RIC8, Gi1/2, LGN and NuMA, during mammalian oogenesis, meiosis and fertilization, we examined whether these proteins were co-localized PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19712481 at afore-mentioned stages. By using immunocytochemical studies, we found that at germinal vesicle stage of oocyte maturation RIC8 and NuMA were both broadly expressed in GV and their localization partially overlapped. After germinal vesicle breakdown NuMA and RIC8 were both starting to condense around chromatin but NuMA expression was much wider compared to RIC8, which had accumulated as discrete spots near chromatin. As expected, LGN was weakly detectable in cytoplasm at GV and GVBD SB203580 web stages but was congregated around chromatin during prometaphase. However, co-localization of RIC8 and LGN in GV or GVBD PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19710468 stages was not detected. Gi1/2, as one of the direct interaction partners of RIC8, was uniformly distributed in cytoplasm of oocyte at GV and GVBD stages, and did not reveal significantly overlapping localization with RIC8. 10 / 19 Dynamics of RIC8 in Oogenesis RIC8 exhibits similar localization pattern with NuMa, LGN and Gi1/2 in meiotic spindle Next, we analyzed the cellular localization of RIC8 protein in relation to its interaction partner Gi1/2 as well as LGN and NuMA at different meiotic stages of oocyte development. At metaphase I RIC8 and LGN showed high extent co-localization at the meiotic spindle apparatus and they both remained associated with spindle during anaphase and telophase. However, the degree of their co-localization in this area decreased as LGN localized more close to chromosomes, whereas RIC8 condensed more at the plus-end of meiotic spindle. Later, in MII metaphase arrested oocytes the extent of RIC8 and LGN co-localization was similar to MI oocytes. The locaton of another regulatory protein NuMA partially overlaps with RIC8 in the spindle during meiosis I. In the anaphase and telophase, when chromosomes segregate, NuMA and RIC8 translocated to the spindle midzone, whereas Gi1/2 remained in the cortex area during meiosis I and II. In parallel with accumulation in the meiotic spindle RIC8 also localized to the cell cortex in a partially overlapping manner with Gi1/2. Gi1/2, on the other hand, was only weakly detectable in the meiotic spindle as compared to RIC8, which was rather abundant in this region. After Fertilization RIC8 Translocates to the Pronuclei The oocyte liberates from the metaphase II arrest after a sperm penetration and resumes to meiosis II. The final stages of oocyte meiosis take place in parallel with the transport of sperm’s nuclear material towards the forming female pronucleus. After the sperm penetration and the completion of meiosis II, RIC8 converged into the traveling male pronucleus and the forming female pronucleus. RIC8 accumulated strongly in the nucleolus precursor bodies, the morphological intermediates of reforming nucleolus. Notably, also the nucleoli of the second polar body contained RIC8 protein at high level. In addition, we examined the possible co-localization of Gi1/2, NuMA and LGN proteins at pronuclear stages with RIC8, and we found that RIC8 was localized in the pronucleus as discrete spots around the nucleoli. Although NuMA was broadly localized to the pronucleus, it showed no overlap with RIC8. LGN, in contrary, was not detectable in pronuclei although it was discernible in the remains of spindle of the 
second polar body, where it co-localized with RIC8. Interestingly, LGN and RIC8 co-localized in the middle area of spindle, but did not
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