Acrosomal reaction and early events at fertilization in Marthasterias glacialis (Echinodermata: Asteroidea)

When the spermatozoon of M glacialis contacts the mature oocyte jelly it adheres to it. Following this, there is a slight tumefaction of the acrosome, which is followed by the disruption of the apical acrosomal vesicle and cytoplasmic membranes. Acrosomal vesicle contents are liberated and spread along the outer surface of the oocyte jelly. Meanwhile, the acrosomal process begins to extend, penetrates all the jelly extension, then the vitelline layer, and finally contacts the cytoplasmic egg membrane. Nevertheless, the sperm cell continues lying at the outer border of the jelly. From the beginning of the acrosome reaction the dense and finely fibrillar subacrosomal material is connected, by some expansions, to the basal acrosomal vesicle membrane. Both nuclear and mitochondrial diameters have diminished.     

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.     

Protease-Induced Formation of the Sperm Acrosomal Filament

Filament extension during the sperm acrosome reaction in Sicyonia ingentis is triggered by an egg trypsin-like protease whose action can be mimicked using trypsin. Using biotinylated trypsin and either a fluorescently-labeled or colloidal gold-labeled antibody to biotin, trypsin binding was localized to the anterior granule of the sperm which is exposed upon acrosomal exocytosis. The binding was to proteinaceous material at the base of the granule juxtaposed to the inner acrosomal membrane. Other labeled proteins also bound in the same pattern but only in the presence of unlabeled trypsin; non-proteolytic proteins did not induce filament formation. Binding of all proteins tested occurred slowly over a period of about 30 min. A minimum of 30 min of trypsin exposure was required in order to trigger filament formation, and increasing trypsin concentration did not reduce this time requirement. These results indicate that the protease slowly uncovers a binding site for itself (or other proteins), and then its proteolytic activity is again required to induce filament formation. The protease kallikrein appeared to be a more potent inducer than trypsin, while thrombin and clostripain had no apparent inducing activity.     

Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy

Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope. The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (20 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) suspended in sea water containing 50 mM excess CaCl2. Before activation of the sperm, the acrosomal region appears as a 1.1-microM diameter sphere, slightly less dense than the rest of the sperm head. Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After approximately 1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material. In this paper, we also consider the meaning of the enhanced DIC image seen in the high-resolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope.     

Acrosomal ATPase in starfish and bivalve mollusk spermatozoa

ATPase activity was found in acrosomes of starfish and bivalve mollusk spermatozoa, using a cytochemical method with electron microscopy. The activity was located in central material of the starfish acrosome and in material lining the acrosomal membrane of the Mytilus acrosome, as well as in the basal part of the starfish acrosome. The ATPase activity in the former material was preferably activated by Ca²⁺, while that in the starfish basal material was preferably activated by Mg²⁺. Both types of activity persisted during and after the acrosome reaction. ATPase activity was also observed in the region of the axial filament complex of the flagella, in centrioles and in a basal matrix. ATPase in the acrosome also hydrolysed other nucleoside triphosphates. However, there was no detectable phosphatase activity, and little pyrophosphatase or 5′-nucleotidase activity. Evidence was obtained that adenylate kinase may be included in the acrosome. A possible role of the ATPase activity in the acrosome reaction is discussed.     

Acrosomal exocytosis, a special type of regulated secretion

The acrosome is a single secretory granule present in the head of mammalian--and other animal groups--sperm. Secretion of this granule is an absolute requirement for physiological fertilization. Acrosome exocytosis is a synchronized and tightly regulated all-or-nothing process, with no recycling of membranes. In the last few years, it has been shown that acrosomal exocytosis is mediated by a molecular mechanism that is homologous to that reported in the secretion of neuroendocrinal cells. Moreover, because of its particular characteristics, acrosomal exocytosis is a unique mammalian model for the study of the different steps of the membrane fusion cascade. Combining results in intact and permeabilized sperm, the following sequence of events has been proposed. In resting sperm, SNARE proteins are locked in inactive cis complexes. Sperm activation causes a calcium increase in the cytoplasm that promotes the production of cAMP and activates Rab3A. Afterwards, NSF and alphaSNAP disassemble cis complexes and the free SNAREs are then able to reassemble in loose trans complexes. Membrane fusion is arrested at this stage until calcium is released from inside the acrosome by inositol 1,4,5-trisphosphate-sensitive calcium channels to trigger the final steps of membrane fusion, which require fully assembled trans SNARE complexes and the calcium sensor synaptotagmin. This working model is still incomplete and tentative. Its improvement will be important to share light on this and other processes of regulated exocytosis. Moreover, it will bring new perspectives into the field of sperm-related fertility and sterility.     

Localization of Acrosomal Proteinase after Vesculation of the Acrosome

The freshly ovulated egg is covered by the zona pellucida and follicular-cell layer. The former is reported to dissolved by acrosomal proteinase and the latter may be dispersed by hyaluronidase. If these two enzymes have different targets, two possibilities can be taken into consideration concerning the timing of release of the two enzymes from the acrosome.
Since hyaluronidase is considered to be released immediately after the acrosomal reaction at the follicular-cell layer, this work was undertaken to examine the timing of release of acrosomal proteinase after vesiculation of the acrosome, by demonstrating the site of the proteinase histochemically. The results indicated that the acrosomal proteinase disappeared immediately after vesiculation of the acrosome. This result suggests that spermatozoa that show vesiculation of the acrosome in the follicular-cell layer probably lose acrosomal proteinase in this layer. Thus the spermatozoon probably does not attack the follicular-cell layer and zona pellucida, one by one by release sequential of hyaluronidase and acrosomal proteinase.     

Acrosomal actin: twists and turns of a versatile filament

A new electron cryomicroscopic reconstruction of an actin-scruin bundle from Limulus sperm reveals details about the enormous structural plasticity within F-actin. The twist and tilt of the actin subunits show very large deviations from ideal F-actin, providing clues about actin dynamics.     

Acrosomal status in fresh and capacitated human ejaculated sperm

The acrosomal status of human sperm was evaluated by immunofluorescence utilizing a specific monoclonal antibody that recognizes target antigen(s) localized in the acrosomal cap region. Spontaneous acrosomal loss was first examined in sperm preparations used for successful in vitro fertilization of human eggs. In these sperm populations, less than 20% of the sperm underwent degenerative or spontaneous acrosomal loss following 24 h of incubation. The correlation of acrosomal loss with changes in motility and viability suggested that sperm senescence was not necessarily coupled to acrosomal loss. Chemical induction of acrosomal loss by calcium ionophore A23187 and lysophosphatidylcholine (LPC) was characterized. Maximal ionophore induction (10 microM A23187 in media containing calcium) was observed in cells exposed to capacitating conditions in vitro; sperm exposed to noncapacitating conditions did not readily acquire the ability to respond to ionophore. The reaction induced by ionophore was slow (60 min), and at least 30% of the cells were always resistant to induction. In contrast, LPC induced rapid, synchronous acrosomal loss in either freshly ejaculated or capacitated sperm in the presence or absence of extracellular calcium, suggesting that this loss was not a physiologic reaction. These studies may provide a basis for evaluating capacitation and ultimately fertility potential in the human male.     

Calcineurin-mediated Dephosphorylation of Synaptotagmin VI Is Necessary for Acrosomal Exocytosis

Regulated secretion is a fundamental process underlying the function of many cell types. In particular, acrosomal exocytosis in mammalian sperm is essential for egg fertilization. In general, exocytosis is initiated by a cytosolic calcium increase. In this report we show that calcium affects several factors during human sperm acrosomal exocytosis. By using an antibody that specifically recognizes synaptotagmin VI phosphorylated at the polybasic region of the C2B domain, we showed that a calcium-dependent dephosphorylation of this protein occurred at early stages of the acrosomal exocytosis in streptolysin O-permeabilized sperm. We identified the phosphatase as calcineurin and showed that the activity of this enzyme is absolutely required during the early steps of the secretory process. When added to sperm, an inhibitor-insensitive, catalytically active domain of calcineurin was able to rescue the effect of the specific calcineurin inhibitor cyclosporin A. This same domain dephosphorylated recombinant synaptotagmin VI C2B domain, validating this protein as a new substrate for calcineurin. When sperm were treated with catalytically active calcineurin before stimulation, exocytosis was inhibited, an effect that was rescued by the phosphomimetic synaptotagmin VI C2B-T418E,T419E mutant domain. These observations indicate that synaptotagmin must be dephosphorylated at a specific window of time and suggest that phosphorylated synaptotagmin has an active role at early stages of the acrosomal exocytosis.     

Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies

An important question in mammalian gamete physiology concerns how capacitation and the occurrence of acrosome reactions in motile sperm relate to fertility. Evaluation of these relationships has been restricted by practical limitations because rapid, quantitative assays are unavailable. We have developed a rapid, reproducible assay for the evaluation of acrosomal status utilizing monoclonal antibodies specific to antigens localized in the acrosomal cap region of the sperm head. Mice were immunized with human ejaculated sperm preparations and the resultant hybridomas producing antisperm antibody were selected by solid-phase radioimmunoassay and indirect immunofluorescence (IIF). Two monoclonal antibodies (HS-19, HS-21) recognized target antigens restricted to the acrosomal cap by IIF, and 87 +/- 8.5% of the sperm in fresh ejaculates from 10 different sperm donors showed positive cap fluorescence with these reagents. Loss of HS-21 binding as measured by IIF was correlated with disappearance of the acrosomal cap as observed directly by transmission electron microscopy. Acrosomal disappearance, artificially induced in vitro using the calcium ionophore A23187, also resulted in a loss of HS-21 binding. The induction of acrosomal loss by ionophore was dependent upon extracellular calcium. The data presented suggest that specific monoclonal antibodies can be used for the rapid evaluation of acrosomal status in mammalian sperm.     

Acrosomal reaction of the Thyone sperm. III. The relationship between actin assembly and water influx during the extension of the acrosomal process

In an attempt to investigate the role of water influx in the extension of the acrosomal process of Thyone sperm, we induced the acrosomal reaction in sea water whose osmolarity varied from 50 to 150% of that of sea water. (a) Video sequences of the elongation of the acrosomal processes were made; plots of the length of the acrosomal process as a function of (time)1/2 produced a straight line except at the beginning of elongation and at the end in both hypotonic and hypertonic sea water (up to 1.33 times the osmolarity of sea water), although the rate of elongation was fastest in hypotonic sea water and was progressively slower as the tonicity was raised. (b) Close examination of the video sequences revealed that regardless of the tonicity of the sea water, the morphology of the acrosomal processes were similar. (c) From thin sections of fixed sperm, the amount of actin polymerization that takes place is roughly coupled to the length of the acrosomal process formed so that sperm with short processes only polymerize a portion of the actin that must be present in those sperm. From these facts we conclude that the influx of water and the release of actin monomers from their storage form in the profilactin (so that these monomers can polymerize) are coupled. The exact role of water influx, why it occurs, and whether it could contribute to the extension of the acrosomal process by a hydrostatic pressure mechanism is discussed.     

十足目甲壳动物精子发生过程顶体形成和细胞核变化

十足目甲壳动物精子发生对于细胞学家是一个长期有趣的主体,早期对十足目甲壳动物精子发生的研究结果,往往把无鞭毛的精子细胞器与可运动精子的归为同类.McCroan使用孚尔根(Feulgen)染色方法观察绿螯虾(Cambarus viridis)的精子发生,首次发现在其辐射臂观察到有核物质[1].     

Acrosomal enzymes of human spermatozoa before and after in vitro capacitation

The effect of in vitro capacitation (events that occur before the acrosome reaction) on the acrosomal enzymes of human spermatozoa was determined. Capacitation of human spermatozoa was assessed by their ability to penetrate denuded hamster oocytes. The activities of a number of enzymes commonly associated with the sperm acrosome, including nonzymogen acrosin, proacrosin, inhibitor-bound acrosin, hyaluronidase, acid phosphatase, beta-glucuronidase, beta-glucosidase, beta-N-acetylglucosaminidase, beta-galactosidase and beta-N-acetylgalactosaminidase were assessed. With the exception of acid phosphatase, no alteration in enzyme activity occurred after 4 h of incubating the spermatozoa under capacitation conditions although gamete fusion took place. The acid phosphatase levels decreased twofold, presumably due to the loss of seminal (prostatic acid phosphatase that loosely adheres to spermatozoa. After 8 h of capacitation, a large decrease in sperm enzyme levels took place only in the case of hyaluronidase, although small decreases were also noted in total acrosin, proacrosin and inhibited acrosin. No new electrophoretically migrating forms of acrosin were observed. Decreases in total acrosin and proacrosin, but not in inhibited acrosin, also occurred when spermatozoa were incubated under noncapacitating conditions for 8 h, indicating that capacitation may specifically cause the release of some acrosin inhibitor from human spermatozoa. It is concluded that, with the possible exception of hyaluronidase, the in vitro capacitation of human spermatozoa does not cause a major change in its acrosomal enzyme content so that these hydrolases are fully present before the acrosome reaction takes place during gamete fusion. Serum albumin appears to protect against the loss of some of these enzymes since the activity of several glycosidases was significantly reduced when the spermatozoa were incubated for 8 h in human serum albumin-free medium.     

Isolation of Acrosomal Vesicles and Their Surrounding Membranes from Starfish Sperm*

Intact acrosomal vesicles and their surrounding membranes were isolated from starfish sperm. The identification of the acrosomal vesicle and confirmation of its purification away from other sperm organelles was made by electron microscopy and SDS-polyacrylamide gel electrophoresis.