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PubMed1990

[Spermatogenic cycle in seals (Callorhinus ursinus L.)].

In the fur-seal germ cells at various stages of development are situated in the spermatic canaliculi as concentric layers in accordance with the stages of the spermatogenic cycle. By means of PAS-reaction 18 stages have been revealed in spermatogenesis of the fur-seal and basing on the first 15 of them, 15 stages of the spermatogenic cycles have been presented. Transformations of the nucleus during the process of spermatids development proceed similar to other animals studied, formation of acrosomes is accompanied with a specific for the given species development of temporal formation--tubulo-bulbar complexes, reduced at terminal stages of spermiogenesis.

Animals↗
PubMed1989

Appearance of lectin-binding sites during vascularization of the primordium of the central nervous system in 10 to 12-day-old mouse embryos.

In the present study lectin-binding sites were investigated for the lectins Ricinus communis agglutinin (RCA I), wheat germ agglutinin (WGA), soya bean agglutinin (SBA), concanavalin A (Con A), Lotus tetragonolobus-(LTA) and Limulus polyphemus agglutinin (LPA) during the initial stages of vasculogenesis of the CNS-anlage in 10 to 12-day-old NMRI mouse embryos. Specific binding sites for the lectins RCA I (sugar specificity: beta-D-galactose, N-acetylgalactosamine), WGA (sugar specificity: N-acetylglucosamine, sialic acid), and SBA (sugar specificity: N-acetylgalactosamine, beta-D-galactose) were detected in the newly formed capillaries within the neuroepithelial cell layer. In contrast, binding sites for Con A, LTA and LPA could not be observed at the start of the vascularization of the CNS-anlage. From these results, the conclusion can be drawn that glycoconjugates containing D-galactose, N-acetylgalactosamine and N-acetyl-glucosamine moieties are involved in the early vasculogenesis of the embryonic CNS-anlage of the mouse.

Animals↗
PubMed2003

Role of carbohydrates in repair of human respiratory epithelium using an in vitro model.

BACKGROUND: The epithelial layer in the conducting airway provides a primary protective barrier. Repair of this barrier normally occurs rapidly after damage, but is compromised in diseases such as asthma. OBJECTIVE: We have developed a human in vitro model system to test our hypothesis that cell surface glycoconjugate-based interactions are required for the normal repair of damaged epithelium. METHODS: Lectins having narrow carbohydrate specificities were used to identify and block specific carbohydrate moieties on human airway-derived epithelial cells in culture. RESULTS: The lectin wheat germ agglutinin bound to N-acetyl glucosamine and inhibited the repair of epithelial damage while having little effect on cell viability. In contrast, other N-acetyl glucosamine binding lectins had no effect even when bound to the cell surface. The involvement of glycoconjugates was confirmed by pre-incubating the lectin with its specific sugar, preventing the inhibition of repair. CONCLUSION: These results indicate that lectin-binding sites are involved in epithelial repair and may be important in the repetitive cycles of injury and repair seen in asthma. This model system provides an insight into the role of glycoconjugates and will help to determine the function of specific carbohydrate groups in epithelial repair. These may present a target for therapeutic intervention in respiratory and other diseases.

Asthma↗
PubMed2003

In vitro differentiation of embryonic stem cells into mineralized osteoblasts.

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid, beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase, an early osteoblast marker, was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining, which detects mineralized calcium. The major noncollagenous component of bone matrix, osteocalcin, was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture, both being characteristic for late stages of osteoblastic differentiation, indicating that at this time of culture the identified cells represent "mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin, both being involved in binding calcium ions and hydroxyapatite during mineralization processes, as well as of collagen type I, representing by far the most predominant collagen in vertebrate organisms, is enhanced at the beginning of the second culture week upon addition of supplements. In the third week of culture, treated cells showed a second peak of osteopontin, osteonectin and collagen type I expression, osteopontin and osteonectin being stimulated 3-4-fold and collagen type I being induced 6-fold over control values. Alkaline phosphatase (ALP) expression was enhanced at the beginning of the third week of culture and was found to be increased again at later stages of culture at days 27-34. The in vitro differentiation of mouse embryonic stem cells into osteoblasts may provide a suitable model for studying the molecular processes of osteoblastic development in vivo.

Animals↗
PubMed1992

Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse.

Fgf-4, initially isolated as a transforming gene from human tumors, is a member of the Fibroblast Growth Factor (FGF) family. It has previously been shown by northern blot hybridization analysis to be expressed in teratocarcinoma and embryonic stem cells, suggesting that it plays a role in embryonic development. We have carried out an RNA in situ hybridization analysis of Fgf-4 expression in the developing mouse embryo, from fertilization through the 14th day of gestation (E14.5). Our results show that Fgf-4 RNA is first detected at the late blastocyst stage in cells that give rise to all of the embryonic lineages (inner cell mass cells). During the early stages of gastrulation, expression becomes restricted to the primitive streak where mesoderm and definitive endoderm are formed. Expression continues in the distal (rostral) two-thirds of the streak through approx. E10, and then is detected in the tail bud, which replaces the streak as the primary source of mesoderm. Additional sites of expression are found after the three primary germ layers are established and organogenesis begins. Fgf-4 RNA is detected transiently in the branchial arch units, the somitic myotome, the apical ectodermal ridge of the developing limb bud and the tooth bud, suggesting that the gene has multiple roles during embryogenesis. These results are compared with the expression patterns of other FGF genes. Taken together, the data suggest that individual members of the gene family are expressed sequentially in developmental pathways such as mesoderm formation and myogenesis, and play a role in specific epithelial-mesenchymal interactions.

Animals↗
PubMed1993

Visualization of lectin-like proteins in human placenta by means of anti-plant lectin antibodies.

Proteins antigenically cross-reactive with lectins were sought in the placenta by immunohistochemistry using polyclonal antibodies raised in rabbit against four well-known lectins: Concanavalin A, Wheat germ agglutinin, Ulex europaeus agglutinin, and Phaseolus vulgaris leukoagglutinin (PHA-L), as well as one antibody raised in goat against PHA-L. Even at high dilutions of the primary antibody, strong staining was obtained after short incubations, in patterns generally resembling those obtained for placental lectins by other means, such as those based on binding capacity for glycosylated probes. One of the immunohistochemical patterns distinguishes with great clarity between the trophoblast cell layers, thus relating to developmental and functional parameters; another localises PHA-L-immunoreactivity to the syncytiotrophoblast. These results underline the validity of the immunohistochemical screening as an approach in its own right. Both positive and negative controls were applied to the immunohistochemical methodology. These controls showed that the staining patterns obtained relate to the specificities of the primary antibodies employed; i.e. to lectins. The PHA-L-like cross-reactivity was analysed immunochemically. In electrophoretically separated and Western-blotted placental extracts there were found anti-PHA-L-binding fractions of apparent molecular weights 30 kDa, 58 kDa and 67 kDa. Control studies of the PHA-L antigen showed anti-PHA-L-binding fractions of approximate molecular weights 32 kDa and 60 kDa. The 30 kDa fraction from placenta and the 32 kDa fraction from PHA-L antigen bound lactosylated BSA but not fucosylated BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed2000

Regulation of primordial germ cell development in the mouse.

Primordial germ cells (PGCs) are the founders of the gametes. They arise at the earliest stages of embryonic development and migrate to the gonadal ridges, where they differentiate into oogonia/oocytes in the ovary, and prospermatogonia in the testis. The present article is a review of the main studies undertaken by the author with the aim of clarifying the mechanisms underlying the development of primordial germ cells. Methods for the isolation and purification of migratory and post-migratory mouse PGCs devised in the author's laboratory are first briefly reviewed. Such methods, together with the primary culture of PGCs onto suitable cell feeder layers, have allowed the analysis of important aspects of the control of their development, concerning in particular survival, proliferation and migration of mouse PGCs. Compounds and growth factors affecting PGC numbers in culture have been identified. These include survival anti-apoptotic factors (SCF, LIF) and positive regulators of proliferation (cAMP, PACAPs, RA). Evidence has been provided that the motility of migrating PGCs relies on integrated signals from extracellular matrix molecules and the surrounding somatic cells. Moreover, homotypic PGC-PGC interaction has been evidenced that might play a role in PGC migration and in regulating their development. Several molecules (i.e. integrins, specific types of oligosaccharides, E-cadherin, the tyrosine kinase receptor c-kit) have been found to be expressed on the surface of PGCs and to mediate adhesive interactions of PGCs with the extracellular matrix, somatic cells and neighbouring PGCs.

Animals↗
PubMedDate not supplied

Homoeobox gene expression in mouse embryos varies with position by the primitive streak stage.

Pattern formation in animal development requires that genes be expressed differentially according to position in the sheets of cells that make up the early embryo. The homoeobox-containing genes of Drosophila are control genes active both in the establishment of a segmentation pattern and in the specification of segment identity. In situ hybridization experiments confirm that these genes are expressed in a segmentally-restricted manner and that their expression presages morphological differentiation of segmental structures. Homoeobox genes have recently been isolated from the mouse and have been shown to be expressed during mouse development. Using in situ hybridization, we show here that expression of the mouse homoeobox gene Mo-10 (ref. 7) is spatially restricted in the developing embryo and that localization of expression is already evident within the germ layers before their morphological differentiation. These findings support the suggestion that the homoeobox genes of mammals, like those of Drosophila, may be important in pattern formation.

Animals↗
PubMed2006

Sustained stromal stem cell self-renewal and osteoblastic differentiation during aging.

We have reported the isolation of a unique subpopulation of human stromal cells from bone marrow termed marrow-isolated adult multilineage inducible (MIAMI) cells. The expression of embryonic stem cell markers SSEA-4, Oct-4, Rex-1, and telomerase reverse transcriptase indicates the developmentally immature status of these cells. They resemble primitive stem cells in their capacity to differentiate, at least in vitro, into mature-like cells from all three germ layers. MIAMI cells are characterized by a unique molecular profile that distinguishes them from other marrow stromal cell populations. Although the frequency of MIAMI cells, among all marrow nucleated cells, decreases from 0.01% at age 3 to 0.0018% at age 45, their numbers remain unchanged after age 45. The level of expression of the markers characteristic of MIAMI cells remains constant independent of age and gender. In long-term in vitro expansion experiments aging increased the population doubling time by about 30%, whereas specific in vitro differentiation of MIAMI cells toward osteoblastic cells was unaffected. Because the oxygen tension in bone marrow ranges from 1% to 7%, we examined the role of oxygen tension in regulating the capacity of MIAMI cells to self-renew and maintain their pluripotentiality during long-term culture. Low oxygen tension upregulated mRNAs for primitive embryonic stem cell markers. Our results suggest that maintaining developmentally primitive human cells in vitro at low oxygen tension is more physiologic and favors stemness. For osteoblastic differentiation, gap-junctional communication mediated by connexin43 is required. Its inhibition not only blocked osteoblastic differentiation but stimulated the adipocytic differentiation.

Bone Marrow Cells↗
PubMed1989

Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells.

Tissue-specific extinguisher 1 (Tse-1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. This locus is clearly active in fibroblasts, as hepatoma cells retaining fibroblast chromosome 11 are extinguished for both tyrosine aminotransferase and phosphoenolpyruvate carboxykinase gene expression. To assess the activity of Tse-1 in other tissues, we transferred mouse chromosome 11 from several different cell types into rat hepatoma recipients. Tse-1 was active in nonhepatic cell lines derived from each primary germ layer, but Tse-1 activity was not apparent in hybrids between hepatoma cells and primary mouse hepatocytes. These differences in the genetic activity of murine Tse-1 were apparently heritable in cis.

Animals↗
PubMed2006

Changes in androgen-producing cell size and circulating 11-ketotestosterone level during female-male sex change of honeycomb grouper Epinephelus merra.

11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011beta-hydroxylase (P45011beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads, P45011beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.

Androgens↗
PubMed2005

RhoA acts downstream of Wnt5 and Wnt11 to regulate convergence and extension movements by involving effectors Rho kinase and Diaphanous: use of zebrafish as an in vivo model for GTPase signaling.

Gastrulation shapes the early embryos by forming three germ layers, ectoderm, mesoderm and endoderm. In vertebrates, this process requires massive cell rearrangement including convergence and extension (CE) movements that involve narrowing and lengthening of embryonic tissues as well as cell elongation. Such polarization and movements require precise reorganization and regulation of the cytoskeleton network and cell adhesion. Rho small GTPases are key regulators for dynamic actin cytoskeleton. However, the signaling mechanisms underlying their functions in CE remain to be further elucidated. We have cloned the zebrafish Danio rerio rhoA and by capitalizing on the specific functional knockdown using morpholinos against rhoA and the availability of CE mutants defective in Wnt signaling, we showed that rhoA morphants were reminiscent to noncanonical wnt mutants with serious disruption in CE movements. Injection of rhoA mRNA effectively rescued such defects in wnt5 and wnt11 mutants. Furthermore, CE defects in rhoA knockdown or wnt mutants can be suppressed through functional bypass after ectopic expression of the two mammalian Rho effectors, the Rho kinase and Diaphanous (mDia). These results provide the first evidence that the RhoA in vivo acts downstream of Wnt5 and Wnt11 to effect, without affecting cell fates, on the CE movements in zebrafish embryos. Significantly, it elicits such effect via both effectors, Rho kinase and Dia. These findings also support the versatility of the zebrafish as a model to further investigate the roles of various classes of small GTPases in regulating cell dynamics in vivo.

Amino Acid Sequence↗
PubMed1992

The in vitro transformation of the miracidium to the mother sporocyst of Schistosoma margrebowiei; changes in the parasite surface and implications for interactions with snail plasma factors.

The in vitro transformation of the miracidium to the mother sporocyst of Schistosoma margrebowiei was initiated by placing the miracidium in mammalian physiological saline. The transformation occurs in stages: the cilia cease beating; the ciliated plates become detached from the intercellular ridges and underlying muscle layers; the intercellular ridges spread over the body surface eventually forming a new tegument; the sporocyst changes from an ovoid to a tubular shape in about 48 h at room temperature. The surfaces of the miracidium, sporocyst and cercaria of S. margrebowiei display stage-specific carbohydrates on their surfaces as indicated by lectin staining. Ricin120 stains the cilia alone of the miracidium whereas peanut agglutinin stains the larval surface except for the cilia. The intercellular ridges of the miracidium stain with concanavalin A and wheat germ agglutinin, and these lectins stain the entire surface of the mature mother sporocyst. The cercaria is the only larval stage which stains positively with asparagus pea lectin. Bulinus nasutus is incompatible with Schistosoma margrebowiei; the haemolymph of this snail contains an agglutinin which agglutinates a wide variety of mammalian erythrocytes including those of human ABO blood groups. The haemagglutinin titre of B. nasutus plasma is reduced after incubation with miracidia of S. margrebowiei indicating that the agglutinin is absorbed onto the surface of this larval stage but not that of the mother sporocyst or cercaria. The possible roles of agglutinins in host-parasite interactions together with the significance of the differences in the surface carbohydrates of the larval stages are discussed.

Adsorption↗
PubMed1991

Effects of the steel gene product on mouse primordial germ cells in culture.

Mutations at the steel (sl) and dominant white spotting (W) loci in the mouse affect primordial germ cells (PGC), melanoblasts and haemopoietic stem cells. The W gene encodes a cell-surface receptor of the tyrosine kinase family, the proto-oncogene c-kit. In situ analysis has shown c-kit messenger RNA expression in PGC in the early genital ridges. The Sl gene encodes the ligand for this receptor, a peptide growth factor, called here stem cell factor (SCF). SCF mRNA is expressed in many regions of the early mouse embryo, including the areas of migration of these cell types. It is important now to identify the role of the Sl-W interaction in the development of these migratory embryonic stem cell populations. Using an in vitro assay system, we show that SCF increases both the overall numbers and colony sizes of migratory PGC isolated from wild-type mouse embryos, and cultured on irradiated feeder layers of STO cells (a mouse embryonic fibroblast line). In the absence of feeder cells, SCF causes a large increase in the initial survival and apparent motility of PGC in culture. But labelling with bromodeoxyuridine shows that SCF is not, by itself, a mitogen for PGC. SCF does not exert a chemotropic effect on PGC in in vitro assays. These results suggest that SCF in vivo is an essential requirement for PGC survival. This demonstrates the control of the early germ-line population by a specific trophic factor.

Animals↗
PubMed1984

A phosphatidylethanolamine-containing complex on human B cells that mediates rosette formation with mouse erythrocytes.

The maturation-associated human B cell rosette receptor (MER) for mouse erythrocytes has been solubilized from B cells by mild trypsinization. It specifically agglutinates mouse red cells. Material with hemagglutinating activity partitioned into the lipid-soluble phase of a Folch partition of the trypsin extract was sensitive to phospholipase C and alkali, and on two-dimensional thin layer chromatography, it co-migrated principally with phosphatidylethanolamine (PE). Phosphatidylcholine, the major lipid present, was inactive. The relationship of phospholipid structure to hemagglutinating activity has been described. PE in the crude trypsin extract was associated with unidentified glycoprotein and albumin. Material containing hemagglutinating lipid bound to a wheat germ lectin-Sepharose column and was released by N-acetylglucosamine, indicating that the PE was complexed with glycoprotein. When the crude trypsin extract or eluate from the lectin column was extracted with aqueous phenol, hemagglutinin in the aqueous phase no longer bound to wheat germ lectin-Sepharose; however, albumin was greatly enriched, indicating that some of the PE exists in a complex with albumin. The molar ratio of PE to albumin was approximately 200:1. After delipidation, this albumin (in molar excess) inhibited hemagglutination by PE in the same way as a recently described subclass of serum albumin. Studies with phospholipase-treated B cells were also consistent with PE being the MER. We conclude that MER is PE, existing in a complex containing glycoprotein and a subclass of albumin. The capacity to form rosettes can be transferred to nonrosetting Raji B cells by the complex, but not pure PE, indicating that the proteins may be involved in orienting PE correctly for it to function as the MER.

Animals↗
PubMed1995

Lesions of area 17 in newborn kittens cause selective changes in the development of area 18.

In the cat, areas 17 and 18 interconnect soon after birth. To test the hypothesis that the normal development of area 18 depends on interactions with area 17, unilateral lesions of area 17 were created in newborn kittens, and the animals allowed to mature. Horseradish peroxidase was then injected into both lateral geniculate nuclei. The major abnormalities of area 18 in the lesioned hemispheres were a thinning of specifically layers 2 and 3 and abnormally faint geniculocortical labelling of layer 4. Cell densities in layers 2 and 3 of the lesioned hemispheres were similar to or lower than normal. Neonatal destruction of area 17 therefore produced a selective loss of cells in layers 2 and 3 in area 18 (the layers that normally interconnect with area 17), and may have reduced thalamic innervation of layer 4.

Animals↗
PubMed2002

From stem cells to tissue-specific differentiation.

Stem cells have been isolated from embryonic, foetal and adult sources. Embryonic stem cells, derived from the pre-implantation embryo, can be expanded indefinitely in vitro. When reintroduced into the blastocyst they contribute to all lineages in vivo. In vitro, differentiated derivatives of embryonic stem cells are obtained by manipulating culture conditions. Embryonic germ cells, derived from the primordial germ cells, display the same degree of pluripotential differentiation. Adult stem cells that repopulate the tissue of origin throughout life possess the ability to differentiate into phenotypes not restricted to the tissue and, in some cases, to the germ layer from which they derive. Although the pluripotential differentiation capability of these stem-cell populations is supported by an increasing amount of evidence, a better understanding of the mechanisms that control differentiation is required for their exploitation in the treatment of human diseases.

Journal Article↗
PubMed2003

Different distribution of immunocompetent cells in the dentogingival junction during root formation in rat molars.

The distribution of immunocompetent cells in the dentogingival junction of rat molars during root formation was investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6-antibody) and monocyte/macrophage lineage cells (ED1-antibody) as well as by histochemical reaction for periodic acid-Schiff (PAS). Two portions (the junctional epithelium in the mesial gingiva of the first molar, and the interdental gingiva between the first and second molars) were selected for observations. At the eruption stage of the first molar (16-18 days after birth), OX6-positive cells, dendritic or oval in shape, were abundantly distributed in the connective tissue between the oral epithelium and tooth germ. Positive cells with slender cell processes were also found beneath the ameloblast layer. At the commencement stage of the first molar occlusion (24-28 days after birth), numerous OX6-positive cells displaying a dendritic fashion existed preferentially in the mesial gingiva, but were fewer in the interdental gingiva. In contrast, the interdental gingiva showed a denser distribution of ED1-positive cells and PAS-reactive polymorphonuclear leukocytes (PMLs) than the mesial gingiva. At the completion stage of root formation (100-120 days after birth), the OX6-immunopositive cells invaded the deeper position of the mesial gingiva with the downgrowth of the epithelium; they had a considerably higher cell density compared with those in the interdental gingiva where PAS-reactive PMLs persisted. These findings indicated that the immunocompetent cells showed a region-specific distribution and cell density by their roles in immune response.

Ameloblasts↗