M.M. and C.A.R. acknowledge the financial support of the Ministero della Salute, Ricerca Finalizzata/giovani ricercatori anno 2009 of the Fondazione IRCCS Ospedale San Matteo, Pavia (I). M.M and C.A.R. wish to thank Marianna Longo for helping with the images preparation.

Este protocolo se lleva a cabo de conformidad con los principios rectores de la Europea (UE) 63/2010 y 26/2014 (italianos) leyes que protegen a los animales utilizados para la investigación científica. La eutanasia Dislocación cervical se utiliza para ovarios aislamiento y es realizado por expertos y personas que figuran en el protocolo aprobado abarca este estudio entrenado. 1. Los animales <ol><li> Mantener adultos de 3 a ratones hembra de 6 semanas de edad, en una habitación con temperatura y humedad controladas (con fases de 12L: 12D y alimentados ad libitum). </li><li> Inyectar ratones por vía intraperitoneal con 2,5 UI de gonadotropina de suero de yegua embarazada (PMSG) para estimular el crecimiento folicular imitando los efectos de la hormona folículo estimulante (FSH). Aislar los ovarios para ovocitos antrales de 48 horas de recogida más tarde. La eutanasia del ratón hembra previamente inyectados con PMSG, de acuerdo con sus directrices institucionales. </li><li> Quite rápidamente los ovarios de la cavidad del cuerpo y los situará en el cont placa de Petriaining medio M2 para el aislamiento ovocitos posterior (véase la sección 3). <ol><li> Hacer una incisión en la piel que cubre la pared abdominal seguido de una incisión en el peritoneo mediante el uso de tijeras de disección estériles. Abra la incisión para exponer el abdomen, mover las agallas en un lado con unas pinzas estériles y localizar las trompas uterinas, formando una forma de V a partir de la vejiga. </li><li> Observar los ovarios ubicados debajo de los riñones. Mantenga cada tubo con pinzas estériles y hacer una pequeña incisión en la parte inferior de los ovarios para liberarlos. Transferencia ambos ovarios en la parte inferior de una placa de Petri que contiene medio de pre-equilibrada. </li></ol></li></ol> 2. Preparación de la hormona <ol><li> Diluir PMSG (disponible en diferentes concentraciones madre) con solución salina isotónica estéril para obtener una concentración de trabajo de 2,5 UI de hormona por 0,1 ml para un volumen de inyección adecuado. NOTA: Las soluciones de hormonas deben alícuotas en tubos de 0,5 ml y se almacenaron a -20 ° C hasta el día de uso. No mantenga alícuotas diluidas de más de tres meses a -20 ° C. Una vez descongelado, si se realizan inyecciones de serie, almacenar las alícuotas de las soluciones de la hormona en el 4 ° C y utilizarlos dentro de 4 días. </li></ol> 3. antro ovocitos Aislamiento Para el aislamiento ovocitos adecuado: <ol><li> Equilibrar medio M2 13 (M2) en 5% de CO 2 y 37 ° C durante al menos 1 hr antes de iniciar el procedimiento de aislamiento ovocitos. Preparar dos placas de Petri (35 mm x 10 mm), una con 1 ml de M2 ​​para perforar ovario y la segunda una con pequeñas gotas (20 l) de M2 ​​cubierto con aceite mineral (~ 1,5 ml) para la transferencia de ovocitos después del aislamiento de la ovario (ver 3.4 a 3.6). Mantenga ambas placas de Petri en la incubadora a 5% de CO 2 y 37 ° C hasta que esté listo para ser utilizado. </li><li> Utilice un microscopio estereoscópico durante los procedimientos de aislamiento de ovocitos. </li><li> Preparar pipetas de vidrio boca delgadas para recoger los ovocitos por stretchhing pipetas Pasteur de vidrio mantienen horizontalmente (la celebración de los extremos de la pipeta con las dos manos), sobre una vertical de la llama procedente del quemador Bunsen (Figura 1). <ol><li> Una vez que el vidrio comienza a derretirse, tome la pipeta de la llama y realizar un movimiento de tracción rápida para obtener la pipeta deseada. Firmemente cortar el exceso de vidrio cerca de la punta de la pipeta con uñas para ajustar la longitud y el diámetro del capilar interno estrecho. Asegúrese de que el capilar fina tiene un diámetro interno de ~ 100 m, ligeramente mayor que el diámetro del ovocito (~ 80 m). </li><li> Para la boca pipeta, conecte un extremo del tubo de aspiración a la pipeta tirado por el uso de una punta adecuada y coloque el otro extremo en la boca, asegurándolo con los dientes. </li></ol></li><li> Recoger los ovarios usando pinzas estériles y depositarlas en la parte inferior de una placa de cultivo celular de Petri (35 mm x 10 mm). Cubra con 1 ml de M2 ​​equilibraron previamente a 37 ° Cy 5% de CO 2 (Figura 2). </li><li> Pinchar suavemente los folículos antrales de los ovarios con una aguja de jeringa de insulina estéril para liberar ovocitos antrales en M2 (Figura 3). </li><li> Suavemente boca pipetear los ovocitos a través de una pipeta Pasteur estrecha para eliminar las células del folículo y cualquier otra restos de tejido sea posible (Figura 4A) y luego conformarse ellos en la parte inferior de las pequeñas gotas (20 l) de M2 cubiertos con aceite mineral adecuado para el cultivo de embriones (anteriormente preparado; Figura 4B). NOTA: Es importante eliminar todas las células del cúmulo unidos a la zona pelúcida de los ovocitos por pipeteo continua a través de varias y diferentes gotas de M2. Realice este paso lo más rápido posible para evitar alteraciones en el pH del medio. </li></ol> 4. antral Los ovocitos tinción con Hoechst33342 NOTA: los ovocitos antrales se puede distinguir en SN (Rodeado Nucleolo), NSN (No Rodeado Nucleolo) oen cualquier otra forma intermedia, en función de su organización de la cromatina después de la tinción con la concentración de supravital Hoechst33342 (o cualquier otro marcadores específicos de bases AT) 14. De hecho, los ovocitos SN muestran un anillo de Hoechst-positivas alrededor del nucléolo, mientras que el NSN no lo hacen, de ahí su nombre. Para la tinción de ovocitos adecuado: <ol><li> La transferencia de los ovocitos utilizando una pipeta de boca en gotas de Hoechst33342 (20 l) diluido en M2 a la concentración supravital de 50 ng / l durante 10 min en oscuridad. </li><li> Después de la incubación con Hoechst33342, transferir cada solo ovocito con una pipeta de boca en la parte inferior de las pequeñas gotas (5 l) de M2 ​​cubiertos con aceite mineral, para la clasificación sobre la base de organización de la cromatina (SN o NSN). Utilice cualquier microscopio de fluorescencia equipado con filtro Hoechst33342 (longitud de onda de emisión de 486 nm) para visualizar la configuración de la cromatina de los ovocitos. NOTA: Dependiendo del tipo de microscopio de fluorescencia utilizado, it puede llegar a ser imperativo el uso de fondo de cristal placa de Petri para la adquisición de imágenes. La microscopía confocal Olympus Fluoview FV10i cuando está equipado con un soporte de muestra para 35 mm plato requiere sólo de abajo a los platos de cristal para la adquisición de imágenes, con un grosor estándar de 0,17 mm y un rango de 0,13 a 0,21 mm aceptables. </li><li> Excite los ovocitos durante no más de 5-10 segundos, probablemente tiempo suficiente para visualizar la presencia o la ausencia de un anillo alrededor del nucléolo (Figura 5). </li><li> Después de la clasificación, la cultura de la SN antral y ovocitos NSN en M2 en el 5% de CO 2 y 37 ° C para inducir la maduración in vitro o mantenerlos en tampones apropiados para los análisis moleculares o celulares. </li></ol>

<ol>
	<li>Parfenov, V., Potchukalina, G., Dudina, L., Kostyuchek, D., Gruzova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+antral+follicles:+oocyte+nucleus+and+the+karyosphere+formation+(electron+microscopic+and+autoradiographic+data).">Human antral follicles: oocyte nucleus and the karyosphere formation (electron microscopic and autoradiographic data).</a> Gamete Res. 22 (2), 219-231 (1989).</li><li>Anderiesz, C., Ferraretti, A. P., Magli, C., Fiorentino, A., Fortini, D., Gianaroli, L., Jones, G. M., Trounson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+recombinant+human+gonadotropins+on+human,+bovine+and+murine+oocyte+meiosis,+fertilization+and+embryonic+development+in+vitro.">Effect of recombinant human gonadotropins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro.</a> Human Reprod. 15 (5), 1140-1148 (2000).</li><li>Mattson, B. A., Albertini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis:+Chromatin+and+microtubule+dynamics+during+meiotic+prophase.">Oogenesis: Chromatin and microtubule dynamics during meiotic prophase.</a> Mol. Reprod. Dev. 25 (4), 374-383 (1990).</li><li>Debey, P., Szollosi, M. S., Szollosi, D., Vautier, D., Girousse, A., Besomber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competent+mouse+oocytes+isolated+from+antral+follicles+exhibit+different+chromatin+organization+and+follow+different+maturation+dynamics.">Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.</a> Mol. Reprod. Dev. 36 (1), 59-74 (1993).</li><li>Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+organisation+during+mouse+oocyte+growth.">Chromatin organisation during mouse oocyte growth.</a> Mol. Reprod. Dev. 41 (1-2), 479-485 (1995).</li><li>Wu, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+oocytes+for+in+vitro+maturation+by+brilliant+cresyl+blue+staining:+a+study+using+the+mouse+model.">Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model.</a> Cell Res. 17, 722-731 (2007).</li><li>Opiela, Y., K&#261;tska-Ksi&#261;&#380;kiewicz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+Brilliant+Cresyl+Blue+(BCB)+staining+of+mammalian+oocytes+used+for+in+vitro+embryo+production+(IVP).">The utility of Brilliant Cresyl Blue (BCB) staining of mammalian oocytes used for in vitro embryo production (IVP).</a> Reproductive Biol. 13, 177-183 (2013).</li><li>Labrecque, R., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+mammalian+oocyte+competence+by+transcriptome+analysis:+progress+and+challenges.">The study of mammalian oocyte competence by transcriptome analysis: progress and challenges.</a> Mol. Human Reprod. 20 (2), 103-116 (2014).</li><li>Monti, M., Zanoni, M., Calligaro, A., Ko, M. S., Mauri, P. L., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+arrest+and+mouse+antral+Not-Surrouded+Nucleolus+oocyte.">Developmental arrest and mouse antral Not-Surrouded Nucleolus oocyte.</a> Biol. Reprod. 88 (1), 1-7 (2013).</li><li>Yurttas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+PADI6+and+the+cytoplasmic+lattices+in+ribosomal+storage+in+oocytes+and+translational+control+in+the+early+mouse+embryo.">Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo.</a> Development. 135 (15), 2627-2636 (2008).</li><li>Li, L., Baibakov, B., Dean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subcortical+maternal+complex+essential+for+preimplantation+mouse+embryogenesis.">A subcortical maternal complex essential for preimplantation mouse embryogenesis.</a> Dev. Cell. 15 (3), 416-425 (2008).</li><li>Ami, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FT-IR+spectra+signatures+of+mouse+antral+oocytes:+molecular+markers+of+oocyte+maturation+and+developmental+competence.">FT-IR spectra signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence.</a> Biochim. Biophys. Acta. 1813 (6), 1220-1222 (2011).</li><li>Fulton, B. P., Whittingam, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+mammalian+oocytes+by+intercellular+injection+of+calcium.">Activation of mammalian oocytes by intercellular injection of calcium.</a> Nature. 273 (5658), 149-151 (1978).</li><li>Luttmer, S. J., Longo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+living+and+fixed+gametes+and+early+embryos+stained+with+supravital+fluorochromes+(Hoechst+33342+and+3,3&#8242;-dihexyloxacarbocyanine+iodide).">Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3&#8242;-dihexyloxacarbocyanine iodide).</a> Gamete Res. 15 (3), 267-283 (1986).</li><li>Monti, M., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biopolitics+of+frozen+embryos.">The biopolitics of frozen embryos.</a> Int. J. Dev. Biol. 55, 243-247 (2011).</li></ol>

The authors have nothing to disclose

Este protocolo describe el método usado para aislar y clasificar antrales ratón ovocitos GV. No hay pasos críticos particulares a subrayar, con pocas excepciones. En primer lugar: este procedimiento debe realizarse lo más rápidamente posible para: a) mantener la vitalidad de los ovocitos y b) evitar los cambios de pH en el medio utilizado. Segunda: los ovocitos deben ser brevemente excitado (5-10 seg) para Hoechst33342 para evitar daños de la cromatina y la posterior muerte de los ovocitos, especialmente si IVM y ...

Antrales ovocitos completamente crecidos (GV, vesícula germinal) aislados del compartimento antral del ovario se utilizan de forma rutinaria para diferentes propósitos, tales como, por ejemplo, el estudio de los mecanismos moleculares y celulares detrás de la maduración in vitro hasta la etapa metafase II. A pesar de sus hermosos apariencia ovocitos más antrales, pero no todos, son capaces de completar la meiosis y llegar a la etapa de blastocisto; de hecho, en muchas especies de mamíferos, incluyendo seres humanos 1,2, aproximadamente 30% de ellos detener su desarrollo en la etapa de 2 células, si se produce la fertilización 3-5. Hasta la fecha, unos parámetros se utilizan para distinguir entre ovocitos capaces de sostener el desarrollo embrionario de los que no pueden hacerlo. Por ejemplo, la tinción de azul de cresilo (BCB) es un método válido para ordenar los ovocitos basados ​​en la actividad de la glucosa-6-fosfato deshidrogenasa-aunque la utilidad de este método de clasificación relacionadas con BCB + o tinción es BCB-todavía depende 6-8 laboratorio. Otro método bien establecido reside en su organización de la cromatina: si se tiñeron con los tintes de intercalación de ADN (como Hoechst33342), el 70% de los ovocitos antrales muestra un anillo de tinte positivo alrededor del nucléolo y se llaman Rodeado Nucleolo (SN), mientras que el restante 30% mostrar unas señales positivas más manchados y se llaman No Rodeado Nucleolo (NSN) 3-5. Recientemente hemos demostrado que la ausencia de celosías citoplasmáticos 9 (CPL, centros de almacenamiento importantes para ARNm y ribosomas maternos en ambos ovocitos de mamíferos y embriones) 10 y la baja regulación de MATER (esencial para la formación de CPL 11), junto con la presencia de gotas de lípidos en sus citoplasmas 9,12, puede haber otras causas relacionadas con el ovocitos incapacidad NSN para continuar más allá de la etapa de 2 células. Desafortunadamente, algunas técnicas se pueden aplicar para ordenar SN antral y ovocitos NSN y,Por esta razón, el desarrollo de un método menos invasivo (sin tinción nuclear, los procedimientos de fijación o la manipulación con pipetas boca) podría llegar a ser muy importante para aumentar las tasas de tanto el desarrollo del embrión humano y animal. En este trabajo se detalle el protocolo simple y rápida utilizó para aislar ad tipo SN y NSN ovocitos antrales de ratones hembras y se dirige a todos los científicos interesados ​​en el estudio de la gametogénesis femenina, la maduración del ovocito y el desarrollo embrionario.

<table><tbody><tr><td>PMSG</td><td>Sigma</td><td>G4527</td><td></td></tr><tr><td>EmbrioMax M2</td><td>Millipore</td><td>MR-015-PD</td><td></td></tr><tr><td>Hoechst33342</td><td>Thermo Scientific</td><td>62249</td><td></td></tr><tr><td>Mineral Oil</td><td>Sigma</td><td>M8410</td><td></td></tr><tr><td>Cell Culture Dish 35mmx10mm</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>&amp;micro;-Dish 35mm, high glass bottom</td><td>Ibidi</td><td>81158</td><td></td></tr><tr><td>Pipette Pasteur Borosilicate</td><td>Corning</td><td>7095D-9</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated micropillary pipettes</td><td>Sigma</td><td>A5177</td><td></td></tr></tbody></table>

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

Las siguientes imágenes muestran el método utilizado para aislar los ovocitos de los ovarios de ratones antrales, a partir de la preparación de las pipetas para la clasificación de los ovocitos &#39;mediante Hoechst33342. Este método es bastante sencillo y se puede realizar en cualquier laboratorio equipado con una incubadora de CO 2, un estereomicroscopio y un microscopio de fluorescencia equipado con el filtro correcto para la observación de la Hoechst33342 Fig...

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

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

Aquí se presenta un protocolo para la caracterización de los ovocitos antrales ratón sobre la base de la organización nucleolar.

Aislamiento y caracterización de ratón antral Los ovocitos Sobre la base de organización de la cromatina nucleolar

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

isolation-characterization-mouse-antral-oocytes-based-on-nucleolar

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

13.3K vistas.  Fondazione IRCCS Policlinico San Matteo.  Aquí se presenta un protocolo para la caracterización de los ovocitos antrales ratón sobre la base de la organización nucleolar. 

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

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Cromatina difundir preparados para el análisis de progresión de ovocitos de ratón de profase a metafase II

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

Investigación

Genética

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Uso de ovocitos de ratón para evaluar la función humana del Gene durante la Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

Análisis de la segregación cromosómica, acetilación de histonas y morfología de huso en ovocitos de caballo

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

Biología del Desarrollo

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

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure. Oocytes are isolated from fully-grown antral follicles and cultured for 15 h inside a microscope equipped for time-lapse analysis at 37 &#176;C and 5% CO2. Pictures are taken at 8 min intervals. The images are analyzed using the Particle Image Velocimetry (PIV) method that calculates, for each oocyte, the profile of Cytoplasmic Movement Velocities (CMVs) occurring throughout the culture period. Finally, the CMVs of each single oocyte are fed through a mathematical classification tool (Feed-forward Artificial Neural Network, FANN), which predicts the probability of a gamete to be developmentally competent or incompetent with an accuracy of 91.03%. This protocol, set up for the mouse, could now be tested on oocytes of other species, including humans.

Here, we present a protocol for non-invasive assessment of oocyte developmental competence performed during their in vitro maturation from the germinal vesicle to the metaphase II stage. This method combines time-lapse imaging with particle image velocimetry (PIV) and neural network analyses.

Una identificación basada en redes neuronal desarrollo competentes o incompetentes adulto de ovocitos de ratón

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Cultivo de tejido ovárico para visualizar fenómenos en ovario de ratón

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

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

Ablación con láser multifotón de centros organizadores de microtúbulos citoplasmáticos en ovocitos de ratón

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

Identificación del daño en el ADN en ovocitos de ratones con profase detenida

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Autor destacado: Comprensión de la respuesta al daño del ADN en ovocitos de mamíferos y embriones preimplantacionales 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Detección de roturas de ADN bicatenario en ovocitos de ratón

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

La microinyección de ovocitos de ratón, Maduración y Evaluación de ploidía

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

Aislamiento y caracterización de ratón antral Los ovocitos Sobre la base de organización de la cromatina nucleolar

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Cultivo de tejido ovárico para visualizar fenómenos en ovario de ratón

Ablación con láser multifotón de centros organizadores de microtúbulos citoplasmáticos en ovocitos de ratón

Detección de roturas de ADN bicatenario en ovocitos de ratón

Detección de roturas de ADN bicatenario en ovocitos de ratón

Detección de roturas de ADN bicatenario en ovocitos de ratón

La microinyección de ovocitos de ratón, Maduración y Evaluación de ploidía