Name: preparation of meiotic chromosome spreads from mouse spermatocytes
Uploaded: 2017-11-22T12:30:00
Description: Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes at JoVE.com

The authors acknowledge the technical assistance of Abigail Harris. They also acknowledge Paula Cohen and Kim Holloway for helping to optimize the protocol of preparing surface-spread nuclei from mouse spermatocytes. The authors also appreciate critical reading of the manuscript by Karen Schindler.
This work was supported by NIH R01 GM106262 to K.M.B.

All methods involving mice utilized high ethical and welfare standards, and were approved by the Institutional Animal Care and Use Committee at Drexel University College of Medicine.
1. Preparation of solutions and instruments needed
<ol>
	<li>Prepare Hypotonic Extraction Buffer (HEB) in 50 mL of ultrapure laboratory grade water, pH 8.2 - 8.4 (Table 1). Make the solution fresh each time, keep on ice, and use within 2 h of preparing spreads; this is necessary to optimize the reducing and prot...

<ol>
	<li>Handel, M. A., Schimenti, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+mammalian+meiosis:+regulation,+dynamics+and+impact+on+fertility.">Genetics of mammalian meiosis: regulation, dynamics and impact on fertility.</a> Nat Rev Genet. 11, 124-136 (2010).</li><li>Peters, A. H., Plug, A. W., van Vugt, M. J., de Boer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+drying-down+technique+for+the+spreading+of+mammalian+meiocytes+from+the+male+and+female+germline.">A drying-down technique for the spreading of mammalian meiocytes from the male and female germline.</a> Chromosome Res. 5, 66-68 (1997).</li><li>Counce, S. J., Meyer, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+synaptonemal+complex+and+the+kinetochore+in+Locusta+spermatocytes+studied+by+whole+mount+electron+microscopy.">Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy.</a> Chromosoma. 44, 231-253 (1973).</li><li>Speed, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiosis+in+the+foetal+mouse+ovary.+I.+An+analysis+at+the+light+microscope+level+using+surface-spreading.">Meiosis in the foetal mouse ovary. I. An analysis at the light microscope level using surface-spreading.</a> Chromosoma. 85, 427-437 (1982).</li><li>Ashley, T., 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=Dynamic+changes+in+Rad51+distribution+on+chromatin+during+meiosis+in+male+and+female+vertebrates.">Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates.</a> Chromosoma. 104, 19-28 (1995).</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, 336-342 (1996).</li><li>Plug, A. W., Xu, J., Reddy, G., Golub, E. I., Ashley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+association+of+Rad51+protein+with+selected+sites+in+meiotic+chromatin.">Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.</a> Proc Natl Acad Sci U S A. 93, 5920-5924 (1996).</li><li>Berkowitz, K. 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=Disruption+of+CHTF18+causes+defective+meiotic+recombination+in+male+mice.">Disruption of CHTF18 causes defective meiotic recombination in male mice.</a> PLoS Genet. 8, e1002996 (2012).</li><li>Gomez, R., 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=Sororin+loads+to+the+synaptonemal+complex+central+region+independently+of+meiotic+cohesin+complexes.">Sororin loads to the synaptonemal complex central region independently of meiotic cohesin complexes.</a> EMBO Rep. 17, 695-707 (2016).</li><li>Brooker, A. S., Berkowitz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+cohesins+in+mitosis,+meiosis,+and+human+health+and+disease.">The roles of cohesins in mitosis, meiosis, and human health and disease.</a> Methods Mol Biol. 1170, 229-266 (2014).</li><li>McNicoll, F., Stevense, M., Jessberger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cohesin+in+gametogenesis.">Cohesin in gametogenesis.</a> Curr Top Dev Biol. 102, 1-34 (2013).</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 J. 282, 2426-2443 (2015).</li><li>Holloway, J. K., Sun, X., Yokoo, R., Villeneuve, A. M., 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=Mammalian+CNTD1+is+critical+for+meiotic+crossover+maturation+and+deselection+of+excess+precrossover+sites.">Mammalian CNTD1 is critical for meiotic crossover maturation and deselection of excess precrossover sites.</a> J Cell Biol. 205, 633-641 (2014).</li><li>La Salle, S., Sun, F., Handel, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+short-term+culture+of+mouse+spermatocytes+for+analysis+of+meiosis.">Isolation and short-term culture of mouse spermatocytes for analysis of meiosis.</a> Methods Mol Biol. 558, 279-297 (2009).</li><li>Wiltshire, T., Park, C., Caldwell, K. A., Handel, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+premature+G2/M-phase+transition+in+pachytene+spermatocytes+includes+events+unique+to+meiosis.">Induced premature G2/M-phase transition in pachytene spermatocytes includes events unique to meiosis.</a> Dev Biol. 169, 557-567 (1995).</li></ol>

To obtain high numbers of well spread, good quality spermatocyte nuclei, the most critical steps of this protocol include appropriate incubation time of seminiferous tubules in HEB, appropriate mincing of seminiferous tubules, and the technique employed to spread the sucrose cell suspension across the fixative coated slide(s). We incubate seminiferous tubules from testes of wild type males ranging from postnatal day 15 to adulthood in HEB for 45 min, while testes from comparably aged Chtf18-null mice are incubat...

Mice are used extensively as a model organism to study meiosis in mammals. Both the normal developmental processes and defects that occur during meiosis can be evaluated. The timeline and progression of salient features that occur during prophase I and substages, as well as a multitude of specific proteins involved in processes crucial to regulation of meiosis can be characterized in both wild type and mutant mice. Several specialized processes that occur during meiosis I can be studied in detail (reviewed in Handel and Schimenti1). These include DNA double-strand break (DSB) formation and repair, recombination, synaptonemal complex formation, and chromosome segregation.
An important aspect of studying meiotic processes is the ability to examine nuclei containing homologous chromosomes that are visually and optically resolved to better distinguish and identify the substages of prophase I. Prophase I is comprised of 5 substages that are characterized by specific features including formation of the synaptonemal complex (SC). The SC is a tripartite protein scaffold that enables pairing and DNA double-strand break repair of homologs. During leptonema, homologs align as axial elements of the SC are laid down. Then attachment of central elements to the synaptonemal complex during zygonema facilitates pairing and physical connection (synapsis) between pairs of homologs. During pachynema, synapsis of homologs becomes complete and DNA crossovers are formed from repair of a select population of DNA double-strand breaks via homologous recombination. The SC disassembles during diplonema, allowing homologs to desynapse but remain attached at their centromeres and at sites of DNA crossovers. Finally during diakinesis, homologs recondense and the transition to metaphase occurs1.
Historically, surface-spreading of meiotic chromatin yielded few nuclei, especially those from early stages of prophase I2. As a result, these methods were modified to improve the separation of cells from tissues (i.e. testis or ovary), the technique of spreading meiotic chromatin, and the yield of high quality meiotic nuclei for evaluation2,3,4. In addition, this method proved to be useful in preserving nuclear and chromatin bound proteins in meiotic nuclei as demonstrated by published immunolocalization techniques5,6,7. Therefore, the method initially described by Peters2 and demonstrated here yields many separate burst spermatocyte nuclei containing homologs undergoing the leptotene, zygotene, pachytene, diplotene, and diakinesis substages of prophase I.
The technique of preparing surface-spread nuclei (also called chromatin spread analysis) is widely used to study meiosis in the fields of reproductive and cell biology. Slides containing surface-spread nuclei can be subsequently immunostained with antibodies to proteins of interest or subjected to silver staining, and then analyzed with microscopy. The method is similar for both male and female mice, although modifications have been described for female mice2. It is crucial not to overmince seminiferous tubules. Nuclei must also be well spread so that following immunostaining homologs and associated proteins are clearly seen with microscopy. Surface-spread nuclei can be prepared in just a few hours and either immunostained immediately or stored at -80 &#176;C for a maximum of 3 weeks before thawing and immunostaining. However, optimal results are best obtained for some proteins if immunostaining is performed immediately or within 2 weeks of preparing surface-spread nuclei. Slides can be immunostained with a standard protocol using antibodies to proteins of interest, followed by incubation with secondary antibodies conjugated to fluorescent proteins or dyes, then imaged with fluorescence microscopy8. At postnatal day 15 - 21 there is sufficient tissue per pair of wild type testes to yield 6 - 10 slides, and older mice (including adults) will yield more slides. However, wild type mice 6 weeks of age and older will also yield more post-meiotic cells (i.e. spermatids) on the slides following immunostaining. In addition, all substages of prophase I can be observed in juvenile and adult mice. Testes from males at postnatal days 10 through 15 will yield many more leptotene and zygotene cells. Mice older than postnatal day 14 to 15 will yield more pachytene and diplotene nuclei.
Here we describe and demonstrate a widely-used method of preparing surface-spread nuclei from mouse spermatocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Photo-Flo 200</td>
			<td>Kodak</td>
			<td>1464510</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (formaldehyde aqueous solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
			<td>Dilute 16% solution into 4% working stocks with 1X PBS and freeze aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Teflon printed 3 ring slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>63418-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Premium uncharged frosted end glass slides</td>
			<td>Several commercial brands</td>
			<td></td>
			<td>Clean slides in ethyl or isopropyl alcohol and allow to drip dry prior to use; label slides on frosted end with a permanent marker or pencil depending on subsequent use of slides</td>
		</tr>
		<tr>
			<td>Surgical scissors (sharp and blunt tips)</td>
			<td>Fine Science Tools</td>
			<td>14001-12</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Fine tip surgical scissors (sharp tips)</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Straight fine tip forceps</td>
			<td>Fine Science Tools</td>
			<td>11050-10</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Curved medium tip forceps</td>
			<td>Fisher</td>
			<td>16100110</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td>Different brand of a similar type will work</td>
		</tr>
		<tr>
			<td>Mouse anti-SYCP3</td>
			<td>Abcam</td>
			<td>ab97672</td>
			<td>Use at 1:300</td>
		</tr>
		<tr>
			<td>Anti-centromere protein (derived from human CREST patient serum)</td>
			<td>Antibodies Incorporated</td>
			<td>15-234-0001</td>
			<td>Use at 1:50</td>
		</tr>
		<tr>
			<td>Humidified chamber</td>
			<td></td>
			<td></td>
			<td>We use Nunc square culture dishes 500 cm2/well with moistened paper towels</td>
		</tr>
		<tr>
			<td>Widefield microscope with epifluorescence</td>
			<td>Leica microsystems</td>
			<td></td>
			<td>Any standard model</td>
		</tr>
		<tr>
			<td>Coplin jars</td>
			<td>Several commercial brands</td>
			<td></td>
			<td>50 ml capacity; 40 mm (1.6 in.) diameter, holds 10 slides (back to back)</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Several commercial brands</td>
			<td></td>
			<td>100 x 15 mm diameter, polystyrene sterile untreated</td>
		</tr>
	</tbody>
</table>

preparation of meiotic chromosome spreads from mouse spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

The ability of this method to provide large numbers of surface spread nuclei containing intact homologs depends on three main factors: 1) appropriate incubation time of seminiferous tubules in hypotonic buffer (i.e. HEB) to obtain adequately swelled spermatocyte nuclei that burst but do not disintegrate from prolonged incubation in HEB, 2) micropipetting the sucrose droplets of cells to obtain a separated suspension of nuclei that are not clumped together, and 3) tilting each fixative coa...

Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes at JoVE.com

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

The overall goal of this protocol is prepare meiotic chromosome spreads from mouse spermatocytes to allow the study of male meiosis. This method can help answer key questions in the reproductive biology field about the mechanisms that regulate meiotic re-combination and chromosome segregation. The main advantage of this technique is that it yields an abundance of spermatocyte nuclei at different stages of meiosis, especially those cells undergoing the sub-stages of prophase 1.Visual demonstration of this method is critical because a technique used to spread the spermatocyte nuclei onto slides is difficult to convey in writing. Begin by weighing paired testes harvested from post-natal to adult aged male mice and placing the weighed specimens in a Petri dish containing a few drops of cold hypotonic extraction buffer. To harvest the tubules, use the tips of a curved forceps to hold one testis against the bottom of the dish and use a straight-edge razor blade to make a small, vertical mid-line incision through the capsule of the testis.Use a second set of curved forceps or a straight-edge razor blade to gently squeeze the seminiferous tubules from the testis and transfer the seminiferous tubules into a 15 milliliter conical tube containing 10 milliliters of cold hypotonic extraction buffer, taking care to avoid collecting pieces of testis capsule with the tubules. When all of the tubules have been collected, placed the conical cubical on ice for 30 to 60 minutes, depending on the size of the testes. While the tubules are incubating, label the frosted ends of pre-cleaned slides with the mouse number, slide number, and date, and place the labeled slides in a Coplin jar containing fixative solution.At the end of the incubation, pour the tubules into a new Petri dish and separate the tubules into approximately three millimeter diameter clumps. Next, place 23 microliters of the 100 minimal or sucrose solution into one ring of a Teflon printed three ring slide and add one clump of tubules to the ring. Using curved tipped forceps to hold the clump, use a size 11 razor blade to gently mince the tubules until the solution becomes cloudy.When the solution becomes cloudy, remove the debris and another 23 microliters of sucrose solution to the ring. Use a micro-pipette to gently pipette the mixture a few times to help separate the cells in the suspension. Then remove a slide from the fixative solution and tilt the slide over the jar so that one generous droplet collects at the bottom corner.Take care that the droplet is large enough that coupled with additional cell suspension the slide will be very wet and be completely covered when the mixture is spread. Add 20 microliters of cells to the droplet and mix the suspension two to three times by pipetting. Next spread the cell suspension along the length of the slide and slowly tilt the slide to the side opposite the droplet.Then slowly tilt the slide back to spread the cell suspension along the width of the slide to cover it completely. To avoid overlapping of the cells take care not to spread the cell suspension over the same area twice. After spreading, immediately place the slide in a humidified chamber and spread the next slide.When the desired number of slides have been prepared cover the chamber and allow the slides to dry at room temperature for two and a half hours. At the end of the covered incubation, remove the cover to allow the slides to air dry completely for an additional 30 minutes. When the slides are completely dry, place them in a Coplin jar containing 0.4%Photo-Flo 200 in PBS for two two five minute incubations with gentle agitation followed by one five minute wash in 0.4%Photo-Flo 200 in water with agitation.After the last wash carefully wipe the edges and bottom of each slide to remove the excess liquid and dry the slides for 15 to 20 minutes in an angled, almost upright position. Then store the slides at minus 80 degrees Celsius in a tightly closed slide box protected from light for up to three weeks before immunostaining. In this image well-spread pachitean spermotocyte nuclei can be observed.These nuclei contain unevenly immunostained homologues that appear tattered due to too along in incubation of the seminiferous tubules in the hypotonic extraction buffer. These nuclei were poorly spread resulting in overlapping cells, while these nuclei contained fragmented homologues, resulting from over-mincing of the seminiferous tubules. If this technique is performed correctly however, a variety of nuclei representing the main five sub-stages of prophase 1 can be observed.Once mastered this protocol including the solution preparation including the solution preparation, drying, and washing steps can be completed in approximately five and a half hours, if it is performed properly. While attempting this procedure it's important to incubate the seminiferous tubules in the hypotonic buffer for the appropriate amount of time, and to tilt the slides at one direction at a time when spreading the cell suspension. Following this procedure, other methods including immunostaining and microscopy are often performed to evaluate fundamental meiotic processes, such as DNA double-stranded break repair and crossover formation.After watching this video you should have a good understanding of how to prepare male meiotic chromosome spreads. A straightforward procedure that requires some practice to gain competence and optimal results. Thanks for watching this video and good luck with this protocol.

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