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

Agradecemos a Dr. Naojiro Minami (Universidad de Kioto) para proporcionar los ratones Oog1pro3.9 . Esta investigación fue apoyada por el JSP (KAKENHI # JP15H06275) y la Fundación de Nitto.

Ratones fueron alojados en una sala de ambiente controlada a 23 ± 1 ° C con un ciclo oscuro h 12 h luz/12. Protocolos de cuidado de los animales y los experimentos se llevaron a cabo con arreglo a las directrices para la experimentación Animal en la Universidad médica de Aichi y fueron aprobados por el Comité de uso y cuidado Animal titular.
1. preparación de medio de cultivo y platos
<ol><li>Para preparar medio de cultivo base, agregar suero bovino fetal (FBS, 5% v/v), FSH (100 mIU/mL), LH (10 mU/mL) y penicilina-estreptomicina (penicilina 100 U/mL, estreptomicina, 100 mg/mL) al mínimo esencial medio (MEM-alfa) y la mezcla en tubos de 50 mL. Nota: Volumen Total requiere de medios de cultivo varía entre experimentos y 1 mL de medio de cultivo es generalmente suficiente para platos de cultura de fondo de cristal de 3,5 cm.</li>
	<li>Verter alícuotas de 1 mL de medio de cultivo mezclado en platos de cultura de fondo de cristal de 3,5 cm y coloque lugar 30 mm de la célula cultura insertos en platos. Precaliente los platos en una incubadora (5% CO2 y 37 ° C) y los medios de comunicación.</li>
</ol>2. preparación del tejido ovárico
<ol><li>Pre-warm fosfato tampón salino (PBS) (−) y MEM-alfa que contiene 5% FBS, 100 U/mL de penicilina y estreptomicina 100 mg/mL en una incubadora (5% CO2 y 37 ° C). Nota: Aproximadamente 3 mL alícuotas de PBS (−) por ovario son suficientes para el uso durante la disección del ovario, y alícuotas de 1 mL de MEM-alfa son suficientes para el almacenamiento transitorio de los ovarios solo en platos de 3,5 cm.</li>
	<li>
Suprimir los ovarios de ratones femeninos 4 semana - el viejo del ICR (nombrado después de Instituto de investigación del cáncer) y de los tejidos que rodean al ovario usando tijeras y pinzas en un microscopio estereoscópico. Reserva los ovarios retirados en medio de cultivo (véase el paso 2.1) hasta su uso.
	<ol>
<li>Lugar los ovarios solo sobre el papel de filtro humedecidos con pre calentado PBS (−) (véase el paso 2.1) y cortar en 4 pedazos con una cuchilla de micrótomo en un microscopio estereoscópico. Nota: Los ovarios de ratones ICR 4 semana - el viejo son unos 2 mm de diámetro. El número de piezas depende del tamaño de ovario; sin embargo, trozos gruesos de alrededor de 500 μm permiten observaciones de folículo apropiado (en piezas más gruesas que 500 mm, se disminuye la transparencia del tejido, en pedazos más finos de 500 mm, se rompen y se perdieron muchos folículos antrales).</li>
		<li>Después de la disección, coloque a las muestras de ovario en rodajas con medio de cultivo en platos de 3,5 cm (vea el paso 2.1).</li>
	</ol>
</li>
</ol>3. ovario cultivo de tejidos
<ol><li>Gota aproximadamente 0,5 μl de medio de cultivo por rebanadas de tejido ovárico en la inserción de la cultura de célula donde estará el tejido ovárico mediante una micropipeta.</li>
	<li>Coloque a cada muestra en rodajas en una gota de medio de cultivo sobre los insertos de la cultura de célula con unas pinzas.</li>
	<li>Cultura de los tejidos del ovario de 5% CO2 a 37 ° C.</li>
	<li>Reemplazar el medio de cultivo con medio fresco precalentado cada 2 días. Nota: El horario para los cambios de medio para el cultivo de secciones de ovario de 4 semana - viejos ratones ICR se presenta en la figura 1.<ol>
<li>Para reproducir la oleada de LH, tratar cultivadas 4 semana - viejos ovarios de ratones ICR con el medio que contiene 100 mU/mL FSH y LH de 12 h cada 4 días (figura 1).</li>
	</ol>
</li>
</ol>4. microscopio imágenes de ovarios cultivados
<ol><li>Iniciar la proyección de imagen los ovarios cultivados después de día 1 cuando los tejidos han adherido en los insertos de la cultura de célula y realizar análisis de microscopio confocal o invertida en intervalos de 24 h para permitir un crecimiento suficiente del folículo entre puntos de tiempo. Nota: La microscopia Confocal es superior a la microscopía invertida para la observación de los folículos en los ovarios todo cultos.</li>
</ol>5. Time-lapse proyección de imagen de ovarios cultivados
<ol><li>
Optimizar las condiciones de proyección de imagen de Time-lapse, incluyendo láser intensidades y tiempos de exposición, para el sistema de proyección de imagen (tabla 1).
	<ol>
<li>Seleccione muestras pareadas ovario de ratones solo para el uso como los grupos tratamiento y control. Variar intensidades láser y tiempo de exposición para lograr las mejores imágenes (tabla 1).</li>
		<li>Comparar el crecimiento del folículo bajo cada condición de cultivo mediante la medición de áreas de folículo en imágenes de muestras de control a intervalos de 24 h y en time-lapse de muestras (ver paso 6). Al mismo tiempo, contar el número de ovuladas ovocitos en cada ovario conjunto y seleccionar las condiciones óptimas de imagen Time-lapse.</li>
	</ol>
</li>
	<li>Capturar imágenes a intervalos de 30 min con el sistema Time-lapse de imágenes bajo las condiciones determinadas (tabla 1).</li>
</ol>6. Análisis de crecimiento de folículo
<ol><li>
Para analizar el desarrollo del folículo, medir las áreas de folículo en las imágenes utilizando el software ImageJ (http://resbweb.nih.gov/ij/).
	<ol>
<li>Inicialmente, establecer la escala de la imagen haciendo clic en "Set escala" debajo de "Analizar" en la barra de herramientas. Introduzca las longitudes de lado de la imagen capturada y el correspondiente número de píxeles en los campos en blanco de "Distancia del conocido" y "Distancia en píxeles", respectivamente.</li>
		<li>Haga clic en "Mano libre" en la barra de herramientas y contorno de los folículos en las imágenes capturadas de campo brillante.</li>
		<li>Haga clic en "Medida" en "Analizar" en la barra de herramientas. Nota: Si desean otros datos de medición, haga clic en "Configurar medición" debajo de "Analizar" en la barra de herramientas y marque las casillas apropiadas en la lista.</li>
	</ol>
</li>
</ol>7. análisis del desarrollo folicular utilizando ratones transgénicos
<ol><li>Recoger los ovarios de postnatal días 0 y 4 (P0 y P4) hembra de ratones transgénicos que contienen los transgenes Oog1pro3.9 R26-H2B-mCherry cultura en partes movibles como se describe en los pasos 1.1, 3.2, excepto no rodaja la P0 y ovarios de P4.</li>
	<li>Reemplazar el medio de cultivo con medio fresco, precalentado en platos de 3,5 cm en una incubadora con 5% CO2 a 37 ° C cada 2 días. Nota: La concentración de LH en el medio de cultivo de ovarios P0 y P4 puede seguir siendo constante debido a las oleadas de LH no ocurre en ratones P0 y P4.</li>
	<li>Ajuste del microscopio para visualizar solamente AcGFP1-positivo primario los folículos en ovarios P4 cultivados (tabla 1). Nota: Sólo primarios y primordiales de los folículos están presentes en ovarios de ratón hembra P4.</li>
	<li>Captura de imágenes de culto P0 Oog1pro3.9R26-H2B-mCherry ovarios de ratones transgénicos en intervalos de 30 minutos utilizando la configuración utilizada para ovarios P4 (ver paso 5.2).</li>
	<li>Rastrear y analizar el desarrollo del folículo de señales AcGFP1 y H2B mCHerry.</li>
</ol>

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Los autores no tienen nada que revelar.

En este estudio, hemos desarrollado dos nuevos métodos para estudiar el desarrollo folicular en ovarios de ratón. El primer método implica el cultivo de los tejidos de ratones adultos ovárico en rodajas seguida por análisis del desarrollo del folículo, y el segundo involucra el uso de Time-lapse de imágenes para visualizar el desarrollo folicular temprano durante la etapa independiente de la gonadotropina. Anteriormente, utiliza el método de cultivo de tejidos de ovario presente para evaluar el efecto del factor ...

Los ovarios de la hembra del ratón contienen varios mil folículos1, y la ovulación periódica madura aproximadamente diez ovocitos en cada ciclo estral. Los folículos se clasifican en varias etapas del desarrollo: primordial, primaria, secundaria, antral y los folículos de Graaf, dependiendo de la forma de la capa celular granulosa que rodean a cada ovocito. La mayoría de los folículos primordiales están inactivos, y algunos de ellos se activan y se convierten en folículos primarios en cada estro ciclo2. Después de la etapa folicular secundaria, desarrollo folicular está regulado principalmente por gonadotropinas, foliculares estimulante hormona (FSH) y hormona luteinizante (LH). Sin embargo, desarrollo del folículo primordial y principal es independiente de la gonadotropina, y los mecanismos de regulación que rigen estas etapas siguen siendo mal inderstood3,4,5. Además de factores de crecimiento y hormonas, el folículo primordial y primario está regulado por las interacciones entre los folículos de6,7. Por lo tanto, se realizaron análisis de la dinámica folicular en los tejidos de ovario de ratón e investigaron los mecanismos de regulación asociados con cultivos de tejido ovárico8,9,10.
Adjunto, presentamos dos métodos del modelo de cultivo de tejido ovárico. El primero se utiliza para analizar el desarrollo del folículo por la medida de áreas foliculares, y la segunda se utiliza para estudiar el mecanismo de regulación durante el desarrollo temprano del folículo de primordial a la etapa de folículo secundario con ratones transgénicos. Para el análisis del desarrollo del folículo, utilizan principalmente ovarios de ratones hembra 4 - semanas de edad debido a que permiten la fácil visualización de los folículos. Para inducir la ovulación periódica y desarrollo de modelos en vivo del folículo, reproducen de LH y la ovulación observada, la atresia del folículo y secreción de estradiol bajo condiciones de cultivo de tejidos. Imágenes de los ovarios cultivados fueron capturados, y los procesos de desarrollo del folículo se analizaron por los cambios de trazado en la zona folicular. Sin embargo, en los análisis de microscopía de campo claro, la distinción entre folículos primordiales y principios primarios era claro. Así, se desarrolló un método para detectar folículos pequeños y distinguir entre el primordial, primario, y folículo secundario en cultiva tejidos ováricos usando Oogenesin1 pro3 (Oog1). 9 y R26-H2B-mCherry ovarios de ratones transgénicos a los 0 y 4 días después del nacimiento11. Oog1 expresión es detectable en ovocitos después de entrada en meiosis y aumenta gradualmente con el desarrollo del folículo, lo que permite la observación de la transición de primordial a primarios folículos con imágenes de Time-lapse de tejido de ovario cultivadas11 ,12. Aunque se han utilizado métodos morfológicos para estudiar factores que activan los folículos primordiales inactivos13,14,15,16, desarrollo fisiológico del folículo en los ovarios es difícil de observar, y los efectos de varios factores siendo desacostumbrados. Los métodos actuales de la cultura fueron diseñados para abordar esta falta en tiempo real Análisis de factores objetivos.
En el presente estudio, con desarrollo folicular utilizando un método de imagen Time-lapse y había caracterizado el proceso de desarrollo folicular. Nuestros nuevos métodos ofrecen una herramienta sin precedentes para la investigación de la fisiología de los ovarios.

<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

Las hembras mamíferas ovulan periódicamente una cantidad casi constante de ovocitos durante cada ciclo estral. Para sostener tal regularidad y periodicidad, regulación ocurre a nivel de eje hipotalámico-pituitario-gonadal y en el desarrollo de los folículos en el ovario. A pesar de estudios activos, mecanismos de desarrollo del folículo no son claros debido a los varios pasos de la activación del folículo primordial inactiva a la ovulación y debido a la complejidad de la regulación que difiere en cada fase folicular. Para investigar los mecanismos de desarrollo folicular y la dinámica de los folículos durante el ciclo estral, se desarrolló un modelo de cultura de tejido ovárico de ratón que puede ser utilizado para observar el desarrollo folicular utilizando un microscopio. Desarrollo sistemático del folículo, la ovulación periódica y atresia del folículo pueden todas reproducidas en el modelo de ovario cultivadas, y las condiciones de cultivo pueden ser moduladas experimentalmente. Aquí, demostramos la utilidad de este método en el estudio de los mecanismos de regulación del desarrollo folicular y otros fenómenos de ovarianos.

La figura 1 muestra el protocolo para los cambios en los medios de comunicación en cultura del tejido ovárica. Siguiendo este programa, ovarios de ratones ICR 4 semanas de edad fueron cultivadas e imagen a intervalos de 24 h mediante microscopía confocal (figura 2). Durante el cultivo de tejidos de ovario durante 3 semanas, más folículos antrales y secundarios fueron degenerados por atresia del folículo y algunos fueron ovu...

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

Cultivo de tejido ovárico para visualizar fenómenos en ovario de ratón

Cultivos de tejido ováricos se puede utilizar como modelos de la atresia de desarrollo, la ovulación y el folículo folículo e indicar mecanismos reguladores de procesos dinámicos de ovarianos.

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

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13.7K vistas.  Aichi Medical University. Cultivos de tejido ováricos se puede utilizar como modelos de la atresia de desarrollo, la ovulación y el folículo folículo e indicar mecanismos reguladores de procesos dinámicos de ovarianos.

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.

Cultura y Co-cultura de ratón ovarios y los folículos ováricos

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

Investigación

Biología del Desarrollo

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.

Un modelo ortotópico de cáncer de ovario seroso en ratones inmunocompetentes para In vivo Imágenes y seguimiento de tumores de las respuestas inmunitarias del tumor

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 ...

Inmunología e Infección

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.

Los hidrogeles de alginato para la cultura del órgano en tres dimensiones de los ovarios y oviductos

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 ...

Bioingeniería

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.

Todo Monte inmunofluorescencia y cuantificación del folículo de los ovarios de ratón cultivadas

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.

Enumeración exacta del folículo en ovarios adultos del ratón

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.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

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

Biología

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.

Inmunofluorescencia de ovario completo, limpieza y microscopía multifotónica para el análisis cuantitativo en 3D de la reserva ovárica en desarrollo en ratones

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.

Reconstrucción tridimensional de la arquitectura Vascular del ovario de ratón pasivo claridad-despejado

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.

Cultivo de tejido de corteza ovárica bovina

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.

Inoculación ortotópica de células de cáncer de ovario en ovarios murinos: un procedimiento para establecer un modelo de ratón de cáncer de ovario para estudiar la diseminación peritoneal de las células