Slanted centriole and transient anchoring apparatus during spermiogenesis of an Oligochaete (Annelida)

An electron microscopic analysis of Tubifex tubifex (Annelida, Oligochaeta) spermiogenesis has revealed the presence of a slanted basal body during the early stages: the centriole has the shape of a cylinder obliquely cut at one extremity. In these stages an anchoring apparatus is also present, similar to the one of primitive spermatozoa, and disappearing in the mature sperm. The slanted centriole in cross section gives images of “incomplete” centrioles similar to the ones reported in the literature and interpreted as images of centriologenesis. The transient anchoring apparatus is interpreted as a rudimentary organelle used during spermiogenesis to maintain the centriole in a correct position. This function in the mature spermatozoon is performed by the mitochondria.     

Centrosome reduction during Rhesus spermiogenesis: gamma-tubulin, centrin, and centriole degeneration

Centrosome reduction during spermiogenesis has been studied using anti-gamma-tubulin and anti-centrin antibodies and electron microscopy in nonhuman primates. Rhesus spermatids possess apparently normal centrosomes comprising a pair of centrioles associated with gamma-tubulin and centrin. However, they do not nucleate detectable microtubules. The spermatids discard gamma-tubulin in the residual bodies during the spermiation stage. Mature sperm do not have any detectable gamma-tubulin. About half of the centrin associated with the distal centriole degenerates during spermiogenesis and the remainder is intimately bound to the centriolar microtubules. The mature sperm possess highly degenerated distal centrioles. The centriolar microtubules degenerate in the rostral region and the ventral side of the sperm. The study indicates that the centrosome is reduced during rhesus spermiogenesis, but not completely as in mice.     

Cyclin O (Ccno) functions during deuterosome-mediated centriole amplification of multiciliated cells

Mucociliary clearance and fluid transport along epithelial surfaces are carried out by multiciliated cells (MCCs). Recently, human mutations in Cyclin O (CCNO) were linked to severe airway disease. Here, we show that Ccno expression is restricted to MCCs and the genetic deletion of Ccno in mouse leads to reduced numbers of multiple motile cilia and characteristic phenotypes of MCC dysfunction including severe hydrocephalus and mucociliary clearance deficits. Reduced cilia numbers are caused by compromised generation of centrioles at deuterosomes, which serve as major amplification platform for centrioles in MCCs. Ccno-deficient MCCs fail to sufficiently generate deuterosomes, and only reduced numbers of fully functional centrioles that undergo maturation to ciliary basal bodies are formed. Collectively, this study implicates CCNO as first known regulator of deuterosome formation and function for the amplification of centrioles in MCCs.     

A cytochemical study of the Golgi apparatus of the spermatid during spermiogenesis in the rat

The reactivity of the various components of the Golgi apparatus of rat spermatids for three phosphatase activities (nicotinamide adenine dinucleotide phosphatase, NADPase; thiamine pyrophosphatase, TPPase; cytidine monophosphatase, CMPase) and the incorporation of 3H-fucose by the spermatids was analyzed at the 19 steps of spermiogenesis, i.e., during and after this organelle elaborated the glycoprotein-rich acrosomic system. During steps 1-3, the Golgi apparatus produced, in addition to the proacrosomic granules, multivesicular bodies that became associated with the chromatoid body. NADPase was located within the four of five intermediate saccules of Golgi stacks, and TPPase was found in the last one or two saccules on the trans aspect of the stacks from steps 1 to 17 of spermiogenesis. CMPase was located within the thick saccular GERL elements found in the trans region of the Golgi apparatus from steps 1 to 7 of spermiogenesis, but the CMPase-positive GERL disappeared from the Golgi apparatus after its detachment from the acrosomic system at step 8. Th acrosomic system itself was reactive from CMPase and TPPase but was negative for NADPase, while the multivesicular bodies were CMPase and NADPase positive but unreactive for TPPase. Tritiated-fucose was readily incorporated within the Golgi apparatus of steps 1-17 spermatids; in steps 1-7 it was subsequently incorporated within the acrosomic system and multivesicular bodies. These various data indicated (1) that the Golgi apparatus of spermatids, although it loses its CMPase-positive GERL element in step 8, retains evidence of functional capacity until it degenerates in step 17; (2) that in early spermatids the various saccular components of the Golgi are specialized with respect to enzymatic activities; and (3) that each Golgi region may contribute in a coordinated fashion to the formation of the acrosomic system and multivesicular bodies.     

Preparing to move: assembly of the MSP amoeboid motility apparatus during spermiogenesis in Ascaris

We exploited the rapid, inducible conversion of non-motile Ascaris spermatids into crawling spermatozoa to examine the pattern of assembly of the MSP motility apparatus that powers sperm locomotion. In live sperm, the first detectable motile activity is the extension of spikes and, later, blebs from the cell surface. However, examination of cells by EM revealed that the formation of surface protrusions is preceded by assembly of MSP filament tails on the membranous organelles in the peripheral cytoplasm. These organelle-associated filament meshworks assemble within 30 sec after induction of spermiogenesis and persist until the membranous organelles are sequestered into the cell body when the lamellipod extends. The filopodia-like spikes, which are packed with bundles of filaments, extend and retract rapidly but last only a few seconds before giving way to, or converting into, blebs. Coalescence of these blebs, each supported by a dense mesh of filaments, often initiates lamellipod extension, which culminates in the formation of the robust, dynamic MSP fiber complexes that generate sperm motility. The same membrane phosphoprotein that orchestrates assembly of the fiber complexes at the leading edge of the lamellipod of mature sperm is also found at all sites of filament assembly during spermiogenesis. The orderly progression of steps that leads to construction of a functional motility apparatus illustrates the precise spatio-temporal control of MSP filament assembly in the developing cell and highlights the remarkable similarity in organization and plasticity shared by the MSP cytoskeleton and the actin filament arrays in conventional crawling cells.     

Poly(ADP-ribosyl)ation during chromatin remodeling steps in rat spermiogenesis

In spermiogenesis, spermatid differentiation is marked by dramatic changes in chromatin density and composition. The extreme condensation of the spermatid nucleus is characterized by an exchange of histones to transition proteins and then to protamines as the major nuclear proteins. Alterations in DNA topology that occur in this process have been shown to require the controlled formation of DNA strand breaks. Poly(ADP-ribosyl)ation is a posttranslational modification of proteins mediated by a family of poly(ADP-ribose) polymerase (PARP) proteins, and two family members, PARP-1 and PARP-2, are activated by DNA strand breaks that are directly detected by the DNA-binding domains of these enzymes. Here, we show for the first time that poly(ADP-ribose) formation, mediated by poly(ADP-ribose) polymerases (PARP-1 and presumably PARP-2), occurs in spermatids of steps 11–14, steps that immediately precede the most pronounced phase of chromatin condensation in spermiogenesis. High levels of ADP-ribose polymer were observed in spermatid steps 12–13 in which the highest rates of chromatin nucleoprotein exchanges take place. We also detected -H2AX, indicating the presence of DNA double-strand breaks during the same steps. Thus, we hypothesize that transient ADP-ribose polymer formation may facilitate DNA strand break management during the chromatin remodeling steps of sperm cell maturation.M.L. Meyer-Ficca and H. Scherthan contributed equally to this work     

Dynamics of F-actin during spermiogenesis in Gampsocleis gratiosa ( Orthoptera: Tettigoniidae)

为阐明F-肌动蛋白在优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl精子形成过程中的动态变化,本研究利用微分干涉相衬技术和免疫荧光技术首次对优雅蝈螽精子形成过程中的F-肌动蛋白进行了细胞定位,利用透射电镜技术从超微水平观察了优雅蝈螽精子顶体复合体的结构.结果显示:精子形成早期,F-肌动蛋白富集于亚顶体区域,形态由“球状”转变为“棒锥状”;精子形成中期,F-肌动蛋白呈“倒Y型”分布于亚顶体区域和细胞核前端两侧;精子形成后期,亚顶体区域的F-肌动蛋白解聚消失,F-肌动蛋白呈“箭头状”,仅分布于顶体复合体扩张的两翼中.F-肌动蛋白动态变化伴随着细胞核和精子头部的形态改变,F-肌动蛋白的动态装配在精子顶体复合体形态构建和细胞核的形变中起着重要的作用.本研究还发现未成熟的精子尾部有一些富含F-肌动蛋白的细胞质微滴,与精子形成过程中多余细胞质和细胞器的外排有关.F-肌动蛋白的动态变化研究为进一步阐明细胞骨架蛋白在昆虫精子形成过程中的功能和作用机制奠定了基础.     

Distribution of microtubules during centriole separation in rat kangaroo (Potorous) cells.

The distribution of microtubule profiles during separation of the centriole duplexes in prophase of rat kangaroo cells (PTK2W) has been investigated by serial section reconstructions. Separation begins with a reduction of the interphase microtubular network which radiates from the region of the duplex to all parts of the cell. As duplexes separate, microtubular elements appear between the duplexes. However, most microtubular elements extend oblique to the axis of separation rather than aligned along the axis of separation. Few microtubules are seen extending directly between duplexes. Longer profiles are seen as separation continues. Prior to the completion of separation, microtubules coalesce to form a discrete band often juxtaposed on the nuclear membrane. Subsequently, the duplexes reach their final position and nuclear envelope dissolution is initiated.     

Ultrastructure of spermiogenesis and synthesis of enigmatic cytoplasmic inclusions in the spirally shaped spermatozoon of the polychaete Spio setosa (Annelida: Spionidae)

The sperm of Spio setosa (Polychaeta, Spionidae) are known to be very unusual in form; here, spermiogenesis and the structure of the spermatozoon in this species are described by transmission electron microscopy. While spermiogenesis is similar to that described for many other polychaetes, two notable exceptions to this process include the synthesis of abundant ring‐shaped and tubular, membrane‐bounded cytoplasmic inclusions in the midpiece, and the differentiation of a spirally shaped sperm head. Spermatids develop as free‐floating tetrads in the male's coelom. A microtubular manchette does not develop during chromatin condensation and nuclear elongation, and the spiral acrosome forms as a single Golgi‐derived vesicle that migrates anteriorly to become housed in a deep anterior nuclear fossa. Early in spermiogenesis, numerous Golgi‐derived, membrane‐bounded cytoplasmic inclusions appear in the cytoplasm; these ultimately occupy the sperm midpiece only. The mature spermatozoon in the male has a 15‐μm‐long head consisting of a nucleus coiled like a spring and a spiral acrosome with differentiated substructure, the posterior two thirds of which sits in an anterior nuclear fossa. The midpiece is wider than the rest of the spermatozoon and contains 9–10 spherical mitochondria surrounding the two centrioles, as well as numerous membrane‐bounded conoid and tubular cytoplasmic inclusions. The axoneme has a 9 + 2 arrangement of microtubules. By contrast, stored sperm in the female's seminal receptacles have lost the midpiece inclusions but contain an abundance of glycogen. The function of the midpiece inclusions remains unresolved, and the significance of their absence in stored sperm within the seminal receptacle and the appearance of midpiece glycogen stores remains unclear and requires additional investigation.     

Indo-Pacific Syllidae (Annelida,Phyllodocida) share an evolutionary history

The geographic distribution of three intriguing genera of Syllidae (Annelida, Phyllodocida), Alcyonosyllis, Paraopisthosyllis and Megasyllis, is restricted to the Pacific Ocean, Indian Ocean and the Red Sea. In this study new material of several species in all three genera is identified, and the distributions of the species and genera refined; four species of Alcyonosyllis were found to have increased ranges. Additionally, one new species of Paraopisthosyllis is described. Paraopisthosyllis pardus sp. nov. is characterized by having long bidentate bladed-chaetae, segments covered by small papillae, and a colour pattern consisting in dark red-brown antennae and dorsal cirri and several transversal dark red-brown lines per segment. The three genera share several striking morphological characteristics, such as alternation in the arrangement of dorsal cirri, wide segments with secondary annuli and bright, contrasting colour patterns. Alcyonosyllis species are found in association with other organisms, most noticeably anthozoans, whereas members of Paraopisthosyllis and Megasyllis are free living. Molecular information (sequences of DNA) from Alcyonosyllis species (including the type species, A. phili), and the type of Megasyllis (M. corruscans) is included herein for the first time in a phylogenetic analysis. A phylogenetic analysis performed through different methods (Maximum parsimony and Maximum likelihood) using sequences of three genes (18S, 16S and COI) reveals that all the three genera form a monophyletic group within Syllidae, with several synapomorphies, and a common ancestor probably from the Indo-Pacific. Their geographic distribution pattern, the relationships between these genera and the rest of syllids, and the symbiosis in Alcyonosyllis are discussed.     

优雅蝈螽精子形成过程中F-肌动蛋白的动态变化

为阐明F-肌动蛋白在优雅蝈螽Gampsocleis gratiosa Brunner von Wattenwyl精子形成过程中的动态变化, 本研究利用微分干涉相衬技术和免疫荧光技术首次对优雅蝈螽精子形成过程中的F-肌动蛋白进行了细胞定位, 利用透射电镜技术从超微水平观察了优雅蝈螽精子顶体复合体的结构。结果显示： 精子形成早期, F-肌动蛋白富集于亚顶体区域, 形态由“球状”转变为“棒锥状”; 精子形成中期, F-肌动蛋白呈“倒Y型”分布于亚顶体区域和细胞核前端两侧; 精子形成后期, 亚顶体区域的F 肌动蛋白解聚消失, F-肌动蛋白呈“箭头状”, 仅分布于顶体复合体扩张的两翼中。F-肌动蛋白动态变化伴随着细胞核和精子头部的形态改变, F-肌动蛋白的动态装配在精子顶体复合体形态构建和细胞核的形变中起着重要的作用。本研究还发现未成熟的精子尾部有一些富含F-肌动蛋白的细胞质微滴, 与精子形成过程中多余细胞质和细胞器的外排有关。F-肌动蛋白的动态变化研究为进一步阐明细胞骨架蛋白在昆虫精子形成过程中的功能和作用机制奠定了基础。     

Chromatin condensation and acrosome development during spermiogenesis of Ensis ensis (Mollusca,Bivalvia)

We describe chromatin condensation and acrosome development during spermiogenesis of Ensis ensis. The overall shape of the mature spermatozoon corresponds to the primitive type. The nucleus is oval and on its superior pole there is an elongated acrosome; the middle piece contains four mitochondria around the centriolar complex. The condensation of the nuclei seems to occur in three steps: first the diameter of chromatin fibers increases slightly from 17 to 20 nm; second, in midspermatids fiber pairs coalesce; and third, the coalescence continues by addition of other fibers until the nuclei become highly compacted. Chromatin changes are related with nuclear protein composition. Small proacrosomal vesicles show two regions of different electron density. At a later stage they fuse to give a single, spherical vesicle in round spermatids, which migrates to the upper pole and transforms into a tapered acrosome (18 μm long) with a central channel filled with finely fibrous material. © 1994 Wiley-Liss, Inc.     

Molecular systematics of polychaetes (Annelida)

Hydrobiologia - Some progress has been made in the field of molecular systematics of polychaetes over the past couple of years. In particular, phylogenetic analyses of sequence data from the 18S...     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): I. Meiotic apparatus formation

The fine structure of the animal pole cortex is examined in the fertilized Tubifex egg undergoing the formation of the second meiotic apparatus (MA). The fully formed MA which orients its axis at right angles to the surface is found at the animal pole about 40 min after formation of the first polar body. It is composed of a spindle and asters at its poles; a centriole is found in the inner aster, but not in the peripheral aster adjacent to the surface. During the formation of the MA, the animal pole surface is lined with a 0.15-μm-thick, electron-dense cortical layer, which is rich in microfilaments. The arrangement of the filaments in the layer changes from a parallel array to a meshwork with progressive formation of the MA. Microtubules of the peripheral aster terminate in the cortical layer. When a jet stream of glycerol/dimethyl sulfoxide solution is applied to an egg fragment glued on a polylysine-coated coverslip, an egg cortex-MA complex is isolated on the coverslip; the MA appears to be tethered to the egg surface by the structural connection between the filamentous cortical layer and microtubules of the peripheral aster. Cytochalasin B (50 μg/ml), when administrated at early phase of the MA formation, does not show any effect on the structure of the cortical layer and the MA; however, if eggs shortly before the termination of the first polar body formation are immersed in the same test solution, the cortical layer of the animal pole becomes thinner, and the filamentous material is not observed in it. Furthermore, in these eggs, the peripheral aster and the spindle are not structurally discernible because of the suppression of microtubule assembly, whereas microtubules on kinetochores and in the inner aster are normally developed. These results are discussed in relation to the role of the animal pole cortex in fixing of the MA to the egg surface and in forming of the MA.     

Characterization of chromatin-condensing proteins during spermiogenesis in a neogastropod mollusc (Murex brandaris)

During the process of chromatin cndensation in the spermiogenesis of the neogastropod mollusc Murex brandaris, the nuclear protein complement undergoes a complex series of changes. These changes lead to the appearance of three small protamines in the ripe sperm nuclei. We have characterized this system electrophoretically and at the compositions with antibodies elicited against a specific spermatozoan protamine. Our results indicate that the complex pattern of chromatin condensation during spermiogenesis in this species (M. brandaris) may be modulated by a series of post-translational (and intranuclear) modifications of DNA-interacting proteins, such as precursors to the sperm protamines. The amino acid composition of each sperm protamine is remarkably simple (lys + arg + gly ≥96 mol%). This system of spermiogenic/spermatozoal proteins in the neogastropod M. brandaris clearly differs from that in patellogastropods and archaeogastropods, and it may be helpful in understanding evolutionary changes in the chromatin condensation pattern during the spermiogenesis of gastropod molluscs. © 1994 Wiley-Liss, Inc.     

Chaetae and chaetogenesis in polychaetes (Annelida)

Annelid chaetae are epidermal extracellular structures that are in general clearly visible from the exterior. Their structure is highly diverse, especially within the Polychaeta, and each species shows a specific pattern of chaetae. Chaetae have therefore gained immense significance for species determination, making them the best studied structures in polychaetes. The shape of chaetae is determined by the temporal and spatial modification of the microvilli pattern of a single cell, the chaetoblast. As chaetae are species specific, the process of their formation must be under strict control and the information needed to form certain chaetae must be highly conservative. It can be assumed that corresponding chaetogenesis is caused by commonly inherited information. Thus, comparative chaetogenesis can help to test hypotheses on the homology of certain types of chaetae and help to unravel the influence of functional constraints on the shape of chaetae. Different types of chaetae are compared here and the present state of our knowledge of their structure and formation is used to present some homology hypotheses. There are some strong arguments for a homology of uncini and certain hooks and hooded hooks. Acicula are compared to other supportive setae and the significance of the arrangement of chaeta for phylogenetic considerations is shown. Coding issues are provided in order to facilitate inclusion of information on chaetae into data matrices.     

Differential expression of lysosomal associated membrane protein (LAMP-1) during mammalian spermiogenesis

The mammalian acrosome is a secretory vesicle of mature sperms that plays an important role in fertilization. Recent evidence had pointed out that some components found at endosomes in somatic cells are associated with the developing acrosome during the early steps of spermiogenesis. Moreover, the mammalian acrosome contains many enzymes found within lysosomes in somatic cells. In this work, we studied the dynamics of some components of the endosome/lysosome system, as a way to understand the complex membrane trafficking circuit established during spermatogenesis. We show that the cation independent-mannose-6-phosphate receptor (CI-MPR) is transiently expressed in the cytoplasm of mid-stage spermatids (steps 5-11). On the other hand, gamma-adaptin, an adaptor molecule of a complex involved in trafficking from the Golgi to lysosomes, was expressed in cytoplasmic vesicles only in pachytene and Cap-phase spermatids (steps 1-5). Our major finding is that the lysosomal protein LAMP-1 is differentially expressed during spermiogenesis. LAMP-1 appears late in spermatogenesis (Acrosome-phase) contrasting with LAMP-2, which is present throughout the complete process. Both proteins appear to be associated with cytoplasmic vesicles and not with the developing acrosome. None of the studied proteins is present in epididymal spermatozoa. Our results suggest that the CI-MPR could be involved in membrane trafficking and/or acrosomal shaping during spermiogenesis.     

Evolution of interstitial Polychaeta (Annelida)

An update of the systematics is given for the eight most important interstitial polychaete families: Diurodrilidae, Nerillidae, Protodrilidae, Protodriloididae, Saccocirridae, Parergodrilidae, Polygordidae and Psammodrilidae. Additional information and new observations are presented for the Diurodrilidae, Nerillidae and Psammodrilidae. Three new supplementary evolutionary hypotheses for these families are here suggested: (I) basal position of Diurodrilidae in Polychaeta, (2) evolution of Nerillidae in mud, and (3) evolution from meio- to macrofaunal forms of Psammodrilidae.     

Extra-Golgi pathway of an acrosomal antigen during spermiogenesis in the rat

Summary A monoclonal antibody (MC41) was produced that specifically recognizes a sperm acrosomal antigen of approximately 165000 dalton in the rat. Rat testis was examined using a pre-embedding immunoperoxidase technique to reveal the pathway of the MC41 antigen to the acrosome during spermiogenesis. The MC41 immunoreaction appeared in several organelles of spermatids in a stage-specific manner: (1) in the endoplasmic reticulum (ER) throughout spermiogenesis, (2) in the outer acrosomal membrane from steps 9 to 19, (3) as a weak immunoreaction in the vesicular structures in the acrosomal matrix from steps 11 to 17, and (4) as a strong immunoreaction in the acrosomal matrix especially at the terminal step of spermiogenesis (step 19). However, no immunoreaction was observed in the Golgi region throughout spermiogenesis. These results suggest that the pathway of the MC41 antigen leads firstly from the ER to the outer acrosomal membrane and secondly to the acrosomal matrix. This pathway does not involve the Golgi apparatus and is referred to as the extra-Golgi pathway.