Differentiation of the acrosomal complex in ostrich (Struthio camelus) spermatids

The acrosomal complex of ostrich sperm consists of a small, cone-shaped acrosome and a slender, cylindrical perforatorium housed within a deep endonuclear canal. The perforatorium is almost exclusively endonuclear in location and is only covered by the acrosome at its point of origin in the apical subacrosomal space. The development of the acrosome is generally similar to that described in other non-passerine birds. Small proacrosomal granules (vesicles) emanating from the Golgi apparatus coalesce to form a large, membrane-bound acrosomal vesicle filled with homogeneous, electron-dense material. The acrosomal vesicle attaches to the nucleus via a shallow depression and subsequently collapses to form the typical cap-like acrosome of non-passerine birds. In ostrich spermatids the endonuclear canal becomes obvious when the collapsed acrosomal vesicle has assumed a dumbbell-shaped appearance. The perforatorium, which originates from moderately electron-dense material contained within the apical subacrosomal space, expands within the deepening endonuclear canal. The material of the perforatorium does not originate in the form of an obvious granule as in chicken and budgerigar spermatids. Indications are that in ostrich spermatids the developing acrosome plays a role in the shaping of the tip of the nucleus. The perforatorium, however, appears to represent a residual structure that has no specifically identified function. © 1996 Wiley-Liss, Inc.     

两种石龙子精子超微结构的比较

利用透射电镜研究多线南蜥和印度蜓蜥附睾精子的超微结构。两种卵胎生石龙子的精子具有一些有鳞类精子的共同特征,即具有顶体囊、顶体下锥、单个核前穿孔器和核喙,无核内管,纤维鞘伸入中段,与双联微管3和8相邻的外周致密纤维具双份纤维结构。多线南蜥和印度蜓蜥精子超微结构的种间差异主要表现在:多线南蜥精子核前方的顶体下锥电子密度较小,顶体囊具单侧嵴,横切面上可见非连续的致密体环或11个线粒体;印度蜓蜥无单侧嵴,横切面上可见连续的致密体环或12个线粒体。迄今未发现石龙子科精子的独征,但该科不同类群的顶体囊、顶体下腔、核前方的顶体下锥电子致密程度、核肩、纵切面线粒体与致密体的排列方式、横切面致密体环形状和线粒体等精子超微结构特征有一定程度的差异。这些差异可为研究石龙子科系统发生提供辅助信息。     

The ultrastructure of the spermatozoa of Epipedobates flavopictus (Amphibia,Anura, Dendrobatidae), with comments on its evolutionary significance

We describe, for the first time, the spermatozoon ultrastructure of a dendrobatid frog, Epipedobates flavopictus. Mature spermatozoa of E. flavopictus are filiform, with a moderately curved head and a proportionally short tail. The acrosomal vesicle is a conical structure that covers the nucleus for a considerable distance. A homogeneous subacrosomal cone lies between the acrosome vesicle and the nucleus. The nucleus contains a nuclear space at its anterior end, and electron-lucent spaces and inclusions. No perforatorium is present. In the midpiece, the proximal centriole is housed inside a deep nuclear fossa. Mitochondria are scattered around the posterior end of the nucleus and inside the undulating membrane in the anterior portion of the tail. In transverse section the tail is formed by an U-shaped axial fiber connected to the axoneme through an axial sheath, which supports the undulating membrane. The juxta-axonemal fiber is absent. The spermatozoon of E. flavopictus has several characteristics not observed before in any anurans, such as a curved axial fiber, absence of a juxta-axonemal fiber, and presence of mitochondria in the typical undulating membrane. Our results endorse the view that, in anurans, the conical perforatorium and subacrosomal cone are homologous and that Dendrobatidae should be grouped within Bufonoidea rather than Ranoidea.     

Sperm Ultrastructure of Tarsius bancanus(Tarsiidae, Primates): Implications for Primate Phylogeny and the Use of Sperm in Systematics

The ultrastructure of the epididymal sperm of Tarsius bancanus is described. The sperm possess the typical eutherian pattern of a dorsoventrally flattened, ovate sperm head, comprising a nucleus capped by a symmetrical acrosome, and a distinct midpiece and principal piece containing a 9 + 9 + 2 arrangement of outer coarse fibres and microtubules. However, unique features are also present. The overall head length (9 μm) equals the greatest for any primate yet examined, and the subacrosomal space ("perforatorium" or "pseudoperforatorium") tilted at 30° to the sagittal axis of the sperm, is described for the first time for mammals. The acrosome extends only a short distance beyond the length of the nucleus of the mature sperm, and a significant reduction in the acrosome to nuclei ratio appears to occur during the final stages of sperm maturation. In contrast to earlier predictions based on the spermatid of Tarsius syrhicta , the mature spermatozoa of Tarsius shows greatest morphological similarity with the sperm of the Anthropoidea, which have a short symmetrical acrosome, than with the Strepsirhini, which have a relatively long acrosome that can be either symmetrical (Lemuriformes) or asymetrical (Lorisiformes). Four proposed phylogenies of the Primates are assessed using comparative sperm ultrastructure. Placing the Tarsiidae as a sister group to the Lorisidae appears the least likely. The sperm data are consistent with the Tarsiidae being a sister group to the Anthropoidea, to the Strepsirhini, or even to the extant primate groups as a whole. Use of sperm morphology to provide characters in phylogenetic systematics of the primates is discussed, and the principle of "total evidence" is preferred to the common practice of "hanging" sperm on phylogenetic hypotheses based on other evidence. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

Ultrastructure of the spermatozoon of the American Alligator,Alligator mississippiensis (Reptilia: Alligatoridae)

This study details the ultrastructure of the spermatozoa of the American Alligator, Alligator mississippiensis. American Alligator spermatozoa are filiform and slightly curved. The acrosome is tapered at its anterior end and surrounded by the acrosome vesicle and an underlying subacrosomal cone, which rests just cephalic to the nuclear rostrum. One endonuclear canal extends from the subacrosomal cone through the rostral nucleus and deep into the nuclear body. The neck region separates the nucleus and midpiece and houses the proximal centriole and pericentriolar material. The distal centriole extends through the midpiece and has 9 × 3 sets of peripheral microtubules with a central doublet pair within the axoneme that is surrounded by a dense sheath. The midpiece is composed of seven to nine rings of mitochondria, which have combinations of concentrically and septate cristae. The principal piece has a dense fibrous sheath that surrounds an axoneme with a 9 + 2 microtubule arrangement. The sheath becomes significantly reduced in size caudally within the principal piece and is completely missing from the endpiece. Dense peripheral fibers, especially those associated with microtubule doublets 3 and 8, penetrate into the anterior portion of the principal piece axoneme. The data reported here hypothesize that sperm morphology is highly conserved in Crocodylia; however, specific morphological differences can exist between species. J. Morphol. 2011. © 2011 Wiley‐Liss, Inc.     

Ultrastructural study of spermiogenesis in a rare desert amphisbaenian Diplometopon zarudnyi

Spermiogenesis, in particular the head differentiation of Diplometopon zarudnyi, was studied at the ultrastructural level by Transmission Electron Microscope (TEM). The process includes acrosomal vesicle development, nuclear elongation, chromatin condensation and exclusion of excess cytoplasm. In stage I, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. This step commences with an acrosome vesicle forming from Golgi transport vesicles; simultaneously, the nucleus begins to move eccentrically. In stage II, the round proacrosomal vesicle is flattened by projection of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle as the fibrous layer forms the subacrosomal cone. Circular manchettes surrounded by mitochondria develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. The nucleus has uniform diffuse chromatin with small indices of heterochromatin. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. In stage III, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; coarse granules replace the small ones within the nucleus. One endonuclear canal is present where the perforatorium resides. In stage IV, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the residues of the cytoplasmic lobes are removed. In stage V, the sperm head matures.     

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.     

乌梢蛇精子头部形成的超微结构

摘要：为了解乌梢蛇（Zaocys dhumnades）精子形成的规律,用透射电镜对其头部超微结构进行了观察。结果表明,乌梢蛇精子头部形成可分为4个阶段：阶段Ⅰ,前顶体囊泡内的颗粒物质融合形成1个顶体颗粒而发育为顶体囊泡,随着顶体囊泡的增大,在顶体囊泡与核膜之间形成了致密的纤维物质层。阶段Ⅱ,顶体囊泡变扁平,顶体颗粒分散...     

北草蜥精子的超微结构--兼评不同类群蜥蜴精子形态的差异

应用透射电镜对北草蜥精子的超微结构研究结果表明,北草蜥精子头部顶体囊始终呈圆形,由皮质和髓质组成;顶体囊单侧脊的皮质与髓质问具电子透亮区;穿孔器1个,无穿孔器基板;具顶体下腔;细胞核长形,核内小管缺,核前电子透亮区缺,核肩圆。尾部颈段具片层结构。中段短,多层膜结构缺;纵切面上具2层线粒体;横切面上每圈线粒体6个;2组致密体,具连续的环状结构;线粒体与环状结构的排列模式：rs1／mi1、rs2／mi2;纤维鞘伸人中段,具终环。主段前面部分具薄的细胞质颗粒区;纤维3和8至主段前端消失;轴丝呈“9＋2”型。蜥蜴科内不同种类的线粒体数目不同,但都具有2组致密体。不同类群蜥蜴的顶体囊、顶体下腔、核前电子透亮区、穿孔器基板、核肩,以及线粒体与致密体的数目和排列方式等精子超微结构特征都为研究蜥蜴的系统发生提供了辅助信息。     

An ultrastructural study of the spermatozoa ofEulalia sp. (Phyllodocidae),Lepidonotus sp. (Polynoidae),Lumbrineris sp. (Lumbrineridae) andOwenia fusiformis (Oweniidae)

The ultrastructure of the mature spermatozoa of four polychaetes is described:Eulalia sp. (Phyllodocidae),Lepidonotus sp. (Polynoidae),Lumbrineris sp. (Lumbrineridae) andOwenia fusiformis (Oweniidae). All the sperm show features typical of externally fertilizing sperm in having a rounded nucleus, a short unmodified midpiece, and a simple flagellum with a 9+2 axoneme.Owenia fusiformis andLepidonotus sp. have a nuclear cone extending into the subacrosomal space that may act to present the inner acrosomal membrane to the egg during fertilization. The acrosome ofLumbrineris sp. is flattened and crenulated. The sperm ofEulalia sp. is unusual in having the four mitochondria of the midpiece ensheathed by a membrane. Comparisons are made with other polychaete sperm, and the use of sperm ultrastructure as a taxonomic tool within the Polychaeta is discussed.     

Spermatozoa of Pseudinae (Amphibia, Anura, Hylidae), with a test of the hypothesis that sperm ultrastructure correlates with reproductive modes in anurans

We describe, for the first time, the sperm ultrastructure of the two genera of Pseudinae. Based on sperm ultrastructure, the five species herein examined can be separated into three groups: one containing Pseudis paradoxa, P. bolbodactyla, and P. tocantins, the second containing P. minuta, and the third containing Lysapsus laevis. The midpiece is similar in all species and auxiliary fibers and the undulating membrane are absent. In Pseudis a subacrosomal cone and a multilaminar structure (P. minuta) or a granular material (P. paradoxa group) are seen above the nucleus. Lysapsus laevis has only remnants of the subacrosomal cone. All species have peripheral fibers associated with the outer doublets of the axoneme. We tested the hypothesis of correlation between the presence of an undulating membrane and fertilization environments in anurans using a concentrated changes test (CCT) based on the Hay et al. (Mol Biol Evol 1995;12:928-937) hypothesis of phylogenetic relationships among anuran families. Only a subset of the resolved topologies derived from the Hay et al. (1995) cladogram, where Ranoidea is the sister-group of Sooglossidae, produced significant probabilities of the CCT. Therefore, support for the correlation between sperm ultrastructure and fertilization environments in anurans is, at best, equivocal.     

Ultrastructure of spermiogenesis and the distribution of spermatozoal nuclear histones in the Japanese mantis shrimp,Oratosquilla oratoria (Crustacea: Stomatopoda)

The Japanese mantis shrimp Oratosquilla oratoria (Stomatopoda; Crustacea) is one of the most economically important aquatic species of Pacific shrimp and it is distributed from Japan to the coast of China, the Philippines, the Malay Peninsula, and the Hawaiian Islands. Early studies described certain characteristics of spermatogenesis and the sperm ultrastructure in Stomatopoda, but the composition of sperm basic nuclear proteins (SBNPs) remains completely unknown. We studied the sperm ultrastructure of O. oratoria using transmission electron microscopy and the histone composition using immunofluorescence and immunoelectron microscopy. We found that the spherical nucleus is adjacent to the electron translucent external coat, which occurs in early spermatids. The acrosomal structure begins to form at the junction of the nucleus and the external coat. At the mid-spermatid stage, part of the chromatin appears to be more electron-dense than the external coat side. The aflagellate sperm of O. oratoria, are rounded or slightly ovoid in shape and have a consistent granular nucleus, an acrosome structure of pushpin shape and a spherical vesicular body in which faintly granular material is scattered. The acrosome consists of an acrosomal vesicle, perforatorium, and subacrosomal material. The sperm contains histones H2A, H2B, H3, H4, H3.3, H2AX, and H2AZ as well as some histone modifications, that is, H3K9me3, H3K4me2, H3S10ph, H4Kac, and H2A + H4S1ph. Histones are localized not only in the nucleus of the sperm but also in other structures outside the nucleus. The results may provide new perspectives for systematic studies of crustaceans and their sperm chromatin components. These findings extend the study of the sperm structure of Stomatopoda and provide basic data to elucidate the epigenetic mechanism of fertilization.     

An ultrastructural analysis of mature sperm from the crayfish Pacifastacus 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.     

A comparison of the spermatozoa of OratosquiMa stephensoni and Squilla mantis (Crustacea, Stomatopoda) with comments on the phylogeny of the Malacostraca

Each stomatopod sperm, aflagellate and obovoid, is surrounded by an electron dense coat. A spermatophore is absent. The discus-shaped acrosome vesicle is penetrated and underlain by a straight, slender acrosome rod (perforatorium) ensheathed, below the vesicle, in subacrosomal material. Feulgen-positive granular material, indicating chromatin, fills most of the length of the cell but there is no certain nuclear membrane. Two centrioles, consisting of doublets each with a radial “foot” as in decapods and peracarids, occur near the acrosome and like it are embedded in the chromatin. Myelin-like membranes are associated with degenerating mitochondria in the posterior region of the cell. Thiéry-positive granules are aggregated as a glycogen body posteriorly in the cell. Spermatozoa1 ultrastructure confirms monophyly of the mysid–amphipod–isopod– cumacean section of the Peracarida but affinities of the tanaids are uncertain. A closer phylogenetic relationship of Phyllocarida to the Branchiopoda than to the Malacostraca is suggested. A weak similarity (synapomorphy?) of syncarids and peracarids is the filiform perforatorium bipassing the nucleus. Stomatopods resemble decapods in their diffuse sperm chromatin but are placed below the syncarid–peracarid–decapod assemblage.     

An ultrastructural comparative study of the sperm of<Emphasis Type="Italic"> Hyla pseudopseudis</Emphasis>,<Emphasis Type="Italic"> Scinax rostratus</Emphasis>, and<Emphasis Type="Italic"> S. squalirostris</Emphasis> (Amphibia: Anura: Hylidae)

We make detailed comparisons of the ultrastructure of the spermatozoon among three species of the family Hylidae, Hyla pseudopseudis, Scinax rostratus, and S. squalirostris. The acrosome complex consists of two conical structures covering the nuclear rostrum, the acrosome vesicle, and the subacrosomal cone. The nucleus has a moderately condensed chromatin with a conical shape in longitudinal sections and a circular shape in cross-sections. In H. pseudopseudis, mitochondria are numerous and circular, and in S. rostratus and S. squalirostris there are fewer mitochondria that are more elongate in longitudinal and transverse sections. In H. pseudopseudis, the mitochondrial collar starts adjacent to the distal centriole, occupying the whole midpiece, whereas in both Scinax species the mitochondrial collar starts only at the posterior one-third of the midpiece. In both Scinax species, the presence of juxta-axonemal fiber, axial sheath, and axial fiber in the tail are seemingly plesiomorphic characters, widespread among bufonoid frogs. In H. pseudopseudis, however, the absence of axial fiber and axial sheath seems to be derived from the typical bufonoid condition. The differences between Hyla and Scinax sperm endorse the separation of the two genera and suggest that sperm ultrastructure can be a useful tool to investigate relationships at the intrafamily level.     

C-terminal kinesin motor KIFC1 participates in acrosome biogenesis and vesicle transport

We have identified a possible role for the KIFC1 motor protein in formation of the acrosome, an organelle unique to spermatogenesis. KIFC1, a C-terminal kinesin motor, first appears on membrane-bounded organelles (MBOs) in the medulla of early spermatids followed by localization to the acrosomal vesicle. KIFC1 continues to be present on the acrosome of elongating spermatids as it flattens on the spermatid nucleus; however, increasing amounts of KIFC1 are found at the caudal aspect of the spermatid head and in distal cytoplasm. The KIFC1 motor is also found in the nucleus of very immature round spermatids just prior to its appearance on the acrosome. In some cases, KIFC1 appears localized just below the nuclear membrane adjacent to the subacrosomal membrane. We demonstrate that KIFC1 is associated with importin beta and colocalizes with this nuclear transport factor on curvilinear structures associated with the spermatid nuclei. These data support a model in which KIFC1, perhaps in association with nuclear factors, assists in the formation and/or elongation of the spermatid acrosome. This article represents the first demonstration of a direct association of a molecular motor with the spermatid acrosome, the formation of which is essential for fertilization.     

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm

Sperm from the American lobster (Homarus americanus) are normally nonmotile. However, during fertilization, the sperm undergo a calcium-dependent acrosome reaction that propels them forward about 18 μMm. The reaction occurs in two phases, eversion and ejection, which take place too quickly to permit analysis by direct observation. The purposes of this study were to examine the structural changes occurring in sperm during the normal acrosome reaction and to determine the rate of the reaction using video microscopy. The reaction was induced in vitro by ionophore A23187 and recorded using a video system attached to a Nikon Nomarski interference microscope. Videotapes were played back frame by frame (30 frames/sec), and images of reactions from 10 sperm were analyzed. The acrosome reaction, including the eversion of the acrosomal vesicle and ejection of the subacrosomal material and nucleus, can be divided into 4 steps: (1) expansion of the apical cap followed by expansion of the remainder of the acrosomal cylinder; expansion of the cylinder begins at its apical end and proceeds toward its base, (2) eversion of the apical half of the acrosomal vesicle and initial contraction of the apical cap, (3) eversion of the basal half of the acrosomal vesicle, continued contraction of the apical cap, and ejection of the subacrosomal material and nucleus, and (4) final contraction of the apical cap and ejection of the acrosomal filament. During steps 2, 3, and 4, the mean forward movement of sperm is 12.7, 3.9, and 1.1 μMm, respectively. Although the time required to complete the reaction ranged from 0.66 to 5.16 s, most sperm reacted in less than 3. s, and these sperm were considered to have typical rates. For sperm that reacted in less than 3 s, both step 1 and step 4 take about 0.2 s and show little variation among sperm. the time required to complete steps 2 and 3 averaged 0.63 and 0.37 s, respectively. Forward movement of the sperm during the acrosome reaction is caused by eversion of the inner and outer acrosomal material and contraction of the apical cap. The protein(s) responsible for this contraction is not yet known. © 1993 Wiley-Liss, Inc.     

Fine structure of the spermatozoon of <Emphasis Type="Italic">Diopatra neapolitana</Emphasis> (Polychaeta,Onuphidae)

The identification of Diopatra species lacks of clear diagnostic features of taxonomic importance and the knowledge of their reproductive characters is scant. The spermatozoa of Diopatra neapolitana were ultrastructurally investigated by electron microscopy in order to correlate the mode of reproduction with sperm cells morphology. The mature male gamete has a depressed subspherical nucleus, a cone-like acrosome, and a long flagellum. The acrosome is conical in shape and radially symmetrical, with a base diameter twice the height. Within the acrosome vesicle, the basal region includes a very electron-dense thickened ring composed of paracrystalline substances. The subacrosomal space is filled with a poorly electron-dense material, with straight filaments axially arranged to form a perforatorium. The nucleus contains the complete axial canal, holding the hind perforatorium region. The middle piece consists of five mitochondria with well-distinct membranes and tubulo-vesicular cristae. Two centrioles are located perpendicularly to each other. The proximal one lies in the central fossa and the distal one, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a 9+2 arrangement of microtubules. In general, the spermatozoon of D. neapolitana conforms exteriorly to the typical ect-aquasperm; the acrosome complex ultrastructure, however, shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning—free-swimming larvae) of D. neapolitana belonging to Santa Gilla population, and may be helpful to solve the taxonomic problems of the D. neapolitana complex as well.     

SPERMOGENESIS AND SPERM STRUCTURE IN THREE SPECIES OF PATELLOIDA AND ONE SPECIES OF NIPPONACMAEA (PATELLOGASTROPODA: ACMAEOIDEA)

The spermatozoa of Patelloida profunda albonotata, P. saccharina,P. pygmaea and Nipponacmaea schrenkii (Lottiidae) are describedby transmission electron microscopy. All have ect-aquasperm,typical of invertebrates using external fertilization. The spermof all four species have a cylindrical nucleus (length: breadth> 4: 1 in P. p. albonotata and P. saccharina; <4: 1 inP. pygmaea and N. schrenkii) which tapers towards the roundedanterior end. All have an acrosome with a posterior acrosomallobe which extends into the centre of the subacrosomal space.In P. p. albonotata and P. saccharina the acrosomal contentsare undifferentiated and the posterior lobe extends to the nucleus,being separated from it by flocculent material. In P. pygmaeathe acrosomal contents are differentiated, the lobe is relativelyshort and the subacrosomal space is filled with material witha fibrous appearance. The acrosome of N. schrenkii is undifferentiatedand the posterior lobe is no more than a bulge. The sperm ofP. p. albonotata and N. sacchrina have a small (0.25 µmlong) cytoplasmic collar which surrounds the axoneme anteriorlywhereas in P. pygmaea and N. schrenkii the cytoplasmic collaris longer (1 µm) and is swollen by an electron-dense vesicle.The composition and function of this vesicle is unknown. Thespermatozoa of Patelloida and Nipponacmaea have structural featureswhich are similar to sperm of the Lottiidae providing some supportfor the placement of these genera in the Lottiidae as proposedby Lindberg & Hedegaard (1996) and Sasaki & Okutani(1993) respectively. The similarities of the sperm of P. pygmaeato N.schrenkii raise some doubts about the tax-onomic statusof the former species. Spermiogenesis in all four species issimilar to that described for other Acmaeoidea and Patelloidea.In P. pygmaea and N. schrenkii, however, in addition to theacrosomal vesicle, the Golgi body produces a number of electron-densevesicles which fuse and eventually form a single vesicle inthe collar of the mid-piece. (Received 24 October 1996; accepted 10 February 1997)