中国科学院昆明动物研究所机构知识库

无脊椎动物和低等脊椎动物的雌性个体卵巢内均存在生殖干细胞来维持雌性配子的持续发生，然而雌性哺乳动物的卵巢内是否也存在配子的再生一直备受争议。传统观点认为雌性哺乳动物出生后卵巢内所有的生殖细胞都已经发育到了原始卵泡阶段，形成了足够其终生可用的卵泡固定池，没有生殖干细胞及其分化而来的卵可以补给卵泡池。近年来，相继有科学家从小鼠及人的卵巢中分选出了一类可长期体外培养的生殖细胞，该类细胞在体外培养时表现出干细胞特征，移植到正常或者经化疗药物处理导致不育的小鼠卵巢中能形成有功能的卵。然而，这些研究都是通过细胞体外培养或者移植实验来证明生殖干细胞的存在，有的科学家认为所谓的生殖干细胞是在分选及培养过程中产生的人工的生殖干细胞，并且对目前广泛使用的卵巢生殖干细胞分选基因Ddx4提出质疑，认为Ddx4并不能真正有效的富集到生殖干细胞。体内世系追踪与细胞体外培养和移植相比，最大的优点是能反映细胞在体内正常生理状态下的真实行为。已有科学家采用Ddx4作为示踪基因或者以Ddx4是否表达作为生殖干细胞的标准在小鼠卵巢内并未检测到生殖干细胞及卵子的再生。因此雌性哺乳动物卵巢中是否存在卵子的再生现象，如果存在，再生现象是否依赖于生殖干细胞，生殖干细胞特异性表达的分子标记是什么仍然是亟待解决的科学问题。相关成果会给临床上治疗卵巢功能失败、建立性细胞转基因动物等提供理论依据。在本论文中，我们通过体内世系追踪系统对成年小鼠卵巢中是否存在卵子再生及生殖干细胞进行了深入探讨，并对收集的不同发育阶段的生殖单细胞样品基因表达初步分析以用于后续卵巢生殖干细胞特异性表达分子标记的筛选。体内示踪系统能否成功检测到干细胞的关键是示踪基因的选择。和前人不同的是，我们选取Oct4作为示踪基因研究小鼠卵巢中是否存在生殖干细胞。Oct4表达于可自我更新的胚胎时期原始生殖细胞及成年小鼠卵巢中较为原始的生殖细胞。在建立的Oct4-MerCreMer+/Tm; R26R-EYFPTm/Tm转基因示踪系统中，被标记上EYFP的细胞都是生殖细胞，我们重点研究尚未形成明显滤泡结构的小生殖细胞在示踪过程中的变化。在示踪1天，3天，2个月及4个月后，我们通过免疫荧光染色在成年小鼠的卵巢中发现了以下4个重要现象。1，示踪不同时间后，卵巢内都存在表达Sycp3及Stra8的小生殖细胞。Sycp3和Stra8是减数第一次分裂前期瞬时表达的基因。经过长达4个月的示踪仍存在表达Sycp3及Stra8的小生殖细胞，提示成年小鼠卵巢中存在减数分裂的重新进入现象。2，示踪不同时间后，卵巢中都存在表达Sycp3及Stra8的原始卵泡，提示卵巢中存在原始滤泡的补充现象。3，示踪不同时间，小鼠卵巢中有极少部分的生殖细胞能进行不同形式的有丝分裂和增殖。4，经过长达4个月的示踪，卵巢中始终存在EYFP+Oct4+Dazl-的小生殖细胞。通过Brdu标记实验，Oct4+Dazl-的小生殖细胞在进行有丝分裂的DNA复制，而Oct4+Dazl+的小生殖细胞在进行减数分裂的DNA复制。因此，通过Oct4驱动的体内世系追踪系统，我们在成年小鼠的卵巢中发现了减数分裂的持续进入和原始卵泡的补充，证明了成年小鼠卵巢中存在卵子再生现象。同时有少数生殖细胞能通过有丝分裂的DNA复制及不同形式的增殖在长期示踪过程中维持自身群体的稳定存在，提示成年小鼠卵子的再生基于卵巢生殖干细胞，并且潜在的生殖干细胞存在于Oct4+Dazl-生殖细胞群中，而不存在于Oct4+Dazl+或者Oct4+Ddx4+细胞群中。在确认小鼠卵巢生殖干细胞的存在之后，我们试图对该类细胞特异表达的分子标记进行筛选。采用单细胞扩增技术结合已建立的示踪系统收集不同发育阶段的生殖单细胞样品，希望通过RNA-seq及对不同发育阶段的生殖细胞进行基因表达分析，找到卵巢生殖干细胞群体以及特异性在卵巢生殖干细胞中表达，在其他分化阶段不表达的基因。通过fluidigm检测对目前收集的单细胞样品进行初步基因表达分析，我们发现有的单细胞样品表达Sycp3、Mlh1、Msh5等减数第一次分裂前期瞬时表达的基因，有的单细胞样品在表达Sycp3等基因的同时还表达原始卵泡标记基因Sohlh1及Sohlh2，再次从RNA水平上证明成年小鼠卵巢存在减数分裂的重新进入与原始滤泡的补充。通过PCA分析可以将样品分为X、Y、Z三大类，其中X类又可以分为m、n、o三群，我们猜测Z类及o群单细胞样品可能含有潜在的卵巢生殖干细胞。同时我们发现卵巢生殖细胞的基因表达具有较大的异质性，提示我们要想确切的了解生殖细胞的发育过程并准确的通过测序的基因表达数据从众多的样品中辨别出卵巢生殖干细胞还需要再扩大样品数量。后续我们将采用Smart-seq2结合细胞标签的扩增技术继续收集样品，以用于高通量样品的测序及分析。采用生殖干细胞特异表达的分子标记建立新的示踪系统可以阐明生殖干细胞对成年小鼠卵巢正常功能维持的意义，并为该类细胞的体外研究提供基础。

Ovarian germ stem cells exist in some invertebrate and lower vertebrate females to maintain the follicle regeneration after birth. In female mammals, whether oogenesis continues after birth has been vigorously debated. The prevailing dogma declares that newborn females possess their life time complement of germ cells that are not replenished by ovarian germ stem cells after birth. Recently, some researches isolate rare germ cells from adult mouse and human ovary that proliferate in dishes and act out stem cell properties. These cells can form developmentally competent oocytes following being transplanted into wild type and infertile mice. However, the researches mentioned above all adopt cell culture in vitro and transplantation technology to prove the exsitence of ovarian germ stem cells. The technology they used reignite debate regarding if germ stem cells were an artifact of purification or culture methods. At the same time, some scientist can not get actual ovarian germ stem cells using Ddx4 as a purification gene. As opposed to cell culture in vitro and transplantation, in vivo genetic cell fate tracing can provide information on how cells behave in the intact tissue under physiological conditions. However some scientists do not detect germ stem cells and neo-oogenesis in adult mouse ovaries when they select Ddx4 as tracing gene to constract Cre-loxp tacing system or identify germ stem cells on the basis of Ddx4 expression. Thus, whether there is follicle regeneration in female mammals remains controversial. If yes, whether the follicle regeneration depends on ovarian germ stem cells and what is the specific molecular marker of these cells? Relevant results can provide the theoretical basis for clinical treatment of ovarian functional failure and establishing transgenic animal with ovaraian germ cells. In this work, we utilize Cre-loxP tacing system to investigate whether there are neo-oogenesis and ovarian germ stem cells in postnatal mouse ovary. We also collect some single germ cells of different developmental stage and preliminarily analyse their gene expression for subsequent specific molecular marker selection of germ stem cells.The key to successfully detect stem cells by genetic tracing is the selection of the gene to drive Cre recombinase. Different from previous studies, we use Oct4 as the tracing gene. Undifferentiated primitive germ cells in fetal ovary and primitive germ cells from postnatal mouse ovary express Oct4. Oct4-MerCreMer mice were crossed with R26R-EYFP mice to establish a tamoxifen inducible tracing system. In this system, all labeled cells are germ cells and we focus on the pre-follicle small germ cells. After tracing germ cells for one day, three days, two months and four months, we find four important phenomenons in adult mouse ovary. A, at different tracing time, some EYFP labeled non-follicular small germ cells always express Sycp3 and Stra8. Sycp3 and Stra8 are transiently expressed in prophase of the first division of meiosis, so Sycp3 and Stra8 expressing small germ cells exist after tracing for four months indicate the persistent meiosis entry in adult mouse ovary. B, at different tracing time, primordial follicles retaining Sycp3 or Stra8 signal can always be detected, indicating that primordial follicle pool can be replenishment in postnatal ovaries. C, at different tracing time, rare small EYFP labeled germ cells are undergoing different types of division. D, at four months post injection, EYFP+Oct4+Dazl- small germ cells still exist in the mouse ovary. By Brdu incorporation assay, we find that Brdu labeling in Oct4+Dazl- small germ cells reflect mitotic DNA replication, whereas Brdu labeling in Oct4+Dazl+ cells reflect pre-meiotic DNA replication. Together, by Oct4-Cre-loxp tracing system, we detect meiosis reentry and promordial follicle replenishment in adult mouse ovary, indicating that there is neo-oogenesis. At the same time, rare germ cells can undergo different types of proliferation and mitotic DNA replication, these cells can maintain their own group during long time tracing by self-renewal capacity, indicating that neo-oogenesis depends on germ stem cells in adult mouse ovary. Our result further suggest that ovarian germ stem cells exist in the Oct4+Dazl- germ cell population not the Oct4+Dazl+ or Oct4+Ddx4+ germ cell population.After illustrating the existence of germ stem cells in postnatal mouse ovary, we try to find out their specific molecular marker. Using single cell cDNA amplification technology combined with Oct4-Cre-loxp tracing system, we want to find out the ovarian germ stem cells population and their specific molecular marker by RNA-seq and gene expression analysis of germ cells in different developmental stages. We collect some single germ cells at different developmental stage and preliminarily analyse their gene expression after fluidigm assay and find that some samples express several transient expressing genes in prophase of the first meiosis division such as Sycp3, Mlh1 and Msh5. Some other samples simultaneously express Sycp3 and Sohlh1 or Sohlh2. These phenomenons suggest that there are meiosis reentry and primordial follicles replenishment in adult mouse ovary in RNA level. All samples we collected can be divided into three types by PCA analysis. The X type can be divided into three populations: m, n and o population. We speculate that the Z type and o population may contain the potential ovarian germ stem cells. However, we find that ovarian germ cells have large degree of heterogeneity regarding genes expression, indicating that we must increase the number of samples if we want to well understand the developmental stages of germ cells and pick out the actual germ stem cells from plenty of samples according to the sequencing data. We will use Smart-seq2 combined with cell barcodes to collect more samples for high-throughput sample sequencing and analysis subsequently. Using the specific molecular marker of germ stem cells to construct a new in vivo Cre-loxp tracing system can illustrate the significance of germ stem cells to normal ovarian function. Further more, the specific molecular marker is the basis of germ stem cells study in vitro.