Characterization of stem cells in sheep OSE smears. ( A ) OCT-4 immunolocalization in OSE smear after 15 hrs of culture with FSH. Note that only stem cells express OCT-4 whereas the somatic epithelial cells are devoid of OCT-4. Scale bar represents 20 μm ( B ) Confocal imaging of ovarian stem cells stained for OCT-4 with PI as the nuclear counterstain. VSELs express nuclear OCT-4 whereas the slightly bigger OGSCs and also the ‘cysts’ express cytoplasmic OCT-4. First column shows OCT-4 staining, middle panel shows PI staining whereas the third column is the merged image. ( C ) Confocal image of a cell surface marker SSEA-4 to show cytoplasmic continuity in a germ cell nest or a ‘cyst’. Z stack imaging of a cell cluster collected after FSH treatment shows cytoplasmic continuity between cells due to incomplete cytokinesis and is a distinct feature of clonal expansion of a stem cell. Magnification 63X with 3X zoom ( D ) qRT-PCR results showing increased relative mRNA expression of pluripotent VSELs specific (Oct-4A and Sox-2) and OGSCs specific (Oct-4) transcripts after 15 hrs of FSH treatment (black bars) compared to without FSH (grey bars). Results are representative of three different experiments.