MOLECULAR REPRODUCTION AND DEVELOPMENT 32~41-50(1992)

A Transient Rise in Intracellular Ca2' Is a Precursor Reaction to the Zona Pellucida-Induced Acrosome Reaction in Mouse Sperm and Is Blocked by the Induced Acrosome Reaction Inhibitor 3-Quinuclidinyl Benzilate BAYARD T. STOREY, CATHERINE L. HOURANI, AND J.B. KIM Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania The acrosome reaction induced by ABSTRACT the zona pellucida in mouse sperm has been shown to proceed in two stages experimentally distinguishable by the fluorescent probe chlortetracycline. Entry into the first stage of sperm bound to isolated,structurally intact zonae pellucidae is blocked by the compound 3-quinuclidinylbenzilate. In this study,we show, utilizing the fluorescent Ca2+ indicator fluo-3, that the first stage of the zona-induced acrosome reaction is characterized by an increase in intracellular Ca", followed by a decrease as the acrosome reaction proceeds. This calcium transient is completely suppressed by 3-quinuclidinylbenzilate. We conclude that the Ca2+ transient is induced by the zona pellucida and is required for the zona-induced acrosome reaction. Blockage of this sperm intracellular Ca2+transient provides a mechanism for the inhibitory action of 3-quinuclidinylbenzilate on the zona-induced acrosome reaction in mouse sperm. 0 1992 Wiley-Liss, lnc.

inhibitor 3-quinuclidiny benzilate (QNB) that allowed sperm to bind normally to the zona pellucida but blocked the acrosome reaction in sperm so bound. This compound was originally used as a n antagonist of the muscarinic subgroup of cholinergic receptors (Yamamura and Snyder, 1974). Other muscarinic antagonists were also found to inhibit the zona-induced acrosome reaction, but differences in the pharmacology of the inhibition indicated that this mode of QNB action was not directly comparable to that of its antagonism of neuroactive muscarinic receptors (Florman and Storey, 1982b). QNB was shown to bind to mouse sperm a t a single site with KD = 5 nM and to inhibit the zonainduced reaction with K, comparable to KD (Florman and Storey, 1981, 1982b). This finding implied that there is a sperm QNB binding protein (SQBP) that may act as a receptor for a zona ligand in the induced reaction, but such a protein has yet to be definitively identified. The subsequent demonstration that only one, ZP3, of Key Words: Fluo-3, lntracellular Ca2+transient, QNB, the three glycoproteins designated ZPl,ZP2, and ZP3 Population kinetics making up the zona pellucida structure (Bleil and Wassarman, 1980a,b) had the ability to mediate both sperm binding and the induction of the acrosome reaction INTRODUCTION (Bleil and Wassarman, 198Oc, 1983) suggested strongly As a model for mammalian fertilization, the mouse t h a t this induction had the characteristics of a ligandhas proved to be useful in both in vivo and in vitro induced secretory reaction (Florman et al., 1982) and studies. The sequence of events, starting from the first that the mechanism of the induction should involve encounter of the sperm with the zona pellucida of the intracellular signal transduction reactions similar to egg and ending with the acrosome reaction of the those observed in agonist-induced reactions in somatic sperm, that enables the sperm to penetrate the zona cells (Kopf, l989,1990a,b). Ca2+ has been implicated in pellucida, has been established over the past years in virtually all ligand-receptor-induced cellular resome detail in this model (see reviews by Wassarman, sponses mediated by cellular signal transduction (Tsien 1987a,b, 1988; Saling, 1989; Kopf and Gerton, 1991; and Tsien, 1990). In somatic cells, the intracellularly Storey and Kopf, 1991). With the fluorescent probe active Ca2' may come either from intracellular stores chlortetracycline (CTC), which gives different fluoresor from the external medium through activation of apcent patterns on acrosome-intact and acrosome-reacted mouse sperm, the sperm was shown to bind to the zona pellucida with its plasma membrane intact (Saling and Storey, 1979; Saling e t al., 1979) and, once bound, to Received September 16,1991; accepted December 16,1991. undergo the acrosome reaction induced by the zona pel- Address reprint requests to Dr. Bayard T. Storey, Department of Oblucida (Florman and Storey, 1982a). The zona-induced stetrics and Gynecology, 339 John Morgan Bldg., University of Pennacrosome reaction was demonstrated with a specific sylvania Medical Center, Philadelphia, PA 19104-6080.

0 1992 WILEY-LISS, INC.

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B.T. STOREY ET AL.

propriate channels or from both (Glossman and Striessnig, 1988; Tsien and Tsien, 1990). In mammalian spermatozoa, Ca2+ plays a central role in the acrosome reaction. Its source is the external medium; mature sperm cells have no identifiable intracellular Ca2+ stores (see reviews by Yanagimachi, 1988; Florman and Babcock, 1991). In human sperm, the acrosome reaction induced by human follicular fluid (Suarez et a]., 1986) was shown to be preceded by a transient influx of Ca2+ (Thomas and Meizel, 1988). Progesterone alone, a t levels consistent with its content in follicular fluid, induces the Ca2+ transient and subsequent acrosome reaction (Thomas and Meizel, 1989; Osman et al., 1989; Blackmore et al., 1990; Meizel e t al., 1990). The zona-induced reaction in bovine sperm has been well characterized (Florman and First, 1988a,b; Florman et al., 1990), and the requirement for a zona-induced Ca2+influx as prerequisite to the acrosome reaction was demonstrated by image-analysis methods with individual sperm cells (Florman et al., 1989). It thus became of importance to ascertain if a n early influx of Ca2+into mouse sperm bound to zonae pellucidae was a precursor reaction required for the zona-induced acrosome reaction. Previous work had implicated such a role for Ca2+by showing that the divalent cation ionophore A23187 markedly accelerated the zona-induced acrosome reaction and that this acceleration was partially inhibited by QNB (Lee and Storey, 1985). The Ca2+ influx into both human and bovine sperm was monitored with the Ca2+ fluorescent indicator fura-2 (Grynkiewicz et al., 1985). Attempts to use fura-2 to monitor Ca2+in mouse sperm failed; the indicator adsorbed to the sperm surface to give a uniform fluorescence that did not change during the zona-induced acrosome reaction (Lee and Storey, 1989).When this surface fluorescence was quenched by addition of exogenous Mn2+,the indicator was found to have been preferentially sequestered intracellularly in the acrosomal compartment and to be absent in the main cytosolic compartment of the cell (Lee and Storey, 1989). The acrosomal fura-2 fluorescence, designated pattern F, was lost in sperm bound to isolated, intact zonae pellucidae, and so undergoing the zona-induced acrosome reaction, with a time course identical to that for the loss of fluorescence from the fluorescent pH indicator N-(n-dodecyl)-9-aminoacridine (NDAA). This latter indicator was also localized in the acrosomal compartment of fresh sperm to give the pattern designated N (Lee and Storey, 1989). The simultaneous loss of pat.. terns F and N indicates the loss of permeability barriers to small cations, e.g., H + and Ca2+,in the plasma and outer acrosomal membranes, and this loss marks the endpoint of the first stage of the zona-induced acrosome reaction (Lee and Storey, 1985; Lee et al., 1987; Kligman et al., 1991) as assessed by CTC fluorescence pattern assay (Ward and Storey, 1984). Fura-2 proved useful in defining the transition reaction of loss of F and N patterns a s a key reaction defining the course of the zona-induced acrosome reaction, but it proved useless in monitoring a n early Caz+ in-

flux. In this paper, we describe the use of two newer fluorescent Ca2+ indicators, rhod-2 and fluo-3 (Minta et al., 1989), and provide evidence that the latter indicator does demonstrate a n early Ca2+ influx that is blocked by QNB.

MATERIALS AND METHODS Reagents and Media Fluo-3 AM, rhod-2 AM, and fura-2 AM were purchased from Molecular Probes (Eugene, OR). The divalent cation ionophore A23187 was from CalBiochem (La Jolla, CAI. HEPES (N-hydroxyethy1piperazine-N'ethanesulfonic acid) was obtained from Research Organics (Cleveland, OH). Water for culture media was obtained from Flow Laboratories (McLean, VA). Glutaraldehyde was obtained a s a 25% aqueous solution from Polysciences, Inc. (Warminster, PA). Hyaluronidase (bovine testicular), human chorionic gonadotropin (hCG), bovine serum albumin (BSA, fraction V powder), and agarose (Type VII) were from Sigma Chemical Co. (St. Louis, MO). Pregnant mare serum gonadotropin (PMSG) was from Diosynth, Inc. (Chicago, IL). N,Ndimethylformamide (DMF) and dimethylsulfoxide (DMSO) were HPLC/Spectro-grade from Peirce (Rockford, IL). RS( 2) 3-quinuclidinyl benzilate (QNB) was from Research Biochemicals Inc. (Natick, MA). All other reagents, from various suppliers, were of analytical grade. QNB and A23187 were all kept as stock solutions in DMF in light-shielded containers a t 4°C. Under these conditions, the reagents were stable for months. Fluo-3 AM, rhod-2 AM, and and fura-2 AM, supplied in packaging in tubes containing 20 kg, W3-e dissolved in DMSO, as recommended by the manufacturer, to give stock solutions of 4 mM for the latter two and 1mM for fluo-3 AM. (The special tubes containing the three indicator dyes were fully resistant to DMSO but appeared to soften in DMF.) Dye solutions were kept no longer than week. The medium (MJB) used in the Of gametes was a modification of the bicarbonate medium (HMB) used in previous studies (Lee and Storey, 1986, 1989).It had the composition 109 mM NaC1,5 mM KCI, 25 mM HEPES, 25 mM NaHCO,, 1.7 mM CaCI,, 1.2 mM MgC1z, 5.6 mM glucosey l.o mM pyruvate, 25.0 mM lactate, and 2.0% (WiV) BSA. Medium MJB was first prepared without pyruvate, CaCI,, BSA, and NaHCO,; it was filter sterilized through a 0.20 (*.mfilter (Nalgene) and frozen at -70°C in 20 ml aliquots for single use. Aliquots were thawed as needed, and pyruvate, CaCl,, BSA, and NaHCO, were added. The medium was then refiltered and sterilized. Collection of Gametes and Isolation of Zonae Pellucidae Spermatozoa were collected from the caudae epididymides of two Swiss Webster retired breeders. The caudae epididymides were excised and transferred to 1.0 ml medium MJB in a covered well of a four-well Nunc dish, and four small incisions were made. The

INTRACELLULAR Ca2+ IN MOUSE SPERM ACROSOME REACTION cauda was held with forceps, and the spermatozoa were very gently squeezed out of the incision by pressure for -5 sec from a second pair of forceps, then allowed to swim out of the epididymal tissue for 15 min. Alternatively, the tissue was gently minced at the rim of the well, and the sperm were allowed to swim away from the tissue for 15 rnin (Lee and Storey, 1985). After removal of the epididymal tissue, the concentrated sperm suspension was immediately checked for cell concentration by appropriate dilution of a n aliquot and counting in a hemacytometer. Enough medium was then added to the original suspension to give a cell concentration of 0.1-1.0 x lo7 cells/ml, depending on the experiment. The sperm were capacitated at the chosen cell concentration for 60 min a t 37°C in humidified 5% CO, in air (Ward and Storey, 1984). Eggs were obtained from virgin 10-12-week-old Swiss Webster mice, which had been superovulated with intraperitoneal injections of 10.0 IU PMSG followed 46-50 h r later by 10.0 IU hCG. Swollen ampullae, collected 13-15 h r after hCG, were ruptured; the cumulus-oophorus masses were teased out into the oil, and the combined masses were transferred to 2.0 ml MJB containing 10 mg/ml hyaluronidase to disperse cumulus cells. After 5 min of incubation for dispersal, the eggs were washed three times with MJB to remove enzyme and residual adherent cells. Zonae pellucidae were isolated by forcing the cumulus-free eggs through a micropipette with id = 0.75 diameter of egg with zona. The isolated zonae were washed three times with MJB, then collected in 0.10 ml aliquots of MJB such that each aliquot contained 100-200 individual zonae. An alternate source of zonae pellucidae was obtained by large-scale preparation of zona fragments purified by Percoll density gradient centrifugation, according to the method of Bleil and Wassarman (1986), with the following modifications. Lima bean trypsin inhibitor was added to the homogenization buffer a t 1mg/ml, and 1%Nonidet P-40 and 1M NaCl were added to the final two washed by centrifugation. The zona fragments were resuspended in a buffer of composition: 25 mM triethanolamine, 150 mM NaC1, 1 mM MgCl,, 1 mM CaCl,, and 1%(w/v) polyvinylpyrrolidone, brought to pH 7.8 with HC1, at -200 zonae/pl for storage at -80°C. The zona fragments were recovered by centrifugation at 15,OOOg for 5 min and resuspension to the desired zona content in medium MJB. In accordance with the report of Bleil and Wassarman (19861, the fragments showed identical properties to whole zonae from ovulated eggs in their ability to induce the acrosome reaction in bound sperm with a characteristic time course.

43

fluorescent probes. Since all three fluorescent probes utilized in this study were Ca2+ indicators with the structure of a tetracarboxylic acid, which, at the experimental pH of 7.4, are present in the charged impermeant tetracarboxylate anion form, the probes must be loaded into the cells as the neutral membrane-permeant tetra-acetoxymethyl (AM) esters of the dyes (Grynkiewicz et al., 1985; Minta e t al., 1989). This was done by incubation of the appropriate AM ester during the capacitation procedure described above. In the experiments utilizing fura-2 and rhod-2 in combination, capacitation was allowed to proceed for 30 min a t 0.81.0 x lo7 cells/ml (Lee and Storey, 1989). Fura-2 AM was then added to a final concentration of 4 pM. After a further 15 rnin interval of capacitation, rhod-2 AM was added. For the 60 min capacitation period, this procedure provided a 30 rnin loading period for fura-2 AM and a 1 5 min loading period for rhod-2 AM. It had been shown in our previous study that, with this combination of sperm cell and fura-2 AM concentrations and loading times, the fura-2 was found trapped only in the acrosomal compartment (Lee and Storey, 1989).With fluo-3 AM, the capacitation was carried out at 0.1 x lo7 cells/ml. The dye was added to a final concentration of 2 pM a t the beginning of the capacitation time and allowed to load for the full 60 min capacitation time. The maximum concentration of DMSO used in these experiments was 0.2%, which had been shown to be totally without effect on either the spontaneous or the zona-induced acrosome reaction in mouse sperm (Lee and Storey, unpublished experiments). After capacitation and concomitant loading of the sperm with fura-2 AM and rhod-2 AM, 3 pl aliquots of sperm in suspension a t O.S--~.O x lo7 cells/ml were added to 0.10 ml volumes containing the isolated zonae or zona fragments, such that the final cell concentration was 2-3 x lo5 cells/ml. With fluo-3 AM, the sperm were capacitated a t 0.1 x lo7 cells/ml, so 30 pl aliquots of sperm suspension were added to 70 p1 volumes containing the isolated zonae or zona fragments to give the desired 2-3 x lo5 celldm1 concentration of sperm in the binding reaction. Sperm were allowed to bind to zonae for 15 min, a t which time the zonae with bound sperm were washed rapidly three times in MJB, using a micropipette with id = -2 egg diameters to remove loosely associated sperm (Lee and Storey, 1985). This washing procedure for sperm bound to isolated, intact zonae was shown to be sufficiently vigorous that loosely associated sperm were removed as judged by complete loss from two-cell embryos (Inoue and Wolf, 1975; Bleil and Wassarman, 1980b; Bleil et al., 1988) and so should be sufficiently vigorous to wash out most of the extraFluorescence Assays: Populations of Sperm cellular dye. The zona fragments utilized from the Bound to Zonae large-scale preparations from ovaries ranged in size The assay of fluorescence pattern changes as a func- from hemizonae of nearly the same diameter as the tion of incubation time in sperm bound to isolated in- intact zona to pieces of about one-fourth the size of the tact zonae pellucidae was carried out essentially a s de- intact zonae. The washing procedure removed the scribed in earlier studies that utilized CTC (Lee and loosely bound sperm from these as effectively a s from Storey, 1985) and NDAA (Lee and Storey, 1989) as the intact zonae.

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B.T. STOREY ET AL.

The end of the washing period was taken as zero time. Different procedures were adopted for assaying the fluorescence patterns exhibited by the fura-21 rhod-2- and the fluo-3-treated sperm. For the sperm loaded with fura-21rhod-2, a t the selected time points postbinding, a 10 pl aliquot containing -20 zonae or zona fragments with bound sperm was mixed with 10 pl MJB containing 1.2% (wlv) agarose on a slide kept a t room temperature (22”C), a coverslip was placed over the drop, and the agarose was allowed to solidify. Type VII agarose proved to be the most useful in this procedure in that it could readily be liquefied as a 1.2% solution a t 65”C, and remained liquid at 3 5 T , but solidified rapidly a s a 0.6% solution after mixing with the sperm suspension on the slide at 22°C. The number of sperm bound to the isolated zonae was assessed by phase-contrast microscopy ( ~ 4 0 0 with ) a Nikon Optiphot microscope equipped with interchangeable phase-contrast and epifluorescence optics. The bound sperm were then first examined by epifluorescence microscopy a t X400 with fluorescence excitation at 405 nm, using the Optiphot “V” filter cassette that gives transmission between 395 and 425 nm. The emission was filtered through the combined DM 445 dichroic mirror of the filter cassette and 470K guard filter that blocks wavelengths shorter than 450 nm and gives maximum transmittance at wavelengths longer than 500 nm. This excitationlemission combination shows green fluorescence from fura-2 and orange-red fluorescence from rhod-2 on the same sperm cell when the cells are assayed at early times postbinding to zonae. The sperm were then examined by epifluorescence with excitation at 546 nm, using the Optiphot “G’ filter cassette that gives transmission between 525 and 555 nm. The emission was filtered through the combined DM 575 dichroic and 580W guard filter that blocks wavelengths shorter than 570 nm and gives maximum transmittance at wavelengths longer than 600 nm. This excitationlemission combination shows fluorescence only from rhod-2. Determinations of the fluorescence patterns were made at 15 and 30 min postbinding, then a t 30 min intervals thereafter to 180 min. In the experiments utilizing fluo-3, the sperm were examined by epifluorescence microscopy at x 400 with fluorescence excitation a t 4661486 nm, using the Optiphot “B” filter cassette that gives transmission between 460 and 490 nm. The emission was filtered through the combined DM 505 dichroic and 515W guard filter that blocks wavelengths shorter than 500 nm and gives maximum transmittance at wavelengths longer than 520 nm. The agarose interfered with the assessment of fluorescent patterns because of excessively bright hazy background emission. Glutaraldehyde at 0.1%, the concentration used successfully in experiments with CTC (Ward and Storey, 1984; Lee and Storey, 1985), also gave excessive background emission. In these experiments, a 10 pl volume containing 20-30 zonae or zonae fragments with bound sperm was placed on a slide inside a thin ring of vaseline 3 - 4 mm in diameter, and a coverslip was applied such that

the liquid filled the entire space bounded by the ring. This resulted in constraint of the sperm between the cover slip and the slide such that most of the sperm were oriented with the side plane of the head parallel to the slide surface. This immobilized the heads, although some flagellar motion could still be observed, and made the fluorescence patterns readily visible. Fading of the fluorescence, presumably due to photobleaching, occurred in 1-2 min, but this allowed sufficient time to distinguish bright- from dark-headed sperm for purposes of scoring.

Fluorescence Assay of a Single Sperm Bound to a Zona The method used to try to assess changes in fluo-3 fluorescence intensity in single sperm cells bound to structurally intact zonae or zona fragments was that described by Lee and Storey (1989) for monitoring changes in NDAA fluorescence, with the sole modification that the excitationlemission filter combinations appropriate to fluo-3, described above, were used. Photographic Recording Photographs of both phase-contrast and fluorescent emission were taken with the Nikon automatic camera unit for the Optiphot microscope, as described by Lee and Storey (1989), using Kodak T-MAX p3200 film.

RESULTS Fura-2/Rhod-2as Ca2+ Indicators Treatment of mouse sperm cells with the membrane permeant tetraacetoxymethyl (AM) ester form of the rhod-2 Ca2’ indicator, rhod-2 AM, with subsequent removal of the exogenous indicator by multiple washing of the sperm to bound to intact zonae, yielded cells with red fluorescence over the entire head and midpiece. There was no detectable change in this fluorescence emission in sperm bound to intact zonae over the time course required for induction of the acrosome reaction as detected with CTC (Lee and Storey, 1985,1989). This observation indicated that the intracellular rhod-2 AM had been hydrolyzed to the membrane-impermeant tetracarboxylate rhod-2 and so did not leak from the cells. The fluorescence was brightest when the excitation light was at 546 nm, near the excitation maximum of the dye (Minta et al., 1989), but excitation with light a t 405 nm also gave readily observable red fluorescence. This wavelength is optimal for excitation of the Ca2+free form of fura-2 (Grynkiewicz et al., 1985). Treatment of mouse sperm with both rhod-2 AM and fura-2 AM, with subsequent removal of both dyes from the suspension medium, yielded cells that showed the green emission from fura-2 over the acrosomal arc region of the sperm head, identical to the previously observed pattern F, and red fluorescence from rhod-2 over the rest of the head and the midpiece, when excitation light a t 405 nm was used. In sperm bound to zonae, the green fluorescence was lost over the same time course observed for loss of pattern F under the conditions of

INTRACELLULAR Ca2+ IN MOUSE SPERM ACROSOME REACTION

45

pattern F with its characteristic time course occurs in membrane-intact cells and in the absence of exogenous Mn2+. 100 80

40 20

0 0

20

40

60

80 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0

lime, post-tJinding,min Fig. 1. Percentage of mouse sperm loaded with the Ca2+indicator dyes fura-2 and rhod-2 showing dual-color fluorescence from both dyes (open squares) and red fluorescence only (solid squares) as a function of time after binding to isolated, structurally intact mouse zonae pellucidae from ovulated eggs. Excitation was at 405 nm, and the emission was observed a t wavelengths longer than 500 nm. The green fluorescence emitted by fura-2 a t this excitation wavelength was clearly observed as a n arc overlying the acrosomal region of the sperm head and corresponded precisely to pattern F seen with fura-2 alone in the presence of exogenous Mn2+(Lee and Storey, 1989).The remainder of the sperm head showed red fluorescence. As the green fluorescence was lost over time, the red fluorescence was retained, so that the cells appeared uniformly red. Only cells showing fluorescence were scored, so the experimental variable was the percentage of cells showing dual color of all the fluorescent cells counted; the result shown is the average of four to six determinations per time point, with error bars giving the standard deviation. The percentage cells showing only red fluorescence was 100% minus the dual color cells, so the points have no error bars shown.

Mn2+ tracer quenching utilized in the previous study (Lee and Storey, 1989), leaving cells that exhibited only red fluorescence. The time course curve for this F pattern loss and increase in percentage of cells exhibiting only red fluorescence is shown in Figure 1. The persistence of rhod-2 fluorescence in the cells as the fura-2 fluorescence is lost shows that the plasma membrane over the rest of the sperm cell remains intact. The loss of patterns F and N were scored in the previous study as loss of fluorescence from the cells under observation. Observation of the loss of pattern F necessitated the inclusion of 100 FM Mn2+ in the outside medium. In principle, this fluorescence loss could arise from damage to the plasma membrane overlying the head as well as from the physiological permeabilization of the membranes overlying the acrosome. Monitoring of sperm motility indicated that such damage was mostly absent or minimal (Lee and Storey, 1989),but the result shown in Figure 1, in which only cells retaining red fluorescence were scored, verifies that the physiological loss of

Fluo-3 as Ca2+Indicator The intensity of the red emission excitated at either 405 nm or 546 nm from sperm loaded with rhod-2 did not change perceptibly over the time course shown in Figure 1,so it was not useful as a reporter for intracellular Ca2' changes. The indicator fluo-3 did show an intensity change. This is shown in Figure 2. Despite a photo-induced loss of fluorescence intensity from the dye occurring within an observation time of 1-2 min, not unexpected from the reported photolability of fluo-3 (Minta et al., 19891, sperm bound to zonae with bright heads (Fig. 2D) and dim heads (Fig. 2B) could be clearly differentiated and readily scored. At early times after binding to zonae, the sperm exhibited mostly dim heads. As the time progressed, the percentage of sperm with bright heads first increased, then decreased. The fluorescence emission from the midpiece changed very little. Unfortunately, the photo-induced fluorescence intensity loss allowed the sperm heads to be scored only as bright or dim and precluded quantitation of the intracellular Ca2+levels corresponding to those two categories. Time Course of Fluo-3 Fluorescence Changes: Inhibition by QNB The time course of the increase and subsequent decrease of fluo-3 fluorescence in the heads of sperm bound to zonae is shown for four representative experiments in Figure 3. Also shown in Figure 3 is the inhibitory effect of 50 FM QNB on this change of fluorescence intensity. The averaged time course curve for the experiments shown in Figure 3 is presented in Figure 4. Under the conditions of the experiments depicted in Figure 3, QNB completely blocks the onset of the first stage of the zona-induced acrosome reaction in mouse sperm (Florman and Storey, 1982a; Lee and Storey, 1985, 1989; Lee et al., 1987; Kligman et al., 1991). A concomitant feature of that blockage is seen from Figure 3 to be complete inhibition of the fluo-3 intensity change seen in the absence of QNB. Since an increase in fluorescence emission intensity of about 30-fold from fluo-3 is observed in going from Ca2+-freet o Ca2+-saturated indicator, with KD of 400 nM (Minta et al., 1989), the transient increase in fho-3 intensity seen in the sperm bound t o the zona may be attributed to an increase in intracellular Ca2+concentration in the head, followed by a decrease. The implication of the transient time course seen averaged in Figure 4 is that acrosomereacted sperm have low intracellular Ca2+ contents in head comparable to those of acrosome-intact sperm at early times after binding to zonae. The intracellular Ca2+ increase may thus be attributed to the initiation of the acrosome reaction, while the initial and endreacted states of the cell's intracellular Ca2' content are similar. Treatment of the sperm in suspension in the absence of zonae with the ionophore A23187 in-

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B.T. STOREY ET AL.

Fig. 2. Paired phase-contrast and fluorescent images of mouse sperm bound to structurally intact mouse zonae pellucida after loading with the Ca2+indicator dye fluo-3. Phase-contrast (A) and epifluorescent (B) photographs of sperm bound to zonae obtained at about 20 min postbinding. Most of the sperm showed dim fluorescence over the head region and intermediate brightness over the midpiece. The fluorescence faded to a uniform dimness over the whole cell in 1-3 min, allowing time for scoring and photography. The cells were unfixed but partially immobilized by constraint between the slide and the coverslip (see Materials and Methods); some cells displayed flagellar mo-

tion, as seen by the blurred midpiece in the lower of the two cells shown. Phase-contrast (C) and epifluorescent (D) photographs of sperm bound to zonae obtained at about 60 min postbinding. Many cells displayed the bright fluorescence over the head as shown, with the fluorescence displayed by the midpiece remaining a t intermediate brightness. The bright head fluorescence seen in D remained for 1 3 min, allowing sufficient time for scoring and photography, and then faded, along with the midpiece fluorescence, as described above. Bar = 10 ym.

creased the percentage of sperm with bright heads over time, consistent with the expected slow increase of intracellular Ca2+ induced by ionophore alone (Florman and Storey, 1982a). In the presence of zonae, treatment with A23187 produced only dim heads within the time required for scoring. This is consistent with the very rapid acrosome reaction induced by zonae plus ionophore, which occurs too quickly to be assessed by the technique of scoring fluorescence patterns in sperm populations (Lee and Storey, 1985). The maximum in the percentage of sperm with bright fluo-3 fluorescence occurs at about 70 min postbinding (Fig. 41, which corresponds to the time of half-maximal loss of pattern F (Fig. 1) after the 30 min lag period characteristic of the zona-induced acrosome reaction occurring on structurally intact zonae (Florman and Storey, 1982a; Florman et al., 1982). If one assumes that the percentage of cells showing bright fluo-3 fluorescence in the presence of QNB represents the baseline value, the time to half-maximal percentage of bright cells during the phase of increasing intracellular Ca2+ is about 30 rnin postbinding, corresponding to the end of the lag period. This would be the time course expected,

if the rise in intracellular Ca2+ were occurring in the cells a s a precursor reaction to the reaction mediating the loss of pattern F. The loss of pattern F has been identified as the monitor of the transition reaction between the two regulated stages of the acrosome reaction (Kligman et al., 1991). The time course curves in Figures 1 and 4 were obtained by scoring populations of sperm bound to zonae for percentages of a given fluorescence pattern. This method has been designated that of “population kinetics” to differentiate i t from the measurement of fluorescence changes on individual cells bound to a n intact zona or a zona fragment, which has been designated that of “single cell kinetics” (Lee and Storey, 1989). All attempts to obtain single cell kinetics with fluo-3 in mouse sperm bound to structurally intact zonae or zona fragments failed because of photo-induced fluorescence loss, which occurred in 1-3 min. This loss occurred even a t the lowest levels of excitation light consistent with recording a fluorescence signal from the single cell in the microscope field by photomultiplier tube detection for quantitation of the levels of intracellular Ca2+ in the individual cells.

INTRACELLULAR Ca2+ IN MOUSE SPERM ACROSOME REACTION

*

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creased, there was an increase in the percentage of bound sperm showing the bright pattern, followed by a decrease in this percentage, indicative of a transient rise in intracellular Ca2+. This rise was completely blocked by QNB under conditions leading to complete inhibition of the zona-induced acrosome a t its earliest stage by this compound (Lee et al., 1987). The simplest interpretation of this result is that a transient rise in intracellular Ca2+is required for the zona-induced acrosome reaction, that this transient rise is itself induced by the zona pellucida, and that the inhibitory effect of QNB on the zona-induced acrosomal exocytosis derives from its blockage of the intracellular Ca2+rise. This interpretation is consistent with the earlier observation of partial inhibition by QNB of the very marked acceleration of the zona-induced acrosome brought I about by A23187. One plausible mechanism for the 0 50 100 150 QNB effect is that the zona-induced acrosome reaction requires activation of a Ca2+ channel mediated by a time, poatandlng,min sperm receptor binding one or more ZP3 ligands and Fig. 3. Percentage of sperm cells showing the bright head fluores- that this channel is blocked, either directly or indicence seen in Figure 2D as a function of time after binding to isolated, rectly, by QNB. Another plausible mechanism is that structurally intact zonae pellucidae fragments in the absence (open QNB prevents interaction of the appropriate ZP3 symbols) and presence (solid symbols) of 50 FM 3-quinuclidinyl benziligands with the sperm receptor itself. Resolution of late (QNB).Four separate experiments are shown to indicate the varithese interpretations should become feasible if the ation in time course found between individual experiments. mouse sperm binding site for QNB with K, = 5 nM, the putative SQBP mentioned above, can be identified as a membrane protein and characterized. This investiga50 I tion is underway. It was pointed out in an earlier study that the time courses obtained for population kinetics in this system 40 are dominated by the lag periods preceding rather rapid single reactions (Lee and Storey, 1989). Kinetic measurements with individual sperm cells bound to zonae 30 gave the result that the loss of pattern N was rapid, with a half-time of 2 min (Lee and Storey, 1989). The much longer time course observed by scoring the fluo20 rescent patterns of populations of cells bound to zonae could be attributed to the variable time lag between binding of the sperm and rapid loss of patterns N and F, with a spread of lag times from 30 to 150 min. The effect is to “stretch out” the time base of the kinetics com’OI pared with the time base of the single cell kinetics. If the reaction is a transient, such as the one detected 0 0 50 100 1 5 0 here in which intracellular Ca2+ rises, and then falls back towards the initial value, population kinetics has time, post-binding,min the additional complication that a sufficient fraction of Fig. 4. Averaged percentage and time points from the four experiments shown in Figure 3 to give a composite time course of the in- cells needs to be in the transient state a t the intermedicrease, followed by the decrease, in the percentage of sperm cells ate sampling time points for the reaction to be detectbound to zonae showing bright head fluorescence from intracellular able. The faster the transient, the lower the percentage fluo-3. Error bars shown are standard deviations. of cells caught in the transient state. The Ca2+influx occurring during the first stage of the zona-induced acrosome reaction in mouse sperm is apparently rapid DISCUSSION enough that a maximum of 40% of the cells scored were Despite its limitations due to photolability, the in the higher Ca2+ state but was not so rapid that it Ca2+indicator fluo-3 exhibited clearly distinguishable could not be detected. It is of interest that a transient bright and dim patterns on the sperm head (Fig. 2>, rise in intracellular Ca2+is the cationic precursor reacwhich made it possible to demonstrate a time-depen- tion to the progesterone-induced acrosome reaction in dent change in these two fluorescence patterns in human sperm (Thomas and Meisel, 19881, while a sussperm bound to zonae. As the time postbinding in- tained rise in intracellular Ca2+ is the precursor reac---t

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rI

48

B.T. STOREY ET AL.

tion t o the zona-induced acrosome reaction in bovine sperm (Florman et al., 1989). It should be emphasized that the results reported here show that a population of cells shows a rise in intracellular Ca2+ prior to the reactions causing the loss of CTC pattern B. In terms of the timing of the changes in mouse sperm intracellular Ca2+,it may well be that that the higher level is maintained until the transition point signalled by the loss of the F and N fluorescent patterns, corresponding to permeabilization of the acrosome compartment due to pore formation in the apposed outer acrosomal and plasma membrane (Storey and Kopf, 1991), after which transition the intracellular Ca2+returns to the initial value. The reaction of Ca2+influx into the sperm as precursor to the zona-induced acrosome reaction would thus be similar to that seen in the zona-induced acrosome reaction in bovine sperm. Fluorescent Ca2+indicators suitable for monitoring this change in single cells would be required for this, and none have been found to date. Examination of Figures 3 and 4 also reveals that the percentage of bright fluorescence sperm at the first time point samples was always higher than the percentage observed with QNB. If the percentage with QNB is taken as background, then the higher percentage of bright sperm seen a t the first time point implies that the transient increase in intracellular Ca2+occurs in some of the cells at very early times. This implication strengthens the case for the Ca2+increase as one of the precursor reactions t o the reaction of acrosome permeabilization that is reported by loss of patterns F and N. Further experimental confirmation of this proposed reaction sequence requires probes to report Ca2+ influx and acrosome permeabilization in the same cell. This problem is currently under investigation. Ligand-receptor induction of increased intracellular Ca2+concentration, often seen as a transient involving a rapid rise and slower decline, has long been recognized as one of the family of reactions that make up the intracellular signal transduction system linking the receptor with the cell’s effectors, which in turn bring about the cell’s response to the agonist ligand (Berridge, 1986). Components associated with intracellular signal transduction are present in mouse sperm, The GTP-binding proteins mediating such transduction, designated G proteins (Casey and Gilman, 1988), have been shown to be present in mouse sperm (Kopf et al., 1986; Glassner et al., 1991) as well as in sperm from other species (Bentley et al., 1986; Kopf et al., 1986). A subgroup of these, designated G, and sensitive to inactivation by the bacterial toxin pertussis toxin (PT) (Ross, 1989;Freissmuth et al., 19891,presumably functions in the induced acrosome reaction, since PT blocks this reaction without affecting sperm binding to zonae (Endo et al., 1987). A 95 kDa protein, designated p95, that binds ZP3 but not ZP2 after isolation by gel electrophoresis, has been identified and has been shown to possess tyrosine kinase activity enhanced by its aggregation caused by ZP3 ligand binding (Leyton and Saling, 1989a,b;Leyton et al., 1990; Saling et al., 1990). A number of somatic cells have receptors possessing this

ligand-induced aggregation-activated tyrosine kinase activity (Yarden and Ullrich, 19881, suggesting that p95 could be the primary sperm receptor mediating the ZP3 ligand-induced acrosome reaction. A similar set of intracellular signal transducing mediators is found in other cells manifesting ligand-induced exocytosis of intracellular granules, in particular, mast cells and basophils, and, in this exocytotic reaction, a transient increase in intracellular Ca2+is observed (Penner, 1988; Koopman and Jackson, 1990; Benhamou et al., 1990).It is of interest that, in these cells, the signal pathways initiated by ligand-induced receptor aggregation with consequent activation of tyrosine kinase (TK) activity and those activated by Ca2+ influx intersect at effector levels a t considerable remove “downstream” from the ligand-induced stimuli (Penner, 1988; Benhamou et al., 1990; Apgar, 1991). Roldan and Harrison (1989, 1990a,b)have reported that Ca2+influx induced by the ionophore A23187 can trigger polyphosphoinositide breakdown in sperm from ram, boar, mouse, human, and guinea pig and that this reaction occurs under conditions in which acrosomal exocytosis is effected by ionophore treatment. This could be one downstream point at which the zona-induced signals may converge; the ionophore treatment, by allowing sufficient Ca2+to enter the cell, may bypass the receptors responding to the zona ligands and activate this process directly. We postulate that an analogous situation occurs in the zonainduced acrosomal exocytosis in mouse sperm, in that ZP3 may activate two independent receptors on the mouse sperm plasma membrane overlying the acrosoma1 region. Both receptors must be activated for the full signal transduction chain to be activated a t a convergence point downstream from the receptors. An analogy is the safe deposit box, which requires the action of two separate keys in concert to open. One receptor is the p95 receptor, whose activation requires aggregation, with consequent action of TK activity leading to protein phosphorylation and possible activation of phospholipase C (Ledbetter et al., 1991). The other is the receptor mediating Ca2+ influx, whose action is blocked by QNB. The involvement of muscarinic receptors in modulation of Ca2+influx through channels in turn regulated by G proteins has been found in somatic cells (Yatani et al., 1987; Lechleiter et al., 1991). It is tempting to draw the analogy between these and the putative sperm cell receptor, SQBP, but it should be remembered that the pharmacology of the inhibition of the zona-induced acrosome reaction by muscarinic antagonists differs significantly from the pharmacology of somatic cell muscarinic receptor antagonism.

CONCLUSIONS We conclude from this study that the acrosome reaction induced in mouse sperm by the mouse zona pellucida proceeds through a series of reactions, which includes a transient rise in intracellular Ca2+ in the sperm head as an early essential reaction. Blockage of this transient rise is accomplished by QNB, under conditions leading to blockage of the zona-induced ac-

INTRACELLULAR Ca2+ IN MOUSE SPERM ACROSOME REACTION rosome reaction at its earliest stage. This observation provides for the first time a mechanism for the action of QNB as inhibitor of the zona-induced acrosome reaction. Details of this mechanism are not yet at hand, but the action of QNB in suppressing the required Ca2’ influx provides a means for studying the interaction of the reaction sequences leading to the zona pellucidainduced acrosome reaction in mouse sperm.

ACKNOWLEDGMENTS This work was supported by NIH Program Project HD06274. We thank Drs. G.S. Kopf and C.R. Ward for help and advice during the entire course of this study.

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