Name: chromatin spread preparations for the analysis of mouse oocyte progression from prophase to metaphase ii
Uploaded: 2018-02-26T13:00:00
Description: Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

This work was supported by NIGMS (R01GM11755) to P.W.J and by a training grant fellowship from the National Cancer Institute (NIH) (CA009110) to G.H. and J.H.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Johns Hopkins University. Experiments were performed on wild-type C57BL/6J mice.
1. Harvesting Fetal or Neonatal Ovaries and Preparation of Prophase Chromatin Spreads
<ol>
	<li>To extract embryos at 14.5&#8211;19.5 days post-coitum (dpc) sacrifice the pregnant females via cervical dislocation or CO2 asphyxiation according to IACUC guidelines. 
	NOTE: For postnatal day 1...

<ol>
	<li>Morelli, M. A., Cohen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+all+germ+cells+are+created+equal:+aspects+of+sexual+dimorphism+in+mammalian+meiosis.">Not all germ cells are created equal: aspects of sexual dimorphism in mammalian meiosis.</a> Reproduction. 130 (6), 761-781 (2005).</li><li>Keeney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spo11+and+the+Formation+of+DNA+Double-Strand+Breaks+in+Meiosis.">Spo11 and the Formation of DNA Double-Strand Breaks in Meiosis.</a> Genome Dyn Stab. 2, 81-123 (2008).</li><li>Zickler, D., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination,+Pairing,+and+Synapsis+of+Homologs+during+Meiosis.">Recombination, Pairing, and Synapsis of Homologs during Meiosis.</a> Cold Spring Harb Perspect Biol. 7 (6), (2015).</li><li>Vries, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Sycp1+functions+in+synaptonemal+complex+assembly,+meiotic+recombination,+and+XY+body+formation.">Mouse Sycp1 functions in synaptonemal complex assembly, meiotic recombination, and XY body formation.</a> Genes Dev. 19 (11), 1376-1389 (2005).</li><li>Baker, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+mouse+Mlh1+in+DNA+mismatch+repair+and+meiotic+crossing+over.">Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.</a> Nat Genet. 13 (3), 336-342 (1996).</li><li>Lipkin, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+arrest+and+aneuploidy+in+MLH3-deficient+mice.">Meiotic arrest and aneuploidy in MLH3-deficient mice.</a> Nat Genet. 31 (4), 385-390 (2002).</li><li>Kolas, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+MMR+proteins+on+meiotic+chromosomes+in+mice+indicates+distinct+functions+during+prophase+I.">Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I.</a> J Cell Biol. 171 (3), 447-458 (2005).</li><li>Rankin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+elaboration:+making+sense+of+meiotic+cohesin+dynamics.">Complex elaboration: making sense of meiotic cohesin dynamics.</a> FEBS Journal. 282 (13), 2426-2443 (2015).</li><li>Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Cohesin+and+Condensin+in+Chromosome+Dynamics+During+Mammalian+Meiosis.">Roles of Cohesin and Condensin in Chromosome Dynamics During Mammalian Meiosis.</a> J. Reprod Dev. 59 (5), 431-436 (2013).</li><li>Verver, D. E., Hwang, G. H., Jordan, P. W., Hamer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+complex+chromosome+structures+during+meiosis:+versatile+deployment+of+Smc5/6.">Resolving complex chromosome structures during meiosis: versatile deployment of Smc5/6.</a> Chromosoma. 125 (1), 15-27 (2016).</li><li>Hopkins, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis-specific+cohesin+component,+Stag3+is+essential+for+maintaining+centromere+chromatid+cohesion,+and+required+for+DNA+repair+and+synapsis+between+homologous+chromosomes.">Meiosis-specific cohesin component, Stag3 is essential for maintaining centromere chromatid cohesion, and required for DNA repair and synapsis between homologous chromosomes.</a> PLoS Genet. 10 (7), e1004413 (2014).</li><li>Hwang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMC5/6+is+required+for+the+formation+of+segregation-competent+bivalent+chromosomes+during+meiosis+I+in+mouse+oocytes.">SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.</a> Development. 144 (9), 1648-1660 (2017).</li><li>Kota, S. K., Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+transitions+in+germ+cell+development+and+meiosis.">Epigenetic transitions in germ cell development and meiosis.</a> Dev Cell. 19 (5), 675-686 (2010).</li><li>Taketo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microspread+ovarian+cell+preparations+for+the+analysis+of+meiotic+prophase+progression+in+oocytes+with+improved+recovery+by+cytospin+centrifugation.">Microspread ovarian cell preparations for the analysis of meiotic prophase progression in oocytes with improved recovery by cytospin centrifugation.</a> Methods Mol Biol. 825 (1), 173-181 (2012).</li><li>Sun, X., Cohen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+recombination+in+mouse+oocytes.">Studying recombination in mouse oocytes.</a> Methods Mol Biol. 957 (1), 1-18 (2013).</li><li>Kim, J. H., Ishiguro, K., Kudo, N., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+meiosis-specific+cohesins+in+mouse+embryonic+oocytes.">Studying meiosis-specific cohesins in mouse embryonic oocytes.</a> Methods Mol Biol. 957 (1), 47-57 (2013).</li><li>Susiarjo, M., Rubio, C., 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=Analyzing+mammalian+female+meiosis.">Analyzing mammalian female meiosis.</a> Methods Mol Biol. 558, 339-354 (2009).</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>Hassold, T., Hall, H., 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=The+origin+of+human+aneuploidy:+where+we+have+been,+where+we+are+going.">The origin of human aneuploidy: where we have been, where we are going.</a> Hum Mol Genet. 16, R203-R208 (2007).</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>Chambon, J. 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Chromatin spread preparations allow researchers to chronologically study female mammalian meiosis and the dynamic localization of proteins involved. The embryonic and neonatal chromatin spreads allow for close analysis of events throughout meiotic prophase. Metaphase I and metaphase II chromatin spreads can be used to distinguish single sister chromatids from paired sisters and paired homologous chromosomes, as well as assess ploidy. By comparison, the protocol described here can be advantageous compared to whole oocyte ...

During spermatogenesis, large semi-synchronous waves of meiotic germ cells are rapidly and continuously replenished in the testis at the onset of puberty and throughout adulthood1. In contrast to males, meiosis in females is initiated solely during fetal development. Following birth, oocytes remain arrested in a prolonged dictyate stage of prophase I with an intact germinal vesicle (GV; nuclear envelope) until puberty. At the onset of puberty, a subset of oocytes are cyclically selected to undergo growth and maturation, marking the initiation of meiotic resumption. Meiotic resumption in fully-grown oocytes is manifested by the disappearance of the GV in a process known as germinal vesicle breakdown (GVBD). The oocyte then undergoes chromosome condensation and segregation, followed by polar body extrusion. Oocytes become arrested upon progression to MII and are stimulated to complete the second and final meiotic division only after fertilization.
Female fertility is highly dependent on the success of meiotic prophase I progression. Key to this is formation of a physical linkage between homologous chromosomes known as the chiasmata, which is mediated by repair of induced DNA double strand breaks (DSBs) via crossover recombination2. This process occurs within the context of a dynamic protein-rich scaffold known as the synaptonemal complex (SC) that forms between homologous chromosomes to facilitate their synapsis3. The SC is a zipper-like tripartite structure that consists of two parallel lateral elements connected by central region proteins that holds homologs together throughout ongoing DNA repair. Prior to synapsis, precursors of the lateral elements, called axial elements, form between sister chromatids. Synaptonemal complex proteins such as SYCP2 and SYCP3 form axial elements that colocalize to the sister-chromatid cohesion axes during early prophase. These later serve as binding sites for the transverse filament protein, SYCP1, which facilitates central element assembly and synapsis between aligned homologs4. In mouse oocytes, complete synapsis is indicated by the presence of 20 completely overlapping SYCP3 and SYCP1 stretches, which can be visualized by using chromatin spread preparations. Synapsis is completed upon entry into the pachytene substage, whereby mature crossovers that are destined to form chiasmata between homologs are decorated with mutL homolog (MLH1/3) dimers to promote their accurate processing5,6,7. The structural maintenance of chromosomes (SMC) complexes, including cohesin, condensin, and the SMC5/6 complex, are important for the regulation of chromosome dynamics and structure throughout meiosis8,9,10,11,12. Collectively, these events ensure proper bi-orientation of homologous chromosomes to opposing spindle poles following disassembly of the SC.
The meiotic cell cycle is a powerful model to examine the roles of various proteins in genome maintenance due to the programmed induction and subsequent repair of DNA DSBs. Furthermore, mammalian meiosis is also a relevant model for the study of epigenetic modifications and imprinting13. However, it is technically difficult to assess these events during female meiosis, which takes place in fetal and neonatal ovaries in mammals (Figure 1). Prophase I can be divided into 5 substages: leptonema, zygonema, pachynema, diplonema and dictyate. Herein, we describe how to isolate and distinguish between fetal and neonatal ovaries and testes (Figure 2). Adapted from previously described methods, this manuscript also outlines a protocol (step 1) with video demonstration for preparation of female meiotic prophase I chromatin spreads14,15,16,17. When coupled with immunolabelling, as described in steps 6-7, this protocol enables detailed microscopic analysis of prophase I events in oocytes.
Oogenesis is error-prone, and chromosome missegregation events during the first meiotic division represent the most common source of genetic disease in progeny18,19. In this manuscript (protocol step 2), we describe a protocol in which mature GV-staged oocytes are extracted from primed ovaries of adult female mice. Under supportive conditions, fully-grown oocytes undergo luteinizing hormone-independent resumption of meiosis following isolation and culture20. Following meiotic resumption, oocytes progress through meiosis I, then arrest at metaphase II. Oocytes remain arrested at metaphase II, unless fertilized. In steps 2&#8211;5, we adapt previously reported protocols with video demonstration to describe how to collect, culture and prepare MI and MII oocytes for chromatin spread preparations21. This chromatin spreading technique allows for clear immunolabelling of proteins associated with chromosomes. Furthermore, this protocol can also be used to distinguish between bivalent and univalent chromosomes, and can further resolve single sister chromatids to facilitate assessment of oocyte ploidy. Therefore, in addition to revealing localization patterns of meiotic proteins, this protocol can also serve as an invaluable tool for elucidating potential causes of chromosome missegregation during MI and MII.

<table>
	<tbody>
		<tr>
			<td>10X Phosphate buffered saline (PBS) pH 7.4</td>
			<td>Quality Biological</td>
			<td>119-069-161</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm x 15 mm petri dishes</td>
			<td>Denville</td>
			<td>T1106</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm x 12mm petri dishes</td>
			<td>Denville</td>
			<td>T1103</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>VWR</td>
			<td>300-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissection scissors</td>
			<td>Ted Pella</td>
			<td>1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160;scissors, sharp tip, 41/2&#34;</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Watch glass square 1 5/8</td>
			<td>Carolina Biological Supply Company</td>
			<td>742300</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S8501</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium Citrate Dihydrate</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>646563</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>50x Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Add to hypotonic buffer immediately before use</td>
		</tr>
		<tr>
			<td>Turberculin syringe with needle (1cc, 27 G x 1/2 in)</td>
			<td>Becton, Dickinson (BD)</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>Gold seal ultra frost glass slides (25X75mm, 1mm thick)</td>
			<td>Thermo</td>
			<td>3063-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP pen, large (liquid blocker)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>71310</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde aqueous</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Detergent in manuscript</td>
		</tr>
		<tr>
			<td>Immuno stain moisture chamber</td>
			<td>Evergreen</td>
			<td>240-9020-Z10</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jars</td>
			<td>Fisher</td>
			<td>08-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-flo 200</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>74257</td>
			<td>Wetting agent in manuscript</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin (PMSG)</td>
			<td>Sigma</td>
			<td>G4877</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Human chorionic gonadotropin (HCG)</td>
			<td>Sigma</td>
			<td>C0434</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>PYREX Spot Plates with nine concave cavities</td>
			<td>Fisher</td>
			<td>13-748B</td>
			<td>Autoclave before each use</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Fisher</td>
			<td>22260943</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Brand Parafilm M</td>
			<td>Sigma</td>
			<td>BR701501</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (3.2 mm inner diameter x 6.4 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5B</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Latex rubber tubing (6.4 mm inner diameter x 11.1 mm outer diameter)</td>
			<td>Fisher</td>
			<td>14-178-5D</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Flexible silicone rubber nosepiece, hard plastic mouthpiece and 15 inches of latex tubing</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Mitutoyo micrometer head</td>
			<td>Mituoyo</td>
			<td>150-208</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD Biosciences</td>
			<td>309646</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Syringe filter 0.45 &#956;m filter</td>
			<td>Corning</td>
			<td>431220</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>Glass Pasteur Pipette 5 3/4&#34;</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>83 x 0.5 mm pipette tip</td>
			<td>Denville</td>
			<td>P3080</td>
			<td>For making oocyte collection needles</td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Invitrogen</td>
			<td>11415049</td>
			<td></td>
		</tr>
		<tr>
			<td>Waymouth&#39;s media</td>
			<td>Life Technologies</td>
			<td>11220035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher</td>
			<td>A3160602</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td>For oocyte culture media</td>
		</tr>
		<tr>
			<td>500ml filter units 0.22um</td>
			<td>Denville</td>
			<td>F5227</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrode&#8217;s solution</td>
			<td>Sigma</td>
			<td>T1788</td>
			<td>Store aliquots at -20C</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H-1270</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A1470</td>
			<td>For ADB/wash buffer</td>
		</tr>
		<tr>
			<td>VECTASHIELD antifade mounting medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slides (22mmx60mm)</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>Amazon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SteREO Discovery.V8</td>
			<td>Zeiss</td>
			<td>495015-0001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Observer Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-MLH1</td>
			<td>Life Technologies</td>
			<td>MA5-15431</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti-SYCP1</td>
			<td>Life Technologies</td>
			<td>PA1-16763</td>
			<td>Dilution factor- 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-SCP3</td>
			<td>Santa Cruz</td>
			<td>sc-20845</td>
			<td>Dilution factor- 1:50</td>
		</tr>
		<tr>
			<td>Human anti-centromere protein</td>
			<td>Antibodies Incorporated</td>
			<td>15-235</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Rabbit anti- TOPOII</td>
			<td>Abcam</td>
			<td>ab109524</td>
			<td>Dilution factor- 1:100</td>
		</tr>
		<tr>
			<td>Mouse anti-Histone H4 (di methyl K20, tri methyl K20)</td>
			<td>Abcam</td>
			<td>ab78517</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Rabbit anti-Rec8</td>
			<td>Courtesy of Dr. Karen Schindler</td>
			<td>N/A</td>
			<td>Dilution factor- 1:10,000</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11057</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) Alexa Fluor 488 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-21202</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) Alexa Fluor 647 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-31573</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11004</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11013</td>
			<td>Dilution factor- 1:500</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Alexa Fluor 568 conjugate</td>
			<td>Thermo-Fisher Scientific</td>
			<td>A-11011</td>
			<td>Dilution factor- 1:500</td>
		</tr>
	</tbody>
</table>

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.

We have described two techniques for visualizing and assessing meiotic chromosomes in oocytes. The first technique is catered toward assessing prophase progression in embryonic and neonatal ovaries. Prophase chromatin spread preparations are incredibly valuable for visualizing numerous dynamic processes during meiosis, including chromosome pairing, synapsis and desynapsis, homologous recombination, and epigenetic chromosome remodeling. Here, we have demonstrated the utility of this method...

Watch this Scientific Journal Video about Chromatin Spread Preparations for the Analysis of Mouse Oocyte Progression from Prophase to Metaphase II at JoVE.com

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

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 ...

The overall goal of this chromatin spread preparation is to chronologically visualize the dynamic localization patterns of chromatin-bound proteins and chromosome morphology of oocytes throughout mammalian oogenesis. This method can help answer key questions in the field of female reproductive biology, such as homologous chromosome pairing, synapsis, DNA repair, and meiotic chromosome segregation. The main advantage of this technique is that it allows for clear immunolabeling and visualization of proteins associated with chromosomes and assessment of oocyte ploidy.Oogenesis is error-prone, and chromosome missegregation often results in genetic disease. This method can be used to improve our understanding of maternal causes of aneuploidy and infertility. After euthanizing a female mouse that is 14 to 19 days post-coitum, make a V-shaped opening into the abdominopelvic cavity.Then remove the maternal uterine horn. Next separate the embryos from the placenta, and sacrifice the embryos by decapitation. Then transfer them to 35-millimeter Petri dish containing three milliliters of PBS at 37 degrees Celsius.Then move one pup to a separate dish also loaded with warm PBS. After euthanizing the fetus, make a cut along the ventral midline of the posterior half, along the anterior half below the forelimbs, and directly above the hindlimbs and tail. Next open the embryo's abdomen using 3.5-inch scissors.Using fine-tipped forceps, displace or remove the liver and loops of bowel, exposing the gonads. Now determine if the gonads are male or female gonads. The ovaries are located immediately below and behind the kidney, toward the posterior wall of the peritoneal cavity.Remove both ovaries using a pair of fine-tipped forceps, and transfer them into a small container containing PBS that is maintained at 37 degrees Celsius. Proceed quickly to collect the ovaries from all of the fetuses. Next in a new small container, completely immerse each separate set of ovaries in half a milliliter of freshly made hypo-extraction buffer.Let them incubate at 25 degrees Celsius for at least 15 minutes, but not more than 30 minutes. During the incubation, use a hydrophobic barrier PAP pen to draw two 22 by 22 millimeter squares on a clean glass slide. Prepare one slide for each set of ovaries.When the incubation is complete, add a 50-microliter drop of 100 millimolar sucrose to a clean slide, and transfer one pair of ovaries into the drop. Now use two 27-gauge needles to tease the ovaries and release the cells therein. With forceps, remove and discard the large pieces of ovary.Then carefully pipette the sucrose solution to disperse the released cells. Next load each square on a slide with 40 microliters of 1%PFA with 0.2%Triton X-100. Spread the solution around the square using a pipette tip or by rocking the slide back and forth.Then for each pool of sucrose solution with cells, load two fixative puddles with 20 microliters of suspended cells so that each slide has cells from one set of ovaries. For either MI or MII oocyte collections, first prepare and euthanize the donor mouse. Then make a large V-shaped opening in the abdominopelvic cavity, and use forceps to displace the intestines to locate each uterine horn.Find the ovaries proximal to the rib cage. Hold the oviduct with fine forceps, and cut the fat superior to the ovary using dissection scissors. While still holding the oviduct, use fine forceps to release the ovary from the bursa, and transfer the ovaries into a collection dish containing medium maintained at 37 degrees Celsius.To collect GV-stage oocytes, use a one-milliliter syringe with a 27-gauge needle to release the cumulus-oocyte complexes by manually puncturing the large antral follicles. To collect MII stage oocytes, tear a hole in the ampulla of the oviduct to release them rather than simply making a puncture. Now collect the oocytes using a mouth-operated glass pipette or capillary, or use a hand-operated micrometer syringe.Some oocytes may be surrounded with cumulus cells, others not. For the MI oocytes, prepare fresh hyaluronidase in MEM-alpha/BSA to denude the oocytes of the surrounding cumulus cells. For each collection of oocytes, treat them in an incubator with 2.5 milliliters of prepared hyaluronidase for three minutes.Then transfer the MI oocytes to freshly made warm MEM-alpha/BSA medium. There, use a mouth-operated glass pipette to provide the shearing force needed to completely detach the cumulus cells. In general, the success of this procedure requires the mastery of oocyte collection and manipulation using mouth-operated glass pipettes or capillaries.Allow the oocytes to recover in the incubator. Meanwhile, into a heated nine-well glass plate, load 300 microliters of warm Tyrode's solution. Load two wells with 300 microliters of warm MEM-alpha/BSA, and load one well with 300 microliters of warm Waymouth's medium.To remove the zona pellucida, transfer five to 10 MI or MII-stage oocytes into the bath of Tyrode's solution for just 30 to 45 seconds while watching them under a dissection microscope. When the zona pellucida is visibly dissolved, immediately transfer the oocytes to the medium using a pipette pre-wet with MEM-alpha/BSA medium. Then transfer the oocytes to a second bath of warmed medium.After the second bath, transfer the oocytes to warm Waymouth's medium, and let them recover in an incubator for 30 minutes. To prepare for a chromatin spread, use a PAP pen to draw an 11 by 44 millimeter rectangle on a glass slide. Next coat the slide with a thin layer of 1%PFA with 0.2%Triton X-100 in water, and then rock the slide back and forth to spread the solution.Then tap the slide to remove the excess. Now siphon between five and 10 oocytes with a minimal amount of medium. Then transfer the oocytes to the prepared slide by simultaneously dragging a pipette tip through the fixative solution and dropping the oocytes onto the slide from about one centimeter above.The oocytes should burst immediately, spreading chromatin on the slide. Now incubate slides at room temperature in a closed humidified chamber. Let them settle overnight so the chromatin adheres to the slide.The described embryonic chromatin collection was used to quantitatively analyze crossover formation in oocytes harvested from C57 black/6J embryos. Prophase chromatin spread preparations were immunolabeled to detect SYCP3, SYCP1, and MLH1, and stained with DAPI. Pachytene-stage chromosomes had 27 MLH1 foci distributed along 20 fully assembled SC structures.When the spread preparation was suboptimal and individual chromosomes were indistinguishable, the preparation was not included in the assessment. In good preparations, SMC6 was enriched at the pericentromeric heterochromatin region and along the chromosome arms, and the MLH1 foci distributed along the SC at the pachytene stage. The number of MLH1-positive crossovers from 15 good wild-type preparations was about 24.The oocyte collection method was used to assess chromatin morphology in oocytes following meiotic resumption. Protein localization, as well as ploidy, was easily assessed, exposing potential causes of chromosome segregation errors. Topoisomerase II-alpha could be seen along the chromosome arms and the pericentromeric heterochromatin.In MII oocytes, paired sister chromatids could be distinguished by chromosome and centromere morphology. Once mastered, these techniques without or after culturing can be completed within one or two hours per mouse. Following this procedure, other complementary methods, like whole cell oocyte immunolabeling and live cell imaging can be performed to assess homologous chromosomes and sister chromatids within the context of the cell.Collectively, chromatin spread methods described here can be easily applied to assess mammalian oogenesis and the sexually dimorphic features of chromosome and protein dynamics during meiosis.

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