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.     

Nuclear fragmentation characterises paraspermiogenesis in Tubifex tubifex (Annelida, Oligochaeta)

It is known that tubificine oligochaetes produce two types of spermatozoa: eusperm, fertilizing sperm with regular haploid DNA content; and parasperm, with a much lower DNA content, protecting and carrying the eusperm. Whereas mature spermatozoa and spermatids of the two lines are easily recognized by their morphology and DNA content, little is known about the first steps of differentiation of the two lines. This subject is addressed here in two ways: we have measured DNA content by a new method based on confocal laser microscopy and found that the total DNA content of parasperm cysts is extremely variable and equal or lower than total DNA content of eusperm cysts. Then we focused on the spermatocytes, and we found that the cells which will form paraspermatids undergo a peculiar kind of nuclear fragmentation which differ greatly from a regular cell division. During fragmentation DNA is distributed unevenly among the spermatids and this gives rise to a great and variable number of parasperm with variable DNA content. Immunocytochemical assays revealed that a proper meiotic spindle is never formed during fragmentation and that actin may play an important role in the chromatin division. Electron micrographs showed that the centrioles undergo a phenomenon of mass reproduction similar to that found in ciliated epithelia which supplies each of the numerous paraspermatids of its basal body. Mol. Reprod. Dev. 59: 442-450, 2001.     

Temporal pattern of the double sperm line production in Tubifex tubifex (Annelida,Oligochaeta)

Laboratory cohort cultures of the tubificid Tubifex tubifexwere carried out at 21 °C in homogeneous conditions. Our aim was to study the temporal pattern of spermatogenesis of the two sperm lines which characterise this species. Starting from the second week after cocoon laying, we analysed the seminal vesicle content. We counted the cysts in the vesicles and arranged them into three classes: premeiotic, paraspermatids and euspermatids. Our results show that spermatogenesis begins early, at week 3, but cysts of spermatids are not found until week 5 for the parasperm line and week 6 for the eusperm line. These data are derived from a preliminary study of complex population dynamics. Nonetheless, they allow us to formulate some hypothesis regarding the mechanism which regulate the production of the two types of sperm.     

The ultrastructure and histochemistry of the spermathecae of Tubifex tubifex (Annelida: Oligochaeta)

The wall of the spermathecal ampulla in Tubifex tubifex consists of epithelial, muscular and peritoneal layers. The epithelial surface contains closely microvilli while lateral and basal plasma membranes are extensively convoluted. Epithelial cytoplasm exhibits a vertical zonation of subcellular components. The distal zone contains filiform secretory particles which are orientated perpendicular to the apical surface; extrusion occurs by their fusion with the plasma membrane between the bases of neighbouring microvilli. Mitochondiral and Golgi zones, the latter containing the nucleus, subtend the distal zone. The basal zone, composed of vertical compartments formed by the folded plasma membrane, is rich in α-glycogen rosettes. The distal epithelium and lumen material contain neutral mucopolysaccharides and carboxylated acid mucopolysaccharides in conjunction with neutral protein. The ultrastructure of the spermathecal duct wall is comparable with that of the ampulla but is characterized by extremely long microvilli and a prominent musculature.     

Cyclic Changes in Shape of A Non-Nucleate Egg Fragment of Tubifex (Annelida, Oligochaeta)

A non-nucleate fragment separated from the fertilized Tubifex egg at metaphase of the second meiosis showed temporary surface deformation at 3–3.5 hr intervals, i.e. , synchronously with the onset of formation of the second polar body and early cleavages in control eggs. From the two-cell stage on, the periodicity of the surface activity in the non-nucleate fragment was found to be synchronous with the cleavage cycles of the CD-cell and its descendants, but not with those of the AB-cell. This surface deformation was completely inhibited by cytochalasin B (50 μg/ml). Electron microscopy shows that microfilaments are present exclusively in the cortical layer of the deforming fragments. Cycloheximide-treated egg fragments commenced surface deformation after a delay of 1–2 hrs; pulse-treatment indicated that the surface deformation requires proteins synthesized specifically during the period of the previous surface deformation. These results are discussed in relation to the nucleus-independent cytoplasmic rhythm and asynchronous cleavage of Tubifex eggs.     

Pattern of body-wall muscle differentiation during embryonic development of Enchytraeus coronatus (Annelida: Oligochaeta; Enchytraeidae)

The plesiomorphic arrangement of body-wall musculature within the annelids is still under discussion. While polychaete groups show a great variety of patterns in their somatic muscles, the musculature of soil-living oligochaetes was thought to represent the characteristic pattern in annelids. Oligochaete body-wall muscles consist of an outer continuous layer of circular and an inner continuous layer of longitudinal muscles, forming a closed tube. Since designs of adult body musculature are influenced by evolutionary changes, additional patterns found during embryogenesis can give further information about possible plesiomorphic features. In oligochaetes, detailed cell-lineage analyses document the origin of the mesoderm and consequently the muscles, but later processes of muscle formation remain unclear. In the present work, body-wall muscle differentiation was monitored during embryogenesis of thesoil-living oligochaete Enchytraeus coronatus (Annelida) by phalloidin staining. Primary circular muscles form in a discrete anterior-to-posterior segmental pattern, whereas emerging longitudinal muscles are restricted to one ventral and one dorsal pair of primary strands, which continuously elongate towards posterior. These primary muscles establish an initial muscle-template. Secondary circular and longitudinal muscles subsequently differentiate in the previous spaces later in development. The prominent ventral primary longitudinal muscle strands on both sides eventually meet at the ventral midline due to neurulation, which moves the ventral nerve cord into a coelomic position, closing the muscle layers into a complete tube. This early embryonic pattern in E. coronatus resembles the adult body-wall muscle arrangements in several polychaete groups as well as muscle differentiation during embryonic development of the polychaete Capitella sp. I.     

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.     

Polar lobe formation and cytokinesis in fertilized eggs of Ilyanassa obsoleta. II. Large bleb formation caused by high concentrations of exogenous calcium ions

Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl₂, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl₂. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na⁺, Mg²⁺, or Mn²⁺ are present in addition to Ca²⁺, but not if comparable concentrations of K⁺ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K⁺ but not exogenous Ca²⁺. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl₂ has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca²⁺ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K⁺ antagonizes this Ca²⁺-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca²⁺ to intracellular K⁺ is critical in the control of polar lobe formation and cytokinesis.     

Localization of putative biochemical messengers during Eisenia foetida (Annelida, Oligochaeta) development

This histochemical-immunohistochemical study was performed on the earthworm Eisenia foetida at different developmental stages, to investigate the presence and distribution of cholinergic molecules (AChE, BuChE, alpha-bungarotoxin-binding sites), several biogenic amines (5HT and catecholamines), and some immunologically-related peptides (somatostatin. FMRF-amide, VIP, substance-P, bombesin). The results showed that the pattern of labelling for the markers is different at different stages. In summary, cholinesterases appeared widely distributed in the early embryonic stages. They then were localized in particular areas of the developing nerve and muscle tissues, whereas in newborn and adult earthworms they were restricted to ventral muscular fibers and to some CNS cells. Biogenic amines were constantly present in the embryonic and adult nervous tissues. Immunologically-related peptides were detectable after organogenesis. Our results provide indirect evidence for a role of cholinesterases in regulating early intercellular communications, neurogenesis and myogenesis, and support the hypothesis that some conservative sequences of messenger peptides arose very early in evolution.     

Reorganization of peptidergic systems during brain regeneration in Eisenia fetida (Oligochaeta, Annelida)

After the extirpation of the brain reorganization of the peptidergic (FMRFamide, neuropeptide Y, proctolin) systems was studied in the newly forming cerebral ganglion of the annelid Eisenia fetida. During regeneration, all immunoreactive fibres appear on the 1st-2nd postoperative day. At the beginning of regeneration, immunoreactive neurons and fibres form a mixed structure in the wound tissue. On the 3rd postoperative day, FMRFamide positive and neuropeptide Y-immunoreactive, while on the 7th postoperative day proctolin-immunoreactive neurons appear in the loose wound tissue. From the 25th postoperative day a capsule gradually develops around it. The neurons of the preganglion move to the surface of the newly appearing preganglion. The number of these cells gradually increase, and by the 72th-80th postoperative days the localization and number of peptide-immunoreactive neurons is similar to that in the intact one. The neurons of all examined peptidergic systems may originate from the neuroblasts, situated on the inner and outer surface of the intact ganglia (e.g. suboesophageal and ventral cord ganglia). In addition FMRFamide and proctolin immunoreactive neurons may take their derive by mitotic proliferation from the pharyngeal neurons, too.     

Nervous system dynamics during fragmentation and regeneration in Enchytraeus japonensis (Oligochaeta, Annelida)

Enchytraeus japonensis is a small terrestrial oligochaete which primarily reproduces asexually by fragmentation and regeneration. In order to introduce a molecular approach to the study of regeneration we developed a whole-mount immunostaining procedure for the worm. Using an antibody directed against acetylated tubulin in conjunction with confocal laser-scanning microscopy, we succeeded in clarifying the three- dimensional structure of the entire nervous system in the full-grown worm and its dynamics during the fragmentation and regeneration process. In addition, we examined the expression of neurotransmitters and neuropeptides in the worm using a fluorescently-labeled antagonist and various antibodies. In particular, we found two circumferential structures in the body wall muscle of each segment that react strongly with α-bungarotoxin, an antagonist of nicotinic acetylcholine receptors, and detected nerve fibers just underneath these structures. During the fragmentation process, the circular body wall muscles contract near one of these circumferential structures in the middle of the segment, which causes constriction and results in fission of the body. This α-bungarotoxin-positive structure was designated the neuromuscular junction of the circular muscle. During the regeneration process nerve fibers grow from the remaining ventral nerve cord and gradually form networks in both the anterior and posterior regeneration buds. The growing fibers extend to the prostomium (a sensory organ) at the anterior end prior to connecting to the presumptive brain rudiment. A neural network appears around the pygidium, and this is followed by growth of the body at the posterior end. The nervous system appears to play an important role in both anterior and posterior regeneration. Received: 9 June 1999 / Accepted: 30 December 1999     

External structures used during attachment and sperm transfer in tubificids (Annelida,Oligochaeta)

The genital region of seven species of Tubificidae has been studied by SEM (Scanning Electron Microscopy). The form and the position of penial and spermathecal chaetae, male and spermathecal pores and other special structures have been examined. Peristodrilus montanus shows a special system to hold the partner: the penial chaetae anchor in an elaborated structure of the body wall formed between the spermathecal pores, the `anchorage bridge'. Protuberodrilus tourenqui has a long glandular porophore with the male pores at the tip, allowing contact with the spermathecal pores which are located in deep, close to the mid-ventral line of the body. The grooved and strongly curved penial chaetae of Rhyacodrilus falciformis seem to be used both for attachment and for sperm transfer, entering into the lateral spermathecal pores. The embrace of the partners, as suggested by observations on Psammoryctides barbatus, Potamothrix bavaricus, Potamothrix hammoniensis and Potamothrix heuscheri, seems to be another important mechanism to fix contact between male and spermathecal pores and allow sperm transfer. The spermathecal chaetae could be interpreted as piercing chaetae with a chemical or mechanical stimulating role. Sensitive cilia near the penial chaetae seem to be used by the three rhyacodrilines studied to find the correct anchorage place. There is a great variety of structures which appear to be used for attachment and sperm transfer in tubificids, and consequently their role in the evolution of the whole family may be profound.     

Thyroglobulin-like immunoreactivity in the nervous system of Eisenia foetida (Annelida,Oligochaeta)

Summary Immunohistochemical studies were performed by use of specific rabbit antisera and purified antibodies to human Tg on cephalic and body sections of Eisenia foetida and on cephalic sections of Lumbricus terrestris. Secondary antisera, either fluorescein- or peroxidase-conjugated, were used to identify the immunoreaction. Immunoreactive perikarya and some immunoreactive nerve fibres were detected in both the cerebral ganglion and the ventral nerve cord of E. foetida. From 8 to 19 Tg-like positive neurons per frontal section were observed in the brain, mainly in the dorsal zone. From 2 to 4 positive perikarya per ganglion were found in sagittal sections of the ventral nerve cord with a repetitive distribution. Numerous positive neurons were also found in the cephalic segments of L. terrestris. The present results indicate that a substance immunologically related to mammalian Tg is synthesized in earthworms. This suggests that some conservative sequences of Tg structure arose very early in evolution and supports the idea of a common evolutionary origin for endocrine and nervous systems.     

Body wall muscles in the haplotaxidsHaplotaxis gordioides andPelodrilus leruthi (Annelida,Oligochaeta)

Summary The muscle organization of the body wall in two species of Haplotaxida is described.Haplotaxis gordioides andPelodrilus leruthi show significant differences in the longitudinal muscle layer. The observations suggest that inH. gordioides, the muscle fibres could foreshadow the flat circomyarian organization of microdrile muscles, while theP. leruthi organization recalls the pinnate arrangement of megadrile body wall.