An ultrastructural study of the cuticle in the marine annelid Heterodrilus (Tubificidae, Clitellata)

The ultrastructure of the cuticle in four species of the marine Heterodrilus (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus) is investigated with transmission electron microscopy. The noncellular cuticle consists of several parts; closest to the epidermis is a thick zone of collagen fibers embedded in a matrix. The matrix continues outside the fiber zone, forming a layered epicuticle. The external surface of the epicuticle is covered by evenly distributed, membrane-bound bodies, termed epicuticular projections. The epicuticular projections have their longitudinal axis perpendicular to the surface of the cuticle and are attached to the surface by either the surrounding membrane itself or by short pedestals. Microvilli, extensions from the epidermal cells, penetrate and sometimes pass completely through the cuticle. There is interspecific variation in the morphology of the cuticle. The four studied species differ in the arrangement of the collagen fibers, from irregularly distributed fibril bundles to orthogonally arranged fiber layers, as well as in the number and density of layers in the epicuticle. One of the studied species, H. paucifascis, shows intraspecific variation, which is associated with sample locality. The Bahamian specimens of H. paucifascis have four layers in the epicuticle, club-shaped epicuticular projections, and collagen fibers forming a less defined orthogonal grid, while the Belizean specimens have three layers in the epicuticle, epicuticular projections with a bulging part at midlevel, and a distinct orthogonal grid. Based on these findings the variation in the morphology of the cuticle appears to be dependent on both phylogenetic constraints, and functional and environmental factors.     

Morphology and ultrastructure of Siberian sturgeon (Acipenser baerii) spermatozoa using scanning and transmission electron microscopy

BACKGROUND INFORMATION: Available data concerning the sperm morphology of teleost fishes demonstrate wide variation. In the present study, the spermatozoa of Siberian sturgeon (Acipenser baerii Brandt, 1869), a chondrostean fish, was investigated. In contrast with teleost fish, chondrostean spermatozoa have a head with a distinct acrosome, whereas other structures, such as a midpiece and a single flagellum, are present in spermatozoa of most species. RESULTS: The average length of the head including the acrosome and the midpiece was 7.01+/-0.83 microm. Ten posterolateral projections derived from the acrosome were present on a subacrosomal region, with mean lengths of 0.94+/-0.15 microm and widths of 0.93+/-0.11 microm. The nucleus consisted of electrodense homogeneous nuclear chromatin. Three intertwining endonuclear canals, bound by membranes, traversed the nucleus longitudinally from the acrosomal end to the basal nuclear fossa region. There were between three and six mitochondria, two types of centrioles (proximal and distal) in the midpiece and two vacuoles composed of lipid droplets. The flagellum (44.75+/-4.93 microm in length), originating from the centriolar apparatus, had a typical 9+2 eukaryotic flagellar organization. In addition, there was an extracellular cytoplasm canal between the cytoplasmic sheath and the flagellum. CONCLUSIONS: A principal components analysis explained the individual morphological variation fairly well. Of the total accumulated variance, 41.45% was accounted for by parameters related to the head and midpiece of the sperm and the length of the flagellum. Comparing the present study with previous studies of morphology of sturgeon spermatozoa, there were large inter- or intra-specific differences that could be valuable taxonomically.     

Ultrastructural shape and three-dimensional organization of the intracuticular canal systems in the mineralized cuticle of the green crab Carcinus maenas

Two main self-contained canal systems are present in the crab mineralized cuticle. The first, or fibre canal system, is constituted by simple, unbranched vertical canals containing axially running fibres in close association with myoepidermal junctions. The second, or pore canal system, is composed of procuticular pore canals and epicuticular channels that prolong the procuticular canals. In opposition to widespread opinion, pore canals make up a three-dimensional branched system extending from the apical plasma membrane of the epidermis up to the epicuticle. Branching occurs by projections of lateral horizontal from the vertical canals at the lower level of the pigmented layer and by innumerable ramifications of epicuticular canals. In agreement with Neville's model for insects, vertical procuticular pore canals of crustacean mineralized cuticle, and also fibre canals, exhibit a twisted ribbon structure reflecting the helicoidal arrangement of the horizontal chitin-protein microfibrils.     

Cuticular ultrastructure in some marine oligochaetes (Tubificidae)

Abstract. The ultrastructure of the thin, non-cellular cuticle is described for 6 marine oligochaetes, representing 3 of the subfamilies (Phallodrilinae, Limnodriloidinae, and Rhyacodrilinae) of the Tubificidae. The main components of the cuticle in these 6 species, as in most other oligochaetes examined, are: (1) a fiber zone closest to the epidermis, consisting of collagen fibers embedded in a matrix, (2) an epicuticle, which is a continuation of the matrix outside the fiber zone, and (3) epicuticular projections, which are membrane-bound bodies covering the outer surface of the epicuticle. The projections are probably formed by the microvilli that penetrate the cuticle from the epidermal cells below, but this was confirmed only in the studied limnodriloidines. Three of the species examined, Duridrilus turdus, Olavius vacuus , and Heterodrilus paucifascis , lack microvilli. The morphology of the components in the cuticle differs between the studied species. The collagen fibers may form an "orthogonal grid" (i.e., layers of parallel fibers perpendicular to the layers immediately above and below), or they may form parallel layers, or be irregularly scattered. The number of dense layers in the epicuticle, as well as the shape and internal structure of the epicuticular projections, also vary. All these characters might be useful in future phylogenetic analyses to achieve better hypotheses of relationships within oligochaetes as well as to other groups.     

Ultrastructural study of spermatozoa of the paddlefish, Polyodon spathula

Paddlefish, Polyodon spathula, spermatozoa were examined by transmission electron microscopy. Their structure has the same characteristic architectural features as sturgeon spermatozoa. Paddlefish spermatozoa are of the primitive type and consist of a rod-shaped head, a midpiece and a long flagellum. The head is about 5.15 mm in length and contains the nucleus and an apical acrosomal complex. Inside the nucleus there are three nuclear channels that begin in the subacrosomal area and have a triple helical arrangement. An nuclear fossa is present centrally, at the posterior end of the nucleus. The midpiece contains a pair of centrioles in a perpendicular arrangement, mitochondria and a narrow cytoplasmic sleeve. The flagellum has a central axoneme with a 9 + 2 pattern and two lateral projections or fins.     

Fine structure and morphology of sterlet (Acipenser ruthenus L. 1758) spermatozoa and acrosin localization

Ultrastructure of sterlet Acipenser ruthenus L. 1758 sperm was examined by scanning and transmission electron microscopy, which allowed us to use various methods for visualizations of different parts of sterlet spermatozoa. Sperm cells possess a head with a distinct acrosome, a midpiece and a single flagellum surrounded by the flagellar plasma membrane. The average length of the head including the acrosome and the midpiece was estimated as 5.14+/-0.42 microm. Nine to 10 posterolateral projections were derived from the acrosome. Three inter-twining endonuclear canals bounded by membranes traversed the nucleus in its whole length from the acrosome to the implantation fossa. Acrosin was located in all the three parts (acrosome, endonuclear canals and implantation fossa). The proximal and distal centrioles located in the midpiece compacted of nine peripheral triplets of microtubules. One cut of the midpiece contained from two to six mitochondria with area of 215+/-85 nm(2) in average. The flagellum was 42.47+/-1.89 microm in length with typical eukaryotic organization of one central pair and nine peripheral pairs of microtubules. It passed through a cytoplasmic channel in the midpiece, which was formed by an invagination at the plasmalemma. The flagellum gradually developed two lateral extensions of its plasma membrane, so-called "fins". Detected morphological variation can be described by four principal component axes corresponding to groups of individual morphometric characters defined on the sperm structures. Correlations among the characters indicate that the sperms are variable in their shape rather than size. Significant variation among examined fish individuals was found only in flagellum and nucleus length. Comparison between the present and previous studies of morphology of sturgeon spermatozoa confirmed large inter- and/or intra-specific differences that could be of substantial taxonomic value.     

Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

The structure of the cuticle and sheath of the infective juvenile of Trichostrongylus colubriformis

The infective third-stage juvenile of Trichostrongylus colubriformis is surrounded by its own cuticle as well as the incompletely moulted cuticle of the second-stage juvenile, which is referred to as the sheath. The sheath comprises an outer epicuticle, an amorphous cortical zone, a fibrous basal zone and an inner electron-dense layer. The basal zone of the sheath consists of three layers of fibres; the fibres are parallel within each layer, but the fibre direction of the middle layer is at an angle to that of the inner and outer layers. The cuticle comprises a complex outer epicuticle, an amorphous cortical zone and a striated basal zone. The lateral alae of the cuticle and the sheath are aligned and overlie the lateral hypodermal cords. The lateral alae of the sheath consist of two wing-like expansions of the cortical zone with associated specializations of the inner electron-dense layer which form a groove. The cuticular lateral alae consist of two tube-like expansions of the cortical zone. The lateral alar complex of the cuticle and the sheath may maximise locomotory efficiency and prevent rotation of the juvenile within the sheath.     

Sperm ultrastructure of an oviparous and an ovoviviparous onychophoran species (Peripatopsidae) with some phylogenetic considerations

The spermatozoa of the Australian oviparous Ooperipatellus insignis and the South African ovoviviparous Opisthopatus cinctipes (both: Onychophora, Peripatopsidae) were studied and compared with the spermatozoal patterns already described in the taxon. The spermatozoa of both species conform with the general plan described for the Onychophora: they are filiform cells formed, in sequence, by an elongated, fully condensed nucleus capped by an acrosome and surrounded by several spiral ridges; by a mitochondrial midpiece characteristically interpolated between the nucleus and a characteristic flagellum. Major differences between the spermatozoa of both species concern their acrosome organization. The correlation between the acrosomal pattern and the size and structure of the ovarial eggs (oocytes) in onychophorans has been investigated. A parsimony analysis was performed on 21 spermatozoal characters of the species considered. Its results are congruent with those of the traditional systematics. A new set of autapomorphies characterising onychophoran sperm is suggested and some of the spermatological homologies proposed between Onychophora and Euclitellata spermatozoa are critically discussed. Our analysis suggests that spermatozoal characters are good phylogenetic markers among onychophorans, also at low taxonomic level.     

Morphology and fine structure of Acipenser persicus (Acipenseridae, Chondrostei) spermatozoon: Inter-species comparison in Acipenseriformes

This study describes morphology and fine structure of the Persian sturgeon (Acipenser persicus) (Acipenseridae, Chondrostei) spermatozoon. The results show that the spermatozoon of A. persicus is differentiated into an elongated head (length: mean±SD: 7.1±0.5μm) with an acrosome (length: 1.2±0.2μm), a cylindrical midpiece (length: 1.8±0.5μm), a flagellum (length: 50.3±5.9μm) and a total length of 59.2±6.2μm. Ten posterolateral projections (PLPs) arise from the posterior edge of the acrosome and there were 3 endonuclear canals that traversed the nucleus from the acrosomal end to the basal nuclear fossa region. Three to six mitochondria were in peripheral midpiece and the proximal and distal centrioles were located near to "implantation fossa" and basement of the flagellum. The axoneme has a typical eukaryotic structure composed of 9 peripheral microtubules and a central pair of single microtubule surrounded by the plasma membrane. Lateral fins were observed along the flagellum. The fins started and ended at 0.5-1μm from midpiece and at 4-6μm from the end of flagellum. There were significant differences in the size of almost all measured morphological parameters between males and flagellar, midpiece and nucleus characters were more isolated parameters that can be considered for detecting inter-individual variations. This study showed that sperm morphology and fine structure are similar among sturgeon species, but the dimensions of the parameters may differ.     

Ultrastructural organization of hypodermis and formation of cuticle in pharate larva of Leptotrombidium orientale (Acariformes: Trombiculidae)

The ultrastructural organization of hypodermis and the process of cuticle deposition is described for the pharate larvae of a trombiculid mite, Leptotrombidium orientale, being under the egg-shell and prelarval covering. The thin single-layered hypodermis consists of flattened epithelial cells containing oval or stretched nuclei and smooth basal plasma membrane. The apical membrane forms short scarce microvilli participating in the cuticle deposition. First of all, upper layers of the epicuticle, such as cuticulin lamella, wax and cement layers, are formed above the microvilli with plasma membrane plaques. Cuticulin layer is seen smooth at the early steps of this process. Very soon, however, epicuticle starts to be curved and forms particular high and tightly packed ridges, whereas the surface of hypodermal cells remains flat. Then a thick layer of the protein epicuticle is deposited due to secretory activity of hypodermal cells. Nearly simultaneously the thick lamellar procuticle starts to form through the deposition of their microfibrils at the tips of microvilli of the apical plasma membrane. Procuticle, as such, remains flat, is situated beneath the epicuticular ridges and contains curved pore canals. Cup-like pores in the epicuticle provide augmentation of the protein epicuticle mass due to secretion of particular substances by cells and to their transportation through the pore canals towards these epicuticular pores. The very beginning of the larval cuticle formation apparently indicates the starting point of the larval stage in ontogenesis, even though it remains for some time enveloped by the prelarval covering or sometimes by the egg-shell. When all the processes of formation are over, hungry larvae with a fully formed cuticle are actively hatched from two splitted halves of prelarval covering.     

Detergent treatment of human and rabbit spermatozoa: ultrastructural changes and release of midpiece enzymes

To aid the study of spermatozoa acrosome, ultrastructural changes and midpiece enzyme release were studied in human and rabbit spermatozoa treated with various detergents. Dissolution of the spermatozoa plasma membrane and acrosome was caused by washing with Hyamine 2389 (.15%) and Hyamine 2389 (.075%) with Triton X-100 (.075%). Variable damage to the structures of the tail and midpiece was greater with Hyamine alone than when combined with Triton. Spontaneous midpiece enzyme release and the appearance of aspartate aminotransferase and alanine aminotransferase occurred in the presence of physiological saline. Sonication of the spermatozoa resulted in a greater release of midpiece enzymes than with the detergent treatment. It is concluded that detergent treatment results in more morphologic changes than previously believed, and that although the detergent extracts contain largely acrosomal proteins, ultrastuctural, histochemical and immunohistochemical techniques are necessary for specific localization of a sperm enzyme.     

The ultrastructure of the cuticle of some British lumbricids (Annelida)

The ultrastructure of the cuticle of 11 species of British lumbricids is described. The number of unbanded collagenous fibre layers varies with the species and is roughly proportional to the size of the adult worm. Four zones are discernible in the cuticle matrix in all species except E. foetida where the outermost zone shows subdivision.
Microvilli, cytoplasmic extensions from the epithelial surface, occur. The "long" and "short" microvilli of other workers are shown to be different views of the same structure. The microvilli have an ovoid base with the two poles forming low shoulders on either side of the ascending microvillus. The bases are oriented at right-angles to the longitudinal axis of the worm and the microvilli are arranged in regular staggered rows along the same axis. Details of the spatial alignment of the microvilli are given and the possible role of these regularly arranged structures as factors in the orientation of the collagen fibre unit filaments is discussed, and it is speculated that they might also have some proprioceptive function.
Distally the microvilli terminate among the surface epicuticular projections and are shown to give rise to them. The epicuticular projections are peanut-shell shaped with a distinct substructure consisting of a 8 nm electron dense lining and two parallel dense discs, each 8 nm deep, above the "waist".
The mucous cell pores are lined with electron pale microfibrillar material and at the base of the pore a circlet of 13–15 short microvilli, with prominent tonofilaments, arises from the cytoplasm of the mucous cell. Surrounding the pore microvilli are numerous, small, membrane bound mucous pore particles.     

蓝尾石龙子精子的超微结构

蓝尾石龙子(Eumeces elegans)附睾以2．5％戊二醛和1％锇酸双重固定,按常规制作超薄切片,用H-600透射电镜研究观察精子的超微结构。精子由头部和尾组成,头部由顶体复合体和核组成,尾由颈段、中段、主段和末段组成。头部的顶体囊前部扁平,分为皮质和髓质,顶体下锥由类结晶状的顶体下物质组成,穿孔器顶端尖,、穿孔器基板塞子状,细胞核延长,核内小管缺,核伸展部前端具一电子透明区,核肩圆,核陷窝锥形。颈段具片层结构,近端中心粒和远端中心粒的长轴呈直角,9束外周致密纤维与远端中心粒相应的9束三联微管相联,向后与轴丝相应的9束双联微管相联,中央纤维与2个中央单微管相联。中段短,含有线状嵴的柱状线粒体,由连续的规则小卵状或小梯形致密体组成线粒体间的环状结构,纤维鞘伸入中段,终环紧贴于细胞膜的内表面。线粒体与环状结构的模式为：rs1／mi1,rs2／mi2,rs3／mi3,rs4／mi4,横切面上每圈线粒体数目为10个。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。轴丝复合体呈“9 2”型。蓝尾石龙子精子超微结构与已描述的石龙子科种类比较发现,与蜓蜥群和胎生群的石龙子相似;但没有发现石龙子科精子的独征。     

Ultrastructural morphometry of the human sperm flagellum with a stereological analysis of the lengths of the dense fibres

The dimensions of the various regions of the flagellum and the length of each of the dense fibres has been determined by transmission electron microscopy of a large number of spermatozoa from ten men. The overall mean length of the flagellum was 60.5 micron, and its diameter diminished from 0.88 micron in the midpiece to 0.17 micron at the terminal filament. The midpiece and terminal filament as measured in longitudinal sections had variable lengths among spermatozoa (3.4 +/- 0.5 (S.D.) micron and 3.1 +/- 1.0 micron respectively). Stereological analysis was used to estimate the length of the principal piece (53 micron) and the dense fibres. These latter fibres were of unequal length and extended along 60% of the length of the principal piece. They fell into 3 groups with respect to their lengths: (i) fibres 3 and 8 were short (6 micron); (ii) fibres 4, 2 and 7 were of medium length (17, 18 and 21 micron respectively); and (iii) the longest fibres were 5, 6, 9 (31, 32 and 31 micron respectively) and fibre 1 which was a little longer (35 micron). Although there was variation in the length of the various fibres among spermatozoa, the order of their termination was relatively constant. The relationship between these quantitative data regarding the structural characteristics of the dense fibres and the shape of the flagellar wave is discussed.     

The ultrastructure of spermatozoa and spermiogenesis in pyramidellid gastropods,and its systematic importance

Ultrastructural observations on spermiogenesis and spermatozoa of selected pyramidellid gastropods (species ofTurbonilla, Pyrgulina, Cingulina andHinemoa) are presented. During spermatid developement, the condensing nucleus becomes initially anterio-posteriorly compressed or sometimes cup-shaped. Concurrently, the acrosomal complex attaches to an electrondense layer at the presumptive anterior pole of the nucleus, while at the opposite (posterior) pole of the nucleus a shallow invagination is formed to accommodate the centriolar derivative. Midpiece formation begins soon after these events have taken place, and involves the following processes: (1) the wrapping of individual mitochondria around the axoneme/coarse fibre complex; (2) later internal metamorphosis resulting in replacement of cristae by paracrystalline layers which envelope the matrix material; and (3) formation of a glycogen-filled helix within the mitochondrial derivative (via a secondary wrapping of mitochondria). Advanced stages of nuclear condensation (elongation, transformation of fibres into lamellae, subsequent compaction) and midpiece formation proceed within a microtubular sheath (‘manchette’). Pyramidellid spermatozoa consist of an acrosomal complex (round to ovoid apical vesicle; column-shaped acrosomal pedestal), helically-keeled nucleus (short, 7–10 μm long, shallow basal invagination for axoneme/coarse fibre attachment), elongate helical midpiece (composed of axoneme, coarse fibres, paracrystalline and matrix materials, glycogen-filled helix), glycogen piece (length variable, preceeded by a dense ring structure at junction with midpiece). The features of developing and mature spermatozoa observed in the Pyramidellidae are as observed in opisthobranch and pulmonate gastropods indicating that the Pyramidelloidea should be placed within the Euthyneura/Heterobranchia, most appropriately as a member group of the Opisthobranchia.     

Ultrastructural morphometry of the human sperm flagellum with a stereological analysis of the lengths of the dense fibres

The dimensions of the various regions of the flagellum and the length of each of the dense fibres has been determined by transmission electron microscopy of a large number of spermatozoa from ten men. The overall mean length of the flagellum was 60.5 micron, and its diameter diminished from 0.88 micron in the midpiece to 0.17 micron at the terminal filament. The midpiece and terminal filament as measured in longitudinal sections had variable lengths among spermatozoa (3.4 +/- 0.5 (S.D.) micron and 3.1 +/- 1.0 micron respectively). Stereological analysis was used to estimate the length of the principal piece (53 micron) and the dense fibres. These latter fibres were of unequal length and extended along 60% of the length of the principal piece. They fell into 3 groups with respect to their lengths: (i) fibres 3 and 8 were short (6 micron); (ii) fibres 4, 2 and 7 were of medium length (17, 18 and 21 micron respectively); and (iii) the longest fibres were 5, 6, 9 (31, 32 and 31 micron respectively) and fibre 1 which was a little longer (35 micron). Although there was variation in the length of the various fibres among spermatozoa, the order of their termination was relatively constant. The relationship between these quantitative data regarding the structural characteristics of the dense fibres and the shape of the flagellar wave is discussed.     

Ultrastructure and morphology of spermatozoa in Chinese sturgeon (Acipenser sinensis Gray 1835) using scanning and transmission electron microscopy

The Chinese sturgeon (Acipenser sinensis Gray 1835) is an endangered anadromous sturgeon inhabiting the Yangtze River in China. In this study, the ultrastructure and morphology of spermatozoa was studied using transmission and scanning electron microscopy with a cryo-holder. The spermatozoon consisted of an elongated head with a distinct acrosome and nucleus region, a midpiece and a flagellum. The mean length of the head and midpiece, the flagellum and total length of spermatozoon were 4.48, 33.3 and 37.8 microm, respectively. The nucleus was an elongated trapezoid shape with anterior (acrosome) end narrower than the posterior. Granular material and an actin filament were observed within the anterior acrosome. Three to five endonuclear canals were present. The midpiece was eudipleural along its longitudinal axis. Compared to other sturgeon species, the data from the present study suggest a more recent evolutionary linkage between Chinese sturgeon and white sturgeon (Acipenser transmontanus Richardson 1836).     

Ultrastructure of spermatogenic cells and spermatozoa in Hoplias malabaricus (Teleostei, Characiformes, Erythrinidae)

The differentiation of spermatids in Hoplias malabaricus is characterized by chromatin compaction, flagellum development, nuclear rotation, nuclear fossa formation, and excess cytoplasm elimination. In the resulting spermatozoon, the head is round and the nucleus contains chromatin compacted in thick filaments, peripherically arranged, to a central electron-lucent area. The acrosome is absent. The nuclear fossa is eccentric but not pronounced. The proximal centriole penetrates it and is oblique to the flagellum. The long midpiece has several converging elongate vesicles, forming membranous hoops in the initial segment of the flagellum, but has no cytoplasmic channel. The mitochondria are elongate and branched or C-shaped and located around the initial segment of the axoneme. The lateral flagellum does not show lateral projections. The ultrastructural characteristics of H.malabaricus spermatozoa are similar to the Cypriniformes.