Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 1997. / Typescript. Bibliography: leaves [222]-256.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

A expressão da conexina 32 e 43 nas células gigantes trofoblásticas foi analisada em condições de cultura celular com e sem influência de hormônios sexuais. Placentônios bovinos foram coletados de vacas prenhes em abatedouro nos diferentes períodos gestacionais e divididos em três grupos, primeiro terço (I), segundo terço (II), terceiro terço (III) e transportados ao laboratório em condições assépticas à temperatura de 4ºC em solução de PBS com antibiótico. No laboratório as células foram isoladas e cultivadas em meio D-MEM com 10% SFB por cinco dias. As detecções das conexinas 32 e 43 foram realizadas através de munofluorescencia, pelo método de amplificação da tiramida-fluoresceina, utilizando anticorpo primário policlonal a imunoglobulina de coelho, anti-conexina 32 e 43 de camundongo. Os resultados mostraram que as células gigantes trofoblásticas expressam conexinas 32 e 43 nos três períodos gestacionais com exceção da Cx32 no primeiro terço gestacional sem adição de hormônios, a qual passou a expressar fluorescência após a adição de hormônios. A distribuição da Cx43 evoluiu com a progressão da gestação, permanecendo limitada ao interior das células gigantes trofoblásticas, sem formar junções comunicantes. O terceiro terço gestacional mostra a Cx43 na CGTT localizada no interior do núcleo. A adição de hormônios ao meio de cultura vem confirmar que a progesterona e o estrógeno podem ter um papel no controle da Cx32. Para Cx43 onde se adicionou progesterona os níveis de expressão foram baixos no início aumentando no decorrer da gestação, o mesmo foi encontrado para o conjugado de progesterona/ estrógeno. Para o estrógeno no grupo I os níveis de expressão foram menores se comparados aos três grupos diminuindo no grupo II e voltando a aumentar no grupo III. / The trophoblast cells expression of connexins 32 and 43 was studied in cell culture conditions with or without influence of sexual hormones. Bovine placentomes were collected from pregnant cows in slaughterhouses in different gestational periods and so divided into three groups, first term (I), second term (II), and third term (III), and transported to the laboratory in aseptic conditions at a temperature of 40C in PBS solution with antibiotics. At the laboratory the cells were isolated and cultivated in D-MEM environment with 10% SFB for five days. The detections of connexins 32 and 43 were achieved through immunofluorescence, by the tyramide-fluorescein amplification method, using a rabbit polyclonal primary antibody anti-connexin 32 and 43 of mice. The results showed that the trophoblast cells expressed connexins 32 and 43 in the three gestational periods with an exception of the Cx32 at the first gestational period. However, after the addition of sexual hormones, they began to express the connexins in the cytoplasm in the first stage. The distribution of the Cx43 evolved with the progression of the gestation, remaining limited to the trophoblast`s cells interior, without formation of gap junctions. The third gestational term shows the Cx43 located inside the nuclei of the CGTT. Addition of hormones in the culture environment confirmed that estrogen and progesterone may have an important action controlling Cx32. For Cx43, the expression was low after prosterone was added to the culture medium, but increased as the gestation evolved, the same was found for a combined compost of estrogen\\ progesterone. For estrogen, in group I the expression levels were inferior when compared to the three groups decreasing in group II and increasing again in group III. We may conclude that the sexual hormes, specially estrogen, affect the expression of connexins in trophoblastic cells.

Os bovinos são considerados a maior fonte de proteína animal para o homem, razão pela qual se fazem imprescindíveis pesquisas em diversas áreas de produção desses animais com o intuito de minimizar problemas que possam interferir no desenvolvimento e produtividade dos rebanhos. Nesse cenário, o estudo da placenta e placentação tem por objetivo conhecer o desenvolvimento embrionário e suas interações fisiológicas entre o feto e o organismo materno, caracterizado pela íntima união entre eles. O estudo da placenta e placentação tem por objetivo conhecer o desenvolvimento embrionário e suas interações fisiológicas entre o feto e o organismo materno, caracterizado pela íntima união entre eles. Além disso, a falta de marcadores específicos para células placentárias bovinas e as alterações que essas células sofrem durante o progresso da gestação são as principais barreiras para a determinação do perfil morfológico celular presente na interface materno-fetal, razão pela qual seu monitoramento torna-se importante. Buscando melhor compreender essas mudanças, o presente trabalho estudou a dinâmica populacional de células de placenta bovina a fresco e em cultivo. Foram utilizadas 30 amostras de placentônios, divididos em três grupos, de acordo com o desenvolvimento da gestação, quais sejam: primeiro, segundo e terceiro trimestres (n:10 para cada terço). Células oriundas de placentônios foram examinadas a fresco e pós-cultivo durante dez dias, pela microscopia óptica, microscopia eletrônica de transmissão e citometria de fluxo. Os resultados deste trabalho demonstraram a presença de tipos morfológicos celulares distintos na dependência do progresso da gestação: células gigantes trofoblásticas binucleadas, células gigantes trofoblásticas trinucleadas, células gigantes trofoblásticas mononucleadas, células mononucleadas maiores, células mononucleadas menores e células que apresentam citoplasma amplo, contendo inúmeras vesículas em seu interior. Com a utilização da citometria de fluxo, foi possível identificar nas amostras a fresco três populações distintas em tamanho (FSC) e complexidade (SSC) no primeiro terço, cinco populações no segundo terço e duas populações no terceiro terço gestacional. As células placentárias bovinas apresentaram freqüências variáveis de acordo com o trimestre gestacional e seu tempo de cultivo. / There is a great diversity of cell types in the bovine placenta like mononucleate trophoblast giant cells, binucleate trophoblast giant cells, specialized leucocytes (uterine natural killer, uterine macrophages, etc.) and part of their characterization and function remains unknown or controversial. Besides that, some of these cells change as pregnancy progresses; thus their monitoring is very important. The lack of specific markers to bovine placental cells is a main barrier for the development of an efficient cell tracing. Alternative techniques are being used to study these cells, as well as morphologic observation by optical and electron microscopy methods. The study was carried out to establish a populational kinetics of fresh and cultured bovine placental cells by optical microscopy, by electron microscopy and flow cytometry. Bovine placentomas were sampled at the slaughterhouse from cows with different trimesters of pregnancy (n:10 each). Fragments from the cotiledonary/caruncular interface were collected and cells were mechanically dissociated and their viability was estimated by Trypan Blue exclusion. After that they were incubated (2,5x107 cells per 2 mL) in Dulbecco´s Modified Eagle Medium with 10% FBS. First of all, fresh bovine placental cells analyzed by flow cytometry in order to identity the profiles of populational cells. At the same time, smear of fresh cells was made, fixed by methanol, stained with Giemsa for optical microscopy morphologic analisys and differential counting. After this, cells were analised by flow cytometry and optical microscopy .This process was repeated on the first, fifth and tenth days of the culture. We found seven types of cell: mononucleate trophoblast giant cells, binucleate trophoblast giant cells, trinucleate trophoblast giant cells, large mononucleate cells and small mononucleate cells. The placental cells displayed different frequencies according to the gestation period and culture time length.

O presente trabalho estudou a expressão da conexina 43 nas células trofoblásticas gigantes bovinas, em especial nas células trofoblásticas gigantes binucleadas. Foram utilizadas 12 amostras de placenta bovina, divididas em três grupos segundo a fase gestacional, primeiro terço, segundo terço e terceiro terço da gestação. O tecido placentário foi fixado em solução metacarn e, posteriormente, submetido ao método tradicional de inclusão em parafina. A detecção da conexina 43 foi realizada através de imuno-histoquímica, pelo método de amplificação da tyramida, utilizando como anticorpo primário policlonal a imunoglobulina de coelho anti-conexina 43 de camundongo. Os resultados deste trabalho indicaram que as células trofoblásticas gigantes bovinas são capazes de expressar a conexina 43 nas três fases da gestação. A distribuição da conexina 43 evoluiu com a progressão da gestação, mas ficou limitada ao interior das células trofoblásticas gigantes bovinas, sem formar marcações pontuais na membrana citoplasmática que pudessem indicar a formação de junções comunicantes. A avaliação de diferentes protocolos histológicos e imuno-histoquímicos permitiu estabelecer a melhor metodologia para a preservação do arranjo tecidual da placenta bovina e a identificação da conexina 43 nas células trofoblásticas gigantes bovinas / The present study has researched the expression of Connexin-43 in bovine trophoblast giant cells, especially in binucleate trophoblast giant cells. Twelve samples of bovine placenta were used, divided into three groups according to the gestational phase, first part, second part and third part of gestation. The placental tissue was fixed in Methacarn solution and later submitted to the traditional method, that is, embedded in paraffin. The detection of Connexin-43 was done through immunohistochemistry by the tyramide amplification method, using as polyclonal primary antibody, rabbit immunoglobuline anti-mouse Connexin-43. The results of this study have revealed that the bovine trophoblast giant cells are capable of the expression of Connexin-43 in the three phases of gestation. The distribution of Connexin-43 evolved as gestation progressed, but was limited to the interior of the bovine trophoblast giant cells without punctate markings in the cytoplasmic membrane which could indicate the formation of communicating junctions. The assessment of different histological and immunohistochemical protocols allowed us to establish the best methodology for the preservation of the tissue arrangement of bovine placenta and the identification of Connexin-43 in the bovine trophoblast giant cells

A função da eritrofagocitose observada após o extravasamento de sangue na interface materno-fetal é indefinida em várias espécies, incluindo o búfalo. Na ovelha, este processo foi muito estudado, e ocorre na zona arcada do placentônio (topos dos septos maternos e base dos vilos fetais), região onde o processo é realizado pelo trofoblasto. É possível que o ferro seja transferido para o feto mediante a eritrofagocitose trofoblástica nesta área hemófaga da placenta e nas glândulas endometriais. Para este estudo foram utilizadas placentas de búfalas entre 2-3, 4-5, 6-7, 8-9 e 10 meses de prenhez, fixadas por perfusão com solução aquosa de formaldeído a 10% e paraformaldeído a 4%, para microscopia de luz, e glutaraldeído a 2,5%, para microscopia eletrônica de transmissão, processadas e coradas para microscopia de luz (HE, azul de Toluidina, tricromo de Gomori, Hematoxilina-floxina, Azul de metileno - fucsina básica), histoquímica (reação de Perls, PAS e fosfatase ácida) além de microscopia eletrônica de transmissão. A metodologia utilizada permitiu-nos observar que as áreas hemófagas estavam presentes em determinadas regiões do placentônio, nas quais se identificavam áreas hemorrágicas entre o epitélio uterino e o trofoblástico, nas placentas de 4 a 10 meses de prenhez. Eritrócitos foram encontrados nas células trofoblásticas, elucidando, deste modo, a eritrofagocitose. A reação de PAS foi positiva, marcando substância mucóide, principalmente na base dos vilos fetais, células trofoblásticas binucleadas e nas glândulas endometriais da região interplacentomal. A reação de Perls foi negativa nos placentônios e positiva nas glândulas endometriais. A reação de fosfatase ácida foi positiva tanto nos placentônios, quanto na região interplacentomal. A ultraestrutura da região das áreas hemófagas revelou eritrócitos ingeridos dentro das células trofoblásticas epiteliais em diferentes fases de digestão e eletrondensidades, várias vesículas endocíticas, cavéola, muitas gotículas lipídicas, retículo endoplasmático rugoso bem desenvolvido e a presença de grande quantidade de mitocôndrias. O epitélio das glândulas endometriais da região interplacentomal é do tipo colunar com a presença de microvilos em seu ápice, e núcleos basais. / The function of erytrophagocytosis observed after blood extravasation in the maternal-fetal interface is indefinite various species, including the water buffalo. In ewe, this process had been hardly studied and it occurs in the placentome arcade zone (top of maternal septa), region where the process is performed by the trophoblast. It is possible that iron is being transferred to the fetus through the trophoblastic erytrophagocytosis in the hemophagous areas of the placenta and in the endometrial glands. In our research we have been using placentomes of buffaloes between 2-3, 4-5, 6-7, 8-9 and 10 months of pregnancy, fixed by perfusion with 10% formaldehyde aqueous solution, 4% paraformoldehyde for light microscopy and 2,5% glutaraldehyde for transmission electron microscopy, processed and stained for light microscopy (HE, Toluidine blue, Gomori trichrome, hematoxilin - floxin, methilen blue ? basic fucsin), histochemistry (Perls, PAS and acid phosfatase reaction) and transmission electron microscopy. The methodology used allowed us to observe that the haemophagous areas were present in determined regions of the placentome, in which there were showing haemorragic areas between the trophoblastic and uterine epithelium, in 4-10 months pregnant placentae. Erythrocytes had been found inside trophoblastic cells, thus contributing to explain the erytrophagocytosis. The PAS reaction was positive, staining mucoid substance, mainly in the basis of the fetal villi, trophoblastic binucleate cells and in an endometrial glands in the interplacentomal region. The Perls reaction was negative in the placenton as well as in the endometrial glands. The acid phosphatase reaction was positive in placenton as well as in the interplacentomal region. The ultrastructure of the haemophagous areas revealed ingested erythrocytes inside the epithelial cells of trophoblast in different phases of digestion and electrondensities, various endocitic vesicles, caveolae, many lipid droplets, well-developed rough endoplasmic reticulum and large number of mitochondrias. The endometrial glands epithelium of interplacentomal region is columnar type with microvilli and basal nuclei.

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Dietary polyphenols have been shown to influence cell migration and invasion in models of tumorigenesis and non-cancerous, healthy cells; however, never shown in EVT cells. We hypothesize that polyphenols present in V. angustifolium leaves will promote trophoblast migration and invasion through ERK and AKT activation. Using the HTR-8/SVneo cell line as a model for EVT cells, the leaf extract increased trophoblast migration and invasion, in an ERK- and AKT-independent manner, and had no effect on cell proliferation or viability. One major polyphenol of the leaf extract was identified and may be an active compound. We have demonstrated for the first time that V. angustifolium leaf extract increases EVT migration and invasion in vitro, thus further investigations examining potential therapeutic applications of this extract in the context of placenta-mediated diseases are warranted.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22744号 / 医博第4662号 / 新制||医||1046(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 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

OBJECTIVE: Our purpose was to determine the effects of insulin and glucose on glucose transport and expression of GLUT1 glucose transporter messenger ribonucleic acid in first-trimester human trophoblast-like cells. STUDY DESIGN: First-trimester human trophoblast-like cells were maintained as a continuous cell line. For 2[3H]deoxy-d-glucose uptake and messenger ribonucleic acid studies the cells were incubated in the presence or absence of insulin (10-7 to 10-11 mol/L) or d-glucose (0 to 50 mmol/L) for 0 to 24 hours. Glucose transport was measured by incubating cells with 0.1 mmol/L,2[3H]deoxy-d-glucose for 5 minutes. Specific uptake was determined by incubating companion cultures with 10 μmol/L cytochalasin B. The cells were then solubilized with sodium hydroxide and the radioactivity counted. Data were expressed as nanomoles of 2[3H]deoxy-d-glucose transported per milligram of protein per 5 minutes and analyzed by one-way analysis of variance with post hoc testing by the method of Tukey. GLUT1 messenger ribonucleic acid was measured by Northern blotting of total ribonucleic acid samples hybridized to a phosphorus 32-labeled complementary deoxyribonucleic encoding the rat GLUT1 glucose transporter. As a control for loading efficiency, blots were stripped and rehybridized to a 40-mer phosphorus 32-labeled β-actin oligonucleotide probe. RESULTS: Insulin treatment resulted in a dose-dependent increase in the transport of 2[3H]deoxy-d-glucose at 24 hours (p < 0.001 at 10-7 mol/L). This change was first detected at 12 hours of incubation. These data closely paralled the insulin-induced increase in GLUT1 messenger ribonucleic acid seen in Northern blots. In contrast to insulin, increasing concentrations of d-glucose did not change the transport of 2[3H]deoxy-d-glucose. However, when cells were incubated in low concentrations of d-glucose (0 or 1 mmol/L), an enhancement in the uptake of 2[3H]deoxy-d-glucose (p < 0.001) was observed. Kinetic studies indicated that d-glucose augmentation of 2[3H]deoxy-d-glucose uptake was significant at 9 hours (p < 0.05). The effects of d-glucose on GLUT1 messenger ribonucleic acid expression paralleled the uptake of 2[3H]deoxy-d-glucose, although the modulation of GLUT1 messenger ribonucleic acid levels by glucose was much less pronounced than in insulin-treated cells. CONCLUSION: Although it has been assumed that the placenta has a limited role in influencing glucose transport to the fetus, our in vitro data demonstrate that both insulin and glucose can modulate glucose transport at the cellular level of the placental trophoblast. Thus maternal insulin and glycemic status may influence the expression of GLUT1, the major trophoblast glucose transporter protein, therefore directly affecting first-trimester placental glucose transport. These in vitro data may help explain the association between maternal glucose abnormalities and impaired fetal development during the first trimester when placental GLUT1 messenger ribonucleic acid expression is at its peak.