Em vista das limitações éticas em torno da obtenção de células-tronco de fetos humanos, o cão é uma alternativa para estes estudos. Além disso, a terapia celular proporciona novas expectativas para o tratamento na espécie. Realizamos o estudo comparativo das células isoladas de saco vitelino, fígado e medula óssea de fetos caninos. As células foram analisadas microscopicamente e ultra estruturalmente. O imunofenótipo das células foi avaliado através de marcadores. Caracterizamos a plasticidade, o cariótipo e o potencial teratogênico destas células. Após expansão as células progenitoras formaram colônias com morfologia fibroblastóide. As células progenitoras do saco vitelino e medula óssea são compostas por: células com alta proporção núcleo-citoplasma e células com citoplasma rico em organelas, enquanto que as células progenitoras do fígado eram semelhantes à célula epitelial e células ricas em organelas. As células-progenitoras dos três tecidos fetais foram positivas para os anticorpos nestina e vimentina, mas negativas para CD45 e CD13. Células progenitoras de medula óssea foram positivas para o marcador CD44. Células progenitoras do fígado e medula óssea expressaram a proteína citoqueratina-18, enquanto as do saco vitelino expressaram ve-caderina. Células positivas para Oct3/4 foram detectadas em todas as células progenitoras. As células-progenitoras do saco vitelino e medula óssea diferenciaram-se em tecidos ósseo, cartilaginoso e muscular; já as do fígado para tecido ósseo e muscular. Nenhum tipo celular diferenciou-se em adipócitos. As células progenitoras da medula óssea diferenciaram em células semelhantes a neurônios. Sugere-se a presença de progenitores semelhantes a células mesenquimais e epiteliais. Todas as células mantiveram o cariótipo estável e não formaram tumores. Células progenitoras de medula óssea apresentaram maior capacidade de proliferação e diversidade de diferenciação. Sugere-se que estas células são possíveis candidatas para a terapia celular. / The use the human fetuses for stem cells isolation have ethical limitations. In this context the dog is an excellent candidate to fetal stem cells. Furthermore, these cells can be used in cell therapy of canine diseases We aimed at isolation and comparative characterization of progenitor cells from yolk sac, liver and bone marrow of canine fetuses. Cells were characterized using stem cells antibodies. Differentiation assays as well as karyotype analysis were performed. Teratogenic properties this cells were evaluated. After establishment of primary culture, best proliferation potential was observed in bone marrow progenitor cells. Bone marrow and liver progenitor cells were more efficient in CFU-F assay, then yolk sac progenitor cells. Evidenced by TEM cells with a high nuclear-to-cytoplasmic ratio and cells with cytoplasm rich in organelles. Cells isolated from liver showed epithelial-like morphology and cytoplasm rich in organelles. The yolk sac, liver bone marrow cells reacted positively with nestin and vimentin, being negative to CD45 and CD13 antibodies. Additionally bone marrow progenitor cells were positive to CD44. Bone marrow and liver progenitor cells reacted positively with cytokeratin 18. Yolk sac progenitor cells were positive to ve-cadherin. A few Oct3/4 positive cells were found in yolk sac, liver and bone marrow. Yolk sac and bone marrow progenitor cells showed successful osteogenic, chondrogenic, myogenic differentiation. Differentiation liver progenitor cells were able to bone and muscle cells. The bone marrow progenitor cells were able to produce neuron-like cells. None of progenitor cells showed adipogenic differentiation. The study suggests the presence of mesenchymal-like and epithelial-like progenitor cells. All the karyotype remained and failed to induce the formation of tumors. Stem cells from bone marrow showed high diversity of differentiation than other cell types. It is suggested that these cells are possible candidates for cell therapy.

A placenta é encontrada apenas em mamíferos, e é resultado do sucesso da implantação do blastocisto no útero, representando um órgão funcional de trocas materno-fetais, sendo também um importante órgão endócrino. Na família Cricetidae e Subfamília Sigmodontinae englobam os ratos e camundongos da América do Sul. Devido à semelhança entre a morfologia da placenta e o processo de placentação dos roedores com a placenta humana, estes animais representam um interessante modelo para estudos relacionados à placentação. O objetivo deste estudo foi descrever a morfologia da placenta e a placentação em 5 espécies de sigmodontes (Necromys lasiurus, Oryzomys subflavus, Oryzomys sp., Oryzomys megacephalus e Oligoryzomys sp.). Nas análises macroscópicas constatou-se que a placenta desse grupo apresenta um formato discóide, sendo classificada como zonária discoidal. Na microscopia observou-se que a placenta é do tipo corioalantóidea, labiríntica, hemocorial, com zonas de espongiotrofoblasto e uma região de decídua materna. A placenta vitelina é vilosa, completamente invertida e persistia até o final da gestação. Somente em Oryzomys subflavus foi observada na placenta vitelina uma intensa atividade hemofágica, evidenciada pela técnica de Tricrômo de Masson. A barreira placentária nas espécies Necromys lasiurus e Oryzomys sp. é do tipo hemotricorial, existindo três camadas trofoblásticas adjuntas ao endotélio do capilar fetal separando os sistemas sanguíneos materno e fetal. As células trofoblásticas gigantes foram observadas em diferentes regiões da placenta. Em todas as espécies as células deciduais foram positivas para reação histoquímica de ácido periódico de Schiff (P.A.S.). / Placenta is found only in mammals and it is result of the success of blastocyst implantation in the uterus, representing a functional organ to maternal-fetal exchanges and it is also an important endocrine organ. The Family Cricetidae and Subfamily Sigmodontinae include the rats and mice from South America. Due to the similarity of both the placenta morphology and the placentation between rodents and human, these animals represent an interesting model for studies related to the placenta and placentation. The aim of this research was to describe the placenta morphology and placentation in 5 species of sigmodonts (Necromys lasiurus, Oryzomys subflavus, Oryzomys sp., Oryzomys megacephalus Oryzomys sp. and Oligoryzomys sp.). In the macroscopic analysis the placenta shows a discoid shape, it is classified as discoid type. The microscopy results showed the placenta is chorioallantoic, labyrinth, hemochorial, with areas of spongio zone and a region of maternal decidua. The yolk sac placenta is villous, completely inverted and persisted until the end of pregnancy. Only in Oryzomys subflavus was observed in the yolk sac placenta an intense hemophagous activity, evidenced by the Masson\'s trichrome stain. The placental barrier in Necromys lasiurus and Oryzomys sp. is of the hemotrichorial type, composed by three trophoblast layers more the fetal capillary endothelium to separate the maternal and fetal blood systems. The trophoblast giant cells were observed in different regions of the placenta. In all species the decidual cells were positive to histochemical reaction using periodic acid-Schiff (PAS).

O objetivo deste estudo foi caracterizar morfologicamente o desenvolvimento inicial da gestação bovina proveniente de monta natural, com idades compreendidas entre 15 a 70 dias, e de fecundação in vitro (FIV) com 35 dias, com ênfase no desenvolvimento placentário inicial, e diferenciação das estruturas extra embrionárias. Para tanto, foram utilizados 141 conceptos, provenientes de monta natural, e sete conceptos obtidos pela técnica de FIV. Após as coletas, os conceptos foram dissecados, mensurados macroscopicamente e fotodocumentados. As membranas extra-embrionárias foram cortadas em fragmentos de 5 cm2, e, em seguida fixadas em paraformoldeído 4%, para análise por microscopia de luz, e glutaraldeído 2,5%, para utilização em microscopia eletrônica de varredura e transmissão. As membranas extra-embrionárias e fetais apresentaram graus variáveis de desenvolvimento ao longo dos períodos analisados. O aparecimento macroscópico da vascularização do alantóide, sua tentativa de se fundir com o cório e o aparecimento efetivo dos primeiros cotilédones em desenvolvimento, foram eventos observados em embriões a partir de 1,9±0,27 cm de \"Crown-Rump\" (CR) (30 a 40 dias da gestação). O CR médio, o peso do embrião, o peso do saco gestacional e os comprimentos do cório e âmnio aumentaram gradativamente com o evoluir da gestação. O epitélio alantoidiano apresentou um dimorfismo celular a partir de 0,9 cm de CR (15 a 20 dias de gestação), porém, mostrou-se imaturo até o feto atingir o comprimento de 7,2 cm de CR (60 a 70 dias da gestação). O trofoblasto apresentou células mononucleadas e células gigantes binucleadas em diferentes níveis ao longo da gestação. O saco vitelino persistiu até 70 dias de gestação, e o seu epitélio apresentou indícios de atividade funcional até 50 dias de gestação. De todos os parâmetros mensurados na análise macroscópica, somente o comprimento o CR e o saco vitelino apresentaram diferença significativa entre os conceptos de monta natural e de FIV. Nos conceptos de monta natural, o comprimento do saco vitelino foi de 5,53 cm, em média, e nos conceptos de FIV, de apenas 1,07 cm. Todavia, faz-se necessário analisar um número maior de animais submetidos a FIV para corroborar a diferença encontrada nestas medidas. Os resultados sugerem ainda a existência de uma placenta vitelínica ativa, importante para a manutenção da gestação, que se estabelece temporariamente entre a placenta coriovitelínica e alantovitelínica transitória e a placenta cório-alantóide definitiva. / The main goal of this study was to describe morphology and the early gestational development of 15 to 70 day-old bovine embryos obtained by natural mating and 35 day-old bovine embryos obtained by in vitro fertilization (IFV) technique. One hundred and forty-one concepts originated by natural mating and seven by IVF technique were used. All concepts were dissected, macroscopically measured and photographed. Extraembryonic membranes were cut in 5 cm2 fragments and fixed in 4% paraformoldehyde for light and Scanning Electron Microscopy (SEM) and in 2.5% glutaraldehyde for Transmition Electron Microscopy (TEM). Ali membranes showed different stages of development during analyzed periods. The beginning of allantois macroscopic vascularization, the attempt of fusion between allantois and chorium membranes and the effective development of the first cotyledons were observed in 30-to-40 day-old embryos with 1.9± 0.24 cm of Crown-Rump (CR) length. The average CR, the embryos and gestational sac weight, the chorion and amnion length increased during gestation. The allantoic epithelia showed a cellular dimorphism with 0.9 em CR, however, the maturation has not happened until the foetal length of 7.2 em CR. The trophoblast showed different levels of mononucleate cells and binucleate giant cells. The yolk sac persisted until 70 days of gestation and the epithelium seemed functionally activated until 50 days of gestation. The CR and yolk sac size were the only measured macroscopic parameter showingdifference between natural mating and FIV concepts. Yolk sac size presented average values of 5.53 cm and 1.07 cm for natural mating and FIV concepts, respectively. Therefore, it is necessary to analyze a higher number of FIV embryos to corroborate that observed yolk sac size difference. All results obtained in this study suggest the existent of an active vitelline placenta, important to the gestational maintenance and temporarily establish between the transitory choriovitelline placenta and the permanent chorioallantoic placenta.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22324号 / 医博第4565号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 竹内 理, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

We review morphological features of the amniote egg and embryos in a comparative phylogenetic framework, including all major clades of extant vertebrates. We discuss 40 characters that are relevant for an analysis of the evolutionary history of the vertebrate egg. Special attention is given to the morphology of the cellular yolk sac, the eggshell, and extraembryonic membranes. Many features that are typically assigned to amniotes, such as a large yolk sac, delayed egg deposition, and terrestrial reproduction have evolved independently and convergently in numerous clades of vertebrates. We use phylogenetic character mapping and ancestral character state reconstruction as tools to recognize sequence, order, and patterns of morphological evolution and deduce a hypothesis of the evolutionary history of the amniote egg. Besides amnion and chorioallantois, amniotes ancestrally possess copulatory organs (secondarily reduced in most birds), internal fertilization, and delayed deposition of eggs that contain an embryo in the primitive streak or early somite stage. Except for the amnion, chorioallantois, and amniote type of eggshell, these features evolved convergently in almost all major clades of aquatic vertebrates possibly in response to selective factors such as egg predation, hostile environmental conditions for egg development, or to adjust hatching of young to favorable season. A functionally important feature of the amnion membrane is its myogenic contractility that moves the (early) embryo and prevents adhering of the growing embryo to extraembryonic materials. This function of the amnion membrane and the liquid-filled amnion cavity may have evolved under the requirements of delayed deposition of eggs that contain developing embryos. The chorioallantois is a temporary embryonic exchange organ that supports embryonic development. A possible evolutionary scenario is that the amniote egg presents an exaptation that paved the evolutionary pathway for reproduction on land. As shown by numerous examples from anamniotes, reproduction on land has occurred multiple times among vertebrates—the amniote egg presenting one “solution” that enabled the conquest of land for reproduction.

Development of the yolk sac of squamate reptiles (lizards and snakes) differs from other amniote lineages in the pattern of growth of extraembryonic mesoderm, which produces a cavity, the yolk cleft, within the yolk. The structure of the yolk cleft and the accompanying isolated yolk mass influence development of the allantois and chorioallantoic membrane. The yolk cleft of viviparous species of the Eugongylus group of scincid lizards is the foundation for an elaborate yolk sac placenta; development of the yolk cleft of oviparous species has not been studied. We used light microscopy to describe the yolk sac and chorioallantoic membrane in a developmental series of an oviparous member of this species group, Oligosoma lichenigerum. Topology of the extraembryonic membranes of late stage embryos differs from viviparous species as a result of differences in development of the yolk sac. The chorioallantoic membrane encircles the egg of O. lichenigerum but is confined to the embryonic hemisphere of the egg in viviparous species. Early development of the yolk cleft is similar for both modes of parity, but in contrast to viviparous species, the yolk cleft of O. lichenigerum is transformed into a tube-like structure, which fills with cells. The yolk cleft originates as extraembryonic mesoderm is diverted from the periphery of the egg into the yolk sac cavity. As a result, a bilaminar omphalopleure persists over the abembryonic surface of the yolk. The bilaminar omphalopleure is ultimately displaced by intrusion of allantoic mesoderm between ectodermal and endodermal layers. The resulting chorioallantoic membrane has a similar structure but different developmental history to the chorioallantoic membrane of the embryonic hemisphere of the egg.

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.

In 1926, the German biologist Johanna (Hanni) Hrabowski published a study of the morphology and development of the fetal placenta in lizards that has proven to be of historical importance. Her anatomical descriptions and interpretations identified developmental patterns that differ from other amniotes – features now recognized as unique attributes of squamate (lizards and snakes) development. Her 1926 monograph presented the first histological comparison of fetal membranes in closely-related oviparous and viviparous reptiles, thereby establishing a comparative framework for understanding placental specializations for viviparity. Hrabowski reported that yolk sac development did not differ between oviparous and viviparous species. The novel, shared components of yolk sac development she identified are now recognized as the foundation for the unique yolk sac placenta of reptiles, the omphaloplacenta. In addition, Hrabowski's extensive ontogenetic sampling and the detail and accuracy of her anatomical descriptions set high standards for subsequent studies of comparative evolutionary embryology.

The Aquarius/SAC-D joint international science mission, between the National Aeronautics and Space Administration (NASA) of United States and the Argentine Space Agency (Comision Nacional de Actividades Espaciales, CONAE), was launched on a polarorbiting satellite on June 10, 2011. This mission of discovery will provide measurements of the global sea surface salinity, which contributes to understanding climatic changes in the global water cycle and how these variations influence the general ocean circulation. The Microwave Radiometer (MWR), a three channel Dicke radiometer operating at 23.8 GHz H-Pol and 36.5 GHz V-& H-Pol provided by CONAE, will complement Aquarius (NASA’s L-band radiometer/scatterometer) by providing simultaneous spatially collocated environmental measurements such as water vapor, cloud liquid water, surface wind speed, rain rate and sea ice concentration. This dissertation focuses on the overall radiometric calibration of MWR instrument. Which means establishing a transfer function that relates the instrument output to the antenna brightness temperature (Tb). To achieve this goal, the dissertation describes a microwave radiative transfer model of the instrument and validates it using the laboratory and thermal-vacuum test data. This involves estimation of the losses and physical temperature profile in the path from the receiver to each antenna feed-horn for all the receivers. As the pre-launch laboratory tests can only provide a simulated environment which is very different from the operational environment in space, an on-orbit calibration of the instrument is very important. Inter-satellite radiometric cross-calibration of MWR using the Naval Research iii Laboratory’s multi-frequency polarimetric microwave radiometer, WindSat, on board the Coriolis satellite is also an important part of this dissertation. Cross-calibration between two different satellite instruments require normalization of Tb’s to account for the frequency and incidence angle difference between the instruments. Also inter-satellite calibration helps to determine accurate antenna pattern correction coefficients and other small instrument biases.