An ultrastructural study of reproduction in the sporocysts of <Emphasis Type="Italic">Prosorhynchoides gracilescens</Emphasis> (Digenea: Bucephalidae)

The germinal development in Prosorhynchoides gracilescens sporocysts was studied using electron microscopy. The germinal cells proliferated and developed within multiple floating germinal masses located in the terminal portions of sporocyst branches. The germinal masses were composed of supporting and germinal cells. Supporting cells possessed numerous flattened extensions that spread around and between developing germinal cells to form three-dimensional mesh network, which maintained the integrity of the germinal mass. Morphological evidences of close interactions between supporting and germinal cells were numerous gap junctions between the two cell types and phagocytosis of small fragments of germinal cells cytoplasm by supporting cells. The germinal cells displayed structural differences that seemed to reflect their sequential developmental changes. These changes included (1) cell growth and increase of organelles number, (2) dispersion of nuclear chromatin and increase of nucleolus size, (3) polarization of the cell, (4) appearance of specific structures such as nuage and laminated inclusions. The germinal cells left the germinal masses to finish their differentiation in the body cavity and then cleaved to give rise to cercarial embryos. Ultrastructural features of the germinal elements of P. gracilescens sporocysts are discussed in the light of existing controversy concerning the nature of the germinal sacs reproduction.     

Optical and ultrastructural studies of midgut and salivary glands of first instar of Dermatobia hominis (Diptera: Oestridae)

Midguts and salivary glands of newly hatched larvae (L1) of Dermatobia hominis (L., Jr.) were studied using light and electron microscopy. The larval midgut has a tubular, sinusoidal form and consists of a monolayer of epithelial cells with an underlying basement membrane and a surrounding layer of connective tissue. The fine structure of the midgut shows digestive cells with short microvilli, large nuclei, and cytoplasm containing few visible organelles (mitochondria, rough endoplasmic reticulum, and free ribosomes). In the basal region, the plasma membrane of the cells is folded into a labyrinth area. Hemidesmosomes link the basal surface to the basement membrane and septet junctions are present between adjacent cells. The connective tissue circling the basement membrane contains collagen fibrils, muscle fibers, and tracheal tubes. Prominent nuclei with evident nucleoli occur in the digestive cells. The salivary gland is simple and tubular. It has a monolayer of epithelial cells surrounded by basement membrane and connective tissue. The fine structure of the salivary gland shows epithelial cells, microvilli, secretion into the lumen, septate junctions at the lateral face and a basal labyrinth region. The cell nucleus is large and the cytoplasm contains rough endoplasmic reticulum, ribosomes and mitochondria.     

Oogenesis in the free proglottis ofTrilocularia acanthiaevulgaris (Cestoda,Tetraphyllidea)

The early (ovarian) stages of oogenesis inTrilocularia acanthiaevulgaris have been studied by light microscope histochemistry and transmission electron microscopy. The process proceeds as far as meiotic prophase in the primary oocyte. The oogonia and early immature primary oocytes occupy the anterior and outer edges of the ovary and are typically undifferentiated cells, showing a high nucleo-cytoplasmic ratio. The scant cytoplasm is packed with free ribosomes and contains a small number of mitochondria and a few short strands of granular endoplasmic reticulum (GER). The oogonia undergo a number of mitotic divisions, marked by the presence of centrioles. The immature primary oocytes enter meiotic prophase, as evidenced by the appearance of nuclear synaptonemal complexes. The maturing primary oocytes, which occupy the inner, central region of the ovary, undergo a growth phase that is accompanied by an increased nucleolar volume and export of RNA to the cytoplasm via nuclear pores, and by an increase in the number of mitochondria. A GER network develops and, together with the Golgi complexes, is involved in the production of a small number of cortical granules. The GER often takes the form of concentric cisternae. Numerous lipid droplets are also present in the cytoplasm. The mature primary oocytes predominate in the posterior region of the ovary, near the oviduct. They represent a resting phase in development, in which cellular activity is minimal. Lipid droplets are abundant and the cortical granules remain in more central regions of the cell and do not migrate to the periphery of the cell. The ovary contains a second, non-germinal type of cell, the follicle cell. The cell body is smaller in size than the oogonia, and cytoplasmic processes from it ramify around the periphery of the ovary. The different cell types within the ovary are embedded in a cytoplasmic matrix that contains a number of organelles.     

Germline Cysts and the Formation of the Germinal Epithelium During the Female Gonadal Morphogenesis in Cyprinus carpio (Teleostei: Ostariophysi: Cypriniformes)

The formation of both germline cysts and the germinal epithelium is described during the ovary development in Cyprinus carpio. As in the undifferentiated gonad of mammals, cords of PGCs become oogonia when they are surrounded by somatic cells. Ovarian differentiation is triggered when oogonia proliferate and enter meiosis, becoming oocytes. Proliferation of single oogonium results in clusters of interconnected oocytes, the germline cysts, that are encompassed by somatic prefollicle cells and form cell nests. Both PGCs and cell nests are delimited by a basement membrane. Ovarian follicles originate from the germline cysts, about the time of meiotic arrest, as prefollicle cells surround oocytes, individualizing them. They synthesize a basement membrane and an oocyte forms a follicle. With the formation of the stroma, unspecialized mesenchymal cells differentiate, and encompass each follicle, forming the theca. The follicle, basement membrane, and theca constitute the follicle complex. Along the ventral region of the differentiating ovary, the epithelium invaginates to form the ovigerous lamellae whose developing surface epithelium, the germinal epithelium, is composed of epithelial cells, germline cysts with oogonia, oocytes, and developing follicles. The germinal epithelium rests upon a basement membrane. The follicles complexes are connected to the germinal epithelium by a shared portion of basement membrane. In the differentiated ovary, germ cell proliferation in the epithelium forms nests in which there are the germline cysts. Germline cysts, groups of cells that form from a single founder cell and are joined by intercellular bridges, are conserved throughout the vertebrates, as is the germinal epithelium. Anat Rec 293:1581–1606, 2010. © 2010 Wiley‐Liss, Inc.     

Stem cells: you can't tell a cell by its cover

Embryonic stem (ES) cells are capable of unlimited self-renewal and have the ability to give rise to all tissue types in the body. The use of human ES cells for tissue and cell therapeutics has been suggested, but is limited by ethical concerns as these cells are derived from the inner cell mass of human embryos. In addition, the need for HLA matching of ES cell-derived tissues for allogeneic transplantation would require a bank of several thousand ES cell lines to make tissue therapeutics practical. Recently, adult stem cells-of which those in bone marrow are the best studied-have been shown to be capable of multilineage differentiation into cells of various non-blood tissues. Umbilical cord blood (UCB) haematopoietic stem cells have been shown to be equivalent to bone marrow stem cells for reconstitution of the haematopoietic system. Preliminary studies have also demonstrated that UCB haematopoietic stem cells are multipotent and capable of differentiating into non-blood cell types. This observation raises the exciting possibility of replacing human ES cells for tissue and cell therapeutics with UCB blood haematopoietic stem cells that are normally discarded with the placenta after delivery.     

小鼠胚着床前线粒体的分布和超微结构变化

为了解小鼠着档前细胞中线粒体的分布和超微结构的变化规律，观察了２细胞胚，４细胞胚，８细胞胚，桑椹胚，早期囊胚和晚期囊胚，２细胞期和桑椹胚期，线粒体绕胞核集，在挤紧的８细胞胚中，线粒体在细胞接触面处的胞质边缘密集。囊胚期，滋养层细胞的线粒体在胞核周围较宽的区域中分布。     

胚胎干细胞向成骨、软骨细胞定向分化研究进展

骨、软骨组织工程的热点之一是寻找合适的种子细胞.胚胎干细胞因具有全能性和无限增殖的能力有望成为组织工程中的种子细胞新来源.主要介绍了胚胎干细胞定向诱导分化为成骨、软骨细胞研究所取得的进展,并展望了胚胎干细胞作为组织工程种子细胞的前景和所面临的困难.     

Ultrastructural evidence for completion of the entire miracidial maturation in intrauterine eggs of the digenean Brandesia turgida (Brandes, 1888) (Plagiorchiida: Pleurogenidae)

Results of this TEM study provide ultrastructural evidence that miracidial morphogenesis is fully completed within the intrauterine eggs situated in the most posterior uterine regions of the pleurogenid trematode Brandesia turgida (Brandes, 1888). The ultrastructural characteristic of different larval organelles and cell types of these eggshell-enclosed, but fully formed, cilated miracidia is described. The body wall of the pyriform mature miracidium of B. turgida is composed of ciliated epidermis and underlying peripheral body musculature. Two miracidial flame cells of the protonephridial excretory system are localized in the central region of the ciliated larvae. Three types of miracidial glands were observed: a single apical gland, two lateral glands, and several small vesiculated glands; each gland type contains characteristic, but different types of secretory granules. The anterior end of each miracidium consists of an apical papilla on which are situated the exits of the three main larval glands: an exit of a single apical gland as well as the individual exits of two lateral glands. The exits of vesiculated glands, containing characteristic spherical membrane-bound and highly electron-dense granules, evidently different from the two other types of secretory granules of apical and lateral glands, were not identified. Germinative cells, grouped together in a sac-like germinative follicle, are situated in the medioposterior part of the larva, the germatophore. The germinative cells contain numerous electron-dense heterochromatin islands arranged in the form of a network or chain-like pattern and distributed mainly in the karyoplasm adjacent to the nuclear membrane. The thin layer of granular cytoplasm is rich in free ribosomes and contains a few small mitochondria. Both nuclear and cytoplasmic features if these cells indicate their great developmental potential for further growth and multiplication in postembryonic stages of the life cycle. In the mature eggs, the areas of focal cytoplasmic degradation were frequently observed and may be involved in the autolysis of some embryonic structures. Obtained results are compared with available literature data on the functional ultrastructure of the miracidia of other digeneans.     

The meaning of the anti-cancer antibody CLN-IgG (Pritumumab) generated by human × human hybridoma technology against the cyto-skeletal protein,vimentin, in the course of the treatment of malignancy

Cancer stem cells in a tumor mass form a very small subpopulation ranging from below 0.1% in a brain tumor but they have the crucial ability to become malignant. The goal of cancer therapy has been the total killing of tumor cells. However we should clarify that most of all tumor cells are differentiated cancer cells. Thus the elimination of 99.9% of tumor cells under histological criteria cannot ensure the cancer will be cured. Rather cancer cell biologists should turn their attention to reprogramming cancer stem cells to normal stem cells by which malignancy recuperates normal organogenesis from the aspect of the dichotomy of cancer stem cell. The cue points underlying the reverse cancer stem cell at blastogenesis in inflammation site is depending upon cell-to-cell recognition of the tumor-niche cells. Normalization of tumor-niche promises to lead cancer stem cell into normal stem cell owing to autonomous healing mechanisms that reside in the self-defense mechanisms in immunity and the cell competition mechanisms in the wound healing of the tissue cells. Among the cyto-skeletal proteins, vimentin becomes a target of self-restoration of cancer stem cell by means of immune surveillance. A human monoclonal antibody, CLN-IgG recognizes vimentin expressing on the cell surface of the malignant tumor. Since vimentin network resides in the cytoplasm connecting the plasma membrane with chromatin assembly in the nucleus, it is highly likely vimentin plays an important role in up-regulation and down-regulation through signal transduction between certain membrane receptors and gene expression with respect to the transformation of the cell. Aberrant arrangement of vimentin undergoes malignancy accompanied by epithelial–mesenchymal-transition relating to the aberrant apoptotic cellular behavior in the tumor-niche. Restraint of the aberrant expression of vimentin on the plasma membrane of the malignant cell evokes a pertinent signal transduction pathway for healing that is an indication there must be a reverse path that reprograms cancer stem cells to normal organogenesis.     

Origin and early migration of primordial germ cells in the chick embryo: A study of the stages definitive primitive streak through eight somites

The germinal crescent in the chick embryo is characterized by small, PAS-positive, nonglycogen granules from 1.5 to 5 μ in diameter. The primordial germ cells (PGCs) were found to originate in and separate from the germinal crescent endoderm through stage 7 (2 somites). Shortly after separation most of the granules in the PGCs lost their organization and the PAS-positive material was distributed irregularly throughout the cytoplasm. A few of these granules remained within the cells indefinitely. Glycogen of an agranular nature which had shifted to one pole of the cell was observed at stage four. Granular glycogen which was distributed throughout the cytoplasm was not observed prior to stage 7 or 8. Cell counts on individual embryos showed noticeable variations as to the number of germ cells between embryos of the same stage. For example, in stage 4 embryos the minimum number of cells counted, including attached and free, was 78 and the maimum 169, while in stage 9 the minimum was 83 and the maximum 469 cells. After separation the germ cells were observed almost anywhere between the ectoderm and the endoderm although the majority remained in the area where they originated.     

Salivary glands of second and third instars of Dermatobia hominis (Diptera: Oestridae)

Salivary glands of Dermatobia hominis (L., Jr.) (Diptera: Oestridae) larvae were studied under light and electron microscopy. The salivary glands of second (L2) and third instars (L3) are similar and consist of pairs of translucent tubules. The individual efferent ducts unite to form a single deferent duct, which inserts dorsally into the cephalopharingeal skeleton. Each gland has a monolayer of epithelial cells surrounded by basement membrane and connective tissue. The cellular plasma membrane is enfolded at its base, forming a labyrinthine area. The cell surface is linked to the basement membrane (BM) by hemidesmosomes and to adjacent cells by septet junctions and desmosomes. Irregular channels with several vesicles occur between the cytoplasm and BM. Golgi complex, rough and smooth endoplasmic reticulum, ribosome, lysosomes, multivesicular bodies, and myelin figures are usually present in the cells. The nucleus is large, with diffuse chromatin. The connective tissue circling the BM contains collagen fibrils, muscle fibers and tracheal tubes. Lined cuticle encloses the efferent and deferent ductal cells, which have few, widely dispersed mitochondria, free ribosomes, microtubules, and a large nucleus with diffuse chromatin.     

Ultrastructure of the atrioventricular junctional area in the heart of Molossus molossus Pallas 1766 (Chiroptera: Molossidae)

The atrioventricular junctional area (AVJA) consists of a group of structures that connects the atrial and ventricular myocardium. Five hearts of an insect-eating bat were studied in light and transmission electron microscopy. In M. molossus, the AVJA consists in a mass of muscle fibers intermingled with variable amount of connective tissue and blood vessels surrounded by the adjacent myocardium and the attachment of the right atrioventricular and aortic valves in the fibrous skeleton. In light microscopy, conducting cells of the AV node and bundle can be distinguished from working cells: smaller size, paler staining reaction and the presence of e sheath of connective tissue surrounding each cell (largely composition by type I collagen fibers). Three cell types are observed in the AVJA. Nodal cells are irregular with few cytoplasmic organelles and several slender sarcolemmal modifications. Myofibrils are sparse and not clearly observable. Transitional cells are spindle-shaped and grouped together into bundles. The cytoplasm, poor in glycogen, has scarce electron-density and myofibrils organized into sarcomeres. Caveolae is observed randomly distributed at the periphery of the cell. The AV bundle cells are elongated with clusters of myofibrils organized in the periphery and a glycogen free area around the nucleus. Ventricular cells are bigger than the atrial ones and show well-developed myofibrils in alternated rows with mitochondria. Lipid droplets are seen near mitochondria and glycogen granules. Intercalated discs and T-tubules are found in working cells but not in conducting ones. The fibrous skeleton has collagen fibers intercalated with fibroblasts.     

Domains of high-polarized and low-polarized mitochondria may occur in mouse and human oocytes and early embryos

BACKGROUND: The magnitude of the inner mitochondrial membrane potential (deltapsim) appears to influence the level of certain mitochondrial activities including regulation of ionic fluxes and ATP liberation, activities that are often compartmentalized or location dependent in cells. Recent evidence suggests that within cells, mitochondria can be heterogeneous with respect to deltapsim, and that high-polarized mitochondria (high deltapsim) may occur in the subplasmalemmal cytoplasm where intercellular contact is absent. Here, we investigated whether deltapsim in oocytes and preimplantation embryos was heterogeneous and cell contact-associated. METHODS: Mouse and human oocytes and preimplantation stage embryos stained with mitochondria-specific probes rhodamine 123, MitoTracker Orange, and the deltapsim-sensitive probe JC-1, (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazoylcarbocyanine iodide), were examined by epifluorescence, scanning laser confocal, and transmission electron microscopy. The possibility that intercellular contact and deltapsim are associated was examined for oocytes, where transzonal coronal cell contacts were terminated naturally or experimentally, and for intact, disaggregated, and reconstructed cleavage stage mouse embryos. RESULTS: For both oocytes and embryos, clusters of apparently high-polarized mitochondria occur in the pericortical cytoplasm in regions free from intercellular contact. CONCLUSIONS: The findings suggest that mitochondria in oocytes and preimplantation embryos may be heterogeneous with respect to deltapsim. We propose that high-polarized pericortical mitochondria may have a role in the acquisition of oocyte competence and the regulation of early developmental processes that may be associated with elevated metabolism or intracellular signalling through calcium-induced calcium release pathways.     

Differential expression of the embryo/cancer gene ECSA(DPPA2), the cancer/testis gene BORIS and the pluripotency structural gene OCT4, in human preimplantation development

In this paper, we examine the expression profiles of two new putative pluripotent stem cell genes, the embryo/cancer sequence A gene (ECSA) and the cancer/testis gene Brother Of the Regulator of Imprinted Sites (BORIS), in human oocytes, preimplantation embryos, primordial germ cells (PGCs) and embryo stem (ES) cells. Their expression profiles are compared with that of the well-known pluripotency gene, OCT4, using a primer design that avoids amplification of the multiple OCT4 pseudogenes. As expected, OCT4 is high in human oocytes, down-regulated in early cleavage stages and then expressed de novo in human blastocysts and PGCs. BORIS and ECSA show distinct profiles of expression in that BORIS is predominantly expressed in the early stages of preimplantation development, in oocytes and 4-cell embryos, whereas ECSA is predominantly expressed in the later stages, blastocysts and PGCs. BORIS is not detected in blastocysts, PGCs or other fetal and adult somatic tissue tested. Thus, BORIS and ECSA may be involved in two different aspects of reprogramming in development, viz., in late gametogenesis, and at the time of formation of the ES cells (inner cell mass (ICM) and PGC), respectively. However, in human ES cells, where a deprogrammed stem cell state is stably established in culture, an immunofluoresence study shows that all three genes are co-expressed at the protein level. Thus, following their derivation from ICM cells, ES cells may undergo further transformation in culture to express a number of embryo and germ line stem cell functions, which, in normal development, show different temporal and spatial specificity of expression.     

Cytokinetic pattern in the thoracic spinal cord of chick embryos (incubation day 5-13) using PCNA staining and TUNEL method.

For the cytokinetic studies using spinal cords of chick embryos, chronological patterns of cell proliferation and programmed cell death (apoptosis) should be known. Information in the early stages of chick embryos is available while data on later stages are seldom available. To investigate the chronological patterns of cell proliferation and apoptosis in the thoracic spinal cord of normal chick embryos on incubation day 5, 6, 8, 10 and 13 (Hamburger and Hamilton stage 26-40), proliferating cell nuclear antigen (PCNA) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method were used. Cell proliferation was active at the germinal layer on days 5 and 6. It markedly declined on day 8 and became negligible on day 13. TUNEL-positive cells were mainly found in the germinal layer, the ventrolateral part of the mantle layer and the dorsal root ganglion. Compared to PCNA-positive cells, TUNEL-positive cells were sparse, especially after day 10, when only a few positive cells were scattered. These results will be used as a control data for the studies such as an experimental research for neural tube defects.     

Morphology of mitochondrial permeability transition: morphometric volumetry in apoptotic cells

Here we report on the mitochondrial permeability transition (MPT), which refers to the morphology of mitochondria whose inner membrane has lost its selective permeability. In all types of apoptotic cells so far examined, we found outer mitochondrial membranes that had been ruptured. These mitochondria present a swollen matrix covered by an inner membrane herniating into the cytoplasm through the breached outer membrane. Similarly ruptured outer mitochondrial membranes have been reported in studies on mitochondrial fractions induced to undergo MPT, carried out by others. Our observations were made on five types of rat tissue cells and six different cultured cell lines in the early stages of apoptosis. Samples from the cell lines HL-60, HeLa, WEHI-164, and a special batch of PC-12 cells were subjected to various apoptogenic agents and analyzed morphometrically. Nonapoptotic companion cells with unaltered nuclear structure (CUNS) were also analyzed. The mitochondrial volume in microm(3) and the volume fraction of the cytoplasm occupied by mitochondria in cells with typical nuclear signs of apoptosis and also in CUNS were evaluated. The volume of the mitochondria with ruptured membrane represents at least 69% (47-89%) of the total mitochondrial volume of the apoptotic cells. Thus, a considerable fraction of the cellular mitochondrial mass is or was in the state of permeability transition and probably involved in enhancement of the apoptotic program. In all samples, a fraction of the cells with normal nuclei possessed mitochondria with breached outer membranes as described above. In these cells, MPT occurred before the appearance of the typical nuclear phenotype of the apoptotic cells.     

Sequential alterations of neuronal architecture in nucleus magnocellularis of the developing chicken: An electron microscope study

The development of the auditory nerve endings and their target cells in nucleus magnocellularis was studied by electron microscopy of perfusion-fixed brains from embryonic day 12 to hatching. Embryonic days 12–13: somatic processes extend from the perikaryon. The cytoplasm of the soma and processes contains free ribosomes, mitochondria, lysosomes, rough endoplasmic reticulum, Golgi apparatus and an eccentric, heterochromatic nucleus. Small profiles of auditory nerve fibers containing round, clear vesicles make specialized contacts, including some synapses, on distal somatic processes but rarely on proximal somatic processes or on the soma. The postsynaptic zones contain a flocculent matrix. Days 15–17: somatic processes disappear and occasional attachment plaques are seen between cell bodies. The nucleus appears euchromatic. Cytoplasmic organelles form a dense matrix indicative of intense metabolic activity. Somatic spines are evident. The afferent axons form large, vesiculated profiles located, increasingly, on the cell body and somatic spines, with many points of synaptic contact. Opposite each ending a band of amorphous, flocculent material fills the postsynaptic cytoplasm. Embryonic day 18-hatching: the somatic cytoplasm becomes less dense; stacks of rough endoplasmic reticulum start to condense. Afferent axon terminals mature, especially the synaptic membrane complex and associated densities. The postsynaptic flocculent material diminishes in extent until it is found associated only with somatic spines.The ultrastructural observations on the maturation of nucleus magnocellularis closely corroborate and extend previous results with the Golgi methods. Developing auditory nerve fibers initially synapse on the distal parts of the somatic processes of the immature cells. As the somatic processes disappear or retract, axonal endings move to the soma and develop into large axosomatic end-bulbs. Possibly, the somatic processes as they retract drag the auditory nerve endings to the cell body. The findings also suggest a role of the transiently appearing, flocculent material of the postsynaptic regions in the formation of synapses.     

Ultrastructure of developing germ cells in the fetal rabbit testis

Testicular differentiation, as evidenced by the appearance of a distinct tunica albuginea and formation of plate-like aggregates of germ cells and mesenchymal cells, occurs at 16 days in the rabbit fetus. The germ cells at this stage of development are isolated large, round to oval cells that are easily distinguished from the smaller elongated mesenchymal elements surrounding them. The germ cell nuclei have a smooth spherical contour and a prominent eccentric nucleolus. Cytoplasmic organelles are sparse, consisting of scattered polyribosomes and spherical or ovoid mitochondria distributed singly in the cell. At 18 days, the cell groups form into distinct cords of germ cells and supporting cells, the earliest stage of seminiferous tubule formation. Between 22 and 25 days, proliferating germ cells show increasing condensation of nuclear chromatin and increased nucleolar complexity. The cytoplasm contains groups of mitochondria, dense polyribosomal complexes, lipid aggregates, scattered smooth vesicles and prominent Golgi complexes. Intercellular bridges appear between germ cells on day 22 and increase in number on succeeding days. During the last week of fetal development, germ cells become aligned in rows at the periphery of the primitive tubules. Their arrangement at this time resembles that of spermatogonia in the adult testis. However, the fetal “prespermatogonia” lack some of the characteristic ultrastructural features of adult spermatogonia.