The composition, protein genesis and significance of the inner acrosomal membrane of eutherian sperm

As a consequence of the acrosomal reaction during fertilization, the inner acrosomal membrane (IAM) becomes exposed and forms the leading edge of the sperm for adhesive binding to and subsequent penetration of the zona-pellucida (ZP) of the metaphase-II-arrested oocyte. A premise of this review is that the IAM of spermatozoa anchors receptors and enzymes (on its extracellular side) that are required for sperm attachment to and penetration of the ZP. We propose a sperm cell fractionation strategy that allows for direct access to proteins bound to the extracellular side of the IAM. We review the types of integral and peripheral IAM proteins that have been found by this approach and that have been implicated in ZP recognition and lysis. We also propose a scheme for the origin and assembly of these proteins within the developing acrosome during spermiogenesis. During development, the extravesicular side of the membrane of the acrosomic vesicle is coated by peripheral proteins that transport and bind this secretory vesicle to the spermatid nucleus. The part of the membrane that binds to the nucleus becomes the IAM, while its extravesicular protein coat, which is retained between the IAM and the nuclear envelope of spermatozoa becomes the subacrosomal layer of the perinuclear theca (SAL-PT). Another premise of this review is that the IAM of spermatozoa is bound with proteins (on its intracellular side), namely the SAL-PT proteins, which hold the clue to the mechanism of acrosomal-nuclear docking. We propose a sperm cell fractionation strategy that allows for direct access to SAL-PT proteins. We then review the types of SAL-PT proteins that have been found by this approach and that have been implicated in transporting and binding the acrosome to the sperm nucleus.     

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

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

Arylsulphatase and acid phosphatase activity associated with developing and ripe spermatozoa of the musselMytilus edulis

Summary Arylsulphatase and acid phosphatase activity were demonstrated cytochemically in spermatozoa of the marine musselMytilus edulis. Reaction product resulting from arylsulphatase activity was measured using an integrating microdensitometer and found to increase with incubation time and to be variable according to the pH of the incubation medium. Two peaks in activity, at pH 4.5 and 6.0 were evident for some experimental protocols suggesting the possibility of two isoenzymes; however, studies on the ultrastructural localization of the enzyme showed no difference between sites of activity for the two pH values. Ultrastructural localization of arylsulphatase showed activity associated with the Golgi body of developing spermatids and in particular within the proacrosomal vesicles but limited to the periphery of the acrosomal vesicle which is formed with the fusion of the proacrosomal vesicles. In spawned spermatozoa arylsulphatase activity was localized in association with the axial rod and subacrosomal material; activity also occurred along the outer acrosomal membrane and within the acrosomal vesicle and also associated with the acrosomal process following the acrosome reaction. Sulphate groups were demonstrated cytochemically within the vitelline coat of oocytes in the mantle tissue. These findings suggest that arylsulphatase could be one of the lysins previously demonstrated inM. edulis spermatozoa. Acid phosphatase activity was demonstrated in spawned spermatozoa around the nuclear envelope and along the outer acrosomal mambrane.     

Comparative histochemical observations on the contributions of the acrosomal vesicle and granule to the acrosomal cap in the spermatozoa of mammals

Summary By applying the periodic acid-Schiff technique to frozen sections of material fixed in weak Bouin's solution and extracted with hot pyridine, the contributions of the acrosomal vesicle and granule to the acrosomal cap have been observed in the maturing spermatozoa of the American opossum, guinea pig, rabbit, marmoset and chimpanzee. In the spermatids of the opossum, the acrosomal granule is not developed and the thin, homogeneous carbohydrate layer of the acrosomal cap is derived from the concentrated material of the acrosomal vacuole. In the guinea pig, both the acrosomal vesicle and granule make the greatest contributions to the carbohydrates of the acrosomal cap; these continue to maintain their identity in the head of mature sperm. In the rabbit, marmoset and chimpanzee, the carbohydrate material of acrosomal vesicle origin is less developed and constitutes a thin layer in the lateral portions of the acrosomal cap; the carbohydrate material in its anterior portions is mainly derived from the acrosomal granule which becomes flattened; there is also a slight thickening of the intermediate layer of the acrosomal cap in this region. The distinction between the carbohydrate contributions from the acrosomal vesicle and granule disappears in the fully formed acrosomal cap which shows a homogeneously PAS-positive intermediate layer. In the chimpanzee, the acrosomal vesicle makes a relatively very small contribution to the carbohydrate layer of the acrosomal cap which is mainly formed by spreading of material from the acrosomal granule.     

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.     

Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata)

The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.     

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.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca²⁺ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca²⁺.     

Evidence for the capacitation-associated membrane priming of mouse spermatozoa

An important feature of male fertility is the physiological priming of mammalian spermatozoa by a multifaceted process referred to as capacitation. It is a prerequisite event before spermatozoa can bind to the egg's extracellular coat, the zona pellucida, and undergo a signal transduction cascade. The net result is the fusion of the plasma membrane (PM) and underlying outer acrosomal membrane at multiple sites and the release of acrosomal contents (i.e., glycohydrolases, proteinases, etc.) at the site of sperm-zona binding. In this study, we have used an indirect immunofluorescence (IIF) assay and other staining approaches to examine capacitation-associated membrane priming of mouse spermatozoa. For IIF studies, we used affinity-purified antibodies against two glycohydrolases that cross-reacted with the acrosomal enzymes only when the uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors in vitro capacitation induced membrane priming that allowed the antibodies to cross-react with the acrosomal enzymes in capacitating acrosome-intact spermatozoa without permeabilization, as revealed by the appearance of several distinct fluorescent patterns, including an initial immunopositive lining over the acrosome cap to an intense immunopositive reaction throughout the acrosome. These early immunopositive patterns were followed by the appearance of intense fluorescent spots (droplets) that seem to establish contact with the PM in a time-dependent manner. Inclusion of calmodulin, a 17-kDa Ca(2+)-binding protein which promotes capacitation, in the incubation medium did not alter the overall rate of capacitation; however, its presence accelerated the initial stages of membrane priming. The potential similarities between sperm capacitation and early events of Ca(2+)-triggered membrane fusion among eukaryotes and among various stations of the secretory and endocytotic pathways are discussed.     

三疣梭子蟹精子顶体反应前后胞内Ca~(2+)的变化

应用激光扫描共聚焦显微镜(LSCM)和Fluo-3/AM荧染技术对三疣梭子蟹精子顶体反应前后的胞内Ca2 变化进行了观察和检测.结果显示,在精子顶体反应过程中,胞内Ca2 主要分布在细胞核、穿孔器和胞质膜残存处,胞内Ca2 浓度([Ca2 ]I)总体上呈现先上升后下降的趋势.顶体反应前精子的平均荧光强度为35.95±5.71;穿孔器前伸、顶体囊膜翻转阶段精子的平均荧光强度为66.80±7.35;顶体囊膜脱落、顶体丝形成阶段精子的平均荧光强度为3.87±2.82;上述各阶段间精子荧光强度有极显著差异(P<0.01).顶体反应穿孔器前伸、顶体囊膜翻转阶段的精子相比顶体反应前精子,[Ca2 ]I显著提高;而在顶体囊膜脱落、顶体丝形成阶段,[Ca2 ]I则急剧下降,只在顶体丝基部胞质膜残存处有微量Ca2 存在.初步探讨了三疣梭子蟹精子顶体反应前后胞内Ca2 变化的功能.     

Ultrastructure of spermatozoa correlated with phylogenetic relationship between Heteropneustes fossilis and Rana tigrina

A study using transmission electron microscopy (TEM) of the spermatozoa of Rana tigrina and Heteropneustes fossilis in all phases of the annual reproductive cycle revealed that there was a phylogenetic relationship between them. The spermatozoa of H. fossilis appeared horseshoe-shaped, somewhat oval or wedge-shaped at the anterior end and broader at the posterior end. The horseshoe-shaped spermatozoan nucleus was observed during spermiogenesis of R. tigrina but later changed into a finger shape at maturity. The posterior end of the nuclei of mature spermatozoa of R. tigrina was blunt. The extremely dense homogenized nucleus was capped with an acrosomal vesicle in both species suggesting a definite phylogenetic inter-relationship between them.     

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.     

ELECTRON MICROSCOPE STUDIES ON SPERM DIFFERENTIATION IN MARINE ANNELID WORMS. I. SPERM FORMATION IN IKEDOSOMA GOGOSHIMENSE

The ultrastructur of spermatozoa and the changes through which they are differentiated during sperm formation in an echiuroid were observed under the electron microscope. Many spermatids are connected to one central cytoplasmic mass and the sperm differentiation proceeds synchronously in one sperm-ball. Dense plate-like structures appear in the cytoplasm of early spermatids and disappear soon. In the process of nuclear condensation, many electron-dense aggregates appear in homogeneously textured chromonema and the aggregates are packed together to form a uniformly dense nucleus. Near the centriole at the opposite side from the central mass, the mitochondria fuse together to form one large middle-piece mitochondrion and the acrosomal vesicle is formed from the Golgi-complex. The differentiating acrosome in the late spermatid moves to the anterior tip of the head. In the completed acrosome, a flocculent substance accumulates in the conspicuously expanded invaginated pocket of the acrosomal vesicle and two kinds of material of different electron density fill the inside of the acrosomal vesicle. The spermatozoa remain connected to the central mass at the lateral side of the head until they become fully mature and are packed into the nephridia before spawning.     

Biflagellate spermatozoon of the poison-dart frogs Epipedobates femoralis and Colostethus sp. (Anura,Dendrobatidae)

This study describes the spermatozoa of the dendrobatids Epipedobates femoralis and Colostethus sp. using light and transmission electron microscopy. Both species possess a biflagellate spermatozoon, an unusual characteristic only previously reported in two anuran species belonging to the families Leptodactylidae and Racophoridae. The acrosomal complex of both species consists of a conical acrosomal vesicle and a subacrosomal cone, both of which cover the anterior portion of the nucleus, but to differing extents. In the midpiece, the centrioles are disposed parallel to each other and to the cell axis and give rise to two axonemes. Two paraxonemal rods were also seen entering the nuclear fossa. Both flagella are surrounded by a single mitochondrial collar. Each flagellum is formed by an axial fiber connected to the axoneme by an axial sheath; juxta-axonemal fibers are absent. Our data seem to support that Epipedobates femoralis should be placed in a separate clade possibly related to Colosthetus and that these two genera may not be monophyletic.     

Sperm Bundles in the Seminal Vesicle of the Crematogaster victima (Smith) Adult Males (Hymenoptera: Formicidae)

This study establishes the presence of spermatodesm in the seminal vesicles of sexually mature males of Crematogaster victima (Smith). In this species, the spermatozoa are maintained together by an extracellular matrix in which the acrosomal regions are embedded. This characteristic has not yet been observed in any other Aculeata. However, the sperm morphology in this species is similar to that described for other ants. The spermatozoa measure on average 100 μm in length, and the number of sperm per bundle is up to 256. They are composed of a head formed by the acrosome and nucleus; this is followed by the flagellum, which is formed by the centriolar adjunct, an axoneme with a 9?+?9?+?2 microtubule pattern, two mitochondrial derivatives, and two accessory bodies. The acrosome is formed by the acrosomal vesicle and perforatorium. The nucleus is filled with compact chromatin with many areas of thick and non-compacted filaments. Both mitochondrial derivatives have the same shape and diameters. The presence of sperm bundles in sexually mature males differentiates C. victima from other ants; however, the similarities in the sperm ultrastructure support the monophyly of this insect group.     

Ultrastructure of the spermatozoa of five species of South African bivalves (Mollusca), and an examination of early spermatogenesis

Transmission electron microscopy of the spermatozoa of five species from three families of bivalves has shown that each species has a sperm with unique morphology. However, the morphology of the acrosomes of each species is typical of the subclass of bivalve to which they belong. An examination of spermatogenesis in the five species, along with a re-examination of material from six other species of bivalves, has revealed that pre-spermiogenic cells possess flagella. In addition, acrosome formation begins in the spermatocytes with the formation of proacrosomal vesicles in the Golgi body. During spermiogenesis the proacrosomal vesicles coalesce at the presumptive posterior of the spermatid, with a larger vesicle produced by the Golgi body. The single acrosomal vesicle eventually migrates to the anterior of the spermatid where it assumes its mature form. © 1994 Wiley-Liss, Inc.     

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

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

Comparison of the spermatozoan morphology of Isognomon bicolor and Isognomon alatus (Mollusca, Bivalvia, Isognomonidae)

In this study we used transmission and scanning electron microscopy to examine the spermatozoan structure of Isognomon bicolor and Isognomon alatus. The spermatozoa of both species were of the primitive or ect-aquasperm type. The acrosomal morphologies were essentially similar but the top of the acrosomal vesicle in I. bicolor sperm had a slightly flattened edge whereas the apex of the acrosomal vesicle of I. alatus sperm had a rounded outline. This difference suggested that acrosomal morphology could be an important character for taxonomic differentiation. In the present work, the results demonstrated that the gamete ultrastructure of the two distinct species I. alatus, from Panama, and I. bicolor, from the southeastern region of Brazil, were similar to the other studied species of the superfamily Pterioidea.     

Formation of the cylindrical structure during the acrosome reaction of abalone spermatozoa

Spermatozoa of abalone Haliotis discus were examined before and during the acrosome reaction with special regard to one of the newly formed structures: a cylindrical structure surrounding a part of the elongated acrosomal process near the opening of the acrosomal vesicle. The structure, about 0.2 μm in diameter and about 1 μm in length, was revealed to be composed of a tightly coiled, fine tubular structure about 20 nm in diameter. In the course of the acrosome reaction, a triple-spiral structure appeared in the anterior part of the acrosomal vesicle. Since this spiral structure was also composed of a tightly coiled 20 nm tubule(s), it was concluded that this structure was transformed into the single-walled cylindrical structure by simple stretching in the direction of its longitudinal axis. In the clumps of spermatozoa that underwent acrosome reaction in suspension, the cylindrical structures were frequently found in contact with each other and/or other structures, indicating that they are very sticky.     

Spermatozoal ultrastructure in three Atlantic solenocerid shrimps (Decapoda, Dendrobranchiata)

The spermatozoal ultrastructure in three solenocerid shrimps (Solenocera membranacea, S. africana, and Pleoticus muelleri) from different Atlantic locations was examined with the aim of increasing understanding of the phylogenetic relationships in the Dendrobranchiata. A considerable structural similarity between the sperm of these species and those of penaeid shrimps supports a close affinity between the Penaeidae and Solenoceridae. However, significant differences in the sperm morphology of the previously investigated sicyoniids (namely, a greater complexity of the acrosomal complex) suggest evolutionary separation of the Sicyoniidae from the assemblage Penaeidae-Solenoceridae. Two ultrastructural features distinguish the spermatozoa of the three studied solenocerids from penaeid sperm: 1) separation of the plasma and acrosome membranes at the base of the spike and anterior region of the cap, and 2) asymmetry of the acrosomal cap, which appears to be a synapomorphy of the group. No striking ultrastructural differences were found between the spermatozoa of the closely related species S. membranacea and S. africana, whereas a great number of morphological differences separate the spermatozoa of Pleoticus from those of Solenocera (e.g., shape of the acrosomal cap, structural arrangement of the contents of the whole acrosome vesicle, thickness and distribution of the cytoplasm, and external shape of the spike).