During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas de células distintas, o trofectoderma e a massa celular interna. Esta última sofrerá diferenciação para formar o disco embrionário constituído pelo epiblasto e hipoblasto. Os trabalhos de caracterização morfológica do epiblasto e hipoblasto em bovinos são necessários uma vez que podem ajudar a elucidar as causas de perdas gestacionais, principalmente nos casos de embriões derivados de produção in vitro. Assim, o objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino em diferentes fases do desenvolvimento com ênfase nas células do hipoblasto. Para isso, os embriões bovinos com 7, 14 e 16 dias de gestações derivados de técnicas de produção in vitro foram fixados para processamento e realização de microscopia eletrônica de transmissão. De acordo com os resultados obtidos observou-se que os embriões derivados de transferência nuclear de células somáticas e de partenogênese apresentaram grandes modificações na estrutura macro e microscópica. Nestes embriões, o tamanho estava reduzido, a massa celular interna não se apresentou de forma definida. Além disso, as organelas destes embriões, responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor quantidade e tinham modificações quanto à forma, quando comparados aos resultados vistos nos embriões derivados de fertilização in vitro. Assim, verificamos que os blastocistos D7 derivados de transferência nuclear e partenogênese apresentaram graves modificações morfológicas, assim como verificou-se também nas células do hipoblasto dos embriões D14 e D16. / In cattle, the embryo development is characterized by the apperance of two distinct cell layers, the trophectoderm and the inner cell mass. This latter one will siffer a differentiation to form the embryonic disc constituted by the epiblasto and hypoblast. The works on the morphological characterization of the epiblasto and hypoblast in cattle are needed because they may help to elucidate the causes of pregnancy loss, especially in cases of embryos derived from in vitro production. Considering this, the objective of this study was to characterize ultrastrcturally the bovine embryo at different stages of development with emphasis on the hypoblast cells. To do so, bovine embryos at 7, 14 and 16 days of pregnancies derived from in vitro production techniques were fixed for processing and performing transmission electron microscopy. According to the results observed that embryos derived from the nuclear transfer of somatic cells and from parthenogenesis showed significant changes in the macroscopic and microscopic structure. In these embryos, the size was reduced, the inner cell mass has not show a defined shape. Furthermore, the organelles from such embryos, responsible for the absorption processes, communication, growth and cellular metabolism were fewer in number and have changes in shape, when compared to the results seen in embryos derived from in vitro fertilization. Thus, we observed that the blastocyst D7 derived from nuclear transfer and from the parthenogenesis, showed severe morphological changes, as well as for the hypoblastic cells in the D14 and D16 embryos.

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas de células distintas, o trofectoderma e a massa celular interna. Esta última sofrerá diferenciação para formar o disco embrionário constituído pelo epiblasto e hipoblasto. Os trabalhos de caracterização morfológica do epiblasto e hipoblasto em bovinos são necessários uma vez que podem ajudar a elucidar as causas de perdas gestacionais, principalmente nos casos de embriões derivados de produção in vitro. Assim, o objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino em diferentes fases do desenvolvimento com ênfase nas células do hipoblasto. Para isso, os embriões bovinos com 7, 14 e 16 dias de gestações derivados de técnicas de produção in vitro foram fixados para processamento e realização de microscopia eletrônica de transmissão. De acordo com os resultados obtidos observou-se que os embriões derivados de transferência nuclear de células somáticas e de partenogênese apresentaram grandes modificações na estrutura macro e microscópica. Nestes embriões, o tamanho estava reduzido, a massa celular interna não se apresentou de forma definida. Além disso, as organelas destes embriões, responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor quantidade e tinham modificações quanto à forma, quando comparados aos resultados vistos nos embriões derivados de fertilização in vitro. Assim, verificamos que os blastocistos D7 derivados de transferência nuclear e partenogênese apresentaram graves modificações morfológicas, assim como verificou-se também nas células do hipoblasto dos embriões D14 e D16. / In cattle, the embryo development is characterized by the apperance of two distinct cell layers, the trophectoderm and the inner cell mass. This latter one will siffer a differentiation to form the embryonic disc constituted by the epiblasto and hypoblast. The works on the morphological characterization of the epiblasto and hypoblast in cattle are needed because they may help to elucidate the causes of pregnancy loss, especially in cases of embryos derived from in vitro production. Considering this, the objective of this study was to characterize ultrastrcturally the bovine embryo at different stages of development with emphasis on the hypoblast cells. To do so, bovine embryos at 7, 14 and 16 days of pregnancies derived from in vitro production techniques were fixed for processing and performing transmission electron microscopy. According to the results observed that embryos derived from the nuclear transfer of somatic cells and from parthenogenesis showed significant changes in the macroscopic and microscopic structure. In these embryos, the size was reduced, the inner cell mass has not show a defined shape. Furthermore, the organelles from such embryos, responsible for the absorption processes, communication, growth and cellular metabolism were fewer in number and have changes in shape, when compared to the results seen in embryos derived from in vitro fertilization. Thus, we observed that the blastocyst D7 derived from nuclear transfer and from the parthenogenesis, showed severe morphological changes, as well as for the hypoblastic cells in the D14 and D16 embryos.

During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.