Germ stem cells are active in postnatal mouse ovary under physiological conditions
Germ stem cells are active in postnatal mouse ovary under physiological conditions
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生理条件下，出生后小鼠卵巢中的生殖干细胞活跃
DOI：
10.1093/molehr/gaw015
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发表时间：
2016-05-01
影响因子：
4
通讯作者：
Zheng, Ping
中科院分区：
文献类型：
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作者：
Guo, Kun;Li, Chao-hui;Zheng, Ping
生后小鼠卵巢中是否存在活跃的卵巢生殖细胞？生理条件下成年小鼠卵巢中存在活跃的卵巢生殖细胞并发挥其功能。体外研究表明，小鼠、猪和人的出生后卵巢中均存在生殖细胞。然而，活体研究提供了证据，证明出生后的小鼠卵巢中存在活跃的生殖细胞干细胞。因此，这种生殖细胞在出生后的哺乳动物卵巢中是否真的存在并在体内发挥作用仍存在争议。将八聚体结合转录因子4(Oct4)-MerCreMer转基因小鼠与R26R增强的黄色荧光蛋白(EYFP)小鼠杂交，建立三苯氧胺诱导的示踪系统，以便用EYFP标记年轻成年小鼠(5-6周龄)表达Oct4的潜在卵巢生殖细胞。对注射三苯氧胺后不同时间点(1天、3天、2个月和4个月)的卵巢组织进行免疫荧光染色，观察生殖细胞DNA复制、有丝分裂、进入减数分裂和向原始卵泡期进展的情况。用EYFP标记的单细胞RT-PCR检测减数分裂进入和原始卵泡的形成。5-溴脱氧尿苷三磷酸掺入法检测生殖细胞增殖和有丝分裂。在每个时间点，每组实验使用2-3只动物的卵巢，通过标记表达Oct4的小生殖细胞并追踪它们的命运长达4个月，我们观察了持续的减数分裂进入和原始卵泡的补充。此外，我们捕捉到了有丝分裂DNA复制的瞬时过程以及标记的生殖细胞在追踪后不同时间段的有丝分裂过程。这些证据明确支持出生后卵巢中存在活跃的生殖细胞干细胞，以及它们在生理条件下补充原始卵泡池的功能。此外，我们还指出，在无精子样(DAZL)(-)中缺失的Oct4(+)，而不是Oct4(+)DAZL(+)或Oct4(+)死亡(Asp-Glu-Ala-Asp)盒多肽4(Ddx4)(+)细胞中含有大量的生殖干细胞。目前尚不清楚这些结果是否适用于人类。未来的工作将致力于寻找特异性的卵巢生殖细胞标记物，并评价这些干细胞对正常卵巢功能的意义。阐明出生后哺乳动物卵巢中活性生殖细胞的存在及其功能意义，可以为理解卵巢衰老和衰竭的机制提供新的见解，但不适用。本研究得到国家重点基础研究项目中国(批准号：2012CBA01300)和国家自然科学基金项目中国(31571484)的资助。没有报道称存在利益冲突。
Are active ovarian germ stem cells present in postnatal mouse ovaries under physiological conditions?Active ovarian germ stem cells exist and function in adult mouse ovaries under physiological conditions.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.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.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.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.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.Not applicable.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.