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K Spanel-Borowski

Publications and source records attributed to K Spanel-Borowski.

At least 37 records · Page 2Linked to original sources
PubMed1997

Immunolocalization of CD18-positive cells in the bovine ovary.

The life cycle of follicles and of corpora lutea may be finely tuned by resident leucocytes in control of the angiogenesis, dilatation and permeability of microvessels, remodelling of the extracellular matrix, and phagocytosis of degenerated cells. Since the overall leucocyte distribution has not been studied in the bovine ovary, we located the CD18 molecule in this organ using indirect immunohistochemistry. This molecule represents a subtype of beta 2 integrin and is expressed by all subsets of leucocytes. The microvessels were also identified with an antibody against factor VIIIr antigen. The outer layer of the interstitial cortex, where the primordial follicles were located, contained a few CD18-positive cells and a few microvessels. Intact follicles displayed the CD18-positive cells in the theca. They were aligned in the zone of the basal membrane of preovulatory follicles. This was associated with a dilated inner microvessel layer. CD18-positive cells and probably capillary sprouts were seen in the granulosa layer of freshly ovulated follicles. Regressing follicles exhibited CD18-positive cells in the hypertrophied theca and in the degenerating granulosa layer, together with the pattern of capillary sprouts. At the developmental stage, corpora lutea exhibited many CD18-positive cells in the septa. The microvascular bed was expanding. At the secretory stage, a regular and moderate appearance of CD18-positive cells corresponded to a well-developed microvascular bed. Abundant positive cells were seen at the regressive stage, together with the development of shunt-like arterioles. The semiquantitative evaluation of leucocyte subsets in corpora lutea revealed an increased in the CD18-positive cells between stages of development and regression, which was not observed in the corresponding interstitial cortex. Lymphocytes with the expression of CD2, CD3 or both molecules displayed a similar behaviour. In conclusion, the CD18-positive leucocytes represent a heterologous pool with a distribution that varies in accordance with morphological changes in the follicles and corpora lutea.

Animals↗
PubMed1996

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum.

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").

Animals↗
PubMed1996

Immunolocalization of neurophysin in cytokeratin-positive luteal cells of cows.

We have recently detected a subgroup of cytokeratin (CK)-positive luteal cells in the bovine corpus luteum of the early and mid-luteal phase, but not in that of pregnancy. Since, according to the literature, neurophysin (NP)-positive luteal cells behave comparably, simple immunohistochemistry and double labeling were used to identify in serial sections whether the presence of NP co-incided with that of CK. The numbers of CK-positive cells and NP-positive luteal cells were comparable throughout the estrous cycle, decreasing from early to late luteal phase. While few CK-positive cells were found in the former thecal layer during the early luteal phase, many CK-positive cells appeared in the former granulosal layer. NP-positive cells were only detected in the former granulosal layer. During the mid-luteal phase, the CK-positive cells consisted of small and large luteal cells, but only large NP-positive cells were found. Roughly 80% of the large CK-positive cells contained NP, whereas CK was lacking in more than 50% of the NP-positive cells. The corpora lutea of pregnancy contained neither CK-positive nor NP-positive cells. The significance of the simultaneous occurrence of CK and NP remains to be elucidated.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Simple or repeated induction of superovulation: a study on ovulation rates and microvessel corrosion casts in ovaries of golden hamsters.

Repeatedly stimulated ovaries are reported to decrease the ovulation rate. One cause among others might be that the microvascular bed has been insufficiently developed. Therefore, 30-day-old golden hamsters were superovulated either once or repeatedly. At the light microscopic level, the ovulation rate in serially sectioned ovaries was indirectly determined by the occurrence of corpora lutea and of abnormal follicle rupture with oocyte release into the cortical stroma (IOR). For the study with scanning electron microscopy (SEM), the microvascular bed of the ovaries was cast with a polyester resin, and the corrosion casts of mature follicles observed. The histological sections of once-stimulated ovaries showed a large number of corpora lutea and IOR follicles. This indicated hyperovulation. In corrosion casts of once-stimulated ovaries, large-sized antral follicles with two layers of a dense capillary meshwork were observed. Capillary sprouts were aligned around the antrum 0 to 12 h after administration of human chorionic gonadotrophin (hCG), and these radiated towards the center of the antrum after 12 to 36 h had elapsed. The ovulation site was recognized at the follicle apex by three similarly sized structures which were either a sinusoid, an oocyte replica, or an opening. Repeatedly stimulated ovaries produced a low number of corpora lutea and almost no IOR follicles. This was judged as hypoovulation. The microvessels of mature follicles were reduced in number and incompletely cast. Widespread resin leakages were conspicuous in the follicle wall 36 h after hCG injection, but the capillary sprouts radiated towards the center of the antrum. No ovulation site was detectable. It is concluded, that capillary sprouts are induced before luteinization. The ovulation site is indicated by particular changes in its microvascular bed. An insufficiently developed microvascular bed may be responsible for hypoovulation in repeatedly stimulated ovaries.

Animals↗
PubMed1995

Acetylation of alpha-tubilin in different bovine cell types: implications for microtubule dynamics in interphase and mitosis.

Previous work on five cell types isolated from the bovine corpus luteum showed that the mass of acetylated microtubules (acet-MTs) in interphase differed. Endothelial cells, termed type 3, showed few acet-MTs, whereas the interphase cytoskeleton of granulosal-like cells, termed type 5, was rich in acet-MTs. In the present study, these cultured cells were used to determine whether the degree of alpha-tubulin acetylation in interphase had consequences on mitosis. To this end, the distribution of acet-MTs was determined throughout the cell cycle using a monoclonal antibody, 6-11B-1, directed against acetylated alpha-tubulin. For comparison, tyrosinated MTs were visualized with another monoclonal antibody, YL1/2, detecting tyrosinated alpha-tubulin. Although the amount of acet-MTs in interphase differed significantly between both cell types, major differences in the appearance of acet-MTs during mitosis were only apparent in prophase and during transition from late telophase to interphase. Thus, irrespective of different alpha-tubulin acetylation in interphase, spindle structure is uniform. Since acetylation of alpha-tubulin is believed to indicate the presence of relatively stable MTs, we conclude that MT dynamics is differently controlled in interphase and mitosis. Thereby interphase cells are able to carry out functions which involve stable MTs and the cells progress through mitosis in the presence of more dynamic MTs.

Acetylation↗
PubMed1995

Cytokeratin expression in bovine corpora lutea.

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corpora lutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed "zonation" such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.

Animals↗
PubMed1995

Cytokeratin expression in the developing vagina of the postnatal gerbil (Meriones unguiculatus).

During postnatal development the vaginal epithelium of the Mongolian gerbil is transformed from two to three layers into a stratified, first mucified subsequently keratinized squamous epithelium. Changes in the expression of cytokeratins were studied and the immunohistochemical results compared with the ultrastructural findings at the corresponding stage. The first 10 postnatal days (days pn) were characterized by a moderate, positive immunoreaction for pancytokeratin in all vaginal cell layers. A faint reaction was caused by mAB CK 18.01 against CK 1, 5, 6 and 8. The appearance of mucous granules in the luminal cells after 15 pn seemed to coincide with an increase in cytokeratins. The immunoresponse for pancytokeratin in these cells was very intense compared with the reaction in the basal cell layers. Mucocytes during development and at proestrus were the only cells which reacted faintly positive with mAB against CK 18 alone. The keratinizing epithelium, which differentiates after day 40 pn, reacted strongly positive for pancytokeratin in the keratinizing layers, desquamating, fully keratinized cells, however, showed a negative reaction. The data indicate that mucocytes are not transformed squamous keratinized cells, but represent a cell category with its individual differentiation potential. Vimentin was not expressed. Neither the epithelium of the sinus vagina nor of the Müllerian vagina displayed any response.

Aging↗
PubMed1994

Bovine microvascular endothelial cells of separate morphology differ in growth and response to the action of interferon-gamma.

Five cell types recently isolated from the bovine corpus luteum differed in their epithelioid morphology and their cytoskeleton, but shared common criteria of microvascular endothelial cells. To give strong evidence for the separate entity, the growth rate of the 5 phenotypically different cells was studied. They were seeded at low density on day 0. Most of these cells were treated with 200 to 1000 U recombinant bovine interferon-gamma (IFN-gamma) for 3 days. The untreated remainder served as controls. Cell counts were made for all cultures on days 4, 7, 10 and 13. Morphology: 13 d after treatment with IFN-gamma senescent cells as well as intact cells occurred in cultures of cell types 1 to 4. Cultures of cell type 5 were apparently unchanged and resembled their untreated counterparts. Desmin-positive cells in cultures of cell type 2 developed cell processes. Growth rate: In the absence of IFN-gamma, the growth rate was high for cell types 3 and 4, moderate for cell type 1, and low for cell types 2 and 5. The presence of IFN-gamma caused anti-proliferative effects. These were higher for cell types 3 and 4 than for cell types 1 and 2. IFN-gamma could be cytotoxic on cell type 3. In contrast, the cytokine tended to support the cell growth of cell type 5. These findings substantiate the postulate that endothelial cells exhibiting separate morphology in culture also function differently.

Animals↗
PubMed1994

Isolation of granulosa-like cells from the bovine secretory corpus luteum and their characterization in long-term culture.

BACKGROUND: The isolation of cells termed type 5 from the bovine corpus luteum was recently reported. Since these cells were reminiscent of immature granulosa cells, their morphological and functional relationship requires further investigation in view of the novel concept of corpus luteum growth. It suggests that putative stem cells of unknown origin supply the pool of small luteal cells. METHODS: Bovine corpora lutea were mechanically dispersed, cell suspensions separated over a Percoll density gradient, and type 5 cells purified by colony transfer. Granulosa cells were harvested from small-sized antral follicles. Observations were carried out at the light and electron microscopical level. 3 beta-Hydroxy-steroid-dehydrogenase was localized histochemically in addition to intracellular lipid droplets stained with nile red. Immunolocalization was used to study Factor VIII antigen presence, the architecture of the cytoskeleton, as well as the occurrence of neuronal cell adhesion molecules, and of neuronal cadherin-like molecules. The uptake of acetylated low density lipoprotein was examined. As for progesterone concentration, cells were seeded at low density on day zero. Cell numbers and progesterone levels of supernatants were determined on day 10 in culture. RESULTS AND CONCLUSIONS: Type 5 cells behaved morphologically like immature granulosa cells, yet the total cell number and the progesterone concentration differed for type 5 cells compared to granulosa cells. The addition of LH had no influence on the progesterone concentration as seen for either type 5 cells or for granulosa cells. It is concluded that type 5 cells, which were originally mistaken for microvascular endothelial cells, display similarities with immature granulosa cells. Type 5 cells may play a role in renewal of luteal cells.

Animals↗
PubMed1994

Long-term co-culture of bovine granulosa cells with microvascular endothelial cells: effect on cell growth and cell death.

The intraovarian axis between granulosa cells and thecal cells is regulated by locally produced autocrine and paracrine factors. Until now, microvascular endothelial cells (MVEC) have not been included in such studies. Bovine granulosa cells from medium-sized antral follicles were plated at low density into the lower compartment of 24-well-culture plates on day 0. MVEC derived from bovine corpus luteum were seeded on appropriate inserts and placed as the upper compartment on day 1. Control granulosa cell cultures and MVEC co-cultures were maintained in serum-containing medium. On day 21, control cultures displayed an epithelioid monolayer and the coculture displayed a multilayer. Histochemical staining for 3 beta-HSD activity and for the lipid droplet stain with the fluorescent dye Nile Red were strong, suggesting augmented steroidogenesis in the multilayer. Yet the progesterone levels of supernants corrected for 10,000 cells were similar in monolayers and in multilayers. Co-cultures contained approximately three times more granulosa cells than control cultures as evaluated with a Coulter counter. Additionally, the occurrence of dead cells was quantified with the fluorescent DNA stain, ethidium homodimer, in 11-day-old control cultures and MVEC co-cultures which were deprived of serum, MVEC, or both for an additional 40 h. Serum and MVEC suppressed the occurrence of granulosa cell death. It is concluded that MVEC produce survival factors for the growth and maintenance of granulosa cells.

Animals↗
PubMed1994

Cytokeratin-positive and cytokeratin-negative cultured endothelial cells from bovine aorta and vena cava.

The heterogeneous morphology of microvascular endothelial cells obtained from the bovine corpus luteum was recently attributed to the occurrence of cytokeratin (CK) positive and CK negative endothelial cells. The aim of the present study was to establish comparable differences for bovine macrovascular endothelial cells. For this reason, endothelial cells were scraped from the abdominal aorta as well as the inferior vena cava of cows. At the level of phase contrast microscopy, primary cultures originating from both large vessels could be classified as CK positive or CK negative endothelial cells. After seeding CK positive endothelial cells on Matrigel matrix, a two-dimensional meshwork of so called pseudotubules formed within 2 h. By using immunofluorescence localization CK positive cells were identified by a complex meshwork. It consisted of CK 8, 18 and 19 as displayed by Western blots. The CK negative group showed spindle-shaped or polygonal endothelial cells according to light microscopy. In postconfluent cultures, spindle-shaped cells developed a three-dimensional meshwork of tubules. After seeding spindle-shaped cells on Vitrogen 100 matrix, pseudotubules formed within 1 day. In considering the frequency of occurrence, primary harvests from the vena cava contained less than 1% CK positive cells. With respect to growth, the cell number was two to three times higher for the CK negative group than the CK positive group as judged on day 13 after cell seeding. It is concluded that subpopulations of endothelial cells are derived from large blood vessels.

Animals↗
PubMed1994

The heteromorphology of cultured microvascular endothelial cells.

Endothelial cells alter morphology easily when culture conditions change. One has omitted to test carefully whether this heteromorphology represents constitutional differences which are maintained in culture. Cells were isolated from the microvascular tree of the bovine corpus luteum by mechanical disintegration, i.e. without collagenase treatment. From these cells, 5 phenotypes were purified which formed epithelial monolayers of stable morphology in long-term culture. Each phenotype showed criteria typical of endothelial cells. The criteria were incompletely present in phenotype 5. Differences between the phenotypes related to the structure of the cytoskeleton, the fibronectin matrix and to the expression of cell-cell adhesion molecules from the cadherin family and the NCAM (neuronal cell adhesion molecule) group. Mesothelial cells, pericytes, myocytes as well as luteal cells were considered for differential diagnosis. The present results point to a heterogeneous distribution pattern of endothelial cells within the microvascular tree of a single organ.

Actins↗
PubMed1993

Cultured microvascular endothelial cells (MVEC) differ in cytoskeleton, expression of cadherins and fibronectin matrix. A study under the influence of interferon-gamma.

Endothelial cells are known to undergo transitions in cell shape during long-term culture. Thus, the assumption that the separate phenotypes of microvascular endothelial cells (MVEC) recently isolated from bovine corpus luteum represent constitutively different cell strains cannot automatically be made. For this reason, particular morphological qualities from four of five reported MVEC types were studied. Confluent cultures of MVEC types 1, 3, 4 and 5 were either left untreated or exposed to recombinant bovine interferon-gamma (IFN-gamma; 200 units/0.5 ml culture medium) for 3 days. Paraformaldehyde-fixed monolayers were permeabilized with Triton X-100 prior to the detection of filamentous actin, using phalloidin-FITC. Vimentin filaments, cytokeratin filaments, microtubules, E- and N-cadherins as molecules of cell adhesion plaques, and fibronectin filaments were localized by the application of specific antibodies in combination with epifluorescence microscopy. Cells from untreated single cultures uniformly and reproducibly showed an actin cytoskeleton that distinguished the particular MVEC type. MVEC type 1 presented a circular band of fine actin filaments. MVEC type 3 preferentially had developed a starburst-like actin pattern. MVEC type 4 mainly exhibited a polygonal network. MVEC type 5 showed a prominent circular band of thick microfilament bundles from which short filaments radiated. Cytokeratin filaments were noted in MVEC type 1 only. Vimentin filaments occurred as a dense network constricted to the central area in MVEC type 1, while they were spread out in MVEC types 3 and 4. A wavy path comparable to the course of microtubules was apparent in MVEC type 5. Fibronectin assembled into two differently shaped layers at the basal cell side of each MVEC type. Under IFN-gamma treatment, cytoskeletal diversities were maintained between the MVEC types, yet each MVEC type showed specific modulations to its cytoskeleton and to its fibronectin matrix. Upregulation of anti-E-cadherin labelling was detected in MVEC type 1, showing a fluorescent cell border of linear contour. The upregulation of E-cadherin by IFN-gamma treatment could also be demonstrated by western blotting, which revealed a 135 kDa full-sized molecule and a 95 kDa tryptic fragment characteristic of cadherins. Anti-N-cadherin labelling was evident for MVEC type 5, giving rise to a fluorescent punctate cell margin. Our investigations support the existence of truly separate MVEC types.

Animals↗
PubMed1992

Cultured microvascular endothelial cells derived from the bovine corpus luteum possess NCAM-140.

Previously, five phenotypically different, stable types of microvascular endothelial cells (MVE) were isolated from the bovine corpus and cultured successfully. We found that three out of these five types of MVE express the neural cell adhesion molecule (NCAM). As shown by immunocytochemistry, weak NCAM immunoreactivity occurred mainly in the perinuclear area of cell type 1. Monolayers of types 2 and 5 revealed heavy NCAM immunoreactivity, which was localized predominantly at the lateral cell surface outlining the contact zones of adjacent cells. In contrast, cell types 3 and 4 were not NCAM immunoreactive. Western blot analyses substantiated these results: While cell type 1 showed a weak immunoreactive band, cell types 2 and 5 displayed strong NCAM-immunoreactive bands of a molecular weight of approximately 140 kDa (NCAM-140), which was absent in cell types 3 and 4. These results reveal for the first time that NCAM can be expressed by cultured MVE and may serve in mediating endothelial cell contacts. Since luteal cells also express NCAM-140, this adhesion molecule could in addition be involved in the interactions of luteal cells with MVE.

Animals↗
PubMed1992

Differential adhesion of granulocytes to five distinct phenotypes of cultured microvascular endothelial cells.

Adhesion of isolated human polymorphonuclear granulocytes (PMNs) to five different phenotypes of cultured microvascular endothelial cells derived from bovine corpora lutea was investigated by measuring the myeloperoxidase content of cell lysates. Untreated and interleukin 1 (IL-1) -pretreated confluent monolayers were overlaid with unstimulated and phorbol ester (PMA)-stimulated PMNs in the absence and presence of the monoclonal antibody IB4 recognizing and functionally blocking beta 2 (CD18) of the leukocyte integrins. Unstimulated PMN adhesion was highest on type 4, followed by type 3 and 5 endothelial cells. This adhesion was not inhibited by treatment with IB4. IL-1 pretreatment of endothelial cells resulted in a significant increase of PMN adhesion on types 1, 2, and 4, most of which was also beta 2 integrin-independent. PMA-stimulation of PMNs increased adhesion to maximal values on cell types 1 and 5, which was largely blocked by IB4. Type 2 endothelial cells supported significantly less PMA-stimulated PMN adhesion than all other types. In the presence of IB4, adhesion of PMNs to untreated and IL-1-pretreated type 3 and 4 endothelial cells was significantly reduced by PMA. This reduction of beta 2 integrin-independent adhesion by PMA stimulation is compatible with possible shedding of the lectin-like leukocyte adhesion molecule, L-selectin, from PMNs. Differential PMN adhesion may reflect distinctive expression of endothelial adhesion molecules in different phenotypes of microvascular endothelial cells. Endothelial specialization within the microcirculation may have important functional consequences for the inflammatory response in vivo.

Animals↗
PubMed1992

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.

Acetylation↗
PubMed1992

Endothelial cell helix in small arterioles of human ureters. A study by scanning electron microscopy (SEM).

Microvessel corrosion casts of human ureters were prepared. Portions of casts were longitudinally cut by use of a pulsed Eximer laser beam in order to get insight into microvessel branches studied in SEM. Larger arterioles (greater than 30 microns in diameter) showed oval imprints of endothelial cell nuclei along the vessel axis. Imprints were found in furrows parallel to resin ridges. Smaller arterioles (less than microns in diameter) displayed imprints of nuclei aligned in a helix around the vessel axis. With regard to capillaries, one nucleus imprint was spaced far apart from the other. Venules demonstrated a reticular network of thick, attenuated resin ridges and of small meshes which consisted of round or oval nucleus imprints. It is concluded that smaller arterioles form an endothelial cells helix.

Arterioles↗