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FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

ABSTRACT: Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bound DDX4. Furthermore these cells when isolated were not expressing DDX4, and did not initially possess germline identity. Despite these unremarkable beginnings, they acquired some pre-meiotic markers in culture, including DDX4, but critically never expressed oocyte-specific markers, and furthermore were not immortal but died after a few months. Our results suggest that freshly isolated OSCs are not germ stem cells, and are not being isolated by their DDX4 expression. However it may be that culture induces some pre-meiotic markers. In summary the present study offers weight to the dogma that the adult ovary is populated by a fixed number of oocytes and that adult de novo production is a rare or insignificant event.

SUBMITTER: Zarate-Garcia L

PROVIDER: S-EPMC4908409 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

Zarate-Garcia Larissa L Lane Simon I R SI Merriman Julie A JA Jones Keith T KT

Scientific reports 20160615

Whether the adult mammalian ovary contains oogonial stem cells (OSCs) is controversial. They have been isolated by a live-cell sorting method using the germ cell marker DDX4, which has previously been assumed to be cytoplasmic, not surface-bound. Furthermore their stem cell and germ cell characteristics remain disputed. Here we show that although OSC-like cells can be isolated from the ovary using an antibody to DDX4, there is no good in silico modelling to support the existence of a surface-bou ...[more]

PMID: 27301892

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Project description:Study hypothesisAre active ovarian germ stem cells present in postnatal mouse ovaries under physiological conditions?Study findingActive ovarian germ stem cells exist and function in adult mouse ovaries under physiological conditions.What is known alreadyIn vitro studies suggested the existence of germ stem cells in postnatal ovaries of mouse, pig and human. However, in vivo studies provided evidence against the existence of active germ stem cells in postnatal mouse ovaries. Thus, it remains controversial whether such germ stem cells really exist and function in vivo in postnatal mammalian ovaries.Study design, samples/materials, methodsOctamer-binding transcription factor 4 (Oct4)-MerCreMer transgenic mice were crossed with R26R-enhanced yellow fluorescent protein (EYFP) mice to establish a tamoxifen-inducible tracing system so that Oct4-expressing potential ovarian germ stem cells in young adult mice (5-6 weeks old) can be labeled with EYFP. The germ cell activities of DNA replication, mitotic division, entry into meiosis and progression to primordial follicle stage were investigated by means of immunofluorescent staining of ovarian tissues collected at different time points post-tamoxifen injection (1 day, 3 days, 2 months and 4 months). Meiosis entry and primordial follicle formation were also measured by EYFP-labeled single-cell RT-PCR. Germ cell proliferation and mitotic division were examined through 5-bromodeoxyuridine triphosphate incorporation assay. At each time point, ovaries from two to three animals were used for each set of experiment.Main results and the role of chanceBy labeling the Oct4-expressing small germ cells and tracing their fates for up to 4 months, we observed persistent meiosis entry and primordial follicle replenishment. Furthermore, we captured the transient processes of mitotic DNA replication as well as mitotic division of the marked germ cells at various time periods after tracing. These lines of evidence unambiguously support the presence of active germ stem cells in postnatal ovaries and their function in replenishing primordial follicle pool under physiological conditions. Moreover, we pointed out that Oct4(+) deleted in azoospermia-like (Dazl)(-) but not Oct4(+)Dazl(+) or Oct4(+) DEAD (Asp-Glu-Ala-Asp) Box Polypeptide 4 (Ddx4)(+) cells contain a population of germ stem cells in mouse ovary.Limitations, reasons for cautionThis study was conducted in mice. Whether or not the results are applicable to human remain unclear. The future work should aim at identifying the specific ovarian germ stem cell marker and evaluating the significance of these stem cells to normal ovarian function.Wider implications of the findingsClarifying the existence of active germ stem cells and their functional significance in postnatal mammalian ovaries could provide new insights in understanding the mechanism of ovarian aging and failure.Large scale dataNot applicable.Study funding/competing interestsThis work was supported by the National Key Basic Research Program of China (grant number 2012CBA01300) and the National Natural Science Foundation of China to P.Z. (31571484). No competing interests are reported.

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FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

Publications

FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells.

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