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PubMed2003

Influence of post-ovulatory insemination on sperm distribution, pregnancy and the infiltration by cells of the immune system, and the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium.

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E2 levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro-oestrus was absent. In conclusion, post-ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post-ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.

Animals↗
PubMed2001

The development of the external urethral sphincter in humans.

OBJECTIVE: To assess the hypothesis that during fetal development, the external urethral sphincter changes from a concentric sphincter of undifferentiated muscle fibres to a transient ring of striated muscle which regresses caudo-cranially in the posterior urethra during the first year of life, when the sphincter assumes its omega-shaped configuration. MATERIALS AND METHODS: The anatomy and development of the external urinary sphincter was assessed in human males and females during fetal life. Plastic-embedded sections (transverse, sagittal and frontal planes; 300-700 microm) of the pelvis of 31 females and 31 males (9 weeks of gestation to newborn) were stained with azure II/methylene blue/basic fuchsin and viewed at x 4-80. The sections of interest were taken from the bladder neck to the perineum. The sections of the membranous urethra were reconstructed three-dimensionally using a computer program. RESULTS: In both male and female an omega-shaped external sphincter was apparent in all specimens at > 10 weeks of gestation. In the early fetal period (ninth week), there was undifferentiated mesenchyme; in this period the mesenchyme was more dense in the anterior part and loose in the posterior part of the urethra. In females, there was a close connection between the urethra and the anterior wall of the vagina. CONCLUSION: The omega-shaped configuration of the external urethral sphincter was recognisable from 10 weeks of gestation in both sexes. There was no suggestion of a change from a cylindrical to an omega-shaped sphincter in the fetal period to birth. Also, a transient 'tail' posterior to the sphincter was not apparent. The rectovesical septum was well developed in neonates. There is no reason to assume that the development of the septum leads to an apoptosis of muscle cells in the posterior part of the external sphincter in males after birth. The anatomical development of the external sphincter does not explain transient outlet obstruction during fetal life. The function of the muscle may change during development because of neuronal maturation.

Embryonic and Fetal Development↗
PubMed1999

Stereological estimation of the volume weighted mean volumes of alveoli and acinar pathways in the rat lung to characterise alterations after ischaemia/reperfusion.

The aim of this study was to characterise pulmonary reimplantation injury in isolated, perfused rat lungs following 2 h of cold ischaemia, and 50 min. of in vitro reperfusion. The effects of 2 differently composed lung preservation solutions (low potassium Euro-Collins and Celsior; each n = 5) were examined in comparison with untreated, nonischaemic control lungs (n = 3). After fixation by vascular perfusion and tissue collection by systematic random sampling, the volume weighted mean volume (Vv) of alveoli and acinar pathways was estimated by light microscopic stereology using the method of point sampled intercepts in plastic embedded, Azan-stained material. Significantly higher Vv of alveoli and acinar paths was found in the Celsior group than in Euro-Collins preserved lungs. However, in the controls the size of acinar pathways was similar to Celsior preserved lungs whereas alveolar size was comparable to preservation with Euro-Collins. The between-animal coefficient of variation of alveoli was very low in controls and Celsior preserved but higher in the Euro-Collins group. Size distribution of alveoli and acinar paths in 15 size classes was largely homogeneous in all groups tested. In the Euro-Collins group the fractions of both class 1-alveoli and class 1-acinar paths significantly exceeded those of the other groups. Widely expanded alveoli (size classes 13-15) only occurred after preservation with Celsior whereas wider acinar paths (size class 15) were found in the Celsior group and in the controls. It is concluded that lung preservation with low-potassium Euro-Collins and Celsior solutions may act differently on distinct spaces in the distal gas-exchange regions of lungs. This may be due to selective effects on pulmonary surfactant activity and on elastic tissue elements in the alveolar ducts, respectively. Additionally, the method of point sampled intercepts is considered to be an efficient tool to evaluate the effects of different preservation solutions on lung parenchyma.

Animals↗
PubMed2001

Glomerular cell number in normal subjects and in type 1 diabetic patients.

BACKGROUND: The number of cells in glomeruli has been a challenging measure, especially in human kidneys, with only a small amount of tissue obtained by biopsy. However, the number of cells and their function are important determinants of renal function in health and disease. METHODS: Modern morphometric techniques have now provided the means to determine the numerical density (Nv) and number (with a measure of glomerular volume) of endothelial cells, mesangial cells, and podocytes in plastic-embedded renal tissue biopsied from nondiabetic subjects (N = 36) and type 1 diabetic patients (N = 46) over an extended age range from childhood through late adult. RESULTS: Nv values for all glomerular cells varied only slightly with age and did not change within the range of glomerular lesions of diabetes studied. Thus, the increase in glomerular volume during childhood to a steady level thereafter was the primary determinant of total glomerular cell number. The number of mesangial cells and endothelial cells increased with age, reflecting the increase in all cells, while the podocytes remained unchanged in number over all ages studied (10 to 69 years). Numbers of total glomerular cells, mesangial cells, and endothelial cells were not changed with diabetes, while podocytes were fewer in number in diabetic patients of all ages, with reduced podocyte numbers even in diabetes of short duration. CONCLUSIONS: The essentially constant glomerular cell density in nondiabetic and diabetic subjects under different circumstances possibly indicates an underlying propensity for the glomerulus to regulate its architecture to maintain a constant number of cells per volume, no matter the size of the glomerulus or the severity of diabetic nephropathy studied in this set of patients. The reductions in podocyte numbers in both younger and older diabetic patients indicate a significant risk for functional abnormalities as diabetic nephropathy progresses. Moreover, these observations do not support the suggestion of marked increases in glomerular cell number (and especially mesangial cells) with the development and progression of diabetic nephropathy.

Adolescent↗
PubMed1999

A lectin binding analysis of glycosylation patterns during development of the equine placenta.

The glycosylation of the equine interhaemal barrier and areola was studied throughout the period of gestation. Placentae of 35, 37, 50, 119, 152, 200, 280 and 300 days gestation were investigated, using semithin plastic embedded sections and a panel of 15 biotinylated lectins with an avidin-peroxidase revealing system. Glycosylation of the trophoblast and maternal epithelium showed the most change during the first 50 days of gestation, being associated with the initial stages of adhesion and attachment. In the trophoblast, non-bisected tri/tetraantennary complex N-glycan was only evident after day 37 and terminal N-acetyl galactosamine, alpha2,3- and alpha2,6-linked sialic acids disappeared at the same time. The areolar trophoblast exhibited some differences from microcotyledonary areas, especially with respect to 2-deoxy, 2-acetamido alpha-galactose and tri/tetraantennary, non-bisected complex N-glycan, suggesting that the differences in function between microcotyledonary and areolar trophoblast are reflected at both the morphological and the biochemical level. Granules of the maternal uterine epithelium bound many lectins, particularly those with specificity for bisected and non-bisected bi/triantennary N-linked glycan, 2-deoxy, 2-acetamido alpha-galactosyl, beta-galactosyl and some fucosylated termini. Binding to sialic acids in alpha2,3- and alpha2,6-linkage was sparse. Maternal and fetal capillaries showed little change in glycan expression over the period studied, being rich in bisected and non-bisected bi/triantennary N-linked glycan and sialic acids, with some terminal N-acetyl galactosamine and no detectable terminal fucosyl residues.

Acetylgalactosamine↗
PubMed1999

Equine placental cup cells show glycan expression distinct from that of both chorionic girdle progenitor cells and early allantochorionic trophoblast of the placenta.

Using lectin histochemistry on plastic-embedded material, the glycosylation patterns of equine girdle and cup cells, and associated endometrial glands, have been investigated from 37 to 67 days gestation. Results were compared with the glycosylation of the 50-day allantochorionic trophoblast of the established equine placenta that will later form the microcotyledons. The differentiated cup cells, which secrete equine chorionic gonadotropin (eCG), showed a pattern of glycosylation that was distinct both from the progenitor girdle cells and the allantochorionic trophoblast, with granules that bound lectins indicating high levels of alpha2,6 and alpha2,3-linked sialic acid, N-acetyllactosamine and bi/tri antennary non-bisected and bisected complex N-glycan. This is consistent with the known carbohydrate content of eCG. In contrast, the allantochorionic trophoblast at 50 days lacked detectable amounts of sialic acid and showed high levels of tri/tetra-antennary non-bisected complex N-glycan and N-acetyl galactosamine which was absent in the cup cells. During the process of girdle cell migration into maternal tissues, the uterine glands became greatly enlarged and dilated basally, with increased amounts of glycosylated secretory products revealed by lectins, which often seeped out into the extracellular space via ruptures in the apical regions of the gland wall.

Animals↗
PubMed1987

Morphometric estimation of the numbers of granulosa cells in preovulatory follicles of the ewe.

Several lines of evidence suggest that follicular granulosa cells give rise to the large luteal cells of the corpus luteum in the sheep. To further investigate this suggestion, numbers of granulosa cells in preovulatory follicles were estimated by morphometric methods for comparison with a previous estimate of numbers of large luteal cells (9.6 +/- 0.9 x 10(6)). Preovulatory follicles from five Corriedale ewes were obtained after synchronization of the oestrous cycle with the prostaglandin analogue cloprostenol. Morphometry was undertaken using light microscopy of plastic-embedded tissue sectioned at 1 micron. Mitotic index in the membrana granulosa was 0.05 +/- s.e.m. 0.05%. Mean follicular diameter was 6.25 +/- 0.25 mm and there were 7.68 +/- 0.53 x 10(6) granulosa cells per follicle. These results demonstrate a similarity between the number of granulosa cells per follicle and the number of large luteal cells per corpus luteum and thus support the hypothesis that large luteal cells are derived from granulosa cells.

Animals↗
PubMed1985

Isolation of the non-glycosylated proteins of desmosomes and immunolocalization of a third plaque protein: desmoplakin III.

The cytoplasmic plaque of the spot desmosome or macula adhaerens mediates the attachment of bundles of intermediate filaments to the plasma membrane. We have isolated from a bovine epidermal desmosome preparation a fraction that is highly enriched in the non-glycosylated desmosomal proteins. Plastic-embedded and thin-sectioned high-speed pellets of this fraction reveal closely packed filaments that resemble plaque regions of the low pH whole desmosome preparation from which they are derived. NaDodSO4/polyacrylamide gel electrophoresis reveals four major, non-glycosylated proteins of 240, 210, 81, and 77 kDa. In agreement with a previous study, we find the 240- and 210-kDa proteins (desmoplakins I and II) to be closely related, whereas the 81- and 77-kDa proteins are unique. This is shown both immunologically and by one-dimensional proteolytic peptide mapping. Monospecific, polyclonal rabbit antibodies were prepared against the 81-kDa protein and used, in conjunction with protein A-complexed colloidal gold particles (PAG), to immunolocalize this antigen on ultrathin sections of bovine muzzle epidermis. On antibody-labeled sections, PAG particles were associated principally with the desmosomal cytoplasmic plaque. Sections exposed to preimmune serum showed little or no labeling. We conclude that the 81-kDa protein, like the 240/210-kDa protein family, is one of the major components of the desmosomal plaque. We designate it as "desmoplakin III." The location of the 77-kDa protein remains to be definitively established.

Animals↗
PubMed1995

Rethinking cell structure.

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure.

Animals↗
PubMed2000

Electron microscopic contrast of the cytoskeleton and junctional complexes of intestinal epithelial cells by ethanolic phosphotungstic acid.

After glutaraldehyde fixation and treatment with ethanolic phosphotungstic acid (E-PTA) before plastic embedding, sections of rat large intestine showed a characteristic electron contrasting pattern in epithelial cells. The axis of microvilli, terminal web, a thin band below the luminal plasma membrane, centrioles and junctional complexes (tight junctions, adherens junctions, and desmosomes) appeared highly contrasted. In addition to protein components of microfilaments and intermediate filaments, proteins from the junctional complexes could also be implicated in the contrasting reaction with E-PTA. Mitochondrial membranes, chromatin masses, and nucleoli of enterocytes showed considerable electron density, whereas no reaction was found in the glycocalyx and mucin content of goblet cells. The clear visualization of cytoskeleton elements and junctional complexes by E-PTA contrasting represents a simple and valuable method for studies on the normal and pathological organization of these structures in epithelial cells.

Animals↗
PubMed2003

Mitochondrial and nuclear localization of kanadaptin.

Kanadaptin has originally been isolated as a kidney Cl-/HCO3- anion exchanger 1 (kAE1)-binding protein. Initial studies suggested, that in the kidney of the rabbit kanadaptin is expressed exclusively in all epithelial cells of the collecting duct. Transcripts of kanadaptin were also found in tissues not expressing kAE1, indicating additional roles for kanadaptin. With respect to this, we could recently demonstrate translocation of kanadaptin into the nucleus of mammalian cells in a nuclear localization sequence- and importin-dependent manner (Hübner et al., Biochem. J. 361, 287-296, 2002). In this study, we provide evidence, that kanadaptin is widely expressed in many tissues and that expression of kanadaptin in the mouse occurs early in embryonic development. In rat kidney we found the most intense immunofluorescence for kanadaptin in cells of the proximal tubule, consistent with the detection by in situ hybridization of high amounts of kanadaptin messenger RNA in proximal tubule cells. Immunostaining revealed localization of kanadaptin in two subcellular locations, nuclei and mitochondria. Whereas nuclear localization was demonstrated in virtually all cells, mitochondrial staining was restricted to certain cell types. Nuclear staining was only seen in cryosections, whereas mitochondrial staining was observed in both cryosections and semithin sections of freeze-dried plastic-embedded tissue. In the kidney mitochondrial staining was particularly prominent in proximal tubular epithelium. Most surprisingly, in the collecting duct epithelium (including acid-secreting intercalated cells) only negligible immunostaining, if at all, could be observed. Immunoelectron microscopy showed immunolabelling of the entire cross-sectional profile of mitochondria (matrix/inner membrane). Mitochondrial localization of kanadaptin was further documented by immunoblotting of mitochondria-enriched cellular fractions. Utilizing an interspecies heterokaryon assay, we could further demonstrate that kanadaptin has nuclear export activity. Thus, kanadaptin can be regarded to be a highly mobile nucleocytoplasmic shuttling and multilocalizing protein, but its role in mammalian cells remains still obscure.

Animals↗
PubMedDate not supplied

A fast histochemical staining method to identify hyaline droplets in the rat kidney.

Hyaline droplet formation in the proximal tubular cells of the kidney commonly occurs under different pathological conditions in experimental animals. In rats, intracytoplasmic hyaline droplet formation is predominantly associated with accumulation of the male rat-specific alpha(2u)-globulin, whereas under other (pathological) conditions (eg, histiocytic sarcoma and chronic progressive nephropathy [CPN]) other proteins are involved. Staining methods to visualize hyaline droplets either need plastic embedded material or time-consuming (immuno)histochemical methods. A fast Chromotrope-Aniline-Blue-staining on formalin-fixed paraffin-embedded kidneys taking only 30 minutes is described. Using this method, hyaline droplets consisting of different types of proteins are easily recognized by their bright-red color.

Alpha-Globulins↗
PubMed2005

Megakaryocytopoiesis and apoptosis in patients with myelodysplastic syndromes.

In order to investigate simultaneously the megakaryocytopoiesis and apoptotic characteristics in bone marrow in patients with myelodysplastic syndromes (MDS), we used CD41 immunoenzyme (alkaline phosphatase anti-alkaline phosphatase) and DNA in situ end-labeling techniques on plastic embedded bone marrow biopsy sections of 29 MDS patients. Fourteen patients with iron deficiency anemia served as controls. The results showed that CD41-positive cells in MDS marrow numbered 26.2 +/- 18.2/mm2 (mean +/- standard deviation) compared with 15.6 +/- 7.1/mm2 in controls (P < 0.05). Numbers of cells with the morphology of micro-megakaryocytes in MDS marrow were significantly higher than in controls (P < 0.01). Furthermore, megakaryocytes in MDS marrow were frequently distributed along trabeculae (in 27 cases) and formed clusters (in 25 cases). Apoptotic megakaryocytes in MDS marrow accounted for just 4.4 and 9.3% of all CD41-positive cells and all apoptotic cells, respectively (P > 0.05 compared with controls), but apoptosis occurred only in micro-megakaryocytes. Based on these observations, we conclude that megakaryocytosis and dysmegakaryocytosis are the features of dyshematopoiesis in MDS marrow. Decreased thrombocyte production and thrombocyte release coming from increased dys(micro)megakaryocytes and abnormally located megakaryocytes perhaps play a more important role in peripheral thrombocytopenia than megakaryocytic apoptosis itself. Apoptosis of micro-megakaryocytes may be a protective biological mechanism to remove useless megakaryocytes.

Adolescent↗
PubMed2002

Visualization of in vivo olfactory uptake and transfer using fluorescein dextran.

Nasal administration of a 3 kDa fluorescein dextran (FD3) solution to rats resulted in transcellular absorption across the olfactory epithelium and transfer to the olfactory bulb within 15 min. After entering the lamina propria, FD3 was transferred in the connective tissue surrounding the olfactory nerve bundles to the olfactory bulb of the brain. More FD3 was absorbed across the olfactory epithelium than across the respiratory epithelium and to the nasal associated lymphoid tissue. Further, the amount of FD3 crossing the olfactory epithelium was region-dependent, with higher amounts absorbed in the turbinates than in the nasal septum. Plastic embedding and sectioning followed by fluorescence microscopy, enabled simultaneous visualization of FD3 in the mucosa and olfactory bulb, as well as the opportunity to store the tissue blocks for a prolonged period of time.

Administration, Intranasal↗
PubMed1967

High-resolution radioautography of phlorizin-3H in rings of hamster intestine.

Quantitative light microscope radioautographs of galactose-(3)H and phlorizin-(3)H were prepared from freeze-dried plastic-embedded hamster small intestine incubated in vitro. The usual uphill epithelial cell accumulation of galactose accompanied by a somewhat smaller lamina propria accumulation was observed in control tissue incubated 3 min in 1 mM galactose-(3)H. The addition of 5 x 10(-4)M phlorizin to the medium blocked uphill accumulation, but did not prevent galactose equilibration with the epithelial cells. The galactose content of the lamina propria was considerably less than the galactose content of the epithelial cell. Varying the phlorizin-(3)H content of the medium from 0.6 to 60 microM revealed a brush border binding of phlorizin which followed a Langmuir adsorption isotherm with a half-saturation constant of 13 microM and a maximum binding of 84 micromoles of phlorizin/liter of microvilli or 2.6 x 10(6) sites/epithelial cell. The phlorizin content of the epithelial cell compartment, excluding microvilli, never exceeded 10% that of the medium after 20 min of incubation. These findings directly support the view that phlorizin is a nontransported inhibitor which binds glucose-galactose carriers at the surface of epithelial cell microvilli.

Animals↗
PubMed1971

Improved techniques for the preparation of ultrathin frozen sections.

Ultrathin frozen sections of biological tissues for electron microscopy provide certain advantages in cytochemical studies in which the penetration of cells by large molecules is necessary and in morphological studies of cellular constituents which are dissolved by the reagents employed in routine plastic embedding. The recent introduction of several types of commercially available cryo-ultramicrotomes makes it possible for many laboratories to employ this valuable tool. This paper summarizes recent improvements in the methods developed in this laboratory for preparing ultrathin frozen sections and reviews some of the inherent problems involved in their use. These procedures may serve as a baseline for other investigators who can then modify or adapt them for their specific purposes.

Adrenal Glands↗
PubMed1979

Intermediate (skeletin) filaments in heart Purkinje fibers. A correlative morphological and biochemical identification with evidence of a cytoskeletal function.

Cow Purkinje fibers contain a population of free cytoplasmic filaments which consistently differ in ultrastructural appearance from actin and myosin filaments, irrespective of preparation technique. The fixation and staining techniques, however, influenced the filament diameter, which was found to be 7.4--9.5 nm for filaments in plastic-embedded material, and 7.0 nm in cryo-sectioned material, thus intermediate as compared to actin and myosin filaments. Cross-sectional profiles suggested that the intermediate-sized filaments are composed of four subfilaments. To provide a basis for further biochemical investigations on the filaments, extraction procedures were carried out to remove other cell organelles. Electron microscopy showed that undulating bundles of intermediate filaments converging towards desmosomes still remained, after the extractions, together with Z-disk material. In spite of the extensive extraction, the shape of the individual cells and the assemblies of cell bundles remained intact. This confirms that the intermediate filaments of cow Purkinje fibers together with desmosomes do in fact have a cytoskeletal function. On account of (a) the cytoskeletal function of the filaments, (b) the similarities to the smooth muscle "100-A filament" protein subunit skeletin, and (c) the inadequate and confusing existing terminology, we suggest that the filaments be named "skeletin filaments."

Animals↗
PubMed1979

Concanavalin A-induced endocytosis in rabbit reticulocytes, and its decrease with reticulocyte maturation.

Concanavalin A (Con A) was taken up to a limited extent by endocytosis in rabbit reticulocytes but not in rabbit erythrocytes. This process was observed by the use of ferritin-labeled Con A and transmission electron microscopy of thin sections of plastic-embedded cells. Furthermore, the extent of endocytosis among the reticulocytes decreased with the extent of their maturation, reticulocyte age being measured by ribosome configurations. These results are consistent with the proposal that there are domains in the membranes of reticulocytes in which the Con A receptors are laterally mobile, and can be clustered and endocytosed. These mobile domains exist, or are formed, within a larger framework of immobile membrane. During reticulocyte maturation, these domains are gradually eliminated, eventually disappearing upon formation of the mature erythrocyte. Possible molecular mechanisms for this proposed elimination process are discussed.

Animals↗