Kun Guo1, Chao-Hui Li1, Xin-Yi Wang1, Da-Jian He1, Ping Zheng2. 1. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming 650223, Yunnan, China Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming 650223, Yunnan, China Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650204, Yunnan, China. 2. State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming 650223, Yunnan, China Yunnan Key Laboratory of Animal Reproduction, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiao Chang Dong Lu, Kunming 650223, Yunnan, China zhengp@mail.kiz.ac.cn.
Abstract
STUDY HYPOTHESIS: Are active ovarian germ stem cells present in postnatal mouse ovaries under physiological conditions? STUDY FINDING: Active ovarian germ stem cells exist and function in adult mouse ovaries under physiological conditions. WHAT IS KNOWN ALREADY: In 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, METHODS: Octamer-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 CHANCE: By 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 CAUTION: This 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 FINDINGS: Clarifying 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 DATA: Not applicable. STUDY FUNDING/COMPETING INTERESTS: This 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.
STUDY HYPOTHESIS: Are active ovarian germ stem cells present in postnatal mouse ovaries under physiological conditions? STUDY FINDING: Active ovarian germ stem cells exist and function in adult mouse ovaries under physiological conditions. WHAT IS KNOWN ALREADY: In 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, METHODS:Octamer-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 CHANCE: By 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 CAUTION: This 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 FINDINGS: Clarifying 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 DATA: Not applicable. STUDY FUNDING/COMPETING INTERESTS: This 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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