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 protease inhibiting activities of DTT and PMSF, respectively. Use 10 mL of HEB for each pair of mouse testes.</li>
	<li>Prepare a 100 mM sucrose solution (fresh each time) by adding 0.342 g of sucrose to 10 mL of ultrapure laboratory grade water. Adjust the pH to 8.2</li>
	<li>Prepare fixative solution (1% paraformaldehyde (PFA), 0.15% Triton X-100, pH adjusted to 9.2 with 1 N HCl or 1 N NaOH) fresh monthly and store it at room temperature protected from light. Dilute a 16% commercially available PFA starting stock to 4% in 1X PBS for use as a working stock, and then store 4% aliquots at -20 &#176;C.
	<ol>
		<li>To prepare the fixative solution, add 10 mL of 4% PFA solution and 600 &#181;L of 10% Triton X-100 to 30 mL of 1X PBS, mix well, and adjust the pH to 9.2 with 1 N HCl or 1 N NaOH. Store the fixative solution in a 50-mL conical tube wrapped in aluminum foil at room temperature (for no more than 4 weeks).</li>
	</ol>
	</li>
	<li>Prepare wash solutions (while slides are drying the last 30 min) with Photo-Flo 200: 0.4% Photo-Flo 200/PBS (320 &#181;L of Photo-Flo 200 in 80 mL of 1X PBS) and 0.4% Photo-Flo 200/dH2O (160 &#181;L of Photo-Flo 200 in 40 mL of ultrapure laboratory grade water). Use a 50 mL Coplin jar filled with 40 mL of solutions to wash 10 or fewer slides at a time.</li>
	<li>Prepare the following instruments: 1 pair of surgical scissors (to incise the skin), 1 pair of fine tip surgical scissors (to incise the peritoneum), 2 pairs of straight fine tip forceps (to dissect out each testis), 1 straight edge razor blade (to incise each testis), 2 pairs of curved tip forceps (to immobilize each testis and squeeze out the seminiferous tubules), 50 mL Coplin jars, Teflon printed 3 ring slides, 1 scalpel with size 11 blade, humidified chamber, 100 x 15 mm Petri dishes, fine tip permanent marker or pencil, and a platform rotating shaker.
	<ol>
		<li>Place premium uncharged precleaned frosted end glass slides upright in a 50 mL Coplin jar filled with fixative solution until use. 
		NOTE: Pencil is preferred if slides will be used subsequently in procedures requiring ethanol, such as fluorescence in situ hybridization.</li>
	</ol>
	</li>
</ol>
2. Spermatocyte nuclei spreading
<ol>
	<li>Euthanize a male mouse according to institutional guidelines (e.g. cervical dislocation with or without CO2 asphyxiation).</li>
	<li>Use P10 - P20 males to evaluate meiotic cells in the first wave of spermatogenesis; we routinely use P15 - P17 males to obtain cells from all substages of prophase I. Adult testes will provide cells from prophase I too, but they will also yield more postmeiotic cells, such as spermatids.</li>
	<li>Harvest and weigh the testes (record the paired weights); then place them into a Petri dish containing a few drops of cold HEB to keep them moist.</li>
	<li>Hold the testis against the bottom of the dish with curved tip forceps and use a straight edge razor blade to make a small vertical midline incision through the capsule of the testis. While still holding the testis against the bottom of the dish with one forceps, use a second set of curved tip forceps or straight edge razor blade to gently squeeze the seminiferous tubules from the testis.
	<ol>
		<li>Place the tubules into a 15 mL conical tube containing 10 mL of cold HEB, taking care to avoid putting pieces of capsule into the tube. Repeat these steps with the second testis.</li>
	</ol>
	</li>
	<li>Incubate the decapsulated seminiferous tubules in the conical tube containing HEB on ice for 30 - 60 min, depending on the size of the testes. Incubate seminiferous tubules from a pair of testes weighing 30 mg or less for 30 min, tubules from a pair of testes weighing 31 to 50 mg for 30 - 45 minutes, and larger testes for 60 minutes. Do not alter the times for a single testis. 
	NOTE: The optimal incubation time may vary depending on the experience of the experimenter, the reagents used, and the mice being analyzed.</li>
	<li>While seminiferous tubules are incubating in HEB, use a marker or pencil to label the frosted ends of precleaned slides with mouse number, slide number, and date. Then place the pre-cleaned slides in a Coplin jar containing the fixative solution.</li>
	<li>Following incubation, pour tubules and HEB into a clean Petri dish, and separate tubules into approximately 3 mm diameter clumps. Each clump yields 2 slides and the number of slides yielded depends on the size of testes. 
	NOTE: We obtain 8 slides from wild type and 6 slides from subfertile Chtf18-null P16 testes (which are smaller due to decreased numbers of meiotic and mitotic germ cells8). One adult testis weighing 100 mg or more would be expected to yield 16 slides.</li>
	<li>Place 23 &#181;L of 100 mM sucrose solution into one ring of a Teflon printed, 3 ring slide and add one clump of seminiferous tubules to the ring.&#160; Then using curved tip forceps to hold the clump, gently mince the tubules with a size 11 razor blade until the solution becomes cloudy. 
	NOTE: Do not overmince as this will result in fragmented chromosomes. Note that fragmented chromosomes could also be indicative of a mutant phenotype.</li>
	<li>Remove debris, add another 23 &#181;L of sucrose solution, and use a micropipette to gently pipette the mixture up and down a few times to help separate the cells in the suspension.</li>
	<li>Remove a slide soaking in the Coplin jar containing fixative solution and tilt the slide over the jar so that one generous droplet collects at the bottom corner of the slide.
	<ol>
		<li>Pipet 20 &#181;L of sucrose cell suspension from the ring of the Teflon printed 3 ring slide into the fixative droplet on a premium frosted glass slide, and pipet the suspension up and down 2 - 3 times. 
		NOTE: The fixative droplet should be large enough in volume so that addition of 20 &#956;L of the sucrose cell suspension to it allows coverage across the entire slide when the mixture is spread (see 2.11 below).</li>
	</ol>
	</li>
	<li>Slowly tilt the slide to the side opposite the droplet to spread the cell suspension along the length of the slide. Then slowly tilt the slide back to spread the cell suspension along the width of the slide to cover it completely. Avoid spreading the suspension over the same area of the slide more than once otherwise the cells will overlap with each other.</li>
	<li>Immediately place slide flat in a humidified chamber and repeat steps 2.8 to 2.11 until all clumps of seminiferous tubules have been used to make slides. It is important not to move the humidified chamber during the drying process to avoid tearing and overlapping cells.</li>
	<li>Allow the slides to incubate in a covered humidified chamber at room temperature for 2.5 h so that they dry slowly and not completely. High humidity and slow drying in this step are important to ensure adequate spreading of the chromatin. Then remove the cover of the chamber and allow slides to air-dry completely an additional 30 min.</li>
	<li>Place slides (back to back or in a zigzag pattern) in a Coplin jar containing 0.4% Photo-Flo 200/1X PBS solution and wash the slides twice, 5 min each, by gently agitating the Coplin jar on a platform rotating shaker. Use fresh solution for each wash and keep slides from different mice in separate jars for each of the washes.</li>
	<li>Wash the slides in a Coplin jar containing 0.4% Photo-Flo 200/dH2O solution for 5 min once while agitating.</li>
	<li>Remove slides from the Coplin jar (last wash), carefully wipe the edges and bottoms of the slides to remove excess liquid, and place slides at an angled, almost-upright position to dry. An experimenter who is proficient in this technique can process and prepare surface-spread nuclei from 4 or 5 mice in one day.</li>
	<li>Once dry (15 - 20 min), process slides immediately for immunofluorescence staining or store at -80 &#176;C in a tightly closed slide box protected from light for a maximum of 3 weeks (depending on the target proteins being evaluated; optimal results may be obtained if stained the same day or within 2 weeks).
	<ol>
		<li>If stored at -80 &#176;C, then allow slides to come to room temperature and wash in a Coplin jar containing 1X PBS or Block for 5 min prior to staining. 
		NOTE: Many different immunofluorescence staining protocols will provide good results; refer to Berkowitz et al. for an immunostaining protocol8.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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><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 &amp;deg;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 ...

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Unit 1

Meiosis

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11.7K Views.  Drexel University College of Medicine. Meiose is het ontwikkelings proces waaraan gameten worden gevormd door een ronde van DNA-replicatie en twee opeenvolgende ronden van chromosoom segregatie. Zoogdieren meiose kan worden onderzocht met behulp van een techniek ter voorbereiding Meiotische chromosoom spreads. Hier tonen we een methode voor te bereiden oppervlak-spread kernen van muis spermatocyten.

Video: Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Developmental Biology

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.

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 ...

Genetics

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

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