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 é realizado em conformidade com os princípios orientadores da Europeia (UE) e 63/2010 (26/2014) leis italianas que protegem animais usados ​​para pesquisa científica. Cervical eutanásia deslocamento é utilizado para isolamento e ovários é realizada por peritos e indivíduos listados no protocolo aprovado cobrindo este estudo treinado. 1. Os animais <ol><li> Manter adulto de 3 a 6 semanas de idade ratos fêmeas em uma sala com temperatura e umidade controladas (com fases de 12L: 12E e alimentados ad libitum). </li><li> Injetar camundongos por via intraperitoneal com 2,5 UI de gonadotrofina de égua prenhe (PMSG) para estimular o crescimento folicular, imitando os efeitos do hormônio folículo estimulante (FSH). Isolar os ovários para oócitos antrais 48hrs coleção posteriores. Euthanize o mouse fêmea anteriormente injectados com PMSG, de acordo com suas diretrizes institucionais. </li><li> Remover rapidamente os ovários da cavidade do corpo e colocá-los para o cont placa de Petriaining meio M2, para posterior isolamento oócitos (ver secção 3). <ol><li> Realizar uma incisão na pele que cobre a parede abdominal, seguida por uma incisão no peritoneu utilizando tesouras de dissecação estéreis. Abra a incisão para expor o abdómen, mover os intestinos de um lado usando pinças estéreis e localizar os tubos uterinos, formando uma forma em V a partir da bexiga. </li><li> Observar os ovários localizados abaixo dos rins. Mantenha cada tubo com pinça estéril e fazer uma pequena incisão na parte inferior dos ovários a libertá-los. Transferir ambos os ovários, na parte inferior de uma placa de Petri contendo meio de pré-equilibrada. </li></ol></li></ol> 2. Preparação Hormone <ol><li> Diluir PMSG (disponível em diferentes concentrações de estoque) com solução salina isotónica estéril para se obter uma concentração de trabalho de 2,5 UI por hormona de 0,1 ml para um volume de injecção apropriado. NOTA: As soluções da hormona deve ser dividido em alíquotas em tubos de 0,5 ml e armazenados a -20 ° C até ao dia da utilização. Não manter partes aliquotas diluídas com mais de três meses a -20 ° C. Uma vez descongelado, se injecções são realizadas em série, armazenar as alíquotas de soluções de hormonas a +4 ° C e utilizá-los dentro de 4 dias. </li></ol> 3. Antral Oócitos Isolamento Para o isolamento de oócitos adequada: <ol><li> Equilibrar meio M2 13 (M2) com 5% de CO 2 e 37 ° C durante pelo menos 1 h antes de iniciar o procedimento de isolamento de oócitos. Preparar duas placas de Petri (35 mm x 10 mm), um deles com 1 ml de M2 ​​para perfurar ovário e o segundo com pequenas gotas (20 ul) de M2 ​​coberta com óleo mineral (~ 1,5 ml) para transferência de oócitos após isolamento a partir da ovário (ver 3.4-3.6). Manter ambos os pratos de Petri em incubadora a 5% de CO 2 e 37 ° C até estar pronto para ser utilizado. </li><li> Use um microscópio estereoscópico durante procedimentos de isolamento oócitos. </li><li> Prepare finas pipetas de vidro boca para recolher os ovócitos com fitahing pipetas de Pasteur de vidro mantido na horizontal (segurando as extremidades da pipeta com as duas mãos), ao longo de um vertical da chama proveniente do bico de Bunsen (Figura 1). <ol><li> Uma vez que o vidro começa a derreter, pegue a pipeta para fora da chama e realizar um movimento de puxar rápido para obter a pipeta desejado. Firmemente cortar o vidro em excesso perto do ponto estreito da pipeta com unhas para ajustar o comprimento e o diâmetro interno do capilar. Assegure-se que a fina capilar tem um diâmetro interno de ~ 100 pm, ligeiramente maior do que o diâmetro do oócito (~ 80? M). </li><li> Para boca pipeta, ligar-se uma extremidade do tubo de aspiração para a pipeta puxado utilizando uma ponta apropriada e colocar a outra extremidade na boca, prendendo-o com os dentes. </li></ol></li><li> Recolher os ovários usando pinças estéreis e depositá-los na parte inferior de um prato de cultura de células de petri (35 mm x 10 mm). Cobri-los com 1 ml de M2, previamente equilibrada a 37 ° Ce 5% de CO 2 (Figura 2). </li><li> Suavemente perfurar os folículos antrais dos ovários com uma agulha de seringa estéril para libertar insulina oócitos antrais em M2 (Figura 3). </li><li> Suavemente boca pipetar os oócitos através de uma pipeta de Pasteur estreita para remover as células foliculares e qualquer outro restos de tecido possível (Figura 4A) e em seguida resolver-los na parte inferior de pequenas gotas (20 ul) de m2 de área coberta com óleo mineral adequado para a cultura de embriões (anteriormente preparados; A Figura 4B). NOTA: É importante remover todas as células de cumulus ligados à zona pelúcida dos oócitos por pipetagem contínua através de vários e diferentes gotas de M2. Execute esta etapa o mais rápido possível para evitar alterações no pH do meio. </li></ol> 4. Antral Oócitos Coloração com Hoechst33342 NOTA: ovócitos antrais podem ser distinguidos na SN (Cercado nucléolo), NSN (Não Rodeado nucléolo) ouem qualquer outra forma intermédia, com base na sua organização da cromatina após coloração com concentração de Hoechst33342 supravital (ou quaisquer outros marcadores específicos de bases AT 14). Na verdade, os oócitos SN mostrar um anel Hoechst-positiva em torno do nucléolo enquanto a NSN não, daí o seu nome. Para a coloração oócitos adequada: <ol><li> Transferir os oócitos utilizando uma pipeta de boca em gotas de Hoechst33342 (20 ul), diluído em M2 na concentração supravital de 50 ng / mL durante 10 min no escuro. </li><li> Após a incubação com Hoechst33342, transferir cada um único oócito com uma pipeta de boca na parte inferior de pequenas gotas (5 ul) de m2 de área coberta com óleo mineral, para classificação com base na organização da cromatina (SN ou NSN). Use qualquer microscópio de fluorescência equipado com filtro Hoechst33342 (comprimento de onda de emissão 486 nm) para visualizar a configuração da cromatina dos oócitos. NOTA: Dependendo do tipo de microscópio de fluorescência usado, it pode tornar-se imperativa a utilização de vidro de fundo placa de Petri para aquisição de imagem. A microscopia confocal Olympus Fluoview FV10i quando equipado com um pegador de amostra para 35 milímetros prato requer apenas com fundo de vidro-pratos para aquisição de imagem, com uma espessura padrão de 0,17 milímetros e um intervalo de 0,13 a 0,21 milímetros aceitáveis. </li><li> Excitar os oócitos durante não mais do que 5-10 seg, provavelmente tempo suficiente para visualizar a presença ou a ausência de um anel em torno do nucléolo (Figura 5). </li><li> Após a separação, a cultura SN antral e oócitos NSN em M2 em 5% de CO 2 e 37 ° C, para induzir a maturação in vitro ou mantê-los em tampões apropriados para análises moleculares ou celulares. </li></ol>

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The authors have nothing to disclose

Este protocolo descreve o método usado para isolar e classificar rato antrais oócitos GV. Não há passos críticos particular, sublinhar, com poucas exceções. Primeiro: este procedimento deve ser realizado o mais rapidamente possível: a) manter a vitalidade dos oócitos e b) evitar mudanças de pH no meio utilizado. Segunda: oócitos deve ser animado brevemente (5-10 s) a Hoechst33342 para evitar danos da cromatina e a posterior morte dos oócitos, especialmente se IVM e FIV são os seguintes passos. Até à data,...

Antrais oócitos GV (totalmente crescidas, vesícula germinal) isolados a partir do compartimento do antro do ovário são rotineiramente usadas para diferentes fins, tais como, por exemplo, no estudo dos mecanismos moleculares e celulares subjacentes a maturação in vitro até estádio de metáfase II. Apesar de suas aparência bonita oócitos mais antrais, mas não todos, são capazes de completar a meiose e atingir o estágio de blastocisto; De facto, em muitas espécies de mamíferos, incluindo seres humanos 1,2, cerca de 30% deles deter o seu desenvolvimento na fase de 2 células, se ocorre a fertilização 3-5. Até à data, alguns parâmetros são usados ​​para distinguir entre oócitos capazes de sustentar o desenvolvimento embrionário de pessoas incapazes de fazê-lo. Por exemplo, coloração com azul de Cresyl (BCB) é um método válido para classificar os oócitos com base na actividade da glucose-6-fosfato-desidrogenase, embora a utilidade deste método de classificação relacionada com BC + ou coloração é BCB-Ainda laboratório dependentes 6-8. Outro método bem estabelecido reside na sua organização da cromatina: se coradas com quaisquer corantes intercalantes de ADN (como Hoechst33342), 70% dos oócitos antrais mostrar um anel de corante-positiva em torno do núcleo e são chamados rodeado nucléolo (SN), enquanto que os restantes 30% mostrar um mais manchado sinais positivos e são chamados Não Rodeado nucléolo (NSN) 3-5. Recentemente, mostrou que a ausência de reticulados citoplasmáticos 9 (CPLs, locais de armazenagem importantes para ARNm de origem materna e ribossomas em ambos os oócitos de mamíferos e embriões) 10 e a sub-regulação de MATER (essencial para a formação CPLs 11), juntamente com a presença de gotículas lipídicas em seus citoplasmas 9,12, pode haver outras causas relacionadas com a incapacidade NSN oócitos de proceder para além do estádio de 2 células. Infelizmente, algumas técnicas podem ser aplicadas para classificar SN antral e oócitos e NSN,Por esta razão, o desenvolvimento de um método menos invasivo (sem coloração nuclear, os processos de fixação ou manipulação com pipetas de boca) pode tornar-se muito importante para aumentar as taxas de desenvolvimento embrionário, tanto humana e animal. Neste artigo, detalhamos o protocolo simples e rápida usada para isolar anúncio SN tipo e NSN oócitos antrais de camundongos fêmeas e é dirigida a todos os cientistas interessados ​​no estudo da gametogênese do sexo feminino, a maturação eo desenvolvimento do embrião.

<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.

As imagens seguintes mostram o método usado para isolar oócitos antrais a partir de ovários de rato, a partir de pipetas preparação para triagem dos ovócitos por meio de Hoechst33342. Este método é muito simples e pode ser realizado em qualquer laboratório equipado com uma incubadora de CO 2, um estereomicroscópio e um microscópio de fluorescência equipado com o filtro correcta para a observação do Hoechst33342 Figura 1 mostra a primeira etapa de...

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

Aqui apresentamos um protocolo para a caracterização de oócitos antrais de rato com base na organização nucleolar.

Isolamento e Caracterização do mouse Antral Oócitos Baseado em Nucleolar organização da cromatina

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

Research

JoVE Journal

Developmental Biology

13.3K visualizações.  Fondazione IRCCS Policlinico San Matteo.  Aqui apresentamos um protocolo para a caracterização de oócitos antrais de rato com base na organização nucleolar. 

Vídeo: 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.

Preparações de espalhamento de cromatina para a análise da progressão de oócitos de camundongo da prófase para a metáfase II

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

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.

Usando oócitos de camundongo para avaliar a função de genes humanos durante a meiose 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.

Análise da segregação cromossômica, acetilação de histona e morfologia do fuso em ovócitos de cavalo

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

Biologia do Desenvolvimento

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.

Uma identificação baseado em Rede Neural de ovócitos já crescido mentalmente competente ou incompetente do Mouse

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.

Cultura do tecido ovariano Visualizar fenômenos no ovário de Mouse

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.

Ablação a Laser Multifóton de Centros Organizadores de Microtúbulos Citoplasmáticos em Oócitos de Camundongos

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

Identificando danos ao DNA em oócitos de camundongos com prisão de prófase

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 em destaque: Compreendendo a resposta a danos no DNA em oócitos de mamíferos e embriões pré-implantação 

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.

Detecção de quebras de fita dupla de DNA em ovócitos de camundongos

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.

Avaliação do mouse microinjeção de oócitos, maturação e ploidia

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

Isolamento e Caracterização do mouse Antral Oócitos Baseado em Nucleolar organização da cromatina

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