Name: Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization (Video) | JoVE Journal
Uploaded: 2016-01-07T12:00:01.000+00:00
Duration: 7 min 16 s
Description: Learn to characterize mouse antral oocytes by nucleolar chromatin organization using Hoechst 33342 staining. This method distinguishes SN and NSN oocyte types for developmental biology research.

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5 minutes

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).</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 48hrs later. Euthanize the female mouse previously injected with PMSG, according to your institutional 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. 
	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 medium13 (M2) at 5% CO2 and 37 &#176;C for at least 1 hr 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 3.4-3.6). Keep both petri dishes in the incubator at 5% CO2 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 (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; Figure 4B). 
	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>
4. Antral Oocytes Staining with Hoechst33342
NOTE: Antral oocytes can be distinguished in SN (Surrounded Nucleolus), NSN (Not Surrounded Nucleolus) or in any other intermediate form, based on their chromatin organization after staining with supravital concentration of Hoechst33342 (or any other markers specific for bases A-T)14. In fact, the SN oocytes show a Hoechst-positive ring around the nucleolus while the NSN do not, hence its name.
For proper oocytes staining:
<ol>
	<li>Transfer the oocytes using a mouth pipette into drops of Hoechst33342 (20 &#181;l) diluted in M2 at the supravital concentration of 50 ng/&#181;l for 10 min&#160;in dark.</li>
	<li>After the incubation with Hoechst33342, transfer each single oocyte with a mouth pipette at the bottom of small drops (5 &#181;l) of M2 covered with mineral oil, for sorting based on chromatin organization (SN or NSN). Use any fluorescence microscope equipped with Hoechst33342 filter (emission wavelength 486 nm) to visualize the chromatin configuration of the oocytes. 
	NOTE: Depending on the type of fluorescence microscope used, it may become imperative to use glass bottom petri dish for image acquisition. The confocal microscopy Olympus Fluoview Fv10i when equipped with a specimen holder for 35 mm dish requires only glass bottom-dishes for image acquisition, with a 0.17 mm standard thickness and a range from 0.13 to 0.21 mm acceptable.</li>
	<li>Excite the oocytes for no longer than 5-10 sec, likely enough time to visualize the presence or the absence of a ring around the nucleolus (Figure 5).</li>
	<li>After sorting, culture the antral SN and NSN oocytes in M2 at 5% CO2 and 37 &#176;C to induce in vitro maturation or keep them in appropriate buffers for molecular or cellular analyses.</li>
</ol>

<ol><li>Parfenov, V., Potchukalina, G., Dudina, L., Kostyuchek, D., Gruzova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+antral+follicles%3A+oocyte+nucleus+and+the+karyosphere+formation+%28electron+microscopic+and+autoradiographic+data%29%5BTitle%5D)+AND+%22Gamete+Res%22%5BJournal%5D)">Human antral follicles: oocyte nucleus and the karyosphere formation (electron microscopic and autoradiographic data).</a> Gamete Res. 22 (2), 219-231 (1989).</li>
<li>Anderiesz, C., Ferraretti, A. P., Magli, C., Fiorentino, A., Fortini, D., Gianaroli, L., Jones, G. M., Trounson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+recombinant+human+gonadotropins+on+human%2C+bovine+and+murine+oocyte+meiosis%2C+fertilization+and+embryonic+development+in+vitro%5BTitle%5D)+AND+%22Human+Reprod%22%5BJournal%5D)">Effect of recombinant human gonadotropins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro.</a> Human Reprod. 15 (5), 1140-1148 (2000).</li>
<li>Mattson, B. A., Albertini, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oogenesis%3A+Chromatin+and+microtubule+dynamics+during+meiotic+prophase%5BTitle%5D)+AND+%22Mol.+Reprod.+Dev%22%5BJournal%5D)">Oogenesis: Chromatin and microtubule dynamics during meiotic prophase.</a> Mol. Reprod. Dev. 25 (4), 374-383 (1990).</li>
<li>Debey, P., Szollosi, M. S., Szollosi, D., Vautier, D., Girousse, A., Besomber, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Competent+mouse+oocytes+isolated+from+antral+follicles+exhibit+different+chromatin+organization+and+follow+different+maturation+dynamics%5BTitle%5D)+AND+%22Mol.+Reprod.+Dev%22%5BJournal%5D)">Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.</a> Mol. Reprod. Dev. 36 (1), 59-74 (1993).</li>
<li>Zuccotti, M., Piccinelli, A., Giorgi Rossi, P., Garagna, S., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+organisation+during+mouse+oocyte+growth%5BTitle%5D)+AND+%22Mol.+Reprod.+Dev%22%5BJournal%5D)">Chromatin organisation during mouse oocyte growth.</a> Mol. Reprod. Dev. 41 (1-2), 479-485 (1995).</li>
<li>Wu, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selection+of+oocytes+for+in+vitro+maturation+by+brilliant+cresyl+blue+staining%3A+a+study+using+the+mouse+model%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">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., Kątska-Książkiewicz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+utility+of+Brilliant+Cresyl+Blue+%28BCB%29+staining+of+mammalian+oocytes+used+for+in+vitro+embryo+production+%28IVP%29%5BTitle%5D)+AND+%22Reproductive+Biol%22%5BJournal%5D)">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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+study+of+mammalian+oocyte+competence+by+transcriptome+analysis%3A+progress+and+challenges%5BTitle%5D)+AND+%22Mol.+Human+Reprod%22%5BJournal%5D)">The study of mammalian oocyte competence by transcriptome analysis: progress and challenges.</a> Mol. Human Reprod. 20 (2), 103-116 (2014).</li>
<li>Monti, M., Zanoni, M., Calligaro, A., Ko, M. S., Mauri, P. L., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+arrest+and+mouse+antral+Not-Surrouded+Nucleolus+oocyte%5BTitle%5D)+AND+%22Biol.+Reprod%22%5BJournal%5D)">Developmental arrest and mouse antral Not-Surrouded Nucleolus oocyte.</a> Biol. Reprod. 88 (1), 1-7 (2013).</li>
<li>Yurttas, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+for+PADI6+and+the+cytoplasmic+lattices+in+ribosomal+storage+in+oocytes+and+translational+control+in+the+early+mouse+embryo%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Role for PADI6 and the cytoplasmic lattices in ribosomal storage in oocytes and translational control in the early mouse embryo.</a> Development. 135 (15), 2627-2636 (2008).</li>
<li>Li, L., Baibakov, B., Dean, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+subcortical+maternal+complex+essential+for+preimplantation+mouse+embryogenesis%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">A subcortical maternal complex essential for preimplantation mouse embryogenesis.</a> Dev. Cell. 15 (3), 416-425 (2008).</li>
<li>Ami, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FT-IR+spectra+signatures+of+mouse+antral+oocytes%3A+molecular+markers+of+oocyte+maturation+and+developmental+competence%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">FT-IR spectra signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence.</a> Biochim. Biophys. Acta. 1813 (6), 1220-1222 (2011).</li>
<li>Fulton, B. P., Whittingam, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+mammalian+oocytes+by+intercellular+injection+of+calcium%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Activation of mammalian oocytes by intercellular injection of calcium.</a> Nature. 273 (5658), 149-151 (1978).</li>
<li>Luttmer, S. J., Longo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Examination+of+living+and+fixed+gametes+and+early+embryos+stained+with+supravital+fluorochromes+%28Hoechst+33342+and+3%2C3%E2%80%B2-dihexyloxacarbocyanine+iodide%29%5BTitle%5D)+AND+%22Gamete+Res%22%5BJournal%5D)">Examination of living and fixed gametes and early embryos stained with supravital fluorochromes (Hoechst 33342 and 3,3′-dihexyloxacarbocyanine iodide).</a> Gamete Res. 15 (3), 267-283 (1986).</li>
<li>Monti, M., Redi, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+biopolitics+of+frozen+embryos%5BTitle%5D)+AND+%22Int.+J.+Dev.+Biol%22%5BJournal%5D)">The biopolitics of frozen embryos.</a> Int. J. Dev. Biol. 55, 243-247 (2011).</li>
</ol>

The authors have nothing to disclose

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>&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

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' 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 glass ...

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 ...

The overall goal of this procedure is to detail all of the steps necessary for characterizing mouse antral oocytes based on their chromatin organization, according to their ability to fully sustain a prospective embryonic development. This method can help answer key questions in the field of developmental biology, such as how to distinguish and isolate two different kinds of antral ooyctes, SN, surrounded nucleolus, and NSN, non-surrounded nucleolus, residing in the antral compartment of the mammalian ovary. To harvest the ovaries, begin by injecting three to six 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 micriliter 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 degree 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's important to have the right pipette diameter. If it's too narrow, the ooyctes 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 one 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. To stain the oocytes, use a mouth pipette to transfer the cells into 20 microliter drops of Hoechst 33342, diluted in M2 medium. Then transfer the oocytes into a new Petri dish containing five microliter drops of M2 medium covered with mineral oil, taking care to settle the oocytes individually at the bottom of each drop.When all of the oocytes have been transferred, place the dish under the fluorescent microscope and excite the cells just long enough to visualize the presence or absence of a ring around the nucleolus. Take care not to overexpose the oocytes, as the chromatin can be damaged and the oocytes can die if they are exposed too long. After the SN and NSN oocytes have been identified, sort the cells into the appropriate corresponding Petri dish in the relevant reagent for the desired downstream analysis, according to their chromatin organization.Hoechst 33342 staining for chromatin organization is probably the most critical step of the procedure. Five to 10 seconds of excitation should be long enough to sort the oocytes based on the presence or absence of chromatin surrounding the nucleolus of each oocyte. Once mastered, this technique can be completed in less than three hours if performed properly.While attempting this procedure, it is important to work at room temperature and to remember to treat both ovaries and oocytes gently. Following this procedure, the oocytes can then be used for in vitro maturation and in vitro fertilization. After its development, this technique paved the way for researchers in the field of developmental biology to answer to a still-open question, how to recognize and to distinguish between two kinds of antral oocytes residing in the mammalian ovaries.After watching this video, you will have a good understanding on how to make and use your own mouth pipette, how to isolate and sort antral oocytes by means of Hoechst 33342 staining. Don't forget that working with animal and cells requires special precautions, like wearing your personal protective equipment and using sterilized instruments.

isolation-characterization-mouse-antral-oocytes-based-on-nucleolar

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Learn to characterize mouse antral oocytes by nucleolar chromatin organization using Hoechst 33342 staining. This method distinguishes SN and NSN oocyte types for developmental biology research.

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

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