Sperm surface changes during the acrosome reaction as observed by freeze-fracture

The mammalian acrosome reaction is an exocytotic process that can be analyzed by the technique of freeze-fracture; only sperm cells capacitated in vitro or treated to elicit the acrosome reaction in vitro have been studied, and all pictures published are from material fixed before freezing. All the authors point out the appearance of particle-free areas in the plasma membrane of the acrosomal region during capacitation and before any fusion. This is interpreted as an increase in membrane fluidity as suggested by studies on membrane lipid composition in guinea-pig sperm. We have recently described the induced acrosome reaction in ram spermatozoa. Fusion starts at the limit of the anterior and equatorial segments and progresses forward in the anterior segment along ramified paths, resulting in a fenestration gradient of the acrosomal cap. Fusion propagation may be controlled by fluidity increase in the plasma membrane of the anterior segment, and it is probably inhibited in the equatorial segment by the ordered structure of the acrosomal membrane.     

Electron microscopical studies of membrane injuries in blue fox spermatozoa subjected to the process of freezing and thawing

Disintegration of blue fox sperm membranes is studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In unfrozen spermatozoa studied by SEM, the plasmalemma and the acrosome appeared to be intact, except for a few cases of disruption of the former structure at the anterior part of the head. In semen frozen in 0.5-ml plastic straws by use of N2 vapor after dilution with Tris-fructose-citric acid with 8 vol % glycerol and 20 vol % egg yolk and thawed at 70 degrees C for 8 sec, the spermatozoa displayed different degrees of membrane damage. These alterations could be classified into three main categories of which the first included only minor changes in the plasmalemma, but vesiculation and disintegration of the outer part of the acrosomal membrane. In the second category (also the most frequent one) the outer part of the acrosomal membrane was extensively vesiculated, and the plasmalemma was discharged proximal to the equatorial segment. Extensive loss of plasmalemma and complete absence of the outer part of the acrosomal membrane characterized the last category of membrane damage. The functional implications of the three categories of membrane alterations are discussed.     

Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.     

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.     

Equatorial segment protein defines a discrete acrosomal subcompartment persisting throughout acrosomal biogenesis

The equatorial segment of the acrosome underlies the domain of the sperm that fuses with the egg membrane during fertilization. Equatorial segment protein (ESP), a novel 349-amino acid concanavalin-A-binding protein encoded by a two-exon gene (SP-ESP) located on chromosome 15 at q22, has been localized to the equatorial segment of ejaculated human sperm. Light microscopic immunofluorescent observations revealed that during acrosome biogenesis ESP first appears in the nascent acrosomal vesicle in early round spermatids and subsequently segregates to the periphery of the expanding acrosomal vesicle, thereby defining a peripheral equatorial segment compartment within flattened acrosomal vesicles and in the acrosomes of early and late cap phase, elongating, and mature spermatids. Electron microscopic examination revealed that ESP segregates to an electron-lucent subdomain of the condensing acrosomal matrix in Golgi phase round spermatids and persists in a similar electron-lucent subdomain within cap phase spermatids. Subsequently, ESP was localized to electron-dense regions of the equatorial segment and the expanded equatorial bulb in elongating spermatids and mature sperm. ESP is the earliest known protein to be recognized as a marker for the specification of the equatorial segment, and it allows this region to be traced through all phases of acrosomal biogenesis. Based on these observations, we propose a new model of acrosome biogenesis in which the equatorial segment is defined as a discrete domain within the acrosomal vesicle as early as the Golgi phase of acrosome biogenesis.     

Ultrastructure of Octodon degus spermatozoon with special reference to the acrosome

The ultrastructure of spermatozoa from the cauda epididymidis and vas deferens of Octodon degus-a Chilean hystricomorph rodent-is presented. The head of spermatozoa measured 7.7 micrometer long by 5.9 micrometer wide and the tail was 41 micrometer long. The head was flattened dorso-ventrally and ovate in outline. The acrosome was the most distinctive feature of O. degus spermatozoa. In a frontal view of the head, the rim of the acrosome surrounding the nucleus had the shape of an inverted U. The acrosomal region covering the plane of the flattened head exhibited dome-shaped protrusions. Transverse or sagittal sections of acrosomal protrusions showed that the plasma membrane and outer acrosomal membrane were evaginated, while the inner acrosomal membrane followed the contour of the nucleus. The protrusions were not distributed at random and they were absent in the equatorial segment and in the rim of the acrosome. In frontal views, near the boundary between the acrosome and post-acrosomal region, fine rods about 170 nm long ran obliquely on the caudal part of the equatorial segment. Behind the same boundary, the post-acrosomal region showed a serrated border. Phosphotungstic acid treatment at pH 0.3 produced staining at the surface of the sperm as well as within a superficial layer of the marginal thickening of the acrosome and on the acrosomal protuberances.     

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。     

The Acrosome Reaction in Ciona intestinalis (Ascidia, Tunicata)

An acrosome reaction occurs by fusion between the acrosomal outer membrane and the plasmalemma enclosing the acrosome in Ciona intestinalis spermatozoa. The fusion seems to proceed along the peripheral margin of the acrosome, which causes vesiculation. The membrane bound vesicle formed by this process is probably shed by the sperm. The acrosomal inner membrane is exposed and becomes a part of the plasmalemma enclosing the anterior region of the sperm head. During this process, any acrosomal substance might be released through the opening formed by membrane fusion. The acrosome reaction most likely occurs in C. intestinalis spermatozoa, via vesiculation, in fundamentally the same way as observed in mammalian spermatozoa.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates

The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.     

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

A quantitative ultramorphological approach for systematic assessment of sperm head regions: an example in rams

Examination of the type and frequency of damage to the head of spermatozoa using electron microscopy can be used to evaluate the quality of differently treated sperm. This report describes a systematic approach based on 29 morphological categories of sperm heads assessed from discrete regions in raw, chilled and frozen-thawed spermatozoa. Injury occurred principally at the plasma membrane and could be present or absent in all regions. In the anterior segment, when the plasma membrane is present, it can be intact, dilated, very dilated, disrupted, or contain vesicles characteristic of acrosomal reaction-like capacitation changes. When the plasma membrane is absent, the acrosome may be intact, exhibit a complete loss of contents, or retain some contents of the apical ridge and present a very dilated outer acrosomal membrane. The plasma membrane in the equatorial segment and the boundary between regions can be intact, dilated, very dilated or disrupted. The post-acrosomal plasma membrane is classified as intact, dilated or very dilated, whereas the dense lamina is intact, dilated or fragmented. The morphology of the heads most frequently observed in chilled spermatozoa consists of anterior and equatorial segments with a dilated, or dilated and disrupted plasma membrane; a boundary between regions with an intact and dilated plasma membrane; and a post-acrosomal region with an intact plasma membrane and dense lamina, both dilated. In frozen-thawed spermatozoa, the morphology of the heads is more frequently characterised by no plasma membrane and an acrosome showing complete or some loss of contents in the apical ridge and very dilated outer acrosomal membrane, presenting mostly dilated and fragmented dense lamina in the post-acrosomal region. These findings are consistent with the conclusion that the freezing process produces an increase in the degree of damage to the cells when they are subjected to increasing degrees of cold shock. There are still difficulties in developing a good diluent and process for preserving the plasma membrane in ram spermatozoa. This systematisation, using different categories, allows characterisation of multiple transmission electron microscopy images. Thus, the different changes observed due to cryopreservation may be correlated.     

Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment

Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.     

MEMBRANE DIFFERENTIATIONS IN FREEZE-FRACTURED MAMMALIAN SPERM

A correlated thin-sectioning and freeze-fracturing study has been made of guinea pig and rat spermatozoa. In sections, the cell membrane over the acrosome has a concanavalin A and ruthenium red reactive glycocalyx which exhibits an ordered pattern related to the lattice of crystalline domains within the plane of the membrane revealed by freeze-fracturing. The cleaved acrosomal membrane also shows a finer linear periodicity in some areas. The membrane over the equatorial segment of the guinea pig acrosome is marked by a palisade of oblique ridges not observed in the rat. The plasmalemma of the postacrosomal region is rich in membrane intercalated particles, many randomly dispersed, others clustered in rectilinear arrays. A particle-poor zone is found just anterior to the posterior ring. The fold of redundant nuclear envelope posterior to the ring has many nuclear pores in close hexagonal array. The nuclear envelope lining the implantation fossa is devoid of pores. When cleaved it has a particle-free central area surrounded by a broad zone of large, closely packed, hollow particles. The membrane of the mid-piece in the guinea pig (but not the rat) contains linear strands of 6–8-nm particles oriented circumferentially. The membrane investing the principal piece exhibits the usual randomly distributed particles but in addition, a double row of larger (9 nm) particles runs longitudinally within the membrane over outer dense fiber 1. In the corresponding position in thin sections a local thickening of the membrane is discernible. These observations form a basis for further studies on the functional correlates of these regional specializations of the sperm membrane.     

Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon

Experiments to bind fluorescein-conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) to washed human spermatozoa demonstrated that this lectin binds to the acrosome region in air-dried preparations. Since there was no binding when labelling was performed in suspension, and comparable labelling to that seen in air-dried preparations was seen when spermatozoa treated with saponin (to lyse the plasma membrane) were labelled in suspension, the lectin must bind to an intracellular structure, probably the outer acrosomal membrane. This was confirmed by ultrastructural localization of colloidal gold-conjugated lectin in saponin-treated spermatozoa. Treatment of spermatozoa with the detergent Nonidet P-40 caused a marked change in the binding pattern: more spermatozoa showed binding in the equatorial segment of the acrosome with no binding in the anterior cap region. A comparable, less marked, change was seen when spermatozoa were incubated overnight under conditions known to support the capacitation and spontaneous acrosome reactions. Treatment with the calcium ionophore A23187 for 1 h to induce acrosome reactions artificially in uncapacitated spermatozoa resulted in the appearance of patchy acrosome fluorescence. From these experiments it is concluded that PNA binds specifically to the outer acrosomal membrane, and that FITC-PNA-labelling may be used to monitor the human sperm acrosome reaction.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis

Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.     

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.     

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.