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a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been shown that thapsigargin, a highly specific inhibitor of the microsomal Ca2+ pump, can induce elevation of intracellular free calcium [Ca2+]i [8, 9] and initiate the acrosome reaction (AR) [9, 10] in human and bovine spermatozoa. Putative sites for thapsigargin-sensitive intracellular Ca2+ stores include the cytoplasmic droplet, the sperm nucleus, and the acrosome. In a more recent study, it was shown that inositol 1,4,5-tris-phosphate receptors (IP3-R) are selectively localized to the acrosomes of rat, hamster, mouse, and dog sperm, suggesting that the acrosome is an intracellular Ca2+ store [11]. Moreover, working with isolated acrosomal membranes, we recently observed that these membranes possess an ATP-dependent Ca2+ pump that is inhibited by thapsigargin [12].
Walensky and Snyder [11] studied permeabilized rat sperm treated with sodium azide to follow ATP-dependent Ca2+ loading into nonmitochondrial Ca2+ stores. However, using azide to block the mitochondrial electron transport chain does not prevent the buildup of a proton gradient in the mitochondria due to ATP hydrolysis, resulting in active Ca2+ transport into the mitochondria.
In the present study, we used a mitochondrial uncoupler to prevent the buildup of a proton gradient; therefore no active accumulation of Ca2+ in the mitochondria occurred. Thus, this is the first definitive demonstration of the presence of an intracellular nonmitochondrial ATP-dependent Ca2+ pump in mammalian spermatozoa. A role for this internal calcium store in capacitation and in the AR is suggested.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thapsigargin, rabbit anti-IP3-R polyclonal antibody, and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibodies were purchased from Calbiochem (San Diego, CA); 45CaCl2 from New England Nuclear (Boston, MA); and Fura-2/AM and Fluo-3/AM from Molecular Probes (Eugene, OR). All other materials were purchased from Sigma Chemical Co. (St. Louis, MO).
Sperm Preparation
Ejaculated bull and ram spermatozoa were obtained using an artificial vagina. The semen was washed three times by centrifugation (780 x g, 10 min at 25°C) in NKM buffer containing 110 mM NaCl, 5 mM KCl, and 10 mM 3-[N-morpholino]propanesulfonic acid, pH 7.4. The washed cells were suspended in NKM buffer to a concentration of 109 cells/ml and were maintained at room temperature until use.
Capacitation and AR
In vitro capacitation of bovine sperm was accomplished by the method of Parrish et al. [13]. Briefly, sperm pellets were resuspended to a final concentration of 108 cells/ml in glucose-free TALP medium containing (in mM) 100 NaCl, 3.1 KCl, 1.5 MgCl2, 25 NaHCO3, 0.29 KH2PO4, 21.6 sodium lactate, 0.1 sodium pyruvate, 2 CaCl2, 20 Hepes (pH 7.4) and 50 µg/ml BSA, 10 U/ml penicillin, and 20 µg/ml heparin. The cells were incubated in this capacitation medium for 4 h at 39°C with 5% CO2. At the end of the capacitation, the AR inducer thapsigargin or the Ca2+ ionophore A23187 was added for an additional 20 min of incubation. The occurrence of AR was determined using two methods: first, assay of the release of acrosin from acrosome-reacted cells [14]; second, staining of cells by Pisum sativum agglutinin (PSA), whereby intact cells are stained and acrosome reacted cells are not [15]. For determination of acrosin release from the cells, the cells were pelleted by centrifugation (7500 x g, 5 min 4°C), and the supernatant was adjusted to pH 3.0 with HCl; acrosin activity was determined by the esterolytic assay using benzoylarginine ethyl ester as substrate and recording the optical density increase at 259 nm with time. The molar absorption coefficient was taken as 1150. All the values are given after subtraction of the spontaneous acrosin release.
Staining with PSA was performed using a modification of the method described by Mendoza et al. [15]. Samples of cells treated to induce the AR were smeared on microscope slides. After air drying, sperm smears were dipped in absolute methanol for 30 sec and allowed to dry rapidly. Methanol-fixed smears were incubated with blocking solution (PBS containing 1% BSA) for 10 min, then with biotin-conjugated PSA (50 µg/ml) in PBS containing 1% BSA for 30 min, and finally with peroxidase-conjugated extravidin (1:400) for 10 min. All incubations were performed in a humid chamber. The slides were washed between incubations by dipping in PBS for 5 min. The substrate (AEC from Histostain-SP kit; Zymed Laboratories, South San Francisco, CA) was then added for 10 min; this was followed by washing with distilled water. Hematoxylin was usually used for counterstaining (3 min). The slides were mounted with GVA mounting medium (Zymed) and examined under a brightfield microscope. Cells with red staining over the acrosomal cap were considered acrosome intact, and cells with equatorial red staining or no staining at all were considered acrosome reacted; 200 cells were counted per slide.
45Ca2+ Loading Into Permeabilized Cells
Washed spermatozoa in NKM buffer (4 x 108 cells/ml) were incubated with 10 µM digitonin for 2 min. These permeabilized cells were washed twice at 4°C in buffer M comprising 250 mM mannitol, 70 mM sucrose, and 10 mM Hepes, pH 7.4 (the pH was adjusted by tetraethylammonium) and kept on ice until use. For the determination of ATP-dependent Ca2+ loading, the permeabilized cells were resuspended in buffer B containing 75 mM KCl, 1 mM MgCl2, 5 mM NaH2PO4, 20 mM Hepes, pH 7.4 (with KOH), 10 mM dithiothreitol, 3% polyethylene glycol (average Mr 8000), 10 µM carbonyl cyanide p-trifluoromethoxy-phenylhydrazine (FCCP), 0.05 mM CaCl2 (free Ca2+ 
1 µM), and 3 µCi/ml 45CaCl2. The cell suspension was incubated at 37°C, and the reaction was started by addition of 2 mM ATP. At appropriate time intervals, a 0.1-ml aliquot was removed and vacuum-filtered on GF/C filters. The cells trapped on filters were washed three times with 5 ml of buffer containing 100 mM KCl, 10 mM Hepes, pH 7.4, 5 mM MgCl2, and 1 mM EGTA. Radioactivity of dry filters was determined by liquid scintillation in 4 ml of EcoLume (ICN, Costa Mesa, CA).
45Ca2+ Loading Into Isolated Plasma Membrane Vesicles
Preparation of sperm plasma membrane vesicles and ATP-dependent Ca2+ uptake into these vesicles were performed as described by us previously [6].
Determination of Intracellular Calcium
The intracellular concentration of free Ca2+ was assessed using the fluorescent calcium indicator Fura-2 [16]. Washed cells (4 x 108/ml) were incubated in buffer A for 60 min, at 37°C, with 4 µM Fura-2/AM. The loaded cells were then washed by centrifugation at 780 x g for 10 min to remove extracellular Fura-2, incubated for another 30 min, and washed twice as described above. The cells were used immediately for fluorescence measurements using a Shimadzu (Columbia, MD) RF-5000 spectrofluorophotometer, with the dual excitation wavelength set of 340 nm and 380 nm and emission of 500 nm. During fluorescence measurements, sperm suspensions were stirred at 37°C. The concentration of [Ca2+]i was calculated using an equation from Tsien et al. [17], [Ca2+]i = Kd(F - Fmin)/(Fmax = F), where Kd = 224 nM. Fmax and Fmin were recorded at the end of each incubation. Fmax was determined after the addition of 1% Triton X-100. Fmin was determined in the presence of 2 mM MnCl2. All values were corrected for autofluorescence of the cells.
The spatial distribution of intracellular calcium was determined using a fluorescence microscopy imaging system as described by us recently [18]. Sperm cells were incubated on 25-mm glass coverslips covered with poly-L-lysine with Fluo-3/AM (2 µM) and pluronic acid (1.5 µM) for 30 min in glucose-enriched PBS in 37°C, 5% CO2. After incubation, each dish was rinsed twice with glucose-enriched PBS, and the coverslip was placed in the microscope holder. Cells were covered with 1 ml glucose-enriched PBS. Intracellular calcium measurements were performed using a fluorescence microscopic system consisting of an inverted epifluorescence microscope (Axiovert 135M; Zeiss, Oberkochen, Germany), an intensified charge-coupled device C2400 camera (Hamamatsu, Hamamatsu City, Japan), and frame-grabbing software (Galai, Migdal-Haemeck, Israel). A 75-W xenon lamp served as the source for excitation. A 510 long-pass emission filter was used to select fluorescence emission. The first pictures were taken to estimate basal levels of fluorescence. Afterward, cells were irradiated in situ and the fluorescence was measured every 30 sec for 10 min. Analysis of intracellular calcium levels was performed employing Scan Array 2 software (Galai).
Immunocytochemistry and Indirect Immunofluorescence
Sperm cells were collected on glass slides in a cytocentrifuge (1000 rpm, 5 min). The cells were fixed and permeabilized with cold acetone and methanol (10 min each). The slides were then washed in TBS (137 mM NaCl, 20 mM Tris-HCl, pH 7.6) three times, 5 min each, and nonspecific sites were blocked for 30 min with 1% BSA in TBS. For indirect immunofluorescence staining, slides were incubated with polyclonal anti-IP3-R antibody (rabbit) at concentration of 1 µg/ml for 2 h at room temperature. Controls for IP3-R staining were performed using normal rabbit serum. Slides were washed in TBS three times (5 min each), air dried, and incubated for 10 min in the dark with 1:10 000-diluted FITC-conjugated goat anti-rabbit IgG secondary antibody; they were then washed again in TBS three times, 5 min each. Coverslips were placed with fluoro-guard antifade reagent, and the slides were examined by Venox (Olympus, Melville, NY) AHBT3 immunofluorescence microscopy.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thapsigargin selectively inhibits the Ca2+-Mg2+ ATPase (Ca2+ pump) of intracellular membranes and does not affect the Ca2+ pump of the plasma membrane [19]. To determine whether the AR is induced by mobilization of internal calcium stores, we determined the ability of thapsigargin to induce this reaction. Addition of increased concentrations of thapsigargin to capacitated bovine sperm at a concentration range of 1–30 µM significantly enhanced the AR (Fig. 1). This effect of thapsigargin could not be found in noncapacitated bovine sperm. The occurrence of AR was determined by two different assays, morphological and biochemical, and similar results were obtained. At 15 µM thapsigargin, a maximal effect was seen in which 58% of the cells were acrosome-reacted. A similar effect of thapsigargin on the AR in ram sperm was observed, but here the maximal effect on AR was seen at 30 µM thapsigargin (Table 1). Preincubation of spermatozoa with the calcium channel blocker La3+ (250 µM) completely inhibited thapsigargin-stimulated AR (Table 1).
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Determination of Intracellular ATP-Dependent Ca2+ Pump Activity
The stimulation of AR induced by thapsigargin suggests that spermatozoa contain an intracellular, membrane-associated ATP-dependent Ca2+ pump. We used digitonin-permeabilized ram and bull sperm to follow ATP-dependent accumulation of Ca2+ in intracellular nonmitochondrial Ca2+ stores. Added ATP can be hydrolyzed by the sperm mitochondria to create membrane potential via the H+-ATPase, resulting in active accumulation of Ca2+ in the mitochondria. In order to eliminate this effect, it is not sufficient to block the respiratory chain as performed by Walensky and Snyder [11], since under these conditions the mitochondrial H+-ATPase can still create membrane potential. Therefore, in this study we used the mitochondrial uncoupler FCCP, which dissipates the membrane potential, preventing active transport of Ca2+ into the mitochondria.
It can be seen in Figure 2 that ATP-dependent Ca2+ uptake into permeabilized sperm was strongly inhibited by FCCP, indicating that ATP-dependent Ca2+ uptake by the sperm mitochondria occurs in this system. After 20 min of incubation, about 11 nmol Ca2+/108 cells was taken up in absence of FCCP, whereas in the presence of the uncoupler, only about 4 nmol Ca2+/108 cells was taken up. These results indicate that 64% of the ATP-dependent Ca2+ uptake was accumulated in the mitochondria and only 36% in nonmitochondrial Ca2+ stores.
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A time-dependent increase in ATP-dependent Ca2+ uptake into nonmitochondrial Ca2+ stores in permeabilized spermatozoa is seen in Figure 3. Very little Ca2+ uptake was observed in the absence of ATP or in the presence of the Ca2+ ionophore A23187, and the ATP-dependent Ca2+ uptake was highly inhibited by thapsigargin.
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The addition of the Ca2+ ionophore A23187 to Ca2+-loaded cells after 20-min incubation caused fast release of the accumulated Ca2+, suggesting that Ca2+ is moved into nonmitochondrial Ca2+ stores by an active transport process. Thapsigargin did not inhibit ATP-dependent Ca2+ uptake in isolated plasma membrane vesicles from bovine sperm (Fig. 4), which indicates its specificity for intracellular Ca2+ pumps.
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Elevation of [Ca2+]i by Thapsigargin
Intracellular Ca2+ concentrations were determined in Fura-2-loaded noncapacitated sperm. Low concentrations of thapsigargin (3 µM) caused a fast and significant increase in [Ca2+]i in ram and bull spermatozoa (Fig. 5). This increase in [Ca2+]i could not be seen without addition of Ca2+ in the presence of the Ca2+ chelator EGTA (Fig. 6). These findings indicate that thapsigargin induces Ca2+ influx into the cells from the surrounding medium.
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The effect of thapsigargin on the spatial distribution of intracellular Ca2+ was evaluated in Fluo-3-loaded intact, noncapacitated sperm using a fluorescence microscopy imaging system (Fig. 7). It can be seen that 150 sec after addition of Ca2+, there was a significant increase of intracellular Ca2+ in the sperm head. The Ca2+ concentration started to increase in the midpiece where the sperm mitochondria are located only after 160 sec. After 200 sec, the [Ca2+]i in the head and the midpiece reached its maximal value (Fig. 7, see red color at 400 sec), which did not change during the next 40 sec. The increase in [Ca2+]i in the head under these conditions reflects the induction of AR by thapsigargin. The measurements of changes in [Ca2+]i in these experiments are summarized graphically in Figure 8.
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Localization of the IP3-R in the Sperm
Localization of IP3-R in the spermatozoon should provide information concerning the organelle that accumulates Ca2+ via the ATP-dependent Ca2+ pump described here. Immunocytochemical analysis using polyclonal antibodies that recognize the cytoplasmatic C-terminal of IP3-R revealed extensive staining in the acrosomal and postacrosomal regions of the head and along the tail, but not in the midpiece of the sperm (Fig. 9). The acrosomal region was not stained in sperm without acrosome (see arrowhead in Fig. 9), indicating that the antibody specifically stained the acrosome. The localization of IP3-R in the acrosome cap was previously demonstrated in hamster, rat, mouse, and dog sperm [11].
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Changes in [Ca2+]i in Capacitated Sperm
Spectrofluorometric measurements of [Ca2+]i using Fura-2-loaded capacitated or noncapacitated bovine sperm revealed an increase in [Ca2+]i after the addition of thapsigargin (Fig. 10). In noncapacitated cells, the addition of Ca2+ caused a very slight increase in [Ca2+]i; and the addition of thapsigargin was followed by a lag time of about 30 sec and then a fast increase in [Ca2+]i (Fig. 10A). In capacitated cells, the initial concentration of Ca2+ was higher than that in noncapacitated cells (60 ± 20 vs. 25 ± 10 nM); addition of Ca2+ to the medium induced an almost 2.5-fold increase in [Ca2+]i (160 ± 40 nM), followed by a 7-fold increase after addition of thapsigargin (1100 ± 250 nM) (Fig. 10B). The response to thapsigargin was different between noncapacitated and capacitated sperm. In capacitated sperm there was no lag time, and the increase in Ca2+ was 3.9 times slower; but the final [Ca2+]i reached was twice as high (1100 ± 250 vs. 500 ± 150 nM). These data clearly demonstrate that significant changes in the mechanisms involved in Ca2+ regulation occur during sperm capacitation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The first evidence for the existence of an intracellular ATP-dependent Ca2+ pump in rat sperm was suggested by Walensky and Snyder [11]. They followed ATP-dependent Ca2+ uptake in permeabilized sperm and attempted to prevent Ca2+ accumulation in the mitochondria by blocking mitochondrial respiration with sodium azide. However, under these conditions, the added ATP can be hydrolyzed by the mitochondrial H+-ATPase, resulting in the creation of membrane potential and active accumulation of Ca2+ into the mitochondria. In the present study, we used the mitochondrial uncoupler FCCP and showed that under these conditions, the Ca2+ uptake into the permeabilized cells is much lower (Fig. 2). It is well known that in the presence of uncoupler, the mitochondrial membrane potential is dissipated—conditions under which no ATP synthesis or active Ca2+ uptake can take place. Thus the remaining Ca2+ uptake is highly suggestive of nonmitochondrial Ca2+ stores. The ATP-dependent Ca2+ uptake in permeabilized sperm is strongly inhibited by thapsigargin, and very little uptake can be seen in the presence of the Ca2+ ionophore A23187 (Fig. 3), indicating that Ca2+ is accumulated in intracellular nonmitochondrial Ca2+ stores.
In intact bull and ram spermatozoa, thapsigargin induces the acrosomal reaction (Fig. 1) as well as an increase in [Ca2+]i (Fig. 5). These effects are found only in the presence of extracellular Ca2+; they could not be seen when Ca2+ influx was blocked by the calcium blocker La3+ (Table 1) or when extracellular Ca2+ was chelated by EGTA (Fig. 6). Since the ATP-dependent Ca2+ pump of the plasma membrane is not affected by thapsigargin (Fig. 4), we suggest that the effect of thapsigargin on AR and [Ca2+]i is due to the inhibition of an intracellular Ca2+ pump. It is known that AR is a calcium-dependent process that requires extracellular Ca2+ [21]. In a wide range of somatic cells, release of calcium from internal stores also promotes extracellular calcium influx across the plasma membrane [22]. This phenomenon is called capacitative calcium entry. The mechanism for this phenomenon is not completely understood, although it is accepted that the opening of a calcium channel in the plasma membrane is due to reduction of Ca2+ in the internal stores [23]. Thus, the inhibition of internal Ca2+ pump by thapsigargin in the sperm will cause reduction of Ca2+ in this store, resulting in the opening of Ca2+ channel in the plasma membrane.
In many secretory cells, the initial focal release of Ca2+ from internal stores has a priming effect on exocytosis [24, 25]. Since acrosomal exocytosis can be induced by thapsigargin, it is likely that the internal Ca2+ store in sperm would be localized in close proximity to the region in which acrosomal exocytosis occurs, i.e., in the acrosomal region of the sperm head. In many somatic cell types, calcium stored in internal membranes is released via an IP3-gated Ca2+ channel [22]. Thus, the localization of IP3-R in the acrosome region of bull and ram sperm (Fig. 9) suggests that this organelle may function as a calcium store. This IP3-R was clearly localized in the acrosome of rat, hamster, mouse, and dog sperm, suggesting that the acrosome is the internal calcium store [11]. These authors also showed that ATP-dependent Ca2+ uptake in permeabilized rat sperm is inhibited approximately 50% by inclusion of IP3 in the incubation medium, suggesting that Ca2+ is released from the store by IP3. The fact that they found only 50% inhibition by IP3 suggests that under the Ca2+ uptake conditions used, Ca2+ is also accumulated in IP3-insensitive stores, possibly in the mitochondria, as suggested earlier in this discussion.
Working with isolated bull sperm acrosomes, we found that these membranes possess an ATP-dependent Ca2+ uptake activity, which is blocked by thapsigargin [12]. In addition, these membranes possess a cAMP/protein kinase A (PKA)-dependent Ca2+ channel, suggesting the existence on the acrosomal membrane of either a cAMP-gated calcium channel [26] or a channel opened upon phosphorylation by PKA [27]. Moreover, sperm acrosomes are rich in calreticulin, a high-capacity Ca2+-binding storage protein [28] that has been reported to copurify with an IP3-sensitive Ca2+ store [29]. This information further supports our notion concerning the function of the acrosome as an intraspermatozoal calcium store.
During sperm capacitation, [Ca2+]i is enhanced in the acrosomal region of the head and in the tail [30]. In capacitated cells, thapsigargin induces a large increase in [Ca2+]i (Fig. 10), leading to acrosomal exocytosis. Thus, the data suggest that the internal Ca2+ pump is active during the capacitation process.
The kinetics of Ca2+ influx and the maximal [Ca2+]i induced by thapsigargin in capacitated cells differ from those found in noncapacitated sperm (Fig. 10). In capacitated cells, the maximal [Ca2+]i induced by thapsigargin is 1100 ± 100 nM, while in noncapacitated sperm the [Ca2+]i reached only 500 ± 60 nM. Since [Ca2+]i is higher in capacitated cells, and it accumulated in the acrosomal region, it is possible that a more significant reduction of acrosomal Ca2+ induced in capacitated cells by thapsigargin causes greater influx of Ca2+. It is not clear why, after thapsigargin is added to noncapacitated cells, there is a lag time followed by a fast increase in [Ca2+]i, whereas in capacitated cells there is no lag time and the rate of Ca2+ influx is much slower (Fig. 10). Similar results were obtained by Parrish et al. [9], who determined [Ca2+]i in individual bovine sperm using the imaging technique. Since we suggest that Ca2+ in the acrosome of capacitated cells is higher than in noncapacitated cells, it takes longer to deplete the stores sufficiently to trigger calcium entry from the extracellular medium, and this is the rate-limiting step for activating the capacitative calcium entry mechanism.
Our suggestion concerning the possible accumulation of Ca2+ in the acrosome during capacitation is further supported by the findings that phospholipase C (PLC), which hydrolyzes phosphoinositides to produce IP3, is activated by the egg zona pellucida (ZP3), indicating that IP3-dependent Ca2+ release takes place only at the end of the capacitation process [31]. It was also shown that PLC
is translocated to the sperm plasma membrane during capacitation [31, 32] prior to its activation by the egg zona pellucida. Thus, the formation of IP3 just before the occurrence of AR would keep the acrosomal IP3-gated Ca2+ channel inactive during capacitation, allowing the accumulation of Ca2+ in the acrosome during the capacitation process.
Recently it was suggested that binding of sperm to zona pellucida may activate adenylyl cyclase to form cAMP that activates PKA to open a Ca2+ channel in the acrosomal membrane [1]. As a result, the [Ca2+]i in the sperm cytosol is increased and PLC is activated to produce IP3 and diacylglycerol. IP3 would cause further release of acrosomal Ca2+ via the IP3-gated Ca2+ channel, resulting in Ca2+ depletion in the acrosome and activating the capacitative calcium entry mechanism. This final step would cause a relatively high elevation of [Ca2+]i thereby activating actin-severing proteins, leading to dispersion of the F-actin barrier intervening between the outer acrosomal and the overlying plasma membrane [32]. The primed membranes would then be able to come into contact and fuse, releasing the acrosomal contents and completing the AR.
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FOOTNOTES |
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2 Correspondence. FAX: 972 3 5344766; breith{at}mail.biu.ac.il
Accepted: June 17, 1999.
Received: April 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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