PKA activation is linked to a specific AKAP that induces tail-specific tyrosine phosphorylation, whereas in the sperm head, this PKA activation is linked to another specific AKAP inducing increased PL scrambling. This idea is supported by the fact that, first, PLSCR was homogeneously distributed over the sperm surface but its PKA-dependent activation only took place at the apical sperm head area and, second, complete tyrosine phosphorylation of all tyrosine kinase substrates would end up in homogeneous phosphotyrosine labeling of human sperm cells. Therefore, to unravel specific sperm-signaling routes, it is important to dissect the sperm cell into a tail and a head with specific disruption techniques.
Bicarbonate-induced PE and PS exposure not only correlated with increased tyrosine phosphorylation in the sperm tail but also with increased subpopulations showing capacitation-specific CTC staining. Although, CTC staining is routinely used to assess sperm capacitation in humans [24] and other mammals [23], its principle at the molecular level is not really understood. Nevertheless, strong correlation between capacitation-specific CTC staining and aminophospholipid exposure further supports our idea that bicarbonate-triggered PL scrambling in the apical sperm plasma membrane is an early indicator for human and mammalian sperm capacitation (see also Gadella and Harrison [7]). This correlation of PE and PS exposure and capacitation-specific CTC staining fits with what has been described for equine sperm [23]. Furthermore, the link of PL scrambling to merocyanine 540-detectable membrane changes in sperm from different species [7, 23] may link the induction of PL scrambling directly to the preparation of the acrosome reaction, because both merocyanine 540 and the closely related probe FM1-43 not only detect PL scrambling but also are used to monitor endocytosis and exocytosis [65]. In this regard, the restricted area of PE and PS exposure matches the surface area where plasma membrane and outer acrosomal membrane will fuse at multiple foci on the zona pellucida-triggered acrosome reaction, rendering the equatorial membranes intact, which is essential for the later gamete fusion [2].
The PLSCR has been reported to cause cell surface exposure of PE and PS [14, 18] by enhancing the bidirectional transbilayer movement of PL in the plasma membranes of activated cells [15]. We have used previously described antibodies raised against human PLSCR [16, 18, 40] to demonstrate the presence of a PLSCR isoform in human sperm cells. Western blot and immunoprecipitation experiments revealed a 25-kDa protein PLSCR isoform. Future studies may elucidate how bicarbonate modulates PL asymmetry on sperm capacitation (e.g., by PKA-dependent phosphorylation of PLSCR). For PLSCR1, it is already established that posttranslational modifications such as a tyrosine and/or threonine phosphorylation [40, 66] and multiple palmitoylation [18] can take place. Although PLSCR has been shown to be involved in PL scrambling, the exact role is not understood [67]. Although it is far from clear how scrambling of PL is modulated in the apical region of the sperm plasma membrane, it is tempting to speculate that the bicarbonate/AC/PKA-signaling pathway is involved in activation of the 25-kDa human sperm PLSCR.
In conclusion, bicarbonate triggers a PKA-dependent signaling cascade that induces a relatively fast-mode (30-min) PL scrambling in the apical sperm head plasma membrane of responsive sperm cells. This event seems to be important for further downstream capacitation of sperm cells that is essential to finally fertilize the oocyte.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: B.M. Gadella, Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands. FAX: 31 30 2535492; b.gadella{at}vet.uu.nl
Received: 18 October 2002.
First decision: 12 November 2002.
Accepted: 13 January 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Blood 2000 95:2593-2539
, HBT-control), HBT containing 15 mM bicarbonate (
, HBT-Bic), HTF (
, HTF-control), or HTF containing 24 mM bicarbonate (•, HTF-Bic). A) The relative proportion of intact cells that exposed PS (i.e., that stained positive for annexin V-FITC). B) The relative proportion of intact cells that exposed PE (i.e., that stained positive for Ro-SA-FITC). C) Note that PE exposure was detected earlier than PS exposure (e.g., after 30 min of bicarbonate stimulation; filled bars) but that the proportion of cells showing the bicarbonate-dependent aminophospholipid exposure was identical for PS and PE after longer incubation times (90 min, hatched bars; see also A and B for longer incubation times). Mean values ± SD are incubated from eight ejaculates (n = 8), each of which was measured in triplicate
and dashed line) to assess PE and PS exposure, respectively. Parallel samples were stained with CTC to assess sperm capacitation [