Data were expressed as a percentage with respect sham-mice

These results indicate that Ric8 siRNA efficiently dowregulated Ric8 expression and also RIC8 protein level. Moreover, we also observed that for Ric8 siRNA oocytes maturation from GV-stage to meiosis II metaphase took more time than for the control group. In order to clarify whether downregulation of RIC8 also affects the localization of its interaction partners LGN and Gi1/2 in maturing oocytes, we performed immunofluorescence analysis. The localization pattern of LGN and Gi1/2 was not influenced by Ric8 siRNA treatment of oocytes, but we noticed that the accumulation of Gi1/2 in the cortex region was lower than in control oocytes. Quantification of the Gi1/2 immunofluorescence signal of did not reveal any differences in Gi1/2 levels in the cytoplasm region between the Ric8 siRNA treated and control oocytes. However, the cortical signal of Gi1/2 was significantly lower in Ric8 siRNA treated oocytes compared to control ones. Therefore, our results indicate that RIC8 is required for the maintenance of Gi1/2 levels and its targeting to the cortical region of maturating oocytes. Discussion Microarray analysis of mouse ovaries, at embryonic day 13.5 when oocytes stop dividing mitotically and enter meiosis revealed that the expression of Ric8 gene was LY341495 price upregulated with the onset of meiosis. In order to assess the function of RIC8 in later steps of oogenesis, in meiosis, we mapped its localization during mouse oocyte growth and maturation, fertilization and initial cleavage steps. We demonstrate that at the earlier stages–primary PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710065 oocytes at the diplotene of the first meiotic prophase and during folliculogenesis until preantral follicle stages RIC8 localizes in the cytoplasm. However, at later stages of folliculogenesis, RIC8 also accumulates to the germinal vesicle at the partial rim stage. When chromatin condenses around the nucleolus at the full rim stage, RIC8 surrounds chromatin in the germinal vesicle as characteristic spots. The localization of RIC8 in specific foci is reminiscent of centromere pattern, and in support with that RIC8 has been shown to co-localize with centromeres in HeLa cells. Upon meiotic spindle formation RIC8 shifted to the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19711918 spindle in metaphase and remained there during the anaphase and telophase of meiosis I and II. At the initial steps of oocyte first cleavage, RIC8 accumulated in the region of chromosomes and microtubules, and in the cell cortex. Interestingly, at two-cell stage where interaction with -catenin and E-cadherin mediate adhesion of the blastomeres, RIC8 was specifically excluded from the cell cortex between blastomeres. In analogy, RIC8 is shown to localize to the cortex, spindle poles, centromeres, central spindle and midbody in HeLa cells depending on the phase of cell cycle. Rather similar pattern of RIC8 localization and redistribution has been observed in various model organisms. For example in the early C. elegans embryo it has been mapped in the cell cortex and on the asters of the mitotic spindle, but also on the central spindle, at the nuclear envelope, around the 14 / 19 Dynamics of RIC8 in Oogenesis Fig 8. RIC8 downregulation interferes with localization of Gi1/2 protein to the oocyte cortex. RIC8 protein expression in mouse oocytes was downregulated by microinjection of Ric8 siRNA. DNA was stained with DAPI. Downregulation of Ric8 mRNA expression with siRNA in microinjected oocytes quantified by qRT-PCR. The relative intensity of RIC8 immunofluorescence signal measured by AutoQuant X3. Lo