Name: using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes
Uploaded: 2017-12-06T15:00:00
Description: Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

This work was supported by NIH Grant GM59354 awarded to Sharon E. Bickel. We thank Huy Q. Nguyen for assistance in developing the protocol for generating fluorescent arm probes, Ann Lavanway for help with confocal microscopy, and J. Emiliano Reed for technical assistance. We also thank numerous colleagues in the Drosophila community for helpful discussions and advice.

1. Preparations
<ol>
	<li>Prepare solutions for fluorescence in situ hybridization (FISH). Prepare all solutions using ultrapure water.
	<ol>
		<li>Prepare 5x&#160;Modified Robb&#39;s buffer: 275 mM potassium acetate, 200 mM sodium acetate, 500 mM sucrose, 50 mM glucose, 6 mM magnesium chloride, 5 mM calcium chloride, 500 mM HEPES pH = 7.4. Bring the pH to 7.4 using 10 N NaOH. Filter sterilize and store at -20 &#176;C. Thaw and dilute to 1x&#160;as needed and store 1x&#160;aliquots at -20 &#17...

<ol>
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B., 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=Disjunction+of+homologous+chromosomes+in+meiosis+I+depends+on+proteolytic+cleavage+of+the+meiotic+cohesin+Rec8+by+separin.">Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.</a> Cell. 103 (3), 387-398 (2000).</li><li>Bickel, S. E., Orr-Weaver, T., Balicky, 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+sister-chromatid+cohesion+protein+ORD+is+required+for+chiasma+maintenance+in+Drosophila+oocytes.">The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes.</a> Curr. Biol. 12 (11), 925-929 (2002).</li><li>Hodges, C. A., Revenkova, E., Jessberger, R., Hassold, T. J., Hunt, P. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+cohesin+is+associated+with+age-dependent+misaligned+chromosomes+in+oocytes.">Defective cohesin is associated with age-dependent misaligned chromosomes in oocytes.</a> Reprod Biomed Online. 16 (1), 103-112 (2008).</li><li>Subramanian, V. V., Bickel, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+predisposes+oocytes+to+meiotic+nondisjunction+when+the+cohesin+subunit+SMC1+is+reduced.">Aging predisposes oocytes to meiotic nondisjunction when the cohesin subunit SMC1 is reduced.</a> PLoS Genet. 4 (11), e1000263 (2008).</li><li>Chiang, T., Duncan, F. E., Schindler, K., Schultz, R. M., Lampson, M. 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The use of FISH probes to assess the state of arm cohesion in prometaphase I and metaphase I Drosophila oocytes is a significant advancement in the field of Drosophila meiosis. Historically, Drosophila researchers have been limited to genetic tests to infer premature loss of arm cohesion in mature oocytes11,18,19. Now, with the methods presented here, the state of arm cohesion can be assayed directly u...

Proper segregation of chromosomes during mitosis and meiosis requires that sister chromatid cohesion be established, maintained, and released in a coordinated fashion1,2. Cohesion is established during S phase and is mediated by the cohesin complex, which forms physical linkages that hold the sister chromatids together. In meiosis, cohesion distal to a crossover also functions to hold recombinant homologs together and this physical association helps ensure proper orientation of the bivalent on the metaphase I spindle (Figure 1)3,4,5. Release of arm cohesion at anaphase I allows the homologs to segregate to opposite spindle poles. However, if arm cohesion is lost prematurely, recombinant homologs will lose their physical connection and segregate randomly, which can result in aneuploid gametes (Figure 1).
In human oocytes, errors in chromosome segregation are the leading cause of miscarriages and birth defects, such as Down Syndrome6, and their incidence increases exponentially with maternal age7. Sister chromatid cohesion is established in fetal oocytes and meiotic recombination is completed before birth. Oocytes then arrest in mid-prophase I until ovulation and during this arrest, the continued physical association of recombinant homologs relies on sister chromatid cohesion. Therefore, accurate segregation during meiosis and normal pregnancy outcomes require that cohesion remain intact for up to five decades.
Premature loss of cohesion during the prolonged meiotic arrest of human oocytes has been proposed to contribute to the maternal age effect and multiple lines of evidence support this hypothesis8,9. However, given the challenges of studying meiotic cohesion in human oocytes, much of our understanding of this phenomenon relies on the use of model organisms5,10,11,12,13,14,15.
Drosophila melanogaster oocytes offer numerous advantages for the study of meiotic cohesion and chromosome segregation. A simple genetic assay allows one to recover progeny from aneuploid gametes and measure the fidelity of X-chromosome segregation on a large scale11,16,17. Moreover, one may also determine whether chromosome segregation errors arise because recombinant homologs missegregate during meiosis I, a phenotype that is consistent with premature loss of arm cohesion11,18,19. Direct observation of the state of meiotic cohesion in Drosophila oocytes is also possible using fluorescence in situ hybridization (FISH). Although fluorescent oligonucleotides that hybridize to repetitive satellite sequences have been used for over a decade to monitor pericentromeric cohesion in mature Drosophila oocytes4,20, analysis of arm cohesion has been much more challenging. Visualization of the state of arm cohesion requires a probe that spans a large region of single copy sequences and is bright enough to result in visible signals for individual sister chromatids when arm cohesion is absent. In addition, the oocyte fixation conditions and size of the labeled DNAs must facilitate penetration21 into the large mature Drosophila oocyte (200 &#181;m wide by 500 &#181;m long). Recently, an arm probe was successfully utilized to visualize Drosophila oocyte chromatids during anaphase I, but the authors stated that they could not detect a signal in prometaphase or metaphase I arrested oocytes22. Here we provide a detailed protocol for the generation of X-chromosome arm FISH probes and oocyte preparation conditions that have allowed us to assay for premature loss of sister chromatid cohesion in prometaphase I and metaphase I oocytes. These techniques, which have enabled us to identify gene products that are required for the maintenance of meiotic cohesion, will allow others to assay for sister chromatid cohesion defects in mature Drosophila oocytes of different genotypes.

<table>
	<tbody>
		<tr>
			<td>Kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midi Prep kit</td>
			<td>Qiagen</td>
			<td>12143</td>
			<td>Prep BAC clone DNA</td>
		</tr>
		<tr>
			<td>GenomePlex Complete Whole Genome Amplification (WGA) Kit</td>
			<td>Sigma</td>
			<td>WGA2</td>
			<td>Amplify BAC clone DNA</td>
		</tr>
		<tr>
			<td>ARES Alexa Fluor 647 DNA labeling kit</td>
			<td>Invitrogen</td>
			<td>A21676</td>
			<td>Label BAC clone DNA</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Remove non-conjugated dye following labeling of BAC clone DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals &#38; Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td>Prepare 10% stock 
			Freeze aliquots</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C75-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>Toxic: wear appropriate protection. Prepare 100&#181;g/ml stock in 100% ethanol, store in aliquots at -20 &#176;C. Prepare 1 &#181;g/ml solution in 2X SSCT before use.</td>
		</tr>
		<tr>
			<td>Dextran sulfate</td>
			<td>Sigma</td>
			<td>D-8906</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite</td>
			<td>Drierite Company</td>
			<td>23001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Invitrogen</td>
			<td>15508-013</td>
			<td>Prepare 10 mM stock</td>
		</tr>
		<tr>
			<td>dTTP (10 &#181;mol, 100 &#181;l)</td>
			<td>Boehringer Mannheim</td>
			<td>1277049</td>
			<td>Prepare 1 mM stock</td>
		</tr>
		<tr>
			<td>EDTA (Disodium ethylenediamine tetraacetic acid)</td>
			<td>Fisher Scientific</td>
			<td>S311-500</td>
			<td>Prepare 250 mM stock</td>
		</tr>
		<tr>
			<td>100% ethanol (molecular grade, 200 proof)</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Ultra Pure)</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA (Ethylenebis(oxyethylenenitrilo)tetraacetic acid)</td>
			<td>Sigma</td>
			<td>E-3889</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Ted Pella, Inc.</td>
			<td>18505</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>AM9342</td>
			<td>Toxic: wear appropriate protection</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>901393</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Boehringer Mannheim</td>
			<td>737-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>Fisher Scientific</td>
			<td>H350-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Millipore</td>
			<td>HX0603-4</td>
			<td>Toxic: wear appropriate protection.</td>
		</tr>
		<tr>
			<td>Hydroxylamine</td>
			<td>Sigma</td>
			<td>438227</td>
			<td>Prepare 3 M stock</td>
		</tr>
		<tr>
			<td>4.9 M Magnesium chloride</td>
			<td>Sigma</td>
			<td>104-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 &#376; 7H2O</td>
			<td>Fisher Scientific</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 &#376; 2H2O</td>
			<td>Fisher Scientific</td>
			<td>S369-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (0.1mg/ml)</td>
			<td>Sigma</td>
			<td>P8920-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Fisher Scientific</td>
			<td>BP364-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Fisher Scientific</td>
			<td>S209-500</td>
			<td>Prepare 3M stock</td>
		</tr>
		<tr>
			<td>Sodium cacodylate</td>
			<td>Polysciences, Inc.</td>
			<td>1131</td>
			<td>Toxic: wear appropriate protection. Prepare 400mM stock</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td>BP327-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Tween 20</td>
			<td>Thermo Scientific</td>
			<td>28320</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>10% Triton X-100</td>
			<td>Thermo Scientific</td>
			<td>28314</td>
			<td>Surfact-Amps</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Note: All solutions are prepared using sterile ultrapure water and should be sterilized either by autoclave or filter sterilization.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TE buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris, 1 mM EDTA, pH = 8.0</td>
		</tr>
		<tr>
			<td>20X SSC (Saline Sodium Citrate)</td>
			<td></td>
			<td></td>
			<td>3 M NaCl, 300 mM sodium citrate</td>
		</tr>
		<tr>
			<td>2X cacodylate fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 200 mM sodium cacodylate, 200 mM sucrose, 80 mM sodium acetate, 20 mM EGTA</td>
		</tr>
		<tr>
			<td>1.1X Hybridization buffer</td>
			<td></td>
			<td></td>
			<td>3.3X SSC, 55% formamide, 11% dextran sulfate</td>
		</tr>
		<tr>
			<td>Fix solution</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 4% formaldehyde, 1X cacodylate fix solution</td>
		</tr>
		<tr>
			<td>PBSBTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 0.5% BSA, 0.1% Trition X-100</td>
		</tr>
		<tr>
			<td>PBSTx</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100</td>
		</tr>
		<tr>
			<td>Extraction buffer (PBSTx + Rnase)</td>
			<td></td>
			<td></td>
			<td>1X PBS, 1% Trition X-100, 100 &#181;g/mL RNase</td>
		</tr>
		<tr>
			<td>2X SSCT</td>
			<td></td>
			<td></td>
			<td>2X SSC, 0.1% Tween 20</td>
		</tr>
		<tr>
			<td>2X SSCT + 20% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 20% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 40% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 40% formamide</td>
		</tr>
		<tr>
			<td>2X SSCT + 50% formamide</td>
			<td></td>
			<td></td>
			<td>Toxic: wear appropriate protection. 2X SSC, 0.1% Tween 20, 50% formamide</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Enzymes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AluI</td>
			<td>New England Biolabs</td>
			<td>R0137S</td>
			<td></td>
		</tr>
		<tr>
			<td>HaeIII</td>
			<td>New England Biolabs</td>
			<td>R0108S</td>
			<td></td>
		</tr>
		<tr>
			<td>MseI</td>
			<td>New England Biolabs</td>
			<td>R0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>MspI</td>
			<td>New England Biolabs</td>
			<td>R0106S</td>
			<td></td>
		</tr>
		<tr>
			<td>RsaI</td>
			<td>New England Biolabs</td>
			<td>R0167S</td>
			<td></td>
		</tr>
		<tr>
			<td>BfuCI</td>
			<td>New England Biolabs</td>
			<td>R0636S</td>
			<td></td>
		</tr>
		<tr>
			<td>100X BSA</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>10X NEB buffer #2</td>
			<td>New England Biolabs</td>
			<td></td>
			<td>Restriction enzyme digestion buffer. Comes with NEB enzymes</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase (TdT) 400 U/&#181;l</td>
			<td>Roche/Sigma</td>
			<td>3333566001</td>
			<td></td>
		</tr>
		<tr>
			<td>TdT buffer</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>Cobalt chloride</td>
			<td>Roche/Sigma</td>
			<td></td>
			<td>Toxic: wear appropriate protection. Comes with TdT enzyme</td>
		</tr>
		<tr>
			<td>RNase A (10 mg/mL)</td>
			<td>Thermo-Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cytology Tools etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td></td>
			<td>#5 INOX, Biologie</td>
		</tr>
		<tr>
			<td>9&#8221; Disposable glass Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td>Autoclave to sterilize</td>
		</tr>
		<tr>
			<td>Shallow glass dissecting dish</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Deep well dish (3 wells)</td>
			<td>Pyrex</td>
			<td>7223-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium microscope slides</td>
			<td>Fisher Scientific</td>
			<td>22-038-104</td>
			<td>Used to cover deep well dishes</td>
		</tr>
		<tr>
			<td>Frosted glass slides, 25 x 75 mm</td>
			<td>VWR Scientific</td>
			<td>48312-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, 3 x 1 in, 1 mm thick</td>
			<td>Thermo-Scientific</td>
			<td>3051</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 10 x 10 mm, No. 1.5</td>
			<td>Thermo-Scientific</td>
			<td>3405</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needle</td>
			<td></td>
			<td></td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Prolong GOLD mounting media</td>
			<td>Molecular Probes</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed-air in can</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop 2000, spectrophotometer</td>
			<td>Thermo-Scientific</td>
			<td></td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at room temperature</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Table top microfuge at 4 &#176;C</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven or incubator with rotator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A1RSi laser scanning confoal</td>
			<td>Nikon</td>
			<td></td>
			<td>40X oil Plan Fluor DIC (NA 1.3)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables etc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>500 &#181;l microfuge tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;l PCR tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic container with tight fitting lid</td>
			<td>Various</td>
			<td></td>
			<td>To hold Drierite</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Various</td>
			<td></td>
			<td>disposable wipes</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Various</td>
			<td></td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC purified 5&#39;-labeled oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>Cy3-labeled probes that recognize the 359 bp satellite repeat of the X chromosome</td>
		</tr>
		<tr>
			<td>Volocity 3D Image Analysis Software</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Version 6.3</td>
		</tr>
	</tbody>
</table>

using fluorescence in situ hybridization (fish) to monitor the state of arm cohesion in prometaphase and metaphase i drosophila oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Figure 5 presents images obtained with an arm probe that hybridizes to cytological region 6E-7B on the X chromosome. This probe results in a signal that co-localizes with that of DAPI, is easily distinguishable from the background, and has been used successfully to quantify arm cohesion defects in different genotypes19. Quantification of cohesion defects was restricted to prometaphase I and metaphase I stages; oocytes prior to...

Watch this Scientific Journal Video about Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes at JoVE.com

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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