FINE STRUCTURE OF THE SPERMATOZOON OF HYDROIDES HEXAGONUS (ANNELIDA), WITH SPECIAL REFERENCE TO THE ACROSOMAL REGION

This paper describes in some detail the structure of the acrosomal region of the spermatozoon of Hydroides as a basis for subsequent papers which will deal with the structural changes which this region undergoes during fertilization. The material was osmium-fixed and mild centrifugation was used to aggregate the spermatozoa from collection to final embedding. The studies concern also the acrosomal regions of frozen-thawed sperm prepared by a method which previously had yielded extracts with egg membrane lytic activity. The plasma membrane closely envelops four readily recognizable regions of the spermatozoon: acrosomal, nuclear, mitochondrial, and flagellar. The acrosome consists of an acrosomal vesicle which is bounded by a single continuous membrane, and its periphery is distinguishable into inner, intermediate, and outer zones. The inner and intermediate zones form a pocket into which the narrowed apex of the nucleus intrudes. Granular material adjoins the inner surface of the acrosomal membrane, and this material is characteristically different for each zone. Centrally, the acrosomal vesicle is spanned by an acrosomal granule: its base is at the inner zone and its apex at the outer zone. The apex of the acrosomal granule flares out and touches the acrosomal membrane over a limited area. In this limited area the adjoining granular material of the outer zone is lacking. The acrosomal membrane of the inner zone is invaginated into about fifteen short tubules. The acrosomal membrane of the outer zone is closely surrounded by the plasma membrane. At the apex of the acrosomal region a small apical vesicle is sandwiched between the plasma membrane and the acrosomal membrane. Numerous frozen-thawed specimens and occasional specimens not so treated show acrosomal regions at the apex of which there is a well defined opening or orifice. Around the rim or lip of this orifice plasma and acrosomal membranes may even be fused into a continuum. The evidence indicates that the apical vesicle and the parts of the plasma and acrosomal membranes which surround it constitute a lid, and the rim of this lid constitutes a natural "fracture line" or rim of dehiscence. Should fracture occur, the lid would be removed and the acrosomal vesicle would be open to the exterior.     

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : I. The Acrosomal Region and Its Changes in Early Stages of Fertilization

Previous electron microscope studies of sperm-egg association in the annelid Hydroides revealed novel aspects with respect to the acrosomal region. To determine whether these aspects were unique, a comparable study was made of a species belonging to a widely separated phylum, Hemichordata. Osmium tetroxide-fixed polyspermic material of the enteropneust, Saccoglossus, was used. The acrosomal region includes the membrane-bounded acrosome, with its large acrosomal granule and shallow adnuclear invagination, and the periacrosomal material which surrounds the acrosome except at the apex; here, the acrosomal membrane lies very close to the enclosing sperm plasma membrane. After reaching the egg envelope, the spermatozoon is activated and undergoes a series of changes: the apex dehisces and around the resulting orifice the acrosomal and sperm plasma membranes form a continuous mosaic membrane. The acrosomal granule disappears. Within 7 seconds the invagination becomes the acrosomal tubule, spans the egg envelopes, and meets the egg plasma membrane. The rest of the acrosomal vesicle everts. The periacrosomal mass changes profoundly: part becomes a fibrous core (possibly equivalent to a perforatorium); part remains as a peripheral ring. The basic pattern of structure and sperm-egg association in Saccoglossus is the same as in Hydroides. Previous evidence from four other phyla as interpreted here also indicates conformity to this pattern. The major role of the acrosome is apparently to deliver the sperm plasma membrane to the egg plasma membrane.     

CHANGES IN THE SPERMATOZOON DURING FERTILIZATION IN HYDROIDES HEXAGONUS (ANNELIDA) : II. Incorporation with the Egg

This, the last of a series of three papers, deals with the final events which lead to the incorporation of the spermatozoon with the egg. The material used consisted of moderately polyspermic eggs of Hydroides hexagonus, osmium-fixed at various times up to five minutes after insemination. The first direct contact of sperm head with egg proper is by means of the acrosomal tubules. These deeply indent the egg plasma membrane, and consequently at the apex of the sperm head the surfaces of the two gametes become interdigitated. But at first the sperm and egg plasma membranes maintain their identity and a cross-section through the region of interdigitation shows these two membranes as a number of sets of two closely concentric rings. The egg plasma membrane rises to form a cone which starts to project into the hole which the spermatozoon earlier had produced in the vitelline membrane by means of lysis. But the cone does not literally engulf the sperm head. Instead, where they come into contact, sperm plasma membrane and egg plasma membrane fuse to form one continuous membranous sheet. At this juncture the two gametes have in effect become mutually incorporated and have formed a single fertilized cell with one continuous bounding membrane. At this time, at least, the membrane is a mosaic of mostly egg plasma membrane and a patch of sperm plasma membrane. The evidence indicates that the fusion of the two membranes results from vesiculation of the sperm and egg plasma membranes in the region at which they come to adjoin. Once this fusion of membranes is accomplished, the egg cytoplasm intrudes between the now common membrane and the internal sperm structures, such as the nucleus, and even extends into the flagellum; finally these sperm structures come to lie in the main body of the egg. The vesiculation suggested above appears possibly to resemble pinocytosis, with the difference that the vesicles are formed from the plasma membranes of two cells. At no time, however, is the sperm as a whole engulfed and brought to the interior of the egg within a large vesicle.     

ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA) : II. Zygote Formation by Gamete Membrane Fusion

An earlier paper showed that in Saccoglossus the acrosomal tubule makes contact with the egg plasma membrane. The present paper includes evidence that the sperm and egg plasma membranes fuse to establish the single continuous zygote membrane which, consequently, is a mosaic. Contrary to the general hypothesis of Tyler, pinocytosis or phagocytosis plays no role in zygote formation. Contact between the gametes is actually between two newly exposed surfaces: in the spermatozoon, the surface was formerly the interior of the acrosomal vesicle; in the egg, it was membrane previously covered by the egg envelopes. The concept that all the events of fertilization are mediated by a fertilizin-antifertilizin reaction seems an oversimplification of events actually observed: rather, the evidence indicates that a series of specific biochemical interactions probably would be involved. Gamete membrane fusion permits sperm periacrosomal material to meet the egg cytoplasm; if an activating substance exists in the spermatozoon it probably is periacrosomal rather than acrosomal in origin. The contents of the acrosome are expended in the process of delivering the sperm plasma membrane to the egg plasma membrane. After these membranes coalesce, the sperm nucleus and other internal sperm structures move into the egg cytoplasm.     

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)     

THE ACROSOME REACTION IN MYTILUS EDULIS : I. Fine Structure of the Intact Acrosome

The intact acrosome of the Mytilus edulis spermatozoon consists of a conical vesicle, the basal side of which is deeply invaginated so that the whole vesicle forms a sheath around a very slender axial rod, about 2.7 µ long, inserted in a tube passing through the nucleus. The annular base of the acrosomal vesical is filled with a homogeneous substance; the outer wall of the vesicle is lined with a somewhat irregular layer of a particulate substance interspersed with very fine tubular elements, and its lumen is nearly filled by a strand of material which extends from the inner tip of the invagination to the apex of the acrosome. The lumen of the invagination appears empty except for the rod and a delicate sleeve-like structure which surrounds it. The plasma membrane of the sperm cell lies in immediate contact with the acrosomal membrane over its whole outer surface. In its general organization, this molluscan acrosome shows a rather close homology with that of the annelid Hydroides.     

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.     

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.     

The acrosomal region of the spermatozoon of Ciona intestinalis: Its relationship with the binding to the vitelline coat of the egg

This paper describes a study of the apical region of the spermatozoon of Ciona intestinalis before and during its binding to the vitelline coat of the egg. A combination of the techniques of thin sectioning, negative staining, and freeze fracture has revealed that in the apical-most region, where a small acrosomal vesicle lies on the flat tip of the nucleus, there is a cap-like region almost completely free of particles on the P face of the plasma membrane. The particle-free area is surrounded by two circlets of orderly arranged particles. Upon binding to the vitelline coat the particles of the distal circlet show a partial displacement, while the particles of the apical circlet remain unaltered. The relationship between the apical circlet and the binding process is discussed. The final step of the acrosome reaction, which occurs in only a few of the bound spermatozoa, consists in the fusion of the plasma membrane with the acrosomal membrane, in the dehiscence of the acrosomal contents and finally in the formation of membrane tubules.     

THE ACROSOME REACTION IN MYTILUS EDULIS : II. Stages in the Reaction,Observed in Supernumerary and Calcium-Treated Spermatozoa

Suspensions of Mytilus edulis eggs were fixed with osmium tetroxide at various intervals between 1 and 10 seconds after heavy insemination, and sectioned for electron microscopy to follow the natural process of acrosome reaction in the spermatozoa around the eggs. Sperm suspensions were also fixed after the addition of 10 per cent by volume of M/3 calcium chloride. Within the first second after the acrosome is stimulated to react, an opening appears at its apex, around which the plasma and acrosomal membranes fuse to each other, and the resulting membrane complex is reflected backward, presumably by the swelling of material lining it. At the same time the other material within the now open vesicle disappears, and the rudiment of the acrosomal process, consisting of a short axial rod loosely surrounded by the invaginated part of the acrosomal membrane, is exposed at the anterior side of the sperm head. Within another second this rudiment is extended by elongation of the axial rod and expansion of the surrounding membrane. If the spermatozoon has reacted close to the egg surface, the elongation may be very slight, whereas in suspended spermatozoa the process may reach a length of 13 µ. Possible mechanisms underlying these changes are suggested.     

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.     

MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13).     

Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity: Scanning electron microscopy and time-lapse video microscopy during fertilization of the sea urchin Lytechinus variegatus

Sperm incorporation and the formation of the fertilization cone with its associated microvilli were investigated by scanning electron microscopy of eggs denuded of their vitelline layers with dithiothreitol or stripped of their elevating fertilization coats by physical methods. The activity of the elongating microvilli which appear to engulf the entering spermatozoon was recorded in living untreated eggs with time-lapse video microscopy. Following the acrosome reaction, the elongated acrosomal process connects the sperm head to the egg surface. About 15 microvilli adjacent to the attached sperm elongate at a rate of 2.6 μm/min and appear to engulf the sperm head, midpiece, and sperm tail. These elongate microvilli swell to form the fertilization cone (average height, 6.7 ± 2.0 μm) and are resorbed as the sperm tail enters the egg cytoplasm 10 min after insemination. Cytochalasin B, an inhibitor of microfilament motility, completely inhibits the observed egg plasma membrane surface activity in both control and denuded eggs. These results argue for a role of the microfilaments found in the egg cortex and microvilli as necessary for the engulfment of the sperm during incorporation and indicate that cytochalasin interferes with the fertilization process at this site.     

Fine structure of an elongated dorso-ventrally compressed echinoderm (holothuroidea) spermatozoon

The spermatozoon of Cucumaria pseudocurata is unique among those of the echinoderms in that it is tabloid in shape, i.e., elongated and dorsoventrally compressed. The sperm consists of a dorsal surface which contains an extensive striated rootlet-like structure located within a dorsal groove and a ventral surface which contains a medially situated acrosome. A single mitochondrion lies at the base of the nucleus. The flagellum is unusual in that a 9 + 3 tubular arrangement is observed in the mid-tail region. The acrosome consists of an acrosomal granule bounded by a limiting membrane and a surrounding periacrosomal layer. The granule is irregular in shape with the anterior-posterior surfaces flaring out, forming pockets in the periacrosomal material. The ventral granule surface bulges forming a close association with the plasma membrane. The dorsal surface is indented. Ventral to the depression (within the granule) is a small area containing a particulate-fibrous material. To the inside of the granule limiting membrane there is a second membrane-like structure (incomplete) which extends from the anterior-posterior surfaces around the dorsal face of the granule. Dorso-medial to the granule the periacrosomal layer contains a particulate-fibrous region lodged within the granule depression. This material is presumably the precursor of the acrosomal filament. Prominent cytoplasmic folds extend off from the basal flagellar region. The proximal and distal centrioles are situated perpendicular to one another within the mitochondrion. Centriolar satellite materials are associated with both centrioles. Toward the base of the tail the satellite of the distal centriole consists of nine radiating arms extending at an angle of 45° to the axis of the centriole. Each arm terminates in a dense thickening. The striated rootlet extends anteriorly from the distal centriole to just below the level of the acrosome.     

Ultrastructure of Sperm in <Emphasis Type="Italic">Mercenaria stimpsoni</Emphasis> and <Emphasis Type="Italic">Mactra chinensis</Emphasis> (Mollusca: Bivalvia) from the Sea of Japan

The ultrastructure of the sperm of the common bivalve species Mercenaria stimpsoni and Mactra chinensis from Peter the Great Bay is described. The sperm structure is typical for animals with external insemination. The sperm consists of a head, middle part, and flagellum. The sperm head of M. stimpsoni has a curved crescent form and includes the nucleus and acrosome; the head length is 9.8 μm. The acrosome is subdivided to the acrosome granule and the periacrosomal material. There are 4 mitochondria of about 0.8 μm in size in the middle part of the spermatozoon. The mitochondria surround the centriolar apparatus, which consists of proximal and distal centrioles located at a right angle. The axoneme originates from the distal centriole. The sperm of M. chinensis is barrel-shaped, with a head length of 3.2 μm. The acrosome is relatively larger, and its height is 1–1.2 μm. There are also 4 mitochondria 0.6–0.8 μm in the middle part of the spermatozoon. The sperm structure of the described species is typical of the families to which the mollusks belong, with insignificant variations.     

Ubiquitin signals in the developing acrosome during spermatogenesis of rat testis: an immunoelectron microscopic study.

The localization of ubiquitin (UB) signals in the acrosomes of rat spermiogenic cells was investigated by immunoelectron microscopy using two anti-UB antibodies: UB1, reacting with ubiquitinated proteins and free UB; and FK1, recognizing polyubiquitinated proteins but not monoubiquitinated proteins or free UB. Labeling of UB by UB1 (UB1 signal) was detected in the acrosomes at any stage of differentiation. In step 1 spermatids, UB1 signals were detected on the cytoplasmic surface and in the matrix of transport vesicles located between the trans-Golgi network and the acrosome. Weak signals were detected in acrosomal granules within acrosome vesicles that had not yet attached to the nucleus. In step 4-5 spermatids, the acrosome vesicles had enlarged and attached to the nucleus. Strong gold labeling was noted in a narrow space between the outer acrosomal membrane and the developing acrosomal granule, where a dense fibrous material was observed on routine electron microscopy, whereas the acrosomal granule was weakly stained by UB1 antibody. In step 6-8 spermatids, UB1 signals were detected in the fibrous material that expanded laterally to form a narrow electronless dense zone between the acrosomal granule and the outer acrosomal membrane. Labeling in the acrosomal granule increased. In step 9-11 spermatids, UB1 signals were confined to the narrow zone from the tip of the head to the periphery of the ventral fin. The matrix of the acrosome was weakly stained. In epididymal sperm, UB1 labeling in the acrosome decreased without any pretreatment, whereas staining was noted in a spot in the neck region and in the dorsal fin after trypsin digestion. On the other hand, the staining pattern with FK1 was quite different from that with UB1. The trans-Golgi network was weakly stained but the cis-Golgi network was strongly stained. The dense fibrous material just beneath the outer membrane was never stained with FK1. The results suggest that UB on the surface of transport vesicles is involved in anterograde transport from the Golgi apparatus to the acrosome. The physiological role of UB in acrosomes is not clear. Two candidates for monoubiquitinated proteins in the acrosome, which have a UB-interacting motif, were found by cyber screening.     

Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis.

The localization of proacrosin was determined by using colloidal gold labeling and electron microscopy of boar germ cells during spermiogenesis to post-ejaculation. Proacrosin was first localized in round spermatids during the Golgi phase of spermiogenesis; it was associated with the electron-dense granule, or acrosomal granule that was conspicuous within the acrosome. It remained within the acrosomal granule during the cap and acrosome phases of spermiogenesis. At these stages, there was no apparent association of the proacrosin molecule with the acrosomal membranes. During the maturation phase of spermiogenesis, proacrosin was seen to become dispersed into all regions of the acrosome except the equatorial segment. When sperm from different segments of the epididymis and ejaculated sperm were examined, localization was observed throughout the acrosome except for the equatorial segment. Here proacrosin appeared to be localized on both the inner and outer acrosomal membranes as well as with the acrosomal matrix, although further studies are required to verify the membrane localization. No labeling was seen on the plasma membrane. These data suggest that the synthesis and movement of proacrosin to sites in the acrosome are controlled by an as yet unknown process. The absence of proacrosin on the plasma membrane of mature ejaculated sperm makes it unlikely that this enzyme plays a role in sperm-zona adhesion prior to capacitation.     

Membrane differentiations in spermatozoa of the squid,Loligo pealeii

Regional differences in the structure of the plasma membrane and acrosome membrane of squid spermatozoa were studied by freeze-fracture and thin section electron microscopy. In regions of close apposition the plasma membrane and acrosome membrane are adjoined to one another by regularly spaced linkages. These linkage sites, overlie a set of fibers located at the inner face of the acrosomal membrane. The acrosomal fibers terminate in a layer of granular material located at the base of the acrosome. Detergent treatment of sperm releases the fibers and granular material as an interconnected complex. Freeze-fracture replicas reveal a random arrangement of intramembranous particles in the plasma membrane over the sperm head and linear aggregates of intramembranous particles in the acrosomal membrane. Several regional differences in the structure of the flagellar plasma membrane are present. The thickness of the glycocalyx is progressively reduced distally along the flagellum. Freeze-fracture replicas show evenly spaced linear arrays of intramembranous particles which extend parallel t o the flagellar long axis. Examination of spermatozoa extracted to disrupt flagellar geometry suggest that the dense fiber-doublet microtubule complexes are attached to the plasma membrane. The possible functional role of these membrane differentiations and their relationship t o membrane structures in mammalian spermatozoa are discussed.     

Fertilization in a crab: I. Early events in the ovary,and cytological aspects of the acrosome reaction and gamete contacts

Early events in fertilization were studied in Carcinus maenas by in vitro experiments and ultrastructural analysis; some were found to occur in the lumen of ripe ovaries. The acrosome reaction generally conformed to the usual Reptantia Decapoda pattern. However, a prominent membrane system continuous with the nuclear envelope and located close to the base of the acrosome tubule characterized the type of spermatozoon observed in Carcinus maenas. Such complex anatomical connections linking the three parts of the reacted spermatozoon (acrosome tubule, membrane system and nucleus envelope) may be significant in relation to the membrane system's contribution to the acrosome reaction. The outer layer of the everted acrosomal vesicle was found to comprise tubular elements ending in bell-shaped corpuscles, deeply interdigitated with the oolemma microvilli during the establishment of the initial contacts between the reacted spermatozoon and the egg plasma membrane. At the site of contact, the oolemma formed a minute fertilization cone, locally depressed by the acrosome tubule. During these early fertilization events, the nucleus, like the other spermatozoon components, was seen to penetrate the egg coatings first, and later to be located near the oolemma.