The results are expressed as the mean6standard error of the mean

rature, stained with rabbit polyclonal anti-NuMA, rabbit polyclonal anti-LGN or mouse monoclonal anti–tubulin antibody for 4 h at room PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19712481 temperature. After washes in PBS, secondary antibody Alexa Fluor 555 goat anti-rabbit or Alexa Fluor 633 goat antimouse was added. After washes in PBS, oocytes were stained with DAPI and mounted with Floromount. For negative controls primary antibodies were omitted and no staining was observed. Quantitative RT-PCR Quantitative RT-PCR was performed using cDNA from oocytes. The level of Ric8 downregulation was estimated from 3 separate siRNA injection experiments using 10 oocytes per experiment. The reaction was carried out for 40 cycles of 15 seconds at 95C and 1 minute at 60C in an optimized ready-to-use solution.The relative Ric8 expression was calculated using Ct method. 4 / 19 Dynamics of RIC8 in Oogenesis Confocal Microscopy Images were captured by Olympus IX81 inverted microscope equipped with the FluoView FV1000 confocal system, using excitation at 405 nm, 488 nm, 559 nm and 635 nm and analyzed by Olympus FV1000 software. For Alexa Flour 594 or 555 and Alexa Flour 633 lasers were run in sequential mode to avoid the spectral overlap. Images were LY-411575 chemical information processed with Adobe Photoshop CS4. Quantification of RIC8 and Gi1/2 protein in maturing oocyte Fluorescence intensity estimation was used for quantification of RIC8 and Gi1/2 expression in oocyte cortex and cytoplasm by using AutoQuant X3. Average intensity of RIC8 and Gi1/2 proteins signal was measured in the cell cortex and cytoplasm of three equatorial confocal sections of each cell. To determine the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 relative expression level of RIC8 and Gi1/2 protein, the average fluorescence intensity was normalized to the selected surface area. The presented results show the relative level of protein per unit area. Statistical analysis Student t test was used to estimate the differences between groups. The differences of P<0.05 were considered significant. Results were expressed as mean SEM. Results Subcellular Localization of RIC8 During Folliculogenesis and in the Reproductive Tract The dynamics of RIC8 localization during mitosis in the cultured mammalian cells was characterized recently, but its expression pattern in the mammalian meiosis has not been assessed so far. Therefore, we analyzed RIC8 localization in the course of oogenesis in adult mouse until fertilization by using immunohistochemistry. Mammalian oocytes are arrested at the diplotene stage of the first meiotic prophase. During the process of folliculogenesis, oocytes enter the growth phase and finally resume meiosis. At the primordial and primary follicle stage, when the oocyte enters the growth phase, RIC8 is localized to the cytoplasm of the primary oocyte. At the later preantral and antral secondary follicle stages, RIC8 retains its cytoplasmic localization. However, at the antral secondary follicle stage, when the chromatin starts to fold into surrounded nucleolus configuration, RIC8 is also targeted to germinal vesicle. At the preovulatory stage, when the chromatin is fully condensed around the nucleolus, in the SN configuration, RIC8 localizes in the germinal vesicle. In parallel RIC8 accumulates from cytoplasm to the cell cortex. RIC8 is also present at detectable level in the cytoplasm of the surrounding cumulus cells and mural granulosa cells. To assess the expression and localization of RIC8 protein in the female reproductive tract we analyzed the cross-sections from different areas of the