Name: analysis of chromosome segregation, histone acetylation, and spindle morphology in horse oocytes
Uploaded: 2017-05-11T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Chromosome Segregation, Histone Acetylation, and Spindle Morphology in Horse Oocytes at JoVE.com

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 ...

<ol>
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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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">human chorionic gonadotrophin&#160;</td>
			<td>centravet</td>
			<td>CHO004</td>
			<td>1500unit/animal IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">detomidine</td>
			<td>centravet</td>
			<td>MED010</td>
			<td>9-15&#181;g/kg IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">butylscopolamine bromure&#160;</td>
			<td>centravet</td>
			<td>EST001</td>
			<td>0,2mg/kg IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stereomicroscope</td>
			<td style="width:159px;">NIKON</td>
			<td style="width:113px;">SMZ-2B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">butorphanol</td>
			<td>centravet</td>
			<td>DOL003</td>
			<td>10&#181;g/kg IV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">benzyl-penicillin</td>
			<td>centravet</td>
			<td>DEP203 IM</td>
			<td>15000UI/animal</td>
		</tr>
		<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>
			<td></td>
		</tr>
		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">YOPRO1</td>
			<td>Thermofisher Scientific</td>
			<td>Y3603</td>
			<td></td>
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			<td height="21" style="height:21px;">confocal laser scanning microscope&#160;</td>
			<td>LSM 780&#160;</td>
			<td>Zeiss</td>
			<td></td>
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		<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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			<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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			<td height="21" style="height:21px;">mouse anti-alpha-tubulin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8203</td>
			<td></td>
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			<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;">centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
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		<tr height="21">
			<td height="21" style="height:21px;">thermal-cycler&#160;</td>
			<td>BioRad</td>
			<td>MyiQ&#160;</td>
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			<td height="21" style="height:21px;">mouse monoclonal anti-CENPA</td>
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			<td>Abcam</td>
			<td>ab3609</td>
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	</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

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 ...

The overall goals of this experimental approach are to, first, morphologically and biochemically characterize horse oocytes and, second, to compare gametes from the same animal that were processed in vitro or in vivo. This experimental approach can help answer key questions in oocyte biology and fertility in monovulatory species. It might also improve the safety of gamete manipulation procedures.This approach was developed to overcome the restricted sample availability of oocytes, especially from monovulatory species. Demonstrating the OPU procedures will be Professor Stefan Deleuze of the University of Liege, Cecile Douet, the lab manager of my lab, and Fabrice Reigner, the manager of the equine unit at the INRA Centre Val de Loire. On a daily basis, assess the diameter of the ovarian follicles in a cohort of adult mares using ultrasound.Search for a large follicle with a diameter greater than 33 millimeters. Once found, prepare to inject 1, 500 units of human chorionic gonadotropin to induce the maturation of the oocyte into a dominant follicle. Next, locate the jugular furrow, and swab the injection site with alcohol.Then, to make the vein rise, apply some force two or three centimeters below the site of injection. Insert the needle almost parallel to the neck, aspirate slightly to ensure that the needle is in the vein, and inject the hormone. Prepare an ultrasound probe for vaginal access.Then, connect the needle to a suction system. 35 hours after hCG injection, set up the instrumentation for OPU, and sedate the mare. Good analgesia is extremely important for the animal's and the operator's safety.An animal that does not feel pain or discomfort will allow manipulation of the ovary, making punctures and flushes easier to perform. Once the needle probe is inserted, one operator must manipulate the ovary to face the probe while the other operator manipulates the needle. Locate the dominant follicle in the echographic image.Then, use the needle to pierce the vaginal wall and the wall of the dominant follicle. Once the follicle space is accessed, aspirate the follicular fluid. Rotate the needle to scrape the follicular wall and attach the cumulus-oocyte complex.Then, pour the fluid into a large petri dish. And under a stereoscope, search for the COC. Meanwhile, flush out the follicle with warm Dulbecco's modified PBS containing heparin.It may take several flushes to collect the COC. After the COC is found, change the needle to a double-lumen needle and collect the immature COCs from all of the follicles that are between five and 25 millimeters in diameter. These will be used for in vitro maturation.Once all of the follicles from one ovary are aspirated, repeat the same procedures to collect the immature COCs from the other ovary. Now, choose between the COCs. Keep only COCs with several complete layers of cumulus cells and a brown, finely-granulated ooplasm.Discard the others. At the end of the procedure, inject the mare with 15, 000 units of benzyl-penicillin to prevent infection. Then, house the mare in a quiet, clean stable for at least two hours while she recovers.Before the OPU starts, warm a bottle of supplemented HEPES-buffered TCM-199 to 37 degrees Celsius. Next, prepare the IVM medium. Dispense 500 microliters of supplemented sodium bicarbonate-buffered TCM-199 into the wells of a four-well dish.Then, incubate the dish at 38.5 degrees Celsius for at least two hours. Transfer the recovered immature COCs to a 3.5-centimeter dish filled with two milliliters of the warmed HEPES TCM-199. Move the COCs through different regions of the dish to gently wash away surrounding debris.Now, transfer the COCs to the IVM medium, and incubate them for 28 hours at 38.5 degrees Celsius. To perform a chromosome count on mature COCs, incubate them in 100-micromolar monastrol for an hour at 38.5 degrees Celsius. Next, remove the cumulus cells by treating the COCs with 0.5%hyaluronidase in HEPES-buffered TCM-199.Monitor this reaction on a heated stage set to 38 degrees Celsius. Within about five minutes, remove all of the cells. Use gentle pipetting to remove the last few stubborn cells.Next, remove the zona pellucida using 0.2%pronase in HEPES-buffered TCM-199. Monitor this reaction on a heated stage as well. It should take about five minutes to complete.Then, fix the oocytes in 4%paraformaldehyde in DPBS for 15 minutes at 38.5 degrees Celsius, followed by 45 more minutes of fixation at four degrees Celsius. After the fixation, wash the oocytes by sequentially transferring them between three wells of 0.1%polyvinyl alcohol in DPBS. Use about half a milliliter of solution per well.Then, transfer the oocytes to a well filled with 0.3%Triton X-100 in PBS, and let them incubate for 10 minutes at room temperature. To block non-specific signals, bathe the cells in DPBS with 1%BSA and 10%normal donkey serum. Incubate the cells for at least 30 minutes at room temperature.After 30 minutes, apply the primary antibody, and then incubate the cells at four degrees Celsius overnight. The following day, wash the oocytes, and apply the secondary antibody for an hour at room temperature, shielded from light exposure. After applying the secondary, wash the oocytes again.Then, before mounting, attach double-sided tape strips to slides to serve as coverslip supports. Now, mount the oocytes three to four per slide in a drop of non-hardening, antifade mounting medium supplemented with 20 micromolar of YO-PRO-1. Allow the oocytes to settle for about 10 minutes, and then apply the coverslips.Out of 32 COCs retrieved by OPU from dominant follicles, 28 were in metaphase II.Using in vitro maturation, 37 of 44 COCs collected from non-dominant follicles were matured to metaphase II.The chromosomes of both groups were then compared. The in vitro-matured oocytes were often affected by aneuploidy, mostly hyperploidy. This was not observed in the in vivo-matured oocytes.Next, the pole-to-pole spindle length and the diameter of the spindle at its maximum were both measured. The in vitro-matured oocytes were significantly longer and wider than the in vivo-matured oocytes. In the same samples, the global acetylation level of H4K16 was measured.The results were as expected. Acetylation of H4K16 was significantly lower in in vitro-matured oocytes than in in vivo-matured oocytes. A well-trained team can process five mares in a morning of OPU with an 80%retrieval rate for the preovulatory oocyte and 50 to 60%retrieval of the immature oocytes.During the OPU, always monitor the ess and well-being of the animal, and be ready to provide adequate veterinary care if needed. The experimental approach can be applied to oocytes of other species as well and is especially applicable where samples availability is an issue, such as with humans. Based on our results, studies must be undertaken to better understand the culture conditions needed for proper chromosome segregation and epigenetic changes.The overarching goal is to improve in vitro maturation efficiency for both veterinary medicine and human fertility treatment.

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