Name: Video: Mouse Antral Oocyte Isolation: A Method to Isolate Antral Oocytes from Freshly Harvested Mouse Ovaries - Concept
Uploaded: 2023-04-30T14:50:20.000+00:00
Duration: 1 min 51 s
Description: 1.7K Views. Source: Monti, M. et al. Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization. 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.

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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>

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

Source: Monti, M. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20757/20757_Figure3.jpg" /> 
Figure 3. Initial isolation of antral oocytes from antral follicles by means of a sterile needle.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20757/20757_Figure4.jpg" /> 
Figure 4. Transfer of the freshly isolated oocytes with a mouth pipette from the Petri dish (A) to a different Petri dish containing small drops of pre-warmed and equilibrated M2 medium (B).

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

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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1.7K Views. Source: Monti, M. et al. Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization. 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. 

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

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.

Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus ...

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Developmental Biology

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

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Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...

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

Transcriptie

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

A Neural Network-Based Identification of Developmentally Competent or Incompetent Mouse Fully-Grown Oocytes

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

Fertility Preservation in Patients with Severe Ovarian Dysfunction