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.

This protocol is conducted in accordance with the guiding principles of European (EU 63/2010) and Italian (26/2014) laws protecting animals used for scientific research. Cervical dislocation euthanasia is used for ovaries isolation and it is performed by expert and trained individuals listed in the approved protocol covering this study.&#160;
1. Animals
<ol>
	<li>Maintain adult 3- to 6-week-old female mice in a room with controlled temperature and humidity (with phases of 12L:12D and fed ad libitum).</l...

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This protocol describes the method used to isolate and classify mouse antral GV oocytes. There are no particular critical steps to underline, with few exceptions. First: this procedure should be performed as quickly as possible to: a) maintain the vitality of the oocytes and b) avoid pH changes in the medium used. Second: oocytes should be briefly excited (5-10 sec) to Hoechst33342 to avoid chromatin damage and the subsequent death of the oocytes, especially if IVM and IVF are the following steps. To date, this is the qu...

Antral fully grown oocytes (GV, germinal vesicle) isolated from the antral compartment of the ovary are routinely used for different purposes such as, for example, the study of the molecular and cellular mechanisms behind in vitro maturation till Metaphase II stage.
Despite their beautiful appearance most antral oocytes, but not all, are able to complete meiosis and reach the blastocyst stage; in fact, in many mammalian species, including humans1,2, roughly 30% of them arrest their development at the 2-cell stage, if fertilization occurs3-5. To date, few parameters are used to distinguish between oocytes able to sustain the embryonic development from those unable to do so.
For example, Cresyl blue staining (BCB) is a valid method to sort the oocytes based on the activity of glucose-6-phosphate-dehydrogenase although the utility of this sorting method related to BCB+ or BCB- staining is still laboratory dependent6-8.
Another well-established method resides in their chromatin organization: if stained with any DNA intercalating dyes (like Hoechst33342), 70% of antral oocytes show a dye-positive ring around the nucleolus and are called Surrounded Nucleolus (SN) while the remaining 30% show a more spotted positive signals and are called Not Surrounded Nucleolus (NSN)3-5. Recently we showed that the absence of cytoplasmic lattices9 (CPLs, important storage sites for maternally derived mRNA and ribosomes in both mammalian oocytes and embryos)10 and the down-regulation of MATER (essential for CPLs formation11), together with the presence of lipid droplets in their cytoplasms9,12, can be other causes related to the NSN oocytes inability to proceed beyond the 2-cell stage.
Unfortunately, few techniques can be applied to sort antral SN and NSN oocytes and, for this reason, the development of a less-invasive method (without nuclear staining, fixation procedures or manipulation with mouth pipettes) could become very important to increase the rates of both human and animal embryo development.
In this paper, we detail the simple and quick protocol used to isolate ad sort SN and NSN antral oocytes from female mice and it is addressed to all the scientists interested in the study of female gametogenesis, oocyte maturation and embryo development.

<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.

The following pictures show the method used to isolate antral oocytes from mouse ovaries, starting from pipettes preparation to the oocytes&#39; sorting by means of Hoechst33342. This method is quite simple and can be performed in any laboratory equipped with a CO2 incubator, a stereomicroscope and a fluorescence microscope equipped with the correct filter for the observation of the Hoechst33342. Figure 1 shows the initial step of pipette preparation: as the gl...

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

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar 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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