Regulation of acrosomal matrix dispersion in digitonin-permeabilized guinea pig spermatozoa

Digitonin-permeabilized guinea pig spermatozoa undergo acrosomal matrix dispersion in response to 2.0 mM CaCl2. In this report, the effects of pH and metal ions on matrix dispersion in permeabilized spermatozoa are examined. Calcium-induced dispersion of the acrosomal matrix was dependent on the calcium concentration; the response was not observed at concentrations of CaCl2 less than 50 microM. Magnesium could not substitute for calcium and, in fact, had a retarding effect on the calcium-induced response. Matrix dispersion was also found to be pH-dependent. The induction of matrix dispersion was inhibited at pH 5.6 and pH 9.5 relative to the responses observed at pH 6.3 and pH 7.8. Nigericin induced acrosomal matrix dispersion in the absence of added calcium, indicating a possible role of Na+/H+ exchange across the outer acrosomal membrane in initiating the matrix modification. Sodium was required for the action of nigericin; the ionophore was ineffective in medium in which choline chloride or sucrose was substituted for NaCl. In contrast, the calcium-induced dispersion of the acrosomal matrix occurred in the absence of sodium. Furthermore, low concentrations of calcium inhibited an adenosine triphosphatase activity associated with isolated acrosomal apical segments. These data are consistent with the hypothesis that calcium induces alkalinization of the acrosome, leading to matrix dispersion. However, permeabilized spermatozoa incubated at either pH 9.5 or in the presence of 50 mM NH4Cl at pH 7.5 failed to undergo spontaneous matrix dispersion, suggesting that elevated intraacrosomal pH alone was not sufficient to initiate the reaction. The proposed alternative hypothesis is that calcium initiates matrix dispersion by a mechanism in which elevated intraacrosomal pH may be a secondary response.     

Calcium-induced modification of the acrosomal matrix in digitonin-permeabilized guinea pig spermatozoa

In this report we describe and partially characterize a preparation of digitonin-permeabilized guinea pig spermatozoa that undergo a rapid and synchronous modification of the acrosomal matrix in response to calcium. Permeabilization of cauda epididymal spermatozoa by digitonin was monitored by using adenylate cyclase activity as an indicator. Spermatozoa (5 x 10(7) cells) treated with 0.005% digitonin for 15 s exhibited maximal adenylate cyclase activity but generally retained their structural morphology, as examined by phase-contrast and transmission electron microscopy. The ratio fo cell number to detergent concentration was the critical factor for determining both the efficiency of permeabilization and the maintenance of structural integrity. When permeabilized spermatozoa were treated with 2 mM CaCl2, the cells underwent a rapid and synchronous modification of the acrosomal matrix (AM). As observed by phase-contrast microscopy, the response to CaCl2 was characterized by events that occurred in the following temporal sequence: disruption of the sperm rouleaux, the loss of refractility by the apical segment of the sperm acrosome, and detachment of the apical segment from the spermatozoa. Transmission electron microscopy indicated that the loss of refractility from the sperm apical segment was coincident with a calcium-induced dispersion of the AM. Analysis of the proteins released during this response, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that a specific subset of sperm proteins was released from the spermatozoa, including a major = staining, 45,000 Mr protein apparently generated from a higher molecular weight precursor during the acrosome reaction.     

Purification and characterization of the primary acrosomal autoantigen of guinea pig epididymal spermatozoa

Previous studies showed that sperm auto- and alloantigens participate in guinea pig (GP) fertilization. In an effort to determine how alloantibodies to GP sperm acrosomal contents (AC) inhibit fertilization, we identified acrosomal auto- and alloantigens using Western blots. The predominant autoantigens migrated with Mr = 25,000, Mr = 51,000, and Mr = 55,000 under nonreducing conditions. The primary (Mr = 25,000) acrosomal autoantigen, AA1, was purified to homogeneity from AC by gel filtration, cation-exchange chromatography, chromatofocusing, and a final gel filtration. We also purified AA1 from an acidic glycerol extract of spermatozoa by gel filtration, chromatofocusing, and high-performance liquid chromatography on hydroxylapatite. AA1 is a protein and shares at least one antigenic determinant with a 51,000 Mr acrosomal component. AA1 is acrosome-specific, as determined by immunoabsorption and by indirect immunofluorescence on testicular cells. By quantitative enzyme-linked immunosorbent assay, AA1 comprises 6.4% of acrosomal protein in GP spermatozoa. On the basis of its physiochemical properties and localization, we conclude that AA1 is a unique sperm autoantigen. Surprisingly, several antibody preparations, including allo- and heteroantibodies with high anti-AA1 titers, did not inhibit fertilization in vitro. Thus, the mechanism by which alloantibodies to AC inhibit GP fertilization in vitro is not by binding to AA1.     

Outer acrosomal membrane of guinea pig spermatozoa: Isolation and structural characterization

We describe a protocol to isolate a highly enriched fraction of outer acrosomal membrane from guinea pig spermatozoa and present new data on the ultrastructure of this membrane domain. Cauda epididymal spermatozoa were suspended into a low ionic strength buffer and subjected to brief homogenization; this stripped the plasma membrane from the spermatozoa and severed the acrosomal apical segment from the spermatozoon. The crescent-shaped apical segments retained the outer acrosomal membrane and specific components of the acrosomal matrix. Enriched fractions of apical segments were isolated on discontinuous sucrose gradients and the outer acrosomal membrane purified by subsequent centrifugation onto Percoll density gradients. The isolated outer acrosomal membrane did not form vesicles, but instead rolled up into spiral sheets. Both thin section and negatively stained specimens revealed a paracrystalline arrangement of filaments associated with the luminal surface of the membrane. The isolated outer acrosomal membrane revealed a limited number of polypeptides by SDS-PAGE, and the polypeptide pattern was distinct from the plasma membrane fraction. The isolated acrosomal membranes possessed no oubain sensitive Na+, K+-ATPase activity, whereas about 20% of the ATPase activity of the plasma membrane enriched fraction was inhibited by oubain. The potential function of the structural differentiations of the outer acrosomal membrane in the membrane fusion events of the acrosome reaction is discussed.     

Zona pellucida induces activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

Phospholipase A(2) (PLA(2)) is activated in spermatozoa in response to progesterone and Ca(2+) ionophores, but to our knowledge, no study has yet reported zona pellucida (ZP)-induced activation of PLA(2). We investigated whether PLA(2) is involved in ZP-stimulated acrosomal exocytosis, if Ca(2+) is required for activation of PLA(2), and signal transduction pathways modulating PLA(2) using guinea pig sperm as a model. Spermatozoa were capacitated and labeled in low-Ca(2+) medium with [(14)C]choline chloride or [(14)C]arachidonic acid and were then exposed to millimolar Ca(2+) and various reagents and stimulated with ZP. Precapacitated spermatozoa exposed to millimolar Ca(2+) and stimulated with ZP experienced increases in arachidonic acid (AA) and lysophosphatidylcholine (lysoPC) levels and a parallel decrease in phosphatidylcholine level; these changes are indicative of PLA(2) activation. Simulation with ZP also led to acrosomal exocytosis in a high proportion of spermatozoa. Lipid changes and exocytosis were prevented if spermatozoa were exposed to aristolochic acid, a PLA(2) inhibitor, before treatment with ZP. Stimulation with ZP in medium without added Ca(2+) or in medium with millimolar Ca(2+) and EGTA or La(3+) resulted in no lipid changes or exocytosis. Pretreatment with pertussis toxin, a G(i) protein inhibitor, before stimulation with ZP blocked the release of AA and lysoPC as well as acrosomal exocytosis. Exposure of spermatozoa to the diacylglycerol (DAG) kinase inhibitor R59022 before ZP stimulation led to a significant increase in generation of lysoPC and exocytosis. Taken together, these results indicate very strongly that PLA(2) plays an essential role in ZP-induced exocytosis in spermatozoa, that PLA(2) activation requires Ca(2+) internalization, and that PLA(2) activation is regulated by signal transduction pathways involving G proteins and DAG.     

On the role of glucose in capacitation and acrosomal reaction of guinea pig sperm

In order to determine whether metabolizable sugars delayed capacitation of guinea pig spermatozoa, these cells were pre-incubated in Tyrode's pyruvate lactate glucose medium (T-PLG) or Tyrode's glucose solution (T-G). They were then transferred to minimal culture medium containing pyruvate and lactate (MCM-PL) and the occurrence of acrosomal reactions (AR) was determined by light microscopic observations of wet mount aliquots. The percentage of acrosomal reactions was quantitated in fixed samples and occurrence of a true AR was confirmed by electron microscopy. Activated acrosome-reacted spermatozoa were observed within 5 min when cells were transferred to MCM-PL solution, after preincubating them for 60–120 min either in T-PLG or T-G media. By 15 min in MCM-PL the percentage of acrosome-reacted spermatozoa reached values similar to those obtained in cells pre-incubated from the beginning in MCM-PL medium (P > 0.05 in both) but significantly different from T-PLG and T-G controls (P < 0.0005 in both). The acrosomal reaction was external calcium dependent and independent of the Tyrode's media pH ranging from 7.2 to 8.0. The results obtained suggested that capacitation occurred in T-PLG and that it was not delayed by glucose; the results also suggested that capacitation could occur within a short time with glucose as the only exogenous substrate, but that the acrosome reaction could have been arrested by a glucose metabolite. Data are presented which suggest that intracellular levels of glucose-6-phosphate (as 2-deoxyglucose-6-phosphate)could play a key role in the expression of the acrosome reaction in sperm already able to perform it. A new hypothesis is suggested for the development of the fertilizing potential of guinea pig sperm when in the female genital tract.     

Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Requirement of monovalent cations in the acrosome reaction of guinea pig spermatozoa

The majority of the spermatozoa precapacitated in Ca²⁺-free medium underwent the acrosome raction rapidly when they were transferred to Ca²⁺-containing medium. The presence of Na⁺ and Ca²⁺ in the medium was essential for the acrosome reaction. The vast majority of spermatozoa failed to undergo the reaction in Ca²⁺ medium lacking monovalent ions, although they remained motile. At the concentration of 140 mM, Na⁺, K⁺, Rb⁺, and Cs⁺ all supported the reaction at the maximum level, but at 50 mM the latter three ions were not as effective as Na⁺. Li⁺ was least effective in supporting the reaction. Virtually no acrosome reactions took place when precapacitated spermatozoa were first exposed to Na⁺ medium (no Ca²⁺) and then to Ca²⁺ medium (no Na⁺). On the other hand, a considerably higher proportion of spermatozoa acrosome reacted when they were exposed to these media in the reverse order. The most efficient acrosome reactions took place when the medium contained both a monovalent ion (Na⁺) and Ca²⁺ simultaneously. Possible mechanisms by which monovalent and divalent cations participate in the acrosome reaction are discussed.     

Induction of the acrosome reaction in guinea pig spermatozoa by cGMP analogues

The effect of cyclic nucleotide analgoues upon the immediate induction of the guinea pig acrosome reaction (AR) was studied. Dibutyryl (dB) CGMP and 8-bromo-cGMP, when added to sperm suspensions after varying periods of preincubation in glucose-free BWW medium (NaCl 94.59 mM, KCl 4.7 mM, CaCl2 1.71 mM, KH2PO4 1.19 mM, MgSO4 1.19 mM, NaHCO3 25.07 mM, pyruvate 0.25 mM, lactate 21.58 mM, and bovine seru albumen 1 g/liter), induced the AR in a large proportion of spermatozoa relative to controls. The proportion of ARs induced upon the addition of dB cGMP or 8-bromo-cGMP (10mM) at 1 h was equivalent to that obtained after a 5-h incubation in glucose-free BWW alone. The effect of cGMP analogues was concentration dependent over the tested range of 2-12 mM (less than 1-20%). The simultaneous addition if imidazole (10 mM), a cAMP phosphodiesterase stimulator, potentiated the effect (imidazole + 12 mM 8-bromo-cGMP: 73%). cAMP analogues were without effect. The presence of extracellular Ca++ was required, and it is suggested that a rise in the cGMP/CAMP ratio triggers Ca++ influx and the AR.     

Progesterone primes zona pellucida-induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.     

Involvement of an F-actin skeleton on the acrosome reaction in guinea pig spermatozoa

The acrosome reaction (AR) is a regulated exocytotic process. In several cell types, an actin network situated under the plasma membrane (PM) acts as a physical barrier to prevent this exocytosis. In seeking a function for a cortical skeleton in guinea pig spermatozoa, the PM and the outer acrosomal membrane (OAM) were investigated for the presence of F-actin and spectrin, proteins generally found in cell cortical skeletons. Both membrane types were visualized in whole-mount preparations by electron microscopy. PM proteins gave positive reaction to the Na(+),K(+)-ATPase antibody and the OAM proteins did not react to the antibody. Furthermore, a Triton X-100-resistant skeleton was obtained from both membrane types. Using gold immunoelectron microscopy, F-actin was visualized in the PM and in the OAM skeletons, while spectrin was only detected in the PM skeleton. The presence of an F-actin cortical skeleton in the sperm PM suggests that F-actin may be involved in the AR. The significantly higher number of AR elicited by cytochalasin D (Cyt-D) treatment(P<0.005) and data showing a significant (P>0.03) decrease in F-actin relative concentration in capacitating spermatozoa, agree with this suggestion. Furthermore, the proposal is strengthened by the fact that stabilization of F-actin by phalloidin (Ph) significantly (P>0.01) diminished AR induced by Ca(2+) in a streptolysin O (SLO)-permeabilized sperm model.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay

We have described the different patterns of chlortetracycline (CTC) binding to ram spermatozoa, immediately after ejaculation and upon in vitro capacitation and calcium ionophore-induced acrosomal exocytosis. Four different forms of CTC distribution were found. Form I showed an even distribution of fluorescence over the entire head, with a brighter band in the equatorial region. In Form II, uniform fluorescence was observed without equatorial band. Form III consisted of fluorescence in the anterior portion of the head. Form IV showed no fluorescence over the head. In all cases, fluorescence in the middle piece of the flagellum was observed as well. Immediately after ejaculation, Form I was the most abundant one (78%) in fresh semen with Forms II and III being relatively scarce (less than 15%). Form IV was virtually absent or appeared only occasionally. Incubation under in vitro capacitating conditions led to a significant decrease in Form I and to a significant increase in Forms II and III. Form II was mainly associated to intact acrosomes, while most spermatozoa in Form III showed intermediate forms of acrosomal status. Incubation of spermatozoa with the calcium ionophore A23187 resulted in 55% of spermatozoa showing Form IV, suggesting that it represents the acrosome-reacted stage. Form I was abruptly decreased at 30 min of incubation and was neglectible after 60 min. In contrast, Forms II and III increased at 30 min but decreased later on, suggesting that both forms represent intermediate stages before the acrosomal exocytosis. Analysis of acrosomal status in spermatozoa from individual CTC forms revealed that all spermatozoa that remained in Form II after incubation had intact acrosomes. Intermediate stages were predominant in Form III-spermatozoa, while most Form IV-spermatozoa underwent full acrosomal exocytosis. These results show that CTC binding can be used to monitor changes in ram spermatozoa during capacitation and acrosome-reaction.     

Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm

In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa

Actin cytoskeleton remodeling is a critical process for the acquisition of fertilizing capacity by spermatozoa during capacitation. However, the molecular mechanism that regulates this process has not been fully elucidated. In somatic cells, Ras-related C3 botulinum toxin substrate 1 protein (Rac1) promotes the polymerization of actin by participating in the modeling of two structures: lamellipodia and adhesion complexes linked with the plasma membrane. Rac1 is expressed in mammalian spermatozoa; however, the role of Rac1 in sperm physiology is unknown. This study aimed to elucidate the participation of Rac1 in capacitation and acrosome reaction (AR). Rac1 was found to be dispersed throughout the acrosome and without changes in the middle piece. After 60 minutes of capacitation, Rac1 was found in the apical region of the acrosome only, which concurred with an increase in Rac1-GTP. Rac1 inhibition prevented such changes. In the middle piece, Rac1 localization remained unchanged. Besides, Rac1 inhibition blocked capacitation and AR. The present study demonstrates that Rac1 participates only in the actin cytoskeleton remodeling that occurs in the acrosomal apical region during capacitation, a region where a large amount of actin is polymerized and shaped in a diadem-like structure. Our data also show that this actin cytoskeleton organized by Rac1 interacts with filamin-1, and such interaction was blocked by the inhibition of Rac1, which led to a different organization of the actin cytoskeleton. All these outcomes imply that the formation of an F-actin cytoskeleton in the acrosomal apical region is a necessary event for capacitation and AR, and which is Rac1 driven.     

pH and protease control of acrosomal content stasis and release during the guinea pig sperm acrosome reaction

The purpose of this study was to examine how trypsin inhibitors affect the guinea pig sperm acrosome reaction in vitro. Using spermatozoa pretreated with lysophosphatidyl choline, we found that both naturally occurring high molecular weight and the smaller synthetic trypsin inhibitor p-aminobenzamidine (PAB) delayed the onset of the acrosome reaction as monitored by light microscopy. Examination with electron microscopy revealed that acrosomal matrix dispersal rather than membrane fusion was affected. Despite the morphologic delay in acrosomal content release, PAB unexpectedly permitted 96% of soluble acrosomal antigen to be released into the supernatant. In addition, total acrosin release in the presence of PAB was 74% of control, with the vast majority as latent rather than active enzyme. A morphologically intact but membrane-free target of acrosomal matrix (AM), which is sensitive to trypsin inhibitor, was partially purified using Triton-x-100 at pH 5.2. AM remained morphologically stable at pH 5.2; however, shift up to pH 7 resulted in rapid dissolution within several minutes as monitored by light and electron microscopy and light scattering. Trypsin inhibitor prevented dispersion of AM at pH 7. The results suggest that, during the acrosome reaction, one distinct region of the acrosomal contents disperses after membrane vesiculation in a pH and trypsin inhibitor-insensitive fashion while a pH sensitive trypsin-like activity (acrosin?) disperses another discrete region of acrosomal matrix.     

GABA/progesterone-induced polyphosphoinositide (PPI) breakdown and its role in the acrosome reaction of guinea pig spermatozoa in vitro

To investigate whether GABA/progesterone (P4) stimulates PPI breakdown and its role in the acrosome reaction (AR), spermatozoa of guinea pig were preincubated in MCM-LCa2+ for 5.5 h and then labeled with [32P]pi for 1 h. Samples were washed through a three-step gradient Percoll, adjusted to 5×107 cells/mL and exposed to 2 mmol/L Ca2+, 5 μmol/L GABA, 10 μmol/L P4 and other agents. Lipids were separated by t.l.c. and radioactivity in spots determined by scintillation counting. The AR was assessed by phase-contrast microscopy. The results showed that (i) when spermatozoa were treated with GABA, 32P-label diminished rapidly in phosphatidylinositol 4, 5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and increased in phosphatidic acid (PA). The loss of label from PPI was almost completed by 10 min. The time-course of the AR was much slower than PPI when spermatozoa reached a maximal response by 15 min; (ii) the pattern of PPI hydrolysis and stimulation of AR was similar for the three agonists