ABSTRACTS
Distribution and chemical coding of neurons in intramural ganglia of the porcine urinary bladder trigone
Zenon Pidsudko
Department of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury, Olsztyn, Poland
Abstract: This study presents the distribution and chemical coding of neurons in the porcine intramural ganglia of the urinary bladder trigone (IG-UBT) demonstrated using combined retrograde tracing and double-labelling immunohistochemistry. Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of both the left and right side of the bladder trigone during laparotomy performed under pentobarbital anaesthesia. Ten-µm-thick cryostat sections were processed for double-labelling immunofluorescence with antibodies against tyrosine hydroxylase (TH), dopamine b -hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP), Leu5-enkephalin (LENK) and choline acetyltransferase (ChAT). IG-UBT neurons formed characteristic clusters (from a few to tens neuronal cells) found under visceral peritoneum or in the outer muscular layer. Immunohistochemistry revealed four main populations of IG-UBT neurons: SOM- (ca. 35%), SP (ca. 32%), ChAT- and NPY- immunoreactive (-IR) (ca. 23%) as well as non-adrenergic non-cholinergic nerve cells (ca. 6%). This study has demonstrated a relatively large population of differently coded IG-UBT neurons, which constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.
Author's e-mail: zenekp@moskit.uwm.edu.pl
A. Carpino1 , B. Bilińska3, L. Siciliano1, M. Maggiolini2 and V. Rago
Department of 1Cell Biology and 2Pharmaco-Biology, Faculty of Pharmacy, University of Calabria, Italy; 3Endocrinology and Tissue Culture Laboratory, Institute of Zoology, Jagiellonian University, Cracow, Poland
Abstract: A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERa /ERb ). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERb in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERb as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERb , whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERb protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs re vealed a strong immunoreactivity for ERb , and (2) ERb expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERb has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function.
Autor's e-mail: am_carpino@yahoo.it
Mariola Marchlewicz1, Barbara Wiszniewska1 , Rafał Kurzawa2 and Lidia Wenda-Różewicka1
1Department of Histology and Embryology and 2Clinic for Reproduction and Gynecology, Pomeranian Medical University, Szczecin, Poland
Abstract: The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17b -estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.
Author's e-mail: barbwisz@sci.pam.szczecin.pl
Krystyna Kozłowska1 , Mirosława Cichorek1 , Małgorzata Zarzeczna1 and Sławomir Wójcik2
Departments of 1Embryology and 2 Anatomy and Neurobiology, Medical University, Gdańsk, Poland
Abstract: In the present work it was investigated if a spontaneous alteration of the native melanotic transplantable melanoma form into amelanotic form, connected with the tumor progression, is accompanied by changes of CD44 surface glycoprotein expression. We also tried to find out if there exists any correlation between changes in CD44 expression and IL-6, TNF-a , and IL-10 secretion. Cells of two hamster transplantable melanoma lines: melanotic and amelanotic were used. The levels of TNF-a , IL-6, IL-10 in supernatants were determined by the ELISA test. For the detection of CD44 expression by flow cytometry, isolated melanoma cells were stained with the rat anti-mouse CD44 monoclonal antibody. The stained cells were also examined using a fluorescence microscope and a confocal microscopy system. The obtained results indicate that a spontaneous alteration of the native melanotic form into amelanotic form and the associated tumor progression was accompanied by a decrease in CD44 glycoprotein expression on the cell surface and a decrease in IL-6, TNF-a and especially IL-10 secretion by amelanotic melanoma cells. Our observations suggest a relationship between CD44 expression and locally secreted cytokines in the course of transplantable melanoma progression.
Author's e-mail: cecylia@amedec.amg.gda.pl
Anna Gruszka1, Jolanta Kunert-Radek2 and Marek Pawlikowski1
1Department of Experimental Endocrinology and Hormone Diagnostics; 2 Department of Clinical Endocrinology, Institute of Endocrinology, Medical University, Łódź, Poland
Abstract: It is well established that disruption of apoptosis may lead to tumor initiation, progression or metastasis. It is also well documented that many anticancer drugs induce apoptosis. In the earlier studies, the dopamine D2 receptor agonist bromocriptine (BC) and somatostatin analog octreotide (OCT) were found to inhibit the growth of the estrogen-induced rat prolactinoma. Our previous investigations, applying the TUNEL method showed the involvement of the pro-apoptotic effect in the action of BC, and to a lesser degree, in the action of OCT. The aim of the present study was to investigate whether the pro-apoptotic action of these drugs involves the increased expression of Bax – a member of Bcl-2 protein family which is known to play an important role in the regulation of apoptosis. Male four-week Fisher 344 rats were used in the experiment. Capsules containing diethylstilboestrol (DES) were implanted subcutaneously. Six weeks after the implantation the rats were given OCT (2 x 25 m g/animal/24), BC (3 mg/kg b.w./24 h) or OCT and BC at the above doses for 10 days. Bax expression was detected by immunohistochemistry. Prolactin (PRL) in blood serum was measured by radioimmunoassay (RIA). It has been found that both OCT and BC, alone or in combination, significantly reduce the tumor weight. Both OCT and BC suppressed PRL levels, but the inhibitory effect of BC was stronger than that of OCT. It has been found that the treatment with OCT and BC, alone or in combination, causes a significant increase in Bax expression in the rat prolactinoma cells. Our findings indicate that anti-tumoral action of bromocriptine and to some extent the action of octreotide in the experimental rat prolactinoma is connected with the induction of apoptosis and is associated with increased Bax expression.
Author's e-mail: agruszka@mp.pl
Agnieszka Jó źwik1 , Monika Soroczyńska1, Jacek M. Witkowski1 and Ewa Bryl2
1Department of Pathophysiology and 2 Department of Immunopathology, Medical University, Gdańsk, Poland
Abstract: CD3 antigen is a crucial moleculefactor in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3low (217.6), CD3+(217.9) or CD3low/+ (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor – chelerythrine or calcineurin blocker – cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones.
Author's e-mail: ebryl@amedec.amg.gda.pl
Kamila Wojas1, Jacek Tabarkiewicz1 , Małgorzata Jankiewicz2 and Jacek Roliński1
1Department of Clinical Immunology, University School of Medicine and 2Center of Oncology, Lublin, Poland
Abstract: Dendritic cells (DCs) are regarded as the most potent antigen presenting cells that are well suited to activate T cells toward various antigens, such as tumor-associated antigens, due to their costimulatory activity. There is evidence that DCs are of diverse origin, with at least two types of myeloid and lymphoid precursors implicated in their generation. The recent reports demonstrated that the number and function of dendritic cells might change dramatically in cancer patients. In the present study we evaluated the percentage of myeloid and lymphoid DCs in patients with breast cancer, non-small cell lung cancer (NSCLC) and in the healthy donors. The percentage of both DC populations was significantly lower in patients with NSCLC than in the control group. In patients with breast cancer, the number of lymphoid DCs was significantly higher than in NSCLC patients. The obtained results suggest influence of pathological states on host immune system. The decrease in the number of DCs in the peripheral blood from cancer’s patients may be closely correlated with the type of tumour.
Author's e-mail: kamilawojas@poczta.onet.pl
L. Fraser and J. Strzeżek
Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, Warmia and Mazury University, Olsztyn, Poland
Abstract: The comet assay, under neutral conditions, allows the assessment of DNA integrity influenced by sperm ageing, which is manifested in DNA double-strand breaks. Here, we attempted to use a modified neutral comet assay test (single-cell gel electrophoresis), to our knowledge for the first time, to assess DNA integrity of boar spermatozoa during liquid storage for 96 h at 5° C and 16° C. In this comet assay protocol we used 2% b -mercaptoethanol prior to the lysis procedure, to aid in removing nuclear proteins. Ejaculates from 3 boars (designated A, C and G) were diluted with a standard semen extender, Kortowo-3 (K-3), which was supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh) or ostrich egg yolk (LPFo). Irrespective of the extender type, the percentage of comet-detected spermatozoa with damaged DNA increased gradually during prolonged storage at 5° C and 16° C. Spermatozoa stored in K-3 extender exhibited elevated levels of DNA damage at both storage temperatures. Significant differences in DNA damage among the boars were more pronounced during storage in LPF-based extenders at 5° C: spermatozoa of boars A and G were less susceptible to DNA damage. The percent of tail DNA in comets was lower in LPF-based extenders, and there were individual variations among the boars. We observed that changes in DNA integrity were dependent on the extender type and storage temperature. A higher level of DNA instability was observed in K-3 extended semen compared with K-3/LPFh or K-3/LPFo extended semen during storage at 5° C. No significant difference in the level of DNA damage between K-3/LPFh and K-3/LPFo was observed. It seems that a long-term storage can affect genomic integrity of boar spermatozoa. The modified neutral comet assay can be used to detect low levels of DNA damage in boar spermatozoa during liquid preservation. Therefore, screening for sperm DNA damage may be used as an additional test of sperm function that can have diagnostic value in practice.
Author's e-mail: kbz@uwm.edu.pl
Dariusz Stępiński
Department of Cytophysiology, University of Łódź, Poland
Abstract: Ultrastructural and autoradiographic studies of nucleoli in soybean root meristematic cells in seedlings: (1) grown for 3 days at 25oC (control), (2) grown for three days at 25oC and for 4 days at 10oC, and (3) grown as in (2) and recovered for 1 day at 25oC were carried out. Control nucleoli had dense structure and a few small nucleolar vacuoles. Chilled plant nucleoli had less dense structure and no vacuoles. Nucleoli of plants recovered at 25oC had big nucleolar vacuoles. In autoradiograms of squashed preparations, the labeling of nucleoli and cytoplasm after 20-min incubation in 3H-uridine was 5- and 6-fold stronger, respectively, in control than in chilled roots. Following recovery, the labeling of nucleoli and cytoplasm was much stronger than after chilling or even than in control roots. After 80-min postincubation in non-radioactive medium, average labeling of particular areas of cells was the highest in recovered plants which indicated intensification of rRNA synthesis, maturation and transport into cytoplasm resulting from the resumption of optimal conditions which was correlated with the appearance of big nucleolar vacuoles. In autoradiograms of semi-thin sections from roots of seedlings chilled for 4 days then recovered and incubated for 20 min in 3H-uridine, practically only extravacuolar parts of nucleoli were labeled. After 80-min postincubation, the labeling of nucleolar vacuoles was observed. Thus, during postincubation the labeled molecules were translocated from the nucleolar periphery into nucleolar vacuoles in cells where intensive transport of these molecules to the cytoplasm takes place. On the basis of these results, a hypothesis has been put forward that nucleolar vacuoles may be involved in the intensification of pre-ribosome transport outside nucleolus.
Author's e-mail: dareks@biol.uni.lodz.pl