Name: isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization
Uploaded: 2016-01-07T12:00:01
Description: Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

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.

Denne protokol gennemføres i overensstemmelse med de ledende principper for europæisk (EU 63/2010) og italiensk (26/2014) love, der beskytter dyr, der anvendes til videnskabelig forskning. Cervikal dislokation eutanasi bruges til æggestokke isolering og det udføres af ekspert og uddannede personer, der er opført i den godkendte protokol, der dækker denne undersøgelse. 1. Dyr <ol><li> Bevar voksen 3- til 6 uger gamle hunmus i et rum med kontrolleret temperatur og fugtighed (med faser 12L: 12D og fodr...

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+oocytes+for+in+vitro+maturation+by+brilliant+cresyl+blue+staining:+a+study+using+the+mouse+model.">Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model.</a> Cell Res. 17, 722-731 (2007).</li><li>Opiela, Y., K&#261;tska-Ksi&#261;&#380;kiewicz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+Brilliant+Cresyl+Blue+(BCB)+staining+of+mammalian+oocytes+used+for+in+vitro+embryo+production+(IVP).">The utility of Brilliant Cresyl Blue (BCB) staining of mammalian oocytes used for in vitro embryo production (IVP).</a> Reproductive Biol. 13, 177-183 (2013).</li><li>Labrecque, R., Sirard, M. 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Denne protokol beskriver den anvendte metode til at isolere og klassificere musen antrale GV oocytter. Der er ingen særlige kritiske skridt til at understrege, med få undtagelser. Første: Denne procedure skal udføres så hurtigt som muligt til: a) at bevare vitaliteten af ​​oocytter og b) at undgå pH-ændringer i mediet anvendes. Andet: oocytter skal være kortvarigt ophidset (5-10 sek) til Hoechst33342 at undgå kromatin skader og efterfølgende død af oocytter, især hvis IVM og IVF er følgende trin. Til da...

Antrale fuldvoksne oocytter (GV, kimblære) isoleret fra antrale rum i æggestokkene rutinemæssigt anvendes til forskellige formål, såsom for eksempel undersøgelse af de molekylære og cellulære mekanismer bag in vitro modning Til Metafase II fase. På trods af deres smukke udseende mest antrale oocytter, men ikke alle, er i stand til at fuldføre meiose og nå blastocyst-stadiet; faktisk i mange pattedyrarter, herunder mennesker 1,2, ca. 30% af dem standse deres udvikling ved 2-celle stadiet, hvis befrugtning sker 3-5. Til dato anvendes få parametre til at skelne mellem oocytter stand til at opretholde den embryoniske udvikling fra dem i stand hertil. For eksempel Cresyl blåfarvning (BCB) er en gyldig metode til at sortere oocytter baseret på aktiviteten af ​​glucose-6-phosphat dehydrogenase skønt anvendeligheden af ​​denne sorteringsmetode relateret til BCB + eller BCB- farvningen erstadig laboratorium afhængige 6-8. En anden veletableret metode ligger i deres kromatin organisation: hvis farves med eventuelle DNA-interkalerende farvestoffer (som Hoechst33342), 70% af antrale oocytter viser et farvestof-positiv ring omkring nucleolus og kaldes Omgivet nucleolus (SN), mens de resterende 30% viser en mere plettet positive signaler og kaldes Ikke Omgivet nucleolus (NSN) 3-5. For nylig viste vi, at fraværet af cytoplasmatiske gitre 9 (cpls, vigtige lagringssteder for maternelle mRNA og ribosomer i både pattedyr oocyter og embryoner) 10 og nedregulering af MATER (afgørende for cpls dannelse 11), sammen med tilstedeværelsen af lipiddråber i deres cytoplasmaer 9,12, kan være andre årsager i forbindelse med NSN oocytter manglende evne til at gå ud over den 2-celle stadiet. Desværre kan få teknikker anvendes til at sortere antral SN og NSN oocyter ogaf denne grund, kan udviklingen af ​​en mindre invasiv metode (uden nuklear farvning, fikseringen eller manipulation med munden pipetter) bliver meget vigtigt at øge satserne for både udvikling af menneskelige og dyrs foster. I dette papir, vi detalje den enkle og hurtige protokol, der bruges til at isolere annonce sortere SN og NSN antrale oocytter fra hunmus, og den er rettet til alle de forskere interesseret i studiet af kvindelige gametogenese, oocytmodning og udvikling foster.

<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>&#181;-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.

De følgende billeder viser den anvendte metode til at isolere antrale oocytter fra mus æggestokke, startende fra forberedelse pipetter til oocytterne &#39;sortering ved hjælp af Hoechst33342. Denne metode er ganske enkel og kan udføres i alle laboratorier udstyret med en CO2-inkubator, et stereomikroskop og et fluorescensmikroskop udstyret med den korrekte filter til observation af Hoechst33342 Figur 1 viser det indledende trin af pipette præparatet:. Som...

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

Her præsenterer vi en protokol til karakterisering af muse antral oocytter baseret på nucleolar organisation.

Isolering og karakterisering af muse Antral Oocyter Baseret på 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 ...

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