Ovary organization and ultrastructure in six species of Amynthas and Metaphire earthworms (Annelida, Crassiclitellata, Megascolecidae).

Ovaries in earthworms belonging to the family Megascolecidae are paired structures attached to the septum in the anterior part of the XIII segment. They are fan to rosette shaped with numerous rows of growing oocytes, known as egg strings, radiating from the ovary center towards the segmental cavity. The histological and ultrastructural ovary organization in megascolecids and the course of oogenesis remain unknown. The paper presents the results of light and electron microscopy analyses of ovaries in six megascolecid species, three from the genus Amynthas and three from Metaphire. Both parthenogenetic and sexually reproducing species were included in the study. The organization and ultrastructure of ovaries in all studied species are broadly similar. Considering the histological organization of ovaries, they could be divided into two zones. Zone I (proximal, close to the connection with the septum) is tightly packed with germline and somatic cells. Germ cells are interconnected via intercellular bridges and thin strands of the central cytoplasm (known as cytophore) and form syncytial cysts. Cysts unite oogonia, early meiotic cells (till diplotene), and clustering cells develop synchronously. During diplotene, interconnected cells lose developmental synchrony; most probably, one cell per cyst grows faster than others, detaches from the cysts, and becomes an oocyte. The remaining cells grow slightly and are still interconnected via the thin and reticular cytophore; these cells are considered nurse cells. Zone II has a form of egg strings where growing oocytes are isolated one from another by thin somatic cells and form short cords. We present the ultrastructural details of germline and somatic cells. We propose the term "Amynthas" type of ovaries for this ovary organization. We suppose that such ovaries are characteristic of other megascolecids and related families.

Ovary micromorphology in hormogastrid earthworms with a particular emphasis on the organization of the germline cysts.

There is a gap in our knowledge of microorganization and the functioning of ovaries in earthworms (Crassiclitellata) and allied taxa. Recent analyses of ovaries in microdriles and leech-like taxa revealed that they are composed of syncytial germline cysts accompanied by somatic cells. Although the pattern of cyst organization is conserved across Clitellata - each cell is connected via one intercellular bridge (ring canal) to the central and anuclear cytoplasmic mass termed the cytophore - this system shows high evolutionary plasticity. In Crassiclitellata, only the gross morphology of ovaries and their segmental localization is well known, whereas ultrastructural data are limited to lumbricids like Dendrobaena veneta. Here we present the first report about ovarian histology and ultrastructure in Hormogastridae, a small family of earthworms inhabiting the western parts of the Mediterranean sea basin. We analyzed three species from three different genera and showed that the pattern of ovary organization is the same within this taxon. Ovaries are cone-like, with a broad part connected to the septum and a narrow distal end forming an egg string. Ovaries are composed of numerous cysts uniting a small number of cells, eight in Carpetania matritensis. There is a gradient of cysts development along the long ovary axis, and three zones can be distinguished. In zone I, cysts develop in complete synchrony and unite oogonia and early meiotic cells (till diplotene). Then (zone II), the synchrony is lost, and one cell (prospective oocyte) grows faster than the rest (prospective nurse cells). In zone III, oocytes pass the growth phase and gather nutrients; at this time, their contact with the cytophore is lost. Nurse cells grow slightly, eventually die via apoptosis, and are removed by coelomocytes. The most characteristic feature of hormogastrid germ cysts is the inconspicuous cytophore in the form of thread-like thin cytoplasmic strands (reticular cytophore). We found that the ovary organization in studied hormogastrids is very similar to that described for D. veneta and propose the term "Dendrobaena" type of ovaries. We expect the same microorganization of ovaries will be found in other hormogastrids and lumbricids.

A novel mechanism of bulk cytoplasmic transport by cortical dynein in Drosophila ovary.

Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. We have previously shown that cytoplasmic dynein is required for Drosophila oocyte growth and assumed that it simply transports cargoes along microtubule tracks from nurse cells to the oocyte. Here, we report that instead of transporting individual cargoes along stationary microtubules into the oocyte, cortical dynein actively moves microtubules within nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. This robust microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein performs bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. We propose that the dynein-driven microtubule flow could serve as a novel mode of fast cytoplasmic transport.

Zebrafish dazl regulates cystogenesis and germline stem cell specification during the primordial germ cell to germline stem cell transition.

Fertility and gamete reserves are maintained by asymmetric divisions of the germline stem cells to produce new stem cells or daughters that differentiate as gametes. Before entering meiosis, differentiating germ cells (GCs) of sexual animals typically undergo cystogenesis. This evolutionarily conserved process involves synchronous and incomplete mitotic divisions of a GC daughter (cystoblast) to generate sister cells connected by intercellular bridges that facilitate the exchange of materials to support rapid expansion of the gamete progenitor population. Here, we investigated cystogenesis in zebrafish and found that early GCs are connected by ring canals, and show that Deleted in azoospermia-like (Dazl), a conserved vertebrate RNA-binding protein (Rbp), is a regulator of this process. Analysis of dazl mutants revealed the essential role of Dazl in regulating incomplete cytokinesis, germline cyst formation and germline stem cell specification before the meiotic transition. Accordingly, dazl mutant GCs form defective ring canals, and ultimately remain as individual cells that fail to differentiate as meiocytes. In addition to promoting cystoblast divisions and meiotic entry, dazl is required for germline stem cell establishment and fertility.

A novel pattern of germ cell divisions in the production of hymenopteran insect eggs.

Egg development is a defining process of reproduction in higher eukaryotes. In the fruit fly, Drosophila melanogaster, this process begins with four mitotic divisions starting from a single germ cell, producing a cyst of 16 cystocytes; one of these cells will become the oocyte and the others supporting nurse cells. These mitotic divisions are exceptional because cytokinesis is incomplete, resulting in the formation of cytoplasmic bridges known as ring canals that interconnect the cystocytes. This organization allows all cystocytes to divide synchronously during each mitotic round, resulting in a final, power-of-2 number of germ cells. Given that numerous insects obey this power-of-2 rule, we investigated if strict cell doubling is a universal, underlying cause. Using confocal microscopy, we found striking departures from this paradigm in three different power-of-2 insects belonging to the Apocrita suborder (ants, bees and wasps). In these insects, the earliest-formed cystocytes cease to divide during the latter mitotic cycles while their descendants undergo further division, thereby producing a 'radial' direction of division activity. Such cystocyte division patterns that depart from strict cell doubling may be 'fine-tuned' in order to maintain a final, power-of-2 germ cell number.

Architecture and Life History of Female Germ-Line Cysts in Clitellate Annelids.

Animal female and male germ-line cells often form syncytial units termed cysts, clusters, or clones. Within these cysts, the cells remain interconnected by specific cell junctions known as intercellular bridges or ring canals, which enable cytoplasm to be shared and macromolecules and organelles to be exchanged between cells. Numerous analyses have shown that the spatial organization of cysts and their functioning may differ between the sexes and taxa. The vast majority of our knowledge about the formation and functioning of germ-line cysts comes from studies of model species (mainly Drosophila melanogaster); the other systems of the cyst organization and functioning are much less known and are sometimes overlooked. Here, we present the current state of the knowledge of female germ-line cysts in clitellate annelids (Clitellata), which is a monophyletic taxon of segmented worms (Annelida). The organization of germ-line cysts in clitellates differs markedly from that of the fruit fly and vertebrates. In Clitellata, germ cells are not directly connected one to another, but, as a rule, each cell has one ring canal that connects it to an anuclear central cytoplasmic core, a cytophore. Thus, this pattern of cell distribution is similar to the germ-line cysts of Caenorhabditis elegans. The last decade of studies has revealed that although clitellate female germ-line cysts have a strong morphological plasticity, e.g., cysts may contain from 16 to as many as 2500 cells, the oogenesis always shows a meroistic mode, i.e., the interconnected cells take on different fates; a few (sometimes only one) become oocytes, whereas the rest play the role of supporting (nurse) cells and do not continue oogenesis.This is the first comprehensive summary of the current knowledge on the organization and functioning of female germ-line cysts in clitellate annelids.

Expression of Hbs, Kirre, and Rst during Drosophila ovarian development.

The Irre cell-recognition module (IRM) is a group of evolutionarily conserved and structurally related transmembrane glycoproteins of the immunoglobulin superfamily. In Drosophila melanogaster, it comprises the products of the genes roughest (rst; also known as irreC-rst), kin-of-irre (kirre; also known as duf), sticks-and-stones (sns), and hibris (hbs). In this model organism, the behavior of this group of proteins as a partly redundant functional unit mediating selective cell recognition was demonstrated in a variety of developmental contexts, but their possible involvement in ovarian development and oogenesis has not been investigated, notwithstanding the fact that some rst mutant alleles are also female sterile. Here, we show that IRM genes are dynamically and, to some extent, coordinately transcribed in both pupal and adult ovaries. Additionally, the spatial distribution of Hbs, Kirre, and Rst proteins indicates that they perform cooperative, although largely nonredundant, functions. Finally, phenotypical characterization of three different female sterile rst alleles uncovered two temporally separated and functionally distinct requirements for this locus in ovarian development: one in pupa, essential for the organization of peritoneal and epithelial sheaths that maintain the structural integrity of the adult organ and another, in mature ovarioles, needed for the progression of oogenesis beyond stage 10.

Ovaries of the white worm (Enchytraeus albidus, Annelida, Clitellata) are composed of 16-celled meroistic germ-line cysts.

The paired ovaries of E. albidus are like a bunch of grapes and are composed of clearly separated units, syncytial germ cysts (clusters), which are surrounded by a thin layer of somatic cells. Each cyst maintains the connection with the ovary by an extended stalk that is composed of somatic cells. The spatial architecture of the germ-line cysts found in E. albidus is the same as in other clitellate annelids that have been studied to date. As a rule, germ cells are located at the cyst periphery and each has only one ring canal that connects it to the common and centrally located cytoplasmic mass, the cytophore. Here we present data about the F-actin and microtubular cytoskeleton and some molecular components of the germ-line cysts. We show that the ring canals have an inner rim that is enriched with microfilaments and proteins that contain phosphotyrosine. The microtubules form a loose network in the cytoplasm of the oocyte and nurse cells; moreover, some of them pass through the ring canals to the cytophore. Numerous microtubules are also located in the somatic cells. The germ-line cysts in E. albidus ovaries consist of 16 cells, which is the lowest known number of interconnected germ cells within clitellate annelids. During oogenesis, the fate of interconnected germ cells differentiates and only one cell develops as the future egg, while the other 15 become nurse cells. This differentiation means ovary meroism. The nurse cells gather cell organelles and storage material that then pass through the ring canals and cytophore moving towards the growing oocyte. At the end of oogenesis, the vitellogenic oocyte surrounds the siblings' cells together with the cytophore and engulfs their remnants into the ooplasm. No morphological or molecular markers of the apoptosis of the nurse cells were found. Moreover, the nurse cells did not undergo polyploidisation. The measured DNA level was 4C, which indicates that these cells are not highly-specialised.

Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth.

Intercellular bridges called "ring canals" (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs' anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin-containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs' anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin-dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth.

The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

The spatial arrangement of germ line cells in ovarian follicles of the mutantdicephalic inDrosophila melanogaster.

The pattern of intercellular connections between germ line cells has been studied in follicles of the mutantdicephalic (dic), which possess nurse cell clusters at both poles. Staining of follicles with a fluorescent rhodamine conjugate of phalloidin reveals ring canals and cell membranes and thus allows us to reconstruct the spatial organization of the follicle. Each germ line cell can be identified by the pattern of cell-cell connections which reflect the mitotic history of individual cells in the 16-cell cluster. The results indicate that in both wild-type anddicephalic cystocyte clusters one of the two cells with four ring canals normally becomes the pro-oocyte. However, in some follicles (dicephalic and wild-type) oocytes were found with fewer or more than four ring canals. Indic follicles, one or several nurse cells may become disconnected from the other cells during oocyte growth at stage 9-10. Such disconnected cells cannot later on empty their cytoplasm into the oocyte. This, in turn, might be of consequence for the determination of axial polarity of the embryo.