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

Forfatterne vil gerne takke Fabrice Vincent for supporten med laser scanning confocal microscopy (LSCM) , og Philippe Barrière og Thierry Blard for at udføre daglig ultralyd ovariescanning og hCG-injektion. Dette arbejde blev delvist støttet af &quot;Regione Sardegna og Regione Lombardia&quot; -projektet &quot;Ex Ovo Omnia&quot; (tilskud nr. 26096200 til AML); &quot;L&#39;Oreal Italia per le Donne e la Scienza 2012&quot; fællesskab (kontrakt 2012 til FF), FP7-PEOPLE-2011- CIG, forskningsforvaltningsorganet (REA) &quot;Pro-Ovum&quot; (tilskud nr. 303640 til VL); Og af Postdoktorskolen for Landbrug og Veterinærmedicin, medfinansieret af Den Europæiske Socialfond, Sektorielt Operationelt Program for Menneskelige Ressourceudvikling 2007-2013 (Kontrakt nr. POSDRU / 89 / 1.5 / S / 62371 til IM). In vivo- oocytopsamlingen blev finansieret af Institut Français du Cheval et de l&#39;Equitation.

Alle procedurerne blev godkendt af Dyrepleje- og brugskomitéen CEEA Val de Loire nummer 19 og blev udført i overensstemmelse med de vejledende principper for pleje og brug af laboratoriedyr. 1. Oocytopsamling og In vitro modning <ol><li> Ovum pick-up <ol><li> Dagligt vurder ved ultralyd diameteren af ​​æggestokkene i en kohorte af voksne hopper. Ved fremkomsten af ​​en follikel ≥33 mm (dominerende follikel) injiceres hoppen med 1500 IE af humant chori...

<ol>
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Selv om IVM er blevet udført på heste i mere end 20 år 16 , ved vi endnu ikke, om oocyten kan være oprindelsen af ​​embryonal aneuploidi, som det er blevet foreslået for mennesker 17 . Årsagen er sandsynligvis, at fremstillingen af ​​oocyt-spredninger til kromosometælling resulterer i et betydeligt prøveab. Med dette i betragtning blev en undersøgelse af metoderne til undersøgelse af fejl i kromosomsegregation udført for at søge den mest hensigtsmæssig...

En bred vifte af assisterede reproduktionsteknikker er blevet udviklet til behandling af infertilitet hos kvinder, ledsagere og truede arter. En af de mest almindelige procedurer i kliniske indstillinger er hentning af metafase II (MII) -stadieocytter fra ovariefolliklerne ved ultralydstyret transvaginal aspiration, opsamling af æg (OPU) 1 . Disse oocytter befrugtes derefter in vitro (IVF), med det resulterende embryo (er) implanteret i en recipient uterus. MII-stadie (modne) oocytter hentes efter administration af eksogene gonadotropiner. Denne behandling er imidlertid forbundet, hos nogle patienter, med udviklingen af ​​ovarie hyperstimulationssyndrom (OHSS) 2 . At drage fordel af den egentlige evne hos fuldt dyrkede, umodne oocytter (GV-stadium) til spontant at genoptage meioser, når de er isoleret fra deres follikler, er det muligt at opnå modne oocytter uden administrationGonadotropin 3 . Denne procedure kaldes oocyt in vitro modning (IVM) og repræsenterer en mindre lægemiddelorienteret, billigere og mere patientvenlig tilgang til assisteret reproduktiv teknologi. Succesen med embryonudvikling med in vitro- modnede oocytter er imidlertid generelt lavere end hos in vivo modnede oocytter 4 , 5 . En mulig forklaring er, at in vitro- modnede oocytter påvirkes mere af fejl i kromosomsegregation, og den resulterende aneuploidi nedsætter normal embryonal udvikling 6 . Forståelse af det molekylære grundlag for kromosom mis-segregering under IVM vil i sidste ende afsløre det fulde potentiale ved denne teknik. I denne vene anvendte den eksperimentelle tilgang til at undersøge de morfologiske og biokemiske egenskaber hos in vitro- modnede oocytter sammenlignet med in vivo modEd oocytter er her beskrevet 7 , 8 . Specifikt er procedurerne for OPU af umodne og modne oocytter og IVM af umodne oocytter illustreret under anvendelse af voksne og naturligt cykliske heste som en eksperimentel model. Derefter anvendes immunofluorescens og billedanalyse til at undersøge chromosomsegregation, spindelmorfologi og det globale mønster af histonacylering på disse gameter. Endelig beskrives en protokol for revers-transkription og kvantitativ PCR til analyse af mRNA-ekspression. Sammenlignet med gnaverdyrmodeller tillader heste ikke genetisk manipulation, er det mindre let at manipulere og kræver dyr vedligeholdelse. Denne model får imidlertid stor interesse for undersøgelsen af ​​oocytmodning 9 , 10 på grund af ligheden med menneskelig ovariefysiologi 11 , 12 &lt;/ Sup&gt;. Desuden har udviklingen af ​​pålidelige protokoller af IVM-IVF i hesten en væsentlig økonomisk interesse, da det ville muliggøre en forøgelse af antallet af føl fra marer af høj genetisk værdi. En af begrænsningerne ved at udføre forsøg på oocytter, især i monovulatoriske arter, er den begrænsede prøve tilgængelighed. Denne grænse er blevet overvundet her ved at justere en fremgangsmåde, der tidligere er udviklet i mus, til hesteocyter 13 , 14 for at udføre kromosometælling, der minimerer prøveforløbet (se diskussionen for en sammenligning med andre tilgængelige teknikker). Desuden er en triple-fluorescensfarvningsprotokol optimeret til at udføre flere analyser på den samme prøve, og q-PCR-analyser blev kun udført på puljer med kun 2 oocytter. Samlet set beskriver den foreliggende undersøgelse en eksperimentel tilgang, der har til formål at morfologisk og biokemisk chaRacterize hest oocyt, en celletype, der er ekstremt udfordrende at studere på grund af den lave prøve tilgængelighed. Det kan dog udvide vores viden om reproduktiv biologi og infertilitet hos monovulatoriske arter.

<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>
		</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>
		</tr>
		<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>
		</tr>
		<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>
		</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>
		</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

Området for assisteret reproduktion er udviklet til behandling af infertilitet hos kvinder, ledsagere og truede arter. I hesten tillader assisteret reproduktion også produktion af embryoner fra højtydende uden at afbryde deres sports karriere og bidrager til en stigning i antallet af føl fra marer af høj genetisk værdi. Det nuværende manuskript beskriver de procedurer, der anvendes til at indsamle umodne og modne oocytter fra hestevacci ved anvendelse af ægoptagelse (OPU). Disse oocytter blev derefter brugt til at undersøge forekomsten af ​​aneuploidi ved at tilpasse en protokol, der tidligere var udviklet hos mus. Specifikt blev kromosomer og centromerer af metafase II (MII) oocytter mærket fluorescens og talt på sekventielle fokalplaner efter konfokal lasermikroskop scanning. Denne analyse afslørede en højere forekomst i aneuploiditetsfrekvensen, når umodne oocytter blev opsamlet fra folliklerne og modnet in vitro i forhold tilIn vivo. Immunostaining for tubulin og den acetylerede form af histon 4 ved specifikke lysinrester afslørede også forskelle i den meotiske spindels morfologi og i det globale mønster af histonacylering. Endelig blev ekspression af mRNA&#39;er kodende for histon-deacetylaser (HDAC&#39;er) og acetyltransferaser (HAT&#39;er) undersøgt ved revers transkription og kvantitativ-PCR (q-PCR). Ingen forskelle i det relative ekspression af transkripter blev observeret mellem in vitro og in vivo modnede oocytter. I overensstemmelse med en generel afstødning af transkriptionsaktiviteten under oocytmodning kan analysen af ​​det totale transkriptionsbeløb kun afsløre mRNA-stabilitet eller nedbrydning. Disse resultater viser derfor, at andre oversættelses- og posttranslationelle bestemmelser kan påvirkes. Samlet set beskriver den foreliggende undersøgelse en eksperimentel tilgang til morfologisk og biokemisk karakterisering af hesteocyten, en ceLl type, der er ekstremt udfordrende at studere på grund af lav sample tilgængelighed. Det kan dog udvide vores viden om reproduktiv biologi og infertilitet hos monovulatoriske arter.

De oprindelige fund af disse eksperimenter blev beskrevet i dybden tidligere 7 og er her beskrevet som et eksempel på resultater, som kan opnås under anvendelse af de beskrevne protokoller. Modningshastighed Af de 32 COC&#39;er, der blev hentet af OPU fra dominerende follikler, var 28 (88%) på MII-stadiet. Fjorten af ​​de 58 COC&#39;er op...

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

Dette manuskript beskriver en eksperimentel tilgang til morfologisk og biokemisk karakterisering af hesteocyter. Specifikt illustrerer det foreliggende arbejde, hvordan man opsamler umodne og modne hesteocyter ved hjælp af ultralydstyret ægoptagelse (OPU) og hvordan man undersøger kromosomsegregation, spindelmorfologi, global histonacetylering og mRNA-ekspression.

Analyse af kromosomsegregation, histonacetylering og spindelmorfologi i hesteococytter

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

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

Ekspression af fluorescerende proteiner i<em&gt; Branchiostoma lanceolatum</em&gt; Af mRNA Injektion i befrugtede oocytter

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

Analyse af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

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

Isolering og karakterisering af muse Antral Oocyter Baseret på 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 ...

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

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

Rumlig og tidsmæssig analyse af Active ERK i<em&gt; C. elegans</em&gt; Kimcellelinje

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

Generering af genetisk modificerede mus gennem mikroinjektion af oocytter

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

Levende celle billeddannelse af kromosom Segregation under mitosen

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

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Evaluering af gødskning tilstand ved at spore sperm nuklear morfologi i Arabidopsis dobbelt befrugtning

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

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

Visualisering og analyse af Pharyngeal Arch Arterier ved hjælp af fuld-mount Immunohistochemistry og 3D Rekonstruktion

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

Minimum volumen vitrifikation af umodne Feline Oocytter

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

In Vitro Kultur strategi for Oocytter fra Early Antral Follikel hos Kvæg

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