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.

Dieses Protokoll wird in Übereinstimmung mit den Leitlinien der europäischen (EU 63/2010) und Italienisch (26/2014) Gesetze zum Schutz von Tieren für die wissenschaftliche Forschung verwendet werden, durchgeführt. Genickbruch der Euthanasie ist für Eierstöcke Isolierung verwendet und es wird von Experten durchgeführt und ausgebildeten Personen in der genehmigten Protokoll über diese Studie aufgeführt. 1. Tiere <ol><li> Pflegen Erwachsenen 3- bis 6 Wochen alte weibliche Mäuse in einem Raum mit kontrollierter Temperatur und Feuchtigkeit (mit Phasen der 12L: 12D und ad libitum gefüttert). </li><li> Spritzen Sie Mäusen intraperitoneal mit 2,5 IU pregnant mare serum gonadotropin (PMSG) bis Follikelwachstum durch Nachahmung der Wirkungen von follikelstimulierendem Hormon (FSH) zu stimulieren. Isolieren Sie die Eierstöcke für antralen Oozyten Sammlung von 48 Stunden später. Euthanize die weibliche Maus zuvor mit PMSG injiziert, nach Ihren Richtlinien des Instituts. </li><li> Schnelles Entfernen der Eierstöcke aus der Körperhöhle und legen Sie sie in die Petrischale containing M2 Medium für spätere Oozyten Isolierung (siehe Abschnitt 3). <ol><li> Einen Einschnitt in der Haut, welche die Bauchwand, gefolgt von einem Einschnitt in der Bauchfells unter Verwendung sterilen Sektions Schere. Öffnen Sie die Schnittführung, um den Bauch freizulegen, bewegen Sie den Mut, auf der einen Seite mit einer sterilen Pinzette und lokalisieren die Eileiter und bilden eine V-Form, ausgehend von der Blase. </li><li> Beachten Sie die Eierstöcke unterhalb der Nieren entfernt. Halten Sie jede Röhre mit steriler Pinzette und machen einen kleinen Schnitt an der Unterseite der Eierstöcke, um sie freizugeben. Übertragen beide Ovarien am Boden einer Petrischale mit voräquilibriert Medium. </li></ol></li></ol> 2. Hormonpräparat <ol><li> Verdünnen Sie PMSG (in verschiedenen Aktien Konzentrationen) mit steriler isotonischer Kochsalzlösung, um eine Arbeitskonzentration von 2,5 IU Hormon pro 0,1 ml für eine geeignete Injektionsvolumen zu erhalten. HINWEIS: Hormon-Lösungen müssen in 0,5 ml Röhrchen aliquotiert und gelagert bei -20 ° C bis zum Tag der Verwendung. Bewahren Sie keine verdünnten Aliquots länger als drei Monate bei -20 ° C. Einmal aufgetaut, wenn serielle Injektionen durchgeführt werden, speichern die Teilmengen von Hormon-Lösungen bei +4 ° C und verwenden Sie sie innerhalb von 4 Tagen. </li></ol> 3. Die Eizellen Antrum Isolation Für die richtige Oozyten Trennung: <ol><li> Äquilibrieren M2 Medium 13 (M2) bei 5% CO 2 und 37 ° C für mindestens 1 Stunde vor dem Beginn der Oozyten Isolierungsverfahren. Bereiten zwei Petrischalen (35 mm x 10 mm), eines mit 1 ml M2 für Ovar Punktierung und die zweite mit kleinen Tropfen (20 ul) von M2 mit Mineralöl (~ 1,5 ml) für Oozyten Übertragung nach Isolierung aus der überdachten Eierstock (siehe 3,4-3,6). Halten Sie beide Petrischalen im Inkubator bei 5% CO 2 und 37 ° C bis zur Verwendung bereit. </li><li> Verwenden Sie ein Stereomikroskop bei Oozyten Isolierungsverfahren. </li><li> Bereiten dünnen Mundglaspipetten, um die Eizellen mit Strechfolie sammelnHing Glas Pasteurpipetten horizontal gehalten (Halten der Enden der Pipette mit beiden Händen), über eine vertikale Flamme aus dem Bunsenbrenner (Abbildung 1). <ol><li> Sobald das Glas zu schmelzen beginnt, nehmen Sie die Pipette aus der Flamme, und führen Sie einen schnellen Zugbewegung um das gewünschte Pipette zu erhalten. Fest schneiden das überschüssige Glas in der Nähe der Engstelle der Pipette mit den Fingernägeln, die Länge und der Durchmesser der inneren Kapillare einzustellen. Sicherzustellen, dass die dünne Kapillare einen Innendurchmesser von ~ 100 &amp; mgr; m, der etwas größer als der Durchmesser der Eizelle (~ 80 &amp; mgr; m). </li><li> Zu-Mund-Pipette, schließen Sie ein Ende des Ansaugrohr auf die Pipette gezogen wird unter Verwendung einer geeigneten Spitze und das andere Ende in den Mund, befestigen Sie sie mit den Zähnen. </li></ol></li><li> Sammeln der Eierstöcke mit sterilen Pinzetten und legen sie an der Unterseite eines Zellkulturpetrischale (35 mm x 10 mm). Deckt sie mit 1 ml M2 zuvor bei 37 ° C äquilibriertund 5% CO 2 (Abbildung 2). </li><li> Sanft durchstechen die Antralfollikeln der Eierstöcke mit einem sterilen Insulinspritze Nadel antralen Oozyten in M2 (Figur 3) zu lösen. </li><li> Gently Mund pipettieren die Eizellen durch eine enge Pasteur-Pipette, um Follikelzellen und andere mögliche Gewebetrümmer (4A) zu entfernen und setzen sie an der Unterseite der kleinen Tropfen (20 ul) von M2 mit Mineralöl für Embryokultur überzogen (vorher dann hergestellt; 4B). HINWEIS: Es ist wichtig, alle Cumuluszellen an die Zona pellucida der Eizellen durch kontinuierliches Pipettieren angebracht über mehrere und unterschiedliche Tropfen M2 entfernen. Führen Sie diesen Schritt so schnell wie möglich, um Veränderungen im pH des Mediums zu vermeiden. </li></ol> 4. Antral Oozyten Färbung mit Hoechst33342 HINWEIS: Antral Eizellen können in SN (Umgeben Nucleolus), NSN (Nicht Umgeben Nucleolus) oder unterschieden werdenin einer anderen Zwischenform auf der Grundlage ihrer Chromatinorganisation nach Färbung mit Supravital Konzentration Hoechst33342 (oder andere spezifische Marker für Basen AT) 14. In der Tat zeigen die SN Oozyten eine Hoechst-positive Ring um den Nukleolus, während der NSN nicht, daher der Name. Für die richtige Oozyten-Färbung: <ol><li> Übertragen die Oozyten mit einer Mundpipette in Tropfen Hoechst33342 (20 ul) in M2 verdünnt im supravitalen Konzentration von 50 ng / &amp; mgr; l für 10 Minuten im Dunkeln. </li><li> Nach der Inkubation mit Hoechst33342 Transfer jeder einzelnen Oocyte mit einem Mundpipette an der Unterseite der kleinen Tropfen (5 ul) von M2 mit Mineralöl bedeckt, zum Sortieren basierend auf Chromatinorganisation (SN oder NSN). Verwenden Sie eine beliebige Fluoreszenzmikroskop mit Hoechst33342 Filter (Emissionswellenlänge 486 nm) ausgestattet, um die Chromatin-Konfiguration der Eizellen zu visualisieren. HINWEIS: In Abhängigkeit von der Art des Fluoreszenzmikroskopes, it kann sich zwingend notwendig, um Glasboden Petrischale für die Bildaufnahme verwenden. Die konfokale Mikroskopie Olympus FluoView Fv10i, wenn mit einem Probenhalter für 35 mm-Schale ausgestattet benötigt nur Glasboden-Gerichte für die Bilderfassung, mit einer 0,17 mm Standard-Dicke und einem Bereich von 0,13 bis 0,21 mm akzeptabel. </li><li> Erregt die Oozyten auf nicht mehr als 5-10 sec, wahrscheinlich genügend Zeit, um die Anwesenheit oder die Abwesenheit von einem Ring um den Nukleolus (Abbildung 5) zu visualisieren. </li><li> Nach dem Sortieren, der Kultur der antralen SN und NSN Oozyten in M2 bei 5% CO 2 und 37 ° C, um in vitro-Reifung induzieren oder halten sie in geeigneten Puffern für die molekulare oder zelluläre Analysen. </li></ol>

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The authors have nothing to disclose

Dieses Protokoll beschreibt die verwendet werden, um zu isolieren und zu klassifizieren Maus antralen GV-Oozyten-Methode. Es gibt keine besonderen kritischen Schritte, um zu unterstreichen, mit wenigen Ausnahmen. Erstens: a) Erhaltung der Vitalität der Oozyten und b) zu vermeiden, den pH-Wert des verwendeten Mediums: Dieses Verfahren sollte so schnell wie möglich durchgeführt werden. Zweitens: Eizellen sollten kurz angeregt werden (5-10 sec), um Hoechst33342 Chromatin Schäden und der anschließenden Tod der Eizellen...

Antralen ausgewachsene Oocyten (GV, Keimbläschen) aus dem Antrum Fach der Ovarien isoliert werden routinemäßig für verschiedene Zwecke verwendet, wie zum Beispiel die Untersuchung der molekularen und zellulären Mechanismen, die in vitro-Reifung bis Metaphase II Stufe. Trotz ihres schönen Aussehens meisten antralen Eizellen, aber nicht alle sind in der Lage, um die Meiose zu vervollständigen und erreichen das Blastozystenstadium; in der Tat, in vielen Säugetierspezies, einschließlich Menschen, 1,2, etwa 30% von ihnen verhaften ihre Entwicklung in der 2-Zell-Stadium, wenn die Befruchtung stattfindet 3-5. Bisher sind nur wenige Parameter verwendet werden, um zwischen den Oozyten der Lage, die Embryonalentwicklung von denen nicht in der Lage, dies zu tun aufrecht zu unterscheiden. Zum Beispiel ist Cresyl-Blau-Färbung (BCB) einen gültigen Methode die Oozyten basierend auf der Aktivität der Glucose-6-phosphat-dehydrogenase zu sortieren, obwohl der Nutzen dieses Sortierverfahren den BCB + oder BCB- Färbung zusammenhängtnoch Labor abhängig 6-8. Eine andere gut etablierte Methode liegt in ihrer Chromatinorganisation: Wenn bei allen DNA interkalierende Farbstoffe (wie Hoechst33342) gefärbt, 70% der antralen Oozyten zeigen eine farbstoff positive Ring um den Nukleolus und sind umgeben Nucleolus (SN) aufgerufen wird, während die restlichen 30% zeigen eine gefleckte positive Signale und werden als Nicht Umgeben Nucleolus (NSN) 3-5. Kürzlich haben wir gezeigt, dass das Fehlen von zytoplasmatischen Gittern 9 (CPLs, wichtige Lagerstätten für maternalen mRNA und Ribosom in beiden Säugetier Eizellen und Embryonen) 10 und der Herunterregulierung der MATER (wichtig für CPLs Bildung 11) zusammen mit der Anwesenheit von Lipidtröpfchen in ihrem Zytoplasma 9,12, können andere Ursachen auf den NSN Oozyten Unfähigkeit, die auf über die 2-Zell-Stadium fortfahren. Leider können einige Techniken angewendet, um antralen SN und NSN Eizellen und sortiert werden,Aus diesem Grund könnte die Entwicklung einer weniger invasiven Verfahren (ohne Kernfärbung, der Fixierung oder Manipulation mit Mund-Pipetten) sehr wichtig geworden, um die Raten der menschlichen und tierischen Embryo-Entwicklung zu erhöhen. In diesem Papier haben wir ausführlich die einfache und schnelle Protokoll für die Anzeige Art SN und NSN antralen Eizellen von weiblichen Mäusen zu isolieren, und es wird an alle Wissenschaftler interessieren sich für das Studium der weiblichen Gametogenese, Eizellreifung und Embryoentwicklung gerichtet.

<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.

Die folgenden Bilder zeigen die verwendet werden, um antralen Eizellen aus den Eierstöcken der Maus zu isolieren, ausgehend von Pipetten Zubereitung der Oozyten &quot;Sortierung mittels Hoechst33342 Methode. Diese Methode ist sehr einfach und kann in jedem Labor mit einem CO 2 -Inkubator, einem Stereomikroskop und einem Fluoreszenzmikroskop mit dem korrekten Filter für die Beobachtung des Hoechst33342 ausgestattet erfolgen Abbildung 1 zeigt den Anfangsschrit...

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

Hier präsentieren wir ein Protokoll für die Charakterisierung von Maus antralen Oozyten basierend auf nucleolar Organisation.

Isolierung und Charakterisierung von Maus Antral Oozyten Basierend auf Nucleolar Chromatinorganisation

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 Aufrufe.  Fondazione IRCCS Policlinico San Matteo.  Hier präsentieren wir ein Protokoll für die Charakterisierung von Maus antralen Oozyten basierend auf nucleolar Organisation. 

Serie: 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.

Chromatin verteilt die Vorbereitungen für die Analyse der Maus Eizelle Progression von Prophase, Metaphase II

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

Forschung

Genetik

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.

Mit Maus Eizellen menschliche Gen-Funktion während der Meiose zu beurteilen ich

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.

Analyse der Chromosomensegregation, Histonacetylierung und Spindelmorphologie in Pferdeozyten

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

Entwicklungsbiologie

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.

Eine neuronale Netzwerk-basierte Identifikation der entwicklungsgeschichtlich zuständigen oder inkompetent Maus ausgewachsene Eizellen

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.

Ovarian Tissue Culture Phänomene in der Maus Eierstock zu visualisieren

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.

Multiphotonen-Laserablation von zytoplasmatischen Mikrotubuli-Organisationszentren in Maus-Eizellen

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

Identifizierung von DNA-Schäden in prophase-arrestierten Eizellen von Mäusen

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 im Rampenlicht: Verständnis der DNA-Schadensreaktion in Säugetier-Eizellen und Präimplantationsembryonen 

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.

Nachweis von DNA-Doppelstrangbrüchen in Maus-Eizellen

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.

Maus Oocyte Mikroinjektion, Reifung und Ploidie Beurteilung

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

Isolierung und Charakterisierung von Maus Antral Oozyten Basierend auf Nucleolar Chromatinorganisation

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