Abstract
BackgroundRecent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Two distinct populations of spherical stem cells with high nucleo-cytoplasmic ratio have been recently identified in the adult mammalian ovary surface epithelium (OSE) including nuclear OCT-4A positive very small embryonic-like (VSELs) and cytoplasmic OCT-4 expressing ovarian germ stem cells (OGSCs). Three weeks culture of scraped OSE cells results in spontaneous differentiation of the stem cells into oocyte-like, parthenote-like, embryoid body-like structures and also embryonic stem cell-like colonies whereas epithelial cells attach and transform into a bed of mesenchymal cells. Present study was undertaken, to further characterize ovarian stem cells and to comprehend better the process of spontaneous differentiation of ovarian stem cells into oocyte-like structures in vitro.MethodsOvarian stem cells were enriched by immunomagnetic sorting using SSEA-4 as a cell surface marker and were further characterized. Stem cells and clusters of OGSCs (reminiscent of germ cell nests in fetal ovaries), were characterized by immuno-localization for stem and germ cell specific markers and spontaneous differentiation in OSE cultures was studied by live cell imaging.ResultsDifferential expression of markers specific for pluripotent VSELs (nuclear OCT-4A, SSEA-4, CD133), OGSCs (cytoplasmic OCT-4) primordial germ cells (FRAGILIS, STELLA, VASA) and germ cells (DAZL, GDF-9, SCP-3) were studied. Within one week of culture, stem cells became bigger in size, developed abundant cytoplasm, differentiated into germ cells, revealed presence of Balbiani body-like structure (mitochondrial cloud) and exhibited characteristic cytoplasmic streaming.ConclusionsPresence of germ cell nests, Balbiani body-like structures and cytoplasmic streaming extensively described during fetal ovary development, are indeed well recapitulated during in vitro oogenesis in adult OSE cultures along with characteristic expression of stem/germ cell/oocyte markers. Further studies are required to assess the genetic integrity of in vitro derived oocytes before harnessing their clinical potential. Advance in our knowledge about germ cell differentiation from stem cells will enable researchers to design better in vitro strategies which in turn may have relevance to reproductive biology and regenerative medicine.
Highlights
Recent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology
We further proposed that the ovarian surface epithelium (OSE) cells undergo epithelial-mesenchymal transition in vitro into mesenchymal fibroblasts which may act like supporting somatic granulosa cells and provide the niche to the differentiating stem cells since developing oocytes were invariably found in close association with the mesenchymal fibroblasts [12]
Stage-specific embryonic antigen-4 (SSEA-4) based immunomagnetic separation and enrichment of OSE stem cells followed by characterization of m-Ribonucleic acid (RNA) transcripts and protein for markers of pluripotent stem cells was performed in sheep as human ovarian tissue is not readily available
Summary
Recent studies suggest that ovarian germ line stem cells replenish oocyte-pool in adult stage, and challenge the central doctrine of ‘fixed germ cell pool’ in mammalian reproductive biology. Our group has reported two distinct stem cell populations namely, pluripotent very small embryonic like stem cells (VSELs) with nuclear OCT-4A and slightly bigger progenitor stem cells termed as ovarian germ stem cells (OGSCs) with cytoplasmic OCT-4 in peri-menopausal women and other higher mammalian species [12] similar to our earlier results in adult human [13] and mice [14] testes These putative stem cells differentiated spontaneously in cultures to give rise to oocyte-like structures and parthenotes [12] and expressed both pluripotent (OCT-4, OCT-4A, SSEA-4, NANOG, SOX-2, TERT and STAT-3) and germ (C-KIT, DAZL, GDF-9, VASA) cell markers. Our group has gathered evidence that FSH modulates ovarian stem cells through alternatively spliced variant of FSH receptor, that these stem cells undergo potential self-renewal and clonal expansion as ‘germ cell cysts’ in sheep OSE in vitro [19] and that PMSG treatment results in augmented stem cell activity, neo-oogenesis and primordial follicle assembly in mice [17]
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