Dirofilaria immitis and Wolbachia-derived antigens: its effect on endothelial mammal cells

Antigens of both Dirofilaria immitis and Wolbachia symbiont bacteria are implicated in the inflammatory pathology of heartworm infection. The aim of the present study was to compare the stimulatory capacity of in vitro cultures of vascular endothelial cells by the adult somatic antigens of D. immitis (DiSA) and the recombinant form of the Wolbachia surface protein (rWSP), during the first 24h of stimulation. Our results indicate a different stimulatory activity of the two antigens. Both the DiSA and rWSP stimulate the production of the enzymes responsible of the arachidonic acid metabolism, cyclooxygenase-2, 5-lipoxygenase (5-LO), and leukotriene B4. Only DiSA stimulates the production of prostaglandin E2. Related to the adhesion molecules, the DiSA stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas rWSP stimulates ICAM-1, PECAM-1, and vascular cell adhesion molecule-1 (VCAM-1). Expression of E-cadherin and vascular endothelial growth factor also were stimulated by rWSP. Neither of the two antigens altered the basic physiological mechanisms of endothelial cells, such as cell proliferation, cell cycle, or apoptosis. The biological and pathological significance of these finding are discussed.     

Changes in expression and localization of GPRC5B and RARalpha in the placenta and yolk sac during middle to late gestation in mice

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.     

Morphological aspects of prenatal haematopoietic development in the cat

The appearance of haematopoietic cells and the development of haematopoiesis in certain embryonic organs of the cat were studied using light microscopic (cytological smears and paraffin embedded tissues) and transmission electron microscopic methods. Primitive erythropoiesis occurred predominantly in the yolk sac whereas definitive erythropoiesis occurred in the yolk sac, liver, bone marrow and, to a lesser extent, in the spleen. In the yolk sac, erythropoiesis was predominantly intravascular whereas in the liver and bone marrow it was usually extravascular. Granulopoiesis occurred mainly in the liver and bone marrow. In the liver it was predominantly extravascular and occurred around vessels, bile ducts and in perisinusoidal spaces. Megakaryocytopoiesis occurred in the yolk sac, liver, bone marrow and spleen. The megakaryocytic line of cells were similar to those occurring in adult cats except that in the yolk sac unusual small, mononuclear to binuclear thrombocytogenic megakaryocytes were present also. The relative contributions of the embryonic organs of the cat to haematopoiesis were similar to those described for man and certain other mammals but the results for the sites of development and the appearance of early forms in the erythrocytic, granulocytic and megakaryocytic lines varied at times from those reported for man and other mammals.     

Cell adhesion molecules, leukocyte trafficking, and strategies to reduce leukocyte infiltration

Leukocyte-endothelial cell interactions are mediated by various cell adhesion molecules. These interactions are important for leukocyte extravasation and trafficking in all domestic animal species. An initial slowing of leukocytes on the vascular endothelium is mediated by selectins. This event is followed by (1) activation of beta2 integrins after leukocyte exposure to cytokines and pro-inflammatory mediators, (2) adherence of leukocyte beta2 integrins to vascular endothelial ligands (eg, intercellular adhesion molecule-1 [ICAM-1]), (3) extravasation of leukocytes into tissues through tight junctions of endothelial cells mediated by platelet and endothelial cell adhesion molecule-1 (PECAM-1), and (4) perivascular migration through the extracellular matrix via beta1 integrins. Inhibiting excessive leukocyte egress and subsequent free radical-mediated damage caused by leukocyte components may attenuate or eliminate tissue damage. Several methods have been used to modify leukocyte infiltration in various animal models. These methods include nonspecific inhibition of pro-inflammatory mediators and adhesion molecules by nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, inhibition of cytokines and cytokine receptors, and inhibition of specific types of cell adhesion molecules, with inhibitors such as peptides and antibodies to beta2 integrins, and inhibitors of selectins, ICAMs, and vascular cell adhesion molecule-1 (VCAM-1). By understanding the cellular and molecular events in leukocyte-endothelial cell interactions, therapeutic strategies are being developed in several animal models and diseases in domestic animal species. Such therapies may have clinical benefit in the future to overcome tissue damage induced by excessive leukocyte infiltration.     

In situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in calves with acute Pasteurella haemolytica pneumonia.

The in situ expression of intercellular adhesion molecule-1 (ICAM-1) mRNA in normal and pneumonic lung tissues of Holstein calves with bovine leukocyte adhesion deficiency (BLAD) was compared with that of age-matched non-BLAD Holstein calves by in situ hybridization. Twenty-four Holstein calves (both BLAD and non-BLAD) were randomly assigned to one of two experimental groups and inoculated intrabronchially with Pasteurella haemolytica or pyrogen-free saline. Lung tissues were collected and fixed in 10% neutral formalin at 2 or 4 hours postinoculation (PI). The expression and distribution of ICAM-1 mRNA in the different cell types of the lung tissue was detected by in situ hybridization with a 307-base-pair bovine ICAM-1 riboprobe. In lungs of both non-BLAD and BLAD saline-inoculated calves, ICAM-1 expression was present in epithelial cells but occurred in <30% of cells in bronchi, bronchioles, and alveoli. ICAM-1 expression in vascular endothelial cells was present in <30% of cells in pulmonary arteries and veins. The expression of ICAM-1 was significantly greater (>60% of cells) in bronchiolar and alveolar epithelial cells and pulmonary endothelial cells of arteries and veins in both BLAD and non-BLAD calves inoculated with P. haemolytica. Bronchiolar epithelium had the highest intensity of mRNA expression and highest percentage of cells that were stained, whereas bronchial epithelium had the lowest intensity and percentage of cells stained. Most alveolar macrophages and neutrophils in infected lungs also expressed ICAM-1. ICAM-1 expression was generally increased in infected BLAD calves at 2 hours PI as compared with non-BLAD calves but not at 4 hours PI. The increased expression of ICAM-1 during acute P. haemolytica pneumonia in calves suggests that ICAM-1 is upregulated and may play a role in leukocyte infiltration. The extent of ICAM-1 expression in P. haemolytica-inoculated calves with BLAD was initially enhanced but otherwise similar to that in non-BLAD calves.     

Immunohistochemical characterization of macrophage and dendritic cell subpopulations of the spleen,thymus, tongue and heart in cyclophosphamide-induced immunosuppressed rat

This study was undertaken to investigate the immunohistochemical characterization of different subpopulations of macrophages and dendritic cells (DCs) of the spleen, thymus, tongue and heart in cyclophosphamide (CY)-induced immunosuppressed rat. After CY treatment, remarkably, ED1+, ED2+ and ED3+ macrophage subpopulations, in general exhibited signs of cellular activation such as an increase in number and size of cell, and an upregulation of the ED1, ED2 and ED3 reactive surface molecule expression in all the organs studied, except for some macrophage subpopulations including ED1+ macrophages in the non-lymphoid tissues. Subpopulations of DCs showed a differential sensitivity to CY. Lymphoid DCs were more sensitive to CY than non-lymphoid interstitial DCs. CY induced a conspicuous upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in the vascular endothelial cells, splenic marginal zone and thymic cortex. In this study, we demonstrated the in vivo effects of CY treatment on subpopulations of macrophages and DCs as well as on ICAM-1 expression in the rat spleen, thymus, tongue and heart. Moreover, our results shed more light on the activation effects of CY on certain subpopulations of macrophages, on the differential sensitivity of DCs to CY between the immature and mature ones, on the functional role of different subpopulations of macrophages, and on the significance of upregulated ICAM-1 expression in the splenic marginal zone and thymic cortex after CY treatment.     

Comparative evaluation of the effect of two maternal diets on fatty acids, vitamin E and carotenoids in the chick embryo.

1. The fatty acid profile of egg yolk and vitamin E and carotenoid accumulation in the egg yolk and embryonic tissues were investigated in relation to the maternal diet. 2. Two hundred fertile eggs (Ross 308 Broiler Breeder), obtained from hens fed on a maize-based (M-diet) or a wheat-based diet (W-diet), were incubated using standard conditions. 3. The egg yolk and embryo tissues (residual yolk, yolk sac membrane, liver, kidney, lung, muscles, adipose tissue and plasma) were collected on d 18 of incubation and on d 21 (newly-hatched chicks) and analysed for fatty acids, vitamin E and carotenoids. 4. The diets did not differ in terms of fatty acid or alpha-tocopherol concentrations. The concentration of carotenoids in the M-diet was 11.8 mg/kg and in the W-diet was 5.6 mg/kg with lutein and zeaxanthin being major carotenoids. 5. Eggs from the M-group contained higher (P<0.01) concentrations of beta+gamma-tocopherols, total carotenoids, lutein and zeaxanthin. Chickens hatched from those eggs were characterised by the increased concentrations of total carotenoids and zeaxanthin in all the tissues studied. The concentration of beta+gamma-tocopherol was enhanced only in the liver and yolk sac membrane. 6. It is concluded that the maternal diet plays an important role in antioxidant systems formation during chick embryonic development; the M-diet can increase the antioxidant potential of the egg yolk and embryonic tissues compared to the antioxidant potential provided by parent birds fed the W-diet.     

Yolk sac, body dimensions and hatchling quality of ducklings, chicks and poults

1. One-day-old chicks of Pekin duck, turkey, layer fowl and broiler fowl were examined for bacteria in the yolk sac and yolk fluid. 2. Whole hatchling, yolk-free hatchling and yolk sac weights were recorded for all species along with crown-rump length and beak-tip to toe-tip length. 3. Bacteriology revealed positive results for the whole yolk sacs of 43 to 64% of the birds in the sample of ducklings, poults and layer chicks. Broiler chicks had a 6.6% incidence of bacteria isolated from the whole yolk sac. By contrast, there were very few positive results from swabs of yolk fluid for any of the bird types. 4. The presence of bacteria in the yolk sac of hatchlings suggests that there is colonisation, rather than infection, of the yolk sac membrane during the hatching period or the first few hours post-hatching. Isolation of bacteria from the yolk sacs of young chicks might no longer be considered as solely indicative of yolk sac infection but further research is required to confirm this result. 5. Contrary to what is being suggested in commercial practice relationships between linear dimensions and hatchling weight suggest that measurement of chick length is at best a very crude measure of chick quality.     

Growth-promoting effects of different fractions of extra-embryonic coelomic fluid on embryonic development

In the early stages of embryonic development, many growth-promoting molecules must be provided by the maternal system. These factors may be supplied locally to the embryo, by the decidua, the placenta, or the yolk sac. In this study the growth-promoting potential of extra-embryonic coelomic fluid (EECF) and its fractions was investigated. The embryonic requirement of growth-promoting molecules may be studied by reducing the growth-supporting capacity of serum. Thus, ultrafiltration of rat serum was carried out for 8 h using Millipore filters with a molecular weight exclusion of 30 kDa. Rat embryos at 9.5 days of age were cultured for 8 days for anembryonic yolk sacs, and then EECF was collected and divided into three different molecular weight fractions by ultrafiltration. Rat embryos were cultured for 48 h in whole rat serum and the serum retenate (which has low growth-supporting capacity) in the presence and absence of EECF, its fractions, or in EECF only. Embryos grown in retenate showed severe growth retardation, and the addition of EECF significantly improved embryonic growth. The fraction which contained the molecules with molecular weight between 10 and 30 kDa had significantly more effect on embryonic development than the other fractions. This fraction of EECF was analysed by gel electrophoresis. Three of the four protein bands observed in this fraction were identified by amino-terminal sequencing as alpha-fetoprotein precursor (22 kDa), apolipoprotein A1 precursor (24 kDa) and fetal haemoglobin Y2 chain (14 kDa), none of which are likely to be responsible for the growth-promoting activity. To further investigate growth-promoting proteins, EECF was Western-blotted to nitrocellulose membranes and probed with antisera against rat prolactin, epidermal growth factor, insulin-like growth factors I and II and human placental lactogen. No immunoreactive bands were detected in the EECF, suggesting that either these proteins are not present or are present at levels too low to be detected. Although the growth-promoting effect of the EECF was demonstrated in this study, the molecules responsible remain uncharacterized.     

Evaluation of an early granulocytic response of chick embryos inoculated with herpesvirus of turkeys

1. Early granulocytic response was evaluated in chick embryos inoculated with herpesvirus of turkeys (HVT). 2. Fifty 10-d-old specific pathogen-free embryos were divided into two groups, inoculated via yolk sac. Group 1 were inoculated with a complete dose of HVT and group 2 with vaccinediluent only. 3. Samples were taken for histological evaluation of yolk sac, liver, chorioallantoic membrane, brain and heart from 5 embryos per group on days 12, 14, 16, 18 and 20 of embryonic life. 4. Increases in numbers of granulocytes were detected on days 14 and 16 in the yolk sac, and on d 14 and 20 in the liver of in embryos, which received HVT. In addition, the chorioallantoic membrane was infiltrated with granulocytes. 5. The results confirm that granulopoiesis in the yolk sac is stimulated in the early stages of incubation if a viral antigen is present. The virus also appears to trigger the presence of granulocytes in embryonic liver and chorioallantoic membranes.     

Highly metastatic ovarian yolk sac carcinoma in a rat

We investigated a highly metastatic ovarian yolk sac carcinoma in a 52-week-old female Crl:CD(SD) rat. Macroscopically, the present case had severe ascites, bilateral ovarian masses and numerous nodules in the abdominal and thoracic cavities. Histopathologically, these masses and nodules were generally composed of two types of cells mimicking a parietal and visceral yolk sac. The parietal cells were round to polygonal, contained eosinophilic droplets and were arranged in nests and cords in the eosinophilic matrix. Both the intracytoplasmic droplets and the matrix were stained positively with PAS. The visceral cells were cylindriform, and proliferated in papillary and tubular patterns and occasionally formed Shiller-Duval body-like structures. In the dissemination sites, the neoplastic cells proliferated on the surface of the various tissues and often infiltrated into deeper parts of the tissues. Immunohistochemically, both neoplastic cells were positive for α-fetoprotein and keratin, and the eosinophilic matrix was positive for laminin. Ultrastructurally, the parietal cells had dilated rough endoplasmic reticulums, which were filled with electron-lucent laminated structures. The visceral cells had poorly to moderately developed intracytoplasmic organelles and were interconnected with desmosomes. Taken together, the present tumor was diagnosed as yolk sac carcinoma arising from the ovary and was characterized by not only high metastasis but also invasive infiltration with biphasic proliferation of the parietal and visceral cells.     

Effects of continuous low-dose infusion of lipopolysaccharide on expression of E-selectin and intercellular adhesion molecule-1 messenger RNA and neutrophil accumulation in specific organs in dogs

OBJECTIVE: To determine the effects of continuous low-dose infusion of lipopolysaccharide (LPS) on the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) mRNA and neutrophil accumulation in the lungs, liver, spleen, small intestine, and pancreas in dogs. ANIMALS: 11 healthy adult Beagles. PROCEDURE: Dogs received a continuous infusion of a low dose (10 microg/kg/h, i.v.) of LPS (Escherichia coli 055:B5) or saline (0.9% NaCI) solution (20 mL/kg/h, i.v.) for 8 hours. Activity levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (1L-6) and the number of WBCs in circulation were examined before and 1, 2, 4, and 8 hours after the onset of LPS infusion. Expression of E-selectin and ICAM-1 mRNA and the number of neutrophils in each tissue were examined. RESULTS: After the onset of LPS infusion, serum TNF-alpha and IL-1beta activities transiently increased. Thereafter, IL-6 activity increased, and high IL-6 activity was maintained throughout the experiment. In dogs in the LPS group, expression of E-selectin mRNA increased only in the lungs, and expression of ICAM-1 mRNA increased in the lungs and liver; the number of neutrophils in the tissue increased in the lungs and liver. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that expression of E-selectin and ICAM-1 mRNA increased during sepsis, particularly in the lungs and liver, and that this increase was associated with neutrophil accumulation. Hence, inhibiting the activation of endothelial cells in the lung and liver may decrease organ damage caused by accumulated neutrophils and help regulate multiple-organ dysfunction.     

Desmoplakin and Plakoglobin – Specific Markers of Lymphatic Vessels in the Skin?

Monoclonal antibodies against Desmoplakin and Plakoglobin were tested for their suitability as specific markers of lymphatic vessels. The tissue samples were taken from horse skin in an attempt to establish the horse as a model for human lymphatic diseases. To obtain a clear, positive identification of blood and lymphatic vessels, immunohistochemical staining with antibodies against vascular endothelial growth factor receptor 3 (VEGFR-3) and platelet endothelial adhesion molecule (PECAM-1, CD31), was compared with Desmoplakin and Plakoglobin. Because anti-VEGFR-3 is specific for lymphatic vessels in the skin while anti-CD31 stains blood and lymphatic vessels as well, it can be concluded that VEGFR-3(-)/CD31(+) vessels are blood vessels and VEGFR-3(+)/CD31(+) vessels are lymphatic vessels. It was documented on serial sections that Plakoglobin stains both blood and lymphatic vessels. However, Desmoplakin did not stain several positively identified lymphatic vessels. Therefore, Desmoplakin and Plakoglobin antibodies are not specific markers of lymphatic vessels in the skin and the staining pattern is tissues and species dependent.     

Fetotoxicity of some anticoccidial drugs in chickens

Fetotoxic effects induced by three anticoccidial drugs: robenidine, salinomycin and arprinocid were elucidated in the chicken. Different doses of these drugs were inoculated in groups of embryonated chicken eggs by the yolk sac route. After inoculation, candling of the eggs was performed daily and embryonic or fetal mortalities were recorded. At 19 days old, alive fetuses were collected, weighed, measured and examined morphologically for abnormalities. A group of eggs was kept non-inoculated as a control and another was inoculated with the solvent of the tested drugs. Inoculation of 0.09-9.75 mg robenidine/egg, 0.06-6.75 mg salinomycin/egg or 0.08-8.25 mg arprinocid/egg into the yolk sac of 7 days old embryos caused a dose-dependent fetal death. Arprinocid was the most lethal to chicken fetuses, followed by salinomycin while robenidine was the least. Dead fetuses were usually haemorrhagic, dwarfish and friable. Surviving fetuses showed a dose-dependent reduction in body weight and length, insignificant decrease in leg and wing lengths as well as some developmental abnormalities.     

Effects of breeder hen age and dietary L-carnitine on progeny embryogenesis

1. Ross 308 broiler breeder hens were given diets containing 0 or 25 mg L-carnitine/kg (8 replications per treatment) from 21 weeks of age. 2. Hens were inseminated with semen from Ross broiler breeder males. In a common facility, subsequent progeny hatchability and embryonic mortality at 25, 30, 32, and 38 weeks of breeder age were evaluated. 3. Subsequent egg component weights, incubational egg water loss, progeny embryo growth, and embryo, yolk sac and liver composition through 18 d of incubation at 27, 32, and 38 weeks of breeder age were evaluated. 4. Calculated additions of L-carnitine were in agreement with analysed contents of 3.5 and 31.1 mg free L-carnitine/kg of diet, respectively, and total L-carnitine concentrations increased by 48.6, 21.7, and 10.0% in 0-d yolk, 18-d yolk sac, and 18-d liver samples, respectively, due to the addition of dietary L-carnitine. 5. Supplemental L-carnitine resulted in increased (0.6%) relative 0-d egg yolk weight across weeks 27, 32, and 38, and reduced (0.38%) 18-d yolk sac palmitoleic acid concentration at week 27 without altering embryogenesis. 6. In conclusion, dietary L-carnitine (25 mg/kg of the diet) was deposited in the yolks of broiler breeder hens and was subsequently transferred to the embryonic liver via yolk sac absorption through 18 d of incubation. Furthermore, dietary L-carnitine supplementation increased ovarian follicle yolk deposition in 27-, 32-, and 38-week-old breeder hens, and influenced yolk sac fatty acid beta-oxidation in embryos from 27-week-old breeder hens causing yolk sac palmitoleic acid concentrations to be reduced by 18 d of incubation.     

Pathogenesis of Brucella abortus in chicken embryos

Chicken embryos inoculated with Brucella abortus at 6, 10, and 12 days of incubation were examined by light and electron microscopy. B. abortus was identified by avidin-biotin immunoperoxidase and immunogold techniques. Death occurred from 2 to 5 days post-inoculation, depending on age of the embryo and route of inoculation. B. abortus was recovered from all infected eggs. Brucellae had spread throughout all tissues and localized preferentially within cells of mesodermal derivation. Organ distribution and degree of bacterial replication varied with age of the embryo at time of inoculation. In 6-day-old embryos, B. abortus localized preferentially in endoderm and mesoderm of yolk sac wall, extra- and intraembryonic serosal epithelia, and glomeruli of the mesonephros. In 10- and 12-day-old embryos, B. abortus spread to all tissues; renal glomeruli, liver, spleen, and heart were most severely infected. Intracellular B. abortus was within the rough endoplasmic reticulum of mesenchymal, mesothelial, yolk endodermal, and hepatic cells. In mononuclear phagocytes, endothelial cells, and granulocytes, bacteria were within membrane-bound vacuoles. Intracellular replication of B. abortus in embryonic tissues, especially yolk endoderm, closely resembled that in experimental infections of trophoblasts.     

Effect of dietary manipulation and vaccination of turkey breeder hens on immunoglobulin levels of yolk,yolk sac and neonate poults

Two hundred turkey breeder hens and 24 viable toms of 30–35 weeks age of small white variety were distributed into two treatment groups having four replicates of 25 hens and three toms in each treatment. First four replicates were offered a turkey breeder diet (Diet A) (Nutrient requirements of poultry, 1994, National Academic Press, Washington, DC) and the rest four replicates were maintained on a higher plane of nutrition (Diet B) for 8‐week duration. After 6 weeks of experimental feeding, two replicates from each treatment groups were vaccinated with ND (R₂B) vaccine. Yolk sac of embryo from birds fed Diet B had a significantly higher (p < .05) IgG, IgM level and HI titre (log 2) than those fed Diet A. HI titre values of embryonic yolk sac from the vaccinated birds fed Diet B were significantly higher (p < .05) than that of the control groups. In addition, HI titre values were significantly higher (p < .05) in the day‐old poults of the birds fed Diet B than that of those fed Diet A. There was significantly (p < .01) positive correlation between serum IgG and IgM of the breeder birds and day‐old chicks. Similarly, there was significantly (p < .05) positive correlation between yolk IgG and IgM after 1‐month experimental feeding and yolk sac IgG and IgM. Positive correlation (p < .05) also existed between yolk sac IgM and day‐old chick serum IgM. Furthermore, the HI titres of breeder birds' serum at 14 days post‐vaccination were positively correlated with their egg yolk after 10 and 15 days post‐vaccination, yolk sac and day‐old chicks. Thus, the study envisaged that a higher immunity in neonate poults from turkey breeders maintained on a higher plane of nutrition may be elicited as there was maternal transfer of antibodies from the serum of breeder birds to their offsprings through their yolk sac.     

Die Entwicklung des Dottersackes beim Wiederkäuer (Schaf und Rind)

Yolk sac development was investigated in 69 ovine and 10 bovine embryos from the blastocyst stage to the 7th week of gestation. Light and electron microscopical findings are reported. The yolk sac in sheep and cattle is composed of an enlarged sac-like portion lying below the embryo and two ends which follow the elongated course of the trophoblast. In sheep, an open connection exists between the intestines and the yolk sac up to a crown-rump length (CRL) of 9 mm. It is closed by 12 mm CRL. The wall of the yolk sac is especially well vascularized in the enlarged, sac-like portion. Primary erythropoiesis occurs within the blood capillaries. In the blastocyst, the yolk sac entoderm is made up of elongated, flat cells. It becomes cuboidal in the 3 mm embryo (ovine) and later columnar. The up to 20 microns tall cells stain darkly and contain numerous light-colored vesicles. At 4.5 mm CRL light cells appear between the dark ones. Both cells are rich in rough endoplasmic reticulum (rER). The increased staining of the darker cells is due to an osmophilic cytoplasm and numerous, often parallel lamella of rER. The rER of the light cells is enlarged to irregularly-shaped cisternae, which nearly fill the entire cytoplasm and give them a rounded appearance. The dark cells contain polygonal nuclei, whereas those in the light cells are round with one or two nucleoli. The oval mitochondria have only a few peripheral cristae. Golgi fields are not very common. Cells of the entoderm are connected to one another over zonulae occludentes. They possess microvilli on the luminal surface and are supported by a basement membrane. From 5 mm CRL onwards (ovine), the yolk sac entoderm folds itself between the capillaries, thereby becoming stratified. The intercellular space between the cells expands as projections between neighboring cells interlock. Canaliculi arise between adjacent epithelia. The wall of the yolk sac thickens as a result of this infolding and the densely packed capillaries. Infoldings are especially predominant in the sac-like portion of the yolk sac, and only suggested in the ends. Involution of the yolk sac begins in the peripheral end segments and proceeds centripetally. Numerous glycogen particles appear in the yolk sac entoderm cells of the ovine fetus at a CRL of 36 mm, and by a CRL of 42 mm, the sac-like portion has also begun to show signs of degeneration. Mesenchyme is very sparse within the wall of the yolk sac throughout the entire period of development.(ABSTRACT TRUNCATED AT 400 WORDS)     

巴马小型猪骨髓来源内皮祖细胞的培养鉴定及生物学特性分析简

为探索并建立巴马小型猪骨髓来源内皮祖细胞(EPCs)的体外培养及鉴定方法,本研究在健康猪髂后上棘取骨髓,采用密度梯度分离法分离单个核细胞,按2×10⁷个/孔的密度加入预先包被人纤维连结素（HFN）的6孔细胞培养板中,用含内皮细胞生长因子的选择性培养基诱导培养,逐日在倒置相差显微镜下观察,MTT法检测EPCs增殖情况;流式细胞术（FACs）检测EPCs中AC133和vWF的表达纯度;免疫荧光法双染检测EPCs表面相关抗原ac-LDL和UEA-1。结果发现,从巴马小型猪骨髓分离的EPCs具有增殖能力;MTT结果显示细胞于48 h换液二次贴壁后3～5 d增殖明显,7～10 d增殖加速并出现条索状结构;FACs检测结果显示,二次贴壁细胞vWF阳性细胞百分率74.91%,AC133阳性细胞百分率79.54%,而未经诱导的二次贴壁细胞vWF和AC133阳性率分别是33.65%和39.87%;细胞经免疫荧光双染后Dil-ac-LDL和FITC-UEA-1表达呈阳性,vWF和AC133免疫荧光染色阳性。由此可见,分离培养的细胞具有自我增殖和分化潜能,是内皮祖细胞。