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.

Questo protocollo è condotto in conformità con i principi ispiratori del Europea (UE 63/2010) e Italiano (26/2014) leggi che proteggono gli animali utilizzati per la ricerca scientifica. Cervicale dislocazione l&#39;eutanasia viene utilizzato per l&#39;isolamento ovaie ed è eseguita da esperti e soggetti elencati nel protocollo approvato che copre questo studio addestrato. 1. Gli animali <ol><li> Mantenere adulti da 3 a 6 settimane di età topi femmina in una stanza a temperatura e umidità controllate (con fasi di 12L: 12D e alimentati ad libitum). </li><li> Iniettare topi per via intraperitoneale con 2.5 UI di gonadotropina del siero di cavalla incinta (PMSG) per stimolare la crescita follicolare imitando gli effetti dell&#39;ormone follicolo-stimolante (FSH). Isolare le ovaie di ovociti antrali di raccolta 48 ore successive. Euthanize il mouse femminile precedentemente iniettato con PMSG, in base alle proprie linee guida istituzionali. </li><li> Rimuovere rapidamente le ovaie dalla cavità del corpo e metterli nel piatto cont Petriaining M2 mezzo per successivo isolamento ovociti (vedi sezione 3). <ol><li> Effettuare un&#39;incisione sulla pelle che copre la parete addominale seguita da un&#39;incisione nel peritoneo utilizzando forbici dissezione sterili. Aprire l&#39;incisione per esporre l&#39;addome, spostare le viscere da una parte con una pinzetta sterile e localizzare i tubi uterine, formando una forma a V a partire dalla vescica. </li><li> Osservare le ovaie si trovano al di sotto dei reni. Tenere ogni tubo con pinzetta sterile e fare una piccola incisione nella parte inferiore delle ovaie a rilasciarli. Trasferire entrambe le ovaie nella parte inferiore di una piastra di Petri contenenti terreno pre-equilibrata. </li></ol></li></ol> 2. Ormone Preparazione <ol><li> Diluire PMSG (disponibile in diverse concentrazioni archivi) con soluzione salina isotonica sterile per ottenere una concentrazione di lavoro di 2,5 UI di ormone per 0,1 ml di un volume di iniezione appropriata. NOTA: Le soluzioni di ormoni devono essere aliquotati in provette da 0,5 ml e conservati a -20 ° C fino al giorno di utilizzo. Non tenere aliquote diluito superiori a tre mesi a -20 ° C. Una volta scongelato, se si eseguono iniezioni seriali, memorizzare le aliquote delle soluzioni ormonali a +4 ° C e utilizzare entro 4 giorni. </li></ol> Isolamento 3. Antral Ovociti Per un corretto isolamento ovociti: <ol><li> Equilibrare M2 medio 13 (M2) al 5% di CO 2 e 37 ° C per almeno 1 ora prima di avviare la procedura di isolamento oociti. Preparare due piastre di Petri (35 mm x 10 mm), una con 1 ml di m2 per punzonamento ovaie e la seconda con piccole gocce (20 ml) di M2 coperto con olio minerale (~ 1,5 ml) per il trasferimento ovociti dopo isolamento dal ovaia (vedi 3,4-3,6). Tenere entrambe le capsule di Petri in incubatore al 5% di CO 2 e 37 ° C fino al momento di essere utilizzati. </li><li> Utilizzare uno stereomicroscopio durante le procedure di isolamento ovociti. </li><li> Preparare sottili pipette di vetro bocca per raccogliere gli ovociti stetchhing pipette Pasteur in vetro tenute orizzontalmente (tenendo le estremità della pipetta con entrambe le mani), a fiamma verticale proveniente dal bruciatore Bunsen (Figura 1). <ol><li> Una volta che il vetro inizia a fondere, prendere la pipetta della fiamma ed eseguire un movimento di trazione rapido avere la pipetta desiderata. Saldamente tagliare il vetro in eccesso vicino al punto stretto della pipetta con unghie per regolare la lunghezza e il diametro interno del capillare. Assicurarsi che il sottile capillare ha un diametro interno di ~ 100 micron, leggermente maggiore del diametro del ovocita (~ 80 micron). </li><li> Per bocca pipetta, collegare un&#39;estremità del tubo di aspirazione alla pipetta tirato utilizzando una punta appropriata e posizionare l&#39;altra estremità in bocca, fissandolo con i denti. </li></ol></li><li> Raccogliere ovaie usando pinzette sterili e depositarli sul fondo di un piatto di coltura cellulare di Petri (35 mm x 10 mm). Coprire con 1 ml di M2 precedentemente equilibrata a 37 ° Ce 5% CO 2 (Figura 2). </li><li> Forare delicatamente i follicoli antrali delle ovaie con un ago sterile siringa da insulina per liberare oociti antrali a M2 (Figura 3). </li><li> Delicatamente bocca pipettare gli ovociti attraverso uno stretto pipetta Pasteur per rimuovere le cellule follicolari e qualsiasi altri detriti tessuto possibile (Figura 4A) e poi depositarsi sul fondo di piccole gocce (20 mL) di M2 coperti con olio minerale adatto a cultura dell&#39;embrione (precedentemente preparati; Figura 4B). NOTA: E &#39;importante rimuovere tutte le cellule del cumulo collegati alla zona pellucida degli ovociti pipettando continuo attraverso diversi e diverse gocce di M2. Eseguire questo passaggio il più velocemente possibile per evitare alterazioni del pH del mezzo. </li></ol> 4. Antral ovociti colorazione con Hoechst33342 NOTA: ovociti Antral può essere distinta in SN (Circondato Nucleolo), NSN (Non Circondato Nucleolo) oin qualsiasi altra forma intermedia, in base alla loro organizzazione della cromatina dopo colorazione con concentrazione sopravitale di Hoechst33342 (o qualsiasi altro marker specifici per le basi AT) 14. In effetti, gli ovociti SN mostrano un anello Hoechst-positivo attorno al nucleolo mentre la NSN non lo fanno, da qui il suo nome. Per una corretta colorazione ovociti: <ol><li> Trasferire gli ovociti usando una pipetta bocca in gocce di Hoechst33342 (20 ml) diluito in M2 alla concentrazione sopravitale di 50 ng / ml per 10 min in scuro. </li><li> Dopo l&#39;incubazione con Hoechst33342, trasferire ogni singolo ovocita con una pipetta a bocca in fondo piccole gocce (5 mL) di M2 coperti con olio minerale, per l&#39;ordinamento basato su organizzazione della cromatina (SN o NSN). Utilizzare qualsiasi microscopio a fluorescenza dotato di filtro Hoechst33342 (lunghezza d&#39;onda di emissione 486 nm) per visualizzare la configurazione della cromatina degli ovociti. NOTA: A seconda del tipo di microscopio a fluorescenza utilizzato, it può diventare indispensabile utilizzare vetro Petri fondo per l&#39;acquisizione delle immagini. La microscopia confocale Olympus Fluoview Fv10i quando dotata di supporto del campione per 35 millimetri piatto richiede solo vetro bottom-piatti per l&#39;acquisizione di immagini, con spessore standard 0,17 millimetri e un range da 0,13 a 0,21 millimetri accettabili. </li><li> Eccitare gli ovociti per non più di 5-10 sec, probabilmente abbastanza tempo per visualizzare la presenza o l&#39;assenza di un anello attorno al nucleolo (Figura 5). </li><li> Dopo la cernita, la cultura SN antrale e ovociti NSN in M2 al 5% di CO 2 e 37 ° C per indurre la maturazione in vitro o tenerli in buffer appropriate per analisi molecolari e cellulari. </li></ol>

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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. 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The authors have nothing to disclose

Questo protocollo descrive il metodo utilizzato per isolare e classificare il mouse antrali GV ovociti. Non vi sono particolari passaggi critici per sottolineare, con poche eccezioni. Primo: questa procedura deve essere eseguita il più rapidamente possibile: a) mantenere la vitalità degli ovociti e b) evitare variazioni di pH del mezzo utilizzato. Secondo: gli ovociti devono essere brevemente eccitato (5-10 sec) per Hoechst33342 per evitare danni cromatina e la successiva morte degli ovociti, soprattutto se IVM e IVF ...

Antrale ovociti completamente sviluppati (GV, vescicola germinale) isolati dal vano antrale dell&#39;ovario sono abitualmente utilizzati per scopi diversi, come, per esempio, lo studio dei meccanismi cellulari e molecolari alla base maturazione in vitro fino metafase II. Nonostante le loro bella apparenza oociti antrali più, ma non tutti, sono in grado di completare la meiosi e raggiungere lo stadio di blastocisti; infatti, in molte specie di mammiferi, compreso l&#39;uomo 1,2, circa il 30% del loro arresto loro sviluppo nella fase 2 cellule, se avviene la fecondazione 3-5. Ad oggi, alcuni parametri sono utilizzati per distinguere tra gli ovociti in grado di sostenere lo sviluppo embrionale da quelli in grado di farlo. Ad esempio, cresolo colorazione blu (BCB) è un valido metodo per ordinare gli ovociti basati sull&#39;attività di glucosio-6-fosfato deidrogenasi, sebbene l&#39;utilità di questo metodo di ordinamento correlate a BCB + o colorazione è BCB-ancora dipendente 6-8 laboratorio. Un altro metodo consolidato risiede nella loro organizzazione della cromatina: se macchiata di eventuali coloranti intercalanti del DNA (come Hoechst33342), il 70% degli ovociti antrali mostrano un anello colorante positivo attorno al nucleolo e sono chiamati Circondato Nucleolo (SN), mentre il restante 30% mostrare un segnali positivi più maculati e sono chiamate Non Circondato Nucleolo (NSN) 3-5. Recentemente abbiamo dimostrato che l&#39;assenza di reticoli citoplasmatici 9 (CPL, siti di stoccaggio importanti per mRNA di origine materna e ribosomi sia in ovociti di mammiferi ed embrioni) 10 e la down-regolazione di MATER (essenziale per CPL formazione 11), unitamente alla presenza di gocce lipidiche nei loro cytoplasms 9,12, possono essere altre cause legate alla ovociti incapacità NSN a procedere oltre la fase 2 cellule. Purtroppo, poche tecniche possono essere applicate per ordinare SN antrale e oociti NSN e,per questa ragione, lo sviluppo di un metodo meno invasivo (senza colorazione nucleare, le procedure di fissaggio o di manipolazione con pipette bocca) potrebbe diventare molto importante aumentare i tassi di entrambi sviluppo embrionale umano e animale. In questo lavoro, abbiamo dettaglio il protocollo semplice e veloce utilizzato per isolare annuncio sorta SN e NSN oociti antrali di topi di sesso femminile e si rivolge a tutti gli scienziati interessati allo studio della gametogenesi femminile, la maturazione degli ovociti e sviluppo embrionale.

<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.

Le seguenti immagini mostrano il metodo utilizzato per isolare oociti antrali da ovaie di topo, a partire dalle pipette preparazione alla selezione degli ovociti &#39;per mezzo di Hoechst33342. Questo metodo è molto semplice e può essere eseguita in qualsiasi laboratorio dotato di CO 2 incubatore, uno stereomicroscopio e un microscopio a fluorescenza equipaggiato con il filtro corretto per l&#39;osservazione del Hoechst33342 Figura 1 mostra la fase iniziale ...

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

Qui vi presentiamo un protocollo per la caratterizzazione di oociti antrali di topo sulla base di organizzazione nucleolare.

Isolamento e caratterizzazione di mouse Antral ovociti Sulla base Nucleolare cromatina Organizzazione

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 Views.  Fondazione IRCCS Policlinico San Matteo.  Qui vi presentiamo un protocollo per la caratterizzazione di oociti antrali di topo sulla base di organizzazione nucleolare. 

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 diffusa i preparativi per l'analisi della progressione di ovocita Mouse dalla profase alla metafase II

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

Ricerca

Genetica

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 i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi 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.

Analisi della segregazione dei cromosomi, acetilazione istonica e morfologia del mandrino nei cavalli ovociti

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

Biologia dello sviluppo

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.

Un'identificazione basato su rete neurale di ovociti adulto Mouse inerente allo sviluppo competente o incompetente

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.

Coltura del tessuto ovarico per visualizzare fenomeni nell'ovaia di 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.

Ablazione laser multi-fotone di centri di organizzazione di microtubuli citoplasmatici in ovociti di topo

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

Identificazione del danno al DNA negli ovociti arrestati con profase di topi

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.

Autore in primo piano: Comprendere la risposta al danno al DNA negli ovociti dei mammiferi e negli embrioni preimpianto 

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.

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

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.

Microiniezione degli ovociti mouse, Maturazione e Ploidia valutazione

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

Isolamento e caratterizzazione di mouse Antral ovociti Sulla base Nucleolare cromatina Organizzazione

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Cromatina diffusa i preparativi per l'analisi della progressione di ovocita Mouse dalla profase alla metafase II

Usando i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi I

Analisi della segregazione dei cromosomi, acetilazione istonica e morfologia del mandrino nei cavalli ovociti

Un'identificazione basato su rete neurale di ovociti adulto Mouse inerente allo sviluppo competente o incompetente

Coltura del tessuto ovarico per visualizzare fenomeni nell'ovaia di Mouse

Ablazione laser multi-fotone di centri di organizzazione di microtubuli citoplasmatici in ovociti di topo

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Rilevamento di rotture a doppio filamento di DNA in ovociti di topo

Microiniezione degli ovociti mouse, Maturazione e Ploidia valutazione