MOLECULAR REPRODUCTION AND DEVELOPMENT 3 1 : 7 H 6 (1992)

A Guanine Nucleotide-BindingRegulatory Protein in Human Sperm Mediates Acrosomal Exocytosis Induced by the Human Zona Pellucida MICHAEL A. LEE,l JEROME H. CHECK,' AND GREGORY S. KOPF2 'Department of Obstetrics and Gynecology, Cooper HospitallUniversity Medical Center, Robert Wood Johnson Medical School at Camden, University of Medicine and Dentistry of New Jersey, Camden, New Jersey; 'Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania ABSTRACT Guanine nucleotide-binding regulatory proteins play key intermediary roles in regulating zona pellucida-mediated acrosomal exocytosis in mouse and bull sperm. Since human sperm possess a G,-like protein and undergo the acrosome reaction in response to the human zona pellucida, we investigated whether this G protein plays a regulatory role in this exocytotic process. Zonae pellucidae isolated from eggs that had been inseminated but had shown no signs of fertilization after retrieval for in vitro fertilization and embryo transfer were pooled into groups of 250 in order to reduce variability in biological responses due to the possible presence of ZP that had undergone modifications associated with the polyspermy block. Acid-solubilized zonae pellucidae were incubated with capacitated sperm, and the sperm then assessed for the acrosome reaction using both the P. sativurn agglutinin and chlortetracycline fluorescence assays; both assays gave similar results. Sperm incubated with solubilized zonae pellucidae at a final concentration of 2, 4, or 6 ZP/pl underwent acrosomal exocytosis to a similar extent as compared with A-23187. Sperm were incubated with 1 pglml pertussis toxin during capacitation to functionally inactivate the G,-likeprotein. Pertussis toxin treatment of sperm did not affect sperm motility and the ability of the cells to bind to structurally intact zonae pellucidae. Pertussis toxin, however, completely inhibited the percentage acrosome reactions induced by solubilized zonae pellucidae. By contrast, the A-23187-inducedacrosome reaction was insensitive to PT treatment. Pertussis toxin inhibition of the zona pellucida-induced acrosome reaction occurred in a concentration-dependentmanner with maximal effects observed at 100 ng/ml PT. These data suggest that the pertussis toxin-sensitive G,-like protein in human sperm plays an important regulatory role in the acrosome reaction induced by the human zona pellucida. Key Words: G protein, Pertussis toxin, ADP-ribosylation, Human sperm, Acrosome reaction, Zona pellucida INTRODUCTION Sperm-egg interaction in the mammal first occurs at the level of the zona pellucida (ZP), a n egg-associated product that can be considered to be a n extracellular

0 1992 WILEY-LISS, INC.

matrix (Wassarman, 1988; Kopf and Gerton, 1990). It has been demonstrated in a number of species that the ZP functions to bind sperm in a species-specific fashion and to induce the acrosome reaction of those bound sperm (Kopf and Gerton, 1990). In the mouse, these two biological functions of the ZP are conferred by the ZP glycoprotein, ZP3 (Wassarman, 1988). The nature of the ZP glycoprotein(s) responsible for these biological functions in other mammals is either not known or has only begun to be examined (Moller et al., 1990). Since gamete recognition and binding appears to be mediated by complementary molecules associated with both the ZP of the egg and the plasma membrane of the sperm (Saling, 1989; Kopfand Gerton, 1990), i t is likely that sperm-ZP binding and subsequent acrosomal exocytosis has some similarities to receptor-mediated signaling systems that control cellular function in somatic cells. In the mouse, for example, ZP3 binding sites have been demonstrated to exist on the acrosomal cap region of acrosome-intact sperm (Bleil and Wassarman, 1986), and a number of molecules have been postulated to serve as sperm-associated receptors for ZP3 (Macek and Shur, 1988; Leyton and Saling, 1989; Bleil and Wassarman, 1990). Also consistent with the idea of receptormediated signaling is the demonstration that the ZP3induced acrosome reaction in mouse sperm (Endo et al., 1988) and the ZP-induced acrosome reaction in bovine sperm (Florman et al., 1989) are mediated by a spermassociated guanine nucleotide-binding regulatory protein(s) (G protein) of the Gi type. This particular class of G proteins are substrates for pertussis toxin (PT)-catalyzed ADP-ribosylation, and are functionally inactivated by such a covalent modification. G proteins play important intermediary roles as signal transducing elements in coupling many ligand-receptor interactions to intracellular second messenger cascades/ionic changes (Ross, 19891, and all mammalian sperm stud-

Received August 1,1991; accepted September 5,1991. Address reprint requests to Dr. Gregory S. Kopf, Room 313, John Morgan Building, Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6080.

G P R O T E I N S A N D ZONA P E L L U C I D A - I N D U C E D A C R O S O M E R E A C T I O N ied thus far, including the human, contain G,-like proteins (Bentley et al., 1986; Kopf et al., 1986). ZP-mediated signal transduction leading to acrosomal exocytosis in two species (mouse, bull), therefore, occurs through the action of these transmembrane signaling molecules. Little is known about ZP-mediated sperm signal transduction in the human due, for the most part, to a n inability to obtain sufficient quantities of human ZP for experimental purposes. The human ZP has been shown to bind human sperm and to induce the acrosome reaction of sperm (Cross e t al., 1988; Morales et al., 1989). The human ZP from both unfertilized and fertilized eggs has also been initially characterized (Shabanowitz and O'Rand, 1988; Shabanowitz, 1990). Since human sperm possess a Gi-like protein (Kopf et al., 1986) we have initiated studies to determine whether the ZPinduced acrosome reaction in human sperm is, similar to mouse and bull, regulated by a functional sperm Gi protein. We demonstrate that PT treatment of human sperm, under conditions where Gi is covalently modified by ADP-ribosylation, does not affect the ability of these cells to bind to structurally-intact human ZP, but inhibits the ability of the sperm to undergo ZP-mediated acrosomal exocytosis. Acrosomal exocytosis induced by the divalent cation ionophore A-23187, in contrast, is not inhibited by PT treatment. These data suggest that ZP-induced acrosomal exocytosis in the human also appears to be mediated through a spermassociated Gi protein.

79

ysis was performed using a Cell Soft semen analyzer (Cryo Resources, Inc.) at the end of the 5-hr incubation period according to the manufacturer's recommendations. Human zonae pellucidae were obtained from eggs that failed to show signs of fertilization following insemination for the purposes of in vitro fertilization. The controlled ovarian hyperstimulation regimen using leuprolide acetate in the mid-luteal phase for a minimum of 10 days prior to human menopausal gonadotropin (hMG) administration was modified from the technique described by Meldrum et al. (1989). Follicular aspiration was performed under vaginal ultrasound guidance 32-34 h r after hCG administration. Human tuba1 fluid medium (HTF; Irvine Scientific) and HTF supplemented with 5 mg/ml BSA was used for follicle washings and for the storage of the retrieved eggs. Following culture and assessment of maturity, the eggs were inseminated, cultured, and examined for signs of fertilization (Check et al., 1990). Only those eggs that did not display signs of fertilization for a period of 72 h r after insemination were used for the isolation of ZP.

Preparation of Zonae Pellucidae and Incubation With Spermatozoa ZP were isolated by the following procedure. Eggs were placed in 100-pl droplet of a solution containing 10 mM Hepes-buffered HTF (modified HTF; MHTF), and the eggs then washed through three additional 100-p1droplets of MHTF to remove residual cells of the cumulus oophorus and BSA. The eggs were then transferred to a fourth droplet of MHTF containing 1 mg/ml MATERIALS AND METHODS EDTA and 10 pg/ml lima bean trypsin inhibitor Collection and Preparation of Gametes (Sigma), and drawn through a micropipet with a diamHuman sperm were obtained from healthy, fertile eter 3/4 of the diameter of the egg. This insured the donors after 36-48 h r of abstinence, and the ejaculates rupture of the ZP-enclosed egg. The intact ZP were then then allowed to liquify at room temperature for 15-30 washed four times in the MHTF/EDTA/lima bean min. All ejaculates had initial parameters of at least trypsin inhibitor medium and placed in a medium con65% motility, 60% viability, 60% normal morphology, taining 1% Triton X-100,lM NaC1,l mM benzamidine, acid) (ADA), pH and sperm concentrations of 35 x 106/ml or greater. 10 mM (N-[2-acetamidoliminodiacetic After liquefaction, the semen was mixed with 3 vol 6.6 for 1 min. After the ZP were allowed to settle, they Ham's F-10 medium (Gibco Laboratories) containing 5 were then transferred through four washes containing mg/ml bovine serum albumin (BSA) and the semen 1 mM benzamidine in distilled water, followed by two washed by centrifugation in 12-ml conical tubes a t 600 washes in distilled water alone. The ZP were then g for 10 min. The supernatant was removed, the sperm stored a t 4°C in a solution containing 50% glycerol and pellet gently overlaid with medium, and the tube in- 20 mg/ml BSA until needed in the assays. Prior to their use in the experiments, the washed and clined a t 45" to allow the motile sperm to swim out of the pellet for 30 min. Sperm obtained by this procedure stored ZP were pooled into groups of 2 5 0 in order to displayed percentages of motility >go%. Cell concen- reduce variability in biological responses due to the trations were adjusted to 25-50 x 106/ml and the sus- possible presence of ZP that had undergone modificapensions then incubated for a n additional 5 h r at 37°C tions associated with the polyspermy block. The pooled in a humidified atmosphere of 5% COz in air to promote intact ZP were then washed by centrifugation three capacitation. In experiments where sperm were capaci- times using MHTF minus BSA. The supernatants were tated in the presence of PT, the PT (List Biologicals, removed and the pelleted ZP resuspended with acidiInc.) was added to the appropriate final concentration fied (pH 4.0) Ham's F-10 medium minus BSA to a final a t the onset of the capacitation period, and incubations concentration of 10 ZP/pl. The ZP were incubated at carried out as described above. In some experiments 37°C for 1 h r to solubilize the ZP. The solubilized ZP sperm were capacitated in the presence of cholera toxin were immediately added to the capacitated sperm a t a (Schwarz-Mann Biochemicals). In experiments in final concentration ranging from 2 to 6 ZP/pl, the suswhich semen parameters were monitored, semen anal- pension incubated for a n additional 2 h r at 37"C, and

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Fig. 1. Fluorescence photomicrographs of acrosome-intact and acrosome-reacted human sperm as assessed by both the chlortetracycline (CTC) and Pisum sativum lectin assay. A, B: Acrosome-intact and acrosome-reacted sperm, respectively, using the CTC assay. Note the loss of CTC fluorescence overlying the acrosome in the

acrosome-reacted sperm designated by the arrow in panel B. C, D: Acrosome-intact and acrosome-reacted sperm, respectively, using the Pisum sativum lectin assay. Assays were performed as described under Materials and Methods.

the sperm then processed for acrosomal status. In most experiments, capacitated sperm were incubated with solubilized ZP at a final concentration of 2 ZP/pl, a concentration previously shown to be effective in inducing acrosome reactions (Cross et al., 1988). For those experiments performed with A-23187, a stock solution of the ionophore was added to the sperm suspension to a final concentration of 10 pM. The final concentration of the solvent (dimethylformamide; DMF) never exceeded 0.1%. Control incubations consisted of sperm incubated with a n equivalent concentration of DMF. The sperm were then incubated for a n additional 2 h r prior to being processed for acrosomal status. At the end of the incubation periods, 5-pl aliquots of the sperm suspension were removed from each sample for the chlortetracycline (CTC) assay of acrosomal status (as described below). The remainder of the sample was prepared for the FITC-Pisum sativum agglutinin assay for acrosoma1 status (as described below).

sativum lectin (Vector Laboratories, Inc.), using the basic protocol of Cross et al. (1986). Both assays yielded similar results, and examples of the fluorescence patterns are shown in Figure 1. A minimum of 200 sperm were scored for each determination a t the different time points.

Sperm Binding Assay Sperm suspensions were capacitated a s described above in both the absence or presence of 1 pg/ml PT. The sperm suspensions were then then added to 100-pl droplets of HTF plus 5 mg/ml BSA containing 10 structurally intact ZP to a final sperm concentration of 0.41 x lo6 motile sperm/ml. The sperm were allowed to bind to the Z P for 10 min and the ZP then transferred three times through 100-p1 droplets of HTF plus 5 mgiml BSA using a glass micropipet with a bore twotimes the diameter of the ZP to dislodge loosely attached sperm. ZP with bound sperm were placed on a glass microscope slide in 10 p1 of media and then covDetermination of Acrosomal Status ered with a coverglass. The slides were examined imA fluorescence assay using the probe CTC (Sigma) mediately for the number of sperm bound per ZP using was used to assess capacitation and acrosomal status of x 200 phase-contrast optics. human sperm incubated with solubilized ZP or with ADP-Ribosylation A-23187 (Ward and Storey, 1984; Lee and Storey, 1985; Lee et al.. 1987). Acrosomal status was also assessed PT-catalyzed ADP-ribosylation using [32P]-NADwas using fluorescein isothiocyanate (F1TC)-labeledPisum carried out by the method of Kopf e t al. (1986).

G PROTEINS AND ZONA PELLUCIDA-INDUCED ACROSOME REACTION [32P]NADwas synthesized by the method of Cassel and Pfeuffer (19781, except that a n ATP regenerating system (pyruvate kinase and phosphoenolpyruvate) was included. Positive controls included plasma membranes derived from normal rat kidney cells (Woolkalis and Manning, 1987) and were a gift from Dr. David Manning (Department of Pharmacology, University of Pennsylvania). In experiments in which sperm were first capacitated in the presence of PT and the cells then subjected to in vitro ADP-ribosylation using [32P]NAD,the following protocol was used. Sperm (25 x lo6 cellsiml) were incubated with PT for 3 hr, and the PT then removed by centrifugation of the cells at 1,OOOg for 10 min. The resultant sperm pellet was extracted, the Lubrol PXsoluble fraction prepared, and the fraction then subjected to PT-catalyzed [32Pl-ADP-ribosylation as previously described (Endo et al., 1987). To quantify the incorporation of radioactivity into the PT substrates, the gel was scanned with a Betascope (Betagen, Waltham, MA) and cpm in the 41,000-M, band were determined after background subtraction.

T

ZP ( /PI) A-23187

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6

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Miscellaneous Methods ZP(/pI) 0 2 4 6 0 A-23187 - + Samples subjected to PT-catalyzed ADP-ribosylation were analyzed by discontinuous one-dimensional soFig. 2. Concentration-dependent effects of human solubilized zonae dium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis using 10% acrylamide and highly purified pellucidae and of 10 pM A-23187 on the human sperm acrosome reaction assessed by both the CTC (A) and Pisurn satiuum lectin (B) asSDS (>98% C,, alkyl sulfates) (Laemmli, 1970). [32Pl- says. Experiments were designed as described under Materials and ADP-ribosylated proteins were detected by autoradiog- Methods, and data represent the mean (?SD) of five individual experraphy at -70°C using Kodak X AR5 X-ray film with iments using different preparations of zonae pellucidae. intensifying screens. Protein concentrations were determined by either the method of Bradford (1976) or the bicinchoninic acid protein assay (Smith et al., 1985) 1987) or Pisum sativum lectin (Fig. 2B) (Cross e t al., using BSA a s a standard. 1986) assays. The percentage of acrosome reactions induced by the solubilized ZP were also similar to those RESULTS observed with the divalent cation ionophore A23187 Induction of the Acrosome Reaction by (Fig. 2). Unless otherwise noted, all subsequent experiSolubilized Zonae Pellucidae Isolated From ments examining the acrosome reaction utilized solubiInseminated but Unfertilized Eggs lized ZP a t a final concentration of 2 ZP/pl. ZP isolated from human eggs that were inseminated, Effects of Pertussis Toxin Treatment of Sperm but classified as unfertilized, were pooled into groups of on the Ability of Sperm to Bind to the Zona >50 in order to reduce variability in biological rePellucida and Undergo the Acrosome Reaction sponses due to the possible presence of ZP that had Human sperm were capacitated in the presence of PT undergone modifications associated with the polyspermy block either through spontaneous activation of in order to determine whether functional inactivation the egg or stress-induced effects. The ZP were then used of sperm Gi affected the ability of the treated cells to in either the structurally intact state for sperm binding bind to the human ZP and undergo the acrosome reacassays or were acid-solubilized to assess whether they tion; PT-catalyzed ADP-ribosylation of Gi proteins rewould induce acrosomal exocytosis in capacitated sults in their functional inactivation (Freissmuth et al., 1989). In many cell types, including mouse sperm and sperm. Figure 2 shows that capacitated sperm, when incu- oocytes, PT added to the incubation medium can enter bated with various concentrations of solubilized human the cells and catalyze the in situ ADP-ribosylation of ZP, undergo significant numbers of acrosome reactions cellular G proteins using endogenous cellular NADi when compared to controls. Maximal acrosome reac- pools (Sekura e t al., 1985;Endo et al., 1987; Jones and tions were observed with 2 ZP/pl, a concentration previ- Schultz, 1990). When sperm were capacitated in the presence of 1 ously found to induce significant numbers of acrosome reactions (Cross e t al., 1988), and similar results were pg/ml PT, the toxin was able to cross the sperm memobtained using either the CTC (Fig. 2A) (Lee et al., brane and ADP ribosylate endogenous Gi. This was as-

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M. A. LEE ET AL.

M~x 10-3

123 4

F

205 11666 45 -

--

--

-41

29 -

*

4

Fig. 3. Autoradiogram demonstrating the effects of capacitation of human sperm in either the absence or presence of pertussis toxin on the subsequent ability of pertussis toxin to catalyze the in vitro C3”PlADP-ribosylationof the human sperm M , = 41,000 substrate obtained from resultant Lubrol PX-soluble cell extracts. Equal numbers of control and PT-treated cells were extracted and equal volumes of the extracts applied to the gel. The experiment was carried out under the conditions outlined under Materials and Methods. Lane 1 , 5 kg membrane protein from normal rat kidney cells; lane 2, Lubrol PX-soluble extract from sperm capacitated in the absence of pertussis toxin; lane 3, Lubrol PX-soluble extract from sperm capacitated in the presence of 1 kg/ml pertussis toxin. Scanning of the gel with a Betascope revealed that sperm capacitated in the presence of pertussis toxin (lane 3) incorporated 52% fewer counts into the M , = 41,000 region after in vitro pertussis toxin-catalyzed [32P]-ADP-ribosylationthan did sperm capacitated in the absence of the toxin (lane 2). The experiment was performed three times with similar results. Numbers on the left side of the autoradiogram represent the molecular weight standards ( x Number on the right side of the autoradiogram represents the position of the M , = 41,000 PT substrates. Chevrons designate the beginning and end of the gel.

In contrast to the lack of a n effect on sperm binding to the ZP, PT treatment of sperm inhibited the ability of the cells to undergo acrosomal exocytosis in the presence of solubilized ZP (Fig. 4A,B); similar inhibitory effects were seen using both assays to assess acrosomal status (compare Fig. 4A and 4B). Acrosome reactions induced either spontaneously (observed in the absence of ZP) or nonspecifically (in the presence of A23187), in contrast, were completely insensitive to PT treatment of the sperm (Fig. 4A,B). The inhibitory effect of PT on the ZP-induced acrosome reaction was dependent on the concentration of PT present during the capacitation period (Fig. 5); acrosome reactions induced spontaneously or nonspecifically (with A23187), a s expected, were not affected at any PT concentration used. Further evidence for the specificity of the PT effect on the ZP-induced acrosome reaction is shown in Figure 6. Inactivation of PT by repeated cycles of freeze-thawing totally abolished the inhibitory effect of the toxin on the ZP-induced acrosome reaction (Fig. 6A). Cholera toxin, which does not ADP-ribosylate Gi but does ADP-ribosylate and activate the stimulatory G protein (GJ, had no effect on either the spontaneous or ZP-induced acrosome reactions (Fig. 6B). The lack of effect of cholera toxin is consistent with the apparent absence of functional G, in sperm (Hildebrandt e t al., 1985; Kopf et al., 1986; Bentley et al., 1986).

DISCUSSION A major problem that arises for many investigations of the interaction of human sperm with the human ZP is the inability to obtain sufficient quantities of ZP to carry out detailed studies. Up to the present time, most studies have been performed using material obtained from ovarian sources obtained from pathology cases (surgeries or cadavers). ZP from human eggs that have failed to undergo fertilization after insemination have been used for the characterization of the composition of this extracellular matrix (Shabanowitz and O’Rand, 1988), a s well a s the production of ZP antibodies (Shabanowitz, 1990). Since the major electrophoretic patterns of ZP isolated from inseminated and unfertilized human eggs are identical to ZP obtained from eggs that are not inseminated (Shabanowitz and O’Rand, 1988), we examined whether ZP from eggs that were inseminated, but classified as unfertilized, could be used for both sperm binding assays and for the induction of the acrosome reaction. We demonstrate in this report that ZP from insemisessed by a reduction in the ability of PT to catalyze the in vitro [32P]-ADP-ribosylationof Giusing [32P]-NADf nated human eggs that do not display any morphologiin extracts obtained from the previously PT-treated cal signs of fertilization can be used for studies of husperm when compared to sperm that had not been man sperm-ZP binding and induction of the acrosome treated with the toxin (Fig. 3); this reduction was gen- reaction. Although the percentage of ZP that have unerally on the order of 50-70% that of the control level of dergone biochemical modifications that would lead to radiolabeling. PT treatment of sperm under these con- the loss of biological activity is not known, attempts ditions had no adverse effect on any motility parameter were made to circumvent this potential problem by measured (Table 1).The ability of PT-treated sperm to pooling large quantities of ZP prior to use in our experbind to structurally intact ZP was, likewise, unaffected iments. This approach has enabled us to demonstrate [3.6 k 1 vs. 4.9 t 1.2 sperm bound/ZP in control and that the human sperm acrosome reaction induced by PT-treated sperm, respectively; (mean k SD); n = 51. the human ZP appears to be regulated by a sperm-

G PROTEINS AND ZONA PELLUCIDA-INDUCED ACROSOME REACTION

83

TABLE 1. Effects of Pertussis Toxin on Motility Parameters of Human Sperm Assessed by Computer-Assisted Semen Analysis (CASA)" Motility parameterb Percent motile (250%) Mean velocity (pm/sec) (240 pm/sec) Mean linearity (25.5) Amplitude of lateral head displacement (max.). (um) .. (22.3 pm) Amditude of lateral head displacement (mean) (pm) (21.8 pm) Beat/cross frequency (Hz/sec) (513 Hdsec)

Control

Incubation conditionC 50 ng/ml PT 1 pg/ml PT

81.4 rt 8.6d 68.6 f 6.7

79 f 12.7 71.2 f 8.6

65.3 f 10.4 65.0 f 16.2

7.3 f 0.7 3.23 f 0.5

7.5 f 1.0 3.19 f 0.6

6.23 f 1.04 3.48 f 0.25

2.75 f 0.5

2.64 f 0.62

2.94 f 0.21

18.6 f 1.3

18.73 f 1.8

18.3 f 3.3 ~

~

~~~

'Semen analyses were performed with a CellSoft CASA system according to the manufacturer's recommendations. bNormal ranges for these parameters are shown within the parentheses following each motility parameter. CSpermfrom patients displaying normal semen analyses were capacitated for 5 hr in either the absence (Control) or presence of the indicated concentrations of PT and the parameters monitored by CASA. dData represent the mean f S D of six patients who were assayed at random times. There were no significant differences in any of the motility parameters between the control and experimental groups (P < 0.01). C

.-0

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Pertussis Toxin (nglml) PT

ZP A-23187

-+

-+

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--

+ +

Fig. 5. Concentration-dependent effects o f pertussis toxin on the spontaneous, zona pellucida-induced, and A23187-induced acrosome reaction of human sperm. Acrosomal exocytosis was assessed by the Pisurn satiuurn lectin assay. In these experiments, the zona pellucida concentration was 2 ZPipl and the A23187 concentration was 10 pM. 0, control; 0 , zonae pellucidae; a ,A23187. Data represent the mean ?SD o f five individual experiments.

40

30

PT ZP A-23187

-+

-+

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+ +

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Fig. 4. Effects of pertussis toxin treatment of human sperm on the acrosome reaction induced by either human solubilized zonae pellucidae or A23187. In these experiments, the zona pellucida concentration was 2 ZPipl, the A23187 concentration was 10 pM,and the pertussis toxin concentration was 1 pg/ml. The acrosome reaction was assessed by both the CTC (A) and Pisurn satiuurn lectin (B) assays. Experiments were designed as described under Materials and Methods, and data represent the mean (tSD) of five individual experiments using different preparations o f zonae pellucidae.

associated G protein of t h e Gi class. The involvement of this class of signal-transducing proteins in this exocytotic event is similar to previous observations made in both the murine (Endo e t al., 1987,1988; Vazquez e t al., 1989; Glassner et al., 1991) and bovine (Florman et al., 1989) systems and is consistent with t h e notion t h a t ZP (or ZP3bmediated signal transduction through spermassociated G proteins is a common mechanism by which acrosomal exocytosis is regulated in mammals. I n all three cases, functional inactivation of Gi by PT does not alter t h e ability of the sperm to bind to the ZP but inhibits downstream events leading to exocytosis. In t h e mouse, PT blocks early stages of t h e ZP-induced

84

M. A. LEE ET AL. C

ZP

-

PT Freeze-ThawPT [ PT 1 (n9/ml)

- -

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1

1

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B

+ + + - +

+

100 100

T

T

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0

CT ZP

-

-

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Fig. 6. Effects of pertussis toxin, inactivated pertussis toxin and cholera toxin on the zona pellucida-induced acrosome reaction of human sperm. A Sperm were capacitated in the presence of buffer, 1 ngiml, or 100 ngiml pertussis toxin. In some cases (noted in the figure) the pertussis toxin was inactivated by repetitive cycles of freezing and thawing (3 cycles). Acrosomal exocytosis was then determined after incubation with either buffer or solubilized human zonae pellucidae ( 2 ZPipl final concentration). B: Sperm were capacitated in either the absence or presence of 1 pgiml cholera toxin, and acrosome reactions then determined after incubation with either buffer or solubilized human zonae pellucidae ( 2 ZPipl final concentration). Acrosomal exocytosis was assessed by the Pisum sativum lectin assay. Data represent the mean ?SD of four individual experiments.

acrosome reaction (Endo et al., 1987), including the loss of a transmembrane pH gradient that represents one of the earliest transitions of this regulated exocytotic event (Endo et al., 1988; Kligman et al., 1991). Similarly, PT inhibits ZP-induced elevations of Caz+ and pH, that accompany the acrosome reaction in bovine sperm (Florman et al., 1989). The relationship between sperm G, proteins and these ionic changes are unclear. There is some debate as to whether human sperm which undergo acrosomal exocytosis on the ZP surface represent the fertilizing sperm (Kopf and Gerton, 1990), since both acrosome-intact and acrosome-reacted human sperm can initiate binding to the human ZP (Morales et al., 1989); this issue is currently unresolved. Our results, however, could be interpreted to support the idea that the ZP-induced acrosome reaction is the physiologically relevant exocytotic event since it is the ZP-induced acrosome reaction, and not the spontaneous or A23187-induced acrosome reaction (Figs. 4, 51, which appears to be mediated through a G proteinmediated signal transduction process t h a t may couple

putative sperm plasma membrane-associated ZP receptors to intracellular effectors (Kopf and Gerton, 1990). The observation that prior treatment of the sperm with PT markedly reduces the extent to which subsequent ADP-ribosylation of sperm extracts with L3'PlNAD can be effected by PT (Fig. 3) provides direct evidence that intracellular ADP-ribosylation of G, is occurring during the preincubation period and that the sperm GI is being functionally inactivated. Since GI is the only known substrate for PT-catalyzed ADP-ribosylation in human sperm, it is likely that the target of PT action in these experiments is the sperm G, protein. The lack of effect of cholera toxin is fully consistent with the absence of G, in sperm, and further supports the idea that these toxins are acting in a specific manner. It is of interest that preincubation of the sperm with PT gives essentially complete inhibition of the ZP-induced acrosome reaction at concentrations that do not result in complete ADP-ribosylation of GI (cf. Fig. 3 with Figs. 4,5). This could be explained by differences in the methods used to carry out these two types of experiments. The sperm concentration used for the [32P]-ADPribosylation experiments (Fig. 3) was significantly higher (25-fold higher) than the concentration of sperm capacitated in the presence of PT prior to dilution for the ZP binding and acrosome reaction studies (Figs. 4, 5). These higher sperm concentrations were necessary, since a significant amount of sperm protein is required to obtain a detectable signal of [32P]-ADPribosylation by autoradiography. If the PT had not been in excess in these particular experiments, it is possible that only a fraction of the total number of G,, subunits would be covalently modified by the toxin when the intact sperm were incubated with PT. As a result, one might not expect to get a complete abolition of [ 32P]-ADP-ribosylation in vitro. The specific function of the human sperm GI protein in the acrosome reaction induced by the human zona pellucida is not known at this time. GI, in other cells, has been implicated in receptor-mediated regulation of Ca2+mobilization (Gomperts, 1983; Molski e t al., 1984; Haslam and Davidson, 1984; Murayama and Ui, 1985), K+ channels (Yatani et al., 1988), phospholipase C activity (Cockcroft and Gomperts, 1985; Brandt et al., 1985; Smith e t al., 1985; Wallace and Fain, 1985), and adenylyl cyclase activity (Gilman, 1984; Smigel et al., 1985). All these processes have been implicated in the regulation of the mammalian sperm acrosome reaction (Kopf and Gerton, 1990). The task at hand will be to define those processes that are regulated by this particular class of signal-transducing proteins.

ACKNOWLEDGMENTS This research was supported by a grant from the NIH (HD 06274 to G.S.K.). The authors wish to thank Lavinia Muncy (Univ. of Connecticut) for performing the beta-scans of the autoradiograms. The authors would like to thank Jeffrey S. Chase, Jung Choe, Ahmed Nazari, Kosrow Nowroozi, Althea O'Shaughnessey, and Robert Weiss for performing the

G PROTEINS AND ZONA PELLUCIDA-INDUCED ACROSOME REACTION ovulation inductions and aspirations of the oocytesi eggs. The authors also wish to thank Bayard Storey and Andre Denis for critically reviewing the manuscript.

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