Abstract Type XIII collagen is a type II transmembrane protein which is expressed in many tissues throughout development and adult life. It is located in focal adhesions of cultured cells and in the adhesive structures of tissues such as the myotendinous junctions in muscle, intercalated discs in the heart and the cell-basement membrane interphases. To further characterize the function of this protein, we generated transgenic mice overexpressing it in normal and mutant forms. A large in-frame deletion in the COL2 domain of type XIII collagen led to synthesis of truncated α1(XIII) chains in transgenic mice, disrupting the assembly of normal type XIII collagen trimers. Fibroblasts derived from the mutant mice expressed shortened α1(XIII) chains, and no intracellular accumulation of the mutant protein was detected, suggesting that the mutant molecules were expressed on the cell surface. Transgene expression led to an embryonally lethal phenotype in offspring from heterozygous mating at two distinct stages of development. The early phenotype fetuses died due to the lack of chorioallantoic fusion and functioning placenta at 10.5 dpc, while the death of the late phenotype fetuses was caused by cardiac and placental defects around 13.5 dpc. The phenotype resembles closely several other cell adhesion molecule mutants, indicating that type XIII collagen has an essential role in certain adhesive interactions that are necessary for normal development. Mice overexpressing type XIII collagen with or without a point mutation developed postnatally an unexpected skeletal phenotype marked by a massive increase in bone mass. The cortical bone cross-sectional area and volumetric bone mineral density were highly increased, but trabecular bone volume was not significantly altered. The bone formation rate was several times higher in the mutant mice than in their normal littermates, while the osteoclast number and resorption activity were normal. Type XIII collagen was expressed highly in primary osteoblasts derived from the transgenic mice. Overexpression of type XIII collagen in osteoblasts enhanced both cell proliferation and differentiation while lack of it had opposite effects. Furthermore, mutant cells responded to mechanical strain differently than wild-type cells. The findings suggest that type XIII collagen has an important role in bone modeling, and it may in particular have a function in coupling the regulation of bone mass to mechanical usage.

Les biomatériaux interviennent dans de nombreuses applications médicales. La connaissance de leur évolution une fois implantés dans l’organisme est primordiale pour les améliorer et en créer de nouveaux. Dans cette optique, nous avons réalisé une modélisation à deux dimensions de la transformation d’un biomatériau en os. Pour cette modélisation, nous utilisons la théorie de la percolation. Celle-ci traite de la transmission d’information à travers un milieu où sont distribués un très grand nombre de sites pouvant localement relayer cette information. Nous présentons un modèle de double percolation sites-liens, pour prendre en compte d’une part la vascularisation (et/ou résorption) du biomatériau de l’implant dans un os, et d’autre part sa continuité mécanique. Nous identifions les paramètres pertinents pour d´écrire l’implant et son évolution, qu’ils soient d’origine biologique, chimique ou physique. Les différents phénomènes sont classés suivant deux régimes, percolant ou non-percolant, qui rendent compte des phases avant et après vascularisation de l’implant. Nous avons testé notre simulation en reproduisant les données expérimentales obtenues pour des implants de corail. Nous avons réalisé une étude des différents paramètres de notre modèle, pour déterminer l’influence de ceux-ci sur chaque phase du processus. Cette simulation est aussi adaptable à différents systèmes d’implants. Nous montrons la faisabilité d’une modélisation à trois dimensions en transposant la partie statique de notre simulation. / Biomaterials play an important role in many medical applications. To know how they evolve once inserted in the human body is essential to improve them and to create new ones. For this purpose, we have elaborated a two dimensional model for the transformation of biomaterial into bone. For this model, we have used the percolation theory. This general theory accounts for the transmision of information across an environment in wich a huge number of sites relay localy this piece of information. We present a double site-bond percolation model to account, on the one hand, for the vascularization (and/or resorption) of biomaterial implant in bones and, on the other hand, for its mechanical continuity. We identify the relevant parameters to describe the implant and its evolution, and separate their biological or chimical origin from their physical one. We classify the various phenomena in two regimes, percolating or non-percolating, which concern the two stages before and after the vascularization of the implant. We have tested our simulation by comparing them with experimental results obtained withcoral implants. We have studied how the various parameters of our model can influence each stage of the process. This simulation can also be applied to different types of implants. We show that a three dimensional model is possible by transposing the static part of our simulation.

Although the RING finger protein 121 (RNF121) is a highly conserved E3 ubiquitin ligase from Caenorhabditis elegans to human, its function is poorly understood and in higher eukaryotes it has been studied only at in vitro level. RNF121 has been described to have various functions: i) it was ascribed to function as a broad regulator of NF-κB activation, ii) it was shown to control intracellular trafficking of various membrane proteins, and iii) its downregulation leads to apoptosis. Moreover, RNF121 might have a role in cancer as its expression was found to be 16.4-fold higher in patients suffering from Barrett esophagus (precancerous lesion of esophageal adenocarcinoma) and was even more increased in esophageal adenocarcinoma comparing to healthy population. In addition, RNF121 gene is localized in the candidate region containing breast cancer susceptibility genes. To gain insight into physiological functions of RNF121, Rnf121 knockout mice (Rnf121tm1b(EUCOMM)Hmgu ) were generated in the Czech Centre for Phenogenomics and further studied in our laboratory. Rnf121+ /- intercross breedings showed a prenatal lethal phenotype of Rnf121-/- embryos, which were dying prior embryonic day (E) 11.5. Preliminary experiments carried out in our laboratory showed numerous vascular defects in null mutant embryo,...

One of the major technical challenges with creating 3D artificial tissue constructs is the lack of simple and effective methods to integrate vascular networks within them. Without these vascular-like networks, the cells embedded within the constructs quickly become necrotic. This thesis details the use of a commercially available, low-cost, 3D printer modified with a microfluidic printhead in order to generate instantly perfusable vascular-like networks integrated within gel scaffolds seeded with cells. The printhead featured a coaxial nozzle that allowed the fabrication of hollow, gel tubes (500µm–2mm) that can be easily patterned to create single or multi-layered constructs. Media perfusion of the channels caused a significant increase in cell viability. This microfluidic nozzle design was further modified to allow for multi-axial extrusion in order to 3D print and pattern bi- and tri-layered hollow channel structures. Most available methodologies lack the ability to create multi-layered concentric conduits inside natural extracellular matrices, which would more accurately replicate the hierarchal architecture of biological blood vessels. The nozzle used in this work allowed, for the first time, for these hierarchal structures to be embedded within layers of gels in a fast, simple and low cost manner. This scalable design allowed for versatility in material incorporation, thereby creating heterogeneous structures that contained distinct concentric layers of different cell types and biomaterials. This thesis also demonstrates the use of non-extrusion based 3D biofabrication involving planar processing by means of hydrogel adhesion. There remains a lack of effective adhesives capable of composite layer fusion without affecting the integrity of patterned features. Here, silicon carbide was found for the first time to be an effective and cytocompatible adhesive to achieve strong bonding (0.39±0.03kPa) between hybrid hydrogel films. Multi-layered, heterogeneous constructs with embedded high-resolution microchannels (150µm-1mm) were fabricated in this way. With the new 3D fabrication technology developed in this thesis, gel constructs with embedded arrays of hollow channels can be created and used as potential substitutes for blood vessel networks as well as in applications such as drug discovery models and biological studies. / Thesis / Doctor of Philosophy (PhD) / Additive manufacturing (AM) involves any three-dimensional (3D) fabrication technologies that is used to produce a solid model of a predetermined design. AM techniques have recently been used in tissue engineering applications for fabrication of 3D artificial tissues that resemble architectures and material properties similar to that of the native tissue. Utilizing AM for this purpose presents the advantage of increased control in feature patterning, which leads to the realization of more complex geometries. However, there still remains a lack of simple and effective methods to integrate vascular networks within these 3D artificially engineered scaffolds and tissue constructs. Without these vascular-like networks, the cells embedded within the constructs would quickly die due to a lack of nutrient delivery and waste transport. This remains one of the biggest challenges in true 3D tissue engineering. This thesis presents a number of fast, effective and low-cost AM biofabrication techniques to address this challenge.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24812号 / 工博第5155号 / 新制||工||1985(附属図書館) / 京都大学大学院工学研究科マイクロエンジニアリング専攻 / (主査)教授 横川 隆司, 教授 安達 泰治, 教授 永樂 元次 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

La reparation de defauts osseux par les techniques de l’ingenierie tissulaire osseuse (ITO) est consideree comme une alternative aux greffes conventionnelles. L’objectif de ce projet de these fut de developper des materiaux destines a servir de scaffolds pour le comblement et la regeneration osseuse, ces derniers etant sous la forme d’hydrogels injectables d’une part, et d’eponges, d’autre part. Ces deux types de materiaux ont ete obtenus par melange de chitosane (CHT, cationique), et de polymere de cyclodextrine reticule par l’acide citrique (PCD, anionique), interagissant via des liaisons ioniques et formant des complexes polyélectrolytes. La premiere partie de la these a ete consacree au developpement et caracterisation d’une eponge CHT/PCDs qui a ete chargee avec le facteur de croissance de l’endothelium vasculaire (VEFG) dans le but de favoriser sa vascularisation. Le second volet de la these a eu pour objectif d’optimiser la formulation d’un hydrogel injectable destine a la chirurgie mini-invasive, compose de CHT et de PCD sous sa forme soluble (PCDs) et insoluble (PCDi) [CHT/PCDi/PCDs]. L'etude a ete concentree sur l’optimisation et la caracterisation des proprietes rheologiques de l’hydrogel. Enfin, une etude prospective sur le developpement de l'hydrogel/eponge composite en ajoutant une phase minerale - l'hydroxyapatite (HAp) dans la formulation a ete realisee afin d'ameliorer les proprietes mecaniques et osteoconductrices.L’eponges CHT/PCDs a ratio 3 :3 a ete obtenue par lyophilisation des hydrogels et a subi un traitement thermique (TT) afin d’ameliorer leur stabilite par la formation des liaisons covalentes. L’eponge CHT/PCDs avec un TT a 160°C a montre des proprietes de gonflement eleve (~600%) et une biodegradation ralenti induite par le lysozyme (~12% perte masse dans un mois). Sa microstructure, ses proprietes mecaniques de compression et sa cytocompatibilite avec deux types de cellules (pre-osteoblastes (MC3T3-E1) et endotheliales primaires (HUVECs) ont ete etudiees. Une porosite elevee (~87%) avec des pores interconnectes a ete observee par microtomographie de rayons X, ainsi qu’une bonne adhesion et colonisation cellulaire au sein de l’eponge par microscopie electronique a balayage (MEB). Le VEGF a ete incorpore dans l’eponge, et son profil de liberation a ete suivi, ainsi que la bio-activite du VEGF libere. La liberation du VEGF a ete rapide pendant les trois premiers jours, puis ralenti jusqu'a devenir non-detectable par la methode ELISA jusqu’a 7 jours. Le VEGF libere pendant les deux premiers jours a montre un effet pro-proliferation et pro-migration significatif sur les HUVECs.Les hydrogels injectables de CHT/PCDi/PCDs a differents ratios ont ete optimises et caracterises en fonction de leurs proprietes rheologiques, leur injectabilite, et leur cytotoxicite. L’impact de l’ajoute du PCDi dans l’hydrogel a ete clairement observe par analyses rheologiques Ainsi, l'hydrogel CHT/PCD, compose a parts egales de PCDi et de PCDs, a demontre le meilleur compromis entre stabilite structurelle, proprietes rheofluidifiantes et autoreparantes, et injectabilite. En plus, l’hydrogel a montre une excellente cytocompatibilite vis-avis les cellules pre-osteoblastes MC3T3-E1.Bases sur la formulation optimisee, l’HAp a ete incorporee a differentes concentrations dans l’hydrogel. L’ajout de la phase minerale n’a pas perturbe la formation ni la stabilite structurelle des hydrogels, mais a ameliore les proprietes viscoelastiques. Les eponges composites, elaborees par lyophilisation de ces hydrogels, ont montre que les particules de HAp etaient dispersees de maniere homogene dans la structure macroporeuse de l'eponge. Ces resultats encourageants ont montre qu'il etait possible de fournir un hydrogel injectable ou une eponge composite comme scaffold pour l’ITO [...] / Repair of bone defects by bone tissue engineering (BTE) methods is considered as an alternative to conventional grafts. The aim of this PhD project was to develop two types of BTE scaffolds for bone regeneration: one is in the form of injectable hydrogel, and the other is in the form of sponge. Both scaffolds based on the formation of polyelectrolyte complexes by mixing chitosan (CHT, cationic) and polymer of cyclodextrin (PCD, anionic). Besides developing the sponge scaffold, the vascularization of 3D scaffold (a challenge of BTE) was specially investigated in the first part of the work, for which vascular endothelial growth factor (VEFG) was loaded on the CHT/PCDs sponge to promote the vascularization. The second part of the thesis was dedicated to the elaboration of an injectable CHT/PCD hydrogel, which was intended for minimally invasive surgery. The formulation optimization of hydrogel was performed by tuning the composition ratios of two PCD components: soluble-form PCD (PCDs) and insoluble-form PCD (PCDi), in order to better reach the specific requirement (e.g. rheological properties) of injectable hydrogel for regenerative medicine. Finally, a prospective study on developing the composite hydrogel/sponge by adding a mineral phase - hydroxyapatite (HAp) in the formulation was realized to improve the mechanical and osteoconductive properties.CHT/PCDs sponges were obtained by freeze-drying the hydrogels CHT/PCDs 3:3. The thermal treatment (TT) at different temperatures was further applied on the sponge to improve the mechanical stability. The CHT/PCDs sponge treated at 160°C was opted for further study thanks to high swelling capacity (~ 600%) and moderate lysozyme-induced biodegradation rate in vitro (~ 12% mass loss 21 days). This sponge of choice was further evaluated for the microstructure, the mechanical property (compressive strength) and the cytocompatibility with pre-osteoblasts (MC3T3-E1) and endothelial cells (HUVEC). Results of X-ray microtomography showed a high porosity (~87%) in the sponge with interconnected pores. Good cell adhesion and in-growth (colonization) in the sponge were observed by scanning electron microscopy (SEM). After loading VEGF on the sponge, the release profile of VEGF and the bioactivity of released VEGF were thoroughly studied. It showed that the release of VEGF was rapid (burst) during the first two days, then slowed down up to non-detectable by ELISA method after 7 days. The released VEGF during the first two days showed a significant pro-proliferation and pro-migration effect on HUVECs.For the injectable CHT/PCDi/PCDs hydrogels, optimization of composition ratio was based on evaluating their rheological properties, injectability, and cytotoxicity. The beneficial effect of combining both PCDi and PCDs in the formula of the hydrogel was clearly observed on the properties of hydrogel. Namely, the CHT/PCD hydrogel, composed of equal quantity of PCDi and PCDs, demonstrated the best compromise between structural stability, shearthinning and self-healing properties, and injectability. An excellent cytocompatibility with preosteoblast cells (MC3T3-E1) was also confirmed for the hydrogel with this composition.Based on the optimized formulation, HAp was incorporated at different concentrations, which didn’t disturb the formation or the structural stability of the hydrogels, but improved the viscoelastic properties. The composite sponges, elaborated by lyophilization of these hydrogels, showed that the HAp particles homogeneously dispersed within the macroporous structure of the sponge. These encouraging results showed the feasibility of providing an injectable hydrogel or a composite sponge for BTE scaffold [...]

Euphorbiaceae s.l. are distributed in the most varied types of vegetation and habitat, being one of the biggests, most complexs and diversified families in the angiosperms. Its classification was discussed during long time by many authors and with the phylogenetic analyses was proved its polyphyletic origin, bearing the dissolution in six distinct families: Phyllanthaceae, Picrodendraceae, Putranjivaceae, Pandaceae, Peraceae e Euphorbiaceae s.s. Considering the floral diversity of these families, fours species were selected to this study, aiming to sample the different groups: Phyllanthus urinaria (Phyllanthaceae), Piranhea trifoliate (Picrodendraceae), Alchornea sidifolia (Euphorbiaceae s.s.) and Microdesmis caseariifolia (Pandaceae). There are few detailed literature about the floral structure of the representants from the allied families of Euphorbiaceae s.l., which makes difficult the accurate usage of the floral characters in studies about systematics and evolution of these groups. Furthermore, information on the functional implications of these characteristics in the biology of the species are limited. In this context, the aim of this work was analyse the floral morphology, with an emphasis on the development patterns, vascularization and secretory structures. For this purpose, floral buds and flowers in different stages of development were fixed, dehydrated, embedded in Paraplast or historesin, sectioned and stained, besides the SEM and TEM analysis that helped on the investigation of the floral ontogenesis and the nectariferous tissue. As main results of the structural analysis, we found that Phyllanthus urinaria has sepals and petals, as well Piranhea trifoliate, both from the clade Phyllanthaceae + Picrodendraceae. Then, flowers from these groups should be studied from its origin and vascularization to have a better understanding of the evolutionary path of the perianth in these families. Piranhea trifoliate also have staminodes that may indicate a transitional process during the evolution of the group. In Alchornea sidifolia we found development patterns that explain the formation of characteristics of anemophilous plant, such as the production of more flowers in the male inflorescences than the more compact and fewer flowered female inflorescences, the presence of unisexual flowers with reduced perianth, absence of nectaries, and female flowers with extensive stigmatic receptive surface. This species also has a peculiar characteristic regarding the number of carpels, which is generally three in the Euphorbiaceae and Malpighiales, even though A. sidifolia has two carpels. Microdesmis caseariifolia displayed variation in the number of stamens, shown a placental obturator and a pistilode, and all these features are novelties to the Pandaceae. This study adds to the floral knowledge of the different families, as well as enable to raise issues to be addressed in future studies about the structure and evolution of floral characters in Malpighiales / As Euphorbiaceae s.l. distribuem-se nos mais variados tipos de vegetação e habitat, sendo uma das maiores, mais complexas e diversificadas famílias das angiospermas. Sua classificação foi discutida durante muito tempo por diversos autores e, atualmente, as análises filogenéticas, comprovaram seu polifiletismo, dando suporte ao desmembramento em seis famílias distintas: Phyllanthaceae, Picrodendraceae, Putranjivaceae, Pandaceae, Peraceae e Euphorbiaceae s.s. Considerando a diversidade floral destas famílias, quatro espécies foram selecionadas para este trabalho, visando amostrar os diferentes grupos: Phyllanthus urinaria (Phyllanthaceae), Piranhea trifoliata (Picrodendraceae), Alchornea sidifolia (Euphorbiaceae s.s.) e Microdesmis caseariifolia (Pandaceae). Poucos são os estudos detalhados sobre a estrutura floral de representantes das famílias segregadas de Euphorbiaceae s.l., o que dificulta o uso acurado dos caracteres florais em estudos sobre sistemática e evolução dos grupos em questão. Além disso, as informações sobre as implicações funcionais dessas características na biologia das espécies são limitadas. Nesse contexto, o objetivo desse estudo foi analisar a morfologia da floral, com ênfase nos padrões de desenvolvimento, vascularização e estruturas secretoras. Para isso, botões florais e flores em diversos estágios de desenvolvimento foram fixados, desidratados, incluídos em parafina ou historresina, seccionados e corados, além das análises em MEV e MET que auxiliaram na investigação da ontogênese floral e tecido nectarífero. Como principais resultados das análises estruturais, encontramos que Phyllanthus urinaria possui sépalas e pétalas, bem como Piranhea trifoliata, espécies pertencentes ao clado Phyllanthaceae + Picrodendraceae. Logo, flores desses grupos devem ser analisadas a partir de sua origem e vascularização para que haja um melhor entendimento da trajetória evolutiva do perianto nessas famílias. Piranhea trifoliata também apresentou estaminódios que podem indicar um processo transicional durante a evolução do grupo. Em Alchornea sidifolia nós encontramos padrões de desenvolvimento que explicam a formação de características de plantas anemófilas, como a produção de mais flores nas inflorescências masculinas do que nas femininas, flores unissexuais com perianto reduzido, ausência de nectários e flores femininas com uma extensa região estigmática. Essa espécie também apresenta uma característica peculiar em relação ao número de carpelos, que geralmente são três nas Euphorbiaceae e na das Malpighiales, mas A. sidifolia apresenta dois carpelos. Microdesmis caseariifolia mostrou variação no número de estames, presença de obturador placentário e pistilódio, sendo todas essas características novidades para a família. Este estudo contribuiu com o conhecimento floral das diferentes famílias, bem como permitiu criar hipóteses a serem abordadas em futuros estudos sobre a estrutura e evolução de caracteres florais em Malpighiales

Les modèles numériques présents dans les salles de chirurgie requièrent simultanément une précision millimétrique, une vitesse de résolution importante, tout en prenant en compte la variabilité inter-patient. Dans l’étude proposée, nous développons un modèle numérique du foie, intégrant des lois de comportement hyper-élastiques ainsi que l’impact mécanique de la vascularisation. Une fois constitué, le modèle est traité par des outils mathématiques de réduction de modèles et d’apprentissage afin de fournir une réponse en temps réel. Pour cela, des données mécaniques sont extraites d’indentations numériques puis homogénéisées, pour construire un modèle numérique intégrant l’impact de la vascularisation. Une fois validé sur un échantillon réel les déformations simulées sont utilisées par une méthode d’apprentissage pour construire une réponse fonctionnelle. Une fois intégrée dans un outil chirurgical, ce dernier permet de fournir une réponse en temps réel des déformations du foie. / Numerical models used for surgical application require simultaneously a precision close to the millimeter, high speed resolution and to account for the patient variabilities. In the present study, we develop a numerical model of the liver, which relies on hyper-elastic mechanical behavior completed with the vascularization impact on the macroscopic level. Once completed and implemented, the model is treated with model reduction and learning tools in order to provide a real-time response. Mechanical properties are extracted from numerical indentations and homogeneised to build a model accounting for the impact of the vascularization. Once validated on a real sample, simulated deformations are used as input to a learning solution to build a functional solution. Finally, the function is integrated in a surgical tool, to provide a quick and precise representation of the liver deformations.

Durante a vida fetal e no período neonatal, o timo é órgão de grande importância imunológica e, anatomicamente é o maior órgão linfático e com alta atividade linfopoiética, constando como precursor da linfopoiese. No que diz respeito à irrigação do timo em cães, a literatura é escassa, visto que os autores quando se reportam ao assunto fazem-no na sua maioria de maneira genérica. Com isso, aspectos morfológicos como número de vasos, tamanho do órgão foram estudados considerando as variáveis sexo e faixas etárias. Para este estudo, foram utilizados 24 fetos de cães domésticos, sem raça definida, machos e fêmeas, divididos em quatro grupos etários. Os timos foram processados para o estudo da microscopia de luz, e as análises estereológicas foram realizadas utilizando o método do disector físico associado com o princípio de ConnEulor. O volume do órgão (Vref), comprimento, espessura e largura aumentaram gradativamente com o desenvolvimento, sendo maior nos machos do que nas fêmeas. As variáveis estereológicas analisadas (densidade de comprimento do vaso - Lv, comprimento do vaso - L, densidade de superfície de área - Sv, superfície de área - S, estimação da densidade numérica vascular ? Nv(vasc) número total de vasos no órgão - N (vasc) ), tiveram um aumento gradativo, sendo que o Lv foi maior nas fêmeas e as demais os machos apresentaram maior valor. Aumento na Nv(vasc) e N (vasc) foi observado nos animais do grupo IV. / During phoetal life and neonatal period, the thymus is the organ that has a wide immunological relevance and, anatomically speaking is the largest lymphoid organ presenting high limphopoietic activity presented as the predecessor of limphopoiesis. The literature about thymus irrigation in dogs is scarce, becouse most authors refer to the subject in a general way. Then, morphological aspects as number, shape, size, irrigation, and quantitative were study considering sexy and differents ages of development of the dogs. To the study, were used twenty-four fetus of the mongrel domestics dogs, males and females, divided into four different well defined aged groups (fetus 30, 40, 50 and 60 days). The thymus were processed for the light microscopy study, and the stereological analyses were done using the physical disector method associated with the ConnEulor principle. The volume of the organ, length, thickness and wide increased gradually with the development, in males is great that female. The stereological variables analysed (length density ? Lv, length od vascullar ? L, surface area density ? Sv, surface area ? S, vascullar number density ? N.v(vasc) , and vascullar total number - N. (vasc) ), had the gradual high, the Lv was more in female and the others variables were more in males. The rise of N.v(vasc) and N. (vasc) was observed on the group four animals.

O corpo lúteo é uma glândula endócrina temporária que regula tanto o ciclo estral quanto a prenhez, apresentando extrema dependência de aporte sanguíneo adequado. O tratamento superovulatório aumenta a concentração sérica de progesterona (P4) e conseqüentemente as taxas de concepção e de prenhez. Esse trabalho objetivou quantificar a vascularização dos corpora lútea (CLL) de animais controle e superovulados, correlacionando-a com a P4 sérica e expressão de VEGF e seus receptores. Foram utilizadas 30 búfalas, cujos CLL foram divididos em cinco grupos: superovulados (receberam 400 mg de FSH divididos em doses diárias decrescentes: 80 mg, 60 mg, 40 mg e 20mg a cada 12 horas durante 4 dias), corpos hemorrágicos (CH), corpo lúteo maduro (CL), CL em regressão (CR) e corpo albicans (CA), que não receberam nenhum tratamento. Três CLL de cada grupo foram fixados em formol tamponado para quantificação da densidade vascular e imunolocalização de VEGF, VEGFR-1 e VEGFR-2. Os três restantes foram injetados com resina Mercox para análise da microvascularização. Amostras de sangue foram coletadas e a P4 mensurada através de RIA convencional. A densidade capilar média encontrada foi de 37,78 ± 8,89; 17,8 ± 3,33; 11,92 ± 3,57; 10,83 ± 2,42 e 3,46 ± 1,66 vasos/ mm2 respectivamente para os cinco grupos, indicando maior vascularização (p<0,001) para o grupo superovulado. A microvasculatura apresentou comportamento semelhante para ambos os grupos, revelando apenas maior densidade da rede capilar dos CLL de animais superovulados, o que se refletiu nos valores séricos de progesterona que foram significativamente maiores (p<0,05) para estes animais, com concentração média de 5,58 ( 0,97ng/ml vs 2,02 ( 0,16 ng/ml para os animais controle na mesma fase. O VEGF, bem como seus receptores (VEGFR-1 e VEGFR-2) foram encontrados imunohistoquimicamente no CLL de búfalas. A imunoreatividade para o VEGF e receptores, pode ser observada nas células endoteliais e luteínicas a partir do 2° dia após a ovulação (p.o.) até a fase de corpo lúteo em regressão (17° dia p.o.), com forte reação nas fase luteínicas inicial e média. A imunoreatividade foi mais intensa nos animais submetidos a superovulação. / Corpus luteum is a temporary organ, which regulates the estrous cycle and pregnancy; it is extremely dependent on vascularization. During corpus luteum life span P4 production is associated with an increase of capillary number known as angiogenesis. The angiogenic process is modulated by many factors, including VEGF (vascular endothelial growth factor), which is considered the most important of them. Hyperstimulation of ovarian function, a largely spread technique employed in cattle raising, is associated with high levels of estradiol and P4, as well as an increase of capillary invasion of the new formed CL. This study intended to quantify the vascularization of normal and superovulated corpora lutea (CLL), trying to correlate this parameter with blood P4 concentrations and expression of VEGF and its receptors. For that purpose thirty buffalo cows were divided into five groups: superovulated (received 400 mg FSH divided in decreasing doses of 80 mg, 60 mg, 40 mg e 20mg 12/12h during 4 days), corpus hemorragicans (CH), mature corpus luteum (CL), regression CL (CR) e corpo albicans (CA), that received no treatment and were slaughtered at days 2, 9, 17 and 26 after ovulation. After slaughter ovaries were collected and corpora lutea prepared as follows: three animals from each group had their CLL fixed in buffered formol solution, cut into 0,5 cm pieces, dehydrated in increasing ethanol concentrations, cleared in xylene and embedded in paraffin using conventional procedures. Five µm slices were prepared for quantification of vascular density and immunolocalization of VEGF, VEGFR-1 and VEGFR-2. The ovaries of the remaining animals were injected with Mercox resin through ovarian artery in order to have the microvascularization evaluated by electron scanning microscopy. Blood samples were collected and P4 measured through conventional RIE. Capillary density during CL life span was 37,78 ± 8,89; 17,8 ± 3,33; 11,9 ± 3,57; 10,83 ± 2,42 and 3,46 ± 1,66 vessels/ mm2 respectively for superovulated (CS), CH, CL, CR and CA, indicated higher vascularization (p<0,001) for superovulated group. The microvasculature showed similar behavior: the density of capillary network was higher in CLL of superovulated animals. Values of serum progesterone were significantly higher (p<0,05) for CS animals: 5,58 ± 0,97 ng/ml vs 2,02 ± 0,16 ng/ml for control animals in the same stage of estrous cycle. The VEGF system and its receptors (VEGFR-1 e VEGFR-2) were immunolocalized in CLL of buffalo cows. The immunoreactivity could be detected in endothelial and luteal cells since day two post ovulation (p.o.) until the stage of regression corpus luteum (Day 17 p.o.), with strong immunostaining at the early and midluteal phase. The immunostaining was more intense in CLL of animals submitted to superovulation.