TY - GEN TI - Single cell expression analysis of genes with potential mrna gradient in mouse oocytes T3 - XXIInd Symposium of biology and immunology of reproduction with international participation AU - Dorosh, Andriy AB - In frogs, there are clearly visible differently pigmented animal and vegetal poles of the egg determined before fertilization and leading to asymmetrical divisions. Mammalian egg does not show any comparable differentiation and it has been generally accepted that even the individual blastomeres in 2-cell and 4-cell embryos are homogenous. However, recent findings suggest that those blastomeres display different gene expression patterns and might already possess some inclinations to specific cell lineages. We therefore raised a question, whether there could be any mRNA or protein gradients in pre-fertilization oocytes similar to a previously described amphibian egg one. In mammalian eggs, there is a membrane region that is poor in microvilli, cortical granules are absent beneath plasma membrane and sperm cells generally do not bind to this location. This microvilli free region also covers the egg nucleus, and cytoskeleton localization differs markedly to the rest of the cortical space, forming actin –myosin II cortical cap/ring and is considered as animal pole. The purpose of this study was to determine gene products that can be detected at single cell level using qPCR and display gradient like distribution in mature oocytes. We checked expression of 12 selected genes in a pool of 10 oocytes and single mature oocytes. Then, we analysed gene expression in fixed intact oocytes and those undergoing laser capture microdissection procedure (LCMD). Eventually, we have determined six candidate genes for the study of intracellular spatial gene expression in mature mammalian oocytes by subcellular qPCR and in situ hybridization. UR - http://www.nusl.cz/ntk/nusl-260989 UR - http://hdl.handle.net/11104/0262903 A2 - Vodička, Martin A2 - Margaryan, Hasmik A2 - Dvořáková-Hortová, Kateřina A2 - Šídová, Monika A2 - Ergang, Peter LA - eng KW - oocytes KW - single cell expresion analysis KW - RT-qPCR KW - Laser capture microdissection KW - in situ hybridization PY - 2016 PB - Biotechnologický ústav, Vídeňská 1083, Praha 4, 142 20, http://www.ibt.cas.cz/cs ER -