Name: a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes
Uploaded: 2018-03-03T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

サポートのおかげで可能に作られたこの作品: パヴィア大学ドイツ連邦共和国 2016;パルマ大学フィル 2014、2016;本研究の遂行に必要なプラスチック製品を供給するためのキネシス。博士シェーン ウィンザー (工学部、ブリストル大学、イギリス) は、Cell_PIV ソフトウェアを提供することを感謝いたします。

動物を含むすべてのプロシージャは、制度的動物のケアし使用とパヴィア大学の倫理委員会によって承認されました。動物は、22 ° C、湿度 60% の空気と 12:12 時間の明暗サイクルの条件の下で維持されました。
1. 卵巣の分離
<ol><li>腹腔内 2 4-11 週齢 CD1 雌マウス 10 U の卵胞刺激ホルモン滅菌 1 mL インスリン注射器を注入します。</li>
	<li>46-48 h を待ちます。</li>
	<li>マウスの重さし、Zoletil (Tiletamina、Zolazepan cloridrate) の 50 mg/kg の筋肉内注射で麻酔。頚部転位によって安楽死させます。</li>
	<li>剥離鉗子のペアを使用して腹壁を覆っている皮膚を把握します。はさみで皮膚と体壁をカットし、鉗子で切開を開けます。</li>
	<li>ピンセットのペアを使用して、移動勇気はさておき、正確な子宮角と四肢の運動で卵巣をローカライズします。</li>
	<li>時計鉗子を用いた卵巣優しく押しながら子宮にその靭帯をカットするはさみを使用します。他の卵巣を繰り返します。</li>
	<li>37 ° c、5% CO2空気中で加温 M2 分離培地 1 mL を含む 35 mm 細胞培養シャーレに収集した卵巣を転送します。</li>
	<li>脂肪と顕微鏡下で高級はさみを使用して卵管を取り外します。</li>
</ol>2. 卵丘卵子複合体の単離
<ol><li>幽門洞卵丘卵子複合体 (Coc) が受動的に解放されるまで、滅菌ピンセットや 1 G 滅菌インスリン針を使用して卵巣の表面を穴をあけます。</li>
	<li>卵丘細胞口制御の手を引っ張ってパスツール ピペット (直径 200 μ m) を使用しての 3 つ以上の層で Coc を収集し、培 M2 300 μ L ドロップにそれらを転送します。</li>
	<li>数秒間に軽くピペッティングして手を引いたパスツール ピペット (80 μ m) を使用して卵母細胞を囲む積雲セルを削除します。</li>
	<li>繰り返し卵母細胞から転送 M2 中の 20 μ L ドロップを別卵丘細胞が完全に除去されるまで。</li>
</ol>3. クロマチン組織ベースの卵母細胞の分類
<ol><li>最終濃度 5 mg/mL の 1x PBS で希釈の母液から M2 を中で 0.05 μ G/ml ヘキスト 33342 染色液を準備します。</li>
	<li>ヘキスト染色液 35 mm シャーレの蓋の下部の 3.5 μ の微小液滴を配置します。</li>
	<li>ドロップを染色ヘキストにそれぞれの単一の卵を転送、予め温めておいた (37 ° C) 加熱プレートの上に皿を置き、光博覧会を避けるために暗いふた付きカバーします。</li>
	<li>常温静置後の 10 分は後より 1 〜 2 秒の紫外線 (330-385 nm) の下で 10 倍の倍率で蛍光顕微鏡で卵母細胞を観察します。</li>
	<li>ヘキスト正クロマチン核小体を周囲のリングを呈する場合は、卵母細胞に囲まれた核小体 (SN) (図 1 a)、として分類します。</li>
	<li>彼らの核小体がヘキスト正クロマチンで囲まれていないとショーを分散させる (図 1 b) 核内多色スポットない囲まれた核小体 (NSN) として卵母細胞を分類します。</li>
</ol>4. ニューラル ネットワーク ベースの卵母細胞の分類
<ol><li>4 つの準備 5% 熱不活化牛胎児血清 2 mM L グルタミン 5 mM タウリン、100 U/mL ペニシリン、75 μ g/mL ストレプトマイシン、35 ミリメートルのガラスに 23.5 μ g/μ L ナトリウム ピルビン酸と α MEM 培 2 μ L 水滴シャーレの底し、でそれらをカバー予め温めておいた (37 ° C) および培地の蒸発を避けるためにあらかじめ釣合い鉱物油。 生きている細胞スクリーニング システムを使用している場合注意: 滴特定の距離をガイドラインとして 7 mm × 7 mm の描画グリッドを使用します。</li>
	<li>転送 3-4 卵にドロップします。</li>
	<li>コマ撮り録画ソフトウェアを搭載したライブ細胞スクリーニング システム ガラス底ペトリ皿に配置 (参照材料表)、安定した温度 (37 ° C) と CO2 環境で卵成熟過程の観測が可能濃度 (5%)。</li>
	<li>コマ撮り録画ソフトウェアを開きます。画面の左側にある卵子と右側にあるすべての設定ボタンのライブ ショット ウィンドウが表示されます。</li>
	<li>画面中央に位置する卵子のセンターを選択します。必要な場合は、フォーカスを調整します。</li>
	<li>Phのボタンを選択し、192; DIA ランプを設定露出時間 1/125 s;得るために1.99;1600 ×1200の解像度。</li>
	<li>FL2を選択し、ダイヤ ランプを5に設定露出時間 3 s;得るために2.37;1600 ×1200の解像度。</li>
	<li>文化ドロップ内卵子の位置を記録するために新しいポイントボタンを選択します。各卵母細胞文化ドロップ内の同じルーチンを繰り返します。</li>
	<li>時間を選択し、 8 分と合計時間 15 hまでに買収のサイクルを設定します。</li>
	<li>記録サイクルを開始するコマ撮りを開始を選択します。</li>
	<li>MATLAB ソフトウェアを開き、書き込み >>cell_piv;PIV ツールを選択します。</li>
	<li>記録映画が含まれているフォルダーを選択し、全体像をロードする .jpeg ファイルのいずれかを選択します。各卵母細胞領域ツールを選択をクリックして利益 (率 ROI) の領域を選択します。</li>
	<li>[ファイル] メニューでPIV プロセスを選択します。ビューアー ツールボタンを選択します。 注: プログラム ROI 内の速度ベクトルを計算し、.mat ファイルとして保存、データ ファイルが自動的に生成されます。</li>
	<li>.Mat ファイルを開くを選択します。ボタン選択 ROI ツールを選択し、卵母細胞の輪郭に沿って円を描きます。ファイル] メニューの [保存] をクリックします。 注: この地域でベクトルが表示されます、グラフ ウィンドウで振幅データ更新この新しい投資収益率。</li>
	<li>オープン全体時間経過フレームの収集した振幅データ記録と、フレーム シーケンスの行を列のスプレッドシートに各単一卵母細胞を分析します。</li>
	<li>10 サブグループに NSN と SN の卵母細胞のデータをまとめます。</li>
	<li>繰り返しフィード フォワード人工ニューラル ネットワークを訓練する 9 のサブグループを使用 (ファン) とブラインド テスト 1 サブグループ。<ol>
<li>別に繰り返し 9 回ブラインド テストのサンプル (10 倍クロス検証) を変更します。</li>
	</ol>
</li>
	<li>MATLAB を開き、カスタム スクリプトメインを実行し、リクエストでは、訓練データ (train.txt)、(通常から分析するトレーニングとインターバル撮影の間隔の使用 NSN と SN の卵子の数を持つファイルの名前を挿入フレーム フレーム # 112-113 に # 1-2)。</li>
	<li>トレーニングを実行する入力を押します。</li>
	<li>テスト データ (test.txt) を含むファイルの名前を挿入します。</li>
	<li>MATLAB のワークスペースでベクトルtest_outputs_cytを開きます。 注: 最初の行、それぞれ NSN と SN の卵子の本当の陽性 (TP または True NSN) および偽陽性 (FP または False NSN) を入手する確率を示す画面上にウィンドウが表示されます。代わりに、2 行目で、偽陰性 (FN または False SN) および NSN と SN の卵子の真のネガ (テネシー州または True SN) をそれぞれ取得する確率。</li>
	<li>数式を使用して: (TP+TN)/(TP+FP+FN+TN)、ファンの確度、パーセンテージを計算します。</li>
</ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+configurations+in+the+germinal+vesicle+of+mammalian+oocytes.">Chromatin configurations in the germinal vesicle of mammalian oocytes.</a> Mol. Hum. Reprod. 15 (1), 1-9 (2009).</li><li>Vigone, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+based+identification+of+mouse+cumulus+cell+markers+that+predict+the+developmental+competence+of+their+enclosed+antral+oocytes.">Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes.</a> BMC Genomics. 14, 380 (2013).</li><li>Bui, T. 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著者が明らかに何もありません。

1 つはこのプロトコル マウス卵母細胞と他の種のそれらの実行中の世話をする必要がありますいくつかの重要な手順があります。コンパニオンの積雲からの分離細胞のトリガーに卵母細胞必要がありますすぐに記録の低下に転送一度彼らの卵胞から分離、GV の MII 遷移の開始。議定書への可能な変更が Coc 分離に使われる M2 媒体に 3-イソブチル-1-メチルキサンチン (IBMX) の付加であります。IBM...

女性の不妊は、女性数の増加に影響を与える検査です。世界保健機関によると約カップルの 20% が不妊、女性不妊原因の 40% です。さらに、がん治療を受ける女性の 3 分の 1 (300,000/年、米国やイタリアで 30,000/年それぞれ) 早期の卵巣の失敗を開発。
がん患者の不妊を防ぐため戦略は、MII ステージ (GV-MII 切り替え) に GV 卵子の体外成熟 (IVM) 続いて、がん治療前に卵胞の凍結と分離です。受精と発生過程と全体的な妊娠成功1,2卵子の発生能の非侵襲的なマーカーの可用性が向上します。
ヘキスト 33342、觀螢光色素で染色後に観察されたクロマチン構成に基づいて完全育てられた卵母細胞が囲まれた核小体 (SN) やない囲まれた核小体 (NSN)3のいずれかに分類されます。異質染色質組織以外にも卵母細胞のこれらの 2 つのタイプ表示多く形態的, 機能的違い3,4,5,6,7,8 ,9, 減数分裂と発達能力を含みます。体外成熟および卵巣から分離されたとき両方が MII 期卵子に達するの入力精子受精後 2 細胞期に開発、用語9SN クロマチン組織にのみ発展するかもしれない。有能と無能な卵母細胞を選択するための分類法として良い, 主な欠点は螢光色素自体と、上記のすべての検出に使用される紫外線が細胞に突然変異誘起効果は。
これらすべての理由から、我々 は同じ高い分類精度を維持しながらヘキストの使用を置き換えることができます SN または NSN のクロマチン構造に関連付けられている他の非侵襲的なマーカーの検索。細胞質の運動速度 (CMVs) の時間経過の観察は、細胞状態の特徴的な機能として現れています。たとえば、最近の研究は、CMVs の間の関連付けは、受精の時間と能力をマウスとヒトの受精卵の着床前の完全なと完全な長期的開発10,11で記録を示した。
これらの以前の研究に基づいて、ここで述べたい発達有能か無能なマウス完全育てられた卵5,6,7,8の認識のためのプラットフォーム。プラットフォームは、3 つの主な手順に基づいて: 1) から胞状卵胞由来卵子は最初囲まれて核小体 (SN) またはない囲まれた核小体 (NSN); としてクロマチン構成いずれかに基づいて分類されます各単一卵子の GV の MII の移行中に発生した CMVs の 2) コマ撮り画像が撮影され、粒子画像流速測定法 (PIV); と分析・ 3) PIV による解析で、フィード フォワード人工ニューラル ネットワーク (ファン) ブラインド分類12,13。私たちは、テストおよび他の哺乳類種 (例えば牛、猿、人間) ために使用する利用可能な準備ができているし、マウス用に設計されたプロシージャの最も重要な手順の詳細を与えます。

<table><tbody><tr><td>Folligon</td><td>Intervet</td><td>A201A02</td><td>Hormonal treatment</td></tr><tr><td>Hoechst 33342&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B2261</td><td>For oocyte heterochromatin staining</td></tr><tr><td>Cell culture Petri-dish 35 mm x 10 mm&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>430165</td><td>For COCs isolation</td></tr><tr><td>EmbryoMax M2 Medium (1X), Liquid, with phenol red</td><td>Merck-Millipore</td><td>MR-015-D</td><td>For COCs isolation</td></tr><tr><td>MEM Alpha medium (1X) + Glutamax&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M4526</td><td>For oocyte in vitro maturation</td></tr><tr><td>Cell culture Petri-dish 35 mm glass-bottom&amp;nbsp;</td><td>WillCo&amp;nbsp;</td><td>GWSt-3522</td><td>For imaging experiments</td></tr><tr><td>BioStation IM-LM&amp;nbsp;</td><td>Nikon</td><td>MFA91001</td><td>Live cell screening system&amp;nbsp;</td></tr><tr><td>Pasteur pipette</td><td>Delchimica Scientific Glassware</td><td>6709230</td><td>For follicles manipulation</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td>To prevent contamination and medium evaporation</td></tr><tr><td>Penicillin / Streptomycin</td><td>Life Technologies</td><td>15070063</td><td>To prevent medium contamination&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>ML16141079</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>Life Technologies</td><td>25030</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T0625</td><td>For making up &amp;alpha;MEM medium&amp;nbsp;</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3310</td><td>For making up &amp;alpha;MEM media</td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>&amp;nbsp;P4562</td><td>For making up &amp;alpha;MEM media</td></tr><tr><td>Zoletil (Tiletamina and Zolazepan cloridrate)</td><td>Virbac Srl</td><td>QN01AX9</td><td>For mice anesthesia</td></tr><tr><td>Cell_PIV sofware&amp;nbsp;</td><td>Kindly provided by Dr. Shane Windsor, University of Bristol, UK</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td></tr><tr><td>MATLAB</td><td>The MathWorks, Natick, MA&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; &amp;nbsp;-</td><td>For multi-paradigm numerical computing</td></tr></tbody></table>

a neural network-based identification of developmentally competent or incompetent mouse fully-grown oocytes

不妊クリニック発達有能と無能な卵全体の妊娠の結果を向上させる非侵襲的な手順を使用してを選択する機能の恩恵。我々 は最近、卵核胞 (GV) から体外成熟細胞運動の分析に続いて、中期 II ステージ中にマウス卵母細胞の顕微鏡のライブ観察に基づく分類方法を開発この時間経過の期間中に発生しています。ここでは、このプロシージャの詳細なプロトコルを提案する.卵は、完全育てられたから胞状卵胞から隔離され、37 ° C、5% CO2で時間経過の分析のため、顕微鏡内 15 時間培養します。写真は、8 分間隔で撮影されます。各卵子の培養期間中に発生する細胞質の運動速度 (CMVs) のプロファイルを計算する粒子画像流速測定法 (PIV) メソッドを使用して画像を分析します。最後に、それぞれの単一の卵母細胞の CMVs は主務が発達 91.03% の精度で無能か配偶子の確率を予測する数理分類ツール (フィード フォワード ニューラル ネットワーク、ファン) を介して供給されます。このプロトコルは、マウスの設定は、人間を含む他の種の卵母細胞に今テストでした。

図 2は、それぞれ先頭 (GV) と IVM プロシージャの終わり (MII) 代表的な発達有能で無能な卵を示します。完全育てられたマウス卵母細胞の IVM 培養 15 時間中に発生します。時間経過の観察は減数分裂の進行を記録し、GVBD と第一極体の押し出しなどを含む、主要な減数分裂イベントを検出します。
分析?...

Watch this Scientific Journal Video about A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes at JoVE.com

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

ここでは、中期 II ステージに卵核胞から体外成熟中に実行される卵発達能力の非侵襲的評価するためのプロトコルを提案する.このメソッドは、粒子画像流速測定法 (PIV) ニューラル ネットワーク解析とタイムラプス イメージングを組み合わせます。

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 ...

a-neural-network-based-identification-developmentally-competent-or

Research

JoVE Journal

Developmental Biology

6.6K ビュー.  University of Pavia. ここでは、中期 II ステージに卵核胞から体外成熟中に実行される卵発達能力の非侵襲的評価するためのプロトコルを提案する.このメソッドは、粒子画像流速測定法 (PIV) ニューラル ネットワーク解析とタイムラプス イメージングを組み合わせます。

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

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.

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

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 ...

発生生物学

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 ...

遺伝学

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, ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.

成人マウス卵巣における正確な卵胞列挙体

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

マウスの発達中の卵巣保護区の定量的3D分析のための全卵巣免疫蛍光、クリアリング、および多光子顕微鏡

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

マウス卵母細胞における紡錘体アセンブリチェックポイント完全性の評価

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

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 ...

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

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この動画の章

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

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

「マウス卵巣における現象を可視化する卵巣の組織培養

成人マウス卵巣における正確な卵胞列挙体

マウスの発達中の卵巣保護区の定量的3D分析のための全卵巣免疫蛍光、クリアリング、および多光子顕微鏡

マウス卵母細胞における紡錘体アセンブリチェックポイント完全性の評価

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

要約

さらに動画を探す

この動画の章

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

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

「マウス卵巣における現象を可視化する卵巣の組織培養

成人マウス卵巣における正確な卵胞列挙体

マウスの発達中の卵巣保護区の定量的3D分析のための全卵巣免疫蛍光、クリアリング、および多光子顕微鏡

マウス卵母細胞における紡錘体アセンブリチェックポイント完全性の評価

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