Comparison of PTHrP expression in the epididymis of juvenile and adult European bisons

The aim of the present study was to compare the expression of PTHrP in the epididymes of adult European bisons, and 12- and 5-month-old calves. The highest PTHrP expression was observed in adult animals in muscle cells and endothelium of large vessels, and in muscle cells of the epididymal duct. In one-year-old calves, the reaction was weaker than in adult bulls, being the weakest in 5-month-old calves. However, in small vessels of adult animals, in vascular cells and smooth muscle cells the reaction for PTHrP was considerably weak, being weaker in one-year-old calves, and negative in 5-month-old calves. A similar trace reaction was observed in muscle cells of the epididymal duct in 5- and 12-month-old calves. The present study has revealed that PTHrP expression in vascular and extravascular smooth muscle cells and endothelial cells in European bison is correlated with the animal age and size of the organ.     

Immunolocalization of PTHrP in the European bison and pine vole testis and epididymis

The present study deals with immunohistochemical localization of PTHrP in European bison and pine vole testis and epididymis. PTHrP immunoreactivity was observed in spermatogenic cells of seminiferous tubules in European bison and pine vole testis, with the strongerst reaction occurring in spermatozoa of pine vole testis and epididymal duct. We also observed PTHrP expression in vascular smooth muscle of epididymis and testis in both animal species, as well as slightly weaker reaction in endothelial cells of European bison epididymis. PTHrP was also expressed in the smooth muscle of the epididymal duct in European bison and pine vole. In conclusion, PTHrP is a multifunctional peptide showing both paracrine and autocrine action. Its presence in vascular endothelium and smooth muscle of testis and epididymis is connected with the regulation of vascular muscle tone, thus affecting blood flow in the vessels. PTHrP expression depends on a number of local factors. Moreover, we suppose that PTHrP also contributes to the proliferation and differentiation of spermatogenic cells.     

Immunolocalization of PTHrP in prepubertal and pubertal testis of European bison

The present study deals with immunohistochemical localization of PTHrP in prepubertal and pubertal testis of European bison. PTHrP immunoreactivity was observed in germinal cells in the testis of both prepubertal and pubertal animals. In calves, PTHrP was found in germinal cells, in seminiferous tubules lacking the lumen. The reaction was strong and regularly distributed within the cytoplasm. In adult animals, the reaction showed differentiation in spermatogenic cells. Some cells were strongly and diffusely stained, others exhibited weaker reaction of granular pattern. Sertoli cells and Leydig cells were PTHrP-negative in calves and adult animals.     

Immune responses of bison to ballistic or hand vaccination with Brucella abortus strain RB51

From January through July of 2000, a study was conducted to evaluate clearance, immunologic responses, and potential shedding of Brucella abortus strain RB51 (SRB51) following ballistic or subcutaneous (SQ) vaccination of 7 mo old bison (Bison bison) calves. Ten bison calves were vaccinated SQ with 1.4 x 10(10) colony-forming units (CFU) of SRB51 and five calves were inoculated SQ with sterile 0.15 M sodium chloride. An additional 10 bison calves were ballistically inoculated in the rear leg musculature with 1 x 10(10) CFU of SRB51 and five calves were ballistically inoculated with an empty Biobullet. Serologic responses were monitored at 0, 2, 4, 6, 8, 12, 18, and 24 wk using the standard tube agglutination test and a dot-blot assay. Swabs from rectal, vaginal, nasal, and ocular mucosal surfaces, and blood were obtained for culture from all bison at 2, 4, 6, and 8 weeks post-inoculation to evaluate potential shedding by vaccinated bison or persistent septicemia. The superficial cervical lymph node was biopsied in eight ballistic and eight hand vaccinated bison at 6 or 12 wk to evaluate clearance of the vaccine strain from lymphatic tissues. Lymphocyte proliferative responses to irradiated SRB51 bacteria were evaluated in peripheral blood mononuclear cells (PBMC) at 4, 6, 8, 12, 18, and 24 wk after inoculation. Serum obtained from hand or ballistically vaccinated bison demonstrated antibody responses on the dot-blot assay that were greater than control bison (saline or empty Biobullet) at 2, 4, 6, and 8 wk after vaccination. Antibody titers of ballistically vaccinated bison did not differ (P > 0.05) from hand vaccinated bison at any sampling time. Blood samples obtained from all bison at 2, 4, 6 and 8 wk after vaccination were negative for SRB51. One colony of SRB51 was recovered from the vaginal swab of one ballistically vaccinated bison at 2 wk after vaccination. All other ocular, vaginal, nasal, and rectal swabs were culture negative for SRB51. Strain RB51 was recovered from superficial cervical lymph nodes of hand and ballistic vaccinated bison at 6 (two of four and two of four bison, respectively) and 12 wk (three of four and one of four bison, respectively). Serologic tests and bacterial culture techniques failed to demonstrate infection of nonvaccinated bison. Peripheral blood mononuclear cells obtained from hand vaccinated bison had greater (P < 0.05) proliferative responses to strain RB51 bacteria when compared to PBMC from nonvaccinated and ballistically vaccinated bison. Proliferative responses of PBMC from ballistically vaccinated bison did not differ (P > 0.05) at any sampling time from proliferative responses of PBMC from control bison. Serum alpha 1-acid glycoprotein concentrations, plasma fibrinogen, and total protein concentrations were not influenced by treatments. Ballistic delivery of SRB51 did not induce adverse effects or influence clearance of the vaccine strain. There were no proliferative responses of PBMC to SRB51 in bison ballistically vaccinated with SRB51; whereas bison inoculated with SRB51 by hand injection had greater proliferative responses than control or ballistically vaccinated bison. Our study suggests that ballistic delivery may require a greater dose of SRB51 to induce cell-mediated immune responses in bison that are comparable to those induced by hand injection, and that ballistic or hand delivery of 1 x 10(10) CFU of SRB51 is safe in bison calves.     

The vasculature of the rat embryo: an electron microscope study

The ultrastructure of portions of the arterial and venous systems of the 11.5 day old Wistar rat embryos has been studied by scanning and transmission electron microscopy. The vessels at this stage of development are in the form of capillaries, and the arterial and venous types can be distinguished by the morphology of the endothelial cells by SEM. The endothelial cells of the arterial vessels gave prominent nuclear bulges and numerous microvilli apart from their spindle shape, whilst those of the veins appear flattened, are polygonal in shape, and have few microvilli. Transmission electron microscopy shows that the endothelial cells of the arteries and veins are identical in structure. The ultrastructure of these cells resembles that of endothelial cells at later stages of development including the adult type in that mature forms of cytoplasmic organelles are obtained. In studies on the intercellular junctions and fenestrations with lanthanum nitrate, the impression is formed that the vessels at this stage are impermeable to small molecular size particles, compared with adult capillaries. This suggests that cytoplasmic vesicles must play a major role in the transport of macromolecules in the 11.5 day embryonic vessels.     

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.     

DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries and neural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1-5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis by in situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.     

Contraception of bison by GnRH vaccine: a possible means of decreasing transmission of brucellosis in bison

Preventing pregnancy in brucellosis-infected bison (Bison bison) provides a potential means of preventing transmission of disease. To determine whether a gonadotropin-releasing hormone (GnRH) vaccine was effective in reducing pregnancy in bison and to study the safety of injecting GnRH in pregnant bison, a study was conducted at the Idaho Fish and Game Wildlife Health Laboratory in Caldwell, Idaho (USA). Four pregnant and two nonpregnant female bison were given a single injection of GnRH vaccine, and five pregnant adult females were given a sham injection that contained only adjuvant. Three of the GnRH-vaccinated bison that were pregnant at the time of vaccination delivered healthy calves. One treated bison had dystocia that resulted in a dead calf. All control bison delivered healthy calves. After calving, females of both groups were exposed to two bulls. Treated bison were palpated 6 wk after exposure to the bulls, and blood was drawn for pregnancy-specific protein B analysis. The six treated bison were not pregnant. The sham-treated bison became pregnant and delivered viable calves. This study demonstrates that a single dose of GnRH vaccine is effective in preventing pregnancy in female bison for at least 1 yr.     

Electron-microscopic immunolabelling of vasoactive substances in human umbilical endothelial cells and their actions in early and late pregnancy

Human umbilical vessels are devoid of nerves and therefore endothelial cells may play an important role in the control of feto-placental blood flow. The pharmacological effects of 5-hydroxytryptamine, histamine and endothelin were examined in umbilical arteries and veins from legal terminations (gestational age 8–17 weeks, n=12) and normal term vaginal deliveries (gestational age 38–41, n=12). Immunocytochemistry of human unbilical vessels indicated that 5-hydroxytryptamine, histamine and endothelin were localised in subpopulations of endothelial cells of both artery and vein in late, but not early, pregnancy. 5-Hydroxytryptamine (10 nM–30 μM) caused sustained concentration-dependent contractions in all vessels from early and late pregnancy. Histamine (0.1 μM–30 mM) also caused sustained contractions in all vessels from late pregnancy but only 27% of arteries and 41% of veins from early pregnancy responded. Endothelin (10 pM–30 nM) caused slow long-lasting contractions in all vessels from early and late pregnancy. Atrial natriuretic peptide and neuropeptide Y did not alter vascular tone. The endothelium may thus play an autocrine/paracrine role, by synthesizing and releasing the above reactive substances in late pregnancy to influence feto-placental blood flow. Received: 23 May 1995 / Accepted: 13 October 1995     

Influence of environmental factors on the deep and skin temperature in the European bison,Bison bonasus (L.)

• 1.1. In 43 European bison divided into three groups (Group A, 3–8-month-old calves; Group B, 18-month-7-year-old young bison; Group C, 12–24-year-old bison) the rectal, humerus region and abdomen region temperatures were measured.
• 2.2. The experiments were carried out in winter months, from mid-December to mid-March.
• 3.3. The mean rectal temperatures changed from 38.55°C in calves to 38.15°C in the oldest bison.
• 4.4. The mean temperatures of the humerus region changed from 20.69°C in calves to 21.49°C in older bison.
• 5.5. The mean temperatures of the abdomen region changed from 20.79°C in calves to 22.17°C in older bison (Gr. B).
• 6.6. The cluster analysis divided the bison into four groups named hot, warm, cool and cold bison.
• 7.7. Only air temperature measured 2 m above the ground and snow cover influenced the integrated bison temperature. Age, sex and mass as well as some environmental factors had no influence.
• 8.8. Measurements made 1 to nearly 4hr after a bison's death showed a drop in rectal temperature and mostly increases in temperatures of the humerus and abdomen regions.

     

Differential expression of VEGFA, TIE2, and ANG2 but not ADAMTS1 in rat mesenteric microvascular arteries and veins

Microvessels respond to metabolic stimuli (e.g. pO(2)) and hemodynamic forces (e.g. shear stress and wall stress) with structural adaptations including angiogenesis, remodeling and pruning. These responses could be mediated by differential gene expression in endothelial and smooth muscle cells. Therefore, rat mesenteric arteries and veins were excised by microsurgery, and mRNA expression of four angioadaptation-related genes was quantified by real time duplex RT-PCR in equal amounts of total RNA, correlated to two different house keeping genes (beta-actin, GAPDH). The results show higher expression of VEGFA, TIE2, and ANG2 in arteries than in veins, but equal expression of ADAMTS1. Higher availability of VEGFA mRNA in endothelial cells of arteries shown here could contribute to the maintenance of mechanically stressed blood vessels and counteract pressure-induced vasoconstriction.     

Stem cell antigen-1 expression in the pulmonary vascular endothelium

Although the function of the cell surface protein stem cell antigen-1 (Sca-1) has not been identified, expression of this molecule is a characteristic of bone marrow-derived hematopoietic stem cell populations. Expression of Sca-1, however, is not restricted to hematopoietic tissue. By RT-PCR and Western analysis, we found that Sca-1 is expressed in the adult mouse lung. Sca-1 immunohistochemistry revealed a linear staining pattern on the endothelial surface of large and small pulmonary arteries and veins and alveolar capillaries. Expression of Sca-1 in the pulmonary endothelium was confirmed by dual fluorescent microscopy on lung sections and by fluorescence-activated cell sorting analysis of digested lung tissue; each of these methods showed colocalization with the endothelial marker platelet/endothelial cell adhesion molecule-1. In the kidney, Sca-1 expression was also noted in large vessels, but, in contrast to the lung, was not observed in capillaries. Overall, our data indicate that Sca-1 expression helps define the surface phenotype of endothelial cells throughout the pulmonary vasculature.     

Vascular endothelial expression of indoleamine 2,3-dioxygenase 1 forms a positive gradient towards the feto-maternal interface

We describe the distribution of indoleamine 2,3-dioxygenase 1 (IDO1) in vascular endothelium of human first-trimester and term placenta. Expression of IDO1 protein on the fetal side of the interface extended from almost exclusively sub-trophoblastic capillaries in first-trimester placenta to a nearly general presence on villous vascular endothelia at term, including also most bigger vessels such as villous arteries and veins of stem villi and vessels of the chorionic plate. Umbilical cord vessels were generally negative for IDO1 protein. In the fetal part of the placenta positivity for IDO1 was restricted to vascular endothelium, which did not co-express HLA-DR. This finding paralleled detectability of IDO1 mRNA in first trimester and term tissue and a high increase in the kynurenine to tryptophan ratio in chorionic villous tissue from first trimester to term placenta. Endothelial cells isolated from the chorionic plate of term placenta expressed IDO1 mRNA in contrast to endothelial cells originating from human umbilical vein, iliac vein or aorta. In first trimester decidua we found endothelium of arteries rather than veins expressing IDO1, which was complementory to expression of HLA-DR. An estimation of IDO activity on the basis of the ratio of kynurenine and tryptophan in blood taken from vessels of the chorionic plate of term placenta indicated far higher values than those found in the peripheral blood of adults. Thus, a gradient of vascular endothelial IDO1 expression is present at both sides of the feto-maternal interface.     

Production of prostacyclin, 6-keto-PGF1α and thromboxane B2 by incubated samples of umbilical vessels: A comparison of methods for expressing the reults on dry weight, wet weight, protein or DNA bases

The production of PG12 (determined by abioassay), and of 6-keto-PGF1α and TXB2 (determined by radioummunoassay) by samples of human umbilical vessels have been measured. The results have been calculated on four bases: dry weigt, wet weight, protein and DNA.There was a higher production of PG12 and 6-keto-PGF1α by umbilical veins than by umbilical arteries; no significant difference in TXB2 production was observed between umbilical veins and arteries. The ratio of 6-keto-PGF1α: TXB2 production was about 100 for the samples of veins and about a40 for the samples of arteries.The best methods of expressing the results were on the bases of protein and DNA, the latter basis being marginally the best. The least satisfactory method for expressing the results was that based on dry weight.The physiological and practical implications of the results are discussed.     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo

Remodeling of the primary vascular system of the embryo into arteries and veins has long been thought to depend largely on the influence of hemodynamic forces. This view was recently challenged by the discovery of several molecules specifically expressed by arterial or venous endothelial cells. We here analysed the expression of neuropilin-1 and TIE2, two transmembrane receptors known to play a role in vascular development. In birds, neuropilin-1 was expressed by arterial endothelium and wall cells, but absent from veins. TIE2 was strongly expressed in embryonic veins, but only weakly transcribed in most arteries. To examine whether endothelial cells are committed to an arterial or venous fate once they express these specific receptors, we constructed quail-chick chimeras. The dorsal aorta, carotid artery and the cardinal and jugular veins were isolated together with the vessel wall from quail embryos between embryonic day 2 to 15 and grafted into the coelom of chick hosts. Until embryonic day 7, all grafts yielded endothelial cells that colonized both host arteries and veins. After embryonic day 7, endothelial plasticity was progressively lost and from embryonic day 11 grafts of arteries yielded endothelial cells that colonized only chick arteries and rarely reached the host veins, while grafts of jugular veins colonized mainly host veins. When isolated from the vessel wall, quail aortic endothelial cells from embryonic day 11 embryos were able to colonize both host arteries and veins. Our results show that despite the expression of arterial or venous markers the endothelium remains plastic with regard to arterial-venous differentiation until late in embryonic development and point to a role for the vessel wall in endothelial plasticity and vessel identity.     

Distribution of immune cells and expression of interleukin receptors in ileal Peyer’s patches of calves

Newborn calves lack a mature immune system. The immune system develops with age, but the role of the expression of cytokine receptors in the development of immune cells of Peyer’s patches (PPs) in the intestines of calves in the first 2 months has not yet been elucidated. In this study, the distribution of immune cells and the expression of interleukin (IL) receptors (R) in the ileal PPs of newborn and 2-month-old calves were investigated immunohistochemically with monoclonal antibodies against bovine CD4, CD8, IgM, γδTCR, T19, WC3, WC5, and WC6 antigens. The expression of ILRs was examined with antibodies against CD25 (IL-2Rα), IL-2Rγ, IL-4R, IL-6R, IL-10R, and IL-13R antigens. CD4⁺, CD8⁺, γδTCR⁺, T19⁺, and WC6⁺ cells were found to be more widely distributed in the ileal PPs of 2-month-old calves than in those of newborn calves. Moreover, the expression of CD25 (IL-2Rα), IL-4R, and IL-13R in the ileal PPs of 2-month-old calves was more prominent than that in newborn calves. These data suggest that the immune system of calves at 2 months of age is developed by reactions to foreign antigens and aging.     

Absence of Anaplasma marginale infection in American Bison raised in an anaplasmosis endemic area.

Blood was collected at slaughter from 132 adult American bison (Bison bison) raised in an anaplasmosis endemic area where the vector Dermacentor andersoni (equals venustus) is indigenous. Hematologic studies revealed no indication of clinical anaplasmosis. Card agglutination and complement-fixation tests on all bison serums were negative. Eleven anaplasmosis-susceptible calves each inoculated with 204 ml of blood pooled from 12 bison did not develop anaplasmosis. Results of this study indicate American bison have resistance to natural A. marginale infection.     

reg6 is required for branching morphogenesis during blood vessel regeneration in zebrafish caudal fins

Postnatal neovascularization is essential for wound healing, cancer progression, and many other physiological functions. However, its genetic mechanism is largely unknown. In this report, we study neovascularization in regenerating adult zebrafish fins using transgenic fish that express EGFP in blood vessel endothelial cells. We first describe the morphogenesis of regenerating vessels in wild-type animals and then the phenotypic analysis of a genetic mutation that disrupts blood vessel regeneration. In wild-type zebrafish caudal fins, amputated blood vessels heal their ends by 24 h postamputation (hpa) and then reconnect arteries and veins via anastomosis, to resume blood flow at wound sites by 48 hpa. The truncated vessels regenerate by first growing excess vessels to form unstructured plexuses, resembling the primary capillary plexuses formed during embryonic vasculogenesis. Interestingly, this mode of vessel growth switches by 8 days postamputation (dpa) to growth without a plexus intermediate. During blood vessel regeneration, vessel remodeling begins during early plexus formation and continues until the original vasculature pattern is reestablished at approximately 35 dpa. Temperature-sensitive mutants for reg6 have profound defects in blood vessel regeneration. At the restrictive temperature, reg6 regenerating blood vessels first fail to make reconnections between severed arteries and veins, and then form enlarged vascular sinuses rather than branched vascular plexuses. Reciprocal temperature-shift experiments show that reg6 function is required throughout plexus formation, but not during later growth. Our results suggest that the reg6 mutation causes defects in branch formation and/or angiogenic sprouting.     

Production of prostacyclin, 6-keto-PGF1 alpha and thromboxane B2 by incubated samples of umbilical vessels: a comparison of methods for expressing the results on dry weight, wet weight, protein or DNA bases

The production of PGI2 (determined by bioassay), and of 6-keto-PGF1 alpha and TXB2 (determined by radioimmunoassay) by samples of human umbilical vessels have been measured. The results have been calculated on four bases: dry weight, wet weight, protein and DNA. There was a higher production of PGI2 and 6-keto-PGF1 alpha by umbilical veins than by umbilical arteries; no significant difference in TXB2 production was observed between umbilical veins and arteries. The ratio of 6-keto-PGF1 alpha: TXB2 production was about 100 for the samples of veins and about 40 for the samples of arteries. The best methods of expressing the results were on the bases of protein and DNA, the latter basis being marginally the best. The least satisfactory method for expressing the results was that based on dry weight. The physiological and practical implications of the results are discussed.