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Doctoral Thesis
DOI
10.11606/T.10.2016.tde-10052016-120531
Document
Author
Full name
Naira Caroline Godoy Pieri
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Martins, Daniele dos Santos (President)
Bressan, Fabiana Fernandes
Lopes, Maria Denise
Perecin, Felipe
Portal, Maria Josefina Illera Del
Title in Portuguese
Espermatogênese xenogênica pós-transplante de células tronco caninas no testículo de camundongos
Keywords in Portuguese
Cão
Células tronco
Espermatogônias
SSCs
Abstract in Portuguese
As células tronco espermatogoniais (SSCs) são caracterizadas pela capacidade de autorrenovação, proliferação e transmissão das informações genéticas. Em caninos a primeira tentativa de xenotransplante não obteve o sucesso da produção de espermatozoides, no entanto, há evidências de que as células testiculares xenogênicas podem ser transplantadas no testículo do animal hospedeiro, e gerar espermatozoides viáveis do doador. Portanto, este estudo tem como objetivo realizar o xenotransplante das células germinativas caninas em camundongos imunosuprimidos, e com isto promover à produção de espermatozoides caninos viáveis, geneticamente modificados. E por meio desta técnica, analisar a eficiência da espermatogênese pós-transplante. Células germinativas testiculares foram caracterizadas, isoladas e cultivadas de cães pré-púberes, por meio de sistemas de cultura de enriquecimento e fatores de crescimento. As células foram transduzidas com um gene repórter GFP e LacZ, e por um vetor lentiviral para indentificar as SSCs nos testículos receptores. As SSCs transduzidas foram transplantadas nos testículos de camundongos (C57BL/6) tratados com Busulfan, após diferentes períodos os animais receptores foram eutanasiados e analisados. Aos 10 dias de cultivo as células germinativas adultas foram positivas para CD49f, CD117, e com 5 dias uma expressão semelhante de GFRA1 e DAZL, demonstrando a presença de SSCs e algumas células em meiose. Transplantamos 105 células e 20-43% das células transplantadas foram identificadas na membrana basal dos túbulos seminíferos do animal receptor. Portanto, o transplante das células germinativas caninas, mostrou que a purificação e o cultivo realizados são possíveis para obter SSCs caninas, as quais colonizaram os túbulos seminíferos dos camundongos imunodeficientes e mantiveram-se vivas na membrana basal por 90 dias após transplante, mesmo que estes animais tenham distância filogenética
Title in English
Spermatogenesis xenogeneic after stem cell transplantation in canine testis of mice
Keywords in English
Dog
Spermatogonia
SSCs
Stem cells
Abstract in English
The spermatogonial stem cells (SSCs) are characterized by the capacity for self-renewal, proliferation and transmission of genetic information. In canines the first attempt to xenotransplantation did not achieve the success of sperm production. However, there is evidence that testicular xenogeneic cells can be transplanted into the testis of the host animal, and generate viable sperm donor. Therefore, this study aims conduct xenotransplantation of the canine germ cells in immunosuppressed mice, and thereby promote the production of viable sperm canines, genetically modified. And by this technique, analyze the efficiency of post-transplant spermatogenesis. Testicular germ cells were identified, isolated and cultured prepuberes dogs through enrichment culture systems and growth factors. Cells were modificated with a reporter gene GFP and LacZ. The SSCs canine was transplanted in mice (C57Bl/6), after different period and then the recipient animals were euthanized and analyzed. After 10 days in culture the germ cells were positive for CD49f, CD117, and 5 days a similar expression of GFRA1 and DAZL was observed, demonstrating the presence of SSCs and some cells in meiosis. 105 cells were transplanted and 20-43% of the transplanted cells were identified in the basement membrane of the seminiferous tubules after 90 days after transplantation. Therefore, the transplantation of canine germ cells showed that the cultivation and purification are performed possible for canine SSCs, it can colonize the seminiferous tubules the mice infertility remained alive in the basement membrane for 90 days after transplanting, even though these animals have phylogenetic distance
 
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Publishing Date
2016-06-10
 
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