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 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19703425 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 Fallopian tube and uterus by immunohistochemistry. The oviduct epithelium of adult female mouse consists of two types of epithelial cells, which are unevenly distributed. Ciliated cells are predominant in the region of infundibulum and ampulla, and secretory cells in the isthmus. RIC8 was present in all regions of the Fallopian tube. More precisely, in the area of ampulla, it was clearly localized in the basal layer of the cilia of the ciliated cells. RIC8 was also detectable at high level in the epithelium of the isthmus. Interestingly, the localization of RIC8 seemed to be polarized as 5 / 19 Dynamics of RIC8 in Oogenesis Fig 1. RIC8 in folliculogenesis and in the reproductive tract of adult mouse. RIC8 was visualized with RIC8 antibody, and cell nuclei were visualized with DAPI. Transversal cryosections of ovary with oocytes in different follicular stages starting from primordial follicle to Graafian follicle and different regions of oviduct are shown. Higher magnification of the region of ampulla and isthmus. Abbreviations: A, antrum; Amp, ampulla region of oviduct; Cb, basal layer of cilia; Cc, ciliated cell; Ci, cilia; Co, cumulus oophorus; Cr, corona radiata; Cx, cell cortex, Ec, epithelial cells; Fc, follicular cell; Gc, granulosa cells; Gf, Graafian follicle; GV, germinal vesicle; Ist, isthmus region of oviduct; Lp, lamina propria; Lu, lumen; Pc, peg cell; Pf, primary follicle; Pmf, primordial follicle; PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19705070 Po, primary oocyte; Sf, secondary follicle. Scale bars: 50 m. doi:10.1371/journal.pone.0129131.g001 it accumulated more in the apical cell cortex than in basal. RIC8 expression was also detectable in the endometrium of uterus but at the markedly lower level. During Oocyte Maturation RIC8 Accumulates on Chromatin and Meiotic PR 619 site Spindle The final phase of folliculogenesis comprises the resumption of meiosis and 
germinal vesicle breakdown, which are triggered by a surge of luteinizing hormone. In order to map the RIC8 protein localization after meiotic resumption, we characterized the oocytes at different stages 6 / 19 Dynamics of RIC8 in Oogenesis Fig 2. Dynamic changes in localization of RIC8 in the mouse oocyte during meiosis. RIC8 was visualized with RIC8 antibody, microtubules with antibody to tubulin, chromatin with DAPI and F-actin with phalloidin. GV- stage oocyte; GVBD stage oocytes; oocytes undergoing meiosis I or mei
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