Name: ovarian tissue culture to visualize phenomena in mouse ovary
Uploaded: 2018-06-19T14:30:00.000+00:00
Duration: 4 min 30 s
Description: Watch this Scientific Journal Video about Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary at JoVE.com

Dr. Naojiro Minami (Kyoto Üniversitesi) Oog1pro3.9 fareler verdiğiniz için teşekkür ederiz. Bu araştırma JSP'ler (KAKENHI # JP15H06275) ve Nitto Vakfı tarafından desteklenmiştir.

Fareler 23 ±1 ° C 12 h ışık/12 s karanlık döngüsü ile çevre kontrollü bir odada muhafaza. Hayvan bakım protokolleri ve deneyler kılavuzlarınıza uygun olarak Aichi Tıp Üniversitesi'nde hayvan deneme için yapılmıştır ve görevdeki hayvan bakım ve kullanım Komitesi tarafından kabul edildi.
1. hazırlanması kültür orta ve yemekleri
<ol><li>Temel kültür ortamı hazırlamak için fetal Sığır serum (FBS, % 5 v/v), FSH ekleyin (100 mIU/mL), LH (10 mU/mL) ve penisilin-streptomisin (penisilin, 100 U/mL; streptomisin, 100 mg/mL) asgari temel orta Alfa (MEM-alfa) ve mix 50 mL tüpler için. Not: Gerekli kültür ortamının toplam birimleri deneyler arasında değişir, ancak kültür orta 1 mL 3.5 cm cam-alt kültür yemekleri için genellikle yeterli oluyor.</li>
	<li>1 mL aliquots karma kültür ortamının 3.5 cm cam-alt kültür yemekleri dökün ve yer 30 mm hücre kültür ekler yemekleri ayarlayın. Önceden sıcak ortam ve yemekleri bir kuluçka (%5 CO2 ve 37 ° C).</li>
</ol>2. yumurtalık dokusu hazırlanması
<ol><li>Önceden sıcak fosfat arabelleğe alınmış serum (PBS) (−) ve MEM-Alfa %5 FBS, 100 U/mL penisilin ve 100 mg/mL streptomisin bir kuluçka (%5 CO2 ve 37 ° C) içeren. Not: Yaklaşık 3 mL aliquots yumurtalık PBS (−) yumurtalık diseksiyonu sırasında kullanım için yeterli ve 1 mL aliquots MEM-Alfa 3.5 cm yemeklerinde tek yumurtalık geçici depolama için yeterlidir.</li>
	<li>
Yumurtalık 4 - hafta eski kadın (-den sonra Kanser Araştırma Enstitüsü olarak adlandırılır) ICR fareler gelen tüketim ve makas ve stereoskopik mikroskop altında Cımbız kullanarak yumurtalık çevreleyen dokuların trim. Rezerv kültür orta kaldırılan yumurtalıklarda (bkz. Adım 2.1) kullanımı kadar.
	<ol>
<li>Filtre kağıdı üzerine tek yumurtalık nemlendirilmiş ile yer önceden ısınmış PBS (−) (bkz. Adım 2.1) ve 4 adet stereoskopik bir mikroskop altında microtome bıçak kullanma dilim. Not: 4 - hafta eski ICR fareler yumurtalık yaklaşık 2 mm çapında geçerlidir. Parça sayısı yumurtalık boyutuna bağlıdır; Ancak, yaklaşık 500 µm kalınlığında parçalar uygun folikül gözlemler için izin (500 mm daha kalın parçalar halinde doku şeffaflık azalır; 500 mm ince parçalar halinde birçok antral folikül kırık ve kaybetti).</li>
		<li>Diseksiyon sonra önceden ısıtılmış kültür orta (bkz. Adım 2.1) 3.5 cm yemeklerinde dilimlenmiş yumurtalık numuneler yerleştirin.</li>
	</ol>
</li>
</ol>3. yumurtalık doku kültürü
<ol><li>Yumurtalık dokusu yumurtalık dokusu nerede olacak hücre kültür Ekle dilimlenmiş kültür orta yaklaşık 0.5 µL damla bir micropipette kullanarak ayarlayın.</li>
	<li>Kültür orta Cımbız kullanarak hücre kültür ekler üzerinde bir damla her dilimlenmiş numune yerleştirin.</li>
	<li>37 ° C'de % 5 CO2 yumurtalık dokularda kültür</li>
	<li>Kültür orta ile taze önceden ısıtılmış orta yerine 2 günde. Not: 4 - hafta eski ICR fareler bölümlerden yumurtalık kültür için orta değişiklikler için zamanlamayı Şekil 1' de sunulmuştur.<ol>
<li>LH dalgalanma çoğaltmak, kültürlü 4 - hafta eski ICR fareler yumurtalık 100 mU/mL FSH ve LH 12 h için her 4 gün (Şekil 1) içeren orta ile tedavi.</li>
	</ol>
</li>
</ol>4. mikroskop görüntüleri kültürlü yumurtalık
<ol><li>1. ne zaman belgili tanımlık doku hücre kültür ekler yapıştırılır gün sonra kültürlü yumurtalık görüntüleme başlatın ve zaman puan arasında yeterli folikül büyüme sağlamak için 24 saat aralıklarla confocal veya ters mikroskop çözümlemesi. Not: Confocal mikroskobu köklerinin bütün kültürlü yumurtalık içinde gözlemlemek için ters mikroskobu üstündür.</li>
</ol>5. kültürlü yumurtalık hızlandırılmış görüntüleme
<ol><li>
Hızlandırılmış görüntüleme koşulları, lazer yoğunluklarda ve görüntüleme sistemi (Tablo 1) için pozlama süreleri dahil olmak üzere en iyi duruma getirme.
	<ol>
<li>Eşleştirilmiş yumurtalık numuneler tek fare kullanımı için tedavi ve kontrol grupları olarak seçin. Lazer yoğunluklarda ve en iyi görüntü (Tablo 1) elde etmek için çekim hızı değişir.</li>
		<li>Folikül gelişim her kültür koşul altında folikülü alanları kontrol örnekleri 24 h aralıklarla görüntülerini ve hızlandırılmış görüntüleme örnekleri (bkz. Adım 6) ölçerek karşılaştırın. Aynı anda, her yumurtalık sette ovulated yumurta sayısını saymak ve uygun zaman hata görüntüleme koşulları seçin.</li>
	</ol>
</li>
	<li>Hızlandırılmış görüntüleme sistemi (Tablo 1) belirlenen koşullarda kullanarak 30 dk aralıklarla esir alma imge.</li>
</ol>6. folikülü büyüme Analizi
<ol><li>
Folikül gelişim analiz etmek için ImageJ yazılım (http://resbweb.nih.gov/ij/) kullanarak çektiğiniz resimleri folikül alanlarında ölçmek.
	<ol>
<li>Başlangıçta, araç çubuğunda "Ölçeği Ayarla" altında "Analiz" tıklatarak görüntü ölçeğini ayarlayın. Yakaladığınız görüntüyü ve piksel karşılık gelen sayıların yan uzunlukları sırasıyla "Bilinen mesafe" ve "Uzaklığını piksel", boş alanlara girin.</li>
		<li>"Serbest el" araç çubuğunda tıklatın ve köklerinin yakalanan parlak alan görüntüler anahat.</li>
		<li>"Ölçü" altında "analiz" araç çubuğunda'i tıklatın. Not: diğer ölçüm verileri isterseniz, "ölçü"Analiz"altında araç çubuğunda ayarla"'ı tıklatın ve listeden uygun kutuları işaretleyin.</li>
	</ol>
</li>
</ol>7. analiz transgenik fareler kullanarak folikül gelişimi
<ol><li>Yumurtalık 0 ile 4 (P0 ve P4) içeren transgenik fareler transgenes Oog1pro3.9 ve R26-H2B-mCherrve kültür ekler üzerinde açıklandığı gibi adımlar 1.1-3.2 değil dışında Kadın Doğum sonrası gün dilim P0 ve P4 yumurtalık toplamak.</li>
	<li>Taze, önceden ısıtılmış orta 3.5 cm yemekleri içeren % 5 CO2 37 ° C'de 2 günde bir kuluçka kültür orta yerine. Not: LH dalgalanmaları P0 ve P4 farelerde meydana gelmez çünkü LH konsantrasyonu P0 ve P4 yumurtalık kültür orta sabit kalabilir.</li>
	<li>Sadece AcGFP1 pozitif birincil foliküller kültürlü P4 yumurtalık (Tablo 1) içinde görselleştirmek için mikroskop ayarlayın. Not: Sadece ilkel ve birincil foliküller P4 dişi fare yumurtalıklarda mevcuttur.</li>
	<li>Kültürlü P0 Oog1pro3.9çekim /R26-H2B-mCherry transgenik fareler yumurtalık 30 dk aralıklarla kullanarak (bkz. Adım 5.2) P4 yumurtalık için kullanılan ayarlar.</li>
	<li>İzleme ve folikül gelişim AcGFP1 ve H2B-mCHerry sinyallerini kullanarak çözümleyebilirsiniz.</li>
</ol>

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Yazarlar ifşa gerek yok.

Bu çalışmada, fare yumurtalıklarda folikül gelişiminde eğitim için iki yeni yöntem geliştirdik. İlk yöntem kültür folikül gelişim analizleri tarafından takip dilimlenmiş yumurtalık yetişkin fareler dokuların içerir ve ikinci ön folikül gelişim gonadotropin bağımsız aşamasında görselleştirmek için hızlandırılmış Imaging kullanımını içerir. Daha önce biz lösemi inhibitör faktörü ve progesteron folikül gelişim8,9üzer...

Dişi fare yumurtalık birkaç bin köklerinin1içeren ve düzenli yumurtlama her estrus döngüsü yaklaşık on yumurtalar olgunlaşır. Köklerinin çeşitli gelişim aşamalarında sınıflandırılır: ilkel, İlköğretim, ortaöğretim, antral ve her oosit çevreleyen granulosa hücresel tabaka şeklinde bağlı olarak Graafian köklerinin. En ilkel köklerinin uykuda ve biraz-in onları aktif hale gelir ve her estrus döngüsü2birincil foliküller içine büyümek. İkincil foliküler aşamadan sonra folikül gelişim gonadotropins tarafından uyarıcı hormon (FSH) ve luteinizan hormon (LH) foliküler esas olarak düzenlenir. Ancak, ilkel ve birincil folikül gelişim gonadotropin bağımsız ve bu aşamaların yöneten düzenleyici mekanizmaları kötü inderstood3,4,5kalır. Büyüme faktörleri ve hormonlar ek olarak, ilkel ve birincil folikül köklerinin6,7arasındaki etkileşimler tarafından düzenlenmiştir. Bu nedenle, biz folikül dinamiklerini analiz mouse ovarium dokularda gerçekleştirilen ve yumurtalık dokusu kültürler8,9,10kullanarak ilişkili düzenleyici mekanizmaları araştırıldı.
Burada, biz iki yumurtalık doku kültürü modeli yöntemleri tanıtmak. İlk foliküler alanlarda ölçüm tarafından folikül gelişim analiz etmek için kullanılır ve ikinci transgenik fareler ile ikincil folikül aşamasına gelen ilkel erken folikül gelişimi sırasında düzenleyici mekanizma incelemek için kullanılır. Çünkü onlar köklerinin kolay görselleştirme için izin folikül gelişim analiz için Biz esas olarak yumurtalık 4 - hafta eski dişi farelerin kullanılır. Periyodik yumurtlama ve modeli içinde vivo folikül gelişim ikna etmek için LH dalgalanma ve gözlenen yumurtlama, folikül atrezi ve doku kültürü koşullar altında estradiol salgılanmasını çoğaltılamaz. Kültürlü yumurtalık görüntüleri ele geçirildi ve folikül gelişim süreçleri izleme değişiklikleri foliküler alanında tarafından analiz edildi. Ancak, parlak alan Mikroskobu analizleri, ilkel ve erken birincil foliküller ayrımı belirsiz. Bu nedenle, biz küçük köklerinin algılayıp birincil, ilkel arasında ayırt etmek için bir yöntem geliştirdi ve ikincil folikül kültürlü yumurtalık dokuları kullanarak Oogenesin1 (Oog1) pro3. 9 ve 0 ile 4 gün sonra Doğum11, R26-H2B-mCherry transgenik fareler yumurtalık. Oog1 ifade mayoz girmesinden sonra yumurta algılanabilir ve folikül gelişimi, ilkel geçiş kültürlü yumurtalık dokusunun11 hızlandırılmış görüntüleri kullanarak birincil köklerinin gözlenmesi izin ile yavaş yavaş artar ,12. Morfolojik yöntemleri uyuyan ilkel köklerinin13,14,15,16etkinleştirmek faktörler eğitim için kullanılan, yumurtalık fizyolojik folikül gelişiminde olsa gözlemlemek, zor ve çeşitli faktörlerin etkilerini uncharacterized kalır. Mevcut kültür yöntemleri bu yetersizlik hedef faktörler gerçek zamanlı analizlerde yönelik olarak tasarlanmıştır.
Bu da çalışmanın, biz foliküler gelişim hızlandırılmış bir görüntüleme yöntemi kullanarak izleniyor ve folikül gelişim süreci ile karakterizedir. Roman yöntemlerimiz yumurtalıkların fizyolojisi soruşturma için benzeri görülmemiş bir aracı sağlar.

<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

Memeli kadın düzenli olarak her estrus döngüsü sırasında yumurtalar hemen hemen sabit bir sayıda yumurtlamak. Böyle düzenlilik ve periyodik olarak sürdürmek için düzenleme hipofiz gonad hipotalamik eksen düzeyinde ve follikül yumurtalık içinde geliştirme ortaya çıkar. Etkin çalışmalar rağmen folikül gelişim mekanizmaları çeşitli adımlar uyuyan ilkel folikül etkinleştirme gelen yumurtlama için ve foliküler her aşamada farklı yönetmelik karmaşıklığı yüzünden net değildir. Folikül gelişim mekanizmaları ve köklerinin estrus döngüsü boyunca dinamikleri araştırmak için bir mikroskop kullanarak folikül gelişim gözlemlemek için kullanılabilir bir fare yumurtalık doku kültürü modeli geliştirdi. Sistematik folikül gelişim, periyodik yumurtlama ve folikül atrezi tek kültürlü yumurtalık modelinde çoğaltılabilir ve kültür koşulları deneysel olarak modüle. Burada, biz, folikül gelişim düzenleyici mekanizmaları ve diğer yumurtalık olayları bu yöntemde yararlılığı göstermek.

Şekil 1 yumurtalık doku kültürü sırasında medya iletişim kuralı için değişiklikleri gösterir. Aşağıdaki bu program, 4-hafta-yaşlı ICR fareler yumurtalık kültürlü ve confocal mikroskobu (Şekil 2) kullanarak 24-h aralıklarla görüntüsü. 3 hafta boyunca kültür yumurtalık dokuları sırasında en antral ve ikincil foliküller folikül atrezi tarafından dejenere ve bazı ovulated (Ş...

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

Mouse ovarium olayları görselleştirmek için yumurtalık doku kültürü

Yumurtalık dokusu kültürler folikül gelişim, yumurtlama ve folikül atrezi modelleri olarak kullanılabilir ve düzenleyici mekanizmaları dinamik yumurtalık işlemleri gösterir.

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 Görüntüleme.  Aichi Medical University. Yumurtalık dokusu kültürler folikül gelişim, yumurtlama ve folikül atrezi modelleri olarak kullanılabilir ve düzenleyici mekanizmaları dinamik yumurtalık işlemleri gösterir.

Video: 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.

Kültür ve Co-Kültür Mouse Yumurtalıklar ve Yumurtalık Foliküller

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

Gelişim Biyolojisi

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Immünkompetan Fare Seröz Yumurtalık Kanseri bir Ortotopik Model In vivo</emTümör Bağışıklık Yanıtları> Tümör Görüntüleme ve İzleme

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

İmmünoloji ve Enfeksiyon

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.

Yumurtalıklar ve fallop borularında Üç Boyutlu Organ Kültür Aljinatta Hidrojeller

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 ...

Biyomühendislik

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.

Bütün Dağı ayirt ve kültürlü fare yumurtalıklarda folikül miktar

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.

Yetişkin Fare Yumurtalıklarında Doğru Folikül Numaralandırması

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.

In vivo Görüntüleme ve Yumurtalık Kanseri Ortotopik Fare Modeli Tedavi Tedaviler

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

Biyoloji

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.

Farede Gelişmekte Olan Yumurtalık Rezervinin Kantitatif 3D Analizi için Tüm Yumurtalık İmmünofloresansı, Temizleme ve Multifoton Mikroskobu

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.

Pasif AÇIKLIĞI temizlenmiş Mouse ovarium vasküler mimarisinin üç boyutlu yeniden yapılandırma

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.

Sığır Yumurtalık Korteks Doku Kültürü

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.

Yumurtalık Kanseri Hücrelerinin Murin Yumurtalığına Ortotopik Aşılanması: Hücrelerin Peritoneal Yayılımını İncelemek için Bir Yumurtalık Kanseri Fare Modeli Oluşturma Prosedürü