J-P. Dadoune: New insights into male gametogenesis: what about the spermatogonial stem cell niche? pp. 141-147
A. Minarowska, Ł. Minarowski, A. Karwowska: Regulatory role of cathepsin D in programmed cell death pp. 159-163
New insights into male gametogenesis:
what about the spermatogonial stem cell niche?
Jean-Pierre Dadoune
Service d'Histologie, Biologie de la Reproduction et Cytogénétique, Paris, France
Abstract: The spermatogonial stem cells (SSCs) are at the foundation of spermatogenesis. They can self-renew and generate
a large number of differentiated germ cells. A balance between SSC renewal and differentiation in the adult testis is
essential to maintain normal spermatogenesis and fertility. These two processes are tightly regulated by intrinsic gene
expression in the stem cells and extrinsic signals, including soluble factors and adhesion molecules from the surrounding
microenvironment, known as the SSC niche. Few factors which are only found in germ cells are recognized as indispensable
to SSC maintenance or differentiation. Plzf, a transcriptional repressor protein, and TAF-4b, a germ cell specific component
of the RNA polymerase complex, are considered to be essential for SSC renewal, whereas the transcription factors
Sohlh1 and 2 appear to be crucial for spermatogonial differentiation. By providing glial cell-line-derived neurotrophic factor
(GDNF) and stem cell factor (SCF) for SSCs and segregating them, the Sertoli cell is one of the major contributors to
stem cell niche regulation Another Sertoli cell product, ERM, a transcription factor which is exclusively expressed in mature
Sertoli cells, has been shown to be required for SSC self-renewal and maintenance of spermatogenesis in adult mice. It has
been hypothesized that SSC niche regulation changes with age. SSC self-renewal could be controlled by GDNF during the
perinatal period of development while it is dependent on ERM, during the pubertal period.
Full text
DNA methylation in states of cell physiology and pathology
Anetta Sulewska1, Wieslawa Niklinska2, Miroslaw Kozlowski1, Lukasz Minarowski3
Wojciech Naumnik4, Jacek Niklinski1, Katarzyna Dabrowska3 and Lech Chyczewski3
Departments of: 1Thoracic Surgery, 2Histology and Embryology, 3Clinical Molecular Biology,
4Pneumonology and Tuberculosis, Medical University of Bialystok, Poland
Abstract: DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined
during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of
methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide) while CpG islands located in gene
promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting.
The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis,
may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the
methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA.
Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called
"non-CpG" methylation). The knowledge of a normal methylation process and its aberrations appeared to be useful while
searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique
(using primers for methylated and unmethylated sequences) five new genes which are potential biomarkers of lung cancer
have been presented.
Regulatory role of cathepsin D in apoptosis
Alina Minarowska1, Łukasz Miarowski2, Alicja Karwowska3, Marek Gacko4
13rd Department of Children's Diseases, Medical University of Bialystok, Poland
2Department of Clinical Molecular Biology, Medical University of Bialystok, Poland
3Department of Instrumental Analysis, Medical University of Bialystok, Poland
4Department of Vassal Surgery, Medical University of Bialystok, Poland
Abstract: Cathepsin D (CTSD, EC 3.4.23.5) is well known aspartyl protease. Among different role in cell physiology, a
new function of this enzyme is examined. Cathepsin D is an important regulator of apoptotic pathways in cells. It acts at
different stage of intrinsic and extrinsic pathway of apoptosis. Cathepsin D can either induce apoptosis in presence of cytotoxic
factors, but in certain studies an inhibitory role in apoptosis was also reviewed. Detailed review of involvement of
cathepsin D in cell apoptosis is a purpose of this paper.
The activity of cathepsin D in saliva of cystic fibrosis
patients
Alina Minarowska1, Lukasz Minarowski2, Alicja Karwowska3, Dorota Sands4,
Ewa Dabrowska5
13rd Department of Children's Diseases, Medical University of Bialystok, Bialystok Poland
Departments of: 2Clinical Molecular Biology, 3Instrumental Analysis and 5Social and Preventive
Stomatology Medical University of Bialystok, Bialystok, Poland
4Department of Pediatrics, Mother and Child Institute, Warsaw, Poland
Abstract: Cystic fibrosis (CF) is genetically determined illness, which is caused by the mutation in the CFTR gene. CFTR
protein is also expressed in epithelial cells of parotid glands, therefore parotid glands are also affected in CF patients.
Cathepsin D is one of the proteolitic cascade enzymes. Physiological wearing out result in occurrence of trace quantities of
this enzyme in serum and body fluids, including saliva. Among different enzymes, saliva contains cathepsin D (CTSD, EC
3.4.23.5). The aim of this study was to determine cathepsin D activity in mixed saliva in cystic fibrosis patients and healthy
controls. The study was performed in a group of 26 CF patients (10F, 16M). The results obtained in CF group was compared
with the results of thirty healthy subjects (12F, 14M). From each subject 8 ml of mixed saliva was obtained: before and after
the stimulation of saliva excretion using paraffin pledgets. Protein and glycoprotein content was assessed using Winzler's
method. Protein concentration in controls and CF group before stimulation of excretion was 1.15±0.714 mg/mL and
1.54±0.925 mg/mL. After stimulation protein concentration in saliva has lowered to 0.88±0.77 mg/mL in CF group and
1.24±1.213 mg/mL in controls. Glycoprotein concentration in controls and in CF group was respectively: before stimulation
1.08±0.271 mg/mL and 1.05±0.344 mg/mL; after stimulation 0.92±0.292 mg/mL and 0.86±0.283 mg/mL. The activity
of CTSD in controls was 45.9±24.98 Tyr nmol/mL/4h before stimulation and 109.3±56.94 Tyr nmol/mL/4h after stimulation
of excretion. In CF group CTSD activity before stimulation was 134.5±81.80 Tyr nmol/mL/4h and after stimulation
134.4±62.18 Tyr nmol/mL/4h. Comparing the CTSD activity in both groups statistically significant difference has been
revealed in samples collected before stimulation of excretion (p=0.013). The activity of cathepsin D in saliva of cystic fibrosis
patient is significantly higher than in healthy controls before the stimulation of excretion with paraffin pledgets.
Epstein-Barr virus (EBV) infection in B-cell
non-Hodgkin's lymphomas in children: virus latency
and its correlation with CD21 and CD23 molecules
Aldona Kasprzak1, Rafal Spachacz1, Jacek Wachowiak2, Katarzyna Stefanska2,
Maciej Zabel1,3
1Department of Histology and Embryology, University of Medical Sciences, Poznan;
2Department of Haematology and Paediatric Oncology, University of Medical Sciences, Poznan;
3Department of Histology and Embryology, University of Medical Sciences, Wroc3aw, Poland
Abstract: Epstein Barr virus (EBV) infection of human B lymphocytes in vitro results in immortalisation of the cells and
augmented membranous expression of numerous B-cell activation molecules, including CD23. Other studies demonstrated
that only those B lymphocytes which carry the surface CD21 (EBV receptor) become transformation-competent. Inspired
by the relatively unclear relations between expression of EBV and those of CD21 and CD23 in in vivo conditions we have
decided to define correlations between tissue markers of EBV and of CD21 and CD23 molecules in B-cell non-Hodgkin's
lymphomas (NHLs) in children. The studies were performed on an archival tissue material originating from children with
B-cell NHLs (n=26) using immunocytochemical techniques, in situ hybridisation, and PCR. Our studies confirmed the latent
phase of EBV infection in all of the EBV-positive patients. Viral proteins as well as viral RNAs (EBERs) was found both
in the cytoplasm, in cell nuclei and in cell membranes of mainly the transformed lymphocytes B. Expression of the latent
proteins (EBNA2 and LMP1) and that of EBERs in B-cell NHLs was significantly higher as compared to children with nonneoplastic
lesions. The studies demonstrated reciprocally positive correlations between expressions of CD21 and CD23 in
our children, but no correlation could be demonstrated between expression of EBV tissue markers and that of CD21 and/or
CD23. Positive correlation was confirmed between expression of EBNA2 and LMP1 as well as between expression of the
two proteins and EBERs in B-cell NHLs. Our studies have shown mainly latency III pattern of EBV. We have also demonstrated
a novel form of EBV latency with no EBERs expression. The high detectability of EBV-positive cases both in the
group of B-cell NHLs (77%), and in the group with non-neoplastic lesions (64%) suggested that only more pronounced tissue
expression of EBV markers in B-cell NHLs as compared to the non-neoplastic material may point to a potential role of
EBV in pathogenesis of lymphoma in this group of population in our country.
Bronchial macrophages in asthmatics reveal decreased
CD16 expression and substantial levels of receptors
for IL-10, but not IL-4 and IL-7
Marcin Moniuszko1, Anna Bodzenta-Lukaszyk1, Krzysztof Kowal1
and Milena Dabrowska2
1Department of Allergology and Internal Medicine and 2Department Hematological Diagnostics,
Medical University of Bialystok, Bialystok, Poland
Abstract: The role of different subpopulations of bronchial macrophages (BMs) in asthma pathogenesis has not yet been
completely elucidated. In addition, little is known about potential in vivo responsiveness of BMs to pro- and anti-inflammatory
cytokines present in the bronchial milieu. We aimed to characterize asthmatic patients' BM subpopulations delineated
by common markers of macrophage/monocyte cells, CD16 and CD14, and subsequently to analyze cytokine receptor
expression on those subsets. Subjects included eighteen patients with moderate asthma (six steroid-naive and twelve steroidtreated)
and ten healthy control subjects. Flow cytometry was used to analyze phenotypical features of BMs including
expression of receptors for IL-10, IL-4 and IL-7. Exhaled nitric oxide analysis and induced sputum eosinophil counts were
used to assess airway inflammation. BMs from both steroid-naive and steroid-treated asthmatic patients showed significantly
decreased expression of CD16, as compared to healthy subjects' BMs. CD16, but not CD14, expression inversely correlated
with exhaled nitric oxide levels and sputum eosinophilia. Short-term administration of inhaled cortiocosteroids (ICS)
in steroid-naive asthmatic patients led to significant reduction of CD16 expression and enhancement of CD14 expression.
Next, we analyzed the expression of receptors for IL-10, IL-4 and IL-7 on the surface of BM subpopulations characterized
by different levels of CD14 and CD16 expression. We observed substantial levels of IL-10R on the surface of BMs collected
from asthmatic and healthy subjects. Interestingly, IL-10R was found mostly on those macrophages that co-expressed
CD14. In contrast, independently on co-expression of CD14, the levels of IL-4R and IL-7R on BMs were low in both asthmatic
and healthy subjects. The results suggest that different BM subsets may be differentially involved in regulating the
inflammatory response in allergic asthma.
Serum soluble Fas ligand (sFasL) in patients with
primary squamous cell carcinoma of the esophagus
Miroslaw Kozlowski1, Oksana Kowalczuk2, Anetta Sulewska1, Piotr Dziegielewski1,
Grzegorz Lapuc1, Wojciech Laudanski1, Wieslawa Niklinska3, Lech Chyczewski2,
Jacek Niklinski1 and Jerzy Laudanski1
Departments of: 1Thoracic Surgery, 2Clinical Molecular Biology, 3Histology and Embryology,
Medical University of Bialystok, Bialystok, Poland
Abstract: Esophageal carcinomas have been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host
immune surveillance. Circulating soluble FasL (sFasL) has been suggested to provide protection from Fas-mediated apoptosis.
The aim of this study was to assess serum sFasL levels in esophageal cancer. The pretreatment levels of sFasL in the
serum of 100 patients with esophageal squamous cell cancer and 41 healthy volunteers were determined by ELISA. Probability
of survival was calculated according to the method of Kaplan-Meier. The prognostic influence of high and low level
of sFasL was analyzed with the log-rank test. The mean serum level of sFasL in patients with esophageal cancer was significantly
higher than that in healthy donors (1.567±1.786 vs 0.261±0.435, p<0.0001). The levels of serum sFasL were significantly
higher in advanced stages (II vs IV p<0.034; III vs IV p<0.041; except II vs III p=0.281), patients with lymph
node (N0 vs N1 p<0.0389) or distant (M0 vs. M1 p<0.0388) metastases and significantly lower in patients with well differentiated
tumors (G1 vs G2 p<0.0272). The serum levels of soluble FasL were not related to gender, age, tumor size, Tstage,
tobacco smoking and history of chronic alcohol intake. The survival difference between pretreatment high and low
level of sFasL in surgery and chemio- and/or radiotherapy group was not statistically significant (p=0.525; p=0.840). Our
results indicate that elevated serum sFasL levels might be associated with a disease progression in patients with esophageal
squamous cell carcinoma.
Analysis of expression of MHC class I molecules
and TAP genes in malignant human cell lines
Mariusz Kaczmarek1, Magdalena Frydrychowicz1, Blazej Rubis2,5,
Ewa Mizera-Nyczak1,4, Edyta Nieruchalska3, Jan Sikora1, Elzbieta Kaczmarek3,
Jan Zeromski1
1Chair of Clinical Immunology, 2Chair and Department of Biochemistry and Molecular Biology,
3Laboratory of Morphometry and Medical Image Processing, Chair and Department of Clinical
Patomorphology, University of Medical Sciences, Poznan, Poland
4Department of Tumor Pathology, Wielkopolska Oncology Center, Poznan, Poland
5Department of Clinical Chemistry, University of Medical Sciences, Poznan, Poland
Abstract: TAP proteins (transporters associated with antigen processing) take part in the transport of oligopeptides created
in proteasomes from cytoplasm into endoplasmic reticulum. In the endoplasmic reticulum those oligopeptides are bound to
MHC class I molecules and transported to the cell surface. TAP proteins consist of two subunits: TAP1 and TAP2. It has
been previously shown that TAP protein expression can be decreased in malignant cells, followed by reduced protein expression
or complete lack of MHC class I antigens on the cell surface. The aim of the study was to characterize of MHC class
I protein expression and TAP mRNA synthesis in twenty human malignant tumor cell lines. MHC class I protein expression
was examined by immunohistochemistry and flow cytometry. Expression of TAP genes was studied using RT-PCR and realtime
PCR. All tested cell lines expressed MHC class I molecules. Flow cytometry showed different expression of MHC class
I protein in tested cell lines. Molecular analysis revealed the presence of TAP1 and TAP2 gene transcripts in all cell lines
examined. Quantitative real time PCR analysis showed differences of gene expression among cell lines tested.
The evaluation of vacA gene alleles frequency
in Helicobacter pylori strains in children and adults
in Podlaskie region
Elzbieta Maciorkowska1, Izabela Roszko1, Oksana Kowalczuk3, Maciej Kaczmarski2,
Lech Chyczewski3 and Andrzej Kemona4
Departments of: 1Pediatric Nursing, 2Clinical Molecular Biology, 3General Pathomorphology
and 43rd Department of Children's Diseases, Medical University of Bialystok, Bialystok, Poland
Abstract: The frequency of Helicobacter pylori infection in population can depend on the organism resistance, genetic
condition, and bacterial strains virulence. A vacA gene, of mosaic structure, which encodes vacuolating cytotoxin is one
of the known genes of H. pylori. The existence of several different genotypes of s and m regions enables the formation
of numerous combinations of vacA gene genome. The studies on vacA genotype revealed that the frequency of occurrence
of H. pylori containing s1 or s2, as well as m1 and m2 alleles varies in different parts of the world. The aim of the
studies performed in the group of children and adults was to evaluate the prevalence of particular vacA gene alleles distribution
in the population of the Podlasie province. The allele s1, which occurred in 84.3% of the examined group
(86.8% in children and 81.3% in adults), turned out to be the most frequently observed of the signal encoding region.
Statistically significant differences in s1 and s2 alleles distribution in relation to a dwelling place were not detected. The
allele m2 (42.1% in children and 59% in adults) was the allele of midregion, most frequently occurring in our studies.
The allele m2 was observed more often in H. pylori strains in the inhabitants from the urban areas (data statistically significant).
Morphological aspect of voice disturbances
of aged persons coexisting hypopharynx cancer
Bozena Kosztyla-Hojna1, Anna Andrzejewska2, Ryszard Rutkowski3
and Marek Rogowski1
Departments of: 1Otolaryngology, 2Clinical Pathomorphology, and 3Respiratory Diagnostics
and Bronchofiberoscopy, Medical University of Bialystok, Poland
Abstract: The voice quality in prebysphonia is conditioned by morphological changes in the vocal folds mucosa. The
studies including light microscopy and transmission electron microscopy (TEM) revealed changes within the basal membrane
epithelium and the stroma of the vocal folds mucosa. Age-related changes in thickness of the epithelium and direction
of the basal membrane, increased number of collagenous fibres (C) and fibroblasts and chronic inflammatory process
in the stroma were found. Vacuolated and keratinised epithelial cells, enlarged extracellular spaces and numerous blood
vessels confirm the edematous form of prebysphonia. Thinned epithelium with signs of hyalinization, inflammatory infiltrations
in the stroma with numerous collagenous fibres and small number of blood vessels indicate atrophy of the vocal
folds mucosa. Edematous and atrophic changes in the vocal folds mucosa are most frequently reported form of prebysphonia.
Macrophage-specific RAM11 monoclonal antibody cross-reacts with basal cells of stratified squamous epithelia
Grzegorz J. Lis1, Jan A. Litwin1, Alicja Furgal-Borzych1, Joanna Zarzecka2,
Tadeusz Cichocki1
1Department of Histology, Jagiellonian University Medical College, Krakow, Poland
2Department of Conservative Dentistry and Endodontics, Institute of Stomatology,
Jagiellonian University Medical College, Krakow, Poland
Abstract: RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular
(atheromatous tissue) macrophages. This study demonstrates a cross-reaction of RAM11 with an unknown antigen in rabbit
normal epithelial cells. Formalin-fixed, paraffin sections of the New Zealand White rabbit normal skin, oral mucosa,
esophagus, small intestine and lung were immunostained with RAM11 antibody followed by goat anti-mouse Cy-3-conjugated
antiglobulin. RAM11-positive immunofluorescence was observed in basal layer cells of stratified squamous epithelia
(skin, oral mucosa, esophagus). No RAM11 immunostaining was found in any cells of simple (intestinal, bronchial) epithelia.
These findings show that basal cells of stratified squamous keratinized and non-keratinized epithelia of the rabbit express
an antigenic epitope which is common with that of macrophage antigen recognized by RAM11 monoclonal antibody.
Expression of E-cadherin, ß-catenin and Ki-67 antigen
and their reciprocal relationships in mammary
adenocarcinomas in bitches
Marcin Nowak1, Janusz A. Madej1, Piotr Dziegiel2
1Chair of Pathomorphology, Pathophysiology, Microbiology and Forensic Veterinary Science, Faculty
of Veterinary Medicine, Wroc3aw University of Environmental and Life Sciences, Wroc3aw, Poland
2Chair and Department of Histology and Embryology, University School of Medicine, Wroc3aw, Poland
Abstract: In progression of tumours, resulting from, i.e., release of cells from the parental tumour and development of
metastases, expression of cell adhesion molecules (CAM) plays a significant role. CAM, including E-cadherin and the
linked to it ß-catenin, determine the extent of adhesion between normal and neoplastically altered cells. Moreover, the
unbound form of ß-catenin in a cell nucleus may affect the rate of cell proliferation This study aimed at demonstrating intensity
and localisation of E-cadherin and ß-catenin expression as related to expression of the proliferation-associated antigen,
Ki-67 in mammary adenocarcinomas of bitches. The study was performed on 35 cases of the above mentioned tumours. On
paraffin sections immunohistochemical reactions were performed using monoclonal antibodies directed against E-cadherin,
ß-catenin and Ki-67 antigen. In the studies a membranous expression of E-cadherin, a cytoplasmic-nuclear expression of ß-
catenin and nuclear expression of Ki-67 antigen were demonstrated. Statistical calculations using Spearman's test demonstrated
a pronounced positive correlation between expression of ß-catenin and Ki-67 antigen and absence of correlation
between expression of E-cadherin and Ki-67 antigen. No correlation could be detected between expression intensities of Ecadherin
and ß-catenin.
Comparison of bipyridyl, maltol and kojic acid action
as organic vanadium ligands on activity
of galactosyltransferase (EC 2.4.1.38),
some physiological parameters and ultrastructure
of Golgi complexes in rat hepatocytes
Wojciech Dabros1 and Anna M. Kordowiak2
Departments of: 1Clinical and Experimental Pathomorphology, 2General Biochemistry,
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
Abstract: The biochemical activity and morphology of control and streptozotocin-diabetic rat liver Golgi complexes were
previously investigated by us under influence of some vanadium [V(IV)] compounds. The effectiveness of these derivatives
depends on the kind of complexing ligands. This paper presents the investigation of the effect of bipyridyl, the ligand of a
new vanadium compound, tested by us with maltol and kojic acid (two ligands studied by the present and other authors).
The three ligands alone action was tested under the same experimental conditions as in the case of whole compounds with
vanadium and applied to liver Golgi complexes of control rats. A preliminary study for maltol and kojic acid had been previously
carried out by us parallel with tests of whole vanadium complexes, but valuable differences in biological action
found in our condition of experiments suggested the extension of studies to include the two above-mentioned ligands and
to compare the effects of the three investigated ligands. The supplementary part of the experiment focused mainly on the
ultrastructure of Golgi complexes in hepatocytes. Four groups of animals were used: C - control rats, C + M (maltol), C +
(ka)2 (kojic acid) and C + (bpy)2 (bipyridyl). The control rats received 0.09M NaCl as drinking liquid; all the other animals
were given 3.6 mmol/L of appropriate ligand solution in 0.09M NaCl during 7 days. All the animals survived the experiments.
Only in group C + (bpy)2 did the authors observe statistically significant differences as compared with the controls
(group C). The differences were detected in physiological studies and manifested as body weight decreased by approximately
20% during the experiment, lower liquid (p<0.001) and food (p<0.01) intake and increase of free blood sugar level
(p<0.01). The yield of Golgi membrane isolation decreased in this group (p<0.01). The main investigated biochemical
parameter, i.e. the activity of liver Golgi marker enzyme - galactosyltransferase - was not statistically significantly changed
in comparison with the controls in all the investigated groups of rats; a similar dispersion of individual results were found
in the four groups. In the three experimental groups, ultrastructural observations demonstrated a predominance of cylindrical
Golgi structures, which were haphazardly twisted in the majority of cases. Typically shaped structures were encountered
sporadically. The ligands alone evoked numerous subcellular changes in hepatocytes; these alterations most frequently
involved the mitochondria and endoplasmic reticulum. No such changes had been seen, or else they had been less advanced
when complex vanadium compounds were employed in our earlier experiments. As it follows, the ligands alone were
demonstrated to be much more toxic to morphology of control liver Golgi apparatus as compared to complex compounds,
which showed the ability of the former to normalize Golgi complexes of diabetic animals.
The comparison of multipotential for differentiation
of progenitor mesenchymal-like stem cells obtained
from livers of young and old rats
Maciej Tarnowski1, Halina Koryciak-Komarska1, Piotr Czekaj2, Ryszard Sebesta1,
Tomasz M. Czekaj1, Ksymena Urbanek1, Wirginia Likus1,
Izabela Malinowska-Kolodziej3, Danuta Plewka2, Grazyna Nowaczyk-Dura2,
Ryszard Wiaderkiewicz2, Aleksander L. Sieron1
1Department of General and Molecular Biology and Genetics, Medical University of Silesia, Katowice,
Poland, CoE for Research and Teaching of Molecular Biology of Matrix and Nanotechnology, Network
CoE BioMedTech Silesia,
2Department of Histology; Medical University of Silesia, Katowice, Poland
3Department of Physiology, Medical University of Silesia, Center of Excellence for Research
and Teaching on Molecular Biology of Matrix and Nanotechnology, Katowice, Poland
Abstract: The presence of stem cells differentiating to hepatocytes and cholangiocytes has been previously reported in livers
of young rats. Here, we have isolated, cultured, and characterized mesenchymal stem cells (MSCs) from livers of young
and old rats and tested their multipotential for differentiation. The mesenchymal stem cells in liver sections were identified
by the presence of markers, respectively for primary stem cells Thy-1 and CD34, for differentiation to early cholangiocytes
GST and CK19, and for differentiation to hepatocytes GST? and CK18. Ki67 was detected as the cell proliferation marker.
Cells isolated from livers of either age group were tested in a culture for their viability following storage and were characterized
for the presence of most of the markers detected in cells in situ. The results revealed age-dependent changes in the
number of recovered primary MSCs. In both age groups we have observed cells changing under differentiating conditions
to liver cell lineages, such as cholangiocytes and hepatocytes, as well as to non-liver cells such as adipocytes, astrocytes,
neuroblasts, and osteoblasts. Our data revealed that from the livers of rats 20 months and older the primary MSCs could be
isolated and expanded; however, they were significantly fewer, even though their differentiation multipotential was preserved.
The mechanism involved in the differentiation of liver MSCs seemed to depend on a constellation of signals in Notch
signalling pathways. Thus, our results support the idea of potential use of liver as a source of MSCs, not only for liver reconstruction
but also for cell therapy in general.
Rat epididymal epithelial cells and 17beta-estradiol
synthesis under hCG stimulation in vitro
Malgorzata Swider-Al-Amawi, Mariola Marchlewicz, Agnieszka Kolasa,
Lidia Wenda-Rozewicka and Barbara Wiszniewska
Department of Histology and Embryology, Pomeranian Medical University, Szczecin, Poland
Abstract: Epithelial cells of human and animal epididymis display features of steroidogenic cells. Rat epididymal epithelial
cells in vitro produce androgens which are converted to 17?-estradiol, and released into the medium. The regulation of
the epididymal steroidogenesis is not fully understood but it could be expected that it remains under LH influence. In previous
study we observed that the morphology of rat epididymal epithelial cells in vitro was affected by hCG and the increase
of amount of lipid droplets, glycogen and PAS-positive substances was observed. The present studies show the organelles
which take part in synthesis of steroids in rat epididymal epithelial cells in vitro and the effect of hCG on E2 synthesis. The
cells were cultured in the medium with/without DHT and without DHT in supplementation with hCG. After hCG stimulation
the amount of an active mitochondria were increased when compared to the amount of mitochondria in the epididymal
epithelial cells cultured without DHT. Ultrastructure of the cells was similar to the cells cultured with DHT, while the cytoplasm
of the cells cultured without DHT was disorganized. The synthesis of 17?-estradiol was stimulated by hCG, that
exerted its effect through LH/hCG receptors, localized in the epididymal epithelial cells.
Effect of anabolics on bovine granulosa-luteal cell
primary cultures
Paola Pregel, Enrico Bollo, Francesca Tiziana Cannizzo, Antonella Rampazzo,
Simonetta Appino and Bartolomeo Biolatti
University of Turin, Dipartimento di Patologia Animale, Grugliasco (TO), Italy
Abstract: Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The
use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to
detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells.
Cultures were treated with different concentrations of substances illegally used in cattle (17?-estradiol, clenbuterol and
boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine
the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while
PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations.
The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated
cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17?-estradiol treated cells PCNA
expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could
have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.