Aldo E. Calogero, M.D.,* Nunziatina Burrello, Ph.D.,* Emanuela Ferrara, M.D.,* Jenny Hall, Ph.D., Simon Fishel, Ph.D., and Rosario D Agata, M.D.

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1 FERTILITY AND STERILITY VOL. 71, NO. 5, MAY 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. -Aminobutyric acid (GABA) A and B receptors mediate the stimulatory effects of GABA on the human sperm acrosome reaction: interaction with progesterone Aldo E. Calogero, M.D.,* Nunziatina Burrello, Ph.D.,* Emanuela Ferrara, M.D.,* Jenny Hall, Ph.D., Simon Fishel, Ph.D., and Rosario D Agata, M.D.* University of Catania Medical School, Catania, Italy; Queen s Medical Centre; and Centres for Assisted Reproduction, Park Hospital, Nottingham, Nottinghamshire, United Kingdom Received August 6, 1998; revised and accepted December 8, Reprint requests: Aldo E. Calogero, M.D., Istituto di Medicina Interna e Specialità Internistiche, Cattedra di Andrologia, Ospedale Garibaldi, Piazza S. Maria di Gesù, Catania, Italy (FAX: ; med.int.dagata@tau.it). * Division of Andrology, Department of Internal Medicine, University of Catania Medical School. Department of Obstetrics and Gynaecology, Queen s Medical Centre. Centres for Assisted Reproduction, Park Hospital /99/$20.00 PII S (99) Objective: To evaluate which -aminobutyric acid (GABA) receptor mediates the stimulatory effects of this neurotransmitter on the human sperm acrosome reaction, and to examine the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA A receptor. Design: Prospective study. Setting: A university clinic of andrology. Patient(s): Men with normal sperm analysis parameters. Intervention(s): None. Main Outcome Measure(s): The acrosome reaction of motile spermatozoa. Result(s): The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA A receptor antagonist, and only partially by saclofen, a GABA B receptor antagonist. Accordingly, muscimol, a GABA A receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen, a GABA B receptor agonist, was less effective. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome-reacted spermatozoa compared with progesterone alone. However, this increase was less than a simple addition of effects, suggesting that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the ability of progesterone to induce acrosome reaction was tested in the presence of bicuculline, which suppressed the stimulatory effects of progesterone. Given that the GABA A receptor is linked to the chloride channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Picrotoxin completely suppressed the acrosome reaction induced by progesterone and only partially suppressed that caused by GABA. Conclusion(s): -Aminobutyric acid stimulated the acrosome reaction in human spermatozoa, acting mainly through the GABA A receptor and to a lesser extent through the GABA B receptor. Progesterone interacted with the GABA A receptor to induce the acrosome reaction, and the functional integrity of the chloride channel was vital for this effect. (Fertil Steril 1999;71: by American Society for Reproductive Medicine.) Key Words: GABA, bicuculline, muscimol, saclofen, baclofen, progesterone, picrotoxin, chloride channel -Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central and peripheral nervous systems. Its effects are mediated by two types of receptors, GABA A and GABA B. The GABA A receptor is a plasma membrane multi-subunit receptor complex linked to the chloride channel whose activation results in the opening of this channel, leading to a transmembrane flux of chloride ions and to a consequent membrane hyperpolarization or depolarization, depending on the cell type. Progesterone and its metabolites potentiate the effects of GABA on this receptor (1). The GABA B receptor is not linked to the chloride channel or modulated by progesterone (2). The GABAergic system also is present in the human genital tract. -Aminobutyric acid receptors and the GABA uptake system are present in the human ovary, uterus, and fallopian tubes, and the concentration of GABA in the epithelial tissue of the fallopian tube in- 930

2 creases toward the ampulla (3). -Aminobutyric acid has been shown to modulate the plasma membrane polarization of the human oocyte (4), suggesting that its participation is important to oocyte function. In the male genital tract, GABA is present in the seminal plasma of normozoospermic subjects (5), and a specific binding and transport system is present on the plasma membrane of the human spermatozoon (6). -Aminobutyric acid has been reported to initiate the acrosome reaction of human spermatozoa, an important step in the spermatozoon s fertilizing capability (6 8). We recently showed that GABA modulates sperm cell kinematic parameters and increases the percentage of spermatozoa that move with a hyperactivated motility (9). We undertook the present study to evaluate which GABA receptor mediates GABA s stimulatory effects on the acrosome reaction. Because progesterone, a physiologic inducer of the spermatozoon s acrosome reaction, seems to interact with a sperm GABA A -like receptor to promote the acrosome reaction (7), we also studied the interaction between GABA and progesterone to gather additional evidence that progesterone induces the acrosome reaction by acting on the GABA A receptor in humans. MATERIALS AND METHODS Reagents -Aminobutyric acid, bicuculline, muscimol (5-aminomethyl-3-hydroxyisoxazole), baclofen (4-amino-3-[4-chlorophenyl]-butanoic acid), progesterone, picrotoxin, human serum albumin (HSA), HEPES, and lectin fluorescein isothiocyanate (Arachis hypogaea) were purchased from Sigma Chemical Co. (St. Louis, MO). Saclofen ( -[aminomethyl]- 4-chloro-benzeneethanesulfonic acid) was purchased from Research Biochemicals International (Natick, MA). Percoll was purchased from Pharmacia (Uppsala, Sweden). Medium 199 was purchased from Life Technologies (Paisley, Strathclyde, United Kingdom). Sodium citrate, fructose, and methanol were purchased from Merck (Darmstadt, Germany). Henley slide and Citofluor were purchased from C. A. Henley Ltd. (Loughton, United Kingdom) and Citofluor (London, United Kingdom), respectively. Sperm Preparation Human semen was collected by masturbation after 3 4 days of abstinence from healthy subjects with sperm parameters in the normal range, according to World Health Organization criteria (10). Only samples with a sperm concentration of /ml, total motility of 50%, progressive motility of 40%, viability of 80%, and a leukocyte concentration of 10 6 /ml were used. The protocol was approved by our institutional review board, and informed written consent was obtained from each subject. Samples from each individual subject were tested separately. After 30 minutes of liquefaction at 37 C, motile spermatozoa were isolated as reported previously (11). In brief, seminal plasma was loaded onto a two-step discontinuous Percoll gradient comprising 3 ml of 90% Percoll overlaid with 3 ml of 45% Percoll. Isotonic 90% Percoll was prepared by adding medium 199 with 0.3% HSA (fraction V). Percoll 45% was prepared with Biggers, Whitten, and Wittingham (BWW) medium, supplemented with 20 mm of HEPES and 1% HSA (12). After 20 minutes of centrifugation at 500 g at room temperature, the bottom 0.5-mL fraction of the 90% Percoll was collected, washed with 2 ml of BWW medium, and centrifuged at 250 g for 5 minutes at room temperature. The final pellet was resuspended in BWW at a concentration of spermatozoa per milliliter. Acrosome Reaction Assay Spermatozoa ( /ml) were incubated in BWW medium at 37 C, under 5% CO 2 and 95% air for 2 hours (capacitating conditions). At the end of that time, they were incubated with bicuculline, saclofen, picrotoxin, progesterone, or plain medium for 15 minutes, and then the appropriate concentration of GABA, muscimol, baclofen, and/or progesterone was added and left to incubate for an additional hour. We also tested the effects of the calcium ionophore A23187 in about half the samples, which were selected randomly throughout the study. At the end of the 3 hours and 15 minutes of incubation, the sperm cell suspension was pelleted by centrifugation at 500 g for 5 minutes and resuspended in BWW. The viability of the cells was evaluated with the use of the hypoosmotic swelling test. Fifty microliters of the sperm suspension was added to 450 L of hypoosmotic swelling medium made up of sodium citrate (25 mm) and fructose (75 mm) in distilled water (13, 14). The cells were incubated at 37 C for minutes and then pelleted by centrifugation at 500 g for 5 minutes and resuspended in 50 L of ice-cold methanol. A 10- L aliquot of these cells was pipetted in duplicate onto a four- or eight-spot Henley slide and allowed to dry. The spermatozoa subsequently were overlaid with 10 L of lectin fluorescein isothiocyanate at a concentration of 2 mg/ml. The probe was allowed to incubate with spermatozoa for 15 minutes in the dark (15), after which the slides were washed with phosphate-buffered saline to eliminate the excess lectin. The slides were examined under fluorescent microscopy (Dialux, Leica Instruments, Nussloch, Germany) in the presence of Citofluor, an antiquenching agent, coded, and scored blindly by the same examinor (N.B.). At least 100 spermatozoa were counted for each sample. The acrosome reaction was evaluated according to the criteria published by Mortimer et al. (15): [1] an intact acrosome with uniformly bright fluorescence over the acrosomal region indicated a sperm that was not acrosomereacted; [2] punctate labeling over the acrosomal region indicated a sperm that was partially acrosome-reacted; [3] an equatorial segment that was fluorescent indicated a sperm FERTILITY & STERILITY 931

3 FIGURE 1 Effects of -aminobutyric acid (GABA) on the acrosome reaction of human spermatozoa obtained from 10 normozoospermic subjects. *P.05 (vs. 0); P.05 (vs. GABA 1 M); P.05 (vs. GABA 10 M). FIGURE 2 Effects of bicuculline (BICU) (n 12), a GABA A receptor antagonist (upper panel), or saclofen (SACL) (n 9), a GABA B receptor antagonist (lower panel), on the spontaneous acrosome reaction of human spermatozoa and in response to 10 M of -aminobutyric acid (GABA). *P.05 (vs. no-treatment control); P.05 (vs. GABA 10 M). that was acrosome-reacted; and [4] no labeling observed over the acrosomal region indicated a loss of acrosome membrane. In this study, only spermatozoa that exhibited the third pattern of staining and curled tails were counted as alive and acrosome-reacted. Data Analysis The results are presented as means standard error of the mean throughout the study. The data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan s multiple range test or the paired Student s t-test, as appropriate. The progesterone dose-response curve was analyzed by the four-parameter logistic equation (16). P.05 was defined as statistically significant. RESULTS Effects of GABA The mean ( standard error of the mean) basal rate of the acrosome reaction was 3.1% 0.3% and increased to 26.2% 2.3% after exposure to 5 M of the calcium ionophore A23187 (positive control). -Aminobutyric acid stimulated the acrosome reaction in a concentration-dependent fashion (P.001 determined by ANOVA) (Fig. 1). The effect became statistically significant at a concentration of 10 M (P.05 vs. GABA zero determined by ANOVA followed by Duncan s test), which doubled the percentage of spermatozoa that underwent the acrosome reaction. At a concentration 10 times higher, GABA was significantly more effective (P.05 vs. GABA 10 M determined by ANOVA followed by Duncan s test). The stimulatory effect of 10 M of GABA was antagonized completely by bicuculline, a GABA A receptor antagonist (P.05 vs. GABA alone determined by ANOVA followed by Duncan s test). Bicuculline alone did not have any effect on the rate of the spontaneous acrosome reaction (Fig. 2, upper panel). In addition, the selective GABA B receptor antagonist saclofen antagonized the stimulatory effect of GABA (P.05 vs. GABA alone determined by ANOVA followed by Duncan s test), but only partially, because the percentage of spermatozoa that underwent the acrosome reaction in the presence of GABA (10 M) and saclofen (100 M) was significantly higher than that in the absence of both compounds (P.05 determined by ANOVA followed by Duncan s test). This outcome did not change even when saclofen was used at higher concentrations (data not shown). Saclofen alone tended to increase the rate of the spontaneous acrosome reaction (Fig. 2, lower panel). These results suggest that GABA stimulated the acrosome 932 Calogero et al. GABA receptors and acrosome reaction Vol. 71, No. 5, May 1999

4 TABLE 1 Effects of the GABA A receptor agonist muscimol and the GABA B receptor agonist baclofen on the rate of the acrosome reaction of human sperm obtained from eight normozoospermic subjects. GABA A receptor agonist Change in acrosome reaction (%) Concentration 0 Concentration 10 Muscimol * Baclofen * P.001 (vs. concentration 0) determined by the Student s t-test. P.05 (vs. concentration 0) determined by the Student s t-test. TABLE 2 Interaction between progesterone and -aminobutyric acid (GABA) on the rate of the acrosome reaction of human spermatozoa obtained from seven normozoospermic subjects. Experiment no. Progesterone level GABA level Mean SEM percentage of acrosomereacted spermatozoa Incubation Incubation * Incubation * Incubation * Note: Spermatozoa were incubated with progesterone or vehicle, as indicated, from the beginning, whereas GABA was added after 2 hours and left for another hour. * P.05 (vs. progesterone 0 and GABA 0) determined by ANOVA followed by Duncan s test. P.05 (vs. progesterone alone) determined by ANOVA followed by Duncan s test. reaction by activating the GABA A receptor and, to a lesser extent, the GABA B receptor. To substantiate this finding further, we evaluated the effects of two selective GABA A and GABA B receptor agonists, muscimol and baclofen, respectively. Both muscimol and baclofen, used at a concentration of 10 M, stimulated the acrosome reaction; however, the former was more effective than the latter, and it was as effective as 10 M of GABA (Table 1). Progesterone and GABA Progesterone stimulated the acrosome reaction in a dosedependent fashion with a median effective dose (ED 50 )of M (Fig. 3). In the presence of progesterone, at a concentration of 10 M (approximate ED 20 concentration), the percentage of acrosome-reacted spermatozoa in response to 10 M of GABA increased, but not significantly, compared with GABA alone. This increase was much less than a simple addition of effects (Table 2), suggesting that progesterone and GABA are using the same receptor and/or mechanism of action. To test this hypothesis, we evaluated the effects of progesterone on the acrosome reaction in the presence of increasing concentrations of the GABA A receptor antagonist bicuculline. Bicuculline inhibited, albeit not completely, the progesterone-induced acrosome reaction in a concentrationdependent fashion (P.001 determined by ANOVA) (Fig. 4), suggesting that progesterone interacts with the GABA A receptor to promote the acrosome reaction. Given that the FIGURE 3 Effects of increasing concentrations of progesterone on the acrosome reaction of human spermatozoa obtained from eight normozoospermic subjects. *P.05 (vs. 0); P.05 (vs. progesterone 10 M); P.05 (vs. progesterone 30 M). FIGURE 4 Effects of the GABA A receptor antagonist bicuculline (BICU) on the progesterone (Prog) stimulated acrosome reaction of human spermatozoa obtained from 10 normozoospermic subjects. *P.05 (vs. 0); P.05 (vs. progesterone alone). FERTILITY & STERILITY 933

5 TABLE 3 Effects of the chloride channel blocker picrotoxin on the progesterone- or GABA-induced acrosome reaction of human spermatozoa. Experiment no. Progesterone level GABA level Picrotoxin level Mean SEM percentage of acrosome-reacted spermatozoa Incubation 1 (n 8) Incubation 2 (n 8) * Incubation 3 (n 8) Incubation 4 (n 9) Incubation 5 (n 9) * Incubation 6 (n 9) * * P.05 (vs. no-treatment control) determined by ANOVA followed by Duncan s test. P.05 (vs. progesterone or GABA alone) determined by ANOVA followed by Duncan s test. GABA A receptor is linked to the chloride channel and its activation causes an influx of chloride ions, we tested the effects of progesterone or GABA on the acrosome reaction in the presence of picrotoxin, a chloride channel blocker. Picrotoxin completely suppressed the stimulatory effects of progesterone on the acrosome reaction and only partially suppressed those of GABA (Table 3). Picrotoxin alone did not have any effect on the rate of the spontaneous acrosome reaction (data not shown). DISCUSSION The sperm cell acrosome reaction in mammals is a modified form of exocytosis that involves the fusion of the membrane of the acrosome, a sperm head organelle, with the overlying sperm plasma membrane. The fusion is followed by vesiculation and release of the acrosomal contents, which is necessary to penetrate the zona pellucida and to allow the sperm to fuse with the oocyte s plasma membrane (17). Because failure to undergo the acrosome reaction impairs the spermatozoon s fertilizing capability, a substantial effort has been made to understand the mechanisms that initiate and/or regulate this phenomenon. It is widely accepted that a glycoprotein of the zona pellucida (ZP3) is the in vivo initiator of the acrosome reaction of the fertilizing spermatozoon (18). The present study, by showing that GABA stimulates the acrosome reaction in a concentration-dependent fashion, suggests that this neurotransmitter may be regarded as an inducer of the acrosome reaction of human spermatozoa. This outcome agrees with previous observations that GABA is capable of increasing the percentage of acrosome-reacted spermatozoa (7). Shi et al. (8) recently reported that GABA stimulates the acrosome reaction of human spermatozoa in a concentrationand time-dependent fashion, with a maximum response obtained after a preincubation period of 9 hours. On the other hand, Aanesen et al. (6) were unable to demonstrate any significant effect of GABA (0.1 M and 100 M) on the acrosome reaction of motile spermatozoa separated by the swim-up technique. The reason for this discrepancy is not clear. A stimulatory role of GABA on the acrosome reaction also has been reported in mouse spermatozoa (19). In this species, GABA reaches its maximum effect at a concentration of 0.5 M, suggesting that mouse spermatozoa have a greater sensitivity to GABA than human spermatozoa. We found that the effects of GABA on the human sperm acrosome reaction appeared to be mediated by both GABA A and GABA B receptors. However, the involvement of the GABA A receptor is more pronounced because muscimol, a selective agonist for this receptor, mimicked almost entirely the effects of GABA. Accordingly, GABA A receptors have been found in spermatozoa (20). Although no data are available on the presence of the GABA B receptor in human spermatozoa, the finding that baclofen, a selective agonist for this receptor, induced the acrosome reaction, and that the stimulatory effect of GABA was not overridden completely by the chloride ion channel blocker picrotoxin, shed some light on a possible functional role of this receptor. This is more relevant when we consider that other biologic actions of GABA on human sperm function, such as modulation of kinematic parameters and hyperactivation, are not mediated exclusively by the GABA A receptor, but also by the GABA B receptor (9). A large body of experimental evidence has shown that progesterone, trapped in the matrix of the cumulus oophorus surrounding the oocyte or produced by cumulus cells, is an important physiologic inducer of the acrosome reaction in human spermatozoa (21). In this regard, it has been shown that the acrosome reaction inducing activity of the follicular fluid disappears after the removal of its steroid content and is fully restored by the addition of progesterone (22). The stimulatory effect of progesterone is not mediated by the classic nuclear receptor, but by a receptor located on the plasma membrane. In fact, progesterone, covalently bound to 934 Calogero et al. GABA receptors and acrosome reaction Vol. 71, No. 5, May 1999

6 albumin, forms a complex that is too large to enter an intact sperm membrane but is still able to promote the acrosome reaction (23). However, the nature of this receptor is not fully established. Studies of the postreceptor mechanisms mediating the acrosome reaction inducing property of progesterone have shown that it is mediated by a rapid increase in the intracellular free calcium concentration (24). However, Wistrom and Meizel (7) showed that the transmembrane flux of chloride ions is vital for progesterone to initiate the acrosome reaction; they suggested that progesterone exerts its effect on capacitated human spermatozoa by interacting with a plasma membrane receptor complex resembling the GABA A receptor. Our findings further substantiate this hypothesis. In fact, the simultaneous presence of progesterone and GABA was no more effective than the presence of each compound alone, suggesting that both compounds affect sperm function by acting on the same receptor and/or sharing the same mechanism of action. Further, blockade of the GABA A receptor with picrotoxin or bicuculline did not allow progesterone to induce the acrosome reaction. The efficacy of bicuculline, an antagonist of the GABA binding site on the GABA A receptor, in suppressing the acrosome reaction initiated by progesterone may relate to its ability to block the chloride ion channel (25). However, the interaction of progesterone with a GABA A - like receptor does not explain all the biologic effects of this steroid on sperm function. In this regard, Baldi et al. (26) reported that neither picrotoxin nor bicuculline affected progesterone-induced calcium accumulation in human spermatozoa; they suggested that the effects of progesterone on calcium mobilization are mediated by a receptor other than the GABA A receptor. This conclusion is strengthened by recent studies showing that progesterone-induced calcium and chloride ion fluxes are controlled by two different types of receptors (27 29). These receptors seem to influence each other s activity, however, because the chloride flux may be involved in the regulation of the secondary calcium transient that is essential for the onset of the acrosome reaction (30, 31). A third type of receptor that is associated with protein tyrosine kinase also may mediate some of the effects of progesterone on sperm function (30, 32, 33). In conclusion, GABA stimulated the acrosome reaction through activation of the GABA A receptor and, to a lesser extent, the GABA B receptor. In addition, this study found further circumstantial evidence that progesterone also acts through a GABA A -like, chloride channel linked receptor to promote the acrosome reaction of human spermatozoa. The present findings, in conjunction with the reported presence of the GABA system in the male and female genital tracts (3, 5) and the reported stimulatory effect of GABA on sperm motility and hyperactivation (9), suggest that this neurotransmitter may be regarded as a modulator of sperm function. References 1. Lan NC, Bolger MB, Gee K. Identification and characterization of a pregnane steroid recognition site that is functionally coupled to an expressed GABA A receptor. Neurochem Res 1991;16: Bowery NG, Hill DR, Hudson AL, Price GW, Turnbull WJ, Wilkin GP. Actions and interactions of GABA and benzodiazepine. In: Bowery NG, ed. Heterogeneity of mammalian GABA receptors. New York: Raven Press, 1984: Erdö S. Gestational changes of GABA levels and GABA binding in the human uterus. 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7 cium is independent of the chloride requirement. Mol Cell Endocrinol 1994;101: Sabeur K, Edwards DP, Meizel S. Human sperm plasma membrane progesterone receptor(s) and the acrosome reaction. Biol Reprod 1996; 54: Tesarik J, Carreras A, Mendoza C. Single cell analysis of tyrosine kinase dependent and independent Ca 2 fluxes in progesterone induced acrosome reaction. Mol Hum Reprod 1996;2: Meizel S, Turner KO, Nuccitelli R. Progesterone triggers a wave of increased free calcium during the human sperm acrosome reaction. Dev Biol 1997;182: Mendoza C, Soler A, Tesarik J. Nongenomic steroid action: independent targeting of a plasma membrane calcium channel and a tyrosine kinase. Biochem Biophys Res Commun 1995;210: Murase T, Roldan ER. Progesterone and the zona pellucida activate different transducing pathways in the sequence of events leading to diacylglycerol generation during mouse sperm acrosomal exocytosis. Biochem J 1996;320: Calogero et al. GABA receptors and acrosome reaction Vol. 71, No. 5, May 1999