M.M. and C.A.R. acknowledge the financial support of the Ministero della Salute, Ricerca Finalizzata/giovani ricercatori anno 2009 of the Fondazione IRCCS Ospedale San Matteo, Pavia (I). M.M and C.A.R. wish to thank Marianna Longo for helping with the images preparation.

该协议是按照欧洲的指导原则（EU二千〇十分之六十三）和保护用于科学研究的动物意大利（2014分之26）的法律进行。颈椎脱位安乐死用于卵巢隔离，它是由专家进行和训练有素的已批准的方案涉及本次研究中列出的个人。 1.动物<ol><li>保持成年3至6周大的雌性小鼠中的房间温度和湿度受控（具有12L的阶段:12D，并自由采食）。 </li><li>腹腔注入小鼠2.5 IU孕马血清促性腺激素（PMSG），通过模仿的卵泡刺激素（FSH）的影响，刺激卵泡生长。隔离卵巢的窦收集的卵母细胞48小时后。安乐死的雌性小鼠以前用PMSG注射，根据你的机构的指导方针。 </li><li>从体腔迅速取出卵巢和它们放入培养皿中续癌宁M2培养后续的卵母细胞隔离（见第3节）。 <ol><li>使在皮肤上的覆盖腹壁接着在腹膜切口通过使用无菌解剖剪刀切开。打开切口以暴露腹腔，用无菌镊子将胆一方和本地化子宫管，形成一个V形从膀胱开始。 </li><li>观察位于下方的肾脏卵巢。保持每个管，用无菌镊子和作一小切口在卵巢释放他们的底部。在含预平衡介质的培养皿的底部转移双侧卵巢。 </li></ol></li></ol> 2.激素制剂<ol><li>稀PMSG（在不同的库存浓度可提供）用无菌等渗盐水溶液以获得每0.1 ml的2.5 IU激素一个工作浓度为适当的注射体积。 注:激素溶液必须等分的0.5ml管中，并储存在-20℃下直至使用当天。不要将稀释分装比在-20°C三个月的时间。一旦解冻，如果连续注射执行，存储激素的解决方案的分装置于+4℃，4天之内使用。 </li></ol> 3.胃窦卵母细胞隔离 对于正确的卵母细胞的分离: <ol><li>在开始的卵母细胞的分离步骤之前平衡M2培养基13（M2），在5％的CO 2和37℃下至少1小时。准备两个培养皿（35毫米×10毫米），一个用1ml M2的用于卵巢穿刺和M2的从隔离后覆盖有矿物油（〜1.5毫升）中的卵母细胞转移，第二个具有小滴（20微升）子房（见3.4-3.6）。保持在孵化两培养皿在5％的CO 2和37℃下，直到准备使用。 </li><li>在卵母细胞分离方法使用立体显微镜。 </li><li>准备薄薄的嘴唇玻璃吸管收集的卵母细胞伸缩兴玻璃巴斯德吸液管保持水平（持用双手吸管的两端），在一个垂直的火焰本生灯到来（ 图1）。 <ol><li>一旦玻璃开始熔化，取吸管出来的火焰和执行快速牵引运动，以获得所需吸管。牢牢剪断多余的玻璃靠近窄点用指甲以调节长度和内部毛细管的直径的吸移管的。确保薄毛细管具有〜100微米的内径，比卵母细胞的直径稍大（〜80微米）。 </li><li>口移液管，通过使用适当的尖端连接吸气管至所述拉吸管的一端，并将另一端的嘴，用牙固定它。 </li></ol></li><li>使用无菌镊子收集卵巢和将它们存放在细胞培养的培养皿（35毫米×10毫米）的底部。覆盖它们用1ml M2的先前在37℃下平衡过和5％的CO 2（ 图2）。 </li><li>轻轻穿刺卵巢的窦性卵泡，用无菌胰岛素注射器针头释放胃窦的卵母细胞中的M2（图3）。 </li><li>轻轻口通过一个狭窄的巴斯德吸管除去毛囊细胞和任何其他可能的组织碎片（图4A），然后解决它们在小滴的M2（20微升）覆盖有适于胚胎培养矿物油的底部（先前吸管的卵母细胞制备; 图4B）。 注:通过几个不同滴M2来消除附着在卵母细胞连续移液的透明带的卵丘细胞是非常重要的。执行此步骤尽可能快，以避免在介质的pH的改变。 </li></ol> 4.胃窦卵母细胞染色与Hoechst33342染色注:窦卵母细胞可以在SN（环绕核仁），诺西（不环绕核仁），或者加以区分在任何其他中间形式的基础上，他们的染色质组织，Hoechst33342染色的体外活浓度（或特定的任何其他标记物为基础的AT）14染色后。事实上，SN卵母细胞表现出周围的核仁一个赫斯特阳性的环，而诺西不这样做，因此它的名字。 对于正确的卵母细胞染色: <ol><li>传送用嘴吸管卵母细胞成Hoechst33342染色（20微升）稀释在M2的液滴在50毫微克/微升体外活浓度在黑暗10分钟。 </li><li>用Hoechst33342染色的培养后，在小滴的M2（5微升）覆盖有矿物油，基于染色质组织（SN或NSN）分选的底部转移的每个单个卵母细胞用吸管嘴。使用配备Hoechst33342染色过滤器（发射波长486纳米）的任何荧光显微镜可视化的卵母细胞的染色质结构。 注意:根据所用荧光显微镜的型，it可以成为当务之急使用玻璃底培养皿进行图像采集。在激光共聚焦显微镜奥林巴斯Fluoview Fv10i在配备35毫米盘试样固定器只需要玻璃底菜进行图像采集，用0.17毫米标准厚度和范围内可以接受0.13 0.21毫米。 </li><li>激励的卵母细胞的时间不超过5-10秒，可能有足够的时间来可视化的存在或不存在围绕核仁一个环的（图5）。 </li><li>后整理，培养胃窦SN和诺基亚西门子的卵母细胞中的M2在5％的CO 2和37℃以诱导体外成熟或保持它们在适当的缓冲器，用于分子或细胞的分析。 </li></ol>

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The authors have nothing to disclose

本协议描述用来分离和分类鼠标胃窦GV卵母细胞的方法。有强调，除少数例外，没有特别的关键步骤。第一:此过程应当尽可能快地进行，以:1）保持卵母细胞的活力和b）避免在使用的培养基的pH变化。第二:卵母细胞应该是简要地兴奋（5-10秒），以Hoechst33342染色，以避免染色质损害和卵母细胞的随后死亡，特别是如果IVM和体外受精是以下步骤。迄今为止，这是SN和NSN小鼠卵母细胞胃窦排序的最快方?...

胃窦从卵巢的窦室分离的完全的卵母细胞生长（GV，生发泡）通常用于不同的目的，诸如，例如，在体外成熟直至中期II阶段背后的分子和细胞机制的研究。 尽管他们漂亮的外观最有腔卵母细胞，但不是所有的，都能够完成减数分裂并达到囊胚阶段;事实上，在许多哺乳动物物种，包括人类1,2，其中的大约30％阻止其发展在2-细胞阶段，如果发生受精3-5。迄今为止，只有少数参数用于卵母细胞能够维持从那些无法做到的胚胎发育之间进行区分。 例如，甲酚蓝染色（BCB）是一个有效的方法，以基于葡萄糖-6-磷酸脱氢酶活性的卵母细胞进行排序尽管这种分选方法的效用与BCB +或BCB-染色仍然依赖于实验室6-8。 另一成熟方法驻留在他们的染色质组织:如果沾上任何DNA嵌入染料（如Hoechst33342染色），胃窦的卵母细胞的70％表现出围绕核仁染料正环和被称为环绕核仁（SN）而剩余的30％的显示出更多的斑点积极的信号，被称为不环绕核仁（NSN）3-5。最近我们发现，如果没有细胞质晶格9（的CPL，重要的存储位点在两个哺乳动物卵母细胞和胚胎母体衍生mRNA和核糖体）的10和下调脑膜（必不可少的CPL形成11），加上存在在其胞质9,12脂滴，可以是有关的卵母细胞诺西无法继续进行的2细胞期其他原因。 不幸的是，很少有技术可应用于排序胃窦的SN和NSN和卵母细胞，因为这个原因，较少创伤的方法的开发（没有核染色，固定程序或操纵与嘴移液器）可能成为非常重要的是增加人和动物胚胎发育的速率。 在本文中，我们详细介绍了简单快捷的协议，用于隔离的广告排序SN和雌性小鼠NSN窦卵母细胞，它是给所有有兴趣的女性配子，卵母细胞成熟和胚胎发育的研究中，科学家。

<table><tbody><tr><td>PMSG</td><td>Sigma</td><td>G4527</td><td></td></tr><tr><td>EmbrioMax M2</td><td>Millipore</td><td>MR-015-PD</td><td></td></tr><tr><td>Hoechst33342</td><td>Thermo Scientific</td><td>62249</td><td></td></tr><tr><td>Mineral Oil</td><td>Sigma</td><td>M8410</td><td></td></tr><tr><td>Cell Culture Dish 35mmx10mm</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>&amp;micro;-Dish 35mm, high glass bottom</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Pipette Pasteur Borosilicate</td><td>Corning</td><td>7095D-9</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated micropillary pipettes</td><td>Sigma</td><td>A5177</td><td></td></tr></tbody></table>

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

下面的图片展示用于隔离窦卵母细胞来自小鼠卵巢，用Hoechst33342染色的方式从移液器准备到卵母细胞"排序启动方法。这种方法相当简单，并且可以在装有CO 2培养箱，立体显微镜，配有正确的过滤器，用于观察Hoechst33342染色的荧光显微镜任何实验室中进行图1示出了移液管制备的初始步骤:作为玻璃开始熔化并变软，吸移管的两端必须被拉开，并在同一?...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

在这里，我们提出了一个协议的基础上核仁组织小鼠卵母细胞胃窦表征。

基于核仁染色质组织分离鼠标窦卵母细胞与表征

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

isolation-characterization-mouse-antral-oocytes-based-on-nucleolar

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

13.3K 次观看.  Fondazione IRCCS Policlinico San Matteo. 在这里，我们提出了一个协议的基础上核仁组织小鼠卵母细胞胃窦表征。 

视频: Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

科研

遗传学

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

发育生物学

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

小鼠卵母细胞质微管组织中心的多光子激光消融

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

鉴定小鼠前期停滞卵母细胞中的 DNA 损伤

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

作者聚焦：了解哺乳动物卵母细胞和植入前胚胎的 DNA 损伤反应 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

检测小鼠卵母细胞中的DNA双链断裂

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

小鼠卵母细胞显微注射，成熟和倍性评估

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

基于核仁染色质组织分离鼠标窦卵母细胞与表征

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此视频中的章节

从前期到中期二期小鼠卵母细胞进展的染色质传播制剂分析

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

马卵母细胞染色体分离，组蛋白乙酰化和主轴形态分析

基于神经网络的发育能力或不称职小鼠全生长卵母细胞的识别

卵巢组织培养对小鼠卵巢现象的可视化

小鼠卵母细胞质微管组织中心的多光子激光消融

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

检测小鼠卵母细胞中的DNA双链断裂

小鼠卵母细胞显微注射，成熟和倍性评估