The authors would like to thank Fabrice Vincent for the support with laser scanning confocal microscopy (LSCM), and Philippe Barri&#232;re and Thierry Blard for performing daily ultrasound ovarian scanning and hCG injection. This work was supported in part by the &#34;Regione Sardegna and Regione Lombardia&#34; project &#34;Ex Ovo Omnia&#34; (Grant no. 26096200 to A. M. L.); the &#34;L&#39;Oreal Italia per le Donne e la Scienza 2012&#34; fellowship (Contract 2012 to F. F.), FP7-PEOPLE-2011- CIG, Research Executive Agency (REA) &#34;Pro-Ovum&#34; (Grant no. 303640 to V. L.); and by the Postdoctoral School of Agriculture and Veterinary Medicine, co-financed by the European Social Fund, Sectorial Operational Program for Human Resource Development 2007-2013 (Contract no. POSDRU/89/1.5/S/62371 to I. M.). The in vivo oocyte collection was financed by the Institut Fran&#231;ais du Cheval et de l&#39;Equitation.

All the procedures were approved by the Animal Care and Use Committee CEEA Val de Loire Number 19 and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
1. Oocyte Collection and In Vitro Maturation
<ol>
	<li>Ovum pick-up
	<ol>
		<li>Daily assess by ultrasound the diameter of ovarian follicles in a cohort of adult mares. At the emergence of a follicle &#8805;33 mm (dominant follicle), inject the mare with 1,500 IU of human chorionic gonadotrophin (hCG) in the upper part of the jugular vein (i.v.).
		<ol>
			<li>Before performing the injection, swab the area with alcohol, locate the jugular furrow, and place the thumb 2-3 cm under the site of injection to induce the vein to rise. Insert the needle almost parallel to the neck and aspirate to make sure that the needle is in the vein (the blood will enter the syringe). At this point, inject; hCG will induce the maturation of the oocyte in the dominant follicle.</li>
		</ol>
		</li>
		<li>35 h after the hCG injection, sedate the mare with detomidine (15 &#181;g/kg, i.v.), butorphanol tartrate (10 &#181;g/kg, i.v.) and butylscopolamine bromure (0.2-0.3 mg/kg, 15 mL/mare, i.v.).</li>
		<li>Connect the needle to the ultrasound probe. Insert the ultrasound probe vaginally and hold the ovary transrectally to face the ultrasound probe. Instruct one operator to maneuver the needle and the other to hold the ovary in place, with the follicle facing the ultrasound probe. Start from the ovary with the dominant follicle.</li>
		<li>Puncture the vaginal wall and the wall of the dominant follicle with the needle (ultrasound probes for OPU are equipped with a channel for the needle connected to a suction system); theneedle will be visible in the echographic image.</li>
		<li>Aspirate the follicular fluid in a 50-mL conical tube connected to the suction system. Pour the content of the conical tube into a large petri dish and search for the cumulus-oocyte complex (COC) using a stereomicroscope.</li>
		<li>Since the COC is not always retrieved with the follicular fluid, flush the follicle with Dulbecco&#39;s modified phosphate-buffered saline (DPBS) containing 5 IU/mL heparinat 37 &#176;C. Examine DPBS for COC presence, as previously described for the follicular fluid in step 1.1.5. Flush several times until the COC is retrieved.</li>
		<li>Once the COC from the dominant follicle is retrieved, puncture and flush the follicles &#8805;5 and &#8804;25 mm, as described for the dominant follicle in steps 1.1.3-1.1.4; follicles &#8805;5 and &#8804;25 do not express luteinizing hormone/choriogonadotropin receptor (LHCGR), and therefore, their oocytes will not mature in response to hCG and will be collected at GV stage (immature). Only keep COCs with several complete layers of cumulus cells and brown, finely-granulated ooplasm. Discard the others.</li>
		<li>At the end of the OPU, inject the mare with benzyl-penicillin 15,000 IU/animal to prevent infection.</li>
		<li>House the mare in a quiet, clean stable for at least 2 h. Check signs of awareness by determining the position of the ears, reaction to stimuli (e.g., noise), and gait before returning the mare to the company of other animals.</li>
	</ol>
	</li>
	<li>In vitro maturation
	<ol>
		<li>Warm HEPES-buffered TCM199 (20 mM Hepes) supplemented with 1,790 IU/L heparin, 0.4% bovine serum albumin (BSA), penicillin, and streptomycin to 37 &#176;C. Prepare this medium in advance. Filter-sterilize and keep it at 4 &#176;C for up to 6 months.</li>
		<li>Prepare the IVM medium by supplementing NaHCO3-buffered TCM199 (25 mM NaHCO3) with 50 ng/mL epidermal growth factor (EGF) and 20% calf serum7. Use NaHCO3-buffered TCM199 within 3 weeks of opening a new bottle. Prepare the EGF in advance as 100x stocks, freeze it at -20 &#176;C, and use it within 6 months. Dispense 500 &#181;L in the wells of a 4-well dish and incubate in humidified air with 5% CO2 at 38.5 &#176;C for at least 2 h.</li>
		<li>After the retrieval of the COCs from the &#8805;5 and &#8804;25 mm follicles, put them in a petri dish (3.5-cm diameter) filled with 2 mL of HEPES-TCM199. Move the COCs to different areas of the dish in order to wash away the cellular debris.</li>
		<li>Transfer the COCs to the IVM medium and incubate for 28 h in humidified air with 5% CO2 at 38.5 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Immunofluorescence and Image Analysis
<ol>
	<li>Chromosome count
	<ol>
		<li>After collection from the dominant follicle or at the end of IVM, incubate the COCs with 100 &#181;M monastrol for 1 h in humidified air with 5% CO2 at 38.5 &#176;C. Prepare monastrol in advance as 100x stocks and store it at -20 &#176;C for up to 1 year.</li>
		<li>Remove the cumulus cells by treating them with 0.5% hyaluronidase or by gently pipetting; remove the zona pellucida by treating it in 0.2% pronase. Prepare hyaluronidase and pronase in advance as 10x stocks and store them at -20 &#176;C for up to 1 year.</li>
		<li>Fix the oocytes in 4% paraformaldehyde in DPBS for 15 min at 38.5 &#176;C followed by a further 45 min at 4 &#176;C. 
		CAUTION! Wear personal protective equipment when handling paraformaldehyde, and dispose of contaminated materials in accordance with hazardous waste disposal guidelines.</li>
		<li>Wash the oocytes by sequentially transferring in 3 wells filled with 500 &#181;L of DPBS supplemented with 0.1% polyvinyl alcohol (PVA). Permeabilize in 0.3% Triton X-100 for 10 min at room temperature (RT).</li>
		<li>Block non-specific bindingby incubation in DPBS supplemented with 1% bovine serum albumin (DPBS-1%BSA) and 10% normal donkey serum for at least 30 min at RT. Samples can be kept in blocking solution at 4 &#176;C for 3-4 days.</li>
		<li>For primary staining, incubate the oocytes in rabbit anti-Aurora B phospho-Thr232 in DPBS supplemented with 1% BSA (dilution 1:50) at 4 &#176;C overnight.</li>
		<li>Wash the oocytes as described in step 2.1.4. Incubate the oocytes in a solution of tetra-methylrhodamine isothiocyanate (TRITC)-conjugated donkey anti-rabbit IgG (1:100 in DPBS-1% BSA) for 1 h at RT in the dark.</li>
		<li>Wash the oocytes as described in step 2.1.4, and then place 3-4 oocytes in a drop of non-hardening, anti-fade mounting medium supplemented with 20 &#181;M YOPRO1 on a glass slide. Repeat the procedure until all the oocytes are mounted (3-4 oocytes per slide).</li>
		<li>Allow the oocytes to settle for about 10 min in the mounting medium, and then cover them with a coverslip. To avoid crushing the oocytes, apply two strips of double-sided tape before laying the coverslip on the glass slide. 
		NOTE: This precaution will also ensure that all the samples are equally compressed between the glass slide and the coverslip. Glass slides can be frozen at -20 &#176;C and imaged during the following 2-3 days.</li>
		<li>Image the samples on a confocal laser scanning microscope with a 60X objective using the red (centromeres) and green (chromosomes) channels7,13,14,20,21. Scan the samples spanning the whole metaphasic plate on the z-axis, with steps every 0.35 &#181;m. Save the digitalized images of every single plan.</li>
		<li>Count the centromeres in serial confocal sections using the cell count function of the NIH ImageJ software (available at: https://imagej.nih.gov/ij/plugins/cell-counter.html). 
		NOTE: Avoid a repeated count of centromeres that appear on adjacent sections by identifying the centromeres that appear, stay, and disappear in sequential sections with a numeric code.</li>
		<li>Repeat the chromosomal counting in step 2.1.11 with an independent operator. 
		NOTE: Classify the oocytes as euploid (32 chromosomes), hyperploid (&#62;32), or hypoploid (&#60;32).</li>
	</ol>
	</li>
	<li>Spindle morphology and histone acetylation
	<ol>
		<li>After collection from the dominant follicle or at the end of the IVM, remove the cumulus cells by treatment in 0.5% hyaluronidase or by gentle pipetting, as described in step 2.1.2.</li>
		<li>Wash the oocytes in DPBS-PVA, as in step 2.1.4, and fix them in 2.5% paraformaldehyde for 20 min at 38 &#176;C. Extensively wash, as described in step 2.1.4, and permeabilize in 0.1% Triton X-100 for 5 min at RT. 
		NOTE: Wear personal protective equipment when handling paraformaldehyde and dispose of contaminated materials in accordance with hazardous waste disposal guidelines.</li>
		<li>Block non-specific bindingby incubation in DPBS supplemented with 2% bovine serum albumin (DPBS - 2%BSA), 0.05% saponin, and 10% normal donkey serum for 2 h at RT. 
		NOTE: Samples can be kept in blocking solution at 4 &#176;C for 3 - 4 days.</li>
		<li>For primary staining, incubate the oocytes in mouse anti-alpha-tubulin (1:150) and rabbit anti-acH4K16 (1:250) in DPBS-2% BSA with 0.05% saponin at 4 &#176;C overnight.</li>
		<li>Wash the oocytes as in step 2.1.4. Incubate the oocytes in a solution of green fluorescent dye-conjugated donkey anti-mouse IgG (1:500) and TRITC-conjugated donkey anti-rabbit IgG (1:100) in DPBS-2% BSA with 0.05% saponin for 1 h at RT in the dark.</li>
		<li>Wash the oocytes as in step 2.1.4, and then place 3-4 oocytes in a drop of the non-hardening, anti-fade mounting medium supplemented with 1 &#181;g/mL 4&#39;,6-diamidino-2-phenylindole (DAPI) on a glass slide.</li>
		<li>Mount the oocytes on glass slides as described in step 2.1.8.</li>
		<li>Image the samples on a confocal laser scanning microscope with a 60x objective using the red (acH4K16), green (alpha-tubulin), and blue (DNA) channels. 
		NOTE: Keep the laser settings for the red and blue channels constant throughout the acquisition of all the samples. Scan the samples spanning the whole meiotic spindle and metaphasic plate on the z-axis, with steps every 0.35 &#181;m. Save the digitalized images (single plans and projected 3D image).</li>
		<li>Measure the pole-to-pole spindle length and the spindle diameter at the maximum width on the projected image using the measurement function of the NIH ImageJ software (https://imagej.nih.gov/ij/docs/guide/146-30.html). Repeat each 3 times and calculate the mean.</li>
		<li>Quantify the relative fluorescence of acH4K16 using the integrated density function of the NIH ImageJ software (https://imagej.nih.gov/ij/docs/guide/146-30.html). Normalize it for the integrated density of the DAPI staining.</li>
	</ol>
	</li>
</ol>
3. Analysis of mRNA Expression
<ol>
	<li>After the COCs have been retrieved from the 5 to 25 mm follicles, collect the granulosa cell suspension that remained in the Petri dish and wash it twice in cold DPBS by centrifuging at 10,600 x g for 2 min. Snap freeze in liquid nitrogen. Store the samples at -80 &#176;C for up to 6 months; the cells will serve for the standard curve.</li>
	<li>Carefully remove all the cumulus cells, as described in step 2.1.2, from immature (GV), in vitro matured, and in vivo matured oocytes.</li>
	<li>Wash the oocytes, as described in 2.1.4, in DPBS-PVA, collect them singly in 1 &#181;L volumes, and lay 2 &#181;L RNALater on top. Snap-freeze in liquid nitrogen. Store the samples at -80 &#176;C for up to 6 months.</li>
	<li>Add 2 pg of Luciferase RNA to each sample as a spike-in. Pool the oocytes 2 x 2 and extract the total RNA using a silica membrane filter-based kit suitable to recover RNA from small samples.</li>
	<li>Reverse-transcribe the RNA in 10 &#181;L with 0.25 &#181;g random hexamers and mouse Moloney leukaemia virus reverse transcriptase for 1 h at 37 &#176;C. Store the samples at -20 &#176;C for up to 6 months.</li>
	<li>Dilute the cDNA from the granulosa cells in RNAse-free water to 50 ng/&#181;L. Serially dilute this solution 1:10 to obtain 5 ng/&#181;L, 0.5 ng/&#181;L, 0.05 ng/&#181;L, and 0.005 ng/&#181;L solutions.</li>
	<li>Assemble reactions in triplicate in a total volume of 20 &#181;L per reaction using cDNA equivalent to 0.05 oocytes as the substrate, or 5 &#181;L of the serial dilutions of granulosa cell cDNA, 10 &#181;L of SYBR green supermix, and 0.3 &#181;M of each specific primer (Table 1).</li>
	<li>Run the reaction in a thermal cycler using a 3-step protocol: 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 20 s, repeated 40 times. Finally, acquire the melting curve, starting from 60 &#176;C, and increase the temperature by 0.5 &#176;C every 20 s up to 95 &#176;C.</li>
	<li>Assess the starting quantity (SQ) of the specific mRNA in the oocyte samples using the standard curve15 calculated based on the granulosa cell sample. Express the data as the ratio between the SQ of the gene of interest and the SQ of the housekeeping genes.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocytes+isolated+from+dairy+cows+with+reduced+ovarian+reserve+have+a+high+frequency+of+aneuploidy+and+alterations+in+the+localization+of+progesterone+receptor+membrane+component+1+and+aurora+kinase+B.">Oocytes isolated from dairy cows with reduced ovarian reserve have a high frequency of aneuploidy and alterations in the localization of progesterone receptor membrane component 1 and aurora kinase B.</a> Biol Reprod. 88 (3), 58 (2013).</li><li>Luciano, A. M., Lodde, V., Franciosi, F., Ceciliani, F., Peluso, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+membrane+component+1+expression+and+putative+function+in+bovine+oocyte+maturation,+fertilization,+and+early+embryonic+development.">Progesterone receptor membrane component 1 expression and putative function in bovine oocyte maturation, fertilization, and early embryonic development.</a> Reproduction. 140 (5), 663-672 (2010).</li><li>Terzaghi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGRMC1+participates+in+late+events+of+bovine+granulosa+cells+mitosis+and+oocyte+meiosis.">PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.</a> Cell Cycle. , 1-14 (2016).</li><li>Susor, A., Jansova, D., Anger, M., Kubelka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+in+the+mammalian+oocyte+in+space+and+time.">Translation in the mammalian oocyte in space and time.</a> Cell Tissue Res. 363 (1), 69-84 (2016).</li><li>Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+translation+reveals+a+critical+role+for+deleted+in+azoospermia-like+(Dazl)+at+the+oocyte-to-zygote+transition.">Genome-wide analysis of translation reveals a critical role for deleted in azoospermia-like (Dazl) at the oocyte-to-zygote transition.</a> Genes Dev. 25 (7), 755-766 (2011).</li><li>Ma, J., Flemr, M., Strnad, H., Svoboda, P., Schultz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternally+recruited+DCP1A+and+DCP2+contribute+to+messenger+RNA+degradation+during+oocyte+maturation+and+genome+activation+in+mouse.">Maternally recruited DCP1A and DCP2 contribute to messenger RNA degradation during oocyte maturation and genome activation in mouse.</a> Biol Reprod. 88 (1), 11 (2013).</li></ol>

The authors have nothing to disclose.

Even though IVM has been performed in horses for more than 20 years16, we do not yet know whether the oocyte can be the origin of embryonic aneuploidy, as has been proposed for humans17. The reason is probably that the preparation of oocyte spreads for chromosome counting results in considerable sample loss. With this in mind, a survey of the methods used for investigating errors in chromosome segregation was conducted to search for the most appropriate technique to apply t...

A vast array of assisted reproduction techniques has been developed to treat infertility in women, companion animals, and endangered species. One of the most common procedures in clinical settings is the retrieval of metaphase II (MII)-stage oocytes from the ovarian follicles by ultrasound-guided transvaginal aspiration, ovum pick-up (OPU)1. These oocytes are then fertilized in vitro (IVF), with the resulting embryo(s) implanted in a recipient uterus. The MII-stage (mature) oocytes are retrieved following the administration of exogenous gonadotropins. However, this treatment is associated, in some patients, with the development of ovarian hyperstimulation syndrome (OHSS)2.
Taking advantage of the intrinsic ability of fully-grown, immature oocytes (GV-stage) to spontaneously resume meiosis once isolated from their follicles, it is possible to obtain mature oocytes without administering gonadotropin3. This procedure is called oocyte in vitro maturation (IVM) and represents a less drug-oriented, less expensive, and more patient-friendly approach to assisted reproductive technology. However, the success of embryo development with in vitro-matured oocytes is generally lower than with in vivo matured oocytes4,5. A possible explanation is that in vitro matured oocytes are more affected by errors in chromosome segregation, and the resulting aneuploidy impairs normal embryonic development6.
Understanding the molecular basis of chromosomal mis-segregation during IVM would ultimately disclose the full potential of this technique. In this vein, the experimental approach used to investigate the morphological and biochemical features of in vitro-matured oocytes, compared to in vivo matured oocytes is here described7,8. Specifically, the procedures for the OPU of immature and mature oocytes and the IVM of immature oocytes are illustrated using adult and naturally-cycling horses as an experimental model. Then, immunofluorescence and image analysis are used to investigate chromosome segregation, spindle morphology, and the global pattern of histone acetylation on these gametes. Finally, a protocol of reverse-transcription and quantitative PCR is described for the analysis of mRNA expression.
Compared with rodent animal models, horses do not allow genetic manipulation, are less easy to manipulate, and require expensive maintenance. However, this model is gaining considerable interest for the study of oocyte maturation9,10 due to the similarity to human ovarian physiology11,12. Moreover, the development of reliable protocols of IVM-IVF in the horse has a substantial economic interest, as it would allow for an increase in the number of foals from mares of high genetic value.
One of the limitations of performing experiments on oocytes, especially in monovulatory species, is the restricted sample availability. This limit has been overcome here by adjusting an approach, previously developed in mice, to horse oocytes13,14 in order to conduct chromosome counting that minimizes the sample loss (see the discussion for a comparison with other available techniques). Moreover, a triple-fluorescence staining protocol has been optimized to conduct multiple analyses on the same sample, and q-PCR analyses were performed on pools of 2 oocytes only.
Overall, the present study describes an experimental approach aimed to morphologically and biochemically characterize the horse oocyte, a cell type that is extremely challenging to study due to the low sample availability. However, it can expand our knowledge of the reproductive biology and infertility of monovulatory species.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:369px;">ultrasound probe</td>
			<td style="width:159px;">Aloka</td>
			<td style="width:113px;">UST-5820-7,5</td>
			<td style="width:161px;"></td>
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			<td height="21" style="height:21px;">human chorionic gonadotrophin&#160;</td>
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			<td>CHO004</td>
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			<td>MED010</td>
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			<td height="21" style="height:21px;">butylscopolamine bromure&#160;</td>
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			<td>EST001</td>
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			<td height="21" style="height:21px;">stereomicroscope</td>
			<td style="width:159px;">NIKON</td>
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			<td>DOL003</td>
			<td>10&#181;g/kg IV</td>
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			<td height="21" style="height:21px;">benzyl-penicillin</td>
			<td>centravet</td>
			<td>DEP203 IM</td>
			<td>15000UI/animal</td>
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		<tr height="21">
			<td height="21" style="height:21px;">TCM199</td>
			<td>Sigma-Aldrich</td>
			<td>M3769 10X1L</td>
			<td>powder for hepes-buffered TCM199</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H3784-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heparin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H3149-10KU</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaHCO3-buffered TCM199</td>
			<td>Sigma-Aldrich</td>
			<td>M2154-500ML</td>
			<td>liquid for IVM medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">epidermal growth factor</td>
			<td>Sigma-Aldrich</td>
			<td>E4127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">newborn calf serum</td>
			<td>Sigma-Aldrich</td>
			<td>N4762-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well dishes</td>
			<td>NUNCLON</td>
			<td>144444</td>
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		<tr height="21">
			<td height="21" style="height:21px;">incubator</td>
			<td>Heraeus</td>
			<td>BB6060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">monastrol</td>
			<td>Sigma-Aldrich</td>
			<td>M8515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pronase</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyvinyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>341584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">normal donkey serum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-Aurora B phospho-Thr232&#160;</td>
			<td>BioLegend</td>
			<td>636102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRITC-conjugated donkey anti-rabbit IgG&#160;</td>
			<td>Vector Laboratories</td>
			<td>711-025-152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vecta-Shield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YOPRO1</td>
			<td>Thermofisher Scientific</td>
			<td>Y3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">confocal laser scanning microscope&#160;</td>
			<td>LSM 780&#160;</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">confocal laser scanning microscope&#160;</td>
			<td>LSM 700&#160;</td>
			<td>Zeiss</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ImageJ software</td>
			<td colspan="2">rsb.info. nih.gov/ij/download.html&#160;</td>
			<td>free resource</td>
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		<tr height="21">
			<td height="21" style="height:21px;">mouse anti-alpha-tubulin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit anti-acH4K16</td>
			<td>Upstate Biotechnology</td>
			<td>07-329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AlexaFluor 488-coniugated donkey anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A21202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8417</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
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			<td height="21" style="height:21px;">RNALater</td>
			<td>Invitrogen</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferase RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PicoPure RNA Isolation Kit&#160;</td>
			<td>Applied Biosystems</td>
			<td>12204-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">random hexamers&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>N8080127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse Moloney leukaemia virus reverse transcriptase</td>
			<td>Thermofisher Scientific</td>
			<td>28025013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR green supermix&#160;</td>
			<td>BioRad</td>
			<td>1708880</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">specific primers</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>specific primers were designed using Primer3Plus software (free resource)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thermal-cycler&#160;</td>
			<td>BioRad</td>
			<td>MyiQ&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-CENPA</td>
			<td>Abcam</td>
			<td>ab13939</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mouse monoclonal anti-Aurora B</td>
			<td>Abcam</td>
			<td>ab3609</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The original findings of these experiments were described in depth previously7 and are reported herein as an example of results that can be obtained using the described protocols.
Maturation rate
Of the 32 COCs retrieved by OPU from dominant follicles, 28 (88%) were at the MII stage. Fourteen of the 58 COCs collected from follicles 5-25 mm in size ...

Watch this Scientific Journal Video about Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes at JoVE.com

Analysis of Chromosome Segregation, Histone Acetylation and Spindle Morphology in Horse Oocytes | Developmental Biology | Video

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

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 ...

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10.9K Views.  University of Milan. 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.

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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.

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 ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

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 ...