γ-aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa

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1 Molecular Human Reproduction vol.3 no.8 pp , 1997 γ-aminobutyric acid (GABA) induces the acrosome reaction in human spermatozoa Qi-Xian Shi 1,3, Yu-Ying Yuan 1 and Eduardo R.S.Roldan 2 1 Department of Reproductive Physiology and Toxicology, Zhejiang Academy of Medical Sciences, 60 Tian Mu Shan Street, Hangzhou, Zhejiang , People s Republic of China, and 2 Departamento de Reproducción Animal, Centro de Investigación y Tecnología, INIA, Ctra. de La Coruña km 5.9, Madrid, Spain 3 To whom correspondence should be addressed The sperm acrosome reaction takes place in response to progesterone and zona pellucida. Progesterone may act on more than one type of surface receptor, of which one is a γ-aminobutyric acid (GABA) type A-like receptor. Although there is direct evidence of GABA initiation of mouse sperm acrosome reaction, there are conflicting results regarding GABA-induced exocytosis in human spermatozoa. We have examined whether GABA would initiate exocytosis in human spermatozoa using the chlortetracycline assay and a zona-free hamster oocyte test. Human spermatozoa preincubated for 3 h in Biggers Whitten Whittingham medium with 0.35% bovine serum albumin underwent acrosome reactions in response to GABA, with maximal responses in spermatozoa preincubated for 9 h. The effect was concentration-dependent. Preincubated spermatozoa treated with GABA were able to fertilize a higher proportion of zona-free oocytes, with a higher number of spermatozoa penetrating each oocyte. Exposure of preincubated spermatozoa to GABA and progesterone together resulted in a higher proportion of acrosome reactions than when each agonist was used alone. The effect of GABA was mediated by the influx of extracellular Ca 2 because inclusion of EGTA or the Ca 2 channel antagonist La 3 prevented GABA-induced acrosome reactions. These results indicate that GABA can initiate exocytosis in capacitated human spermatozoa and open up possibilities for studies of signalling mechanisms activated upon occupancy of the GABA A receptor present on the sperm surface. Key words: acrosome reaction/calcium/gaba/progesterone/spermatozoa Introduction At the time of fertilization, mammalian spermatozoa undergo exocytosis of the acrosomal granule (the acrosome reaction ) in response to oocyte-associated agonist(s). This essential exocytotic process results in a release or exposure of enzymes necessary for penetration of the egg vestments, and allows the spermatozoon to fuse with the oocyte s plasma membrane after penetration of the zona pellucida (ZP) (Yanagimachi, 1994). Two main agonists of exocytosis have been identified in the oocyte vestments, progesterone (Meizel et al., 1990) which is trapped in the matrix of the cumulus oophorus and appears to be produced by cumulus cells (Schuetz and Dubin, 1981), and the zona pellucida glycoprotein ZP3 (Bleil and Wassarman, 1980, 1983). Both agonists interact during initiation of exocytosis, with progesterone priming spermatozoa to respond to ZP action (Roldan et al., 1994). It has been shown that progesterone can trigger exocytosis in a variety of species: man (Osman et al., 1989), golden hamster (Meizel et al., 1990; Llanos and Anabalon, 1996), Chinese hamster (Shi et al., 1992), mouse (Roldan et al., 1994), guinea-pig (Shi et al., 1996), pig (Melendrez et al., 1994; Roldan and Vazquez, 1996), and horse (Meyers et al., 1995). Progesterone effects are mediated by membrane receptor(s) located on the sperm head (Blackmore and Latanzio, 1991; Blackmore et al., 1991; Meizel and Turner, 1991; Tesarik et al., 1992; Turner and Meizel, 1995; Sabeur et al., 1996). It appears that the sperm surface receptor(s) mediating progesterone action is different from the intracellular progesterone receptor that mediates the steroid s genomic effects (Baldi et al., 1991; Blackmore et al., 1991). Furthermore, the genomic progesterone receptor has not been detected in human spermatozoa (Castilla et al., 1995). Progesterone prompts a rapid increase in intracellular free Ca 2, which seems to be due entirely to influx from the extracellular space (Thomas and Meizel, 1989; Blackmore et al., 1990, 1991; Wang et al., 1996), and also triggers Cl efflux (Turner and Meizel, 1995; Wang et al., 1996). It seems that the increase in intracellular Ca 2 involves two waves, of which the second is perhaps regulated by protein kinase C (PKC) activity (Tesarik et al., 1996). In fact, PKC activity after progesterone stimulation has been characterized in human spermatozoa, and it seems that PKC activation itself is dependent on Ca 2 influx from the extracellular space (O Toole et al., 1996c). Progesterone stimulation also results in the activation of a protein tyrosine kinase and phosphorylation of a ~95 kdaprotein (Tesarik et al., 1993b; Luconi et al., 1995). The target for this signal transduction pathway is not known. One possibility is that tyrosine kinase could activate phosphoinositidase C, which would then lead to the hydrolysis of the polyphosphoinositides (Thomas and Meizel, 1989; Roldan et al., 1994), and the generation of diacylglycerol (Roldan et al., 1994; O Toole et al., 1996a), that have been observed European Society for Human Reproduction and Embryology 677

2 Q.-X.Shi et al. after stimulation with progesterone. On the other hand, progesterone does not appear to act via G proteins (Tesarik et al., 1993a; Murase and Roldan, 1996). Stimulation of spermatozoa with progesterone also leads to activation of phospholipase A 2 (Baldi et al., 1993; Roldan and Vazquez, 1996), but mechanisms regulating this pathway are also unknown (reviewed in Brucker and Lipford, 1995). It has been suggested that the sperm progesterone receptor located on the cell surface may resemble a γ-aminobutyric acid type A (GABA A ) receptor because acrosomal exocytosis in human and mouse spermatozoa can be inhibited by bicuculline, a GABA A receptor antagonist (Wistrom and Meizel, 1993; Shi and Roldan, 1995). Furthermore, the existence of GABA-specific binding sites in spermatozoa from man (Aanesen et al., 1995) and domestic species (Erdö and Wekerle, 1990), and a GABA A receptor subunit in human spermatozoa (Wistrom and Meizel, 1993) have been revealed. Evidence has been presented showing that GABA and muscimol, two GABA receptor agonists, can induce exocytosis in mouse spermatozoa (Shi and Roldan, 1995). In addition, stimulation with progesterone and GABA or muscimol leads to additive effects on exocytosis (Roldan et al., 1994; Shi and Roldan, 1995), as expected for a GABA A receptor (Robel and Baulieu, 1994). On the other hand, studies on human spermatozoa have shown little or no effect of GABA or muscimol on intracellular Ca 2 rise or exocytosis (Baldi et al., 1991; Wistrom and Meizel, 1993; Aitken et al., 1996b). This study was therefore undertaken to examine the effects of GABA on human spermatozoa. We analysed: (i) whether spermatozoa need to be preincubated under capacitating conditions to become responsive to GABA; (ii) the dose-dependency of this response; (iii) whether GABA enhances the stimulatory action of progesterone; and (iv) if the inhibition of Ca 2 availability (by inclusion of EGTA or the Ca 2 channel antagonist La 3 ) affects the ability of GABA to stimulate acrosomal exocytosis. Materials and methods Materials Reagents were of analytical grade and were purchased from Shanghai Chemical Reagents Co. (Shanghai, P.R. of China). Bovine serum albumin (BSA, fraction V, catalogue number A-7906), sodium pyruvate, sodium lactate, GABA, progesterone, hyaluronidase, trypsin, mineral oil, HEPES and Percoll were from Sigma (St Louis, MO, USA). The medium used throughout this study was Biggers Whitten Whittingham medium (BWW), with some modifications, and with 0.35% BSA (Table I). Semen samples were obtained by masturbation from 12 healthy fertile men after an abstinence of 3 5 days. All samples were examined following World Health Organization (WHO, 1992) criteria. Only samples with motility 70%, viability 85%, and sperm concentration cells/ml were used. The experiments were performed using only motile spermatozoa selected by the swim-up technique. Sperm suspensions were collected and washed twice by centrifugation for 15 min at 600 g. The final pellet was resuspended in BWW and the sperm concentration adjusted to ~ cells/ml. 678 Table I. Composition of BWW medium used with human spermatozoa Component mm NaCl 98.0 KCl 4.78 CaCl 2.2H 2 O 1.71 KH 2 PO MgSO 4.7H 2 O 1.19 NaHCO D-glucose 5.56 Na-pyruvate 0.25 HEPES a 20.0 Phenol Red 5 µg/ml Penicillin G 100 IU/ml Streptomycin sulphate 50 µg/ml Bovine serum albumin (fraction V) 3.5 mg/ml ph was adjusted to 7.6 with 1N NaOH. The osmolality of complete medium was ~300 mosm/kg. a When BWW was used for the sperm penetration assay, NaCl was used instead of HEPES. Sperm treatment To ascertain the capacitation time required for human spermatozoa to respond to GABA, aliquots of 200 µl of sperm suspension in BWW were incubated at 37 C for 0, 1, 3, 5, 7, 9 and 11 h under 5% CO 2 in air. The acrosome reaction was assessed after adding 2.5 µm GABA, unless indicated otherwise. In some experiments, spermatozoa were exposed to 2.5 µm progesterone. As controls, solvents used to dissolve GABA (NaCl 0.15 M) or progesterone [dimethyl sulphoxide (DMSO), final concentration in medium: 0.025%, v/v] were included. Evaluation of acrosomal status by chlortetracycline staining After addition of the agonist, spermatozoa were further incubated for 15 min. Spermatozoa were then stained with the supravital stain Hoechst (which does not stain cells with intact plasma membranes) and chlortetracycline (CTC), as previously described (Ward and Storey, 1984; Adeoya-Osiguwa and Fraser, 1993). Briefly, a 100 mg Hoechst 33258/ml stock solution was made by dissolving the dye in triple-distilled water. The stock solution was stored in a foilwrapped bottle at 4 C, and used within 1 month. Final concentration of dye when added to the sperm suspension was 1 µg/ml. Spermatozoa were stained for 10 min and then washed through 45% Percoll by centrifugation at 600 g for 10 min to remove free dye. The pellet was resuspended in BWW and spermatozoa were then stained with CTC. The number of Hoechst-positive (dead) spermatozoa corresponded closely to the number of immotile cells. Evaluation of fertilizing capacity The sperm penetration assay (SPA) was performed using zona-free hamster oocytes. Ovulation was stimulated in adult female golden hamsters (8 12 weeks old) by i.p. injections of 30 IU pregnant mare s serum gonadotrophin (PMSG), followed 60 h later by injection of 30 IU of human chorionic gonadotrophin (HCG). The animals were killed 16 h after HCG administration. Cumulus oocyte complexes were collected at C. The cumulus masses were subsequently dispersed in 0.1% hyaluronidase in BWW and the denuded ova washed three times in fresh medium before being transferred to 0.05% trypsin to remove the zonae pellucidae. After 1 2 min, the ova were taken out, washed three times with fresh medium, and used for assessment of the fertilizing capacity of human spermatozoa. An aliquot of 50 µl human spermatozoa ( cells/ml), precapacitated for 7 h and treated with GABA for 15 min, was added to 50 µl BWW containing denuded oocytes which had

3 GABA and human sperm acrosome reaction previously been placed in a mm plastic dish (Corning, New York, NY, USA). Thus, the GABA concentration was reduced to half of that present during induction of the acrosome reaction. The drop (final volume, 100 µl) containing spermatozoa and oocytes was immediately overlaid with mineral oil and incubated under 5% CO 2 in air at 37 C. After incubation for 3 h, oocytes were removed, washed three times with BWW and transferred to a slide. Oocytes were then compressed carefully under a coverslip supported by four dots of a paraffin wax vaseline mixture (1:9, v/v). Oocytes were examined at 400 magnification with phase contrast optics (Olympus BH-2) for the presence of decondensed sperm heads with an attached tail within the ooplasm as evidence of fertilization. The percentage of oocytes penetrated by spermatozoa was taken as the fertilization rate. The fertilization index (FI) was the average number of spermatozoa per oocyte that had been penetrated by spermatozoa. Statistical analysis Results are expressed as mean S.D. The significance of results was examined after data transformation [arcsin [root](% acrosome reactions 100)] using analysis of variance and, where appropriate, a Fisher s post-hoc test. P 0.05 was regarded as being statistically significant. Results Human spermatozoa were preincubated for various times in BWW containing 0.35% BSA (at 37 C and under 5% CO 2 in air), and were then challenged with 2.5 µm GABA for 15 min, or left untreated for an equal period of time. Results indicated that some spermatozoa became responsive (i.e. underwent an acrosome reaction) after 1hofpreincubation, with a considerable proportion of sperm cells becoming responsive after 3 h (Figure 1). The proportion of spermatozoa that were capable of undergoing an acrosome reaction in response to GABA increased as preincubation was extended, reaching maximum values after preincubation for 9 h. Spermatozoa that were preincubated for similar periods of time but that were not exposed to GABA (i.e. only to its solvent, as control) did not undergo an acrosome reaction. Motility of spermatozoa remained high during incubation with GABA, but dropped to ~70% after 5 7 h. For this reason, subsequent experiments were carried out preincubating spermatozoa for 7 h. To examine the effect of different concentrations of GABA on the acrosome reaction, spermatozoa were preincubated for 7 h in BWW containing 0.35% BSA at 37 C under 5% CO 2 in air, and were then left unstimulated or stimulated with µm GABA for 15 min. As shown in Figure 2, the effect was biphasic, with a concentration-dependent increase in the proportion of spermatozoa undergoing acrosome reactions at GABA concentrations of up to µm. Further increases in GABA concentration resulted in a reduction in stimulation. The response elicited by GABA was examined further by testing the effect of this agonist on the ability of spermatozoa to penetrate zona-free hamster oocytes (Table II). It is known that only acrosome-reacted spermatozoa can fertilize zona-free oocytes (Yanagimachi, 1994), so this test is a good indicator of the proportion of spermatozoa undergoing acrosome reactions and their fertilizing ability. Treatment of preincubated human spermatozoa with GABA resulted in an increase in the Figure 1. Effect of preincubation on GABA-induced acrosome reactions in human spermatozoa. Spermatozoa were incubated at 37 C in BWW medium under 5% CO 2 in air for various times and were then exposed to 2.5 µm GABA (d) or to its solvent (control) (s). After 15 min, aliquots were analysed for the occurrence of the acrosome reaction. Values are mean SD of five different experiments. Error bars at 7, 9 and 11 h are small and hidden by the symbol. Two factor analysis of variance: treatment, P ; time, P (a) comparison between control and GABA by analysis of variance, no differences at 0 h; at all other time points, P Comparison between time points after GABA treatment (analysis of variance), P ; Fisher s post-hoc test, no differences between 9 h and 11 h; between 0hand 1h,P 0.01; at all other time points, P Figure 2. Effect of different concentrations of GABA on the acrosome reaction of human spermatozoa. Spermatozoa were preincubated at 37 C in BWW medium under 5% CO 2 in air for 7 h and were then exposed to various GABA concentrations for 15 min. Aliquots were then processed for chlortetracycline staining as described in Materials and Methods. Results are means SD of five experiments. Analysis of variance: P ; Fisher s posthoc test: (a) between control (0 µm GABA) and each GABA concentration, P ; (b) between µm and 1.25 µm GABA, P 0.02; (c) between 1.25 µm and 2.5 µm GABA, not significant; (d) between 2.5 µm and 5.0 µm GABA, P ; (e) between 5.0 µm and 10.0 µm GABA, P

4 Q.-X.Shi et al. Table II. Effect of GABA on the penetration of zona-free hamster oocytes by human spermatozoa Sperm No. oocytes No. oocytes Percentage fertilization FI (mean SD) pretreatment examined penetrated (mean SD) Control GABA 2.5 µm a b FI - fertilization index (average no. spermatozoa per penetrated oocyte). Spermatozoa were preincubated at 37 C in BWW medium under 5% CO 2 in air for 7 h and were then treated with 2.5 µm GABA for 15 min. Aliquots of spermatozoa were added to an equal volume of BWW containing zona-free hamster oocytes and incubated for 3h(n 5; see Materials and Methods for details). a P when compared with control (analysis of variance). b P when compared with control (analysis of variance). Figure 3. Effect of GABA and/or progesterone on the occurrence of the acrosome reaction in human spermatozoa. Spermatozoa were preincubated at 37 C in BWW medium under 5% CO 2 in air for 7 h and were then exposed to 1.25 µm GABA or 2.5 µm progesterone (P) or to combinations of these agonists for 15 min. Solvent controls (0.15 M NaCl or 0.025% dimethyl sulphoxide) were also included; there was no significant difference in percentage acrosome reaction between these two controls. At the end the incubations, sub-samples were processed for chlortetracycline staining as described in Materials and Methods. Results are mean SD of three experiments. Analysis of variance: P ; Fisher s post-hoc test: (a) between each treatment and the control, P ; (b) between GABA and progesterone, P ; (c) between GABA progesterone and GABA or progesterone, P proportion of fertilized oocytes, as well as an increase in the fertilization index (i.e. the number of spermatozoa penetrating oocytes), when compared with controls. In order to test whether there was any interaction between progesterone and GABA, spermatozoa were preincubated for 7 h as indicated above, and were then exposed to 1.25 µm GABA, 2.5 µm progesterone, to both agonists, or to their solvents as controls for 15 min. Figure 3 shows that when human spermatozoa were treated with 2.5 µm progesterone, ~30% exhibited an acrosome reaction, whereas after treatment with 1.25 µm GABA, ~40% spermatozoa underwent an acrosome reaction. Exposure of human spermatozoa to both agonists resulted in a proportion of acrosome reactions (~55%) that was significantly higher than that seen after treatment with either agonist separately (Figure 3). In order to assess whether the combination of agonists exhibited an additive effect, the percentage of acrosome reactions seen in controls was subtracted from those recorded after treatment with progesterone, GABA, or both. Data were then compared using analysis of variance. Results showed that when both agonists were included, the proportion of cells that responded ( %) was significantly higher than the proportion of cells responding to each agonist alone (GABA: %; progesterone: %) (analysis of variance, P ; Fisher s post-hoc test: GABA versus GABA progesterone, P 0.001; progesterone versus GABA progesterone, P ). The response stimulated by inclusion of both agonists was, however, slightly lower than what might be expected for a strictly additive effect (GABA progesterone versus sum GABA alone and progesterone alone, P 0.02). This may be due to the fact that the response elicited by GABA alone was near maximal (see Figure 2). Finally, we assessed whether the effect of GABA on the occurrence of the acrosome reaction was related to Ca 2 entry from the extracellular space, and whether this may take place via Ca 2 channels. As seen in Figure 4, inclusion of a Ca 2 chelator (EGTA, 2 mm) in the medium reduced the ability of GABA to stimulate the acrosome reaction. Likewise, inclusion of the Ca 2 channel inhibitor La 3 (100 µm) resulted in a decrease in the proportion of acrosome reactions triggered by GABA. Discussion The results of this study show clearly that GABA can stimulate the occurrence of the acrosome reaction in human spermatozoa, and that these spermatozoa are capable of fusing with zonafree hamster oocytes, thus demonstrating their functional competence. GABA was able to stimulate exocytosis in a high proportion of spermatozoa preincubated for 3 h and, hence, likely to have undergone capacitation. As preincubation proceeded over longer periods of time, more spermatozoa became capable of responding to GABA. This is similar to results obtained in mouse spermatozoa where it was found that only capacitated spermatozoa were able to undergo an acrosome reaction in response to progesterone (Shi and Roldan, 1995). Likewise, among human spermatozoa, only cells preincubated before progesterone stimulation were able to penetrate zona-free hamster oocytes (Aitken et al., 1996a). On the other hand, it has been found that progesterone can elicit another cell 680

5 GABA and human sperm acrosome reaction Figure 4. Effect of La 3 or EGTA on GABA-stimulated acrosome reactions in human spermatozoa. Spermatozoa were preincubated at 37 C in BWW medium under 5% CO 2 in air for 7 h and were then exposed to 100 µm La 3 or 2 mm EGTA for 10 min, followed by treatment with 1.25 µm GABA for 15 min. At the end of this incubation, sub-samples were processed for chlortetracycline staining as described in Materials and Methods. Results are mean SD of three experiments. Analysis of variance: P ; Fisher s post-hoc test: (a) between each treatment and the control, P ; (b) EGTA plus GABA versus GABA, P ; (c) La 3 plus GABA versus GABA, P response, i.e. elevation of intracellular Ca 2, in both noncapacitated and capacitated spermatozoa. It is not entirely clear why progesterone would trigger a Ca 2 rise in both types of spermatozoa, but would induce an acrosome reaction only in capacitated cells. One explanation may relate to phosphorylation changes taking place during capacitation (Luconi et al., 1995; Aitken et al., 1996a) and that may be necessary for the acrosome reaction. We found that GABA clearly induced an acrosome reaction in human spermatozoa, and it enhanced the proportion of sperm cells that penetrated zona-free hamster oocytes. The effect of GABA on the induction of the acrosome reaction was concentration-dependent although, interestingly, high concentrations induced lesser stimulation probably due to saturation of receptors. These results are different from those presented in a recent report where it was described that GABA or muscimol did not induce a Ca 2 transient, or stimulated penetration of zona-free hamster oocytes (Aitken et al., 1996b). It is not clear what are the reasons for this variance and, unfortunately, our results are not directly comparable with those presented in that report because the authors did not examine the effect of reagents directly on the acrosome reaction. On the other hand, our results agree with work showing that GABA would stimulate the acrosome reaction in spermatozoa from mouse (Roldan et al., 1994; Shi and Roldan, 1995; Murase and Roldan, 1996) and guinea pig (Shi et al., 1996), and with a preliminary study indicating that stimulation with GABA and muscimol triggered the acrosome reaction in human spermatozoa (Hall et al., 1996). Earlier studies (Wistrom and Meizel, 1993) have also shown a very small, statistically significant increase in acrosome reactions triggered by GABA in human sperm atozoa. There is additional evidence indicating that human spermatozoa have GABA A receptors on the cell surface, which is consistent with the biological effect of GABA seen in the present study: (i) a GABA A receptor subunit has been detected by Western blotting and immunofluorescence (Wistrom and Meizel, 1993); (ii) GABA binding sites have been revealed (Aanesen et al., 1995); and (iii) progesterone stimulation of the acrosome reaction was inhibited by bicuculline (a GABA A receptor antagonist) and picrotoxin (a blocker of the GABA A receptor/cl channel complex) (Wistrom and Meizel, 1993; Turner and Meizel, 1995). Our study also revealed an interaction between progesterone and GABA in the stimulation of the acrosome reaction. This was revealed by direct examination of acrosomal status where higher numbers of cells responded when both agonists were used together in comparison to exposure to each of the reagents alone. This is in agreement with what is expected from a GABA A receptor, although it seems clear that certain differences exist between the sperm receptor and the neuronal receptor (see discussion in Blackmore et al., 1994; Shi and Roldan, 1995). In addition, spermatozoa seem to have more than one type of progesterone receptor (see Murase and Roldan, 1996 for discussion). One of these receptors may be the GABA A -like receptor (see above), whereas another one may be a receptor linked perhaps to a Ca 2 channel (Roldan et al., 1994; Turner et al., 1994; Shi and Roldan, 1995). There may be yet a third type of receptor linked to protein tyrosine kinase activity (Tesarik et al., 1993b). Therefore, the ability to stimulate acrosome reactions directly with GABA (an agonist of the GABA receptor) opens up the possibility to study signalling pathways activated as a result of the occupancy of this particular receptor (see Murase and Roldan, 1996). The sperm acrosome reaction relies on the presence of extracellular Ca 2 (Roldan and Harrison, 1990) and it is only after this Ca 2 influx is triggered by progesterone that the acrosome reaction takes place (Aitken et al., 1996b; O Toole et al., 1996b). It has been found in previous studies that the progesterone-stimulated acrosome reaction was blocked by the Ca 2 channel antagonist nifedipine (mouse: Shi and Roldan, 1995; man: O Toole et al., 1996b) and, also, that it could be blocked by chelation of extracellular Ca 2 with EGTA (mouse: Roldan et al., 1994; hamster: Llanos and Anabalon, 1996) or by inclusion of the Ca 2 channel antagonists La 3 (mouse: Roldan et al., 1994; hamster: Llanos and Anabalon, 1996). The present study revealed that stimulation of human sperm acrosome reactions with GABA was inhibited if either EGTA or La 3 were included, which agrees well with previous findings in other species. Interestingly, there seems to be some contradiction between the effects of these reagents on the acrosome reaction and their effects on intracellular Ca 2 rises prompted by progesterone. Although chelation of extracellular Ca 2 or inclusion of La 3 blocks progesterone-elicited Ca 2 transients in non-capacitated spermatozoa (Blackmore et al., 1991; Aitken et al., 1996b), Ca 2 channel antagonists appear to have little or no effect on this response in non-capacitated cells [Aitken et al., 1996b, but see McLaughlin and Ford 681

6 Q.-X.Shi et al. (1994) who found that verapamil inhibits progesterone-induced Ca 2 rise]. One possible explanation for these discrepancies may relate to the functional status of spermatozoa, because studies on modulation of Ca 2 levels have always been carried out on non-capacited cells. It could be that changes (e.g. phosphorylation; see Luconi et al., 1995; Aitken et al., 1996a) taking place during capacitation may affect the activity or responsiveness of progesterone receptors and/or Ca 2 channels. Finally, it is noteworthy that, in addition to effects on acrosomal exocytosis, GABA can affect motility and hyperactivation of human spermatozoa (Calogero et al., 1996), and capacitation of ram spermatozoa (de las Heras et al., 1997), which indicates that GABA receptors in spermatozoa may be linked to different aspects of sperm cell function and intracellular signalling. In conclusion, we have shown that GABA can stimulate acrosome reactions in precapacitated human spermatozoa, that the response is concentration-dependent (with a narrow range of concentrations showing a considerable stimulatory effect and higher concentrations being less stimulatory), and that the effect of GABA is dependent on the availability of extracellular Ca 2 for influx. These results could lead to a better understanding of how the GABA A receptor is activated and what mechanisms of intracellular signalling are elicited upon receptor occupancy. Acknowledgements This work was supported by the Natural Science Fund of China ( ). References Aanesen, A., Fried, G., Andersson, E. and Gottlieb, C. (1995) Evidence for γ-aminobutyric acid specific binding sites on human spermatozoa. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, Adeoya-Osiguwa, S.A. and Fraser, L.R. (1993) A biphasic pattern of 45 Ca 2 uptake by mouse spermatozoa in vitro correlates with changing functional potential. J. 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