RESUMO
Inoculation of embryonated chicken eggs has been widely used during the past decades; however, inoculation success rates have not been investigated systematically. In this study named success rates were assessed in brown eggs incubated between 5 and 19 days, which were inoculated with 0.2â ml methylene blue per egg. Inoculations were performed in a simple and fully standardized way. Five embryonic compartments were targeted blindly (amniotic cavity, embryo, allantoic cavity, albumen and yolk) with needles of four different lengths; albumen and yolk were targeted with eggs in upside down position. Three compartments were inoculated within sight (air chamber, chorioallantoic membrane and blood vessel). Twenty embryos were used per incubation day, intended deposition site and needle length. Success rates were assessed by visual inspection after breaking the eggs. The inoculations targeting albumen, yolk, amniotic cavity and embryo yielded low scores. Magnetic resonance imaging was performed to elucidate the reason(s) for these low success rates: needles used were of appropriate length, but embryo and amniotic cavity had variable positions in the eggs, while albumen and yolk rapidly changed position after turning the eggs upside down. The latter led to adjustment of the inoculation method for albumen and yolk. Failures to inoculate compartments within sight were immediately visible; therefore, these eggs could be discarded. Except for the amniotic cavity, full scores (20/20) were obtained for all compartments although not always on every day of incubation. In conclusion, the present study may serve as a guide to more accurately inoculate the various chicken embryo compartments. RESEARCH HIGHLIGHTS Blind inoculation of embryonated egg compartments was successful, except for the amniotic cavity. MRI showed rapid position change of albumen and yolk after turning eggs upside down. In ovo vaccination against Marek's disease might be improved by using 38â mm needles.
Assuntos
Doença de Marek/virologia , Óvulo/ultraestrutura , Alantoide/ultraestrutura , Alantoide/virologia , Âmnio/ultraestrutura , Âmnio/virologia , Animais , Embrião de Galinha , Membrana Corioalantoide/ultraestrutura , Membrana Corioalantoide/virologia , Feminino , Injeções , Imageamento por Ressonância Magnética/veterinária , Masculino , Azul de Metileno , Óvulo/virologiaRESUMO
Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic ("yolk sac") placenta shows evidence of functions in yolk digestion and maternal-fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.
Assuntos
Colubridae/embriologia , Placenta/ultraestrutura , Placentação/fisiologia , Alantoide/embriologia , Alantoide/ultraestrutura , Animais , Evolução Biológica , Córion/embriologia , Córion/ultraestrutura , Feminino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Gravidez , Saco Vitelino/embriologia , Saco Vitelino/ultraestruturaRESUMO
As part of a broad survey of placental structure, function, and evolution in reptilian sauropsids paraffin-section histology was used to study microscopic anatomy of the uterus and fetal membranes of three species of North American watersnakes (Nerodia: Colubridae). The pre-ovulatory uterus is poorly vascularized with inactive shell glands. These shell glands are activated during vitellogenesis but regress during pregnancy. Two placentas develop through apposition of the uterine lining to the chorioallantois and the yolk sac omphalopleure. Fetal and maternal components of the chorioallantoic placenta are progressively vascularized during development. Their epithelia are attenuated, but (contrary to a previous report), epithelia of neither the uterus nor the chorion are eroded. The fetal portion of the yolk sac placenta is an omphalallantois, formed of avascular omphalopleure, isolated yolk mass, and allantois. This placenta is progressively replaced by chorioallantoic placenta during mid- to late-development through depletion of the isolated yolk mass. The chorioallantoic placenta is anatomically specialized for maternal-fetal gas exchange, and its expansion during development reflects the growing needs of the fetus for gas exchange. The yolk sac placenta is morphologically unsuited for gas exchange, but may serve other functions in maternal-fetal exchange.
Assuntos
Colubridae/anatomia & histologia , Placenta/embriologia , Placentação/fisiologia , Alantoide/embriologia , Alantoide/ultraestrutura , Animais , Evolução Biológica , Córion/embriologia , Córion/ultraestrutura , Membranas Extraembrionárias/ultraestrutura , Feminino , Mamíferos , Placenta/ultraestrutura , Gravidez , Estados Unidos , Saco Vitelino/embriologia , Saco Vitelino/ultraestruturaRESUMO
In eggs of oviparous reptiles, fetal membranes maintain developing embryos through the exchange of respiratory gases and provision of water and calcium. As part of a survey of reptilian fetal membranes, we used scanning electron microscopy to study fetal membrane morphology in the oviparous Pueblan milksnake, Lampropeltis triangulum campbelli. The chorioallantois initially is an avascular structure lined by enlarged chorionic and allantoic epithelia. Upon vascularization, the chorionic epithelium becomes greatly attenuated, enhancing the potential for gas exchange; the allantoic epithelium also flattens. The bilaminar omphalopleure of the yolk sac lacks blood vessels, but it becomes vascularized by allantoic capillaries and transformed into an omphalallantois. Upon regression of the isolated yolk mass, this membrane is converted to chorioallantois, equipping it for gas exchange. Allantoic fluid serves as a water reservoir, and we postulate that it facilitates water uptake by establishing an osmotic gradient. Early in development, epithelia of both the chorion and the omphalopleure show apical microvilli that greatly increase the cell surface area available for water uptake. However, these features are incompatible with gas exchange and are lost as oxygen needs take precedence. A comparison of the fetal membranes to those of other squamate species (both oviparous and viviparous) reveals characteristics that are probably ancestral for snakes, some of which are plesiomorphic for Squamata. The widespread phylogenetic distribution of these features reflects their utility as adaptations that serve functional requirements of squamate embryos.
Assuntos
Membrana Corioalantoide/ultraestrutura , Colubridae/embriologia , Membranas Extraembrionárias/ultraestrutura , Alantoide/embriologia , Alantoide/ultraestrutura , Animais , Evolução Biológica , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/embriologia , Membranas Extraembrionárias/embriologia , Oviparidade , Óvulo/crescimento & desenvolvimento , Óvulo/ultraestrutura , Saco Vitelino/embriologia , Saco Vitelino/ultraestruturaRESUMO
The embryonic origin of umbilical cord vestiges is well documented; however, their immunophenotype is unknown. This study was conducted to determine whether vitelline and allantoic remnants can be differentiated using immunohistochemical markers. All allantoic remnants were stained with p63 and were negative for CDX2, whereas the vitelline remnants stained with CDX2 and were negative for p63. An unexpected finding was a small number of morphologically ambiguous cases that stained with both markers in a complimentary manner. The term "hybrid" remnant is proposed for these remnants.
Assuntos
Alantoide/ultraestrutura , Cordão Umbilical/ultraestrutura , Membrana Vitelina/ultraestrutura , Alantoide/metabolismo , Biomarcadores/metabolismo , Fator de Transcrição CDX2 , Amarelo de Eosina-(YS) , Feminino , Expressão Gênica , Hematoxilina , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Coloração e Rotulagem , Cordão Umbilical/metabolismo , Membrana Vitelina/metabolismoRESUMO
Placental membranes mediate maternal-fetal exchange in all viviparous reptilian sauropsids. We used scanning electron microscopy to examine the placental interface in the mountain spiny lizard, Sceloporus jarrovi (Phrynosomatidae). From the late limb bud stage until birth, the conceptus is surrounded by placental membranes formed from the chorioallantois and yolk sac omphalopleure. The chorioallantois lies directly apposed to the uterine lining with no intervening shell membrane. Both fetal and maternal sides of the chorioallantoic placenta are lined by continuous layers of flattened epithelial cells that overlie dense capillary networks. The chorioallantoic placenta shows specializations that enhance respiratory exchange, as well as ultrastructural evidence of maternal secretion and fetal absorption. The yolk sac placenta contains enlarged fetal and maternal epithelia with specializations for histotrophic nutrient transfer. This placenta lacks intrinsic vascularity, although the vascular allantois lies against its inner face, contributing to an omphallantoic placenta. In a specialized region at the abembryonic pole, uterine and fetal tissues are separated by a compact mass of shed shell membrane, yolk droplets, and cellular debris. The omphalopleure in this region develops elongate folds that may contribute to sequestration and absorption of this material. Fetal membrane morphogenesis and composition in S. jarrovi are consistent with those of typical squamates. However, this species exhibits unusual placental specializations characteristic of highly placentotrophic lizards.
Assuntos
Membranas Extraembrionárias/ultraestrutura , Lagartos/anatomia & histologia , Alantoide/embriologia , Alantoide/ultraestrutura , Animais , Membrana Corioalantoide/anatomia & histologia , Membrana Corioalantoide/ultraestrutura , Membranas Extraembrionárias/anatomia & histologia , Feminino , Lagartos/embriologia , Microscopia Eletrônica de Varredura , Útero/embriologia , Útero/ultraestrutura , Viviparidade não Mamífera , Saco Vitelino/anatomia & histologia , Saco Vitelino/ultraestruturaRESUMO
The rodent allantois is thought to be unique amongst mammals in not having an endodermal component. Here, we have investigated the mesothelium, or outer surface, of murine umbilical precursor tissue, the allantois (â¼7.25-8.5 days postcoitum, dpc) to discover whether it exhibits the properties of an epithelium. A combination of morphology, challenge with biotinylated dextran amines (BDAs), and immunohistochemistry revealed that the mesothelium of the mouse allantois exhibits distinct regional properties. By headfold stages (â¼7.75-8.0 dpc), distal mesothelium was generally squamous in shape, and highly permeable to BDA challenge, whereas ventral proximal mesothelium, referred to as "ventral cuboidal mesothelium" (VCM) for the characteristic cuboidal shape of its cells, was relatively impermeable. Although "dorsal cuboidal mesothelium" (DCM) resembled the VCM in cell shape, its permeability to BDA was intermediate between the other two regions. Results of immunostaining for Zonula Occludens-1 (ZO-1) and Epithelial-cadherin (E-cadherin), together with transmission electron microscopy (TEM), suggested that impermeability in the VCM may be due to greater cellular contact area between cells and close packing rather than to maturity of tight junctions, the latter of which, by comparison with the visceral yolk sac, appeared to be rare or absent from the allantoic surface. Both VCM and DCM exhibited an ultrastructure more favorable for protein synthesis than did the distal squamous mesothelium; however, at most stages, VCM exhibited robust afadin (AF-6), whereas the DCM uniquely contained alpha-4-integrin. These observations demonstrate that the allantoic mesothelium is not a conventional epithelium but possesses regional ultrastructural, functional and molecular differences that may play important roles in the correct deployment of the umbilical cord and its associated vascular, hematopoietic, and other cell types.
Assuntos
Alantoide/anatomia & histologia , Alantoide/metabolismo , Alantoide/ultraestrutura , Animais , Caderinas/análise , Epitélio/anatomia & histologia , Epitélio/metabolismo , Epitélio/ultraestrutura , Proteínas de Membrana/análise , Camundongos , Fosfoproteínas/análise , Junções Íntimas/ultraestrutura , Saco Vitelino/anatomia & histologia , Proteína da Zônula de Oclusão-1RESUMO
The aim of this study was to perform a morphological characterization of the initial bovine placental development, between 20 and 70 days post-insemination (p.i.), with emphasis on the differentiation of the allantois and amnion. After collection, the conceptuses were dissected, macroscopically measured and photographically documented. The extraembryonic membranes were cut into fragments measuring 5 cm(2), and then fixed in 4% paraformaldehyde for analysis using light microscopy, and in 2.5% glutaraldehyde for use in scanning and transmission electron microscopy. The extraembryonic and fetal membranes presented variable degrees of development throughout the periods analysed. The macroscopic appearance of vascularization of the allantois, its attempt to merge with the chorium and the effective appearance of the first cotyledons in development were the events observed from 30 to 40 days of pregnancy. The measurements of the amnion increased gradually as gestation developed. The allantoic epithelia presented cellular dimorphism from 20 to 25 days of pregnancy, but was shown to be immature from 60 to 70 days of pregnancy.
Assuntos
Alantoide/crescimento & desenvolvimento , Âmnio/crescimento & desenvolvimento , Bovinos/embriologia , Bovinos/fisiologia , Placentação , Prenhez , Alantoide/ultraestrutura , Âmnio/ultraestrutura , Animais , Desenvolvimento Embrionário/fisiologia , Feminino , Placenta/ultraestrutura , Gravidez , Prenhez/fisiologiaRESUMO
Experiments were performed on the chorio-allantoic membrane (CAM) of the chick to evaluate the effects of bombesin (BN) on vascular neoformation. In morphometrical assays, 10(-13)-10(-4) M BN promoted dose-dependent vascular development. Newly formed vessels converged toward the BN release site in a spoked wheel arrangement, suggesting a diffusion gradient mechanism. Structural and ultrastructural analysis of CAM specimens collected near the BN release site showed that both vasculogenetic and angiogenetic processes cooperated in vascular neoformation that involved committed cells from the mesenchyme (angioblasts) as well as endothelial cells. No pattern of vascular development was detected away from the BN release site. Findings from the present study emphasize the role of BN in vascular net development of respiratory organs.
Assuntos
Alantoide/efeitos dos fármacos , Bombesina/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Alantoide/citologia , Alantoide/fisiologia , Alantoide/ultraestrutura , Animais , Embrião de Galinha , Microscopia Eletrônica de Transmissão , Neurotransmissores/farmacologiaRESUMO
New world lizards of the genus Mabuya have the most specialized level of placentotrophy among reptiles known to date, and related to that, they have the most complex allantoplacenta characterized by a series of morphological specializations that converge with those known for eutherian mammals. One of these specializations is the placentome that is found in the embryonic pole of the incubation chamber. In the mature allantoplacenta, this structure is morphologically the most complex, which could support an important amount of nutrient exchange between mother and fetus. According to the relationship between the chorioallantois and the syncytial uterine epithelia, the placenta of Mabuya populations shows some interesting similarities to the synepitheliochorial type. Recently, cells of chorionic origin have been found invading the syncytial uterine epithelium, and in very close proximity with uterine blood vessels. In this study, we describe the relationship between these invasive chorionic cells, the uterine syncytium, and the subjacent blood vessels of several populations of this genus, by means of high resolution optical microscopy and transmission electron microscopy. Cell groups originating from the chorion, of variable size and shape, penetrate the uterine syncytial epithelium extending complex cytoplasmic projections that come in contact with uterine capillaries and form an extensive and complex double-membrane system that surrounds the capillary. The close relationship between the chorion and the maternal circulation suggests that the Mabuya placentome shows some characteristics of an endotheliochorial placenta. This finding constitutes so far the only documented example of an endotheliochorial placentation in Reptilia.
Assuntos
Córion/citologia , Lagartos/anatomia & histologia , Placenta/citologia , Alantoide/citologia , Alantoide/ultraestrutura , Animais , Proliferação de Células , Córion/ultraestrutura , Endotélio/citologia , Endotélio/ultraestrutura , Feminino , Células Gigantes/citologia , Células Gigantes/ultraestrutura , Placenta/ultraestrutura , GravidezRESUMO
The labyrinthine zone of allantoic placenta was studied after dosated exposure to vibration. The study was carried out in pregnant female Wistar rats (n=68). Rats were subjected to short-term vibration from Day 9 to Day 13 and long-term vibration from the Day 9 to Day 18 of pregnancy (taking into account embryogenesis periodization and the development period of extraembryonic organs). Vibration table with fixed parameters (frequency of 32 Hz and acceleration of 50 m/s2) was used. Placental and fetal mass were measured, labyrinthine zone components were evaluated using the morphometric methods, while labyrinthine cord and fetal vessels along Duval's sinus were studied ultrastructurally. Labyrinth was analyzed from the point of view of rapprochement of maternal and fetal bloodstreams. It was found that the exposure to vibration together with the development of compensatory adaptive mechanisms led to chronic placental insufficiency. Experimental group with short-term vibration exposure (Days 9-13) is of particular interest since chronic placental insufficiency in this group should be considered as decompensated.
Assuntos
Placenta/ultraestrutura , Trofoblastos/ultraestrutura , Vibração/efeitos adversos , Alantoide/irrigação sanguínea , Alantoide/ultraestrutura , Animais , Doença Crônica , Feminino , Masculino , Placenta/irrigação sanguínea , Circulação Placentária , Insuficiência Placentária/etiologia , Insuficiência Placentária/patologia , Gravidez , Ratos , Ratos WistarRESUMO
Mabuya species are highly matrotrophic viviparous lizards with Type IV epitheliochorial allantoplacenta. The allantoplacenta of an Andean population of this genus, currently assigned to Mabuya sp., possesses specializations related to histotrophic nutrition at the embryonic hemisphere (placentome, paraplacentome, and chorionic areolas), while at the abembryonic hemisphere it has a mixed function: histotrophic transfer (absorptive plaques) and hemotrophic nutrition (gas exchange in respiratory segments). These placental specializations were studied using high-resolution light microscopy and transmission electron microscopy, and were compared with those found in other squamate reptiles and eutherian mammals. Cytological features of the placentome suggest that this is an important region for nutritional provision; the paraplacentome also shows characteristics for nutrient transfer, especially lipids. Chorionic areolas allow the absorption of glandular products, as well as uterine and chorionic cellular debris produced by lysis of some cells of both epithelia during areola formation. In the absorptive plaques both uterine and chorionic epithelia are firmly attached and their cellular apices exhibit electron-dense granules that could be related to autocrine and paracrine functions. The short interhemal distance found in the respiratory segments confirms their role in gas exchange. A common feature of all regional specializations in the Mabuya sp. allantoplacenta is the presence of lipids in the interacting chorionic and uterine epithelia, suggesting that lipids are transferred throughout the entire embryonic chamber; placental transfer of lipids may be the principal fetal energy and lipid source in this species. In spite of this feature, each one of the specialized areas of the allantoplacenta has different features suggesting particular functions in the transfer of nutrients (as ions, lipids, proteins, amino acids, sugar, water, and gases), and in the possible synthesis of hormones and proteins. The placental complexity observed in this species of Mabuya is greater than in any other reptile, and resembles that of eutherian mammals: Each one of these specializations of the placental membranes in Mabuya sp. is similar to those found among different eutherian mammals, indicating a very impressive evolutionary convergence at the histological and cytological levels between both clades. However, no eutherian mammal species simultaneously displays all of these specializations in the embryonic chamber as does Mabuya sp.
Assuntos
Alantoide/ultraestrutura , Lagartos , Alantoide/citologia , Animais , Córion/citologia , Córion/ultraestrutura , Epitélio/ultraestrutura , Feminino , Sistema Respiratório/citologia , Sistema Respiratório/ultraestrutura , Viviparidade não MamíferaRESUMO
The ultrastructure of the chorioallantoic placenta of the lesser bush baby (Galago senegalensis) has been studied. The placenta was shown to be of the diffuse, epitheliochorial and adeciduate type. The trophoblasts of the chorionic villi, other than those lining the chorionic vesicles, were characterized by the presence of many lipid droplets. In the later stage of gestation, the fetal capillaries indented the trophoblastic epithelium reducing the distance between fetal and maternal circulations. In addition chorionic vesicles were observed. The trophoblasts lining the chorionic vesicles have outward bulging apical surfaces. There are clefts between these cells and this region is occupied by microvilli of adjacent cells. Several layers of fusiform cells that did not extend up into the cores of the chorionic vesicle villi formed the outer component of the vesicular wall. Granulated cells were observed within the maternal connective tissue and their possible role is discussed.
Assuntos
Alantoide/ultraestrutura , Córion/ultraestrutura , Galago/anatomia & histologia , Animais , Vilosidades Coriônicas/ultraestrutura , Feminino , Galago/fisiologia , Idade Gestacional , Microscopia Eletrônica de Transmissão , Gravidez , Trofoblastos/ultraestruturaRESUMO
The mouse is an extremely useful experimental model for the study of human disease owing to the ease of genetic and physiological manipulation. A more detailed knowledge of murine placental development will, we hope, increase our understanding of the pathogenesis of placentally related complications of human pregnancy. The murine placenta consists of two main fetally derived compartments: the labyrinthine zone and the junctional zone. Exchange in the labyrinthine zone takes place across an interhaemal membrane comprising an outer layer of cytotrophoblast cells and two inner layers of syncytial trophoblast. The cytotrophoblast layer thins as gestation advances, and in addition becomes highly perforated after embryonic day (E)12.5. Furthermore, as gestation advances cytotrophoblast nuclear volume and DNA content increase, suggesting the formation of labyrinthine trophoblast giant cells. The syncytial layers become increasingly microvillous, enlarging the surface area for exchange. Separate basement membranes support the syncytium and the fetal capillary endothelium throughout gestation, although these appear to fuse where the capillaries are closely approximated to the trophoblast. The junctional zone consists of two principal trophoblast cell types, spongiotrophoblasts and invasive glycogen cells, yet the functions of each remain elusive. Spongiotrophoblasts vary in their appearance even when not fully differentiated, but a striking feature is the extensive endoplasmic reticulum of the more mature cells. Early glycogen cells are distinguished by the presence of electron-dense glycogen granules, and large amounts of surrounding extracellular matrix. Later the accumulations of glycogen granules occupy almost all the cytoplasm and there are few organelles. This is the first study to use both scanning and transmission electron microscopy in an ultrastructural description of murine placental development and is complementary to contemporary genetic investigations.
Assuntos
Membranas Extraembrionárias/ultraestrutura , Placentação/fisiologia , Prenhez/fisiologia , Alantoide/ultraestrutura , Animais , Diferenciação Celular , Córion/ultraestrutura , Citoplasma/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Matriz Extracelular/ultraestrutura , Feminino , Glicogênio/análise , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Organelas/ultraestrutura , Placenta/química , Placenta/citologia , Gravidez , Trofoblastos/ultraestruturaRESUMO
Five strains of infectious bronchitis virus (IBV) were isolated from five layer flocks that had nephropathogenic infection in four provinces in China. Among them, three of the five flocks had been vaccinated against infectious bronchitis. Virulence studies indicated that the five Chinese IBV isolates caused 10 to 30% mortality in 15-day-old specific pathogen free chickens and gross lesions were mainly confined to the kidneys in all of the dead chickens. Two oligonucleotide pairs, S1Uni2 and S1Oligo3' or S1Oligo5' and S1Oligo3', were used after propagation of the isolates in embryonated eggs to amplify the S1 protein genes of the spike protein. The cDNA derived by reverse transcriptase-polymerase chain reaction was cloned and sequenced. The nucleotide and amino acid sequence of S1 protein gene had a similar degree of identity (> or =92%) among the five Chinese IBV isolates. The nucleotide and amino acid identity of the S1 protein gene between the five Chinese IBV isolates and 16 strains of other IBVs varied from 60 to 81%. This clearly showed that the five Chinese IBV isolates comprised a separate genotype. These results demonstrated, for the first time, that there is a new genotype of nephropathogenic IBV circulating in vaccinated and non-vaccinated flocks in China.
Assuntos
Galinhas , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/genética , Vacinação/veterinária , Alantoide/ultraestrutura , Animais , Sequência de Bases , China , Infecções por Coronavirus/genética , Infecções por Coronavirus/mortalidade , DNA Complementar/genética , Genótipo , Vírus da Bronquite Infecciosa/patogenicidade , Rim/virologia , Glicoproteínas de Membrana/genética , Microscopia Eletrônica , Dados de Sequência Molecular , Oligonucleotídeos , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da Espécie , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genéticaRESUMO
We have demonstrated previously that new blood vessel formation induced by angiogenic growth factors in onplants placed on the chorioallantoic membrane (CAM) of the chick embryos is critically dependent on the cleavage of fibrillar collagen by a previously unidentified interstitial collagenase. In the present study we have used a quantitative CAM angiogenesis system to search for and functionally characterize host avian collagenases responsible for the collagen remodeling associated with angiogenesis. Among the matrix metalloproteinases (MMPs) identified in the CAM onplant tissue, the chicken MMP-13 (chMMP-13) was the only enzyme whose induction and expression coincided with the onset of angiogenesis and blood vessel formation. The chMMP-13 cDNA has been cloned and recombinantly expressed. The chMMP-13 protein has been purified, characterized in vitro, and examined in situ in the CAM. MMP-13-positive cells appear in the CAM shortly after angiogenic stimulation and then accumulate in the collagen onplant tissue. Morphologically, the chMMP-13-containing cells appear as hematopoietic cells of monocyte/macrophage lineage. In vitro, the chMMP-13 proenzyme is rapidly and efficiently activated through the urokinase plasminogen activator/plasminogen/plasmin cascade into a collagenase capable of cleaving native but not the (r/r) mutant collagenase-resistant collagen. Surprisingly, nanogram levels of purified chMMP-13 elicit an angiogenic response in the CAM onplants comparable with that induced by the angiogenic growth factors. The chMMP-13-mediated response was efficiently blocked by select protease inhibitors indicating that plasmin-activated chMMP-13 can function as an angiogenic factor in vivo. Altogether, the results of this study extend the physiological role of MMP-13, previously associated with cartilage/bone resorption, to the collagen remodeling involved in the angiogenic cascade.
Assuntos
Colágeno/metabolismo , Colagenases/fisiologia , Neovascularização Fisiológica/fisiologia , Alantoide/irrigação sanguínea , Alantoide/metabolismo , Alantoide/ultraestrutura , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Córion/irrigação sanguínea , Córion/metabolismo , Córion/ultraestrutura , Colágeno/genética , Colagenases/biossíntese , Colagenases/genética , Ativação Enzimática , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Metaloproteinase 13 da Matriz , Camundongos , Dados de Sequência Molecular , Plasminogênio/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Diabetes is associated with abnormal angiogenesis. Increased angiogenesis contributes to severe forms of diabetic retinopathy, but angiogenesis is decreased in response to myocardial ischemia in diabetic patients. We evaluated the direct effect of hyperglycemia on angiogenesis in the chicken chorioallantoic membrane assay, a model of active neoangiogenesis. Hyperglycemia, lasting up to 7 days, was induced in 7-day-old chick embryos by a single intravitellus glucose injection. Control embryos received either water (volumic control) or mannitol (osmotic control). Hyperglycemia decreased angiogenesis in this model from the 5th day on. The pattern and expression level of the main vascular growth factors' genes were not altered by hyperglycemia, as assessed by in situ hybridization and semiquantitative RT-PCR. As early as 2 days after hyperglycemia was induced, an increased apoptosis of endothelial cells and pericytes was detected by transferase-mediated deoxyuridine triphosphate nick-end labeling assay and electron microscopy. In the meantime, endothelial cell proliferation was decreased, as assessed by incorporation of bromo-deoxyuridine. Hyperglycemia can therefore impair angiogenesis without altering the expression level of vascular growth factors through induction of apoptosis and decreased proliferation of endothelial cells.
Assuntos
Alantoide/irrigação sanguínea , Córion/irrigação sanguínea , Hiperglicemia/fisiopatologia , Neovascularização Patológica/fisiopatologia , Alantoide/ultraestrutura , Angiopoietina-1/genética , Angiopoietina-2/genética , Animais , Peso Corporal , Embrião de Galinha , Córion/ultraestrutura , Primers do DNA , Modelos Animais de Doenças , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glucose/farmacologia , Hibridização In Situ , Morfogênese , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
An understanding of the evolutionary morphology of extraembryonic membranes in reptiles requires information about oviparous as well as viviparous species. We are studying histology and ultrastructure of the extraembryonic membranes of snakes to clarify the evolutionary history of reptilian fetal membranes, including determination of basal (ancestral) ophidian and squamate patterns. Microscopic anatomy of the membranes of oviparous corn snakes (Elaphe guttata) was examined using light and electron microscopy. At mid-development the inner surface of the eggshell is lined by two extraembryonic membranes, the chorioallantois and the omphalallantoic membrane. The chorioallantois consists of a bilayered cuboidal epithelium that overlies the allantoic blood vessels. During development, allantoic capillaries become more abundant, and the chorionic epithelium thins, decreasing the diffusion distance for respiratory gas exchange. The abembryonic pole of the egg is delimited by a bilaminar omphalopleure and isolated yolk mass, the latter of which is lined on its inner face by the allantois. The isolated yolk mass regresses developmentally, and patches of yolk droplets become isolated and surrounded by allantoic blood vessels. By late development, the abembryonic hemisphere has been fully vascularized by allantoic vessels, forming a "secondary chorioallantois." With regard to its extraembryonic membranes, Elaphe gutatta is similar to viviparous snakes. However, this species exhibits features that have not previously been reported among squamates, perhaps reflecting its oviparous reproductive habits. Morphological evidence for the uptake of eggshell material by epithelia of the chorion and omphalopleure suggests that the potential for absorption by extraembryonic membranes predates the origin of viviparity.
Assuntos
Alantoide/ultraestrutura , Córion/ultraestrutura , Serpentes/anatomia & histologia , Serpentes/embriologia , Saco Vitelino/anatomia & histologia , Alantoide/embriologia , Animais , Evolução Biológica , Córion/embriologia , Casca de Ovo/anatomia & histologia , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/embriologia , Feminino , Gravidez , Especificidade da Espécie , Saco Vitelino/embriologiaRESUMO
The vascularisation of the chorioallantoic membrane (CAM) of avian embryos is influenced by environmental oxygen partial pressure (P(O(2))) on a global level: incubation at high P(O(2)) reduces the density of pre- and post-capillary vessels of the CAM and decelerates the thinning of the blood-gas barrier, and vice versa. This study investigates the effects of local P(O(2)) on vascular development during the formative period of days ten to fifteen, by making half of the egg hypoxic and the other half hyperoxic. The densities of arterioles, venules and capillaries were reduced under the hypoxic side, compared to untreated eggs, but not significantly changed on the hyperoxic side. Harmonic mean thickness of the tissue barrier and total CAM blood volume were not affected by the treatments. Vascular development of the CAM was therefore only partly influenced by local P(O(2)).
Assuntos
Alantoide/irrigação sanguínea , Permeabilidade Capilar/fisiologia , Embrião de Galinha/crescimento & desenvolvimento , Córion/irrigação sanguínea , Casca de Ovo/fisiologia , Condutividade Elétrica , Oxigênio/farmacocinética , Adaptação Fisiológica , Alantoide/crescimento & desenvolvimento , Alantoide/ultraestrutura , Animais , Transporte Biológico , Córion/crescimento & desenvolvimento , Córion/ultraestrutura , Difusão , PermeabilidadeRESUMO
Embryonic tissues, in common with other tissues, including tumours, tend to develop a substantial vasculature when transplanted onto the chorioallantoic membrane (CAM). Studies conducted to date have not examined in any detail the identity of vessels that supply these grafts, although it is known that the survival of transplanted tissues depends on their ability to connect with CAM vessels supplying oxygen and nutrients. We grafted the mesonephros, a challenging model for studies in vascular development, when it was fully developed (HH35). We used reciprocal chick-quail transplantations in order to study the arterial and venous connections and to analyse the cell invasion from the CAM to the organ, whose degeneration in normal conditions is rapid. The revascularization of the grafted mesonephros was produced by the formation of peripheral anastomoses between the graft and previous host vasculatures. The assembly of graft and CAM blood vessels occurred between relatively large arteries or veins, resulting in chimeric vessels of varying morphology depending on their arterial or venous status. Grafts showed an increased angiogenesis from their original vasculature, suggesting that the normal vascular degeneration of the mesonephros was partially inhibited. Three types of isolated host haemangioblast were identified in the mesonephros: migrating angioblast-like cells, indicating vasculogenesis, undifferentiated haematopoietic cells and macrophages, which might have been involved in the angiogenesis. Tomato lectin was found to bind activated macrophages in avian embryos.