Name: isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization
Uploaded: 2016-01-07T12:00:01.000+00:00
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Description: Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

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.

このプロトコルは、欧州（EU 2010分の63）、科学研究のために使用される動物の保護イタリア（2014分の26）法律の指針に従って実施されます。頸椎脱臼を安楽死は、卵巣の単離のために使用され、それは、この研究をカバーする承認されたプロトコルに記載されている個体を専門家によって行われ、訓練されます。 1.動物<ol><li>制御された温度および湿度の部屋で6週齢の雌マウスに大人3を維持（12Lの位相と：12Dおよび摂食自由摂取 ）。 </li><li>卵胞刺激ホルモン（FSH）の効果を模倣することにより卵胞の成長を刺激するために妊馬血清性性腺刺激ホルモン（PMSG）の2.5 IUを腹腔内にマウスを注入。後で洞卵母細胞の収集48時間のための卵巣を分離します。あなたの機関のガイドラインによると、以前にPMSGを注射したマウスを安楽死させる女性。 </li><li>すぐに体腔から卵巣を除去し、ペトリ皿の続きにそれらを置きますその後の卵母細胞の単離のためのM2培地をaining（セクション3を参照）。 <ol><li>無菌解剖ハサミを使用して、腹膜の切開に続いて腹壁をカバーする皮膚の切開を行います。膀胱から始まるV字形状を形成する、腹部を露出するように切開を開き、滅菌ピンセットを用いて、一方の側に勇気を移動させ、子宮管をローカライズ。 </li><li>腎臓の下方に位置する卵巣を観察します。滅菌ピンセットで各チューブを持ち、それらを解放するために卵巣の下部に小さな切開を行います。予め平衡化培地を含むペトリ皿の底に両方の卵巣を転送します。 </li></ol></li></ol> 2.ホルモンの準備<ol><li>適切な注入量0.1 1mlあたり2.5 IUホルモンの作用濃度を得るために、滅菌等張食塩水PMSG（異なるストック濃度で利用可能）を希釈します。 注：ホルモンソリューションを0.5 mlチューブに分注し、で保存する必要があります-2使用日まで0℃。 -20℃で3ヶ月よりも長い希釈アリコートを保管しないでください。解凍したら、シリアル注射が行われた場合、4℃のホルモンソリューションのアリコートを保存し、4日以内にそれらを使用しています。 </li></ol> 3.洞卵母細胞の分離 適切な卵母細胞の単離のために： <ol><li>卵母細胞の単離手順を開始する前に少なくとも1時間、5％CO 2、37℃でM2培地13（M2）を平衡化。 2ペトリ皿（35ミリメートル×10 mm）で、卵巣穿刺からの単離後の卵母細胞の転送のためにミネラルオイル（〜1.5ミリリットル）で覆われたM2の小滴を有する第二1（20μL）のためのM2の1ミリリットルのものを準備します卵巣（3.4から3.6を参照）。準備が使用されるようになるまで、5％CO 2、37℃のインキュベーター内で両方のペトリ皿にしてください。 </li><li>卵母細胞の単離手順中に実体顕微鏡を使用してください。 </li><li>ストレッチにより卵母細胞を収集するために、薄い口のガラスピペットを準備興ガラスパスツールピペットは、ブンゼンバーナーからの垂直燃焼（ 図1）を介して、（両手でピペットの先端を保持する）水平に保ちました。 <ol><li>ガラスが溶融し始めると、炎の外にピペットを取り、目的のピペットを得るために迅速に引き運動を行います。しっかりと長さと内部の毛細血管の直径を調整するための爪とピペットの狭い点に近い過剰ガラスをカット。細い毛細血管が卵母細胞の直径よりも僅かに大きい〜100μmの内径を有することを確認します（〜80ミクロン）。 </li><li>口のピペットには、適切なチップを使用して引っ張っピペットに吸引管の​​一端を接続し、歯とそれを確保、口の中にもう一方の端を置きます。 </li></ol></li><li>滅菌ピンセットを用いて卵巣を採取し、細胞培養用シャーレ（35ミリメートル×10ミリメートル）の底部にそれらを堆積させます。以前は、37℃で平衡化したM2の1mlでそれらをカバー、5％CO 2（ 図2）。 </li><li>ゆっくりM2（ 図3）で胞状卵母細胞を解放するために、滅菌インスリン注射針と卵巣の胞状卵胞を穿刺。 </li><li>そっと口は包細胞および他の可能性のある組織片（ 図4A）を削除し、以前に（胚培養に適したミネラルオイルで覆われたM2の小滴（20μL）の下でそれらを解決するための狭いパスツールピペットを通して卵母細胞をピペット準備; 図4B）。 注：これは、M2のいくつかの異なる滴を通して連続ピペットで卵母細胞の透明帯に接続されているすべての卵丘細胞を除去することが重要です。培地のpHの変化を避けるために、可能な限り迅速にこの手順を実行します。 </li></ol> Hoechst33342 4.胞状卵母細胞の染色注：洞卵母細胞は、SN（囲ま小体）、NSN（小体を囲まれていない）、またはに区別することができます他の中間形式で、Hoechst33342の超生体濃度（又は塩基の具体的な任意の他のマーカーAT）14で染色した後、それらのクロマチン組織に基づきます。 NSNがいない間、実際には、SN卵母細胞は、したがって、その名前、核小体の周りヘキスト陽性リングを示しています。 適切な卵母細胞染色のために： <ol><li>暗所で10分間、50 ngの/μLの濃度で超生体M2に希釈したHoechst33342（20μL）の液滴に口のピペットを用いて卵母細胞を転送します。 </li><li> Hoechst33342とのインキュベーションの後、クロマチン組織（SNまたはNSN）に基づいてソートするため、鉱物油で覆われたM2の小滴（5μL）の底に口のピペットを用いて各単一卵母細胞を転送します。卵母細胞のクロマチン構造を可視化するためにHoechst33342フィルタ（発光波長486ナノメートル）を装備した任意の蛍光顕微鏡を使用してください。 注：使用する蛍光顕微鏡の種類に応じて、私はtは、画像取得用のガラス底ペトリ皿を使用することが不可欠となることができます。 35mm皿のための試料ホルダーを装備した共焦点顕微鏡オリンパスFLUOVIEW Fv10iは0.17ミリメートルの標準的な厚さ0.13から0.21ミリメートル許容可能な範囲で、画像取得のための唯一のガラスボトムディッシュが必要です。 </li><li>そう、もはやよりも5〜10秒のために存在または核小体の周りにリングが存在しない場合（ 図5）を視覚化するのに十分な時間を卵母細胞エキサイト。 </li><li> 体外成熟を ​​誘導するか、または分子または細胞の解析のための適切な緩衝液中でそれらを保つために、5％CO 2、37℃でM2で、文化胞状SNとNSNの卵母細胞をソートした後。 </li></ol>

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The authors have nothing to disclose

このプロトコルは、マウス洞のGV卵母細胞を分離し、分類するために使用される方法について説明します。いくつかの例外を除いて、下線する特段の重要なステップはありません。 A）卵母細胞の活力を維持し、b）に用いられる培地中のpH変化を避ける：まず：にこの手順は、可能な限り迅速に実行する必要があります。第二：卵母細胞は、IVMおよびIVFは、次の手順がある場合は特に、クロマ...

卵巣の胞状コンパートメントから分離された胞状十分に成長した卵母細胞（GV、卵核胞）は日常、などなど、さまざまな目的に使用されている例えば、中期II段階まで体外成熟の背後に分子・細胞メカニズムの研究。 その美しい外観で最も胞状卵母細胞にもかかわらず、すべてではないが、減数分裂を完了し、胚盤胞段階に到達することができます。受精が3-5が発生した場合 、実際には、人間1,2を含む多くの哺乳動物種において、それらのおよそ30％が、2細胞期で、その開発を逮捕します。今日までに、いくつかのパラメータは、そうすることができないものから胚発生を維持することができる卵母細胞を区別するために使用されます。 例えば、クレシルブルー染色（BCB）はBCB +またはBCB-染色に関連し、このソート方法の有用性はあるが、グルコース-6-リン酸デヒドロゲナーゼの活性に基づいて、卵母細胞を分類するための有効な方法であり、まだ研究室に依存6-8。 もう一つのよく確立された方法は、彼らのクロマチン組織に存在する：（Hoechst33342のような）任意のDNAインターカレー色素で染色した場合、胞状卵母細胞の70％が核小体の周りの色素陽性リングを示し、小体（SN）囲ま呼ばれ、残りの30％ながら、より多くの斑点陽性シグナルを示し、仁（NSN）3-5囲まれていないと呼ばれています。最近では、ことを示した細胞質格子9（のCPL、哺乳動物の卵母細胞および胚の両方で母体由来のmRNAとリボソームのための重要な貯蔵部位）の不在10との存在と一緒（のCPL形成の11のために必須）MATERのダウンレギュレーション、その細胞質9,12における脂肪滴は、2細胞期を超えて進行するNSNの卵母細胞の無力に関連する他の原因であることができます。 残念ながら、いくつかのテクニックが胞状SNとNSN卵母細胞とをソートするために適用することができ、このため、（核染色、固定手順や口のピペットとの操作なし）少ない侵襲的方法の開発は、ヒトおよび動物の両方の胚発生の速度を向上させることが非常に重要になる可能性があります。 本稿では、詳細簡単かつ迅速プロトコルは、広告ソートSNと雌マウスからNSN胞状卵母細胞を単離するために使用し、それは女性の配偶子形成、卵成熟と胚発生の研究に興味を持つすべての科学者にアドレス指定されています。

<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.

以下の写真は、Hoechst33342を用いて卵母細胞「ソートにピペットの準備から始めて、マウスの卵巣から胞状卵母細胞を単離するために使用する方法を示しています。この方法は非常に簡単で、CO 2インキュベーター、実体顕微鏡およびHoechst33342の観察のために正しいフィルターを備えた蛍光顕微鏡を備えた任意の実験室で行うことができます。図1は、</stron...

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

ここでは、核小体の組織に基づいて、マウス胞状卵母細胞の特徴付けのためのプロトコルを提示します。

核小体クロマチン組織に基づいてマウス洞卵母細胞の単離とキャラクタリゼーション

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

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13.3K ビュー.  Fondazione IRCCS Policlinico San Matteo. ここでは、核小体の組織に基づいて、マウス胞状卵母細胞の特徴付けのためのプロトコルを提示します。 

ビデオ: 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.

クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

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

遺伝学

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.

マウス卵母細胞を減数分裂の間に人間の遺伝子の機能を評価するために使用私

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.

馬の卵母細胞における染色体分離、ヒストンアセチル化およびスピンドル形態の解析

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

発生生物学

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.

ニューラル ネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定

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.

「マウス卵巣における現象を可視化する卵巣の組織培養

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.

マウス卵母細胞における細胞質微小管組織化中心の多光子レーザーアブレーション

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

マウスの前期停止卵子におけるDNA損傷の同定

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.

著者スポットライト:哺乳類の卵母細胞および着床前胚におけるDNA損傷応答の理解 

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.

マウス卵母細胞におけるDNA二本鎖切断の検出

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.

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

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

核小体クロマチン組織に基づいてマウス洞卵母細胞の単離とキャラクタリゼーション

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クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

マウス卵母細胞を減数分裂の間に人間の遺伝子の機能を評価するために使用私

馬の卵母細胞における染色体分離、ヒストンアセチル化およびスピンドル形態の解析

ニューラルネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定

「マウス卵巣における現象を可視化する卵巣の組織培養

マウス卵母細胞における細胞質微小管組織化中心の多光子レーザーアブレーション

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

核小体クロマチン組織に基づいてマウス洞卵母細胞の単離とキャラクタリゼーション

要約

さらに動画を探す

この動画の章

クロマチン広がるマウス卵母細胞の進行を前期から中期 II の分析のための準備

マウス卵母細胞を減数分裂の間に人間の遺伝子の機能を評価するために使用私

馬の卵母細胞における染色体分離、ヒストンアセチル化およびスピンドル形態の解析

ニューラル ネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定

「マウス卵巣における現象を可視化する卵巣の組織培養

マウス卵母細胞における細胞質微小管組織化中心の多光子レーザーアブレーション

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞におけるDNA二本鎖切断の検出

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

ニューラルネットワークによる発達有能か無能なマウス完全育てられた卵母細胞の同定