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Characterization of Intracellular Ca²⁺ Increase in Response to Progesterone and Cyclic Nucleotides in Mouse Spermatozoa¹

^a Department of Physiology, Tokyo Women's Medical University School of Medicine, Tokyo, Japan ^b Department of Obstetrics and Gynecology, Juntendo University School of Medicine, Tokyo, Japan ^c Laboratory of Intracellular Metabolism, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan

ABSTRACT

Rises in intracellular Ca²⁺ concentration ([Ca²⁺]_i) caused by progesterone, an inducer of the acrosome reaction, or by cyclic nucleotides, possible second messengers, were investigated by Ca²⁺ imaging of the head of individual mouse sperm. Progesterone induced a [Ca²⁺]_i rise in a dose-dependent manner (4–40 µM), primarily in the postacrosomal region. For 20-µM progesterone, Ca²⁺ responses occurred in 42% of sperm, separated into two types: transient type (60% of responding cells; duration, 1–1.5 min; mean amplitude, 335 nM) and prolonged type (40%; >3 min; 730 nM). Prolonged responses required higher doses of progesterone, and their occurrence was enhanced significantly by preincubation for 2–4 h as compared with transient responses. 8-Bromo-cGMP (0.3–3 mM) induced a [Ca²⁺]_i rise more effectively than did 8-bromo-cAMP. For 1-mM 8-bromo-cGMP, 90% of cells exhibited transient Ca²⁺ responses (~1 min; 220 nM), independently of the preincubation time. In Ca²⁺-free medium, most sperm showed no Ca²⁺ response to progesterone and 8-bromo-cGMP. Pimozide, a Ca²⁺ channel blocker, completely blocked prolonged responses and partially inhibited transient responses. These results suggest that progesterone activates at least two distinct Ca²⁺ influx pathways, with fast or slow inactivation kinetics, and some sperm show both types of response. A cyclic nucleotide-mediated process could participate in the progesterone-induced [Ca²⁺]_i rise.

calcium, cAMP, cGMP, progesterone, signal transduction

INTRODUCTION

The acrosome reaction (AR) of sperm, characterized by exocytosis of the acrosomal vesicle (AV), is a prerequisite step for sperm-egg membrane fusion at fertilization. In mammals, the egg zona pellucida (ZP) is generally thought to be the physiological initiator of the AR of the fertilizing sperm, and ZP3, one of the major glycoproteins in the ZP, can induce the AR in vitro in a species-specific manner [1–3]. Progesterone, which is secreted from the cells of the cumulus oophorus and contained in the follicular fluid, is also able to induce the AR [4, 5]. Progesterone could play a physiological role in induction of the AR, because the follicular fluid is incorporated into the oviduct upon ovulation and sperm pass through the cumulus oophorus to reach ovulated eggs. The AR requires an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) [3], as is generally the case in exocytotic events. A [Ca²⁺]_i rise in response to solubilized ZP has been recorded from sperm in suspension [6] or from single sperm of the mouse [7], and spatiotemporal aspects of the [Ca²⁺]_i rise have been precisely analyzed in hamster sperm [8]. The ZP-induced Ca²⁺ response in mouse sperm lasts over 10 min [6, 7]. In human sperm, Ca²⁺ responses are analyzed mainly using progesterone as the AR-inducing factor because of the ethical problem of collecting the ZP from unfertilized human eggs. The progesterone-induced Ca²⁺ response in human sperm takes the form of a transient [Ca²⁺]_i rise that lasts for 1–3 min [9–11], unlike the prolonged ZP-induced Ca²⁺ response in mouse sperm. Progesterone induces the AR in mouse sperm as well [12–14]. The dependence of the AR on Ca²⁺ influx has been shown [12, 13], but Ca²⁺ responses to progesterone in mouse sperm have not been clearly demonstrated.

The signal transduction mechanisms that link stimulation by the ZP or progesterone to the [Ca²⁺]_i rise have been analyzed but not fully established. ZP3 is thought to bind multiple receptors [2], and some signaling pathways in mouse sperm have been proposed, including pertussis toxin-sensitive G proteins that may activate L-type-like Ca²⁺ channels [6, 15, 16] and pertussis toxin-insensitive G proteins coupled to voltage-independent cation channels that may activate T-type Ca²⁺ channels via depolarization [17–19]. Besides Ca²⁺ influx through Ca²⁺ channels, participation of intracellular Ca²⁺ mobilization has been postulated, in particular Ca²⁺ release through inositol 1,4,5-trisphosphate (InsP₃) receptor/Ca²⁺ channels from the AV [16]. For progesterone, there are many reports about various aspects of its action on sperm. One of the pathways for induction of the AR by progesterone in both human and mouse sperm is mediated by gamma amino-butyric acid A (GABA_A) receptor/Cl^- channels that may activate voltage-dependent Ca²⁺ channels via depolarization [11, 13, 20]. The progesterone-induced AR is insensitive to pertussis toxin, whereas the ZP-induced AR is inhibited by pertussis toxin [14]. Other studies have shown that progesterone increases the level of cyclic nucleotides (CNs) in human sperm [21] and that application of membrane-permeable CNs induces Ca²⁺ influx [22, 23] as well as the AR [24, 25]. Cyclic-nucleotide-gated channels have been cloned from bovine sperm [22, 23]. It has been postulated that CNs cause intracellular Ca²⁺ release [26].

In the present study, we recorded Ca²⁺ responses induced by progesterone in individual mouse sperm using a Ca²⁺ imaging method to analyze the mechanism of the [Ca²⁺]_i rise. The temporal pattern of Ca²⁺ responses was investigated in comparison with previously reported progesterone-induced Ca²⁺ responses in human sperm or ZP-induced Ca²⁺ responses in mouse sperm. The mode of Ca²⁺ mobilization leading to the [Ca²⁺]_i rise was identified by removal of external Ca²⁺, and the Ca²⁺ channels responsible for Ca²⁺ influx were analyzed by using Ca²⁺ channel blockers. The possible roles of the second messengers, cGMP and cAMP, were examined similarly.

MATERIALS AND METHODS

Media

M16 medium [27] supplemented with BSA (4 mg/ml) was used for sperm collection and incubation. M2 medium [28] without BSA was used for measurement of [Ca²⁺]_i. Ca²⁺-Free M2 medium was obtained by omitting Ca²⁺ and adding 1 mM EGTA.

Preparation of Sperm

Sperm were obtained from B6D2F₁ (C57BL/6J x DBA/2) male mice (12–16 wk old). Animals were rapidly killed by cervical dislocation. An isolated cauda epididymis was cut in several places with scissors in a 200-µl drop of M16 medium in a plastic dish. Soon after sperm dispersed into the medium, the whole sperm suspension was transferred to a 1.5-ml plastic tube. This time was designated as the zero time of sperm incubation. Sperm swam up during incubation for the first 30 min at 37°C under 5% CO₂ in air.

Before experiments, sperm were loaded with the Ca²⁺-sensitive fluorescent dye fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes, Eugene, OR). The stock solution was 2 mM fura-2 AM in dimethyl sulfoxide (DMSO), stored frozen, and diluted to 5 µM in M16 before use. The upper 100-µl layer of the sperm suspension containing motile spermatozoa was transferred to two 1.5-ml plastic tubes (50 µl in each tube). To one of the tubes, 100 µl of M16 containing 5 µM fura-2 AM and 0.01% detergent pluronic F-127 (Molecular Probes) was added immediately, and the tube was incubated for 30 min. The other tube was treated with fura-2 AM 30–60 min later. After loading the sperm with fura-2, a 50-µl drop of the sperm suspension was placed into two experimental dishes. A few minutes later, the medium was gently replaced by fura-2 AM-free M2 using a fine pipette. Sperm that had not attached to the bottom of the dish were removed by this procedure. The final volume of the drop was either 100 or 80 µl. The drop was covered with oil to avoid evaporation. The two dishes were used for [Ca²⁺]_i measurement with a 15–20-min interval. Thus, experiments were performed after a total incubation time of 60–240 min (including the time for fura-2 loading). During this time, the sperm underwent capacitation [3].

The experimental dish was fabricated as follows. A hole (5 mm in diameter) was made in the bottom of a 35-mm plastic dish, and a glass coverslip (25 x 25 mm) to allow passage of ultraviolet (UV) light was attached from underneath with dental wax. The coverslip was treated with mouse laminin (600 µg/ml; GIBCO, Rockville, MD) to facilitate attachment of sperm to the glass [29]. Poly-L-lysine-coated dishes were not used because more sperm showed higher basal [Ca²⁺]_i, suggesting that sperm tended to be damaged. The dish was placed on the stage of an inverted microscope (IX 70; Olympus, Tokyo, Japan).

Ca²⁺ Imaging

Changes in [Ca²⁺]_i in individual sperm were recorded by a Ca²⁺ imaging system (Argus 50; Hamamatsu Photonics, Hamamatsu, Japan). Ultraviolet light of 340 and 380 nm (UV₃₄₀ and UV₃₈₀) was produced by a xenon lamp and narrow band pass filters of 340 ± 10 nm and 380 ± 10 nm attached to a rotating plate. Fura-2-loaded sperm were irradiated with UV₃₄₀ for 0.25 sec, followed 0.8 sec later by UV₃₈₀ for 0.25 sec, from the bottom of the dish through a x60 objective (NA 1.2, UPlanApo, Olympus). The emitted fluorescence was sent to a silicon intensifier target camera through the same objective and a band pass filter of 510 ± 10 nm. Data sets were stored on the hard disk of the computer as eight-bit digital images and processed to calculate the ratio of fluorescence (R = F₃₄₀/F₃₈₀) with NIH Image (a public domain image processing software for the Macintosh computer). A calibration curve between R and Ca²⁺ was obtained by measuring Rs of Ca²⁺-N-(2-hydroxyethyl) ethylenedinitrilo-triacetic acid buffer solutions. Processed images were presented using pseudocolor to indicate [Ca²⁺]_i.

Experimental Procedure

The experimental dish was kept at 36–37°C by a heated plate. The air was not supplemented with CO₂ because the [Ca²⁺]_i measurements were completed within 15 min. Ultraviolet irradiation was minimized to avoid photo bleaching of fura-2 and damage of the sperm. The basal [Ca²⁺]_i was measured by Ca²⁺ image acquisition (a pair of F₃₄₀ and F₃₈₀) at an interval of 20 sec for the first 3 min. Progesterone was applied from a pipette at about the middle of a 20-sec interval because slight movement due to addition of the medium caused an artifact in the Ca²⁺ images. The sampling interval was changed to 10 sec for the first 60 sec after addition of progesterone and back to 20 sec in the later period. In some experiments using CNs, the sampling interval was set at 2.5 sec for the first 60 sec because the rising phase of [Ca²⁺]_i was faster than that for the progesterone-induced response. The total recording time in a trial was 6.5–13 min. Progesterone (Wako, Tokyo, Japan) was applied by adding 2 µl of M2 containing 0.2–2 mM progesterone (made from 2–20 mM progesterone in DMSO) to the 100-µl drop of the experimental medium. The final concentration of DMSO was 0.2%, irrespective of the progesterone concentration. The effect of CNs was examined by adding 20 µl of M2 containing 1.5–15 mM 8-bromo-cGMP (8-Br-cGMP) or 8-bromo-cAMP (8-Br-cAMP), or 5 mM SpcAMP (adenosine 3',5'-cyclic phosphorothioate-Sp; Calbiochem, La Jolla, CA). The final concentrations of these chemicals were calculated under the assumption of uniform distribution in the drop. To examine the effects of the L-type Ca²⁺ channel blocker verapamil (Sigma, St. Louis, MO) or the T-type Ca²⁺ channel blocker pimozide (RBI, Natick, MA), the experimental medium was replaced by M2 containing these chemicals, and then stimulants were applied.

Selection of Spermatozoa

Ca²⁺ images were acquired from 10–25 spermatozoa in an optical field, and [Ca²⁺]_i was measured in the sperm head. Because the isolated sperm tended to be damaged during preparation procedures, it was essential to select intact spermatozoa. Data were collected from spermatozoa with their heads attached to the bottom of the dish and their tails beating. Nonmotile spermatozoa with straight or curled tails showed poor fura-2 fluorescence in their cytoplasm, suggesting leakage of the dye out of the cell. Spermatozoa with beating but curled tails had higher basal [Ca²⁺]_i and displayed a substantial [Ca²⁺]_i rise in response to addition of medium (without stimulants) from a pipette. The number of nonmotile and/or curled spermatozoa in an optical field was 1–4 out of 10–25 spermatozoa. Data from these damaged or dead spermatozoa were discarded.

Evaluation of Ca²⁺ Responses

For evaluation of Ca²⁺ responses, the amplitude of the [Ca²⁺]_i rise was measured ([Ca²⁺]_i = peak [Ca²⁺]_i - basal [Ca²⁺]_i). A change in [Ca²⁺]_i of <50 nM was not considered significant because the basal [Ca²⁺]_i fluctuated considerably. The effects of stimulants were evaluated by 1) the percentage of responding sperm out of the total motile sperm used for [Ca²⁺]_i measurement and 2) [Ca²⁺]_i in all responding sperm. Values were presented as mean ± SEM in several trials. A Student t-test was used for analysis between groups. A P value of 0.05 was considered significant.

Observation of the Acrosome Reaction

The occurrence of the AR was examined using the chlortetracycline (CTC) method [12, 13] and conditions similar to those as for [Ca²⁺]_i measurement. The sperm were treated with a buffer containing 350 µM CTC (Sigma) 5 min after application of progesterone or 8-Br-cGMP and immediately followed by 1% glutaraldehyde. The sperm were sandwiched between coverslips and kept for 30 min in the dark. The AR was determined by the absence of staining or by very faint staining of the acrosomal cap as viewed with a fluorescence microscope (DIAPHOTO-TMD; Nikon, Tokyo, Japan).

RESULTS

Progesterone-Induced Ca²⁺ Responses

The basal [Ca²⁺]_i in the head of the sperm selected for data analyses (see Materials and Methods) ranged between 200 and 300 nM. Progesterone at the final concentration of 20 µM induced Ca²⁺ responses in 42% ± 6% of cells (eight experiments). Representative Ca²⁺ images are shown in Figure 1 (A and B), and quantitative data are presented in Figure 2. Spermatozoa showing weak flagellar motion were selected for presentation of Ca²⁺ images to avoid artifacts due to movement. The time of progesterone application was defined as the zero time. Values of [Ca²⁺]_i from two images immediately before and after addition of progesterone are connected with a dotted line in Figure 2.

View larger version (11K): [in this window] [in a new window]   FIG. 1. Images of Ca2+ responses in mouse sperm induced by 20 µM progesterone (A and B) or 1 mM 8-Br-cGMP (C). Progesterone-induced Ca2+ responses can be classified as transient (A) or prolonged (B) responses. The zero time was defined as the time of application of a stimulant

View larger version (30K): [in this window] [in a new window]   FIG. 2. Time course of changes in [Ca2+]i in the sperm head caused by 20 µM progesterone. A) Transient responses. B) Prolonged responses. The response indicated by a bold line in A or B corresponds to that shown in Figure 1, A and B, respectively

Spatial pattern Ca²⁺ images showed that a [Ca²⁺]_i increase occurred in the postacrosomal region of the sperm head immediately after application of 20 µM progesterone (Fig. 1, A and B). A smaller increase occurred in the acrosomal cap region. This may indicate a [Ca²⁺]_i rise in the thin region of cytoplasm surrounding the AV, although this could also be an optical artifact resulting from the large increase in fluorescence in the postacrosomal region. Fura-2 showed significant incorporation into the AV, as indicated by fluorescence remaining in the acrosomal cap region of the sperm in which most of fura-2 in the cytoplasm had leaked out (see Materials and Methods). Ca²⁺ in the AV in the resting state did not appear to be particularly high (Fig. 1, left), suggesting that the AV may not be a Ca²⁺ store. [Ca²⁺]_i in the flagellum seemed to be substantially elevated in response to progesterone, but accurate measurement was impossible in the present experiments.

Temporal pattern Although Ca²⁺ responses to 20 µM progesterone varied among sperm in amplitude ([Ca²⁺]_i) and time course (Fig. 2), two temporal patterns were observed. In one pattern, [Ca²⁺]_i reached a peak 10–15 sec after progesterone application and declined nearly to the basal level at 60–80 sec (Figs. 1A and 2A). In the other pattern, [Ca²⁺]_i reached a peak at 25–30 sec and was sustained at the peak level or declined much more slowly than the first pattern (Figs. 1B and 2B). Figure 3 is a histogram of the time between the half-maximal points in a Ca²⁺ response (T1/2; see inset) presented with a bin size of 20 sec. Cases in which T1/2 was over 180 sec or could not be obtained within the time of the [Ca²⁺]_i measurement (3.5–10 min) were categorized as >180 sec. Values of T1/2 were clearly separated into two groups. One group ranged between 15 and 100 sec, with the peak around 40 sec, and the other group was distributed over 180 sec. Only a small number of sperm showed intermediate values between the two groups. The former group was defined as a transient response, and the latter group was defined as a prolonged response.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Histogram showing the distribution of the times between the half-maximal points of the Ca2+ response (T1/2) induced by 20 µM progesterone (see inset). Bin size = 20 sec

Dose-response relationship Figure 4, A and B, shows the percentage of responding sperm and amplitude ([Ca²⁺]_i = peak [Ca²⁺]_i - basal [Ca²⁺]_i), respectively, as a function of the progesterone concentration (three to eight experiments each). In control experiments, a small transient [Ca²⁺]_i rise of ~60 nM occurred in ~5% of cells. Transient Ca²⁺ responses were induced by 4 µM progesterone in 23% of cells, and their mean [Ca²⁺]_i was 155 nM. With 40 µM progesterone, the mean [Ca²⁺]_i was increased to 345 nM, but the percentage of responding sperm was not significantly increased. Prolonged Ca²⁺ responses were not seen with 4 µM progesterone, and only 4% of cells showed prolonged responses to 10 µM progesterone (Fig. 4A). Thus, the minimal effective concentration for induction of prolonged responses was substantially higher than that for transient responses. The mean [Ca²⁺]_i for prolonged responses to 10 or 20 µM progesterone was 665 or 730 µM, respectively (Fig. 4B), indicating that prolonged responses are generally greater than transient responses. However, in some cases, the prolonged Ca²⁺ responses were smaller than relatively large transient responses. The prolonged response pattern is, therefore, an intrinsic characteristic and is not simply due to a delayed decline of the larger [Ca²⁺]_i rise.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Dose-response relationship of progesterone-induced Ca2+ responses. A) Percentage of sperm showing transient Ca2+ responses (; mean ± SEM) or prolonged responses (). The percentage of the total number of responding cells is also presented (). The numbers in parentheses indicate the number of experiments. B) The amplitude of the [Ca2+]i rise in transient (open bars) or prolonged (solid bars) responses ([Ca2+]i = peak [Ca2+]i - basal [Ca2+]i). The numbers in parentheses indicate the number of cells from which Ca2+ responses were obtained

With 40 µM progesterone, 28% of sperm showed prolonged responses, and 16% showed transient responses. The percentage of total responding cells was 43%, the same as with 20 µM progesterone (Fig. 4A). With 40 µM progesterone, the mean [Ca²⁺]_i for prolonged responses was decreased to 540 nM, whereas that of transient responses was unaltered (Fig. 4B). Some fraction of the transient responses might become prolonged responses at this high concentration of progesterone. On the whole, 20 µM was the optimal concentration to evaluate progesterone-induced Ca²⁺ responses. This concentration was used for the following analysis.

Effects of preincubation time The sperm were preincubated for >1 h to induce capacitation, as is usually used for in vitro fertilization. Figure 5 shows that the mean percentage of sperm responding to 20 µM progesterone was higher for longer preincubation times (Fig. 5A), although the amplitude of Ca²⁺ responses was unaltered (Fig. 5B). After 1–2 h of incubation, the frequency of transient and prolonged Ca²⁺ responses was only 11% and 6%, respectively. After 2–3 h of incubation, the frequency of transient responses was significantly increased to 26% (P < 0.05) but was not further elevated after 3–4 h. After 2–3 h of incubation, the frequency of prolonged responses was greater, and after 3–4 h, it was significantly increased to 35% (P < 0.01; Fig. 5A). Thus, preincubation facilitated the responsiveness of sperm to progesterone. The effect was more significant for the prolonged responses. Incubation of >4 h resulted in a higher rate of curled spermatozoa (~30%), in which a [Ca²⁺]_i rise was produced by DMSO alone. Thus, incubation of the sperm for 2–4 h was optimal for evaluating progesterone-induced Ca²⁺ responses.

View larger version (26K): [in this window] [in a new window]   FIG. 5. Effects of preincubation time on Ca2+ responses induced by 20 µM progesterone. Data are presented as in Figure 4. Asterisks indicate values at 2–3 h that are significantly larger than the value at 1–2 h (*P < 0.05 or **P < 0.01) and a value at 3–4 h that is significantly larger than the value at 2–3 h (**P < 0.01)

Induction of the AR Because the AR of the mouse sperm could not be morphologically identified in the same cells that had been used for Ca²⁺ imaging, the induction of the AR was examined by CTC staining under conditions similar to those for Ca²⁺ imaging but without application of fura-2 and UV light. Although definitive identification of the AR of mouse sperm is difficult even using this method, the induction of the AR by 20 µM and 40 µM progesterone was estimated to be 27% and 37%, respectively (about 200 spermatozoa were counted in three experiments for each progesterone concentration). These values were smaller than 42–43% for induction of Ca²⁺ responses.

Cyclic Nucleotide-Induced Ca²⁺ Responses

Effects of cyclic nucleotides Both 8-Br-cGMP and 8-Br-cAMP (1 mM) induced Ca²⁺ responses in 91% ± 4% of cells in six experiments and in 82% ± 4% of cells in four experiments, respectively. All of these Ca²⁺ responses were the transient type. The [Ca²⁺]_i rise was most notable in the postacrosomal region of the sperm head (Fig. 1C). In Figure 6A, the [Ca²⁺]_i rise in response to 8-Br-cGMP appeared to reach a peak within 10 sec after application of 8-Br-cGMP; i.e., the time resolution was insufficient. Therefore, Ca²⁺ images were sampled every 2.5 sec while 8-Br-cGMP was gently added with a microsyringe. Figure 6B shows that Ca²⁺ responses to 3 mM 8-Br-cGMP reached a peak at about 5 sec. [Ca²⁺]_i returned nearly to the basal level within 60 sec. The mean value of T1/2 for the Ca²⁺ responses induced by 1 mM 8-Br-cGMP was 36 ± 0.7 sec (n = 281), significantly smaller than that for the transient responses induced by 20 µM progesterone (48 ± 2 sec, n = 85; P < 0.01). The mean [Ca²⁺]_i for 1 mM 8-Br-cGMP was 220 ± 15 nM (n = 115), which was the maximal value in the dose-response relationship but was significantly smaller than the amplitude of the transient responses induced by 20 µM progesterone (337 ± 29 nM, n = 56; P < 0.01). Ca²⁺ responses induced by 1 mM 8-Br-cAMP had a similar temporal pattern, but their mean [Ca²⁺]_i was much smaller (87 ± 7 nM, n = 27) than the cGMP-induced response (P < 0.01). SpcAMP (1 mM), a specific agonist of cAMP, induced a transient Ca²⁺ response in 88% ± 2% of cells (Fig. 7A). The mean [Ca²⁺]_i induced by 1 mM SpcAMP was intermediate between the values for 8-Br-cGMP and those for 8-Br-cAMP (186 ± 16 nM, n = 41; Fig. 7B).

View larger version (17K): [in this window] [in a new window]   FIG. 6. Time course of changes in [Ca2+]i in the sperm head caused by 1 mM (A) or 3 mM (B) 8-Br-cGMP. [Ca2+]i was measured every 10 sec (A) or 2.5 sec (B). The Ca2+ response indicated by a bold line (A) corresponds to the response shown in Figure 1C

View larger version (26K): [in this window] [in a new window]   FIG. 7. Dose-response relationship of 8-Br-cGMP- and 8-Br-cAMP-induced Ca2+ responses (A and B) and the effect of preincubation time on the 8-Br-cGMP-induced Ca2+ responses (C). Ca2+ responses induced by 1 mM SpcAMP are also included (A and B). Data are presented as in Figures 4 and 5. Asterisks in A indicate significant difference between 8-Br-cGMP and 8-Br-cAMP. Asterisks with horizontal bars in B indicate significant difference between values (**P < 0.01)

Dose-response relationship About 50% of sperm responded to 0.3 mM 8-Br-cGMP (Fig. 7A), although the mean [Ca²⁺]_i was 85 nM (Fig. 7B). The response was increased to 98% and the mean [Ca²⁺]_i was increased to 210 nM by raising the concentration to 3 mM. The mean response of cells to 0.3 mM 8-Br-cAMP was about 25%, half of that for 0.3 mM 8-Br-cGMP (Fig. 7A). The response was >80% at 1 and 3 mM, but the mean [Ca²⁺]_i in the responding cells was significantly smaller than that for 8-Br-cGMP, even when the concentration of 8-Br-cAMP was increased to 3 mM (Fig. 7B). Thus, cAMP is less effective than cGMP.

No effect of preincubation For 8-Br-cGMP-induced Ca²⁺ responses, neither the responsiveness of the sperm nor [Ca²⁺]_i was affected by the preincubation time (1 to 4 h; Fig. 7C), unlike the progesterone-induced Ca²⁺ responses.

Induction of the AR The percentage of sperm that underwent an AR in response to 1 mM and 3 mM 8-Br-cGMP was 23% and 26%, respectively. This response was much smaller than the 91% and 98% of sperm that showed Ca²⁺ responses. Only the relatively large transient Ca²⁺ responses induced by cGMP seemed to cause the AR.

Effects of External Ca²⁺ and Ca²⁺ Channel Blockers

The mode of Ca²⁺ mobilization for progesterone- or cGMP-induced Ca²⁺ responses was examined by removal of Ca²⁺ from the external medium. In Ca²⁺-free medium, the prolonged Ca²⁺ response was never induced by 20 µM progesterone (Fig. 8A), indicating that the prolonged [Ca²⁺]_i rise is due to Ca²⁺ entry from outside of the cell. Transient Ca²⁺ responses were observed in only 6% of sperm (Fig. 8A), and the mean [Ca²⁺]_i was 83 nM (Fig. 8B), in contrast to the values of 26% (P < 0.01) and 335 nM (P < 0.01) in the presence of Ca²⁺. This result indicates that Ca²⁺ influx is also responsible for the transient [Ca²⁺]_i rise. Intracellular Ca²⁺ release appears to contribute to the [Ca²⁺]_i rise in only a small fraction of sperm, and even for these sperm, it is a minor factor. That was also the case for transient Ca²⁺ responses to 1 mM 8-Br-cGMP (Fig. 8, C and D).

View larger version (40K): [in this window] [in a new window]   FIG. 8. Effects of external Ca2+ and Ca2+ channel blockers on Ca2+ responses induced by 20 µM progesterone (A and B) or 1 mM 8-Br-cGMP (C and D). Ca2+ responses were measured in Ca2+-free medium containing 5 mM EGTA or in the presence of 5 µM verapamil or 1 µM pimozide. The preincubation time was 2–3 h in all experiments. Spermatozoa were bathed in the test medium for 10 min before application of the stimulant. Asterisks indicate values that are significantly less than the Ca2+ responses in normal medium (control) (*P < 0.05 or **P < 0.01)

The Ca²⁺ influx pathway activated by progesterone or cGMP was examined by using Ca²⁺ channel blockers. In the presence of 5 µM verapamil, an L-type Ca²⁺ channel blocker, neither the mean percentage of responding sperm nor [Ca²⁺]_i was significantly changed for either transient or prolonged Ca²⁺ responses to 20 µM progesterone, although these values tended to be slightly decreased in comparison with control values (Fig. 8, A and B). Verapamil did not significantly reduce Ca²⁺ responses to 1 mM 8-Br-cGMP (Fig. 8, C and D).

In the presence of 1 µM pimozide, which is a T-type Ca²⁺ channel blocker, prolonged Ca²⁺ responses were never induced by 20 µM progesterone (Fig. 8A). The mean frequency of transient Ca²⁺ responses was decreased from 26% to 16% by pimozide (Fig. 8A), although the difference was not significant (P > 0.1). The mean [Ca²⁺]_i for the transient Ca²⁺ responses was significantly reduced by pimozide (P < 0.05; Fig. 8B), but it was still 220 nM in the presence of pimozide. For Ca²⁺ responses induced by 1 mM 8-Br-cGMP, 1 µM pimozide reduced the mean percentage of responding cells from 91% (control value) to 57% (Fig. 8C) and the mean [Ca²⁺]_i from 220 to 110 nM (Fig. 8D). This inhibition by pimozide was significant (P < 0.01).

DISCUSSION

The present study is the first to demonstrate Ca²⁺ responses of mouse sperm induced by progesterone and possible second messengers, cyclic nucleotides. [Ca²⁺]_i measurement using Ca²⁺ imaging was carefully performed in individual sperm, despite difficulties due to small size, flagellar motion, and sensitivity to mechanical treatment. Appropriate conditions had to be determined in manipulation of the sperm, preincubation, loading with fura-2, elimination of damaged sperm, avoidance of artifacts caused by mechanical stimulation upon application of stimulants or by chemicals such as DMSO, and the optimal concentration of stimulants. Values of the Ca²⁺ response parameters were still considerably scattered among cells. Ca²⁺ responses were induced by 4–40 µM progesterone. The reported concentration of progesterone for induction of the AR in mouse sperm is in the range of 3–15 µM [12]. The progesterone concentration in the follicular fluid has been reported in humans as 1 µg/ml [4] or 1.6 µg/ml [30] (approximately 3–5 µM). However, the effective concentration of progesterone in the follicular fluid is estimated to be at least five-fold higher than that of free progesterone, because corticosteroid-binding globulin contained in the follicular fluid increases the AR-inducing activity of progesterone [31].

Ca²⁺ imaging showed that the progesterone-induced [Ca²⁺]_i rise is greatest in the postacrosomal region, although the present method had low spatiotemporal resolution. High-resolution imaging of hamster sperm has shown that the ZP-induced [Ca²⁺]_i rise begins around the equatorial segment and spreads over the posterior region of the head within 0.6 sec [8]. The AV is postulated to serve as a Ca²⁺ store, as suggested by the presence of InsP₃ receptors on the acrosomal membrane [16]. However, this scenario seems unlikely because Ca²⁺ in the AV was not particularly high in the resting state and no reduction of the Ca²⁺ was detected upon stimulation by progesterone. Similar results were obtained in the ZP-stimulated hamster sperm [8].

The present study revealed that in response to progesterone, sperm exhibit either a transient [Ca²⁺]_i rise lasting for 1–1.5 min or a prolonged [Ca²⁺]_i rise that lasts for at least several minutes. With the present experimental conditions, the percentage of sperm that showed an AR in response to 20–40 µM progesterone was lower than that for Ca²⁺ responses. Most of the prolonged Ca²⁺ responses are probably sufficient to induce the AR. Among the transient Ca²⁺ responses, only the relatively large ones may cause the AR.

The two types of progesterone-induced Ca²⁺ responses showed the following characteristics: 1) both types were attributable to Ca²⁺ influx because they were completely abolished in Ca²⁺-free medium in most sperm; the participation of intracellular Ca²⁺ release, if any, appeared to be quite minor; 2) the transient type was induced by progesterone at lower concentrations and was smaller in amplitude than the prolonged type; 3) both types, particularly the prolonged type, were enhanced by preincubation for 2–4 h, the time required for sperm capacitation [3]; and 4) pimozide completely blocked the prolonged type and significantly inhibited the transient type, whereas verapamil did not significantly affect either type. These results lead us to suggest that the two types of Ca²⁺ responses involve at least two Ca²⁺ influx pathways mediated by distinct types of Ca²⁺-permeable channels with fast or slow inactivation kinetics. It is conceivable that the sperm showing the transient Ca²⁺ response mainly possess fast inactivating Ca²⁺ channels, and the sperm showing the prolonged response express both types of Ca²⁺ channels. The slowly inactivating Ca²⁺ channels (or signaling pathway that activates the channels) may develop more significantly during preincubation of the sperm than fast inactivating Ca²⁺ channels. In human sperm, the progesterone-induced Ca²⁺ response is transient, lasting for 1–3 min [9–11]. Fast inactivating Ca²⁺ channels may be predominantly expressed in human sperm.

For identification of Ca²⁺ channel types, pimozide is known as a blocker of the low threshold T-type voltage-gated Ca²⁺ channel [32, 33]. A pimozide-sensitive inward Ca²⁺ current [7] or [Ca²⁺]_i rise [19] has been shown to be involved in ZP3-induced responses in mouse spermatogenetic cells or spermatozoa, respectively, and is thought to trigger a long-lasting [Ca²⁺]_i rise, although the mechanism is unknown [7, 19]. However, because this Ca²⁺ transient response is as short as 200–300 msec in total duration [19], it is different from the transient response recorded in the present experiments. Pimozide at a concentration of 1 µM inhibits not only T-type Ca²⁺ channel current but also L-type Ca²⁺ channel current in undifferentiated PC12 cells [34] and inhibits cGMP-activated current in rod photoreceptor cells [35]. The pimozide-sensitive Ca²⁺ channels responsible for progesterone-induced Ca²⁺ influx are presumably different from the T-type Ca²⁺ channel.

A CN-induced [Ca²⁺]_i rise was also recorded primarily in the postacrosomal region. cGMP was more effective than cAMP. The [Ca²⁺]_i rise was transient and was smaller and shorter than the progesterone-induced transient responses. The transient Ca²⁺ response produced by application of 8-Br-cGMP alone seemed to be insufficient to induce the AR in the majority of sperm. The response was attributable to Ca²⁺ influx and was inhibited by pimozide but not by verapamil, like the progesterone-induced responses. The CN-induced transient Ca²⁺ response was not affected by preincubation of the sperm, unlike the progesterone-induced responses; the signaling pathway seems to have been established before incubation. These results and the finding of a progesterone-induced increase in intracellular CNs [21] suggest that the progesterone-induced transient Ca²⁺ response could be partly caused by pimozide-sensitive Ca²⁺-permeable channels activated by a CN-mediated process. It has been shown that subunits of CN-gated channels of photoreceptor cells are expressed in the flagellum of bovine sperm and that a [Ca²⁺]_i rise is induced by cGMP, or cAMP to a lesser extent, in the postacrosomal region and the flagellum [23]. The distribution of CN-gated channels in the mouse sperm head remains to be examined in future studies.

The slowly inactivating Ca²⁺ influx responsible for the prolonged Ca²⁺ responses appeared to be a pimozide-sensitive Ca²⁺ channel but not a verapamil-sensitive L-type Ca²⁺ channel. Alternatively, the prolonged response could be generated secondarily as a result of the transient response via, for example, elevated [Ca²⁺]_i or depolarization. In that case, the sensitivity to pimozide would be attributable to CN-activated channels involved in the transient response instead of slowly inactivating Ca²⁺ channels. It is unknown whether the prolonged response is mediated by the same Ca²⁺ influx pathway responsible for the prolonged [Ca²⁺]_i rise induced by ZP3. Further studies are required for precise identification of the Ca²⁺ channels and the functional coupling to progesterone-activated GABA_A receptor/Cl^- channels.

The [Ca²⁺]_i rise that causes the AR in human sperm has been examined using progesterone as the stimulant [9–11]. Studies in mouse sperm will provide additional information, because isolation and characterization of targeted molecules are more feasible in the mouse because of knowledge of the genetic background and the technology of gene-knockout animals.

ACKNOWLEDGMENTS

The authors are grateful to Drs. H. Shirakawa, S. Oda, and T. Awaji and Mr. T. Shikano for advice on the experiments and discussion. They thank Dr. L.A. Jaffe (Department of Physiology, University of Connecticut, Farmington, CT) for improvement of the manuscript. They also thank Mr. T. Oohira and Mr. Y. Konuma for their technical assistance.

FOOTNOTES

First decision: 15 October 1999.

¹ This work was supported by a special research grant for the development of characteristic education from the Japan Private School Promotion Foundation.

² Correspondence: Shunichi Miyazaki, Department of Physiology, Tokyo Women's Medical University, School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. FAX: 81 3 5269 7414; shunm{at}research.twmu.ac.jp

³ Yoshinori Kuwabata passed away on 20 February 2000.

Accepted: February 14, 2000.

Received: September 13, 1999.

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