Deletion of Exoc7, but not Exoc3, in male germ cells causes severe spermatogenesis failure with spermatocyte aggregation in mice.

Vesicular trafficking is essential for the transport of intracellularly produced functional molecules to the plasma membrane and extracellular space. The exocyst complex, composed of eight different proteins, is an important functional machinery for "tethering" in vesicular trafficking. Functional studies have been conducted in laboratory mice to identify the mechanisms by which the deletion of each exocyst factor affect various biological phenomena. Interestingly, each exocyst factor-deficient mutant exhibits a different phenotype. This discrepancy may be due to the function of the exocyst factor beyond its role as a component of the exocyst complex. Male germline-specific conditional knockout (cKO) mice of the Exoc1 gene, which encodes one of the exocyst factors EXOC1 (SEC3), exhibit severe spermatogenesis defects; however, whether this abnormality also occurs in mutants lacking other exocyst factors remains unknown. In this study, we found that exocyst factor EXOC3 (SEC6) was not required for spermatogenesis, but depletion of EXOC7 (EXO70) led to severe spermatogenesis defects. In addition to being a component of the exocyst complex, EXOC1 has other functions. Notably, male germ cell-specific Exoc7 cKO and Exoc1 cKO mice exhibited phenotypic similarities, suggesting the importance of the exocyst complex for spermatogenesis. The results of this study will contribute to further understanding of spermatogenesis from the aspect of vesicular trafficking.

ARF6, a component of intercellular bridges, is essential for spermatogenesis in mice.

Male germ cells are connected by intercellular bridges (ICBs) in a syncytium due to incomplete cytokinesis. Syncytium is thought to be important for synchronized germ cell development by interchange of cytoplasmic factors via ICBs. Mammalian ADP-ribosylation factor 6 (ARF6) is a small GTPase that is involved in many cellular mechanisms including but not limited to regulating cellular structure, motility, vesicle trafficking and cytokinesis. ARF6 localizes to ICBs in spermatogonia and spermatocytes in mice. Here we report that mice with global depletion of ARF6 in adulthood using Ubc-CreERT2 display no observable phenotypes but are male sterile. ARF6-deficient males display a progressive loss of germ cells, including LIN28A-expressing spermatogonia, and ultimately develop Sertoli-cell-only syndrome. Specifically, intercellular bridges are lost in ARF6-deficient testis. Furthermore, germ cell-specific inactivation using the Ddx4-CreERT2 results in the same testicular morphological phenotype, showing the germ cell-intrinsic requirement of ARF6. Therefore, ARF6 is essential for spermatogenesis in mice and this function is conserved from Drosophila to mammals.

Branched germline cysts and female-specific cyst fragmentation facilitate oocyte determination in mice.

During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or symmetrical cell fate occurs in female and male germ cells, respectively. Here, we have identified branched cyst structures in mice, and investigated their formation and function in oocyte determination. In fetal female cysts, 16.8% of the germ cells are connected by three or four bridges, namely branching germ cells. These germ cells are preferentially protected from cell death and cyst fragmentation and accumulate cytoplasm and organelles from sister germ cells to become primary oocytes. Changes in cyst structure and differential cell volumes among cyst germ cells suggest that cytoplasmic transport in germline cysts is conducted in a directional manner, in which cellular content is first transported locally between peripheral germ cells and further enriched in branching germ cells, a process causing selective germ cell loss in cysts. Cyst fragmentation occurs extensively in female cysts, but not in male cysts. Male cysts in fetal and adult testes have branched cyst structures, without differential cell fates between germ cells. During fetal cyst formation, E-cadherin (E-cad) junctions between germ cells position intercellular bridges to form branched cysts. Disrupted junction formation in E-cad-depleted cysts led to an altered ratio in branched cysts. Germ cell-specific E-cad knockout resulted in reductions in primary oocyte number and oocyte size. These findings shed light on how oocyte fate is determined within mouse germline cysts.

Adopted neoplastic cells and the consequences of their existence.

A view that guides the bulk of cancer research and oncology posits that each neoplastic cell in a tumor is a genetic offspring of another neoplastic cell. Yet, analyzing tumors from transplant patients has revealed that some normal migratory cells adopt the phenotype of neoplastic cells without acquiring their genome, thus becoming what I suggest to call adopted neoplastic cells. This commentary reviews the evidence for the existence of adopted neoplastic cells, outlines the consequences of their presence, and discusses what kind of cells can be adopted, how, and why.

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.

Uncoupling cell division and cytokinesis during germline development in metazoans.

The canonical eukaryotic cell cycle ends with cytokinesis, which physically divides the mother cell in two and allows the cycle to resume in the newly individualized daughter cells. However, during germline development in nearly all metazoans, dividing germ cells undergo incomplete cytokinesis and germ cells stay connected by intercellular bridges which allow the exchange of cytoplasm and organelles between cells. The near ubiquity of incomplete cytokinesis in animal germ lines suggests that this is an ancient feature that is fundamental for the development and function of this tissue. While cytokinesis has been studied for several decades, the mechanisms that enable regulated incomplete cytokinesis in germ cells are only beginning to emerge. Here we review the current knowledge on the regulation of germ cell intercellular bridge formation, focusing on findings made using mouse, Drosophila melanogaster and Caenorhabditis elegans as experimental systems.

Ovary micromorphology and oogenesis in a rhyacodriline oligochaete (Clitellata: Naididae, Rhyacodrilinae).

The main goal of the article is to describe the ovary organization and oogenesis in Peristodrilus montanus, an aquatic oligochaete of the subfamily Rhyacodrilinae. The presented analysis will not only enrich the knowledge about how eggs are formed but, because of the suggested conservatism of ovary organization in clitellate annelids, can contribute to disentangling the complex phylogenetic relationships of the rhyacodrilines within Naididae. The paired, conically shaped ovaries are located in segment XI. They are composed of a dozen or so syncytial germ-line cysts, which are associated with somatic cells. Each germ cell in a cyst has one intercellular bridge that joins it to a central and anuclear cytoplasmic mass, the cytophore. This pattern of cyst organization is typical for all clitellates that have been studied to date. Initially, the germ cells in a cyst undergo a synchronous development, however, there is no synchrony between cysts, and therefore there is a developmental gradient (oogonia, pre-diplotene germ cells, germ cells in diplotene) of oogenesis along the long ovary axis. The cysts are composed of a maximum of 32 cells. Cysts with cells in diplotene detach from the ovaries and the extraovarian phase of oogenesis begins. The developmental synchrony is lost, one cell (an oocyte) per cyst starts to gather cell components and yolk and grows considerably. The remaining cells grow to some extent and function as nurse cells. Like in other microdriles, P. montanus oocytes are rich in yolk; other features of oogenesis are also similar to those that are known from other microdrile taxa. The system of ovary organization found in the studied species is broadly similar to the corresponding features known from Naidinae and Phreodrilidae and, to some extent, in Enchytraeidae. However, this system is different from the one that is known in Tubificinae, Limnodriloidinae and Branchiurinae.

Fine intercellular connections in development: TNTs, cytonemes, or intercellular bridges?

Intercellular communication is a fundamental property of multicellular organisms, necessary for their adequate responses to changing environment. Tunneling nanotubes (TNTs) represent a novel means of intercellular communication being a long cell-to-cell conduit. TNTs are actively formed under a broad range of stresses and are also proposed to exist under physiological conditions. Development is a physiological condition of particular interest, as it requires fine coordination. Here we discuss whether protrusions shown to exist during embryonic development of different species could be TNTs or if they represent other types of cell structure, like cytonemes or intercellular bridges, that are suggested to play an important role in development.

Description of ovary organization and oogenesis in a phreodrilid clitellate.

Phreodrilidae is a small family uniting about 50 species of minute freshwater clitellate annelids inhabiting mainly the Southern hemisphere. Other than the male and spermathecal genitalia, their internal organization is poorly known. Here, we present results of our study of the ovaries and oogenesis in Insulodrilus bifidus, a phreodrilid from Western Australia using light and electron microscopy. The ovaries are paired and located in segment XII. They are inconspicuous and composed of several (10-12) spherical germ-line cysts loosely interconnected by flattened somatic cells. The cysts usually comprise 32 germ cells and each cell is connected via a cytoplasmic bridge (ring canal) to the central cytoplasmic mass (the cytophore). In ovaries, germ cells in a given cyst develop in full synchrony. However, there is no synchrony among cysts, so there is a developmental gradient of cysts (from oogonial to early meiotic) along the longitudinal ovary axis. Within the cysts that are located in the distal end of the ovary the synchrony is finally lost and interconnected cells diversify into two morphologically distinct categories: an oocyte and 31 nurse cells. Such cysts detach from the ovaries and further development occurs within the body cavity. The oocyte gathers nutrients, mainly in form of yolk spheres, whereas nurse cells grow slightly and do not gather yolk. Organelles such as ribosomes, mitochondria and endoplasmic reticulum pass freely through the ring canals and are present within the cytophore, which suggests cytoplasmic transfer towards the oocyte. The formation of female germ-line cysts equipped with cytophore and cells differentiated into oocyte and nurse cells matches the general pattern of oogenesis found in clitellates. In details, the ovary organization and oogenesis found in I. bifidus resembles the situation described in some representatives of Naidinae and Enchytraeidae.

Expression analysis of genes encoding TEX11, TEX12, TEX14 and TEX15 in testis tissues of men with non-obstructive azoospermia.

OBJECTIVE: Spermatogenesis is a complex process controlled by a plethora of genes. Changes in expression and function of these genes may thus lead to spermatogenic deficiency and male infertility. TEX11, TEX12, TEX14 and TEX15 are germ cell-specific genes expressed in the testis. TEX11, involved in the initiation and maintenance of chromosome synapses in meiotic chromosomes, has been shown to be essential for meiosis and fertility in males. TEX14, a component of intercellular bridges in germ cells, is required for spermatogenesis and fertility. TEX12 and TEX15 are essential for correct assembly of the synaptonemal complex and thus meiosis progression. METHODS: In order to examine whether changes in expression of these genes is associated with impaired spermatogenesis, expression levels of these genes were quantified by RT-qPCR on samples retrieved from infertile patients submitted to diagnostic testicular biopsy at Royan institute. Samples were divided into two groups of 18 patients with non-obstructive azoospermia considered as case; nine patients with obstructive azoospermia were included in the control group. RESULTS: A significant down-regulation of these genes was observed in the SCOS group when compared to the control group. CONCLUSION: This result suggests that regular expression of TEX11, TEX12, TEX14 and TEX15 is essential for the early stages of spermatogenesis.

Micromorphology of ovaries and oogenesis in Grania postclitellochaeta (Clitellata: Enchytraeidae).

The genus Grania comprises over 70 species of exclusively marine clitellate annelids belonging to the family Enchytraeidae. Morphologically, this genus is well separated from other enchytraeids, with thick cuticles, anterior segments I-IV fused into a "head", chaetal bundles consisting only of one stout chaeta, and reduction of circular musculature. The aim of the present study is to describe the ovary organization and the course of oogenesis in Grania postclitellochaeta, and to compare it with other known systems of ovary organization and oogenesis in clitellate annelids, especially in enchytraeids. Generally, oogenesis in G. postclitellochaeta can be divided into two phases: (i) early stages of oogenesis, occurring within the paired ovaries - each ovary is similar to a bunch of grapes, where each 'lobe' is a germ-line cyst enveloped by flat somatic cells, and (ii) oogenesis proper, which takes place within the body lumen where each growing oocyte is accompanied by its own group of nurse cells. Germ cells are interconnected by cytoplasmic channels (intercellular bridges, ring canals) and form syncytial cysts. As in other clitellate annelids, the cyst center contains a common cytoplasm (cytophore) to which each cell is connected by one ring canal only. Initially, within the ovary, all interconnected cells develop synchronously and are morphologically similar. At the time when the cysts detach from the ovary, one of the interconnected cells begins to gather nutrients, grows and becomes an oocyte, whereas the rest of the cells (nurse cells) do not continue meiosis and instead seem to provide the oocyte with macromolecules and cell organelles. Analysis of serial sections reveals that cysts are always composed of 16 cells - one oocyte and fifteen nurse cells. A comparative analysis showed that almost all features of oogenesis in G. postclitellochaeta are similar to that in other representatives of Enchytraeidae (mainly Enchytraeus albidus), suggesting evolutionary conservation of the process across this family.

Characterization of Tunneling Nanotubes in Wharton's jelly Mesenchymal Stem Cells. An Intercellular Exchange of Components between Neighboring Cells.

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.

Analysis of the cytoskeleton organization and its possible functions in male earthworm germ-line cysts equipped with a cytophore.

We studied the organization of F-actin and the microtubular cytoskeleton in male germ-line cysts in the seminal vesicles of the earthworm Dendrobaena veneta using light, fluorescent and electron microscopy along with both chemically fixed tissue and life cell imaging. Additionally, in order to follow the functioning of the cytoskeleton, we incubated the cysts in colchicine, nocodazole, cytochalasin D and latrunculin A. The male germ-line cells of D. veneta are interconnected via stable intercellular bridges (IB), and form syncytial cysts. Each germ cell has only one IB that connects it to the anuclear central cytoplasmic mass, the cytophore. During the studies, we analyzed the cytoskeleton in spermatogonial, spermatocytic and spermatid cysts. F-actin was detected in the cortical cytoplasm and forms distinct rings in the IBs. The arrangement of the microtubules changed dynamically during spermatogenesis. The microtubules are distributed evenly in whole spermatogonial and spermatocytic cysts; however, they primarily accumulate within the IBs in spermatogonia. In early spermatids, microtubules pass through the IBs and are present in whole cysts. During spermatid elongation, the microtubules form a manchette while they are absent in the cytophore and in the IBs. Use of cytoskeletal drugs did not alter the general morphology of the cysts. Detectable effects-the occurrence of nuclei in the late spermatids and manchette fragments in the cytophore-were observed only after incubation in nocodazole. Our results suggest that the microtubules are responsible for cytoplasmic/organelle transfer between the germ cells and the cytophore during spermatogenesis and for the positioning of the spermatid nuclei.

"Intercellular bridges" in a case of well differentiated squamous carcinoma.

Intercellular bridges may aide in definitive identification of malignant cell origin, especially in squamous cell carcinoma. They are difficult to identify in routine cytologic specimens and are especially rare in smear preparations. Herein, we present images of intercellular bridges from a case of well differentiated squamous cell carcinoma of the esophagus in a cytologic specimen obtained from FNA of a paraesophageal lymph node.

Structural and biochemical insights into the role of testis-expressed gene 14 (TEX14) in forming the stable intercellular bridges of germ cells.

Intercellular bridges are a conserved feature of spermatogenesis in mammalian germ cells and derive from arresting cell abscission at the final stage of cytokinesis. However, it remains to be fully understood how germ cell abscission is arrested in the presence of general cytokinesis components. The TEX14 (testis-expressed gene 14) protein is recruited to the midbody and plays a key role in the inactivation of germ cell abscission. To gain insights into the structural organization of TEX14 at the midbody, we have determined the crystal structures of the EABR [endosomal sorting complex required for transport (ESCRT) and ALIX-binding region] of CEP55 bound to the TEX14 peptide (or its chimeric peptides) and performed functional characterization of the CEP55-TEX14 interaction by multiexperiment analyses. We show that TEX14 interacts with CEP55-EABR via its AxGPPx3Y (Ala793, Gly795, Pro796, Pro797, and Tyr801) and PP (Pro803 and Pro804) sequences, which together form the AxGPPx3YxPP motif. TEX14 competitively binds to CEP55-EABR to prevent the recruitment of ALIX, which is a component of the ESCRT machinery with the AxGPPx3Y motif. We also demonstrate that a high affinity and a low dissociation rate of TEX14 to CEP55, and an increase in the local concentration of TEX14, cooperatively prevent ALIX from recruiting ESCRT complexes to the midbody. The action mechanism of TEX14 suggests a scheme of how to inactivate the abscission of abnormal cells, including cancer cells.

Intercellular bridges are essential for human parthenogenetic cell survival.

Parthenogenetic cells, obtained from in vitro activated mammalian oocytes, display multipolar spindles, chromosome malsegregation and a high incidence of aneuploidy, probably due to the lack of paternal contribution. Despite this, parthenogenetic cells do not show high rates of apoptosis and are able to proliferate in a way comparable to their biparental counterpart. We hypothesize that a series of adaptive mechanisms are present in parthenogenetic cells, allowing a continuous proliferation and ordinate cell differentiation both in vitro and in vivo. Here we identify the presence of intercellular bridges that contribute to the establishment of a wide communication network among human parthenogenetic cells, providing a mutual exchange of missing products. Silencing of two molecules essential for intercellular bridge formation and maintenance demonstrates the key function played by these cytoplasmic passageways that ensure normal cell functions and survival, alleviating the unbalance in cellular component composition.

Gonads and gametogenesis in astigmatic mites (Acariformes: Astigmata).

Astigmatans are a large group of mites living in nearly every environment and exhibiting very diverse reproductive strategies. In spite of an uniform anatomical organization of their reproductive systems, gametogenesis in each sex is highly variable, leading to gamete formation showing many peculiar features and emphasizing the distinct position of Astigmata. This review summarizes the contemporary knowledge on the structure of ovaries and testes in astigmatic mites, the peculiarities of oogenesis and spermatogenesis, as well as provides new data on several species not studied previously. New questions are discussed and approaches for future studies are proposed.

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.

Eccrine poroma on the face: an atypical presentation.

An eccrine poroma is a solitary tumor arising from the eccrine duct epithelium in the epidermis. The lesions commonly occur on the sole of the foot, the hands, and occasionally on the nose, eyelids, neck, and chest. We report a patient who presented with a slow-growing nodular lesion over her left cheek, prompting a diagnosis of basal cell carcinoma or keratoacanthoma. Biopsy from the nodule revealed a well-defined epidermal tumor with uniform small cuboidal cells with rounded deeply basophilic nuclei, few narrow ductal lumina, and occasional cystic spaces confirming the diagnosis of an eccrine poroma.

Tunneling Nanotubes: A new paradigm for studying intercellular communication and therapeutics in cancer.

Tunneling nanotubes are actin-based cytoplasmic extensions that function as intercellular channels in a wide variety of cell types.There is a renewed and keen interest in the examination of modes of intercellular communication in cells of all types, especially in the field of cancer biology. Tunneling nanotubes -which in the literature have also been referred to as "membrane nanotubes," "'intercellular' or 'epithelial' bridges," or "cytoplasmic extensions" - are under active investigation for their role in facilitating direct intercellular communication. These structures have not, until recently, been scrutinized as a unique and previously unrecognized form of direct cell-to-cell transmission of cellular cargo in the context of human cancer. Our recent study of tunneling nanotubes in human malignant pleural mesothelioma and lung adenocarcinomas demonstrated efficient transfer of cellular contents, including proteins, Golgi vesicles, and mitochondria, between cells derived from several well-established cancer cell lines. Further, we provided effective demonstration that such nanotubes can form between primary malignant cells from human patients. For the first time, we also demonstrated the in vivo relevance of these structures in humans, having effectively imaged nanotubes in intact solid tumors from patients. Here we provide further analysis and discussion on our findings, and offer a prospective 'road map' for studying tunneling nanotubes in the context of human cancer. We hope that further understanding of the mechanisms, methods of transfer, and particularly the role of nanotubes in tumor-stromal cross-talk will lead to identification of new selective targets for cancer therapeutics.