Name: isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization
Uploaded: 2016-01-07T12:00:01
Description: Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

M.M. and C.A.R. acknowledge the financial support of the Ministero della Salute, Ricerca Finalizzata/giovani ricercatori anno 2009 of the Fondazione IRCCS Ospedale San Matteo, Pavia (I). M.M and C.A.R. wish to thank Marianna Longo for helping with the images preparation.

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

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G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+oocytes+for+in+vitro+maturation+by+brilliant+cresyl+blue+staining:+a+study+using+the+mouse+model.">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., K&#261;tska-Ksi&#261;&#380;kiewicz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+Brilliant+Cresyl+Blue+(BCB)+staining+of+mammalian+oocytes+used+for+in+vitro+embryo+production+(IVP).">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. 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This protocol describes the method used to isolate and classify mouse antral GV oocytes. There are no particular critical steps to underline, with few exceptions. First: this procedure should be performed as quickly as possible to: a) maintain the vitality of the oocytes and b) avoid pH changes in the medium used. Second: oocytes should be briefly excited (5-10 sec) to Hoechst33342 to avoid chromatin damage and the subsequent death of the oocytes, especially if IVM and IVF are the following steps. To date, this is the qu...

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

<table>
	<tbody>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>G4527</td>
			<td></td>
		</tr>
		<tr>
			<td>EmbrioMax M2</td>
			<td>Millipore</td>
			<td>MR-015-PD</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst33342</td>
			<td>Thermo Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish 35mmx10mm</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;-Dish 35mm, high glass bottom</td>
			<td>Ibidi</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Pasteur Borosilicate</td>
			<td>Corning</td>
			<td>7095D-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assemblies for calibrated micropillary pipettes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of mouse antral oocytes based on nucleolar chromatin organization

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded Nucleolus) or unable (NSN, Not Surrounded Nucleolus) to develop to the blastocyst stage after in vitro maturation (IVM) and in vitro fertilization (IVF). It makes use of Hoeschst33342 (or any other DNA intercalating dye) able to bind to the heterochromatin of the nucleolus showing a ring in the SN oocytes or not, like in the NSN oocytes. This represents the easiest and quickest way to sort both antral oocytes that can be eventually used for IVM or IVF procedures. 
Briefly, the protocol consists of the following steps: hormone injection to stimulate follicular growth; isolation of the oocytes at the GV stage from the antral compartment by puncturing the ovary with a sterile needle; preparation of thin glass pipettes for mouth pipetting of the oocytes; sorting of the oocytes with Hoechst33342 prepared at a supravital concentration; IVM, IVF or any other molecular/cellular analysis.
Unfortunately there are still few evidences to sort SN and NSN oocytes using less invasive techniques. If and once they will be identified, they could be potentially applied to human assisted reproductive technologies, although with several aspects that should be modified. To date, this technique has potential implications to dramatically increase IVM and IVF successful procedures in both endangered and species with economic interest.

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

Watch this Scientific Journal Video about Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization at JoVE.com

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

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

This protocol describes a simple and quick method to isolate and characterize mouse antral GV (Germinal Vesicle) oocytes as able (SN, Surrounded ...

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.

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The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

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The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

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The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

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Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

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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 ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

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;

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...