Biomedical subjects

K Spanel-Borowski

Publications and source records attributed to K Spanel-Borowski.

At least 19 recordsLinked to original sources

PubMed2002

Development and regression of non-capillary vessels in the bovine corpus luteum.

The corpus luteum life cycle is accompanied by capillary growth, maturation and degeneration. Arterial blood vessels are thought to undergo hyperplasia and hypertrophy during the stage of regression, as is the case with non-capillary vessels. In this study, we used morphological studies to show that the development of non-capillary vessels occurs at other corpus luteum stages. Non-capillary vessels were present at the developmental stage of the corpus luteum, and increased markedly in number in the subsequent stages. After double-staining for ASM-1 actin and Ki-67 nuclear antigen, the proliferation of smooth muscle cells (SMCs) was only detected during stages of development and secretion. When the capillaries had disappeared at the regression stage, the arterial blood-vessel walls thickened noticeably. This was attributed to the development of fibroelastosis as shown by staining for collagenous and elastic fibres. In conclusion, the bovine corpus luteum represents a physiological model for studying arteriolization at all stages of development and secretion. At the regression stage, arterioregression sets in.

Animals↗

PubMed2002

Correlation between expression of selectins and migration of eosinophils into the bovine ovary during the periovulatory period.

Leukocytes enter specific ovarian areas at a precisely defined moment, influencing cyclically changing structures such as follicles and corpora lutea. As yet, no studies have been published on the trafficking mechanisms involving the interaction between adhesion molecules on endothelial cells (ECs) and those on leukocytes. First, antibodies against human adhesion molecules were examined by flow cytometry with the aim of identifying the same bovine antigen. Western blot analysis and immunoprecipitation revealed that the molecules had the same molecular weight as their human counterparts. Afterwards, we investigated the distribution of these antigens in various ovarian stages using immunohistology. Among the molecules, P-selectin (CD62P) and L-selectin (CD62L) showed stage-dependent expression, and were thus examined further. In the preovulatory follicle, microvascular ECs were negative for CD62P. Few of the leukocytes expressed CD62L. In a freshly ruptured follicle, CD62P expression was found in the dilated vessels of the former thecal layer. Simultaneously, a large proportion of the rapidly increased numbers of leukocytes, mainly eosinophils, located around the microvessels of the outer thecal layer expressed CD62L. In the early corpus luteum development stage, CD62L showed peak expression with 70%-80% positive cells compared to leukocytes. In the secretory stage, the septal venules showed a consistent, but now weak, staining for CD62P. Few leukocytes expressed CD62L. During regression, the total number of leukocytes, now representing macrophages, increased significantly, but the proportion of CD62L-positive cells remained constant. In summary, we found a strong correlation of CD62P expression on activated ECs and the appearance of CD62L-positive leukocytes in the early corpus luteum development stage, suggesting the participation of both selectins in the migration of eosinophils under physiological conditions.

Animals↗

PubMed2001

The expression of substance P and its neurokinin-1 receptor mRNA in the bovine corpus luteum of early developmental stage.

Related to all leukocytes, 90% of eosinophils are recruited into the bovine corpus luteum of early developmental stage. We here describe a simultaneous appearance of substance P (SP)-positive fibre-like structures and the neurokinin-1 (NK-1) receptor mRNA for SP. Substance P was depicted by using indirect immunohistology and immunofluorescence localization. The dot blot analysis confirmed the presence of SP at the protein level. Using nested reverse transcribed-polymerase chain reaction (RT-PCR) analysis, a 358 bp long partial bovine receptor mRNA for SP (NK-1) was sequenced in the spinal cord. The mRNA for SP and for the NK-1 receptor were then detected in the corpus luteum of early developmental stage with RT-PCR and nested RT-PCR. We conclude: The production of SP and the expression of NK-1 receptor mRNA may be involved in the selective recruitment of eosinophils into the bovine corpus luteum of early developmental stage.

Animals↗

PubMed2001

Increase in final stages of follicular atresia and premature decay of corpora lutea in Insl3-deficient mice.

The insulin-like factor 3 (Insl3), a member of the insulin-like hormone family, is exclusively synthesized in gonads. Our recent analysis of Insl3-deficient mice revealed the regulating role of the Insl3 factor on the gubernaculum development during the transabdominal descent of the testis. Here we define the role of the Insl3 factor by histometric analysis of wild-type and Insl3(-/-) ovaries. Ovaries from 40-day-old- and 6-month-old Insl3(-/-) mice as well as from wild-type littermates were serially sectioned. Sections were stained with periodic acid Schiff reaction (PAS) for counting the number of zonae pellucidae which indicated the final stages of follicular atresia. Corpora lutea were also determined. Some sections were processed using either a modified TUNEL method for in situ detection of apoptosis or a lectin labelling technique with Griffonia simplicifolia I agglutinin (GS I) for endothelial cell occurrence. The number of zonae pellucidae was higher in Insl3-deficient ovaries of both ages than in ovaries of wild-type sisters (P < 0.05 for 40-day-old ovaries; P < 0.01 for 6-month-old ovaries). Additionally, the wild-type mice of both ages possessed threefold more corpora lutea than their Insl3 littermates (P < 0.01 for 40-day-old; P < 0.001 for 6-month-old). In general, wild-type corpora lutea looked healthy, showed GS I-positive endothelial cells and no apoptotic cells. Corpora lutea from mutants were rich in regressing GS I luteal cells, and apoptotic cells appeared. We conclude: Follicular atresia and luteolysis are accelerated in ovaries of Insl3-deficient mice probably because of increased apoptosis. The Insl3 function thus appears to rescue endocrine cells from the apoptotic pathway.

Animals↗

PubMed2001

Limitation of microwave treatment for double immunolabelling with antibodies of the same species and isotype.

Microwave treatment (MW) involves completely blocking contaminating staining in the double-labelling technique, using primary monoclonal antibodies from the same species and the same isotype as well as the same secondary antibody (ab). However, we noticed some limitations when locating proliferating cell types in cryostat and paraffin sections using the advantages presented by MW. Control experiments have shown that MW does not diminish contaminating staining when cytoplasmic (desmin, ASM-1) or nuclear (Ki-67) antigens have been labelled with antibodies in the first round of immunolabelling. In contrast to the cell surface antigen, CD18, where the primary ab had to be crosslinked by a secondary ab to obtain contaminating staining, this was observed for the detection of cytoplasmic or nuclear antigens only labelled with a primary ab. In conclusion, for double immunolabelling with abs from the same species and the same isotype, MW is not able to completely abolish contaminating staining.

Animals↗

PubMed2001

Increase in calcitonin gene related peptide (CGRP) and decrease in mast cells in dihydroepiandrosterone (DHEA)-induced polycystic rat ovaries.

UNLABELLED: The polycystic ovary is reported to correspond with a high density in intraovarian nerve fibers and their sympathetic hyperresponsiveness. Peptidergic nerves may also be involved in this process. An interaction between nerve fibers and mast cells is assumed because of nerve growth-factor production by mast cells. Here we investigated CGRP-positive nerve fibers and mast cells in polycystic ovaries induced in immature rats with dihydroepiandrosterone (DHEA). The DHEA treated ovaries contained less corpora lutea than controls (mean +/- SEM: 4.3 +/- 0.6 versus 11.3 +/- 0.9, P > 0.001) and less intact antral follicles (4.7 +/- 0.7 versus 8.1 +/- 1.1; P < 0.05) according to the histometric approach. By immunolabelling more CGRP-positive nerve fibers were found in the DHEA treated ovaries than in controls (mean +/- SEM per one section: 23.2 +/- 5.8 fibers versus 10.3 +/- 0.9 and 171 +/- 44.7 varicosities versus 84 +/- 9.5). This was confirmed by dot blot analysis, showing a significant higher CGRP signal intensity per microgram homogenized ovaries of the DHEA treated group compared to the untreated (P < 0.05). Toluidine-blue-stained mast cells populated the medulla in both groups, yet had strikingly decreased in the DHEA treated ovaries (23.5 +/- 3.9 versus 89 +/- 5.6, P < 0.005). CONCLUSION: The increase in CGRP-positive nerve fibers and the decrease of toluidine-blue-stained mast cells points to an altered neuroimmune function in DHEA-induced polycystic rat ovaries.

Animals↗

PubMed2001

Increase in nerve fibers and loss of mast cells in polycystic and postmenopausal ovaries.

OBJECTIVE: To quantify nerve fibers and mast cells in human ovaries at different functional stages. DESIGN: Retrospective study. SETTING: Research laboratory of the university. SPECIMEN(S): 8 human ovaries in the follicular (cyclic) phase, 7 polycystic ovaries, and postmenopausal ovaries with (n=5) or without (n=7) hyperthecosis. MAIN OUTCOME MEASURE(S): Single- and double immunohistology for the S100 antigen in glial cells of autonomic nerve fibers, for chymase and tryptase in mast cells, and for the common leukocyte antigen on leukocytes. Histometric evaluation was also performed. INTERVENTION(S): None. RESULT(S): Polycystic ovaries contained significantly more S100-positive nerve fibers in the corticomedullary region than did cyclic ovaries (mean +/- SD per 2-mm(2) area, 476 +/- 136 and 224 +/- 133; P<.01). Postmenopausal ovaries with or without hyperthecosis had the highest density of nerve fibers. In cyclic and polycystic ovaries, more tryptase-positive mast cells than chymase-positive mast cells were found in the interstitial cortex and the medulla. In cyclic ovaries, areas with a moderate density of nerve fibers contained many mast cells. Hence, with increasing nerve fiber density in polycystic ovaries, the number of mast cells decreased strikingly compared with cyclic ovaries (p<.001). Almost no mast cells were seen in postmenopausal ovaries with and without hyperthecosis. The number of leukocyte antigen-positive leukocytes was similar in all groups. CONCLUSION(S): The high density of nerve fibers in polycystic and postmenopausal ovaries, together with a conspicuous decrease in mast cells, indicates altered neuroimmune communication.

Aged↗

PubMed2001

Evidence of leptin expression in normal and polycystic human ovaries.

Leptin, the 'obese' protein, is found in cultured granulosa cells derived from human pre-ovulatory follicles. However, the occurrence of leptin has not been studied in intact ovaries, either normal or polycystic, until now. Paraffin sections from 25 human ovaries of different cycle stages and 25 wedge resections of polycystic ovaries were investigated by means of immunochemistry. Additionally, three ovaries were available for reverse transcription-polymerase chain reaction analysis. Leptin-positive cells were located in the granulosa cells of pre-antral follicles, and distinctly in the thecal layer of intact and regressing antral follicles. In the corpus luteum (CL) in the developmental stage, the former epithelioid leptin-positive thecal cells became fibroblast-like in the septum. In the CL of the secretory stage, single leptin-positive cells were detected between luteal cells. In polycystic ovaries, leptin-positive cells were noted both in the hypertrophied thecal layer and in the luteinized granulosa layer. Our findings on leptin expression at the protein level were confirmed by a positive mRNA signal for leptin in granulosa cells and in the CL. Additionally, mRNA of the full-length leptin receptor OB-R and of the short isoforms B219.1-B219.3 was identified in granulosa cells and the CL, as well as in the cortex and medulla. We conclude that leptin is produced in the ovary and may act in autocrine and paracrine ways.

Adolescent↗

PubMed2001

Leucocyte proliferation in the bovine corpus luteum.

Leucocytes vary in type and number during the lifespan of a corpus luteum. The aim of this study was to determine whether there is an increase in the number of lymphocytes and macrophages as a result of local proliferation. Bovine corpora lutea were classified into stages of development, secretion and regression. A new double immunolabelling method was established for nuclear Ki-67 antigen (a marker for cell proliferation) and for leucocyte surface antigens (detection of CD2-, CD3-, CD4-, CD8-positive lymphocytes and CD14-positive monocytes). Differential cell counting was performed. Between the stages of development and regression there was an increase in the number of T-lymphocytes and macrophages. The percentage of proliferating leucocytes in relation to the total number of proliferating cells was approximately 20% at the stage of advanced secretion and 70% at late regression. The increase in the number of proliferating leucocytes at late regression was due to CD14-positive macrophages. These macrophages migrated from small blood vessels into the septa of corpora lutea at the early stage of regression. Macrophages showed local proliferation in the late stage of regression when capillaries were no longer present. It is concluded that the physiological involution of the corpus luteum is an inflammatory-like condition, which includes local proliferation of monocytes.

Animals↗

PubMed2001

Pulmonary inflammation induced by a recombinant Brugia malayi gamma-glutamyl transpeptidase homolog: involvement of humoral autoimmune responses.

BACKGROUND: A major allergen from the lymphatic filarial parasite Brugia malayi implicated in the pathogenesis of tropical pulmonary eosinophilia (TPE) has recently been cloned and identified as the homolog of the membrane-bound mammalian enzyme gamma-glutamyl transpeptidase (gamma-GT). Patients with acute TPE show autoreactive antibodies against endogenous gamma-GT from the pulmonary epithelium. MATERIALS AND METHODS: Recombinant B. malayi gamma-GT, alone or adsorbed to aluminium hydroxide (AL), was used in a BALB/c mouse model to analyze its antigenic/allergenic potential, its potential to induce pulmonary inflammation, and its capacity to induce autoreacting antibodies. RESULTS: Mice immunized with B. malayi gamma-GT showed significant levels of gamma-GT-specific IgG1, IgG2a, IgG3, IgA, IgE antibodies, and mild blood eosinophilia, even in the absence of adjuvant. Intranasal challenge with B. malayi gamma-GT induced peribronchial and perivascular inflammation characterized by a mixed infiltrate of lymphocytes, neutrophils, eosinophils, and macrophages. Both IL-4 and IFN-gamma were detected in the peripheral blood and in the bronchoalveolar lavage fluid of immunized and intranasally challenged mice. Histological analysis of murine lungs using affinity-purified antibodies from mice immunized with the parasite's gamma-GT revealed the presence of autoimmune antibodies against pulmonary epithelium. Western blot analysis identified the 55 kDa heavy chain subunit of the murine gamma-GT as the target of autoreactive/crossreacting antibodies. CONCLUSION: Our data from the in vivo mouse model demonstrate the potent allergenicity/antigenicity of B. malayi gamma-GT, and its capacity to induce pulmonary inflammation upon intranasal challenge. This leads to breakdown of tolerance against endogenous murine gamma-GT. Thus, humoral autoimmunity against the airways epithelium may contribute to the pathogenesis of TPE.

Animals↗

PubMed2000

The transient disappearance of cytokeratin in human fetal and adult ovaries.

Cells from the inner and outer granulosa cell layers of the ovarian follicles differ in function, probably because of their different origins from the surface epithelium and from the rete. This suggestion has not so far been thoroughly investigated in the human ovary. We examined fetal ovaries from the early, middle and late gestational periods, ovaries from fertile women, and preovulatory follicular cells obtained from patients under in vitro fertilization therapy (IVF). Indirect immunohistology and immunocytology were used to detect the presence of cytokeratin (CK)-positive epithelial cells. In fetal ovaries from the early gestational period, prominent rete tubules (sometimes with oocytes) appeared to be fused with the sex cords and primordial follicles. Both showed CK-positively, detected with the pan-CK antibody Lu-5. Cytokeratin 19 was clearly expressed in the fusion area. In the fetal and adult ovaries, CK-positive follicular or granulosa cells were noted in the primordial and primary follicles as well as the preovulatory follicles. Cytokeratin was not detected in the granulosa cells of growing follicles, CK-positive and -negative luteal cells were identified in the developing corpus luteum. We conclude for the human ovary: (1) the heterogeneous morphology of granulosa cells may be explained by their twofold origin from the surface epithelium and the rete, (2) the rete tubules appear to be involved in folliculogenesis, (3) the transient absence of CK expression in growing follicles compared to resting and mature follicles or to the developing corpus luteum indicates a particular role of CK-positive cells at the periovulatory period.

Adult↗

PubMed2000

Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures.

We evaluated the presence and number of eosinophils at varying stages in the human corpus luteum from 27 ovaries of women at reproductive age. Eosinophils preferentially accumulated in dilated microvessels of the thecal layer transforming into septa of the corpus luteum. The granulosa layer under luteinization, the thecal layer, and haemorrhages in the former antrum each contained low, moderate and high numbers of extravasated eosinophils respectively. Eosinophils decreased rapidly during the stages of secretion and regression. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) systems were used to investigate the expression and regulation of the eosinophil-attracting chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin in granulosa cells obtained from follicular aspirates from women undergoing IVF. Contaminating leukocytes were determined by CD18 mRNA quantification. Granulosa cells expressed RANTES (n = 3; 43 +/- 14 pg/ml, mean +/- SEM). 4ss-phorbol-12-myristate-13-acetate (PMA; 211 +/- 53) and tumour necrosis factor alpha (TNFalpha) (238 +/- 59), but not interleukin (IL)-1 up-regulated RANTES at significant levels. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with lower levels. We found only traces of eotaxin mRNA and no eotaxin secretion, even in stimulated granulosa cell cultures, independently of leukocyte levels. Taken together, this is the first study demonstrating the selective presence of eosinophils in human periovulatory structures. RANTES, but not eotaxin, may play an active process in the accumulation of these cells.

Adult↗

PubMed2000

Difference in localization of eosinophils and mast cells in the bovine ovary.

The bovine ovary contains a considerable number of leucocytes which can be located with an antibody against the CD18 molecule. In the present study, subtyping and cell counting were carried out on histological sections stained with Sirius red for eosinophils and with toluidine blue for mast cells. The CD18(+) cells were identified immunohistologically. Eosinophils and mast cells contributed considerably to the CD18(+) pool. The number of eosinophils in the corpus luteum increased rapidly in early development to approximately 90% of the CD18(+) cells, and decreased to 30% during secretion and to 10% during regression. Mast cells were not detectable in the follicles, the corpus luteum and the periphery of the cortex, but were observed in the interstitial cortical stroma and the medulla. The number of mast cells in these regions, which corresponded to 60-76% of the CD18(+) cells, did not change significantly throughout the oestrous cycle. It is concluded that eosinophils are selectively recruited at the periovulatory period and that mast cells are unevenly distributed.

Animals↗

PubMed1999

Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period.

RANTES may be one of the chemoattractants involved in stimulating eosinophils and macrophages to migrate selectively into bovine dominant follicles and into developing corpora lutea. We sequenced a 736 bp fragment of the bovine RANTES mRNA encoding the complete protein and defined the ovarian source of RANTES mRNA. As demonstrated by competitive RT-PCR, follicle-derived macrophages showed a 100-1000 times higher RANTES mRNA level compared to unpurified granulosa cells or follicle-derived fibroblasts. By means of in situ hybridization, RANTES mRNA positive macrophages were located in the former thecal layer of the developing corpora lutea.

Amino Acid Sequence↗

PubMed1998

A sparsely vascularised zone in the cortex of the bovine ovary.

We consider that the microvascular bed may play a role in the initiation and maintenance of growth from primordial to primary follicles. Therefore, using immunochemistry, we examined microvessels in calf and cow ovaries to identify the presence of factor VIII-related antigen endothelial cells. A vessel-poor zone was observed in the cortex of immature and mature cow ovaries. Primordial and primary follicles were assembled in this zone. It is concluded that follicular dormancy is likely to be maintained by the scarcity of microvessels and thus by the consequent poverty of the blood supply.

Aging↗

PubMed1997

Evidence for the maintenance of macrophage-like cells in long-term bovine granulosa cell cultures.

Ovarian macrophages may be involved in the degradation of the basal membrane after rupture of a preovulatory follicle, or in angiogenesis during follicle and corpus luteum development, or may support steroidogenesis. The present study describes easy access to macrophage-like cells in the ovary and characterizes them. Cells from bovine antral follicles with 1-4 ml of their naturally occurring fluid were plated on day zero. On day 10, the granulosa cell culture showed macrophage-like cells. They developed as non-adherent cells and also adhered to the monolayer as single cells or as hemopoietic-like clones. The macrophage-like cells contained strong activity of non-specific esterase, acid phosphatase or 3beta-hydroxy-steroid dehydrogenase (3beta-HSD). Only the steroid enzyme was absent on day 19. As detected by immunolocalization, macrophage-like cells of various sizes showed a response for the CD18 surface molecule of leukocytes, for the CD45 molecule related to hemopoietic progenitor cells, and for the CD14 molecule selectively expressed on cells of the monocyte-macrophage lineage. The macrophage-like cells exhibited a granular-like actin structure as revealed by fluorochrome-labeled phalloidin. The ultrastructure of small, intermediate or large macrophage-like cells was similar to that of monocytes or of macrophages. In situ immunolabeling of intact or regressing follicles revealed single cells positive for CD14, CD18 or CD45. In order to establish that in vitro proliferation of the macrophage-like cells had occurred, their number was assessed between days 1 to 19 of cultivation. The cultures derived from 23 follicles were classified into groups A, B, or C according to the number of non-adherent cells (<5000, <12 500, or >/=12 500 per 16-mm well) on day 12 of cultivation. The total number of macrophage-like cells (non-adherent and adherent) increased approximately fourfold in all groups between days 1 to 12. The total number decreased to day 5 levels between days 15 to 19. Thus, the macrophage-like cells probably represent a subpopulation of the macrophage family. They proliferate in co-culture with granulosa cells.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Evidence for the development of macrophage-like cells in long-term culture of bovine aortic endothelial cells.

Macrophages are known to be derived from monocytes which proliferate in the bone marrow. The proliferation of monocytes may occur in other places as well. In the present study, we describe the morphological behaviour of macrophage-like cells in endothelial cell cultures obtained from bovine aorta. These cells resembled hemopoietic clones containing progenitor-like cells. Immature and mature macrophage-like cells were rich in acid phosphatase activity, and expressed the CD18 molecule using immunolocalisation. Mature cells contained intracellular lipid droplets. "Actin" globules were apparent only in the peripheral cell areas without lamellipodia or filipodia. At the ultrastructural level, the mature cells were crowded with granules which could be lysosomes, phagolysosomes, or endocytotic vesicles. Multinuclear giant cells which behaved in a different way to the macrophage-like cells were observed. The development and maintenance of macrophage-like cells appears to be dependent on the coculture with endothelial cells. It may signify that endothelial cells are involved in the proliferation of monocytes outside the bone marrow.

Acid Phosphatase↗

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↗