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.     

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.     

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.     

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.     

东方扁虾精子的超微结构

利用电镜研究了东方扁虾（Ｔｈｅｎｕｓ ｏｒｉｅｎｔａｌｉｓ）精子的形态和结构。精子由核、膜复合物区和顶体区３部分组成。核内含非浓缩的染色质、微管及细纤维丝,外被核膜;５～６条辐射臂自核部位伸出,臂内充满微管。膜复合物区位于核与顶体之间,由许多膜片层结构及其衍生的囊泡共同组成。顶体区由顶体囊和围顶体物质组成,顶体结构复杂,由顶体帽、内顶体物质和外顶体物质等构成;围顶体物质呈细颗粒状,主要分布于顶体囊     

ACROSOME DIFFERENTIATION IN THE SPERMATOGENESIS OF CIONA INTESTINALIS*

The process of acrosome formation in the course of spermatogenesis of Ciona intestinalis has been investigated. At the flute-beak-shaped tip of the head of the mature spermatozoon a small acrosomal vesicle(s) is described. The vesicles migrate to a region where the outer and inner nuclear membranes fuse thus giving rise to a “dense plate”. At the same time the chromatin begins to organize into longitudinally oriented strands which become attached to the inner side of the dense plate. The possible relationships between the dense plate, the formation of the acrosome and the orientation of the chromatin is discussed.     

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.     

锯缘青蟹精子发生的超微结构

采用透射电镜观察锯缘青蟹精子发生过程中超微结构的变化,结果表明：精原细胞椭圆形,染色质分布于核膜周围,胞质中具嵴少的线粒体,内质网小泡等。初级精母细胞染色质呈非浓缩状,胞质中具众 内质网小泡,特殊的膜系及晶格状结构。次级精母细胞核质间出现由内质小泡聚集成的腔。     

东方扁虾精子发生的超微结构

应用电镜技术研究了东方扁虾（Thenus orientalis)精子发生的全过程,精原细胞呈椭圆形,其染色质分布较均匀,线粒体集中于细胞一端形成“线粒体区”。初级精母细胞较大,染色质凝聚成块,次级精母细胞核质间常出现大的囊泡,胞质内囊泡丰富而线粒体数量却明显减少,早期精细胞核发生极化、解聚,部分胞质被抛弃。中期精细胞外观呈金字塔形,分为三区;正在形成的顶体位于塔顶,核位于塔基部,居间的细胞质基质内富含膜复合物,后期精细胞顶体进一步分化。形成顶体帽和内、外顶体物质等三个结构组份。成熟精子核呈盘状或碗状,具有5-6条内部充满微管的辐射臂。     

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.     

锯缘青蟹精子超微结构的研究

利用光镜和电镜观察了锯缘青蟹成熟精子的形态和超微结构。精子呈陀螺形，无鞭毛，在较宽的一端环生着１０余辐射臂。精子由球状的顶体、核杯以及核衍生的辐射臂三部分组成。顶体包括顶体管和顶体囊，后者包绕在顶体管的中央管周围，并可分为头帽带，内层和外层区。顶体被杯状的核包裹，仅头帽露于精子表面。成熟的精子中，位于核杯和顶体管之间的核膜出现局部断续或消失，中心粒和一些胞器出现的核杯腔中。     

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).     

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.     

MORPHOLOGICAL OBSERVATIONS ON THE SPERMATOZOA OF ECHINOTHURID SEA URCHINS*

The morphology of the spermatozoa of three species of echinothurid sea urchins, Asthenosoma ijimai, Araeosoma owstoni, Hapalosoma gemmiferum, was investigated by means of transmission and scanning electron microscopy. The spermatozoa of these three species of echinothurid sea urchins have similar fine structure, but they differ in several features from the more familiar regular sea urchins. 1) The external anatomy of the head region of the echinothurid spermatozoon is diagnostic in that it has a highly elongated head. 2) The spermatozoon of echinothurid sea urchins has a very long slender nucleus, protruding on its proximal end, so that the shape of the nucleus resembles a sperhead. 3) The acrosomal granule in the acrosomal vesicle of the echinothurid spermatozoon is not a mass of homogenous particulate material but an electron opaque rod condensed in the central part of the acrosomal vesicle. Scanning electron microscopic examination revealed that echinothurid spermatozoa form acrosomal processes similar to those of other regular sea urchins. 4) The basal body is situated just beneath the middle of the posterior protrusion of the nucleus. The distal centriole is located beside the basal body almost in contact with it. The axis of the distal centriole is almost but not quite parallel to that of the basal body. A satellite complex can be recognized around the posterior part of the proximal centriole.     

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)     

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.     

SPERMIOGENESIS IN THE PULMONATE SNAIL, EUHADRA HICKONIS 1. ACROSOME FORMATION

Electron microscopical observations of the course of acrosomal differentiation in Euhadra hickonis show that the vesicular component of the mature acrosome is produced by early Golgi activity, whereas an equivalent amount of material that forms a basal component is added later to the outside of the vesicle. It is also suggested that similar material which concurrently accumulates against part of the outer surface of the nuclear envelope is finally incorporated into the basal part of the acrosome.
In the early spermatid, which has a highly polymorphic nucleus, material derived from the well-developed Golgi complex accumulates within a network of tubules in its central maturing zone to form a single acrosomal vesicle ca. 150 nm in diameter. The next stage is characterized by the strikingly spherical shape of the nucleus, as well as by the addition of electron-dense material to the outside of the nuclear envelope over the future anterior surface, and to its inside in the posterior region where the centriolar fossa will form.
At mid-spermiogenesis the Golgi complex moves posteriorly away from the acrosomal vesicle, which remains in the anterior cytoplasm. A growing mass of densely filamentous material forms a hollowed hemisphere around one side of the vesicle. This complex approaches the coated anterior part of the nuclear envelope, turning if necessary so that the filamentous material is in the lead, and the latter merges with the electron-dense material at the center of the coated area. As the late spermatid nucleus elongates, this material passes through a series of changes in arrangement and electron density, finally forming a homogeneously particulate element of medium density that surrounds the proximal half of the acrosomal vesicle and caps the slender tip of the nucleus in the mature spermatozoon.     

Fine Structural Changes in the Acrosome Reaction of the Japanese Abalone, Haliotis disus

The spermatozoon of the Japanese abalone, Haliotis discus , and its structural changes during the acrosome reaction were observed by electron microscopy. The spermatozoon has a huge acrosome in the shape of a hanging bell or a forefinger with a deep fossa at the posterior end being filled with a bundle of microfilaments. The membranes of the acrosomal apex, the so-called trigger region, are structurally discernible from those of other acrosomal regions. Following the trigger region, a unique structure under the acrosomal membrane covers the surface of the acrosomal content in the form of a truncated cone.
The acrosome reaction occurs in the jelly layer very close to the egg envelope. First, the membranes at the apex of the acrosome are vesiculated, followed by the formation of a narrow gap between the outer acrosomal membrane and the acrosomal content. Next, the bundle of micro-filaments elongates, running through the center of the acrosome, reaching the trigger region and protruding out of the acrosomal top. Then release of the acrosomal content occurs in two steps, disclosing the "membrane undercoating structure" that comprises globular particles with a fuzzy material connecting them. This resembles the undercoat network found in erythrocytes.     

Spermatogenic ultrastructure in the anomalodesmatan bivalve Myochama anomioides (Mollusca: Myochamidae) – does the nucleus help position the ‘temporary’ acrosome?

Spermatogenic ultrastructure in the marine bivalve mollusc Myochama anomioides (Myochamidae) is described and contrasted with other bivalves, especially other euheterodonts. Small (0.1 μm diameter), primary proacrosomal vesicles produced in spermatocytes give rise to much larger (0.4 μm diameter) secondary proacrosomal vesicles in early spermatids, which in turn form the dished‐shaped, definitive acrosomal vesicle (diameter 1.0 μm) of later spermatids. The acrosomal vesicle acquires a deposit of subacrosomal material and comes to lie close to or in contact with the plasma membrane. The acrosomal complex (acrosomal vesicle + subacrosomal material) initially positions itself at the apex of the condensing, fibrous nucleus (the so‐called temporary acrosome position), but subsequently begins to move posteriorly. The condensing nucleus becomes markedly folded so that its apex is posteriorly orientated towards the migrating acrosomal complex and the midpiece (mitochondria and centrioles). The close spatial relationship of nuclear apex to acrosomal complex during this folding strongly suggests that acrosomal migration in M. anomioides is assisted, at least in part, by movement of the late spermatid nucleus. Similar nuclear folding has previously been demonstrated in an early stage of fertilization in another anomalodesmatan (Laternula limicola) raising the possibility that one event might be a reversal of the other.     

尼罗罗非鱼精子形成中核内囊泡的释放

通过透射电镜观察了尼罗罗非鱼的精子形成过程。尼罗罗非鱼精子细胞在成熟过程中,细胞核中出现由双层生物膜构成的囊泡。囊泡中均匀分布着电子密度低的物质。该囊泡逐渐从细胞核内排到细胞核外。在此过程中细胞核不但排出不参与染色质浓缩的物质,还将多余的核膜排出。进入袖套的囊泡可以留在精子的袖套中,而排到核前方和核侧面的囊泡继续以出芽的方式排出精子细胞。尼罗罗非鱼成熟精子的头部仅有染色质高度浓缩的细胞核。细胞核前