Acrosome reaction in sperm of the toad,bufo bufo japonicus

The acrosome in the sperm of the toad, Bufo bufo japonicus, consists of a membrane-limited acrosomal cap and a fibrous perforatorium. When sperm are incubated with the oviducal pars recta extract (PRE) for 30–60 min, the outer acrosomal membrane fuses with the overlying plasma membrane at several points with concomitant loss of the contents of the acrosomal cap. The inner acrosomal membrane thus exposed fuses with the plasma membrane at the caudal end of the acrosomal region. This PRE-induced acrosome reaction is completely inhibited by soybean trypsin inhibitor. Sperm found in the innermost jelly layer of inseminated eggs possess an intact acrosome, but those either passing through the vitelline coat or localizing in the perivitelline space are acrosome-reacted in the same manner as when treated with PRE. These observations, combined with recent evidence showing involvement of the pars recta substance in fertilization, indicate that the acrosome reaction occurring in a fertilizing sperm at or near the surface of the vitelline coat is a response to a substance that is derived from the pars recta and deposited in the vitelline coat.     

Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze-fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus , and its changes during the acrosome reaction were studied by the freeze-fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and 'truncated cone' regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle-free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.     

Detection of acrosome-reacted toad sperm based on specific lectin binding to the inner acrosomal membrane

When the sperm of the toad Bufo japonicus were treated with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA), soybean agglutinin (SBA), or Dolichos biflorus agglutinin (DBA), a few sperm fluoresced at the acrosomal region. The number of sperm showing this lectin binding to the acrosome increased significantly upon mild sonication of the sperm suspension. Electron microscopy revealed that ferritin-conjugated PNA bind not to the outer acrosomal and overlying plasma membranes, but specifically to the surface of the inner acrosomal membrane exposed by sonication. Both the percentage of FITC-PNA-labeled sperm and the activity of vitelline coat lysin released by sperm increased in good correlation with increasing sonication time, although the PNA-labeled sperm decreased in number upon longer sonication. These results indicate that the binding of FITC-PNA to the sperm provides a reliable measure of the acrosome reaction of Bufo sperm.     

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : I. Passage of the Acrosomal Region through the Vitelline Membrane

In the previous paper the structure of the acrosomal region of the spermatozoon was described. The present paper describes the changes which this region undergoes during passage through the vitelline membrane. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed usually 9 seconds after insemination. There are essentially four major changes in the acrosome during passage of the sperm head through the vitelline membrane. First, the acrosome breaks open apically by a kind of dehiscence which results in the formation of a well defined orifice. Around the lips of the orifice the edges of the plasma and acrosomal membranes are then found to be fused to form a continuous membranous sheet. Second, the walls of the acrosomal vesicle are completely everted, and this appears to be the means by which the apex of the sperm head is moved through the vitelline membrane. The lip of the orifice comes to lie deeper and deeper within the vitelline membrane. At the same time the lip itself is made up of constantly changing material as first the material of the outer zone and then that of the intermediate zone everts. One is reminded of the lip of an amphibian blastopore, which during gastrulation maintains its morphological identity as a lip but is nevertheless made up of constantly changing cells, with constantly changing outline and even constantly changing position. Third, the large acrosomal granule rapidly disappears. This disappearance is closely correlated with a corresponding disappearance of a part of the principal material of the vitelline membrane from before it, and the suggestion is made that the acrosomal granule is the source of the lysin which dissolves this part of the vitelline membrane. Fourth, in the inner zone the fifteen or so short tubular invaginations of the acrosomal membrane, present in the normal unreacted spermatozoon, lengthen considerably to become a tuft of acrosomal tubules. These tubules are the first structures of the advancing sperm head to touch the plasma membrane of the egg. It is notable that the surface of the acrosomal tubules which once faced into the closed acrosomal cavity becomes the first part of the sperm plasma membrane to meet the plasma membrane of the egg. The acrosomal tubules of Hydroides, which arise simply by lengthening of already existing shorter tubules, are considered to represent the acrosome filaments of other species.     

Purification and immunocytochemical localization of the vitelline coat lysin of abalone spermatozoa

Spermatozoa of the abalone Haliotis discus were treated with high-calcium seawater to induce the acrosome reaction. The soluble components released from the sperm acrosomal vesicles showed potent lytic activity on the egg vitelline coat. A vitelline coat lysin was purified by salting-in, preparative polyacrylamide gel electrophoresis, and high-performance liquid chromatography. Its molecular weight was 15,500 and its isoelectric point 9.6. These properties were similar to those of other molluskan vitelline coat lysins. The lysin was immunocytochemically localized using a protein A-gold technique, in the posterior half of the acrosomal vesicle.     

The passage of spermatozoa through the vitelline membrane in the domestic fowl,Gallus gallus

Summary The developing outer layer of the vitelline membrane of the ovum in the posterior part of the infundibulum of the domestic fowl contains many spermatozoa in nearly parallel orientation with its inner layer. When the acrosomal region of a spermatozoon approaches or contacts the inner layer, promptly undergoes the acrosome reaction. The outer acrosomal membrane and overlying plasma membrane fuse together and the apical region of the acrosome opens, so that the acrosomal contents are released. Meanwhile the spermatozoon remains a time in contact with the surface of the inner layer, and the network of the inner layer just under the tip of the sperm head begins to be dissolved. This dissolution extends downward forming a tunnel, approximately 9 m in diameter. The spermatozoon then passes through the inner layer obliquely via the central region of the tunnel and arrives at the perivitelline space.The authors are greatly indebted to assoc. prof. Dr. Osamu Koga for his valuable advices. The authors also wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices. This investigation was supported by a grant from the Ministry of Education of Japan (156185)     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

An ultrastructural analysis of the gametes and early fertilization in two bivalve molluscs,Chama macerophylla and Spisula solidissima with special reference to gamete binding

Summary An ultrastructural investigation of the gametes and their interaction during the early events of fertilization in molluscs has been performed. A gamete binding event involving large numbers of sperm has been identified and examined in detail. The surface of the oocyte is projected into numerous microvilli which extend through the vitelline envelope. Tufts of fibrillar material radiate from the tips of these microvilli, forming a layer external to the vitelline envelope. The acrosomal vesicle of the mature spermatozoon contains two major components, which function differently during fertilization. The vesicle is indented at its adnuclear surface, constituting a preformed acrosomal tubule. This tubule does not elongate during the acrosome reaction. Completion of the reaction results in the formation of an extracellular coat, derived from one component of the acrosomal vesicle, on the anterior surface of the sperm. Sperm-egg binding is accomplished by an association of the extracellular coat on the reacted sperm and the fibrous tufts on the tips of the microvilli of the oocyte. Evidence that gamete membrane fusion occurs by fusion of the acrosomal tubule and a microvillus is presented. These observations provide a generalized pattern of molluscan fertilization.The assistance of Mr. B. Calloway in identifying and obtaining the organisms is gratefully acknowledged. This investigation was supported by NSF grants PCM 76-13459 and PCM 76-09654 and performed at the Bermuda Biological Station with instruments made available through the courtesy of Philips, Inc., DuPont-Sorvall, and L.K.B. Inc. Bermuda Biological Station Contribution No. 709     

THE SURFACE EVENTS AT FERTILIZATION OF THE SEA URCHIN EGG I. EVENTS ON THE SURFACE OF THE VITELLINE COAT1

Sperm-egg interaction during normal fertilization in the sea urchins, Strongylocentrotus intermedius and Hemicentrotus pulcherrimus, was studied by scanning and transmission electron microscopy. Several seconds after insemination, acrosome-reacted spermatozoa were found attached to the surface of the vitelline coat on each egg. Soon, several bulges of the vitelline coat appeared surrounding the fertilizing spermatozoon. These bulges then spread over the surface increasing in number, while they became fewer and disappeared around the sperm head. Thin sections of the bulging areas revealed discharging cortical granules. As the bulging vitelline coat was elevated, the sperm head was incorporated into the perivitelline space, passing through a small hole in the coat that resulted from penetration of the sperm acrosomal process immediately before fusion of the gametes. When the spermatozoon disappeared beneath the fertilization membrane, a hole was left in the membrane and the cortical reaction had finished on the other hemispheric surface. Mechanical removal of the membrane at that time exposed a spermatozoon protruding perpendicularly from the egg plasma membrane surface. The anterior tip of the sperm head was smoothly connected with the egg surface, and neither microvillous projections nor cytoplasmic covering of the egg cytoplasm could be found around the spermatozoon.     

Sialyl glycoprotein distribution on the plasma membrane of ejaculated ram spermatozoa

Localization of sialyl residues on unfixed ejaculated ram sperm membrane using the direct covalent probes of either ferritin hydrazide or latex hydrazide revealed a unique regional distribution on the plasmalemma covering the sperm head only. Three different labelling zones were identified based on the intensity and the nature of the sialyl glycoconjugates: a patchy-like zone which included the plasma membrane overlaying the post-nuclear cap and the convex side of the apical body of the acrosome; highly ordered heavily labelled zones including the plasmalemma adjacent to the concave apical body of the acrosome and to the posterior part of the equatorial acrosomal segment; a paucity-labelling zone which included the plasma membrane underlying the principal acrosomal region and the anterior part of the equatorial acrosomal segment. The possible physiological role of the highly ordered labelled zones is discussed.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

Topographical differences in the distribution of surface coat components and intramembrane particles. A cytochemical and freeze- fracture study in culture forms of Trypanosoma cruzi

A regional specialization of the cell surface of T. cruzi culture forms was found at the cytostome as a localized thick surface coat rich in carbohydrate-containing components. The prominent surface coat was located over a region of the plasma membrane where intramembranous particles were exceedingly low in number. In turn, the particle-poor region was related to specialized submembrane fibrils not present under other regions of the plasma membrane. The cystostome region provides a striking example of a stable regional differentiation of the plasma membrane, involving the outer surface, the membrane interior, and the underlying cytoplasm. In addition, independence of Con A receptors, colloidal iron binding sites, and ruthenium red-stainable surface components from membrane particles was demonstrated at the flagellar membrane.     

Glycosidases,proteases and ascidian fertilization

Spermatozoa should bind to and then penetrate the vitelline coat for fertilization in ascidians and many other animals. There is substantial evidence that the binding of ascidian sperm is mediated by a sperm glycosidase and complementary saccharide chains of glycoproteins in the vitelline coat. Involvement of a sperm proteasome in the binding is also suggested. For the penetration, sperm proteases such as chymotrypsin-like enzyme, acrosin, spermosin and proteasome are suggested to play essential roles. Sperm glycosidase, that is translocated from the tip of sperm head to the surface overlying the mitochondrion, anchors the mitochondrion at the outer surface of vitelline coat. Therefore it assists sperm to penetrate the vitelline coat and traverse the perivitelline space. For fusion with egg plasma membrane, sperm metalloendoprotease seems to be involved. Egg glycosidases and proteases serve for some steps after fertilization, such as the prevention of polyspermy, expansion of perivitelline space and regulation of cell cycle.     

Localization of zona pellucida binding sites on rabbit spermatozoa and induction of the acrosome reaction by solubilized zonae

The binding of mammalian spermatozoa to the egg's extracellular coat, the zona pellucida, is a complex process which culminates in species-specific penetration of the sperm to the egg plasma membrane. To investigate where on the spermatozoon's surface the zona binding sites are located, whole rabbit zonae were labeled with FITC, heat solubilized and used to observe the surface binding patterns on live spermatozoa. Before the acrosome reaction the zona binding sites are located either over the entire head as well as the middle piece or alternatively in patches along the apical ridge of the head. After the acrosome reaction there is a 29% loss of fluorescence and the zona binding sites are present in the posterior aspect of the acrosomal region, the anterior postacrosomal region and the middle piece. These results demonstrate the presence of zona binding sites after the acrosome reaction which would account for the sperm's ability to remain bound to the zona after the acrosome reaction. Further, we report for the first time that solubilized rabbit zonae pellucidae will induce the acrosome reaction in in vitro capacitated rabbit sperm whereas solubilized pig zonae pellucidae will not. Since rabbit sperm bind pig zonae, the induction and specificity of the physiological acrosome reaction must reside in the affinity of the binding rather than the binding itself.     

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.     

小鼠精子表面SBA结合糖复合物的形成与变化

用ＨＲＰ标记的大豆凝集素（ＳＢＡ）对睾丸与附睾切片,以及取自附睾和子宫（交配后）内的精子进行了标记,旨在认识精子在发生、成熟和获能过程中表面糖复合物的形成与变化规律。在睾丸内,精母细胞和早期精子细胞胞质内有一强阳性颗粒,处于精子形成期的精子细胞呈弱阳性标记。附睾管内的精子团呈强阳性,附睾管上皮则仅在游离缘呈弱阳性。交配后１．５小时自子宫内洗出的精子,其顶体区的标记增至强阳性,但随着在子宫内存留时间的延长,标记强度逐渐减弱或消失。结果表明,１）精子表面的ＳＢＡ结合糖复合物出现于精子形成期;２）在成熟和获能过程中精子表面的ＳＢＡ结合糖复合物发生明显的变化     

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.