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>

<ol>
	<li>Dellenbach, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transvaginal,+sonographically+controlled+ovarian+follicle+puncture+for+egg+retrieval.">Transvaginal, sonographically controlled ovarian follicle puncture for egg retrieval.</a> Lancet. 1 (8392), 1467 (1984).</li><li>Humaidan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+hyperstimulation+syndrome:+review+and+new+classification+criteria+for+reporting+in+clinical+trials.">Ovarian hyperstimulation syndrome: review and new classification criteria for reporting in clinical trials.</a> Hum Reprod. , (2016).</li><li>Pincus, G., Enzmann, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Comparative+Behavior+of+Mammalian+Eggs+in+Vivo+and+in+Vitro+:+I.+The+Activation+of+Ovarian+Eggs.">The Comparative Behavior of Mammalian Eggs in Vivo and in Vitro : I. The Activation of Ovarian Eggs.</a> J Exp Med. 62 (5), 665-675 (1935).</li><li>Emery, B. R., Wilcox, A. L., Aoki, V. W., Peterson, C. M., Carrell, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+oocyte+maturation+and+subsequent+delayed+fertilization+is+associated+with+increased+embryo+aneuploidy.">In vitro oocyte maturation and subsequent delayed fertilization is associated with increased embryo aneuploidy.</a> Fertil Steril. 84 (4), 1027-1029 (2005).</li><li>Nichols, S. M., Gierbolini, L., Gonzalez-Martinez, J. A., Bavister, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+in+vitro+maturation+and+age+on+oocyte+quality+in+the+rhesus+macaque+Macaca+mulatta.">Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.</a> Fertil Steril. 93 (5), 1591-1600 (2010).</li><li>Requena, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+in-vitro+maturation+of+oocytes+on+aneuploidy+rate.">The impact of in-vitro maturation of oocytes on aneuploidy rate.</a> Reprod Biomed Online. 18 (6), 777-783 (2009).</li><li>Franciosi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+maturation+affects+chromosome+segregation,+spindle+morphology+and+acetylation+of+lysine+16+on+histone+H4+in+horse+oocytes.">In vitro maturation affects chromosome segregation, spindle morphology and acetylation of lysine 16 on histone H4 in horse oocytes.</a> Reprod Fertil Dev. , (2015).</li><li>Franciosi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+histone+H4+acetylation+during+in+vivo+versus+in+vitro+maturation+of+equine+oocytes.">Changes in histone H4 acetylation during in vivo versus in vitro maturation of equine oocytes.</a> Mol Hum Reprod. 18 (5), 243-252 (2012).</li><li>Choi, Y. H., Gibbons, J. R., Canesin, H. S., Hinrichs, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+medium+variations+(zinc+supplementation+during+oocyte+maturation,+perifertilization+pH,+and+embryo+culture+protein+source)+on+equine+embryo+development+after+intracytoplasmic+sperm+injection.">Effect of medium variations (zinc supplementation during oocyte maturation, perifertilization pH, and embryo culture protein source) on equine embryo development after intracytoplasmic sperm injection.</a> Theriogenology. , (2016).</li><li>Hendriks, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+age+and+in+vitro+culture+affect+mitochondrial+number+and+function+in+equine+oocytes+and+embryos.">Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos.</a> Reprod Fertil Dev. 27 (6), 957-968 (2015).</li><li>Carnevale, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mare+model+for+follicular+maturation+and+reproductive+aging+in+the+woman.">The mare model for follicular maturation and reproductive aging in the woman.</a> Theriogenology. 69 (1), 23-30 (2008).</li><li>Ginther, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mare:+a+1000-pound+guinea+pig+for+study+of+the+ovulatory+follicular+wave+in+women.">The mare: a 1000-pound guinea pig for study of the ovulatory follicular wave in women.</a> Theriogenology. 77 (5), 818-828 (2012).</li><li>Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+weakened+centromere+cohesion+is+a+leading+cause+of+age-related+aneuploidy+in+oocytes.">Evidence that weakened centromere cohesion is a leading cause of age-related aneuploidy in oocytes.</a> Curr Biol. 20 (17), 1522-1528 (2010).</li><li>Duncan, F. E., Chiang, T., Schultz, R. M., Lampson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+a+defective+spindle+assembly+checkpoint+is+not+the+primary+cause+of+maternal+age-associated+aneuploidy+in+mouse+eggs.">Evidence that a defective spindle assembly checkpoint is not the primary cause of maternal age-associated aneuploidy in mouse eggs.</a> Biol Reprod. 81 (4), 768-776 (2009).</li><li>Larionov, A., Krause, A., Miller, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standard+curve+based+method+for+relative+real+time+PCR+data+processing.">A standard curve based method for relative real time PCR data processing.</a> BMC Bioinformatics. 6 (62), (2005).</li><li>Choi, Y. H., Hochi, S., Braun, J., Sato, K., Oguri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+maturation+of+equine+oocytes+collected+by+follicle+aspiration+and+by+the+slicing+of+ovaries.">In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries.</a> Theriogenology. 40 (5), 959-966 (1993).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Akiyama, T., Nagata, M., Aoki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inadequate+histone+deacetylation+during+oocyte+meiosis+causes+aneuploidy+and+embryo+death+in+mice.">Inadequate histone deacetylation during oocyte meiosis causes aneuploidy and embryo death in mice.</a> Proc Natl Acad Sci U S A. 103 (19), 7339-7344 (2006).</li><li>Homer, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad2+prevents+aneuploidy+and+premature+proteolysis+of+cyclin+B+and+securin+during+meiosis+I+in+mouse+oocytes.">Mad2 prevents aneuploidy and premature proteolysis of cyclin B and securin during meiosis I in mouse oocytes.</a> Genes Dev. 19 (2), 202-207 (2005).</li><li>Rambags, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+chromosomal+abnormalities+in+equine+embryos+produced+in+vivo+and+in+vitro.">Numerical chromosomal abnormalities in equine embryos produced in vivo and in vitro.</a> Mol Reprod Dev. 72 (1), 77-87 (2005).</li><li>Nabti, I., Marangos, P., Bormann, J., Kudo, N. R., Carroll, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-mode+regulation+of+the+APC/C+by+CDK1+and+MAPK+controls+meiosis+I+progression+and+fidelity.">Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity.</a> J Cell Biol. 204 (6), 891-900 (2014).</li><li>Shomper, M., Lappa, C., FitzHarris, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetochore+microtubule+establishment+is+defective+in+oocytes+from+aged+mice.">Kinetochore microtubule establishment is defective in oocytes from aged mice.</a> Cell Cycle. 13 (7), 1171-1179 (2014).</li><li>Luzzo, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+fat+diet+induced+developmental+defects+in+the+mouse:+oocyte+meiotic+aneuploidy+and+fetal+growth+retardation/brain+defects.">High fat diet induced developmental defects in the mouse: oocyte meiotic aneuploidy and fetal growth retardation/brain defects.</a> PLoS One. 7 (11), e49217 (2012).</li><li>Ma, 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=Histone+deacetylase+2+(HDAC2)+regulates+chromosome+segregation+and+kinetochore+function+via+H4K16+deacetylation+during+oocyte+maturation+in+mouse.">Histone deacetylase 2 (HDAC2) regulates chromosome segregation and kinetochore function via H4K16 deacetylation during oocyte maturation in mouse.</a> PLoS Genet. 9 (3), e1003377 (2013).</li><li>Yang, F., Baumann, C., Viveiros, M. M., De La Fuente, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+hyperacetylation+during+meiosis+interferes+with+large-scale+chromatin+remodeling,+axial+chromatid+condensation+and+sister+chromatid+separation+in+the+mammalian+oocyte.">Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.</a> Int J Dev Biol. 56 (10-12), 889-899 (2012).</li><li>Luciano, A. 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><tbody><tr><td>ultrasound probe</td><td>Aloka</td><td>UST-5820-7,5</td><td></td></tr><tr><td>human chorionic gonadotrophin</td><td>centravet</td><td>CHO004</td><td>1,500 unit/animal IV</td></tr><tr><td>detomidine</td><td>centravet</td><td>MED010</td><td>9 - 15 &amp;micro;g/kg IV</td></tr><tr><td>butylscopolamine bromure</td><td>centravet</td><td>EST001</td><td>0.2 mg/kg IV</td></tr><tr><td>stereomicroscope</td><td>NIKON</td><td>SMZ-2B</td><td></td></tr><tr><td>butorphanol</td><td>centravet</td><td>DOL003</td><td>10&amp;micro;g/kg IV</td></tr><tr><td>benzyl-penicillin</td><td>centravet</td><td>DEP203 IM</td><td>15,000 UI/animal</td></tr><tr><td>TCM199</td><td>Sigma-Aldrich</td><td>M3769 10X1L</td><td>powder for hepes-buffered TCM199</td></tr><tr><td>Hepes sodium salt</td><td>Sigma-Aldrich</td><td>H3784-100G</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma-Aldrich</td><td>A8806</td><td></td></tr><tr><td>heparin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;-buffered TCM199</td><td>Sigma-Aldrich</td><td>M2154-500ML</td><td>liquid for IVM medium</td></tr><tr><td>epidermal growth factor</td><td>Sigma-Aldrich</td><td>E4127</td><td></td></tr><tr><td>newborn calf serum</td><td>Sigma-Aldrich</td><td>N4762-500ML</td><td></td></tr><tr><td>4-well dishes</td><td>NUNCLON</td><td>144444</td><td></td></tr><tr><td>incubator</td><td>Heraeus</td><td>BB6060</td><td></td></tr><tr><td>monastrol</td><td>Sigma-Aldrich</td><td>M8515</td><td></td></tr><tr><td>hyaluronidase</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>pronase</td><td>Sigma-Aldrich</td><td>P5147</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>polyvinyl alcohol</td><td>Sigma-Aldrich</td><td>341584</td><td></td></tr><tr><td>triton-X 100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>normal donkey serum</td><td>Sigma-Aldrich</td><td>D9663</td><td></td></tr><tr><td>rabbit anti-Aurora B phospho-Thr232</td><td>BioLegend</td><td>636102</td><td></td></tr><tr><td>TRITC-conjugated donkey anti-rabbit IgG</td><td>Vector Laboratories</td><td>711-025-152</td><td></td></tr><tr><td>Vecta-Shield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>YOPRO1</td><td>Thermofisher Scientific</td><td>Y3603</td><td></td></tr><tr><td>confocal laser scanning microscope</td><td>LSM 780</td><td>Zeiss</td><td></td></tr><tr><td>confocal laser scanning microscope</td><td>LSM 700</td><td>Zeiss</td><td></td></tr><tr><td>ImageJ software</td><td>rsb.info. nih.gov/ij/download.html</td><td>free resource</td><td></td></tr><tr><td>mouse anti-alpha-tubulin</td><td>Sigma-Aldrich</td><td>T8203</td><td></td></tr><tr><td>rabbit anti-acH4K16</td><td>Upstate Biotechnology</td><td>07-329</td><td></td></tr><tr><td>AlexaFluor 488-coniugated donkey anti-mouse IgG</td><td>Life Technologies</td><td>A21202</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole</td><td>Sigma-Aldrich</td><td>D8417</td><td>DAPI</td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>RNALater</td><td>Invitrogen</td><td>AM7020</td><td></td></tr><tr><td>Luciferase RNA</td><td>Promega</td><td>L4561</td><td></td></tr><tr><td>PicoPure RNA Isolation Kit</td><td>Applied Biosystems</td><td>12204-01</td><td></td></tr><tr><td>random hexamers</td><td>Thermofisher Scientific</td><td>N8080127</td><td></td></tr><tr><td>mouse Moloney leukaemia virus reverse transcriptase</td><td>Thermofisher Scientific</td><td>28025013</td><td></td></tr><tr><td>SYBR green supermix</td><td>BioRad</td><td>1708880</td><td></td></tr><tr><td>specific primers</td><td>Sigma-Aldrich</td><td></td><td>specific primers were designed using Primer3Plus software (free resource)</td></tr><tr><td>thermal-cycler</td><td>BioRad</td><td>MyiQ</td><td></td></tr><tr><td>mouse monoclonal anti-CENPA</td><td>Abcam</td><td>ab13939</td><td></td></tr><tr><td>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

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

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 ...

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 ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

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 ...

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 ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

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

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Chapters in this video

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

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

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Human Egg Maturity Assessment and Its Clinical Application

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

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

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes