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 quickest way to sort SN and NSN mouse antral oocytes.
Hochest33342 has been proven to be an efficient way to characterize SN and NSN oocytes in female mice (and other mammalian laboratory models) but it can&#39;t be, unfortunately, used to sort antral oocytes in humans for fertility purposes.
However, since in vitro maturation of immature oocytes is routinely used in humans too, it would be extremely important to find a non-invasive way to distinguish the &#34;good&#34; oocytes (i.e., SN) from the &#34;bad&#34; ones (i.e., NSN) to increase the success of in vitro fertilization and subsequent prospective embryos development. Besides, the isolation and culture of SN oocytes from ovarian tissue could be used for fertility preservation in women undergoing cancer treatments or suffering with ovarian pathologies.
By exploiting this natural phenomenon, important data could be provided for the understanding of naturally occurring embryo preimplantation losses which speaks not only to those interested in the basic biology of oocyte development but also to human and animal IVF clinicians.
The implications of this technique for human health, biopolitics of embryos and animal reproduction are significant15. Being able to identify and eliminate &#34;life-incompatible&#34; oocytes would greatly raise the effective level of IVF techniques, leading to cutting-edge solutions for important unmet clinical needs.
In fact, the international scientific community of gamete biologists could contribute to seek the critical improvements needed to apply this protocol to human IVF procedures. Even though the protocol can&#39;t be applied to humans (for clear safety reasons), it still keeps a tremendous capacity to improve the knowledge of the link between chromatin organization (i.e., genome architecture) and gene expression during the final stages of antral maturation, one of the hot topics in human reproduction.

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

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 glass begins to melt and become soft, both ends of the pipette must be pulled apart and, at the same time, the pipette must be taken out of the flame. Alternatively, glass capillary can be pulled in the same way or using a micropipette puller, if properly calibrated. This is a crucial step because it is very important to have the right mouth pipette with the right capillary diameter: if it is too thin, oocytes may be fragmented inside the small capillary and die in few minutes, if it is too big, a proper suction of the medium containing the oocytes can be lost when moving the oocytes, for example, from one drop to another. Figures 2 and 3 show the mechanical isolation of antral oocytes from antral follicles using a sterile insulin needle. It is quite easy to recognize these follicles because they are characterized by a cavity or antrum containing a fluid, that is the medium in which the granulosa cells and oocyte are found and through which different molecules can be exchanged to and from the external environment. Once isolated, antral oocytes must be transferred from one drop to another of M2 medium to remove somatic cells surrounding the oocytes, as shown in Figures 4A and B. Once &#34;denuded&#34;, antral oocytes are ready for classification. Figure 5 shows the last step of this protocol, that is the staining of the isolated antral oocytes with Hoechst33342 for a quick sorting based on their chromatin organization. This marker stains living cells much more rapidly than DAPI, for example, and must be used at supravital concentration (50 ng/&#181;l) to preserve cell viability. Cell staining for chromatin organization is probably the most critical step of this protocol: if the oocytes are excited too long they can be damaged and become unusable for future essays. Overall, if applied to mouse oocytes, this is a simple method that do not require specific precautions (except for those indicated in the method section). All the steps can be performed at RT.
<img alt="Figure 1" src="/files/ftp_upload/53616/53616fig1.jpg" /> 
Figure 1. Preparation of a mouth pipette using glass Pasteur pipettes and a Bunsen burner. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53616/53616fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53616/53616fig2.jpg" /> 
Figure 2. Intact ovaries in petri dish filled with pre-warmed and equilibrated M2 medium. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53616/53616fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53616/53616fig3.jpg" /> 
Figure 3. Initial isolation of antral oocytes from antral follicles by means of a sterile needle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53616/53616fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53616/53616fig4.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 M2medium (B) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53616/53616fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53616/53616fig5.jpg" /> 
Figure 5. Oocytes stained and sorted with Hoechst33342. Single images of NSN and SN oocytes taken at the confocal microscope Olympus Fluoview Fv10i (magnification 60X; bar: 10 &#181;m; PhC: phase contrast). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53616/53616fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.3K Views.  Fondazione IRCCS Policlinico San Matteo.  Hier presenteren we een protocol voor de karakterisering van muis antral eicellen gebaseerd op nucleolair organisatie. 

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

Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for spermatogenesis. These techniques allow for visualization of protein and DNA localization patterns during meiotic events such as homologous chromosome pairing, synapsis and DNA repair. While a few protocols have been described in the literature, general chromatin spread techniques using mammalian prophase oocytes are limited and difficult due to the timing of meiosis initiation in fetal ovaries. In comparison, prophase spermatocytes can be collected from juvenile male mice with higher yields without the need for microdissection. However, it is difficult to obtain a pure synchronized population of cells at specific stages due to the heterogeneity of meiotic and post-meiotic germ cell populations in the juvenile and adult testis. For later stages of meiosis, it is advantageous to assess oocytes undergoing meiosis I (MI) or meiosis II (MII), because groups of mature oocytes can be collected from adult female mice and stimulated to resume meiosis in culture. Here, methods for meiotic chromatin spread preparations using oocytes dissected from fetal, neonatal and adult ovaries are described with accompanying video demonstrations. Chromosome missegregation events in mammalian oocytes are frequent, particularly during MI. These techniques can be used to assess and characterize the effects of different mutations or environmental exposures during various stages of oogenesis. As there are distinct differences between oogenesis and spermatogenesis, the techniques described within are invaluable to increase our understanding of mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

Oogenesis in mammals is known to be error-prone, particularly due to chromosome missegregation. This manuscript describes chromatin spread preparation methods for mouse prophase, metaphase I and II-staged oocytes. These fundamental techniques allow for the study of chromatin-bound proteins and chromosome morphology throughout mammalian oogenesis.

Chromatin spread techniques have been widely used to assess the dynamic localization of various proteins during gametogenesis, particularly for ...

Genetics

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted reproduction also allows for the production of embryos from high performers without interrupting their sports career and contributes to an increase in the number of foals from mares of high genetic value. The present manuscript describes the procedures used for collecting immature and mature oocytes from horse ovaries using ovum pick-up (OPU). These oocytes were then used to investigate the incidence of aneuploidy by adapting a protocol previously developed in mice. Specifically, the chromosomes and the centromeres of metaphase II (MII) oocytes were fluorescently labeled and counted on sequential focal plans after confocal laser microscope scanning. This analysis revealed a higher incidence in the aneuploidy rate when immature oocytes were collected from the follicles and matured in vitro compared to in vivo. Immunostaining for tubulin and the acetylated form of histone four at specific lysine residues also revealed differences in the morphology of the meiotic spindle and in the global pattern of histone acetylation. Finally, the expression of mRNAs coding for histone deacetylases (HDACs) and acetyl-transferases (HATs) was investigated by reverse transcription and quantitative-PCR (q-PCR). No differences in the relative expression of transcripts were observed between in vitro and in vivo matured oocytes. In agreement with a general silencing of the transcriptional activity during oocyte maturation, the analysis of the total transcript amount can only reveal mRNA stability or degradation. Therefore, these findings indicate that other translational and post-translational regulations might be affected.
Overall, the present study describes an experimental approach to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to low sample availability. However, it can expand our knowledge on the reproductive biology and infertility in monovulatory species.

This manuscript describes an experimental approach to morphologically and biochemically characterize horse oocytes. Specifically, the present work illustrates how to collect immature and mature horse oocytes by ultrasound-guided ovum pick-up (OPU) and how to investigate chromosome segregation, spindle morphology, global histone acetylation, and mRNA expression.

The field of assisted reproduction has been developed to treat infertility in women, companion animals, and endangered species. In the horse, assisted ...

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 ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Biology

Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method was performed for the subsequent paired interrogation of the cell-specific ovarian transcriptome and epigenome using a novel transgenic NuTRAP mouse model. The expression of the NuTRAP allele is under the control of a floxed STOP cassette and can be targeted to specific ovarian cell types using promoter-specific Cre lines. Since recent studies have implicated ovarian stromal cells in driving premature aging phenotypes, the NuTRAP expression system was targeted to stromal cells using a Cyp17a1-Cre driver. The induction of the NuTRAP construct was specific to ovarian stromal fibroblasts, and sufficient DNA and RNA for sequencing studies were obtained from a single ovary. The NuTRAP model and methods presented here can be used to study any ovarian cell type with an available Cre line.

In this protocol, the translating ribosome affinity purification (TRAP) method and the isolation of nuclei tagged in specific cell types (INTACT) method were optimized for the paired interrogation of the cell-specific ovarian transcriptome and epigenome using the NuTRAP mouse model crossed to a Cyp17a1-Cre mouse line.

Assessing cell-type-specific epigenomic and transcriptomic changes are key to understanding ovarian aging. To this end, the optimization of the ...

Impact of Cytoskeleton on Nuclear Shape and Condensate Dynamics in Mouse Oocytes

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

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

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