Zygmunt Giżejewski

Divergent evolution in the cytoplasmic domains of PRLR and GHR genes in Artiodactyla

BackgroundProlactin receptor (PRLR) and growth hormone receptor (GHR) belong to the large superfamily of class 1 cytokine receptors. Both of them have been identified as candidate genes affecting key quantitative traits, like growth and reproduction in livestock. We have previously studied the molecular anatomy of the cytoplasmic domain of GHR in different cattle breeds and artiodactyl species. In this study we have analysed the corresponding cytoplasmic signalling region of PRLR.ResultsWe sequenced PRLR gene exon 10, coding for the major part of the cytoplasmic domain, from cattle, American bison, European bison, yak, sheep, pig and wild boar individuals. We found different patterns of variation in the two receptors within and between ruminants and pigs. Pigs and bison species have no variation within GHR exon 10, but show high haplotype diversity for the PRLR exon 10. In cattle, PRLR shows lower diversity than GHR. The Bovinae PRLR haplotype network fits better the known phylogenetic relationships between the species than that of the GHR, where differences within cattle breeds are larger than between the different species in the subfamily. By comparison with the wild boar haplotypes, a high number of subsequent nonsynonymous substitutions seem to have accumulated in the pig PRLR exon 10 after domestication.ConclusionBoth genes affect a multitude of traits that have been targets of selection after domestication. The genes seem to have responded differently to different selection pressures imposed by human artificial selection. The results suggest possible effects of selective sweeps in GHR before domestication in the pig lineage or species divergence in the Bison lineage. The PRLR results may be explained by strong directional selection in pigs or functional switching.

Age‐dependent pattern of connexin43 expression in testes of European bison (Bison bonasus, L.)

Morphology and the expression of connexin43 (Cx43) was investigated in testes of immature, prepubertal, and adult European bison bulls by means of routine histology, immunohistochemistry, and Western blotting, respectively. Testes were collected from culled animals living in Bialowieza and Borecka Forests, Poland. Histological examination of testicular tissue of immature and prepubertal males revealed normal structure, whereas of adult individuals either normal testicular structure with advanced spermatogenesis or varying degrees of tubule and interstitial tissue abnormality were seen. Immunohistochemical studies revealed Cx43 signal mostly at Leydig cell membrane appositions. In testes of immature males heterogeneous staining was observed; its intensity markedly increased in prepubertal males reaching almost two times more intense staining in adults. Strong Cx43 signal between Leydig cells was also observed in testes with structural alterations; however, sporadically it was of weak linear pattern. In the tubules of the latter, the intensity of Cx43 staining was weak to moderate or it was even absent. Immunohistochemical qualitative analysis was confirmed by quantitative image analysis in which the staining intensity was expressed as relative optical density of diaminobenzidine deposits. Data from Western blot analyses confirmed the results obtained by immunohistochemistry; immunodetectable Cx43 protein as a band of 43 kDa was detected in all testes samples. Overall, the increase in Cx43 level in testes along the bison postnatal development may be capable of a better exchange of metabolites and coordinating Leydig cell activity during maturation. A relationship between homozygosity occurring in European bison and altered intercellular communication is suggested.

Immunoexpression of Aromatase in Immature and Adult Males of the European Bison (Bison bonasus, Linnaeus 1758)

Based on recent literature dealing with the role of oestrogens in the male gonad, attempts were undertaken to reveal the site of aromatization within the testis of the European bison (Bison bonasus). Testes were collected from culled animals living in free-ranging populations in Bialowieza Forest, Poland (nine males aged 8 months to 10 years). Moreover, to check for any alterations in the expression of testicular aromatase between American bison (Bison bison) and European bison, testes from one adult 10-year-old individual were also chosen for this study. For immunohistochemistry, 4% formaldehyde fixative was used. Both qualitative and quantitative evaluations of immunohistochemical staining were performed. Leydig cells, Sertoli cells and germ cells exhibited a positive immunoreaction for aromatase in testes of immature and sexually mature bison. A marked increase in aromatase expression was observed in three adult European individuals with impaired spermatogenesis. Consistent with recent data and those of our own, it might be suggested that the strong expression of aromatase negatively affects spermatogenic function in bison testes and may serve as a possible explanation of specific sperm defects observed in European bison bulls. On the contrary, one cannot exclude that differences in the aromatase immunoexpression levels are attributed to the homozygosity, the cause of frequent disease in European bison.

Determination of perfluorinated sulfonate and perfluorinated acids in tissues of free-living European beaver (castor fiber L.) by d-SPE/ micro-UHPLC-MS/MS

Perfluorooctanesulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the main representatives of an rising class of persistent organic pollutants (POPs), perfluorochemicals (PFCs). In this study, determination of selected PFCs concentration in liver, brain, tail, adipose and peritoneum tissues of free-living European beaver (Castor fiber L.) was addressed. Tissue samples, collected from beavers living in Masurian Lakeland (NE Poland), were analyzed by dispersive Solid Phase Extraction (d-SPE) with micro-UHPLC-MS/MS system. In a group of ten selected pefrluorinated compounds only two perfluorinated acids (PFOA and PFNA) and one perfluorinated sulfonate (PFOS) were quantified. PFOA was detected in all analysed tissue samples in both female and male beavers in a range from 0.55 to 0.98ngg(-1) ww whereas PFOS was identified in all analyzed female beaver tissues and only in liver, subcutaneous adipose and peritoneum tissues of male beavers at the concentration level from 0.86 to 5.08ngg(-1) ww. PFNA was only identified in female beaver tissues (liver, subcutaneous adipose and peritoneum) in a range from 1.50 to 6.61ngg(-1) ww. This study demonstrated the bioaccumulation of PFCs in tissue samples collected from beavers living in area known as green lungs of Poland. The results provided in this study indicate for the increasing risk of PFCs occurrence in the environment and the level of PFCs in tissue of free-living European beavers may serve as bioindicator of environmental pollution by these compounds.

Potential role of beavers (Castor fiber) in contamination of water in the Masurian Lake District (north-eastern Poland) with protozoan parasites Cryptosporidium spp. and Giardia duodenalis

Abstract The purpose of this study was to assess the possible influence of beavers on the contamination of lake water with zoonotic parasites Giardia duodenalis and Cryptosporidium spp., with respect to the risk to human health. A total of 79 water samples were taken around the habitats of beavers from 14 localities situated in the recreational Masurian Lake District (north-eastern Poland). Water was sampled in the spring and autumn seasons, at different distances from beavers’ lodges (0-2, 10, 30, and 50 m). The samples were examined for the presence of (oo)cysts of zoonotic protozoa Giardia duodenalis and Cryptosporidium spp. by direct fluorescence assay (DFA) and by nested and real time PCR. By DFA, the presence of Giardia cysts was found in 36 samples (45.6%) and the presence of Cryptosporidium oocysts in 26 samples (32.9%). Numbers of Giardia cysts, Cryptosporidium oocysts, and summarised (oo)cysts of both parasites showed a significant variation depending on locality. The numbers of Giardia cysts significantly decreased with the distance from beavers’ lodges while the numbers of Cryptosporidium oocysts did not show such dependence. The amount of Giardia cysts in samples collected in spring was approximately 3 times higher than in autumn. Conversely, a larger number of Cryptosporidium oocysts were detected in samples collected in autumn than in spring. By PCR, Giardia DNA was found in 38 samples (48.1%) whereas DNA of Cryptosporidium was found in only 7 samples (8.9%). Eleven Giardia isolates were subjected to phylogenetic analysis by restriction fragment length polymorphism PCR or sequencing which evidenced their belonging to zoonotic assemblages: A (3 isolates) and B (8 isolates). In conclusion, water in the vicinity of beavers’ lodges in the tested region was markedly contaminated with (oo)cysts of Giardia duodenalis and Cryptosporidium spp., which confirms the potential role of beavers as a reservoir of these parasites and indicates a need for implementation of appropriate preventive measures to protect tourists’ health.

Persistent Müllerian duct syndrome (PMDS) in the Polish free-ranged bull populations of the European bison (Bison bonasus L.)

This study describes the diversity of vestigial male uteri of the European bison (Eb) examined for: (1) morphology, (2) glycoprotein localization, (3) total protein and glycoprotein profiles, (4) steroid concentrations, and (5) PMDS based on the mutation of AMH and AMHR2 genes. Uteri of adult bulls (5-12 years old) were compared to a uterus of a juvenile female (6 months old). Male uterine proteins were analyzed in parallel to secretory endometrial proteins of pseudo-pregnant pig (PsEND) and BSA used as profile-controls. Hematoxylin/eosin-staining revealed the diversity of male uterine morphology, including lumen size/shape, endometrial (END) gland density, luminal knob-like epithelial structures and multiple intrauterine cells proliferating within the lumen. PAS-staining revealed the presence of glycoproteins restricted to luminal epithelial cells and END glands. Heterologous total protein PAGE-profiles (20-66kDa) revealed two dominant fractions (66 and 45kDa), similar to PAS-profiles (67 and 47kDa) in male and female uterine tissues. In male uterine tissues, androstendione and progesterone, but not testosterone, estrone or estradiol concentrations were lower than in the female. Sequencing of AMH- and AMHR2-like amplicons allowed identification of these gene mutations in Eb. Our results provide novel data regarding PMDS, demonstrating the diversity of uterine morphology, glycoprotein mass/profile, steroid concentration and AMH/AMHR2 mutations in Eb bulls.

Light and Electron Microscopy of the European Beaver (Castor fiber) Stomach Reveal Unique Morphological Features with Possible General Biological Significance

Anatomical, histological, and ultrastructural studies of the European beaver stomach revealed several unique morphological features. The prominent attribute of its gross morphology was the cardiogastric gland (CGG), located near the oesophageal entrance. Light microscopy showed that the CGG was formed by invaginations of the mucosa into the submucosa, which contained densely packed proper gastric glands comprised primarily of parietal and chief cells. Mucous neck cells represented <0.1% of cells in the CGG gastric glands and 22–32% of cells in the proper gastric glands of the mucosa lining the stomach lumen. These data suggest that chief cells in the CGG develop from undifferentiated cells that migrate through the gastric gland neck rather than from mucous neck cells. Classical chief cell formation (i.e., arising from mucous neck cells) occurred in the mucosa lining the stomach lumen, however. The muscularis around the CGG consisted primarily of skeletal muscle tissue. The cardiac region was rudimentary while the fundus/corpus and pyloric regions were equally developed. Another unusual feature of the beaver stomach was the presence of specific mucus with a thickness up to 950 µm (in frozen, unfixed sections) that coated the mucosa. Our observations suggest that the formation of this mucus is complex and includes the secretory granule accumulation in the cytoplasm of pit cells, the granule aggregation inside cells, and the incorporation of degenerating cells into the mucus.

Endoparasites of the European beaver (Castor fiber L. 1758) in north-eastern Poland

Abstract Parasitological examination after necropsies of 48 European beavers from Podlaskie and Warmisko-Mazurskie provinces were performed between April 2011 and November 2012. All helminthes were isolated from the contents of the gastro-intestinal tract and their species were determined. In addition, blood samples and faeces were examined. All beavers were infected with six species of parasites. Stichorchis subtriqetrus trematodes were found in 93.7% of animals. They were localized mainly in the caecum, less in the colon, and single juvenile parasites were found in the small intestine. The intensity of infection ranged from two to 893 parasites. Travassosius rufus nematodes (10-4336 specimens) were present in the stomach of 68.7% of the beavers. In the small intestine of four (8.3%) beavers, two-six specimens of Psilotrema castoris were found. This is the first record of this species in Poland and the third of its discovery in the world. Furthermore, in the small intestine of one beaver, two Trichostrongylus capricola nematodes were detected. In the liver of one beaver, pathological changes caused by hydatid cestode Echinococus granulosus occurred. Inflammatory changes of the gastric mucosa caused by Travassosius rufus and of caecum caused by Stichorchis subtriquertus, were observed. Coproscopy was performed with the use of Baermann, flotation, and decantation methods. All results of Baermann method were negative. Examinations with flotation and decantation methods confirmed necropsy findings. Using the flotation method, single oocysts of Eimeria sprehni in one beaver were detected. A blood test conducted by Kingston and Morton method did not reveal the presence of protozoa or microfilariae.

Variations of selected trace element contents in two layers of red deer antlers

Abstract Introduction: Hard antlers of deer are unique bioindicators of environmental metal pollutions, but sampling methods presented in the literature are inconsistent. Due to the specific growth pattern of antlers and their histological structure, sampling methods described in the literature were reviewed, the suitability of using mixed samples of both antler layers as element bioindicators was assessed, and the codified method of antler sampling used for bioindication was described. Material and Methods: Lead, cadmium, mercury, arsenic, copper, zinc, and iron in trabecular and cortical parts of hard antlers of red deer (Cervus elaphus) were determined using different methods of atomic absorption spectrometry (depending on the element). Results: Mean mercury content in trabecular bone (0.010 ±0.018 mg/kg) was 5 times higher than in cortical bone (0.002 ±0.003 mg/kg). Mean iron concentration was approximately 15 times higher in trabecular (239.83 ±130.15 mg/kg) than in cortical bone (16.17 ±16.44 mg/kg). Concentrations of other analysed elements did not differ statistically between antler layers. Conclusion: In mixed antler samples, concentrations of mercury and iron depend on the particular antler layer contents. This therefore warrants caution when comparing results across studies and specification of the sampling methodology of antlers is highly recommended.

Co-expression pattern of dopamine beta-hydroxylase (DβH) and neuropeptide Y (NPY) within sympathetic innervation of ovary and umbilical cord of the European bison (Bison bonasus L.).

Co-expression of dopamine β-hydroxylase (DβH) and neuropeptide Y (NPY) has never been examined in ovary (OV) and umbilical cord (UC) of the European bison (Eb), the endangered wild species. The OV and UC samples were harvested from seasonally eliminated Eb females (45-120 days post coitum). Frozen histological sections were examined by double fluorescent immunohistochemistry (dF-IHC), using the primary mouse anti-DβH monoclonals and rabbit anti-NPY polyclonals and then the immunocomplexes were visualized with FITC and CY3 fluorophores, respectively. Numerous DβH immunoreactive nerve fibers (DβH-IRs) and a little less frequent NPY-IRs were found in the bundle-like structures, innervating mainly perivascular regions of the OV. The NPY-IRs constantly co-expressed DβH, while some DβH-IRs did not express NPY. This specific pattern of innervation was observed both in the stromal and cortical regions of the OV. The simultaneous co-expression of DβH and NPY were also detected in the UC, in which specific single or bundle-like structures ran along the smooth muscles of blood vessels. The spatial-specific co-expression of DβH and NPY in OV and UC, may suggest that these markers are involved in the control of vascularization that regulates nourishing blood circulation required for proper pregnancy maintenance and efficient embryo/fetus development in the Eb.