Periodic acid-Schiff (PAS) reaction and plastination in whole body slices. A novel technique to identify fascial tissue structures.

Since collagen rich fascial tissue is often very delicate and difficult to discern on native tissue slices, we have developed a method for staining full-body slices using the periodic acid-Schiff (PAS) reaction with subsequent plastination. Since the PAS reaction primarily stains carbohydrates, we could exploit the circumstance that different collagen types vary in carbohydrate content. Contrary to fasciae, tissues such as muscle, bone, nerves and blood vessels exhibit significantly less staining or remain unstained. We have validated the whole-body slice staining results in microscopic tissue slides which were stained with standard extracellular matrix stains such as Masson-Goldner trichrome stain and van-Gieson stain. Furthermore, we have performed immunofluorescence imaging to confirm the presence of collagen in the stained tissue. We achieved very good staining and plastination results and were able to clearly identify even very thin fascia in transversal body slices. This technique may prove useful in advancing our knowledge on the complex topography of fascial structures.

Advantages and Limitations of Salmon-Gal/Tetrazolium Salt Histochemistry for the Detection of LacZ Reporter Gene Activity in Murine Epithelial Tissue.

The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial ß-galactosidase (Bact ß-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact ß-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous ß-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact ß-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine-potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact ß-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

Comments on Methods to Suppress Endogenous ß-Galactosidase Activity in Mouse Tissues Expressing the LacZ Reporter Gene.

The Escherichia coli LacZ gene (encoding ß-galactosidase) is a widely used reporter for gene regulation analysis in transgenic mice. Determination of ß-galactosidase activity is classically performed using 5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside/ferri-/ferrocyanide (X-Gal/FeCN) histochemistry. Uncertainty about the origin of the ß-galactosidase signal is encountered in tissues containing high levels of endogenous ß-galactosidase. Here, we show that reliable results can nevertheless be obtained in these tissues by performing the histochemical reaction under slightly basic pH conditions (pH 8-9). We further demonstrate that in this context, analysis of tissue sections may be advantageous over that of conventional whole-mount tissues because poor dye penetration and remaining tissue acidity are avoided in tissue sections. We also recommend that bacterial debris should always be carefully removed from the luminal surface of gastrointestinal tract specimens unless staining of resident microflora is deliberately used as an internal positive control in the assay. Finally, we show that 6-chloro-3-indolyl-ß-d-galactopyranoside with nitrotetrazolium blue chloride works well as an alternative chromogenic substrate for visualizing LacZ reporter gene expression in cryostat sections. Its use in high endogenous ß-galactosidase-expressing organs is superior over the use of X-Gal/FeCN at slightly basic pH conditions.

A simple method for inducing estrous cycle stage-specific morphological changes in the vaginal epithelium of immature female mice.

The vaginal epithelium of the adult female laboratory rodent changes from mucous secretion to cornification over the course of the estrous cycle. The morphophysiological changes occur with such regularity, accuracy and precision that the specific stage of the estrous cycle in the rat can be determined by inspection of the vaginal opening and/or exfoliative vaginal cytology. However, in the mouse, post-mortem vaginal histology is often required to determine the estrous cycle stage for ensuring the required level of reliability. Consequently, an excess number of female adult mice are needed to allow for the delivery of sufficient numbers of mice in a desired estrous cycle stage. In this study, we demonstrate that the standard procedure for oocyte superovulation and collection in the laboratory mouse (e.g. injection of equine chorionic gonadotropin followed 48 h later by human chorionic gonadotropin) can also be reliably used to induce changes in the epithelium of 3.5-week-old mouse vaginas in an estrous cycle stage-specific manner (e.g. establishment and replacement of a mucous secreting epithelium with a cornified epithelium; induction of cornification-associated loricrin expression). The superovulation protocol thus allows for the efficient and economic induction of estrous cycle stage-specific characteristics in the Müllerian duct-derived vagina thereby avoiding the necessity of post-mortem identification of the estrous cycle stage. In addition, our study indicates that the laboratory mouse vagina is an excellent organ for studying the sequence of events leading to cornification.

The ductal origin of structural and functional heterogeneity between pancreatic islets.

Islets form in the pancreas after the first endocrine cells have arisen as either single cells or small cell clusters in the epithelial cords. These cords constitute the developing pancreas in one of its earliest recognizable stages. Islet formation begins at the time the cords transform into a branching ductal system, continues while the ductal system expands, and finally stops before the exocrine tissue of ducts and acini reaches its final expansion. Thus, islets continuously arise from founder cells located in the branching and ramifying ducts. Islets arising from proximal duct cells locate between the exocrine lobules, develop strong autonomic and sensory innervations, and pass their blood to efferent veins (insulo-venous efferent system). Islets arising from cells of more distal ducts locate within the exocrine lobules, respond to nerve impulses ending at neighbouring blood vessels, and pass their blood to the surrounding acini (insulo-acinar portal system). Consequently, the section of the ductal system from which an islet arises determines to a large extent its future neighbouring tissue, architecture, properties, and functions. We note that islets interlobular in position are frequently found in rodents (rats and mice), whereas intralobularly-located, peripheral duct islets prevail in humans and cattle. Also, we expound on bovine foetal Laguesse islets as a prominent foetal type of type 1 interlobular neuro-insular complexes, similar to neuro-insular associations frequently found in rodents. Finally, we consider the probable physiological and pathophysiological implications of the different islet positions within and between species.

Dual origin, development, and fate of bovine pancreatic islets.

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo-acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of 'perilobular giant islets' are distinguishable from larger numbers of 'intralobular small islets'. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin-synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette-like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the 'interlobular small islets' persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T-cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo-acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy.

The endocannabinoid N-arachidonoyldopamine (NADA) exerts neuroprotective effects after excitotoxic neuronal damage via cannabinoid receptor 1 (CB(1)).

Endocannabinoids exert numerous effects in the CNS under physiological and pathological conditions. The aim of the present study was to examine whether the endocannabinoid N-arachidonoyldopamine (NADA) may protect neurons in excitotoxically lesioned organotypic hippocampal slice cultures (OHSC). OHSC were excitotoxically lesioned by application of N-methyl-d-aspartate (NMDA, 50 µM) for 4 h and subsequently treated with different NADA concentrations (0.1 pM-50 µM) alone or in combination with cannabinoid receptor antagonists. NADA protected dentate gyrus granule cells and caused a slight reduction in the number of microglial cells. The number of degenerated neurons significantly decreased between 100 pM and 10 µM NADA (p < 0.05). To identify the responsive receptor type of NADA mediated neuroprotection, we applied the cannabinoid (CB) receptor 1 (CB(1)) inverse agonist/antagonist AM251, CB(2) inverse agonist/antagonist AM630, abnormal-cannabidiol (abn-CBD)-sensitive receptor antagonist O-1918, transient receptor potential channel V1 (TRPV1) antagonist 6-iodonordihydrocapsaicin and A1 (TRPA1) antagonist HC-030031. Neuroprotective properties of low (1 nM) but not high (10 µM) NADA concentrations were solely blocked by AM251 and were absent in CB(1)(-/-) mice. AM630, O-1918, 6-iodonordihydrocapsaicin and HC-030031 showed no effects at all NADA concentrations applied. Our findings demonstrate that NADA protects dentate gyrus granule cells by acting via CB(1). NADA reduced the number of microglial cells at distinct concentrations. TRPV1 and TRPA1 were not involved in NADA mediated neuroprotection. Thus, our data implicate that NADA mediated activation of neuronal CB(1) may serve as a novel pharmacological target to mitigate symptoms of neuronal damage.

Expression of KIT in the ovary, and the role of somatic precursor cells.

KIT is a type III receptor protein tyrosine kinase, and KITL its cognate ligand. KIT can mediate its effects via several intracellular signalling pathways, or by formation of a cell-cell anchor with its ligand. Through these mechanisms, KIT controls fundamental cellular processes, including migration, proliferation, differentiation and survival. These cellular processes are modulated by soluble KIT, a cleavage product of KIT, generated at the cell membrane. A cell-retained KIT cleavage fragment also arises from this cleavage event. This cleavage fragment must be distinguished from truncated KIT (trKIT), which originates through cryptic promoter usage. The expression of trKIT is highly restricted to postmeiotic germ cells in the testis. In contrast, KIT, together with its cleavage products, is present in somatic cells and germ cells in the gonads of both sexes. A functional KITL/KIT system is mandatory for normal population of the gonads by germ cells. Signalling via the KITL/KIT system promotes the growth, maturation, and survival of germ cells within the gonads, and prevents meiotic entry and progression. In addition to its importance in germ cell biology, the KITL/KIT system is crucial for gonadal stromal differentiation. During foetal life, KIT is expressed by testicular stromal precursor cells, which develop into Leydig cells. In the ovary, stromal cell KIT expression accompanies theca layer development around advanced follicles. After ovulation, KIT-immunopositive cells translocate from the theca layer to the luteal ganulosa where they contribute to a delicate cellular network that extends between the fully luteinised large luteal cells. In the outer regions of the developing corpus luteum, a highly conspicuous subpopulation of KIT/CD14-double-immunopositive cells can be observed. KIT/CD14-double-immunopositive cells are also seen in the haematopoietic-like colonies of long-term granulosa cultures established from late antral follicles. These cultures demonstrate expression of pluripotency marker genes such as octamer binding transcription factor-3/4 and sex determining region Y-box 2. The KIT/CD14-double-immunopositive cells can be purified and enriched by KIT-immunopositive magnetic cell sorting. Subsequent exposure of the KIT-expressing cells to the hanging drop culture method, combined with haematopoietic differentiation medium, provides the signals necessary for their differentiation into endothelial and steroidogenic cells. This suggests that monocyte-derived multipotent cells are involved in ovarian tissue remodelling. In summary, multicelluar KITL/KIT signalling organizes the stroma in the ovary and testis; monocyte-derived multipotent cells may be involved.

The CD34 surface antigen is restricted to glucagon-expressing cells in the early developing bovine pancreas.

Controversy remains regarding the origin of the pancreatic endocrine cells. It is generally accepted that the majority of insulin-secreting cells derive from the endodermal epithelium of the gastrointestinal tract. The aim of this study was to determine the contribution made by a particular cluster of differentiation (CD)-positive cells to the development of the bovine endocrine pancreas. In bovine embryos and foetuses with crown to rump lengths (CRL) ranging from 1 to 47 cm, cells staining positively for CD34 and/or CD133 were always more numerous in the left lobe and body of pancreas than in the right lobe. In the early stages of pancreatic development (CRL <5 cm), CD34 and/or CD133-reactive cells were concentrated within the epithelial cell cords that form the primitive pancreas. In later developmental stages (CRL >5 cm), individual or groups of CD34 and/or CD133-reactive cells were present in newly formed acini, which bulged out from the duct system that had arisen from the cords. Some of the positively stained cells accumulated in focal areas associated with hyperplastic intra-acinar cells. These "acino-insula-like complexes" appeared to enlarge with age and develop into intralobular Islets of Langerhans. Most of the described CD34 and/or CD133-reactive cells displayed co-localisation with glucagon. A negligible number of these cells showed co-localisation with insulin. Glucagon-stained cells were distinct from insulin-stained cells and were more abundant in embryonic and early foetal pancreata. Our data demonstrate that CD34 and/or CD133-reactive cells contribute to the pancreatic alpha cell population during early foetal development in cattle.

Progenitor cells harvested from bovine follicles become endothelial cells.

Hematopoietic-like colonies develop in post-confluent granulosa cell cultures derived from bovine antral follicles. Previously, we had shown that these colonies gave rise to macrophages. In the present study, we validated the presence of somatic KIT-positive (KIT(+)) progenitor cells in colony-containing granulosa cell cultures. The cultures expressed the progenitor cell markers Sox-2, Oct 3/4, KIT, and alkaline phosphatase in western blot analysis. The successful double immunofluorescence localization of KIT and CD14, CD45, CD133, or VEGF-R2 revealed a specific subpopulation of progenitor cells. Flow cytometry showed that cells doubly positive for KIT and CD14 or CD45 comprised less than 10% of the population. The KIT(+) cells were purified by magnetic selection and differentiated with the hanging drop technique using haematopoietic differentiation medium. Pure cultures of either granulosa cells or endothelial cells were obtained. The spindle-shaped and epithelioid phenotypes indicated endothelial cell heterogeneity of microvascular source. We conclude that progenitor cells are obtained from the follicle harvest, which differentiate into endothelial cells. The cells are relevant for findings to angiogenesis and luteinization of the corpus luteum.

Characterization of bovine fetal Leydig cells by KIT expression.

The origin of fetal Leydig cells (FLC) and whether they share a common lineage with adult Leydig cells (ALC) is still under debate, and a marker to reliably track and isolate fetal Leydig precursor cells remains to be identified. We analyzed KIT positive (KIT+) cells in gonads from bovine fetuses with crown-rump-length (CRL) 2.5-85 cm by immunohistochemistry, and found that KIT expression was gender-specific. In female gonads, expression was mainly associated with epithelial cell cords, which extended from the surface epithelium towards the KIT-negative inner stroma. In male gonads of fetuses, after CRL 2.9 cm, KIT expression was strikingly strong in interstitial cells (IC). Only a few KIT+ cells were detected in the epithelial cell cords and in the stromal layer under the surface epithelium after CRL 3.5 cm. In the male fetuses, KIT expression in IC was a continuous and characteristic feature until full term. At all developmental stages KIT+ areas alternated with anti-Müllerian hormone-positive areas. Platelet-derived growth factor receptor alpha production was initiated after the expression of KIT at CRL 4.5 cm. Detection of cytochrome P450 side chain cleavage enzyme and steroidogenic acute regulatory protein in KIT+ IC identified them as FLC. KIT+ cells, isolated from testes by magnetic-activated cell sorting, retained their steroidogenic capacity in vitro. Together, these findings show that KIT+ IC of fetal testis correspond to FLC, which can be successfully cultivated for advanced studies.

Reduction in corpora lutea number in obese melanocortin-4-receptor-deficient mice.

Obese melanocortin-4-receptor-deficient (MC4R-/-) male mice are reported to have erectile dysfunction, while homozygous MC4R-/- female mice are apparently fertile. A recently established obese mouse strain, carrying an inactivating mutation in the MC4R gene, revealed difficulties in breeding for the homozygous female mice. This prompted us to determine the presence of follicles and corpora lutea (CL) in ovaries of MC4R-/- mice aged 3-6 months in comparison to wild type (MC4R+/+) littermates. Serial sections of formaldehyde-fixed ovaries of mice with vaginal signs of estrus and metestrus were assessed for the number of healthy and regressing follicles and CL. The number of CL, as an estimate for the ovulation rate, decreased to zero during aging in MC4R-/- mice. The number of small- (diameter 100-200 micrometer) and large-sized follicles namely antral follicles (diameter >200 micrometer) were slightly increased in MC4R-/- compared to MC4R+/+ mice. Greater differences were found in very large to cystic follicles, which were more numerous in MC4R-/- mice. The number of regressing antral follicles was higher in the MC4R-/- group compared to the MC4R+/+ group. This was associated with a wide range in the number of collapsed zonae pellucidae as the last remnants of regressed follicles. A conspicuous hypertrophy of the interstitial cells was noted in 6-month-old MC4R-/- mice. In conclusion, cystic follicles and the reduction in CL number point to a decreased ovulation rate in obese MC4R-/- mice.

Inflammatory cytokines increase extracellular procathepsin D in permanent and primary endothelial cell cultures.

The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we examined the expression of Cath D in the human endothelial cell line EA.hy 926 and in primary endothelial cells isolated from human umbilical cord veins (HUVEC). After serum-free incubation with or without human interferon-gamma (hIFN-gamma) and/or human tumor necrosis factor-alpha (hTNF-alpha) immature and mature Cath D forms were examined in cell extracts and in cell-conditioned medium concentrates by Western blotting. Lysates of EA.hy 926 cells as well as of HUVEC contained active Cath D as two-chain form, but only negligible amounts of ProCath D and Cath D intermediates. Yet both endothelial cell cultures accumulated ProCath D in their conditioned media in the absence of any stimulus. The treatment with hIFN-gamma and/or hTNF-alpha had little effect on intracellular levels of Cath D, whereas the cytokine stimulation increased the extracellular presence of ProCath D in both endothelial cell cultures. The extracellular increase of ProCath D was not related to induction of apoptosis, as validated by cleaved caspase-3 in cell lysates. Acidification of cytokine-treated media converted ProCath D into Cath D, which was associated with cathepsin-like activity using a fluorogenic substrate-linked assay. We conclude, in vitro, endothelial cells are a cytokine-dependent source for extracellular ProCath D.

The expression of prolactin and its cathepsin D-mediated cleavage in the bovine corpus luteum vary with the estrous cycle.

In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH(2)-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH(2) was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.

The short prolactin receptor predominates in endothelial cells of micro- and macrovascular origin.

BACKGROUND: Controversial reports on prolactin receptors (PRL-R), the long and short form, on endothelial cells (EC) may be explained by the choice of EC derived from the micro- and macrovascular bed of either endocrine and non-endocrine organs. METHODS: We studied here PRL-R expression in organs [bovine corpus luteum (CL), umbilical vein, aorta] and in organ-derived EC cultures. RESULTS: In the intact CL, both PRL-R forms were present at mRNA and protein level throughout the oestrous cycle stages. The short form prevailed as protein. PRL-R-positive EC were noted by immunofluorescent staining in arterial blood vessels of CL septa, in the umbilical vein and the aorta. In EC cultures of micro- and macrovascular origin, transcripts of both PRL-R forms were shown; again the short-form protein prevailed. Blocking experiments with anti-prolactin (PRL) antibody led to a 60% decrease in cell growth. Treatment with PRL had no effect. CONCLUSION: PRL-R expression in micro- and macrovascular EC is associated with the predominant short form.