l for the secondary follicle stage in the ovary. Later, at the full rim stage, when chromatin had condensed densely around nucleolus, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19712481 RIC8 accumulated in spots distributed around condensed chromatin in the germinal vesicle, with some foci attached to the nucleolus. Further, when the nuclear lamina as well as the nucleolus started to disappear, the spots of RIC8 remained in the close vicinity of chromosomes. With the organization of chromosomes to the metaphase plate, RIC8 distributed along the metaphase spindle and co-localized with tubulin. In anaphase I, during the chromosome separation, RIC8 remained in the vicinity of chromosomes and colocalized with the meiotic spindle. After completion of meiosis I, the 7 / 19 Dynamics of RIC8 in Oogenesis Fig 3. Immunological detection of co-localization of RIC8 with Gi1/2, LGN and NuMA proteins during oocyte development. Mouse oocytes at GV or GVBD stages were KU55933 double-labeled with RIC8 antibody and NuMA, LGN or Gi1/2 antibodies respectively. DNA was stained with DAPI. Higher magnification of the region of chromatin. Dotted white line indicates the borders of oocyte. White arrowheads point RIC8 foci at nucleolus and on chromatin. Abbreviations: GV, germinal vesicle; GVBD, germinal vesicle breakdown. Scale bar: 10 m. doi:10.1371/journal.pone.0129131.g003 8 / 19 Dynamics of RIC8 in Oogenesis Fig 4. GEF RIC8 co-localization with LGN during mouse oocyte maturation. Mouse oocytes at meiosis I and at metaphase block of meiosis II were double-labeled with RIC8 antibody and LGN. DNA was stained with DAPI. Localization of meiotic spindle is denoted with PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713490 white arrows and yellow to orange colour in this area indicates the overlapping regions of 
RIC8 and LGN. Dotted white line indicates the borders of oocyte. Abbreviations: Ana/Tel I, anaphase/telophase of meiosis I; Met I or Met II, metaphase of meiosis I or meiosis II respectively; ms, meiotic spindle; pb, polar body. Scale bar: 10 m. During the second arrest, RIC8 was distributed to the metaphase spindle and diffusely in the cytoplasm. After the ovulation, fertilization triggers the resumption and completion of meiosis II and metaphase is rapidly finished. During the final steps of meiosis II RIC8 was located in the spindle. In addition to the above-mentioned, a part of RIC8 protein was also detectable uniformly in the cytoplasm of oocyte at every examined stage, and also in some extent at the cell cortex. 9 / 19 Dynamics of RIC8 in Oogenesis Fig 5. RIC8 protein expression pattern compared to NuMa or Gi1/2 proteins during mouse oocyte maturation. Mouse oocytes at meiosis I were double-labeled with RIC8 antibody and NuMA or Gi1/2 antibodies. DNA was stained with DAPI. The overlapping regions of RIC8 and NuMA or Gi1/2 at meiotic spindle indicated with white arrows. White arrowheads indicated cell cortex regions, where RIC8 and Gi1/2 colocalize. Abbreviations: Ana/Tel I; anaphase/telophase of meiosis I; ms, meiotic spindle; pb, polar body. Scale bar: 10 m. doi:10.1371/journal.pone.0129131.g005 RIC8 co-localizes with NuMA in the germinal vesicle of oocyte Earlier biochemical studies have indicated that RIC8 acts on a ternary complex of Gi-GDP, LGN and NuMa inducing the release of Gi-GTP and dissociation of NuMa from LGN, which may promote the pulling of aster microtubules during cell division. Furthermore, RIC8 is necessary for the assembly of a cortical signaling complex that orients the mitotic spindle during mammalian cell division. In order to characterize