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.     

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.     

Organization of Actin Filaments in the Axial Rod of Abalone Sperm Revealed by Quick Freeze Technique

An axial rod in abalone ( Haliotis discus ) sperm is a structure composed of a bundle of actin filaments, which elongates anteriorly to form the acrosomal process during the acrosome reaction. The ultrastructure of the actin filament bundle constituting the axial rod was examined using quick freeze technique followed by either freeze-substitution or deep-etch electron microscopy. Thin sections of quick freeze and freeze-substituted sperm revealed that the actin filaments in the axial rod are hexagonally packed in a paracrystalline array through its almost entire length with an average center-to-center spacing of 12 nm. Periodic transverse bands were also observed across the actin filament bundle, which may reflect the cross-bridges interconnecting the adjacent filaments. Quick-freeze deep-etch analysis provided the three-dimensional view of the axial rod. Actin filaments exhibiting 5.5–6 nm spaced striations were observed to run in parallel with each other inside the axial rod. The existence of cross-bridging structures was also displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regularly spaced cross-bridges to form a well ordered hexagonally packed bundle, and also cross-linked by fibrous structure to the lateral inner acrosomal membrane which closely surrounds the anterior half of the actin filament bundle.     

Ultrastructure of spermatogenesis and spermatozoa of Cephalodasys maximus (Gastrotricha,Macrodasyida)

Summary Spermatogenesis and sperm ultrastructure of the macrodasyidan gastrotrich Cephalodasys maximus are described by means of transmission electron microscopy. The filiform sperm consists of an acrosomal accessory structure and an acrosomal vesicle, both being surrounded by spiralled material. The successive nuclear helix encloses the spiral-shaped mitochondrion and the axoneme of the flagellum is accompanied by dense strings, three helical elements and peripheral microtubules. During spermiogenesis the acrosomal accessory structure develops first and moves into a cell projection, where the spiral around this acrosomal rod forms. A nuclear section with condensed chromatin and one single fused large mitochondrion follow into the extension, becoming helical. A connecting clasp between nucleus and flagellum shortens to a cap-like structure. Parallel to the acrosomal and nuclear projection the flagellum develops where the spiralled elements and the basal plate form in succession, while the basal body shrinks.     

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.     

Ultrastructure of the Tubificid Acrosome (Annelida, Oligochaeta)

The later morphogenesis of the acrosome of Limnodriloides winckelmanni and Rhyacodrilus arthingtonae is compared with that in Enchytraeus and in earthworms. After superposition of the acrosome on the tip of the nucleus the manchette continues apically beyond the nucleus to ensheath the acrosomal tube. At the posterior limit of, and probably contained in, the spacious/ terminal primary acrosomal vesicle is an electron-dense ring. A domed protrusion into the floor of the primary vesicle is tentatively regarded as the secondary acrosome vesicle. The axial rod when first observed is attached to the vesicle complex. Later, the rod detaches and extends deeply into the acrosome tube. A membrane ensheathes the tubificid axial rod but its exact homology with the complex layers surrounding the lumbricid or megascolecid axial rod is not clear. The domed apical region of the tubificid acrosome is probably a persistence of the primary acrosome vesicle and it is deduced that the acrosome vesicle surrounding the axial rod in lumbricids and megascolecids is a product, by invagination, of the secondary acrosome vesicle only.     

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.     

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.     

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     

Proacrosome and acrosome of the spermatozoon in Acanthobdella peledina (Annelida: Clitellata)

Summary

Proacrosome and acrosome of the primitive leech Acanthobdella peledina are described by means of transmission electron microscopy. The proacrosome develops in early spermatids and has the shape of a pot-bellied urn with an opening towards the nucleus. Its wall is formed by a thin vesicle. In its interior, many sections of tubular structures are visible. This urn is seated atop a short, electron-dense tube. The resultant acrosome is unusually elongated, with a helically coiled acrosomal tube forming its base. Above the tube the thin acrosomal vesicle encloses a central space, within which is the acrosomal rod. The acrosomal structures clearly indicate a sister-group relationship to the Euhirudinea, but do not corroborate the notion of close kinship with the Branchiobdellidae.     

中华绒螯蟹(Eriocheir sinensis)精子顶体反应的研究

分别用卵水、海水、caCl_2或NaCl水溶液对中华绒螯蟹成熟精子进行人工诱导顶体反应,结果表明:精子的生理性成熟、同种卵或Ca~(++)的存在、碱性环境以及与一定的固体接触均为精子顶体反应触发的重要条件。3月份精子诱导率最高。 电镜观察证明,中华绒螯蟹精子的顶体反应可分四个阶段:(1)辐射臂收缩;(2)顶体囊外翻;(3)顶体管前伸;(4)片层结构脱落。     

An actin-like component in spermatocytes of a crane fly (Nephrotoma suturalis Loew)

Actin-like filaments are seen at the cell periphery after crane fly spermatocytes are glycerinated and then treated with rabbit skeletal muscle heavy meromyosin. ATP and pyrophosphate inhibit the reaction with heavy meromyosin. From prometaphase through metaphase the filaments are all parallel to the cell surface, extending 0.5–1 μ. beneath the plasma membrane in a continuous layer of parallel filaments enveloping the cell; considering the poles of the spindle as north and south poles of the cell, the actin-like filaments at the cell periphery are all arranged as meridians. In late-anaphase, too, actin-like filaments are parallel to the cell surface, but here this includes bundles of filaments oriented as parallels in the furrow and adjacent regions of the cell periphery, as well as filaments oriented as meridians in the rest of the cell periphery. — Actin-like filaments are seen in the cellular projections associated with the spindle poles.     

Ultrastructural analysis of spermiogenesis in Iguana iguana (Reptilia: Sauria: Iguanidae)

Spermiogenesis in the lizard, Iguana iguana, was studied by transmission and scanning electron microscopy. During this process, structures such as the acrosomal complex in the spermatid head and the axonemal complex in the mid and principal pieces of the flagellum are formed. The nuclear content is initially compacted into thick, longitudinal chromatin filaments. Nuclear shape is determined by further compaction and by the manchette, a layer of microtubules surrounding the head. The acrosomal complex originates from Golgi vesicles and the interaction between the proacrosomal vesicle and the nucleus. The midpiece consists of a pair of centrioles, surrounded by a fibrous sheath and rings of simple and modified mitochondria. The centrioles sustain the axoneme that appears at the end of the midpiece. The axoneme extends throughout the principal piece of the flagellum with the 9 + 2 pattern, still surrounded by the fibrous sheath. In the endpiece, the axoneme continues, surrounded only by the plasma membrane. In the lumen of seminiferous tubules, immature spermatozoa retain abundant residual cytoplasm.     

Studies on the fine structure of the mammalian testis. I. Differentiation of the spermatids in the cat (Felis domestica)

The differentiation of cat spermatids was studied in thin sections examined with the electron microscope. The Golgi complex of the spermatid consists of a central aggregation of minute vacuoles, partially surrounded by a lamellar arrangement of flattened vesicles. In the formation of the acrosome, one or more moderately dense homogeneous granules arise within vacuoles of the Golgi complex. The coalescence of these vacuoles and their contained granules gives rise to a single acrosomal granule within a sizable membrane-limited vacuole, termed the acrosomal vesicle. This adheres to the nuclear membrane and later becomes closely applied to the anterior two-thirds of the elongating nucleus to form a closed bilaminar head cap. The substance of the acrosomal granule occupies the narrow cleft between the membranous layers of the cap. The caudal sheath is comprised of many straight filaments extending backward from a ring which encircles the nucleus at the posterior margin of the head cap. Attention is directed to the frequent occurrence of pairs of spermatids joined by a protoplasmic bridge and the origin and possible significance of this relationship are discussed.     

Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

Morphological identification of sperm receptors above egg microvilli in the polychaete, Neanthes japonica

A fine-structural study of sperm-egg interactions in the polychaete Neanthes japonica was carried out. Unfertilized eggs are surrounded by a chorion 0.6-0.7 micrometers thick. Oocyte microvilli are inserted into the inner layer of the chorion. The outer layers of the chorion are opened just above the tips of the microvilli, where a membrane vesicle (microvillus tip vesicle, about 0.2 micrometers in diameter) plugs the chorion's opening. During fertilization, the acrosomal process of the sperm fuses with an egg microvillus within 1 min of insemination. All the microvillus tip vesicles disappear from the chorion surface within 5 min of insemination. When eggs, which are prefixed with glutaraldehyde, are inseminated, numerous sperm undergoing the acrosome reactions attach to the eggs. In the majority of these sperm, the tip of acrosomal process which is coated with the acrosomal content, adhere to a microvillus tip vesicle. These findings suggest that the microvillus tip vesicle serves as a sperm receptor, which induces the acrosome reactions and adhere to the sperm acrosomal process. The adhesion of the acrosomal process to the microvillus tip vesicle seems to be a prerequisite event for its fusion with the microvillus.     

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.     

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.     

ZYGOTE FORMATION IN ASCARIS LUMBRICOIDES (NEMATODA)

Ultrastructural observations of the in utero sperm of Ascaris lumbricoides reveal that it consists of a relatively clear, ameboid anterior region and a conical posterior region containing numerous surface membrane specializations, dense mitochondria, a lipid-like refringent body of variable size, and a dense nucleus which lacks an apparent nuclear envelope. No acrosomal complex was observed. Pseudopods emanating from the anterior cytoplasm make first contact with the primary oocytes and appear to be responsible for the localized removal of the extraneous coat covering the oolemma. Subsequently the gamete membranes interdigitate and finally fuse. Because this pseudopodial action appears similar to that reported for the acrosomal filaments in flagellated sperm, the anterior region of the Ascaris sperm is thought to serve an acrosomal function. Following gamete-membrane fusion, the sperm nucleus acquires a particulate appearance and becomes disorganized. Once inside the oocyte, the sperm cytoplasm consists of dense mitochondria, ribosomes, and vesicles derived from the surface membrane specializations. The refringent body, whose contents possibly contribute to the synthesis of ribosomes, is usually absent by the time the sperm cytoplasm attains a central position in the egg.