我々 は博士直次郎南 (京都大学) がOog1pro3.9マウスを提供することをありがちましょう。本研究は、日本学術振興会 (科研費 # JP15H06275) と日東財団によって支えられました。

マウスは、23 ± 1 ° C 12 h 光/12 h 暗いサイクルで環境制御部屋で収容されました。動物のケアのプロトコルおよび実験愛知医科大学動物実験ガイドラインに従って実施し、現職のケアおよび使用委員会によって承認されました。
1. 培地や料理の準備
<ol><li>基本的な文化のメディアの準備、ウシ胎児血清 (FBS、5 %v/v)、FSH を追加 (100 Miu/ml) LH (10 mU/mL) とペニシリン-ストレプトマイシン (ペニシリン、100 U/mL; ストレプトマイシン、100 mg/mL) 最小必須培地 α (MEM-α) とミックスは 50 mL の管に。 注: 合計ボリュームの必要な文化メディア実験の間で変わるが、培養液 1 mL は 3.5 cm ガラス底培養皿だけで十分です。</li>
	<li>3.5 cm ガラス底培養皿に 1 mL 混合培地の因数を注ぐし、料理に場所 30 mm 細胞培養チップを設定します。中古メディアとインキュベーター (5% CO2と 37 ° C) で料理を温めます。</li>
</ol>2. 卵巣組織の準備
<ol><li>あらかじめ暖かいリン酸緩衝生理食塩水 (PBS) (−) と MEM-アルファ 5 %fbs、100 U/mL ペニシリン、インキュベーター (5% CO2と 37 ° C) で 100 mg/mL ストレプトマイシンを含みます。 注: 約 3 mL 卵巣あたり因数 PBS (−) の使用のための十分な卵巣の解剖中に、MEM アルファの 1 mL 因数 3.5 cm 皿で単一の卵巣の一時的なストレージとして十分です。</li>
	<li>
4 週間の古い女性 ICR の (がん研究所の後の名前) マウスから卵巣を切除し、はさみと実体顕微鏡下で、ピンセットを使用して卵巣を周囲の組織をトリムします。培地の除去の卵巣をぜひご予約ください (手順 2.1 参照) に使用されるまで。
	<ol>
<li>PBS (−) で加温で湿らせたろ紙上に単一の卵巣の場所 (手順 2.1 参照) と実体顕微鏡下のミクロトーム刃を使用して 4 枚にスライス。 注: 4 週齢 ICR マウスの卵巣は、直径約 2 mm です。ピースの数によって異なります卵巣サイズ;ただし、約 500 μ m 厚い個できる適切な卵胞観察 (作品は 500 mm よりも厚く、組織透過性が減少; 個 500 ミリメートルよりも薄いの多くから胞状卵胞が壊れており、失われた)。</li>
		<li>郭清後に、予め温めておいた培 (手順 2.1 参照) 3.5 cm 料理にスライスした卵巣片を配置します。</li>
	</ol>
</li>
</ol>3. 卵巣の組織培養
<ol><li>卵巣組織がなる細胞文化挿入時の卵巣組織をスライス培養培地ごとの約 0.5 μ L をドロップ マイクロ ピペットを使用して設定します。</li>
	<li>ピンセットを使用して細胞培養インサート培のドロップに各スライス標本を配置します。</li>
	<li>37 ° C で 5% CO2の卵巣組織を文化します。</li>
	<li>2 日ごとに予め温めておいた培で培養液を置き換えます。 注: 4 週齢 ICR マウスから卵巣部文化の媒体の変更のスケジュールは、図 1に提示されます。<ol>
<li>LH サージを再現するには、100 mU/mL FSH と LH 12 時間ごとの 4 日 (図 1) を含む培地で培養 4 週齢 ICR マウス卵巣を扱います。</li>
	</ol>
</li>
</ol>4. 顕微鏡培養卵巣の画像
<ol><li>組織が細胞培養チップに付着したとき 1 日目後培養の卵巣の画像を開始し、24 h 間隔時間の点の間の十分な卵胞成長を許可する共焦点または倒立顕微鏡解析を実行します。 注: 共焦点顕微鏡倒立顕微鏡全体培養卵巣内の卵胞を観察するために優れています。</li>
</ol>5. タイムラプス イメージング培養卵巣の
<ol><li>
レーザー強度とイメージング システム (表 1) のための露出時間などを含む、タイムラプス撮影条件を最適化します。
	<ol>
<li>処置および制御グループとして使用のための単一のマウスからペア卵巣片を選択します。レーザーの強度と最高の画像 (表 1) を達成するために露光時間を変えます。</li>
		<li>卵胞エリア 24 時間間隔でコントロールのサンプルの画像とタイムラプス イメージング サンプル (手順 6 を参照) を測定することにより各培養条件下で卵胞の成長を比較します。同時に、各卵巣セット排卵卵子の数をカウントし、最適なタイムラプス イメージング条件を選択します。</li>
	</ol>
</li>
	<li>(表 1) の一定の条件の下でタイムラプス イメージング システムを使用して 30 分間隔で画像をキャプチャします。</li>
</ol>6. 卵胞成長解析
<ol><li>
卵胞発育を分析するには、ImageJ ソフトウェア (http://resbweb.nih.gov/ij/) を使用してキャプチャされたイメージに包領域を測定します。
	<ol>
<li>当初は、ツール ・ バーの「分析」の下で「スケールを設定」をクリックしてイメージ スケールを設定します。それぞれの「知られている距離」と「ピクセルの距離」、空白のフィールドにキャプチャしたイメージとピクセルの対応する番号の側面の長さを入力します。</li>
		<li>「フリーハンド」ツール バーでをクリックし、明るいフィールドから取得されたイメージで卵胞の概要を説明します。</li>
		<li>ツール ・ バーの「分析」の下で「測定」をクリックします。 注: 他の測定データが必要な場合「設定測定」「分析」の下で、ツール ・ バーをクリックし、一覧で該当するボックスをチェックします。</li>
	</ol>
</li>
</ol>7. トランスジェニック マウスを用いた卵胞発育の解析
<ol><li>生後 0 および 4 (P0、P4) トランスジェニック マウスを含む挿入遺伝子Oog1pro3.9とR26 H2B mCherr、文化に従って手順 1.1 – 3.2、そうでない点を除いて女性スライス、P0、P4 卵巣から卵巣を収集します。</li>
	<li>含む 5% CO2 37 ° C で 2 日ごとインキュベーターで 3.5 cm 料理で新鮮な予め温めておいた培地で培養液を交換してください。 注: P0、P4 の卵巣の培地の LH の濃度は、LH サージは P0、P4 のマウスで発生しないために定数残ることができます。</li>
	<li>培養の P4 卵巣 (表 1) でのみ AcGFP1 肯定的なプライマリ卵胞を可視化する顕微鏡を設定します。 注: のみ原始、プライマリ卵胞 P4 雌マウスの卵巣ではあります。</li>
	<li>培養 P0 Oog1pro3.9の画像をキャプチャ/R26 H2B mCherryトランスジェニック マウス卵巣 30 分間隔で P4 卵巣 (5.2 を参照) に使用される設定を使用しています。</li>
	<li>トレースし、AcGFP1 と H2B mCHerry 信号を使用して卵胞の発育を分析します。</li>
</ol>

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本研究ではマウス卵巣における卵胞発育を研究するための 2 つの新しい方法を開発しました。最初のメソッドは卵胞発育の解析に続いてスライスした卵巣成熟マウス組織の文化を含み、2 番目には段階では、性腺刺激ホルモンに依存しない初期の卵胞発育を視覚化する時間経過イメージ投射の使用が含まれます。以前は、卵胞開発8,9日白血病の抑制...

雌マウス卵巣いくつか千包1、および周期的な排卵発情サイクルごとに約 10 卵母細胞が成熟します。卵胞は、いくつかの発達段階に分類: 原初、プライマリ、セカンダリ、幽門洞、および各卵子を取り巻く顆粒膜細胞層の形態によって、グラーフ卵胞。ほとんどの原始卵胞が休止状態とそれらのいくつかはアクティブし各発情周期2プライマリ卵胞に成長します。二次濾胞ステージ後卵胞発育は主に性腺刺激ホルモン、卵胞刺激ホルモン (FSH) と黄体形成ホルモン (LH) によって調整されます。しかし、原始、プライマリ卵胞発育、性腺刺激ホルモンとは無関係です、これらの段階を支配する制御機構のまま悪い inderstood3,4,5。成長因子およびホルモンのほか、原始、プライマリ卵胞は卵胞6,7間の相互作用によって調整されます。したがって、マウス卵巣組織の卵胞動力学の解析を実行し、卵巣の組織培養8,9,10を使用して関連付けられている規制メカニズムを検討しました。
ここで、2 つの卵巣組織培養モデルのメソッドを紹介します。最初、卵胞の測定により卵胞発育を分析するため、2 番目はトランスジェニック マウスとセカンダリ卵胞期に原始から初期の卵胞発育中規制機構の研究使用されます。小胞の開発の分析、主に使用しました 4 週齢の雌マウスの卵巣卵胞を簡単に表示できるため。定期的な排卵とモデル生体内で卵胞発育を誘導するには、培養条件下でのエストラジ オールの分泌卵胞閉鎖と観測された排卵 LH サージが再現されております。培養の卵巣のイメージが捕獲されたと濾胞領域でトレースの変更によって卵胞の開発プロセスを分析しました。ただし、明視野顕微鏡解析で原始と初期プライマリ卵胞の区別は明確です。したがって、小卵胞を検出、原初、プライマリ、区別する手法を開発したし、の二次卵胞培養を用いた卵巣組織Oogenesin1 (Oog1) pro3 。9 と 0 と 4 日誕生11 R26 H2B mCherryトランスジェニック マウスの卵巣。Oog1式減数分裂、参入後は、卵母細胞の検出と原初から培養卵巣組織11 時間経過画像を用いたプライマリ卵胞への移行が観察、卵胞の発育とともに徐々 に増大 ,12。形態学的方法は、休止状態の原始卵胞13,14,15,16を活性化する要因を検討し使用されている、卵巣の生理学的な卵胞発育が観察することは困難、様々 な要因の影響残る例を紹介。本培養法は、ターゲット要素のリアルタイム解析でこの不足に対処するため設計されました。
本研究で我々 はタイムラプス イメージング法による卵胞の発育を追跡され、卵胞発育のプロセスが特徴します。私たちの手法は、卵巣の生理学を調査するための前例のないツールを提供しています。

<table><tbody><tr><td>Follicle stimulating hormone from human pituitary</td><td>SIGMA</td><td>F4021</td><td></td></tr><tr><td>Lutenizing hormone from equine pituitary</td><td>SIGMA</td><td>L9773</td><td></td></tr><tr><td>Penicillin-streptomycin solution</td><td>Wako Pure Chemical Industries</td><td>168-23191</td><td></td></tr><tr><td>MEM a, GlutaMax, no nucleotides</td><td>Thermo Fisher</td><td>32561037</td><td></td></tr><tr><td>Glass bottom dish</td><td>MatTek</td><td>P35G-0-10-C</td><td>35mm dish, No. 0 coverslip, 10mm glass diameter</td></tr><tr><td>Millicell cell culture insert</td><td>Merck Millipore</td><td>PICM0RG50</td><td>Diameter: 315 mm, pore size: 0.4 mm, material: hydrophilic PTTE</td></tr><tr><td>3.5cm cell culture dishes</td><td>greiner bio-one</td><td>627160</td><td></td></tr><tr><td>50ml / centrifuge tube with triple seal cap</td><td>IWAKI</td><td>2345-050</td><td></td></tr><tr><td>Low-profile disposable blades 819</td><td>Leica</td><td>14035838925</td><td></td></tr><tr><td>LSM 710</td><td>Carl Zeiss</td><td></td><td>Confocal microscope</td></tr><tr><td>CellVoyager, CV1000</td><td>Yokogawa Electric Corporation</td><td></td><td>Time-lapse imaging</td></tr><tr><td>BZ-X700</td><td>KEYENCE</td><td></td><td>Time-lapse imaging</td></tr></tbody></table>

ovarian tissue culture to visualize phenomena in mouse ovary

哺乳類の女性は、各発情周期中に卵母細胞数をほぼ一定を定期的に排卵します。このような規則性と周期性を維持するためには、視床下部-下垂体-性腺軸レベル、卵巣内の卵胞の開発に規制が発生します。アクティブな研究にもかかわらずいくつか手順休眠原始卵胞活性化から排卵して卵胞の各段階で異なる規制の複雑さのため、卵胞の発達メカニズムは明らかありません。卵胞発育のメカニズムと発情サイクルを通して卵胞のダイナミクスを調べるためには、顕微鏡を用いて卵胞の発育を観察するために使用できるマウス卵巣組織培養モデルを開発しました。体系的な卵胞発育、周期的な排卵と卵胞閉鎖すべてで再現できる卵巣培養モデルと培養条件を実験的変調することができます。ここでは、卵胞発育の調節機構とその他卵巣現象における本法の有用性を示す.

図 1は、卵巣の組織培養中にメディアに変更するためのプロトコルを示します。このプログラム、4 週齢 ICR マウス卵巣培養され、共焦点顕微鏡 (図 2) を使用して 24 h 間隔で撮像されます。3 週間、卵巣組織の培養中最も幽門洞およびセカンダリの卵胞が卵胞閉鎖によって退化したし、いくつかあった排卵 (図...

Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

「マウス卵巣における現象を可視化する卵巣の組織培養

卵巣の組織培養卵胞開発、排卵や卵胞閉鎖のモデルとして使用することができ、卵巣の動的過程の制御機構を示します。

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

ovarian-tissue-culture-to-visualize-phenomena-in-mouse-ovary

Research

JoVE Journal

Developmental Biology

13.7K ビュー.  Aichi Medical University. 卵巣の組織培養卵胞開発、排卵や卵胞閉鎖のモデルとして使用することができ、卵巣の動的過程の制御機構を示します。

ビデオ: Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化と共培養マウスの卵巣と卵胞

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

発生生物学

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巣と卵管の三次元器官培養用アルギン酸ゲル

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工学

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

子宮外エレクトロポレーションおよびマウスの海馬組織の器官型スライス培養

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ovarian fitness is often assessed by visualizing and quantifying follicles and oocytes. Because the ovary is an opaque three-dimensional tissue, traditional approaches require laboriously slicing the tissue into numerous serial sections in order to visualize cells throughout the entire organ. Furthermore, because quantification by this method typically entails scoring only a subset of the sections separated by the approximate diameter of an oocyte, it is prone to inaccuracy. Here, a protocol is described that instead utilizes whole organ tissue clearing and immunofluorescence staining of mouse ovaries to visualize follicles and oocytes. Compared to more traditional approaches, this protocol is advantageous for visualizing cells within the ovary for numerous reasons: 1) the ovary remains intact throughout sample preparation and processing; 2) small ovaries, which are difficult to section, can be examined with ease; 3) cellular quantification is more readily and accurately achieved; and 4) the whole organ imaged.

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

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 ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

受動的な明快さクリア マウス卵巣の血管の 3次元再構築

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

ウシ卵巣皮質組織培養

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

Orthotopic Inoculation of Ovarian Cancer Cells into Murine Ovary: A Procedure to Establish an Ovarian Cancer Mouse Model to Study Peritoneal Dissemination of Cells

To carry out orthotopic inoculation, after anesthetizing an eight-week-old female nude mouse according to the text protocol, perform a toe pinch to verify depth of the anesthesia. Place the anesthetized mouse on an operating stage, belly side down. Then, disinfect the surgical area with several washes using a chlorhexidine-based scrub and alcohol.
Next, make an approximately 2-centimeter vertical incision near the spine, between the right costal arch and the femur. Place a sterile surgical drape around the incision site. Then, fix the incised skin surfaces with clamps to maintain the opening.
Now, make a second 1-centimeter incision at the parietal peritoneum under the first incision to open the abdominal cavity. Then, clamp the incised abdominal surfaces. With a clamp, clip the fat tissue that surrounds the ipsilateral ovary and fallopian tube to remove the ovary from the abdominal cavity.
Then, carefully place the ovary and fallopian tube on gauze before placing the mouse, belly side down, under the dissecting microscope. Transfer the cell suspension to an insulin syringe. Then, immediately and carefully inject 1 to 2 microliters of the prepared cell suspension into the ovary.
For several seconds, keep the needle in the ovary to ensure that the hydrogel sets. After gelation, the injected tumor cells will remain in the ovary. Carefully, return the ovary into the body. With sterile medical silk, suture the second incision before using wound clips to seal the skin.

「マウス卵巣における現象を可視化する卵巣の組織培養

要約

さらに動画を探す

この動画の章

文化と共培養マウスの卵巣と卵胞

卵巣と卵管の三次元器官培養用アルギン酸ゲル

子宮外エレクトロポレーションおよびマウスの海馬組織の器官型スライス培養

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

成人マウス卵巣における正確な卵胞列挙体

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

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

受動的な明快さクリアマウス卵巣の血管の 3次元再構築

ウシ卵巣皮質組織培養

マウス卵巣への卵巣癌細胞の同所性接種:細胞の腹膜播種を研究するための卵巣癌マウスモデルを確立する手順

「マウス卵巣における現象を可視化する卵巣の組織培養

要約

さらに動画を探す

この動画の章

文化と共培養マウスの卵巣と卵胞

卵巣と卵管の三次元器官培養用アルギン酸ゲル

子宮外エレクトロポレーションおよびマウスの海馬組織の器官型スライス培養

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

成人マウス卵巣における正確な卵胞列挙体

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

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

受動的な明快さクリア マウス卵巣の血管の 3次元再構築

ウシ卵巣皮質組織培養

マウス卵巣への卵巣癌細胞の同所性接種:細胞の腹膜播種を研究するための卵巣癌マウスモデルを確立する手順

受動的な明快さクリアマウス卵巣の血管の 3次元再構築