The formation of syncytial groups of germ-line cells (cysts, clusters) is a widespread phenomenon in animal gametogenesis. There are three major types of the spatial organization of germ-line cysts – linear, branched and cysts that have a central cytoplasmic mass (cytoplasmic core). In our studies, we focused on cysts with a central cytoplasm mass because this type of cyst is still poorly understood. We chose the earthworm Dendrobaena veneta as the model organism because it is easy to cultivate and a large number of germ cells clusters can be obtained from a single specimen.

Generally, clustered germ cells are interconnected via broad cell junctions called intercellular bridges or ring canals (Fig. 1, 2 and 3). Ring canals allow the cytoplasm to flow freely from one cell to another in the entire cluster. Ring canals are nothing more than modified contractile rings that do not close during late cytokinesis. The role of ring canals in the proper functioning of germ-cell clusters is best known in such model species as Caenorhabditis elegans, Drosophila melanogaster and Mus musculus, in which any disorders in the construction and functioning of ring canals result in infertility. Not only is the functioning of ring canals well known in the model species but also their structure and molecular composition. Numerous studies have shown that a rich F-actin cytoskeleton is present in the cortical layer of ring canals (Fig. 3) and that it is required to stabilize the bridges in order to keep them unobstructed, which is necessary for correct exchange of the cytoplasm. On the other hand, it is also known that microtubules play an active role in the development and functioning of germ-cell cysts(Fig. 2). Ring canals are places in which F-actin and microtubules cooperate together with other proteins (e.g. anilin) in order to stabilize the cytoplasmic channel and to optimize its functioning.

In clusters with a central cytoplasmic mass (called a cytophore in annelids), each germ cell has only one intercellular bridge that connects it to the cytophore (Fig. 1, 2 and 3). Therefore, the cytophore is an intermediate structure that mediates cytoplasm sharing between clustering cells.

Our previous studies showed that both F-actin (Fig. 3) and microtubules (Fig. 2) are present in D. veneta germ-line clusters. The aim of the presented studies was to investigate the spatial organization of the microtubules within the ring canals of the male germ-cell clusters in D. veneta. To achieve this goal, we used immunofluorescent methods to visualize the F-actin and microtubules and confocal microscopy to collect high-resolution images. Based on the collected images, we reconstructed the cytoskeletal network within the ring canals.

Figures:

Fig. 1 TEM photography of germ-line clusters. The cluster centre is occupied by cytoplasmic mass called cytophore (cyt), cytophore is surrounded by germ cells (c). Each germ cell is connected to the cytophore by one ring canal (ellipse).

Fig. 2 Immunofluorescence staining of tubulin in germ-line cyst. Tubulin (green fluorescence) is evenly distrubuted in whole cluster but within ring canals (arrows) condensation of tubulin bundles which passes from the cells (c) to the cytophore (cyt) can be seen.

Fig. 3 Fluorescence staining of F-actin in a germ-line cyst. F-actin is stained by rhodamine conjugated with phalloidin and can be seen as a red fluorescence. Nuclei were stained with DAPI and can be observe as blue fluorescence. F-actin is distributed in the cortical layer of germ cells (c) and present in the ring canals (arrows). Ring canals connect the cells (c) to the cytophore (cyt).

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EMC Abstracts - https://emc-proceedings.com/abstract/spatial-organization-of-microtubules-within-the-ring-canals-in-the-male-germ-line-clusters-of-dendrobaena-veneta/