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.     

Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction

This study describes investigations of the importance of intraacrosomal pH in the hamster sperm acrosome reaction (AR). Washed cauda epididymal sperm were capacitated in vitro in a medium containing 2 mM Ca2+, 144 mM Na+, and 3 mM K+. Such sperm underwent a significant increase in the number of AR within 10 min after the addition of the Mg2+-ATPase (adenosine triphosphatase) inhibitors DCCD (20 microM) or NBD-Cl (10 microM) or the proton ionophore FCCP (6 micrograms/ml) at 3.5 hr of incubation or after addition of HN4Cl (3 mM) at 4 hr of incubation. Addition of the mitochondrial electron transport inhibitor rotenone (2.5 microM) at 3.5 hr or of NaCl (3 mM) or KCl (3 mM) at 4 hr did not stimulate AR over control levels, suggesting that the stimulation of AR by the other compounds was not directly due to depletion of acrosomal adenosine triphosphate (ATP) or alteration of the acrosomal transmembrane potential. The AR also was not stimulated by either DCCD or FCCP added prior to 3 hr of incubation of sperm, whereas both compounds were increasingly effective at stimulating AR with increasing length of preincubation of sperm before the addition of the test compounds. The intraacrosomal pH of sperm incubated in low [K+] (0.6-0.9 mM) for 3.5 hr rose by at least one pH unit (as measured with the fluorescent dye 9-aminoacridine) within 15-30 min after raising extracellular [K+] to 4.2-4.5 mM. The pH rise occurred even in the presence of the Ca2+-chelator EGTA (2 mM). Either FCCP (8 micrograms/ml) or DCCD (20 microM), but not rotenone (2.5 microM), plus K+ (3.6 mM), raised the intraacrosomal pH of sperm incubated for 3 hr in low [K+] within 10 min after addition. No pH rise occurred in the absence of additional K+. These results demonstrate that the intraacrosomal pH of the hamster sperm becomes more alkaline in a process not requiring high concentrations of external Ca2+, but requiring K+. The results of this and previous studies lead us to suggest here that the intraacrosomal pH rise may be mediated via a change in K+ and H+ permeability of sperm head membranes, which allows K+ influx and H+ efflux, and via inhibition of an acrosomal Mg2+-ATPase proton pump. We propose that the permeability changes and the consequent alkalinization of the acrosomal interior are important steps in late capacitation and/or the mammalian AR.     

Regulation of proacrosin conversion in isolated guinea pig sperm acrosomal apical segments

Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proacrosin has been identified in extracts of intact guinea spermatozoa as a major silver staining band which reacted immunologically with antibodies made against purified proacrosin from guinea pig testis. Proacrosin exhibited an approximate Mr of 50,000 and was rapidly converted to an Mr 45,000 protein following induction of the acrosome reaction with 2.0 mM CaCl2 and 1 micrograms/ml A23187. Apical segments isolated at pH 6.0 from guinea pig spermatozoa also contained a major silver staining band of Mr 50,000 which cross-reacted with antibodies to guinea pig testis proacrosin. Subcellular fractionation of spermatozoa indicated that proacrosin remained in the particulate fraction of homogenized spermatozoa and was enriched within the isolated acrosomal apical segment. When apical segments isolated at pH 6.0 were incubated at pH 7.5, proacrosin was rapidly converted to the Mr 45,000 form observed in spermatozoa undergoing the acrosome reaction. The conversion process in isolated apical segments was inhibited by leupeptin and was accelerated in the presence of calcium, magnesium, and manganese. Zinc completely inhibited the conversion of proacrosin to the Mr 45,000 protein. Neither proacrosin nor the Mr 45,000 protein were released into the supernatant fluid during the incubation of apical segments at pH 7.5. Furthermore, the proteins were resistant to solubilization by 150 mM NaCl and 1% Triton X-100 but were solubilized by treatment of apical segments with 1 M NaCl. These results provide evidence as to the identity and subcellular distribution of proacrosin in intact guinea pig sperm prior to zymogen conversion and suggest that isolated apical segments exhibit a subset of the exocytotic reactions leading to completion of the acrosome reaction.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.     

Role of prostaglandins in calcium-induced corticosteroid secretion by isolated frog interrenal gland

The role of prostaglandins (PGs) in calcium-induced corticosteroid secretion by frog adrenal (interrenal) gland has been examined in vitro using a perifusion technique. Increasing concentrations of CaCl2 (4-10 mM) stimulated in a dose-dependent manner aldosterone, PGE2 and 6-keto-PGF1 alpha production, whereas TXB2 was not affected. The kinetics of the adrenal response to CaCl2 indicated that the increase in PG output always preceded that of steroid. Administration of cobalt (4 mM), a calcium-channel inhibitor, blocked the calcium-induced stimulation of PGs and corticosteroids. Infusion of indomethacin (5 X 10(-6) M), a specific cyclooxygenase inhibitor, significantly decreased the basal production of PGs and steroids, and prevented the stimulatory effect of CaCl2 (6 mM). Infusion of the calcium ionophore A 23187 (10(-6) M), for 20 min, induced a marked stimulation of PG and steroid production. Taken together, these data support the notion that biosynthesis of prostaglandins is associated with calcium-induced corticosteroid secretion in frog adrenal cells.     

The intraacrosomal calcium pool plays a direct role in acrosomal exocytosis

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP(3))-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP(3)-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP(3)-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.     

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.     

Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.     

Evidence for a delta pH-driven Ca2+ uptake in EGTA-treated bovine spermatozoa

Calcium efflux from ejaculated bovine spermatozoa occurred upon incubation in Ca2+/EGTA buffers with Ca2+ ion concentrations ranging from 0.1 microM to 1 nM. Both total cellular calcium and cytosol free Ca2+ concentrations, the latter measured with Quin 2, were inversely correlated with the Ca2+ activity of the medium. An influx of radioactive 45Ca2+ parallel to a net efflux of calcium took place in spermatozoa incubated in 45Ca2+/EGTA buffers with 45Ca2+ activity of 0.01 microM or 0.1 microM. The uptake of the radioactive isotope was higher in spermatozoa incubated at pH 7.8 than that found at pH 6.8, increased in the presence of acetate or amiloride but decreased when ammonium chloride or monensin was added to the incubation mixture. Addition of acetate produced a decrease of the cytoplasmic pH, determined with the indicator carboxyfluorescein, whereas addition of NH4Cl or monensin caused a pH increase. Addition of either nigericin or monensin to spermatozoa suspended in a choline medium containing low concentrations of Na+, K+ and Ca2+ produced a cytosolic acidification, the subsequent addition of Ca2+ caused a cytosolic alkalinization parallel to an increase of the cytosolic free Ca2+. Addition of CaCl2 to EGTA-pretreated spermatozoa resuspended in a poorly buffered medium induced an evident decrease of extracellular pH suggesting a cellular proton extrusion. Both monensin and nigericin caused an increase of the calcium transport in spermatozoa suspended in a choline medium containing a physiological concentration of 1.5 mM CaCl2. Taken together the present results indicate that, under the experimental conditions used, a delta pH-driven Ca2+ uptake occurs in ejaculated bovine spermatozoa and suggest that Ca2+ is taken up in exchange with H+.     

Characterization of isolated acrosomal matrices from hamster spermatozoa

The acrosomal matrix of hamster spermatozoa was enriched and characterized. Acrosomal matrices were released from spermatozoa with shaking in a pH 5.2 buffer containing Triton X-100 and protease inhibitors, and enriched on a glass-bead column. Phase-contrast microscopy indicated that 70-80% of the acrosomal matrices were released from the spermatozoa and only minor contamination from sperm heads was detected. Transmission electron microscopy confirmed the low level of contamination in the preparation and revealed a bilaminar structure similar but not identical to that of guinea-pig acrosomal matrix. One- and two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the acrosomal matrix to be a complex structure enriched for several polypeptides. Proteinase activity was demonstrated by gelatin-SDS-PAGE. The major activity corresponded to bands of relative molecular masses (Mr) of 56,000, 51,000 and 48,000 with two minor bands of Mr 30,000 and 28,000. The lectin Pisum sativum agglutinin (PSA) bound to the anterior head of spermatozoa and isolated acrosomal matrix as judged by fluorescence microscopy using FITC-PSA. Western blots of spermatozoa and acrosomal matrices followed by overlay with biotinylated PSA indicated that there are at least two PSA-binding glycoproteins of Mr 60,000 and 72,000.     

Evaluation of acrosomal status of bovine spermatozoa using concanavalin a lectin

We report here that fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) specifically labels the acrosomal region of acrosome-reacted bovine spermatozoa. This labeling is found to be useful in evaluating the acrosome status of bovine spermatozoa. When fresh bovine spermatozoa that had been fixed with 4% formaldehyde, smeared on glass slides and then air-dried were stained by FITC-ConA, weak fluorescence was observed on the acrosomal region, although almost all the spermatozoa appeared to be acrosome-intact. However, when fresh sperm suspensions were incubated with FITC-ConA and then mounted on glass slides, no fluorescence was observed on the acrosomal region. Therefore, in the ensuing experiments, both the fixation and the FITC-ConA staining of spermatozoa were done in suspension. When ethanol-treated spermatozoa, whose outer membrane may be permeabilized, were stained with FITC-ConA, the fluorescence was extensively observed on the inner acrosomal region. This fluorescence was inhibited in the presence of 0.2 M D-mannose, a competitive sugar, suggesting that FITC-ConA binds specifically to glycocomponents on the inner acrosomal membrane. We next tried to stain fresh or frozen-thawed spermatozoa from 3 different bulls that had been treated with the calcium ionophore A23187, which is known to induce acrosome reaction of bovine spermatozoa, with FITC-ConA. A significant correlation between the percentage of ConA-labeled spermatozoa and that of rose bengal stained negative ones at various time points during A23187 incubation was achieved. Furthermore, suitability of dual staining to distinguish between physiological acrosome reaction (acrosome-lost and live) and degenerative acrosomal loss (acrosome-lost and dead) using FITC-ConA and Hoechst bis-benzimide 33258 (H258) supravital stain was also confirmed. From these results, it was concluded that the FITC-ConA labeling procedure is a feasible and reliable method for the assessment of physiological acrosome reaction of bovine spermatozoa.     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

Effects of extracellular environment factors on the motility in Japanese eel spermatozoa

We have examined the effects in vitro of the ionic composition, pH and temperature on the motility by the spermatozoa of the Japanese eel (Anguilla japonica). Milt was obtained from 10 males that had been artificially matured by repeated injections of hCG. Sperm motility was monitored with a VHS video recorder and a video camera connected to a microscope. The results showed that most of the sperm were highly motile in 250-700 mM NaCl, 250-650 mM KCl and 350-550 mM CaCl2 solution. The longest duration of sperm motility recorded in 500 mM NaCl, 250 mM KCl and 350 mM CaCl2 solution. Sperm was not motile when suspended at pH 2, sperm motility was observed at pH 3, there was a relatively higher percentage of motile sperm in solutions at pH 4-12 (above 80%). The motility and duration increased within 18-24°C and decreased at the range of 24-30°C. Appropriate K+ ion concentration in the active medium could enhance the percent motility and duration of eel spermatozoa.     

Sperm capacitation in vitro in the eld's deer

Sperm capacitation was examined in the endangered Eld's deer (Cervus eldi thamin). Sperm motility and viability (percentage of sperm cells with intact membranes) were assessed in vitro over time after attempting to induce capacitation in TALP alone and TALP supplemented with calcium (10 mM CaCl2), dibutyryl cAMP (1 mM dbcAMP), or fetal calf serum (20% FCS). Sperm aliquots were evaluated at 0, 3, 6, 9, and 12 h for motility, viability, and ability to acrosome react after exposure to calcium ionophore (A23187, CI; 10 microM) or lysophosphatidylcholine (LC; 100 microg/mL). Fresh sperm aliquots in TALP + 10 mM CaCl2 exposed to CI had fewer (P < 0.05) intact acrosomes than the TALP control (TALP alone) or dbcAMP and FCS treatments after 9 h. Mean (+/- SEM) percentage of intact acrosomes of spermatozoa incubated in medium with increased CaCl2 declined (P < 0.05) from 80.2 +/- 2.6% (0 h) to 49.7 +/- 7.3% after prolonged incubation (9 h). The proportion of capacitated fresh spermatozoa was not influenced by LC treatment. Capacitation was not induced (P > 0.05) by any of the presumptive sperm capacitators after freeze-thawing. Likewise, neither CI nor LC induced the acrosome reaction (AR) in these spermatozoa, suggesting that the freeze-thawing process may have caused membrane damage. Results revealed that the supplementation of medium with CaCl2 evokes capacitation in some spermatozoa. However, Eld's deer spermatozoa appear remarkably resistant to conventional stimulators of capacitation and the AR.     

Evidence for the capacitation-associated membrane priming of mouse spermatozoa

An important feature of male fertility is the physiological priming of mammalian spermatozoa by a multifaceted process referred to as capacitation. It is a prerequisite event before spermatozoa can bind to the egg's extracellular coat, the zona pellucida, and undergo a signal transduction cascade. The net result is the fusion of the plasma membrane (PM) and underlying outer acrosomal membrane at multiple sites and the release of acrosomal contents (i.e., glycohydrolases, proteinases, etc.) at the site of sperm-zona binding. In this study, we have used an indirect immunofluorescence (IIF) assay and other staining approaches to examine capacitation-associated membrane priming of mouse spermatozoa. For IIF studies, we used affinity-purified antibodies against two glycohydrolases that cross-reacted with the acrosomal enzymes only when the uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors in vitro capacitation induced membrane priming that allowed the antibodies to cross-react with the acrosomal enzymes in capacitating acrosome-intact spermatozoa without permeabilization, as revealed by the appearance of several distinct fluorescent patterns, including an initial immunopositive lining over the acrosome cap to an intense immunopositive reaction throughout the acrosome. These early immunopositive patterns were followed by the appearance of intense fluorescent spots (droplets) that seem to establish contact with the PM in a time-dependent manner. Inclusion of calmodulin, a 17-kDa Ca(2+)-binding protein which promotes capacitation, in the incubation medium did not alter the overall rate of capacitation; however, its presence accelerated the initial stages of membrane priming. The potential similarities between sperm capacitation and early events of Ca(2+)-triggered membrane fusion among eukaryotes and among various stations of the secretory and endocytotic pathways are discussed.     

Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation

Thyone sperm were induced to undergo the acrosomal reaction with a calcium ionophore A23187 in sea water containing 50 mM excess CaCl2, and the extension of the acrosomal process was recorded with high- resolution, differential interference contrast video microscopy at 60 fields/sec. The length of the acrosomal process was measured at 0.25-s intervals on nine sperm. When the data were plotted as (length)2 vs. time, the points fell exactly on a straight line except for the initial and very final stages of elongation. Cytochalasin B alters the rate of elongation of the acrosomal process in a dose-dependent way, inhibiting the elongation completely at high concentrations (20 micrograms/ml). However, no inhibition was observed unless excess Ca++ was added to sea water. The concentration of actin in the periacrosomal cup of the unreacted sperm is as high as 160 mg/ml; we calculate this concentration from the number and lengths of the actin filaments in a fully reacted sperm, and the volume of the periacrosomal cup in the unreacted sperm. These results are consistent with the hypothesis proposed earlier that monomers add to the ends of the actin filaments situated at the tip of the growing acrosomal process (the preferred end for monomer addition), and that the rate of elongation of the process is limited by diffusion of monomers from the sperm head (periacrosomal cup) to the tip of the elongating process. During the extension of the acrosomal process, a few blebs distributed along its lengths move out with the process. These blebs maintain a constant distance from the tip of the growing process. At maximum length, the straight acrosomal process slackens into a bow, and numerous new blebs appear. A few seconds later, the process suddenly straightens out again and sometimes actually contracts. The behavior of the blebs indicates that membrane is inserted at the base of the growing acrosomal process, and that membrane assembly and water uptake must be coupled to actin assembly during elongation. We discuss how the dynamic balance of forces seems to determine the shape of the growing acrosomal process, and how actin assembly may be controlled during the acrosomal reaction.     

Effect of heparin on in vitro capacitation of boar sperm

Chlortetracycline (CTC) fluorescent pattern, the ability to undergo acrosome reaction (AR) upon exposure to 10 microM calcium ionophore A23187 and vitality estimation were used to investigate the effect of the sulfated glycosaminoglycan heparin on the in vitro capacitation of porcine spermatozoa. Sperm incubation in capacitating medium (CM) supplemented with 10 mM heparin for up to 120 min, showed an increase in the number of capacitated sperm (B pattern) and acrosome reacted sperm (AR pattern), without affecting their viability. In this condition, spermatozoa were incubated in CM depleted of albumin, calcium, bicarbonate or combinations, in the presence of heparin. In either calcium or bicarbonate-free media, capacitation was only basal and did not show variations in the presence of heparin. In absence of albumin the presence of calcium and bicarbonate stimulated capacitation, which was further increased by the addition of heparin. These results suggest that heparin enhances in vitro capacitation of porcine sperm only under capacitating conditions. Additionally, when sperm were incubated with 100 microg/ml biotinylated heparin in the presence or absence of unlabeled heparin, we observed that heparin binding sites were located mostly on the acrosomal region of boar sperm in an specific and saturable manner. The in vitro effect of heparin described in this work indicates that sulfated glycosaminoglycans, which are normally present in the female reproductive tract, might play an important role in the fertilization process in porcines.     

Signaling pathways in sperm capacitation and acrosome reaction.

The binding to the egg's zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa underago in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is cholesterol efflux leading to the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to indirectly phosphorylate certain proteins on tyrosine. During capacitation, there is also an increase in protein tyrosine phosphorylation dependent actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in F-actin dispersion which enable the outer acrosomal and the plasma membrane to come into contact and fuse completing the acrosomal reaction.     

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)