mouse antral oocyte isolation: a method to isolate antral oocytes from freshly harvested mouse ovaries

Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries

To harvest the ovaries, begin by injecting 3- to 6-week-old female mice IP with 2.5 international units of pregnant mare serum gonadotropin. Two days later, about an hour before the harvest, fill one sterile Petri dish with M2 medium and add several 20-microliter drops of M2 medium to a second Petri dish, covering each drop with 1.5 milliliters of mineral oil.
Then, transfer all of the Petri dishes to a 37 degrees Celsius, 5% carbon dioxide incubator to allow the medium to equilibrate. Next, use sterile dissection scissors to make an incision in the skin covering the abdominal wall, followed by an incision in the peritoneum of the first hormone-injected mouse.
Use sterile tweezers to move the intestines aside to localize the uterine tubes, which form a V-shape starting from the bladder. After locating the ovaries below the kidneys, grasp one uterine tube and release the corresponding ovary with a small incision in the bottom of the tissue.
Repeat the process to harvest the other ovary and then place both ovaries in the Petri dish filled with pre-equilibrated M2 medium. Before isolating the antral oocyte, make glass mouth pipettes by grasping both ends of a glass Pasteur pipette and holding the pipette horizontally over a Bunsen burner flame.
When the glass begins to melt, remove the pipette from the flame and quickly pull the instrument apart. Using a fingernail, firmly snip the excess glass close to the narrow point of the pipette to shorten the tip and to achieve an internal capillary diameter of about 100 microns.
It&#39;s important to have the right pipette diameter; if it&#39;s too narrow, the oocytes will be stuck, fragmented, and die.
Then, connect the unmodified end of the pipette to an aspirator tube. Next, transfer the ovaries into the Petri dish containing 1 milliliter of pre-equilibrated M2 medium and use a sterile insulin syringe needle to gently puncture the antral follicles of the ovaries.
As the oocytes are released, gently collect them by mouth pipette, taking care not to aspirate any follicle cells or other tissue debris. Then, quickly pipette each oocyte through multiple pre-equilibrated M2 medium drops to remove all of the cumulus cells attached to the zona pellucida of the oocytes. Once the oocytes have been denuded, carefully settle the oocytes at the bottom of individual drops of fresh equilibrated M2 medium.

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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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&#160;ad libitum).</li>
	<li>Inject mice intraperitoneally with 2.5 IU of Pregnant mare serum gonadotropin (PMSG) to stimulate follicular growth by mimicking the effects of follicle stimulating hormone (FSH). Isolate the ovaries for antral oocytes collection 48 h later. Euthanize the female mouse previously injected with PMSG, as per standard guidelines.</li>
	<li>Quickly remove the ovaries from the body cavity and place them into the Petri dish containing M2 medium for subsequent oocytes isolation (see section 3).
	<ol>
		<li>Make an incision on the skin covering the abdominal wall followed by an incision in the peritoneum by using sterile dissection scissors. Open the incision to expose the abdomen, move the guts on one side using sterile tweezers and localize the uterine tubes, forming a V-shape starting from the bladder.</li>
		<li>Observe the ovaries located below the kidneys. Hold each tube with sterile tweezer and make a small incision at the bottom of the ovaries to release them. Transfer both ovaries at the bottom of a Petri dish containing pre-equilibrated medium.</li>
	</ol>
	</li>
</ol>
2. Hormone Preparation
<ol>
	<li>Dilute PMSG (available in different stock concentrations) with sterile isotonic saline solution to obtain a working concentration of 2.5 I.U. hormone per 0.1 mL for an appropriate injection volume.&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Hormone solutions must be aliquoted in 0.5 mL tubes and stored at -20 &#176;C until the day of use. Do not keep diluted aliquots longer than three months at -20 &#176;C. Once thawed, if serial injections are performed, store the aliquots of hormone solutions at +4 &#176;C and use them within 4 days.</li>
</ol>
3. Antral Oocytes Isolation
For proper oocytes isolation:
<ol>
	<li>Equilibrate M2 medium&#160;(M2) at 5% CO2&#160;and 37 &#176;C for at least 1 h before starting the oocytes isolation procedure. Prepare two Petri dishes (35 mm x 10 mm), one with 1 mL of M2 for ovary puncturing and the second one with small drops (20 &#181;L) of M2 covered with mineral oil (~1.5 mL) for oocytes transfer after isolation from the ovary (see steps 3.4&#8211;3.6). Keep both Petri dishes in the incubator at 5% CO2&#160;and 37 &#176;C until ready to be used.</li>
	<li>Use a stereomicroscope during oocytes isolation procedures.</li>
	<li>Prepare thin mouth glass pipettes to collect the oocytes by stretching glass Pasteur pipettes kept horizontally (holding the ends of the pipette with both hands), over a vertical flame coming from the Bunsen burner (Figure 1).
	<ol>
		<li>Once the glass begins to melt, take the pipet out of the flame, and perform a quick pulling movement to obtain the desired pipette. Firmly cut the excess glass close to the narrow point of the pipette with fingernails to adjust the length and the diameter of the internal capillary. Ensure that the thin capillary has an internal diameter of ~100 &#181;m, slightly greater than the oocyte&#39;s diameter (~80 &#181;m).</li>
		<li>To mouth pipette, connect one end of the aspirator tube to the pulled pipette by using an appropriate tip and place the other end in the mouth, securing it with the teeth.</li>
	</ol>
	</li>
	<li>Collect the ovaries using sterile tweezers and deposit them at the bottom of a cell culture Petri dish (35 mm x 10 mm). Cover them with 1 mL of M2 previously equilibrated at 37 &#176;C and 5% CO2&#160;(Figure 2).</li>
	<li>Gently puncture the antral follicles of the ovaries with a sterile insulin syringe needle to release antral oocytes in M2 (Figure 3).</li>
	<li>Gently mouth pipet the oocytes through a narrow Pasteur pipette to remove follicle cells and any other possible tissue debris (Figure 4A) and then settle them at the bottom of small drops (20 &#181;L) of M2 covered with mineral oil suitable for embryo culture (previously prepared;&#160;Figure 4B).&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: It is important to remove all the cumulus cells attached to the zona pellucida of the oocytes by continuous pipetting through several and different drops of M2. Perform this step as quick as possible to avoid alterations in the pH of the medium.</li>
</ol>

<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>Mineral Oil</td><td>Sigma</td><td>M8410</td><td></td></tr><tr><td>Cell Culture Dish 35 mm x 10 mm</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>&amp;micro;-Dish 35 mm, 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>

Source: Monti, M. et al. <a href="https://www.jove.com/video/53616" target="_blank">Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization</a>. J. Vis. Exp. (2016)
This video describes the isolation of antral oocytes from freshly harvested mouse ovaries. These antral oocytes can be used for further characterization and developmental studies.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20757/20757_Figure1.jpg" /> 
Figure 1. Preparation of a mouth pipette using glass Pasteur pipettes and a Bunsen burner.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20757/20757_Figure2.jpg" /> 
Figure 2. Intact ovaries in Petri dish filled with pre-warmed and equilibrated M2 medium.</stron...

Source: Monti, M. et al. Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization. J. Vis. Exp. (2016)
This video ...

In a mammalian ovary, fully grown oocytes derived from the antral follicles only need a short period of maturation in culture, a crucial factor for in vitro embryogenesis. These oocytes are surrounded by a cluster of somatic cells, which must be removed through denudation to facilitate sperm injection.
To isolate denuded mouse antral oocytes, begin with an anesthetized female mouse pre-injected with pregnant mare serum gonadotropin hormone to stimulate follicular growth. Prep the mouse and incise the lower abdomen.
Exteriorize the intestine to reveal ovaries at the top of V-shaped uterine tubes. Dissect an ovary. Transfer it to a pre-equilibrated culture medium to maintain the viability of oocytes. Under a stereomicroscope, identify the antral follicles by their distinctive fluid-filled cavity.
Using a sterile needle, puncture the antral follicles of the ovary to release oocytes. Using a pipette of an optimum diameter, carefully aspirate the oocytes to protect them from mechanical damage. Avoid aspirating follicular cells and other tissue debris.
Subsequently, transfer the oocytes into multiple media drops covered with mineral oil. This step helps remove the surrounding somatic cells and generate denuded oocytes. Lastly, transfer the denuded oocytes into fresh media drops with mineral oil and preserve them for further experiments.

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Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries

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Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries