Inhibition of the vacuolar H(+)-pump with bafilomycin A1 does not induce acrosome reaction or activate proacrosin in mouse spermatozoa

Acrosomal protease activation is regarded as an important event triggered by acrosomal reaction and leading to sperm passage through zona pellucida. Mammalian acrosome has an internal acid pH that probably helps to maintain inactive proenzymes that otherwise could be precociously activated and prevent normal fertilization. In this work, we have studied the effect of bafilomycin A1, a potent and specific inhibitor of vacuolar H(+)-pump (V-ATPase), on acrosome reaction and proacrosin activation. We used the pH-sensitive probe Lysotracker Green DND-26 to monitor qualitatively intra-acrosomal pH in cauda epididymal mouse spermatozoa. Our results showed that loss of Lysotracker label induced by bafilomycin A1 (acrosome alkalinization) did not induce acrosome reaction or proacrosin activation. We also developed a new technique for imaging the acrosome, and for evaluating the acrosome reaction, in live mouse spermatozoa using Lysotracker DND-26. These results showed that the V-ATPase is a key regulator of mammalian acrosome pH, and that acrosome alkalinization is not the only prerequisite to activate proacrosin under in vivo conditions. Our results suggest that acrosome alkalinization and acrosome reaction are two processes that could be independently regulated during mammalian sperm capacitation.     

Effect of sperm diluents on the acrosome reaction in canine sperm

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.     

Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein.

We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome, a membrane limited organelle overlying the nucleus. The sperm showed normal fertilizing ability and the integrity of their acrosome was easily examined in a non-invasive manner by tracing the GFP in individual 'live' sperm with fluorescent microscopy. The time required for the dispersal of acrosomal contents was demonstrated to be approximately 3 s after the onset of acrosome reaction.     

Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucida

Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor‐mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model. J. Cell. Physiol. 220: 611–620, 2009. © 2009 Wiley‐Liss, Inc.     

Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4–5 hours at 37°C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.     

Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3

The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction.     

Binding of a murine proteinase inhibitor to the acrosome region of the human sperm head

Proteinase inhibitors are present in the various glands, tissues, and secretions of the male reproductive tract. Some of these inhibitors bind to the acrosomal region of the sperm, and their release during in vitro or in utero incubation suggests that they may play a role in capacitation. In the mouse, the binding site for a trypsin-acrosin inhibitor, the acceptor, has been implicated in capacitation, zona binding, and the acrosome reaction. This presentation demonstrates that a component, molecular weight ?T20,000, on the human sperm head may recognize the murine inhibitor. Furthermore, the acrosome reaction can be induced in capacitated human sperm by immunoaggregation of bound murine inhibitor. The data indicate that the proteinase inhibitor binding site on the human sperm head may, as with a similar site on murine sperm, play a role in the early events of fertilization. © 1993 Wiley-Liss, Inc.     

Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region of mammalian sperm, as well as thapsigargin''s induction of the acrosome reaction, implicate IP3-gated calcium release in the mammalian acrosome reaction.     

VLA-6 integrin distribution and calcium signalling in capacitated boar sperm.

Previous investigations showed that VLA-6 integrin present on boar sperm membrane can induce acrosome reaction upon exposure to laminin accumulated in expanded cumuli (Mattioli et al., 1998. To further investigate this novel sperm egg-recognition system, the authors studied the distribution of VLA-6 integrin on the membrane of boar sperm throughout capacitation and following acrosome reaction, and analyzed intracellular Ca(2+) changes occurring in spermatozoa exposed to laminin. Immunofluorescent localisation of VLA-6 revealed a low proportion (nearly 22%) of positive cells in freshly ejaculated sperm, with integrin mainly concentrated in clustered spots. After 3 hr incubation most of the spermatozoa showed integrin molecules on the membrane, with three different labeling patterns: fluorescence localised on the edge of the acrosome (58.2 +/- 14.2% of the cells); fluorescence uniformly spread over the whole sperm head (5.0 +/- 1.9%) and finally fluorescence concentrated in clustered spots (7.6 +/- 5.6%), as recorded in freshly ejaculated sperm. Twenty-nine percent of cells did not show any distinct fluorescence. Following acrosome reaction sperm with fluorescence on the acrosomal region virtually disappeared and the proportion of unstained cells rose from 29.2 +/- 9.2 to 69.0 +/- 10.1%. Electron microscopy demonstrated that VLA-6 integrin was exclusively located on the sperm membrane of intact spermatozoa. Confocal analysis showed that laminin triggers distinct Ca(2+) raises, and that sperm exposed and kept in the presence of laminin fully retained their ability to rise intracellular Ca(2+) in response to zona pellucida proteins. These data indicate that boar sperm accumulate VLA-6 integrin on the membrane and concentrate it on the acrosomal region as capacitation progresses. Probably due to this compartmentalisation, sperm exposed to laminin experience a Ca(2+) raise that originates in the anterior sperm head where it is more adequate for the induction of acrosome reaction. Mol. Reprod. Dev. 59:322-329, 2001.     

A mouse acrosomal cortical matrix protein, MC41, has ZP2-binding activity and forms a complex with a 75-kDa serine protease.

Sperm with a large acrosome such as that of guinea pigs and hamsters have a subdomain structure in the anterior acrosome, but the mouse acrosome looks homogeneous and its matrix has not been precisely analyzed. The intra-acrosomal protein MC41 is localized in the cortical region of the mouse anterior acrosome, suggesting a subdomain structure in the mouse acrosome. Thus, the present study was undertaken to analyze the mouse acrosomal matrix using an anti-MC41 antibody. When mouse sperm were treated with 2% Triton X-100, Triton-insoluble matrix components remained in the acrosomal cortical region. Immunogold for MC41 labeled the Triton X-100 and high-salt-insoluble matrix components, demonstrating that MC41 is a subdomain-specific acrosomal matrix protein. We further examined interactions of MC41 with acrosomal proteases and zona proteins. A serine protease of 75 kDa was associated with MC41 under low-salt conditions, presumably forming a complex. Far Western blotting technique indicated that MC41 bound to both ZP2 and ZP2(f) in the presence of high-salt-soluble sperm proteins. In acrosome-reacting sperm, MC41 was present on the hybrid vesicles formed by the fusion of the plasma and outer acrosomal membranes. Presumably, MC41 has a significant role in secondary sperm-zona binding during the acrosomal reaction.     

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.     

SNAREs in mammalian sperm: possible implications for fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.     

Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian sperm

Previous data showed that complexin I, a SNARE regulatory protein, is localized in and/or around the acrosome and is necessary for the acrosome reaction in sperm. To understand how complexin I regulates the acrosome reaction, we used complexin-GST pulldown assays to identify interacting proteins. We showed that both complexins I and II bound mouse sperm dynamin 2. Dynamin 2 is a 100 kDa GTPase essential to many aspects of endocytosis but its potential role in exocytosis is unknown. Dynamin 2 is expressed in rat testis and widely expressed in other tissues; however, the function of dynamin 2 in germ cells is uncertain. Dynamin 2 protein was detected in mouse testis and was most abundant in or around the developing acrosome of spermatids. In addition, dynamin 2 was co-localized with complexin I in the acrosomal region of mammalian sperm. Its co-localization and interaction with complexin I suggest that dynamin 2 may play a role during acrosome formation and/or acrosomal exocytosis.     

Differential release of guinea pig sperm acrosomal components during exocytosis

The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.     

Effects of bovine oviductal proteins on bull spermatozoal function

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

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.     

The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.     

Redistribution of mouse sperm surface galactosyltransferase after the acrosome reaction

Gamete recognition in the mouse is mediated by galactosyltransferase (GalTase) on the sperm surface, which binds to its appropriate glycoside substrate in the egg zona pellucida (Lopez, L. C., E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur, 1985, J. Cell Biol., 101:1501-1510). GalTase has been localized by indirect immunofluorescence to the dorsal surface of the anterior sperm head overlying the intact acrosome. Sperm binding to the zona pellucida triggers induction of the acrosome reaction, an exocytotic event that results in vesiculation and release of the outer acrosomal and overlying plasma membranes. Consequently, we examined the fate of sperm surface GalTase after the acrosome reaction. Contrary to our expectations, surface GalTase is not lost during the acrosome reaction despite the loss of its membrane domain. Rather, double-label indirect immunofluorescence assays show that GalTase is redistributed to the lateral surface of the sperm, coincident with the acrosome reaction. This apparent redistribution of GalTase was confirmed by direct enzymatic assays, which show that 90% of sperm GalTase activity is retained during the acrosome reaction. No GalTase activity is detectable on plasma membrane vesicles released during the acrosome reaction. In contrast, removal of plasma membranes by nitrogen cavitation releases GalTase activity from the sperm surface, showing that GalTase redistribution requires a physiological acrosome reaction. The selective redistribution of GalTase to a new membrane domain from one that is lost during the acrosome reaction suggests that GalTase is repositioned for some additional function after initial sperm-zona binding.     

Capacitation and acrosome reaction in buffalo bull spermatozoa assessed by chlortetracycline and Pisum sativum agglutinin fluorescence assay

Studies on buffalo sperm capacitation have been limited because of the non-availability of a direct assay system. We describe two methods for detecting the acrosomal status of buffalo spermatozoa, namely chlortetracycline (CTC) fluorescence assay and Pisum sativum agglutinin (FITC-PSA) stain. We also test them under various treatment regimens and simultaneously standardize and calibrate them with transmission electron microscopy. An initial comparison of three physiological media, such as Krebs-Ringer bicarbonate buffer, Tyrode solution and Brackett & Oliphant medium (having different calcium concentrations and osmolality) used for studying the capacitation of buffalo spermatozoa and assessed by CTC, FITC-PSA, Giemsa stain and TEM, revealed Brackett & Oliphant medium to be marginally better than the other two media. When stained with chlortetracycline, three distinct fluorescent patterns were visible in buffalo spermatozoa under capacitating conditions. These were 'F' with fluorescence in the post acrosomal region characteristic of uncapacitated acrosome-intact cells; 'B' with fluorescence on the anterior portion of the sperm head and a dark band in the post-acrosomal region, characteristic of capacitated, acrosome intact cells and 'AR' with a fluorescent band on the posterior portion of the head, characteristic of acrosome-reacted cells. The FITC-PSA intensely labels the acrosomal region of acrosome intact buffalo sperm. Acrosome reacted sperms had diminished acrosomal labelling by both the probes used. Buffalo spermatozoa was not capacitated when calcium was either omitted from the medium or chelated with EGTA. In the presence of Ca2+ ionophore, A23187, 68% at 4 h and 85% at 8 h completed the acrosome reaction. Time course studies revealed a 4 h incubation period at 1.71 mM Ca2+ concentration to be necessary before transformation of 'F' to 'B' cells could take place. Spontaneous acrosome reaction induced at 6 and 8 h incubation of buffalo spermatozoa in KRB medium resulted in conversion of 'B' cells to 'AR' cells while 'F' cells remained unchanged. A simultaneous evaluation of acrosome intact and acrosome-reacted cells using FITC-PSA, Giemsa and TEM gave results similar to examination by CTC stain. Both the assays are rapid, reproducible, reliable and they detect an increase or decrease in physiological acrosome reactions. They thus can be used to study effects of calcium and prove to be good monitoring systems to identify buffalo sperm capacitation and acrosome reaction in individual buffalo bulls for fertility studies.