The occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation

A previous electron microscopic study of the cat testis revealed that spermatids derived from the same spermatogonium are joined together by intercellular bridges. The present paper records the observation of similar connections between spermatocytes and between spermatids in Hydra, fruit-fly, opossum, pigeon, rat, hamster, guinea pig, rabbit, monkey, and man. In view of these findings, it is considered likely that a syncytial relationship within groups of developing male germ cells is of general occurrence and is probably responsible for their synchronous differentiation. When clusters of spermatids, freshly isolated from the germinal epithelium are observed by phase contrast microscopy, the constrictions between the cellular units of the syncytium disappear and the whole group coalesces into a spherical multinucleate mass. The significance of this observation in relation to the occurrence of abnormal spermatozoa in semen and the prevalence of multinucleate giant cells in pathological testes is discussed. In the ectoderm of Hydra, the clusters of cnidoblasts that arise from proliferation of interstitial cells are also connected by intercellular bridges. The development of nematocysts within these groups of conjoined cells is precisely synchronized. Both in the testis of vertebrates and the ectoderm of Hydra, a syncytium results from incomplete cytokinesis in the proliferation of relatively undifferentiated cells. The intercellular bridges between daughter cells are formed when the cleavage furrow encounters the spindle remnant and is arrested by it. The subsequent dissolution of the spindle filaments establishes free communication between the cells. The discovery of intercellular bridges in the two unrelated tissues discussed here suggests that a similar syncytial relationship may be found elsewhere in nature where groups of cells of common origin differentiate synchronously.     

肝片吸虫精子发生中的合体群团方式

本文从细胞形态学和超微结构等方面研究肝片吸虫Fasciola sp.精子发生的合体群团方式。结果表明:精巢直接涂片,经细胞培养、染色体观察和定向切片电镜观察,均证实肝片吸虫精子发生从B型精原细胞、初级精母细胞、次级精母细胞、精细胞直到精子排出前的最后阶段等各期生殖细胞均以由细胞间桥连接的合体群团方式存在。有倍性群团和非倍性群团两种。未见到子细胞数目超过32的群团,非倍性群团应是部分精母细胞和精细胞退化的结果。无脊椎动物肝片吸虫合体群团超微结构与前人在哺乳类中的观察相似。表明精子发生中各子细胞间是否具有由细胞间桥连接而形成的合体群团,是动物生殖细胞发生与体细胞增殖的重要区别,似是有性生殖的一种功能适应。     

Morphological Studies on the Testis of Adults of Moniliformis (Acanthocephala)

The morphology of the testis in young adult males of Moniliformis dubius developing in the rat has been studied with the aid of light and electron microscopes. In one-day-old male worms, the testis is organised as two zones. One zone consists of individual germ-line cells, while the other consists of a supporting syncytium which embeds the cells and forms the boundary of the testis. The surface of the testis is covered by a fibrous non-cytoplasmic coat. In seven-day-old male worms, the syncytium has lost its compact form, breaking down into multinucleate units connected by cytoplasmic processes and apparently forming a loose syncytial network throughout the testis. The germ cells are now randomly distributed and are surrounded by wide spaces among the segments of the supporting syncytium. The fibrous coat, lined internally by an irregular layer of the syncytium, forms the testis envelope. This basic structure is maintained in the testis of 14-day-old and sexually mature male worms.     

Muscle development in the grasshopper embryo. II. Syncytial origin of the extensor tibiae muscle pioneers

The extensor tibiae muscle (ETi) in the metathoracic leg of the grasshopper, which powers the jump, is among the most studied insect muscles. In contrast to many insect muscles which are simple (consisting of only a single bundle of muscle fibers), the ETi is a complex muscle which consists of an array of bundles of muscle fibers, each with a separate site of insertion on the body wall ectoderm and on the ETi apodeme ectoderm. Here we describe the embryonic development of this complex muscle. The ETi muscle develops from a single muscle pioneer (MP) which connects the initial invagination of the ETi apodeme to the wall of the femur. This MP then dramatically expands around the developing apodeme to form a large horseshoe-shaped, multinucleate cell, called the supramuscle pioneer (supra-MP); the number of nuclei in the supra-MP increases by cell fusion rather than by nuclear division. The arms of the supra-MP grow steadily longer and their outer edges begin to appear scalloped, certain areas remaining tightly apposed to the ectoderm of the wall of the leg while adjacent areas lose their adhesion and are pulled away. By about 50% of embryonic development the ETi supra-MP consists of a periodic series of bridges (cytoplasmic extensions) connecting the leg wall ectoderm with the apodeme, and linked into a giant syncytium near their inner, apodeme surface by a thin layer of cytoplasm containing hundreds of nuclei. Each bridge is surrounded by a cluster of many smaller mesoderm cells. Next the syncytium begins to divide such that by 60% the periodic bridges of the supra-MP have lost syncytial contact with each other and now themselves form an array of smaller, individual, multinucleate MPs connecting the body wall to the apodeme, each surrounded by a mass of undifferentiated mesoderm cells. This initial cycle of fusion and division is followed by a second similar cycle in which the individual mesoderm cells surrounding each MP fuse with the MP. At the same time, the MP divides into the initial bundle of smaller muscle fibers. Coincident with this division into muscle fibers is the further development of thick and thin filaments and the T-tubule system.     

Gonads in Histiostoma mites (Acariformes: Astigmata): Structure and development

The development of male and female gonads in arrhenotokous and thelytokous species of Histiostoma was studied using transmission electron microscopy (TEM). All instars were examined: larvae, protonymphs, facultative heteromorphic deutonymphs (=hypopi), tritonymphs, and adults. In testis primordium, spermatogonia surrounding a testicular central cell (TCC) with a gradually enlarging, branched nucleus are present already at the larval stage. Spermatogonia and the TCC are connected via narrow, tubular intercellular bridges revealing that the TCC is a germline cell. Spermatocytes appear at the protonymphal stage. At the heteromorphic deutonymph stage, the testis primordium is similar to that of the protonymph, but in the tritonymph it is much larger and composed as in the adult: spermatids as well as sperm cells are present. The latter are congregated ventrally in the testis at the entrance of the deferent duct.In the larval ovary, an eccentrically located ovarian nutritive cell (ONC) is surrounded by oogonia which are connected with the ONC via tubular intercellular bridges. In later stages, the ovary grows and oocytes appear in the protonymph. Meiotic synaptonemal complexes in oocytes occur from the tritonymph stage. At about the time of the final molting, tubular intercellular bridges transform into peculiar diaphragm-crossed bridges known only in Histiostoma mites. In the adult female, growing oocytes at the end of previtellogenesis lose intercellular bridges and move ventro-laterally to the ovarian periphery towards the oviduct entrance. Vitellogenesis occurs in oviducts.Germinal cells in both the testis and ovary are embedded in a few somatic stroma cells which may be well discernible already in the larval ovary; in the testis, somatic stroma cells are evident not earlier than the end of the tritonymphal stage. The ovary has a thin wall of flat somatic cells, whereas the testis is covered by a basal lamina only.The obtained results suggest that gonads in Histiostoma and other Astigmata originate from two primordial cells only.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

Identification and characterization of RBM44 as a novel intercellular bridge protein

Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.     

The linear clusters of oogonial cells in the development of telotrophic ovarioles in polyphageColeoptera

Summary During the premetamorphic development of coleopteran telotrophic ovaries the culsters of sister oogonial cells, in which the differentiation of nurse cells and oocytes occurs, are arranged in linear chains. This results from a series of mitoses with the consistent orientation of the spindle parallel to the long axis of the ovariole. As a result of incomplete cytokinesis, the oogonial cells in each sibling cluster are linked to each other by intercellular bridges occupied by fusomes. As a rule, at each cluster division the basal cell (i.e. the oocyte progenitor) starts to divide first. From this cell a wave of mitoses spreads toward the anterior end of the cluster, resulting in a mitotic gradient. It is suggested that the failure of the fusomes in adjacent cells to fuse into one continuous fusome (i.e. polyfusome) allows the spindles to orientate with their long axes parallel to the long axis of the sibling cluster. This would explain why the oogonial divisions in coleopteran telotrophic ovaries generate linear chains of cells rather than the cyst-like arrangement which is typical for polytrophic sibling clusters. Dividing sibling clusters within ovarioles are arranged in bundles. The presence of intercellular bridges between sibling clusters seems to be the underlying cause of this nonrandom distribution of the mitotically active clusters. The transverse bridges have been found to occur between the basal cells as well as between the cells located more anteriorly in adjacent sibling clusters. The transverse bridges are filled with typical fusomes, which in more anterior parts of sibling clusters may fuse with the fusomes of adjacent sister oogonial cells into polyfusomes. The transverse bridges between the basal cells are incorporated in the oocytes. The pattern of sibling cluster formation described in this paper apparently occurs widespread in polyphagous Coleoptera, since it has been found in three relatively distantly related families.     

Delta-tubulin is a component of intercellular bridges and both the early and mature perinuclear rings during spermatogenesis

Mammalian spermatogenesis involves drastic morphological changes leading to the development of the mature sperm. Sperm development includes formation of the acrosome and flagellum, translocation of nucleus-acrosome to the cell surface, and condensation and elongation of the nucleus. In addition, spermatogenic cell progenies differentiate as cohorts of units interconnected by intercellular bridges. Little is known about the structural components involved in the establishment of conjoined spermatogenic cells and the mechanism of nuclear shaping of the male gamete. We identified two isoforms of delta-tubulin and found that the long isoform is predominantly expressed in testis, while the short isoform is expressed in all tissues examined. We also found that delta-tubulin forms intercellular bridges conjoining sister spermatogenic cells. In addition, delta-tubulin is a component of the perinuclear ring of the manchette, which acts on translocation and elongation of the nucleus. Furthermore, small rings clearly distinct from the intercellular bridges, which might mature to perinuclear ring of the manchette in later stages of spermatogenesis, were detected on the cell surface of round spermatids. These results suggest that delta-tubulin is a component of two types of ring, the intercellular bridges and the perinuclear rings, which may be involved in morphological changes of spermatid to mature sperm.     

The Fine Structure of the Testis of a Lancelet (=Amphioxus), Branchiostoma floridae (Phylum Chordata: Subphylum Cephalochordata= Acrania)

The germinal and non-germinal cells of the ripe lancelet testis are described by transmission electron microscopy. The visceral peritoneum of the testis is composed of myoepithelial cells, and the haemal layer consists of regions of narrow sinuses and conspicuously thicker blood vessels filled with blood plasma and bounded by basal laminae. Within the germinal epithelium and the testicular lumen, the non-germinal cells, which are not abundant, contain conspicuous lysosomes and mitochondria with tubular cristae, indicating that they may be involved in steroid synthesis. In the ripe testis, the non-germinal cells do not appear to be organized into a blood-testis barrier. Ail types of spermatogenic cells may be flagellated and are joined in small groups by intercellular bridges. During differentiation of the spermatids, the Golgi complex is associated with formation of the acrosomal vesicle near the posterior pole of the cell. A remarkable feature is the dual origin of the subacrosomal material: one component originates at the posterior end of the spermatid, and the other at the anterior end. Subsequently, the two components merge into one after the acrosomal vesicle has migrated to its definitive anterior position in the mature spermatozoon.     

Quantitative analysis of epithelial cell aggregation in the simple metazoan Hydra reveals a switch from homotypic to heterotypic cell interactions

Hydra, a member of the diploblastic phylum Cnidaria, exhibits the most basic type of organized metazoan tissues. Two unicellular sheets of polarized epithelial cells - ectoderm and endoderm - form a double layer throughout the body column. The double layer can be reestablished from single-cell suspensions by tissue-specific cell-sorting processes. However, the underlying pattern of interactions between ectodermal and endodermal epithelial cells responsible for double-layer formation is unclear. By analyzing cell interactions in a quantitative adhesion assay using mechanically dissociated Hydra epithelial cells, we show that aggregation proceeds in two steps. First, homotypic interactions within ectodermal epithelial cells (ecto-ecto) and within endodermal epithelial cells (endo-endo) form homotypic cell clusters. Second, at an aggregate size of about ten epithelial cells/cluster, ectodermal and endodermal clusters start to form heterotypic aggregates. Homotypic ecto-ecto interactions are inhibited by a polyclonal anti-Hydra membrane antiserum, and under these conditions homotypic endo-endo interactions do not proceed beyond a size of about ten epithelial cells/cluster. These data suggest that homotypic cell clusters reduce their initial homotypic affinity and acquire a new heterotypic affinity. A link between cell adhesion and cell signaling in early Hydra aggregates is discussed.     

Intercellular Bridges in Somatic Cells: Cytoplasmic Continuity of Blastoderm Cells of Loligo pealei

Intercellular bridges between blastomeres of the squid blastoderm apparently arise from the midbody and eventually break after becoming occluded by a 'multi-layered membranous plug'. The bridges can be found between two or more cells and frequently occur between cells of presumably different developmental fate. Similar bridges have been described between differentiating gametes but in only one case (cnidoblasts of Hydra ) have they been reported in somatic tissue. Many suggestions have been put forward in the literature concerning the role of the intercellular bridges in cellular differentiation but none of these hypotheses seem to be adequate to explain the possible function of these bridges in the squid blastoderm.     

Formation of germ-line cysts with a central cytoplasmic core is accompanied by specific orientation of mitotic spindles and partitioning of existing intercellular bridges

Animal germ cells tend to form clonal groups known as clusters or cysts. Germ cells within the cyst (cystocytes) are interconnected by intercellular bridges and thus constitute a syncytium. Our knowledge of the mechanisms that control the formation of germ-cell clusters comes from extensive studies carried on model organisms (Drosophila, Xenopus). Germ-cell clusters have also been described in worms (annelids, flat worms and nematodes), although their architecture differs significantly from that known in arthropods or vertebrates. Their peculiar feature is the presence of a central anucleate cytoplasmic core (cytophore, rachis) around which the cystocytes are clustered. Each cystocyte in such a cluster always has one intercellular bridge connecting it to the central cytoplasmic core. The way that such clusters are formed has remained a riddle for decades. By means of light, fluorescence and electron microscopy, we have analysed the formation and architecture of cystocyte clusters during early stages of spermatogenesis and oogenesis in a few species belonging to clitellate (oligochaetous) annelids. Our data indicate that the appearance of germ cells connected via a central cytophore is accompanied by a specific orientation of the mitotic spindles during cystocyte divisions. Spindle long axes are always oriented tangentially to the surface of the cytophore. In consequence, cystocytes divide perpendicularly to the plane of the existing intercellular bridge. Towards the final stages of cytokinesis, the contractile ring of the cleavage furrow merges with the rim of the intercellular bridge that connects the dividing cystocyte with the cytophore and forces partition of the existing bridge into two new bridges. This work was supported by the following research grants: 2P04C004 28 from the Ministry of Science and Informatization (to P. Świątek and J. Klag) and DS/1018/IZ/2007 (to J. Kubrakiewicz).     

Ontogenetic diversity of colonies and intercellular cytoplasmic bridges in the algae of the genus Volvox

In all representatives of the genus Volvox, cells of cleaving embryos are connected by cytoplasmic bridges, which play an important role in the process of young colony inversion. However, during subsequent development, the intercellular bridges are retained not in all species of Volvox; the occurrence of the bridges in an adult colony correlates with the small size of mature gonidia (asexual reproductive cells) and with the presence of cell growth in the intervals between divisions. This complex of ontogenetic features is derived and arises independently in three evolutionary lineages of colonial volvocine algae. A putative role of the syncytial state of adult colonies for the evolution of developmental cycles in Volvox is discussed.     

Characterization of centrosomal proteins Cep55 and pericentrin in intercellular bridges of mouse testes

Centrosomal protein 55 (Cep55), located in the centrosome in interphase cells and recruited to the midbody during cytokinesis, is essential for completion of cell abscission. Northern blot previously showed that a high level of Cep55 is predominantly expressed in the testis. In the present study, we examined the spatial and temporal expression patterns of Cep55 during mouse testis maturation. We found that Cep55, together with pericentrin, another centrosomal protein, were localized to the intercellular bridges (IBs) interconnecting spermatogenic cells in a syncytium. The IBs were elaborated as a double ring structure formed by an inner ring decorated by Cep55 or pericentrin and an outer ring of mitotic kinesin‐like protein 1 (MKLP1) in the male germ cell in early postnatal stages and adulthood. In addition, Cep55 and pericentrin were also localized to the acrosome region and flagellum neck and middle piece in elongated spermatids, respectively. These results suggest that Cep55 and pericentrin are required for the stable bridge between germ cells during spermatogenesis and spermiogenesis. J. Cell. Biochem. 109: 1274–1285, 2010. © 2010 Wiley‐Liss, Inc.     

Syncytia in plants: cell fusion in endosperm—placental syncytium formation in Utricularia (Lentibulariaceae)

The syncytium formed by Utricularia is extremely unusual and perhaps unique among angiosperm syncytia. All typical plant syncytia (articulated laticifers, amoeboid tapetum, the nucellar plasmodium of river weeds) are formed only by fusion of sporophytic cells which possess the same genetic material, unlike Utricularia in which the syncytium possesses nuclei from two different sources: cells of maternal sporophytic nutritive tissue and endosperm haustorium (both maternal and paternal genetic material). How is this kind of syncytium formed and organized and is it similar to other plant syncytial structures? We used light and electron microscopy to reconstruct the step-by-step development of the Utricularia syncytia. The syncytia of Utricularia developed through heterotypic cell fusion involving the digestion of the cell wall, and finally, heterokaryotic multinucleate structures were formed, which possessed different-sized nuclei that were not regularly arranged in the cytoplasm. We showed that these syncytia were characterized by hypertrophy of nuclei, abundant endoplasmic reticulum and organelles, and the occurrence of wall ingrowths. All these characters testify to high activity and may confirm the nutritive and transport functions of the syncytium for the developing embryo. In Utricularia, the formation of the syncytium provides an economical way to redistribute cell components and release nutrients from the digested cell walls, which can now be used for the embryo, and finally to create a large surface for the exchange of nutrients between the placenta and endosperm.     

Intercellular organelle traffic through cytoplasmic bridges in early spermatids of the rat: mechanisms of haploid gene product sharing

Stable cytoplasmic bridges (or ring canals) connecting the clone of spermatids are assumed to facilitate the sharing of haploid gene products and synchronous development of the cells. We have visualized these cytoplasmic bridges under phase-contrast optics and recorded the sharing of cytoplasmic material between the spermatids by a digital time-lapse imaging system ex vivo. A multitude of small (ca. 0.5 microm) granules were seen to move continuously over the bridges, but only 28% of those entering the bridge were actually transported into other cell. The average speed of the granules decreased significantly during the passage. Immunocytochemistry revealed that some of the shared granules contained haploid cell-specific gene product TRA54. We also demonstrate the novel function for the Golgi complex in acrosome system formation by showing that TRA54 is processed in Golgi complex and is transported into acrosome system of neighboring spermatid. In addition, we propose an intercellular transport function for the male germ cell-specific organelle chromatoid body. This mRNA containing organelle, ca. 1.8 microm in diameter, was demonstrated to go over the cytoplasmic bridge from one spermatid to another. Microtubule inhibitors prevented all organelle movements through the bridges and caused a disintegration of the chromatoid body. This is the first direct demonstration of an organelle traffic through cytoplasmic bridges in mammalian spermatogenesis. Golgi-derived haploid gene products are shared between spermatids, and an active involvement of the chromatoid body in intercellular material transport between round spermatids is proposed.