The zona pellucida-induced acrosome reaction of human spermatozoa is mediated by protein kinases*†

Vol. 61, No.3, March 1994

FERTILITY AND STERILITY Copyright

©

1994 The American Fertility Society

Printed on acid-free paper in U. S. A.

The zona pellucida-induced acrosome reaction of human spermatozoa is mediated by protein kinases*t

Peter Bielfeld, M.D.:\: Andre Faridi, M.D.:\: Lourens J.D. Zaneveld, Ph.D.§ Christopher J. De Jonge, Ph.D. II Heinrich Heine Universitat, Dusseldorf, Germany; Rush University, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois; and the University of Nebraska Medical Center, Omaha, Nebraska

Objective: To determine if the solubilized human zona pellucida (ZP)-induced acrosome reaction is mediated by protein kinases. Design: Capacitated spermatozoa were incubated with inhibitors of cyclic adenosine 3':5'monophosphate (cAMP)-dependent kinase (KT5720), Ca2 + - and phospholipid-dependent kinase (Calphostin C), and cyclic guanosine 3':5'-monophosphate (cGMP)-dependent kinase (KT5823) and then treated with a corresponding kinase stimulator (dibutyryl cAMP, phorbol 12-myristate 13-acetate and dibutyryl cGMP, respectively) to determine the effect on the acrosome reaction. Appropriate controls were performed. Zonae obtained from the unfertilized oocytes of women attending an IVF program were solubilized using acidic NaH 2 P0 4 , and the effect of solubilized ZP on the acrosome reaction was tested in dose-response fashion. Comparative studies with solubilized, zona-free oocyte-treated spermatozoa were performed. The effect of the kinase inhibitors on the solubilized ZP-induced acrosome reaction was then determined. Results: No significant stimulation of the acrosome reaction by kinase stimulators occurred when spermatozoa were pretreated with inhibitors of the kinases, in contrast to the controls. Capacitated spermatozoa incubated with 2, 4, and 6 solubilized ZP showed a dose-dependent increase in the acrosome reaction. Solubilized oocytes had no effect on the acrosome reaction. Pretreatment of spermatozoa with kinase inhibitors significantly lowered the acrosome reaction induced by solubilized ZP but not completely. When a "cocktail" of the three inhibitors was used, a significant reduction in the acrosome reaction occurred in comparison with single inhibitor treatment. Conclusion: The present data indicate a role for human ZP-induced activation of multiple second messenger pathways, involving kinases A, C, and G in the human sperm acrosome reaction. Fertil Steril 1994;61:536-41 Key Words: Zona pellucida, acrosome reaction, signal transduction, protein kinase

A requisite occurrence for fertilization in mammals is an exocytotic event occurring in the sper-

Received July 12, 1993; revised and accepted November 9, 1993. * Supported by private funding and by grant HD 19555 to L.J.D.Z. from the National Institutes of Health, Bethesda, Maryland. t Presented at.the Annual Meeting of The American Fertility Society in New Orleans, Louisiana, November 1 to 5, 1992. :j: Department of Obstetrics and Gynecology, Heinrich Heine Universitat.

536

Bielfeid et al.

ZP-induced acrosome reaction

matozoon, termed the acrosome reaction. The reaction involves the fusion and vesiculation of the sperm plasma and outer acrosomal membrane, allowing for the exposure and release of the acrosomal contents. Spermatozoa that have not under-

§ Department of Obstetrics and Gynecology, Rush Medical College. II Reprint requests: Christopher J. De Jonge, Ph.D., Department of Obstetrics and Gynecology, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-3255.

Fertility and Sterility

gone the acrosome reaction cannot penetrate the zona pellucida (ZP). In somatic cells, exocytosis often occurs as a result of ligand-receptor binding, the interaction of which can be transduced to an amplifying enzyme whose activation results in the conversion of a precursor molecule into an intracellular second messenger followed by the activation of a protein kinase. The intracellular mechanism(s) that results in acrosomal exocytosis in human spermatozoa only recently have begun to be elucidated. Data suggest the role for at least three second messenger pathways (1). One acrosome reaction-stimulating pathway involves activation ofthe amplifying enzyme adenylate cyclase, whose activation converts adenosine 5'triphosphate to the second messenger cyclic adenosine 3':5'-monophosphate (cAMP). Cyclic AMP activates cAMP-dependent protein kinase A (PKA) (2). A second pathway involves the activation of the enzyme phospholipase C, which results in the conversion of phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol, together with calcium and phospholipid, activates protein kinase C (PKC) (3). The third pathway involves the activation of guanylyl cyclase, an amplifying enzyme that also has membrane receptor properties, followed by the conversion of guanosine 5'-triphosphate (GTP) to cGMP. Cyclic GMP activates cGMP-dependent kinase (PKG). Kinase activation is followed by protein phosphorylation (1). Intact and solubilized human ZP can induce the acrosome reaction (4). However, the intracellular biochemical signal processes that are involved in the zona-induced acrosome reaction of human spermatozoa have not been defined. The purpose of the present investigation was to determine if second messenger pathways that contain protein kinases A, C, or G become activated as a result of sperm-zona interaction. MATERIALS AND METHODS Chemicals

Dibutyryl cAMP, dibutyryl cGMP, phorbol 12myristate 13-acetate, Ficoll (Type 400), human serum albumin (HSA, fraction V), HEPES, glutaraldehyde, Bismark brown, Rose bengal, and cell culture tested salts were obtained from Sigma Chemical Co. (St. Louis, MO). KT5720, KT5823 Vol. 61, No.3, March 1994

and Calphostin C were obtained from Kamiya Biochemical Co. (Thousand Oaks, CA). All modulators were of the highest quality commercially available. Modulators were dissolved in dimethylsulphoxide (DMSO) «1 % final concentration ofDMSO in capacitation medium), except dibutyryl cAMP and dibutyryl cGMP which were dissolved in distilled water. Biggers, Whitten, and Whittingham (BWW) medium (5), pH 7.4, containing 35 mg/mL HSA was used as capacitation medium for spermatozoa (6). Sperm Preparation

Semen from apparently normal, healthy volunteers (donor pool of 9 individuals) was obtained by masturbation. After complete liquefaction, the ejaculates were examined for the standard semen parameters (7), and only those samples demonstrating a minimum of 70% motile spermatozoa (motility) were used for experimentation. Seminal plasma was removed by layering the semen over 11 % Ficoll in 0.12 M sodium chloride, 0.025 M HEPES buffer at pH 7.4, and centrifugation at 500 X g for 30 minutes. The supernatant fluid was discarded, the sperm pellet resuspended in 1 mL capacitation medium and followed by recentrifugation at 500 X g for 2 minutes. The supernatant fluid was discarded and the sperm pellet resuspended in capacitation medium to 5.0 X 106 spermatozoa/mL. Sperm motility was then reassessed and samples demonstrating > 15% reduction in motility from the original sample and/or <68% motility were eliminated from the experiments. However, this only happened once. Preparation of Zonae

Zona pellucidae were obtained from the unfertilized nonviable oocytes after unsuccessful IVF. The oocytes were either salt stored (8) or frozen in DMSO (9) until required. Using an inverted phasecontrast microscope, the zona was separated from each oocyte by cutting a slit into the zona using a knife attached to a Narishige micromanipulator (Nikon Inc., Garden City, New York). The zona was isolated from the oocyte proper by aspiration into a micro-transfer pipette (10). The zonae and zona-free oocytes were solubilized separately in NaH 2 P0 4 at pH 2.75 before each experiment (11). Procedure for Modulator Testing

The procedure for the testing of modulators, the synchronous acrosome reaction assay (6), was used. Bielfeld et al.

ZP-induced acrosome reaction

537

Washed sperm samples (0.5 mL) were pipetted into 5-mL plastic centrifuge tubes and incubated at 37°C for 3 hours. Stimulatory compounds were added singly to treatment tubes, and one tube was treated with vehicle alone (control). The tubes were incubated for an additional 60 minutes at 37°C. Motility was reassessed, and the reaction stopped upon the addition of 3% glutaraldehyde. In no instance was a significant (P > 0.05) reduction in sperm motility detected. Inhibitory compounds were added at the end of the capacitation period, 5 minutes before the addition of inducer, to assay their effect on the human sperm acrosome reaction. Spermatozoa that were used for zonae testing were pelleted after the capacitation period by centrifugation at 500 X g for 2 minutes. The supernatant was aspirated, the sperm pellet was resuspended in BWW lacking HSA to a final concentration of 2 X 108 /mL, and 2-JLL aliquots added to control and experimental tubes. Procedure for Testing Solubilized Zonae

To determine the effect of zonae or zona-free 00cytes on the acrosome reaction, solubilized zonae or zona-free oocytes in 2 JLL of 5 mM NaH 2 P0 4 were separately mixed with 2 JLL BWW (2X concentration), without HSA, yielding a pH of approximately 7.35. Capacitated spermatozoa (2 X 108 /mL) were pipetted (2 JLL)into tubes containing the following: [1] 2 JLL BWW (2X concentration) and 2 JLL of 5 mM NaH 2 P0 4 (control); [2] 2 JLL BWW (2X concentration) containing six solubilized zona-free 00cytes; or [3] 2 JLL BWW (2X concentration) containing different numbers of solubilized zonae and incubated at 37°C under parraffin oil (Fisher Scientific, Chicago, IL) for 1 hour. After incubation, sperm were processed as described in the following section. Sperm Acrosomal Evaluation

Acrosomal status was evaluated by the double stain technique (6). Glutaraldehyde (3%) was added to tubes containing spermatozoa and incubated for at least 30 minutes at room temperature. Samples were centrifuged at 1,000 X g for 3 minutes, and the supernatant was aspirated, the pellet resuspended in distilled water, and the samples recentrifuged as before. The supernatant was again aspirated, the pellet resuspended in approximately 50 JLL of distilled water, transferred to microscope slides, and smears of the samples prepared. The slides were then incubated for 10 minutes at 37°C 538

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in 0.8% Bismark brown (pH 1.8). After incubation, the slides were immersed 10 times in two washes of distilled water. The slides were subsequently incubated at room temperature in 0.8% Rose bengal, pH 5.3, for 20 to 23 minutes. After incubation, the slides were washed in distilled water as above. Finally, the slides were passed through an alcohol dehydration series (50%, 95%, and 100% ethanol) and cleared in two 5-minute washes in Histo-Clear (National Diagnostics, Manville, NJ). After drying, the slides were examined under oil immersion (l,OOOX), and the acrosomal status of the spermatozoa was evaluated (200 sperm cells per slide). Statistical Analysis

The mean and SDs as well as the 95% confidence limits (CI) were calculated from data collected from at least three experiments per treatment. The frequency data were subjected to arcsin transformation and statistically compared using Bartlett's test for homogeneity, followed by the F-test and then by the paired t-test and/or Dunnett's multiple comparison test.

RESULTS Effect of Stimulatory and Inhibitory Modulators on the Acrosome Reaction

Stimulatory modulators of PKA (dibutyryl cAMP, 1mM), PKC (PMA, 0.1 JLM) and PKG (dibutyryl cGMP, 1 mM) were tested separately for their effect on the acrosome reaction (Table 1). Each compound caused a significant (P < 0.01) stimulation ofthe acrosome reaction when added to capacitated spermatozoa in comparison with the untreated controls. No significant (P > 0.05) stimulation of the acrosome reaction occurred when an inhibitory modulator of each kinase (respectively, KT 5720 for kinase A; Calphostin C for kinase C; and KT 5823 for kinase G) was added to capacitated spermatozoa 5 minutes before the addition of the inducer for the same pathway kinase (Table 1). Acrosome Reaction Dose-Dependency Using Solubilized Human ZP

The effect of solubilized human ZP on the acrosome reaction of capacitated spermatozoa was evaluated (Table 2). Also, the acrosome reaction-inducing ability of six solubilized zona-free oocytes (representing the greatest number of zonae studied) Fertility and Sterility

Table 1

Effect of Stimulatory and Inhibitory Kinase Modulators on the Acrosome Reaction of Capacitated Spermatozoa

Stimulator

Inhibitor*

Mole

Acrosome reactiont

Mole

%

o o

o Dibutyryl Dibutyryl PMA PMA Dibutyryl Dibutyryl

cAMP cAMP cGMP cGMP

ImM ImM O.I/LM O.I/LM ImM ImM

KT 5720

50nM

Calphostin

50nM

o o

16 33 18 35 20 33 20

233nM

KT5823

± ± ± ± ± ±

2 2 3 4 4 3 ±4

(12 to (29 to (10 to (25 to (10 to (26 to (10 to

20):1: 38)§ 26):1: 45)§ 30):1: 40)§ 30):1:

* Protein kinase inhibitor added or not added at the end of the 3-hour capacitation period, 5 minutes before the addition of stimulator. See text for experimental detail. t N = 3, values represent the mean ± SD (95% CI). Percent acrosome reaction values with dissimilar superscripts are significantly (P < 0.01) different. No significant (P> 0.05) change in sperm motility occurred after incubation to induce capacitation or after treatment with modulators.

was tested. A significant (P < 0.02) stimulation of the acrosome reaction was detected for all concentrations of zonae. In addition, the acrosome reaction response was dose-dependent. In contrast, solubilized zona-free oocytes failed to cause a significant (P > 0.05) stimulation of the acrosome reaction. Inhibitory Modulators and the Solubilized ZPInducedAR

KT 5720, Calphostin C, and KT 5823 were first tested separately to determine the effect on the acrosome reaction when spermatozoa were stimulated with four solubilized ZP. Each kinase inhibitor significantly (P < 0.01) reduced the solubilized ZP-induced acrosome reaction in comparison with spermatozoa treated with solubilized ZP alone .

Table 2 Effect of Solubilized ZP Number on the Acrosome Reaction of Capacitated Human Spermatozoa

However, complete inhibition was not detected with any of the inhibitors as compared with the untreated control (Table 3). A combination of inhibitors (PKA-PKC-PKG), using the concentrations previously tested, was also studied. The combination of inhibitors caused a significantly (P < 0.02) greater inhibition of the solubilized ZP-induced acrosome reaction than individual inhibitors (Table 3).

DISCUSSION

The present data demonstrate that the human sperm acrosome reaction can be induced by solubilized

Table 3 Effect of Kinase Inhibitors on the Solubilized ZPInduced Acrosome Reaction of Capacitated Spermatozoa Solubilized ZP*

Inhibitorst

0 4 4 4 4 4

0 0 KT 5720 Calphostin KT5823 Combo

Acrosome reaction:l:

Mole

%

Solubilized ZP*

Acrosome reactiont %

o Oocyte 2 4 6

16 17 37 49 56

± ± ± ± ±

2 1 2 2 2

(14 (14 (32 (47 (52

to 18):1: to 19):1: to 41)§ to 51)~ to 60)~

* Solubilized ZP was added or not added to spermatozoa in BWW. Solubilized product from six oocytes was used for control. See text for experimental detail. t N = 3 for two and six solubilized ZP, n = 4 for the remainder. Values represent the mean ± SD (95% CI). Percent acrosome reaction values with dissimilar superscripts are significantly (P < 0.02) different from each other. No significant (P> 0.05) change in sperm motility occurred after incubation to induce capacitation or after treatment with modulators. Vol. 61, No.3, March 1994

50nM 50nM 233nM 50,50,233

16 48 34 31 26 21

± ± ± ± ± ±

2 1 1 2 2 1

(13 to (46 to (33 to (26 to (21 to (18 to

20)§ 50)~

36)0 36)0'£ 31)£ 24)§

* Solubilized ZP was added to spermatozoa capacitated (3hour incubation) in BWW. See text for experimental detail. t Protein kinase inhibitors, singly or as a combination of all three inhibitors, were added or not added at the end of the 3hour capacitation period 5 minutes before the addition of solubi1ized ZP. :I: N = 3, values represent the mean ± SD (95% CI). Percent acrosome reaction values with dissimilar superscripts are significantly (P < 0.01) different. No significant (P > 0.05) change in sperm motility occurred after incubation to induce capacitation or after treatment with modulators. Bielfeld et aI.

ZP-induced acrosome reaction

539

human zonae and that the magnitude of acrosome reaction is dependent on the number of zonae used for stimulation. In contrast, no stimulatory effect on the acrosome reaction was detected using the solubilized product of zona-free oocytes from which the maximum number (n = 6) of zonae had been tested. When inhibitory modulators of kinases previously shown to have a role in the acrosome reaction were tested for their effect on the zona-induced acrosome reaction, a significant reduction in the acrosome reaction was detected. Furthermore, the percent induced acrosome reaction values were reduced to levels approaching that of the nontreatment control when a combination of the inhibitors was used. In summary, the present data suggest that the human ZP induces the acrosome reaction via stimulation of kinases from three different signaling pathways. The acrosome reaction is initiated during the penetration of the spermatozoon through the follicle cell layer of the oocyte, either just before or after contact with the ZP. At present, the oocyte stimulus or stimuli of the acrosome reaction remain to be firmly established but likely involve one or more of the zona glycoproteins. Cross et al. (11) demonstrated that acrosome reactions could be stimulated in capacitated human spermatozoa using either whole or aciddisaggregated ZP from nonviable human oocytes. The present data confirm those of Cross et al. (11) and expand those observations by demonstrating a dose-dependent response using solubilized ZP, with extracts of six zonae producing an acrosome reaction that is much higher (56%) than that generally reported for any other stimulator such as protein kinase activators (Table 1) (2, 3), P (12), and ionophore A23187 (6). Extracts of four zonae were used for the subsequent studies because this also produced a large increase in the acrosome reaction (49%) while using fewer zonae (human zonae are difficult to obtain). In addition, the inability of ooplasm to induce the acrosome reaction indicates that the ZP contains specific acrosome reaction stimulatory factors, possibly ZP3. The significance of these findings is that additional support is provided for the relevance of the zona as a physiological ligand, effecting specific biochemical processes, for induction of the human sperm acrosome reaction. De Jonge et al. (2, 3) presented evidence for the role of the cAMP-dependent kinase and the Ca2+- and phospholipid-dependent kinase pathways in the human sperm acrosome reaction. In both studies, stimulatory and inhibitory modulators for specific targets in each pathway were tested for their influence on the acrosome reaction. In the present study, some of the 540

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ZP-induced acrosome reaction

same modulators were tested, and similar results were obtained using those previously tested modulator concentrations. In addition, an inhibitor of the cGMP-dependent kinase pathway was tested, the concentration was based upon the literature data, and acrosome reaction stimulation by dibutyryl cGMP was prevented. These confirmatory results provided an experimental mechanism, using the kinase inhibitory modulators, for determining whether solubilized ZP affects second messenger pathways to elicit the acrosome reaction. Using the same dose levels as used previously, inhibitory modulators were tested for their effect on the zona-induced acrosome reaction. All three inhibitors prevented the acrosome reaction, suggesting that the three pathways leading to the activation of PKA, PKC, and PKG are involved in the ZP-induced acrosome reaction. Some differences were observed in the potency of the inhibitors, but this may have been due to their different dose levels and inhibitory potencies and cannot be taken as an indication that one pathway is more relevant than another. It is notable that none of the inhibitors decreased the zona-induced acrosome reaction to control levels, whereas they were able to do so when a specific stimulant of their target kinase was used. This is in contrast to the response seen when human follicular fluid (FF) is used as stimulator and the same inhibitors as tested herein are used (13). Complete inhibition of the reaction is achieved regardless of the type of inhibitor used. This would imply that more than one pathway is involved when the human zona stimulates the acrosome reaction, so that blockage of one pathway decreases but does not completely eliminate the reaction. This also implies that FF does not contain the same stimulatory properties as human zona. Evidence is available in somatic cells that exocytosis can occur as a result of "cross-talk" between various different second messenger pathways (14), and data are available that the same is true for the human sperm acrosome reaction (Doherty C, Tarchala SM, De Jonge CJ, abstract). The present results imply that the zona-induced acrosome reaction also involves several interacting pathways, because a "cocktail" of all three kinase inhibitors was more effective than anyone of the inhibitors. In conclusion, the induction of the human sperm acrosome reaction by the human ZP appears to involve the activation of PKA, PKC and PKG, probably via typical ligand receptor interactions, resulting in the activation of second messenger signaling pathways. Interactions between the pathways via cross-talk may also occur. Fertility and Sterility

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