Subacrosomal Region Research Articles

Male reproductive biology of the red frog crab, Ranina ranina, off Hachijojima, Izu Islands, Japan

Male red frog crabs, Ranina ranina, were collected year round in 1990 and 1991 off Hachijojima for histological study of the reproductive system and cycle. The testis containing the lobules and seminiferous ducts is surrounded by connective tissue. The seminiferous duct connects to the anterior end of the vas deferens, which can be histologically divided into three portions similar to one another in appearance. It was surrounded by fibrous connective tissue, muscle fibrils and columnar epithelium. Muscle fibrils were absent in the anterior portion. Multiple sperm masses were not formed in the vas deferens and ejaculatory duct, but the sperm mass was covered with a capsule composed of two layers. The outer layer of the capsule was periodic acid Schiff (PAS)-positive, but the inner layer was negative. Both layers were Alcian Blue negative, except the vacuoles in the outer layer that were stained blue. The small round androgenic gland was attached to the posterior end of the vas deferens of the coxa of the eighth thoracic appendage. The ejaculatory duct was distinguishable from the vas deferens by the absence of columnar epithelium and the presence of thick longitudinal muscle fibers. Spermatogenesis was histochemically examined. The acrosomal vesicle appeared to be derived from PAS-positive vesicles in the cytoplasm of the spermatid at the early stage of spermiogenesis. The arms were positive to the Feulgen reaction and the subacrosomal region was negative to PAS. Seasonal changes in reproductive cycle were inconspicuous histologically and microscopically. Sperm were always present in the testis and vas deferens throughout the year and occupied 5.1 to 19.6% of testis observed in cross sections. The minimum size of maturity is less than 39 mm carapace length, but the minimum size capable of successful mating was estimated to be ca. 55 mm.

The late events of fertilisation in the penaeoidean shrimp Sicyonia ingentis.

Antibodies to sea urchin beta-tubulin and mammalian heavy neurofilaments were used to study the late events of fertilisation in the penaeoidean shrimp Sicyonia ingentis. The neurofilament antibody fortuitously stained centrosomes in eggs, as well as the subacrosomal region and acrosomal filament in sperm. The neurofilament antibody also stained a cortical site in eggs which was associated with the positioning of the mitotic spindle. During pronuclear migration, a large maternal microtubule aster formed in addition to the sperm aster. The activity of the maternal centrosome disappeared during syngamy, while the sperm centrosomes formed the poles of the first mitotic spindle. Colcemid treatment modulated the size of the mitotic spindle and blocked pronuclear migration.

Structural changes in sperm from the fiddler crab, Uca tangeri (Crustacea, Brachyura), during the acrosome reaction.

The acrosome reaction (AR) was induced in sperm from the brachyuran crustacean Uca tangeri either by mixing male and female gametes in filtered seawater or by treating the spermatozoa with the divalent cation ionophore A23187. This latter method provided a sufficient number of reacted spermatozoa to allow a detailed ultrastructural study of the AR. The process consists of two separate phases: a) initial release of the acrosomal vesicle contents, and b) further elongation of the acrosomal filament, which causes reversal of the rigid capsule limiting the acrosomal vesicle contents. The elongate acrosomal filament consists of an apical perforatorium and a basal columnar structure called here the proximal piece. The former derives from the perforatorium of the uninduced sperm stage with only small ultrastructural changes. The proximal piece forms from myelin-like membrane layers which are initially distributed all around the subacrosomal region and then accumulate in a column at the perforatorial base, thus promoting a sudden forward projection of the perforatorium. The AR in brachyurans is thought to be a passive mechanism that utilizes the negative pressure exerted on the nucleus--caused by emptying of the acrosomal vesicle--for an organized accumulation of membrane-rich material immediately behind the perforatorium, with the final result of the raising of a 3 microns long acrosomal filament.

Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry

The rat perforatorium is the part of the perinuclear theca that underlies the acrosomic system. It appears to be composed of several polypeptides. The main objective of this study was to determine the distribution of seven of these perforatorial polypeptides in the head of the rat spermatozoon. For this purpose, polyclonal antibodies were affinity purified from these polypeptides and tested 1) for their distribution on electron-microscope sections of late spermatids and spermatozoa by immunogold labeling and 2) for their specificity on Western blots of denatured perforatorial polypeptides by immunoblotting. Immunoblotting showed that all seven of the prominent perforatorial polypeptides had epitopes in common. Immunogold labeling of spermatozoa showed that antibodies against the 13, 13.4, and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner zone of the ventral spur. However, antibodies against the 34, 43, 57, and 63 kDa polypeptides reacted with the entire perforatorium but, in addition, reacted with the inner part of the ventral spur and with a portion of the "outer periacrosomal layer" lying between the plasma membrane and the outer acrosomal membrane. These results suggest 1) that there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer, and of the postacrosomal dense lamina; and 2) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, but may also compose portions of the outer periacrosomal layer and postacrosomal dense lamina. Based on both immunoblotting and immunocytochemical results, using an antiactin monoclonal antibody that recognizes all known isoforms of actin, actin was not detected in the perforatorium of step 19 spermatids or spermatozoa. Actin, however, together with the seven perforatorial polypeptides tested, was present in the subacrosomal space of elongating spermatids before the process of condensation of the perforatorium takes place.

Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton

The cytoskeleton exerts a direct effect on the function of sperm by influencing the distribution of subcellular organelles and plasma membrane molecules. We have prepared six monoclonal antibodies to Triton X-100-insoluble components of the bull sperm cytoskeleton. One of the antibodies reacts with a detachable portion of the bull sperm acrosome. The remainder include an antibody that recognizes the principal and end piece of the tail and another that is specific to the middle piece. Two of the antibodies yield dissimilar staining patterns of the neck region and the tail, and the final monoclonal antibody stains the subacrosomal region and a detachable acrosomal domain of bull sperm. The cross reactivities of the antibodies with hamster sperm and PtK2 cells are described, as is the recognition of bull sperm polypeptides on western blots. The results suggest that these antibodies will provide interesting insights concerning the role of the cytoskeleton in sperm development and function.

Ultrastructural Comparison of Sperm from the Crayfishes Cherax Tenuimanus and Cherax Albidus

Aflagellate sperm of the crayfishes Cherax tenuimanus and Cherax albidus are composed of an acrosome, subacrosomal region, collar, and nucleus. The acrosomes are helmet-shaped and PAS positive. In C. tenuimanus, they can be subdivided into a granular inner region and a homogeneous outer region. A series ofelectron-translucent canals runs through the acrosomes of both species. In C. albidus, these canals are arranged in whorling patterns, while in C. tenuimanus they are present near the base of the acrosome and contain a small electrondense component. The subacrosomal regions are metachromatic when stained with methylene blue and contain a network of fibrils and granules. The collars are composed of granular material, which is positioned immediately subjacent to the subacrosomal region, and of highly convoluted membrane lamellae. Mitochondria and microtubules are associated with the membrane lamellae. The nuclei contain uncondensed chromatin and are bounded by a complete nuclear envelope. Sperm are surrounded by a moderately electron-dense matrix containing granules and filaments. Sperm of Cherax tenuimanus have large deposits of an electron-dense material on their surface. In contrast, sperm of C. albidus have long extracellular lamellae associated with the extracellular surface of their plasma membrane, and these lamellae are also found in the extracellular matrix surrounding the sperm of C. albidus. In both species, the sperm and extracellular materials are surrounded by a moderately PAS-positive primary spermatophore layer which is in turn surrounded by a secondary spermatophore layer secreted from the wall of the vas deferens. This layer includes dense granules which are supported in a matrix. In C. tenuimanus, this matrix is homogeneous, while in C. albidus, this matrix contains light and dark staining areas. The darker regions often have a fibrous appearance. A comparison is made between sperm of C. tenuimanus and C. albidus and the sperm of other pleocyematan species. Cherax tenuimanus (marron) and Cherax albidus (koonac) are fresh-water crayfish native to the large streams of southwestern Australia. Cherax tenuimanus is the third largest species of crayfish in the world (the Tasmanian crayfish Astacopsis franklinii and the Murray River crayfish Euastacus serratus are larger), and attains weights of approximately 2 kg (Shireman, 1973). Cherax albidus is smaller than C. tenuimanus, yet is still much larger than the native American genera (Pacifastacus, Procambarus) which weigh less than one fiftieth of a kilogram. Cherax tenuimanus and C. albidus are excellent animals for aquaculturing, because, in addition to their large size, they are highly palatable, do not burrow, have an uncomplicated life cycle, and grow large fairly rapidly (Crook, 1981). Their potential for aquaculture has generated considerable recent interest in these species. A complete understanding of the reproductive biology of species of Cherax will aid in their aquaculture. Until now, the ultrastructure of their gametes has not been studied, though work has been done on the ultrastructure of sperm from other crayfish genera (Moses, 1956, 196 la, b; Meek and Moses, 1961; Yasuzumi et al., 1961; Yasuzumi and Lee, 1966; Anderson and Ellis, 1967; Pochon-Masson, 1968; Lopez-Camps et al., 1981; Dudenhausen and Talbot, 1982). However, in many of the investigations on American crayfish, morphological detail was not completely resolved because of fixation problems. In this study, we have used the improved fixation procedures of Dudenhausen and Talbot (1982) to examine and compare the ultrastructure of sperm from C. tenuimanus and C. albidus.

Characterization of filaments within the subacrosomal space of rat spermatids during spermiogenesis

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid elongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14–17 of spermiogenesis. About 14 nm in diameter (10–15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments, 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.

Sperm maturation and ultrastructure in Scyllarus chacei

The ultrastructure of the sperm of Scyllarus chacei has been examined. The sperm comprises a nucleus, lamellar region and acrosome. The nucleus contains uncondensed microtubules which originate from the subacrosomal region projecting from the body of the sperm. The unique acrosome is structurally complex. It contains homogeneous, scrolled and crystalline areas capped over by an umbrella-like structure having 40 projections.

An ultrastructural analysis of mature sperm from the crayfishPacifastacus leniusculus, Dana

Sperm from the crayfish, Pacifastacus leniusculus, resemble other reptantian sperm in that they are composed of an acrosome, subacrosomal region, nucleus, membrane lamellar complex, and spikes which radiate from the nuclear compartment. The acrosome (PAS positive vesicle) can be subdivided into three regions: the apical cap, crystalline inner acrosomal material, and outer acrosomal material which is homogeneous except for a peripheral electron dense band. The nucleus contains uncondensed chromatin and bundles of microtubules which project into the spikes. The orientation of the microtubule bundles relative to the nuclear envelope near the base of the subacrosomal region suggests that the nuclear envelope may function in the organization of the spike microtubules.

The structure of sperm from the lobster, Homarus americanus

The structure of sperm from the lobster, Homarus americanus, was examined using light and electron microscopy. These sperm consist of an acrosome, subacrosomal region, collar, nucleus, and three spikes which are extensions of the nucleus. The acrosome is a cylinder projecting from the sperm's antierior pole. It is comprised of inner acrosomal material, outer acrosomal material, and an apical cap, and is limited by a single continuous membrane. The subacrosomal material is finely granular with most of it lying in a fossa in the base of the acrosome. The collar, a region subjacent to the subacrosomal material, contains small mitochrondria and a pair of centrioles. The subacrosomal material and collar are in direct continuity with the chromatin of the nucleus. The nucleus itself is limited by a highly folded, thick membrane which probably represents a composite of the plasma membrane and nuclear envelope. The chromatin is granular or fibrillar and is uncondensed. The spikes are compartments of the nucleus and contain microtubules ensheathed by sheets of membrane. Ionophore A23187 which is fluorescent inserts into membranes of cells and was used to study the distribution of spike membranes in sperm with the aid of a fluorescent microscope. The microtubule/membrane complexes of the spikes converge in the region of the collar and interconnect to form a three-sided vault the apex of which terminates immediately beneath the base of the acrosome. These lobster sperm are similar in structure to other reptantian sperm.

Spermiogenesis in the sea cucumber Holothuria Tubulosa

Ultrastructural studies have shown that the spermatozoon of the sea cucumber Holothuria tubulosa has many primitive features, among them the overall spherical shape of the nucleus, a poorly developed centriolar fossa, and a simple mitochondrial ring in the midpiece. The chromatin is not fully condensed and appears as contorted fibrils 200 A in diameter tightly packed in an irregular fashion. This simple structure of the chromatin can be correlated with the slight changes that take place during spermiogenesis in the basic proteins associated with DNA, as shown by cytochemical and biochemical studies. The production of acrosomal material starts already in spermatogonia, where small electronopaque proacrosomal vesicles can be detected. In the spermatid these vesicles coalesce into a single large proacrosomal vesicle. At a later stage, it is invaginated into the nucleus and a subacrosomal region is also formed. As a result, an approximately spherical acrosome is present in ripe spermatozoa.

Sperm differentiation in the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus

The morphology of the testes and the development of sperm cells in the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus have been investigated with techniques of light and electron microscopy. Developing spermatids are connected by intercellular bridges which persist until the sperm cells are nearly mature. Formation of the acrosomal vesicle is associated with the Golgi complex. During differentiation the chromatin of the spermatid nucleus condenses. Concomitantly, the spermatid nucleus becomes morphologically specialized, i.e., its apex becomes indented (subacrosomal region) and its base invaginated (centriolar fossa). The single large mitochondrion which constitutes the middle-piece of the spermatozoon presumably develops by a fusion of the randomly dispersed mitochondria. During early stages of development spoke-like satellites are associated with the proximal and the distal centrioles. The distal centriole, unlike the proximal one, is associated with a bundle of fibers. In the spermatozoon, the proximal centriole is situated in the centriolar fossa; the distal centriole is found between the base of the nucleus and the mitochondrion.