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. Author manuscript; available in PMC: 2021 Jun 30.
Published in final edited form as: Methods Mol Biol. 2018;1861:113–129. doi: 10.1007/978-1-4939-8766-5_10

Chromosome Spread Analyses of Meiotic Sex Chromosome Inactivation

Kris G Alavattam, Hironori Abe, Akihiko Sakashita, Satoshi H Namekawa
PMCID: PMC8243718  NIHMSID: NIHMS1713106  PMID: 30218364

Abstract

A distinct form of X chromosome inactivation takes place during male meiosis, when the male sex chromosomes undergo a phenomenon known as meiotic sex chromosome inactivation (MSCI). MSCI is directed by DNA damage response signaling independent of Xist RNA to silence the transcriptional activity of the sex chromosomes, an essential event in male germ cell development. Here, we present protocols for the preparation and analyses of chromosome spread slides of mouse meiotic spermatocytes, thereby enabling a quick, inexpensive, and powerful cytological method to complement gene expression studies.

Keywords: Cytology, Slide protocol, Immunofluorescence microscopy, Meiosis, Cell staging, Cell suspension, Testis, Chromosomes, Synapsis, DNA damage response, Sex chromosomes, Chromatin

1. Introduction

A distinct form of X chromosome inactivation takes place during meiosis in male mammals, when the male sex chromosomes, X and Y, undergo a phenomenon known as meiotic sex chromosome inactivation (MSCI) [1–5]. An essential event in male germ cell development, MSCI results in the transcriptional inactivation of X and Y independent of Xist RNA. Underscoring its importance, failure to silence X and Y leads to the arrest and elimination of germ cells, and thus male infertility.

During meiosis, homologous autosomes undergo synapsis and recombination to produce haploid sperm. MSCI begins in the pachytene stage of meiotic prophase in response to the unsynapsed status of the sex chromosomes and is directed by DNA damage response (DDR) signaling [3]. DDR signaling catalyzes the phosphorylation of histone variant H2AX (γH2AX), and the establishment of MSCI requires γH2AX to spread from the X and Y chromosome axes through their combined chromatin domain [6, 7]. This spreading is directed by MDC1, a DDR factor and γH2AX-binding partner, as part of an expansive feed-forward mechanism [7, 8]. The presence of a γH2AX domain tightly correlates with transcriptional silencing, enabling the characterization of MSCI based on γH2AX immunolocalization. After the initiation of MSCI in meiotic prophase, extensive epigenetic programming and chromatin remodeling occur on the sex chromosomes, including the establishment of silent and active histone modifications [9–12]. Distinct DDR pathways regulate epigenetic programming of the sex chromosomes, which is required for male fertility [13–16].

Here, we present protocols for the preparation and analyses of mouse meiotic chromosome spread slides, methods that have been instrumental for the study of MSCI. Chromosome spread slides for meiotic spermatocytes are prepared from dissociated testicular tubules that have been soaked in a hypotonic nuclear extraction buffer, gently processed into a cell suspension, and then applied to positively charged slides coated with a solution of low-concentration detergents and formaldehyde. Adapted from a classic method [17], we have optimized these protocols for greater ease in preparation, reproducibility, and a consistently high signal-to-noise ratio in imaging applications. Our methods are well suited for 2D immunolocalization studies using a variety of fluorescence microscopy systems, and they enable the detailed assessment of various nuclear features for precise temporal staging, aspects of DDR signaling, and general chromatin dynamics. This method joins a method that maintains the 3D organization of the nucleus, developed in our laboratory and optimized for fluorescence in situ hybridization, immunolocalization, and large-scale examination of nuclear architecture with 3D imaging systems [9, 18, 19]. Together, these methods enable quick, inexpensive, and powerful cytological strategies that complement the gene expression studies that give a direct readout of MSCI: transcriptional inactivation. We recommend that analyses of chromosome spread slides are performed alongside methods such as RNA-seq analyses of purified germ cell populations, which can be obtained via gravity sedimentation, e.g., the STA-PUT method [15, 20] and/or flow assisted cell sorting [21–24].

2. Materials

Prepare all solutions using purified deionized water (resistivity of 18 MΩ cm at 25 °C), analytical grade reagents, and a magnetic stirrer with magnetic stir bar. Filter-sterilize with 0.22 μm pore size hydrophilic polyethersulfone membranes or, where indicated, autoclave at 121 °C, 100 kPa (15 psi) using an appropriate liquids cycle. Unless otherwise noted, store solutions at room temperature. Follow appropriate waste disposal regulations.

2.1. Preparation of Chromosome Spread Slides

  1. 200 mM sodium tetraborate (Na2B4O7) solution: In 400 mL of water, dissolve 38.1 g sodium tetraborate decahydrate (Na2B4O7·10H2O) powder (see Note 1). Add water to 500 mL. Filter-sterilize or autoclave.

  2. 200 mM boric acid (H3BO3) solution: In 400 mL of water, dissolve 6.2 g H3BO3 powder. Add water to 500 mL. Filter-sterilize or autoclave.

  3. 100 mM sodium borate buffer, pH 9.2: To 200 mL of 200 mM Na2B4O7 solution in the presence of a pH electrode, add 200 mM H3BO3 solution until the pH is 9.2. Add an equal volume of water to reach a final concentration of 100 mM. Filter-sterilize or autoclave.

  4. 200 mM sucrose aliquots: In 40 mL of water, dissolve 3.4 g sucrose powder. Add water to 50 mL. Filter-sterilize. Separate into 1 mL aliquots and store at −20 °C.

  5. 1 M dithiothreitol (DTT) aliquots: Under a fume hood, add 3.09 g DTT powder to 20 mL of 10 mM sodium acetate (NaOAc) at pH 5.2 (see Note 2). Filter-sterilize. Separate into 400 μL aliquots and store at −20 °C.

  6. 50× protease inhibitor aliquots: Dissolve two protease inhibitor tablets (e.g., cOmplete protease inhibitor cocktail, Roche 11697498001) in 2 mL of water. Filter-sterilize. Separate into 100 μL aliquots and store at −20 °C.

  7. Hypotonic buffer stock (HBS) solution, pH 8.2: To 400 mL of water, add 15 mL of 1 M Tris base (approximate pH of 11; 30 mM final concentration), 8.5 mL of 1 M trisodium citrate (Na3C6H5O7; 17 mM final concentration), and 5 mL of 500 mM EDTA (5 mM final concentration). Adjust the pH to 8.2 with HCl or NaOH. Add water to 500 mL. Filter-sterilize.

  8. Hypotonic extraction buffer (HEB): On the day of the experiment, prepare 4 mL of ice-cold solution per testis to be used. To 900 μL of water, add 2 mL of vortexed HBS solution (50% final solution), 1 mL of 200 mM sucrose (50 mM final concentration), 20 μL of 1 M DTT (5 mM final concentration), and 80 μL of 50× protease inhibitor (1× final concentration). Mix by vortexing for several seconds. Keep on ice or at 4 °C until the time of 4-well dish preparation.

  9. Fixation solution: On the day of the experiment, prepare 100 mL of ice-cold solution per testis to be used (see Note 3). To 80 mL of water, add 2 g of PFA powder (2% final concentration) under a fume hood. To dissolve the PFA powder, add 20 μL of 5 M NaOH (1 mM final concentration), heat the mixture to 60 °C using a water bath, and agitate the solution every 15–20 min. Once dissolved, cool the solution to 4 °C. Add 10 mL of 100 mM sodium borate buffer at pH 9.2 (10 mM final concentration), 200 μL of 10% sodium monododecyl sulfate (SDS; 0.02% final concentration), and 100 μL of Triton X-100 (0.1% final concentration). Add water to 100 mL. Keep on ice or at 4 °C until the time of chromosome spread slide preparation.

  10. 0.4% Photo-Flo 200: To 1 L of water, add 4 mL of Photo-Flo 200 (Kodak 146–4510; 0.4% final concentration). Filter-sterilize.

2.2. Immunostaining Chromosome Spread Slides

  1. 10× phosphate-buffered saline (10× PBS), pH 7.4: In 800 mL of water, dissolve the following compounds: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, and 2.4 g KH2PO4. Adjust the pH to 7.4 with HCl. Add water to 1 L. Filter-sterilize or autoclave.

  2. 1× phosphate-buffered saline (1× PBS), pH 7.4: Dilute 10× PBS to 1× PBS, e.g., mix 100 mL of 10× PBS with 900 mL of water.

  3. PBS–Tween 20 (PBST): To 1 L of 1× PBS, add 1 mL Tween 20 (~0.1% final concentration).

  4. Antibody dilution buffer aliquots: In 80 mL of water, dissolve 150 mg of bovine serum albumin (BSA) powder (0.15% final concentration) and 100 μL of Tween 20 (0.1% final concentration). Add 10 mL of 10× PBS, and then add water to 100 mL. Filter-sterilize. Separate into 1 mL aliquots and store at −20 °C.

  5. 1% BSA in 1× PBS aliquots: To 80 mL of water, add 1 g of BSA) powder (1% final concentration). Add 10 mL of 10× PBS, and then add water to 100 mL. Filter-sterilize. Separate into 1 mL aliquots and store at −20 °C.

  6. DAPI (4′,6-diamidino-2-phenylindole; 1 mg/mL) aliquots: To 5 mL of water or 1x PBS, add 5 mg of DAPI powder (1 mg/mL final concentration). Filter-sterilize. Separate into 500 μL aliquots. For long-term storage, store at −20 °C. For regular use, store at 4 °C.

  7. Antifade mountant: ProLong Gold Antifade Mountant (ThermoFisher) or Vectashield (Vector Laboratories).

2.3. Immunostaining Chromosome Spreads with Antibodies Raised in the Same Species

1. 4% PFA in 1× PBS: Prepare 10 mL of solution. To 8 mL of water, add 0.4 g of PFA powder (4% final concentration). To dissolve the PFA powder, add 1 μL of 5 M NaOH (1 mM final concentration), heat the mixture to 60 °C, and agitate the solution every 15–20 min. Once dissolved, cool the solution to 4 °C. Add 1 mL of 10× PBS, then add water to 10 mL. Keep on ice or at 4 °C until time of immunostaining.

3. Methods

Follow acceptable methods and ethical guidelines for euthanasia of laboratory mice. Unless otherwise noted, perform all procedures at room temperature.

3.1. Chromosome Spread Preparation

  1. After euthanizing mice, excise testes and remove extra-testicular tissues, including the tunica albuginea. Place each testis in a 1.7-mL microtube containing 1 mL of ice-cold 1× PBS. Keep on ice or at 4 °C (see Note 4).

  2. For each individual testis to be used, prepare four 4-well dishes (Nunc 4-well dish for IVF, Thermo Scientific 144444; Fig. 1) for the washing and unraveling of testicular tubules, as well as for the hypotonic nuclear extraction of testicular cells:
    1. Fill each 1.9-cm2 well of three 4-well dishes with 1 mL of ice-cold 1× PBS. Keep the 4-well dishes on ice.
    2. Fill each well of a fourth 4-well dish with 1 mL of ice-cold HEB. Keep the 4-well dish on ice (Fig. 1).

  3. Using standard tweezers, gently tear the testis into four chunks of seminiferous tubules of similar size (see Note 5; Fig. 1). Assign each chunk its own well of a 4-well dish prepared in step 2a.

  4. In the first well containing 1 mL of 1× PBS, use tweezers to gently unravel the chunks of seminiferous tubules into small clumps (Fig. 1). Avoid tearing or mincing the seminiferous tubules. Wash the partially unraveled clumps in clean 1× PBS by transferring the seminiferous tubules to assigned wells in the second dish; once there, perform additional unraveling in the clean 1× PBS. Use the third 4-well dish to repeat this wash/unraveling step one more time (see Note 6).

  5. Transfer the unraveled tubules to assigned wells containing ice-cold HEB (Fig. 1), as prepared in step 2b. Using tweezers, carefully expose unraveled tubule surface area to the HEB. Incubate the seminiferous tubules in HEB on ice for approximately 1.5–2 h with gentle, brief agitation every 30–45 min (see Note 7).

  6. 2–4 min before applying samples to slide surfaces, soak positively charged slides (e.g., ProbeOn Plus Slides, Thermo Scientific) in chilled fixation solution.

  7. After incubation, pipet 30 μL of ice-cold sucrose (100 mM) to a dry, clean uncharged microscope slide (e.g., Gold Seal Plain Slides, Thermo Scientific). Using standard tweezers, transfer a group of 4-to-6 seminiferous tubules to the sucrose (Fig. 1). Gently pull and mash the tubules between the tips of the tweezers (Fig. 1). After approximately 15–20 mashes, a semitranslucent cell suspension is formed. Dilute the suspension with an additional 30 μL of sucrose, mixing by slowly pipetting up and down.

  8. Apply 30 μL of the diluted cell suspension to a positively charged slide that has been soaked in chilled fixation solution (see Note 8; Fig. 1). Slowly tilt the slide at slight angles to mix the cell suspension with remaining fixation solution. Apply the remaining 30 μL of diluted cell suspension to a second positively charged slide.

  9. Place the slides in closed humid chambers at room temperature for a minimum incubation period of 4 h to a maximum overnight period of incubation (see Note 9).

  10. Repeat steps 7–9 until a desired number of slides are created (see Note 3).

  11. After incubation, wash the damp slides in 0.4% Photo-Flo 200, a low-concentration surfactant, at room temperature two times for 2 min each (see Note 10).

  12. Air-dry the slides at room temperature for 15–30 min. Once dry, the slides are ready for staining or indefinite storage at −80 °C.

Fig. 1.

Fig. 1

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Overview of chromosome spread slide preparation from testicular tubules (top); an image and list of some key materials for chromosome spread slide preparation (bottom)

3.2. Immunostaining Chromosome Spreads

  1. Incubate chromosome spread slides in PBST for 5–30 min at room temperature.

  2. Block the wet slides in antibody dilution buffer or 1% BSA solution by applying 1 mL of either solution to the slide surfaces. Then, gently cover the slide surfaces with Parafilm cut to slightly less than the dimensions of the slides (see Note 11). Block for 30–60 min in a humid chamber at room temperature.

  3. During the blocking step, prepare primary antibodies for immunostaining by diluting primary antibodies in antibody dilution buffer to appropriate final concentrations. Prepare 100 μL of this primary antibody solution per slide to be immunostained.

  4. Using tweezers, carefully remove the Parafilm from the surfaces of slides. Coat each wet slide surface with 100 μL of primary antibody solution. Perform this step quickly to minimize any potential drying of the slide surfaces. Gently cover the slides with Parafilm cut to slightly less than the dimensions of the slides (see Note 11).

  5. Incubate the slides for a minimum of 4 h, though preferably overnight, in a humid chamber at room temperature or 4 °C (see Note 12).

  6. After incubation with primary antibodies, remove the Parafilm from each slide surface and wash the slides with PBST 3 times, approximately 5 min each.

  7. During the washing steps, dilute secondary antibodies conjugated to fluorophores in antibody dilution buffer to an appropriate final concentration. Prepare 100 μL of this secondary antibody solution per slide to be immunostained. If using secondary antibodies conjugated to fluorophores with low photoresistance, keep the solution in darkness until time of use.

  8. Using tweezers, carefully remove the Parafilm from each slide surface. Coat each wet slide surface with 100 μL of secondary antibody solution. Gently cover the slides with Parafilm cut to slightly less than the dimensions of the slides (see Note 11).

  9. Incubate the slides for 30–60 min in a humid chamber at room temperature and in darkness if using secondary antibodies conjugated to fluorophores with low photoresistance (see Note 13).

  10. After incubation with secondary antibodies, remove the Parafilm from slide surfaces and wash the slides with PBST 3 times, approximately 5 min each. If using secondary antibodies conjugated to fluorophores with low photoresistance, be careful to minimize exposure to light.

  11. Counterstain the slides with an appropriate fluorescent intercalating agent such as DAPI (see Note 14).

  12. After counterstaining, briefly wash the slides in 1× PBS in darkness.

  13. Dab off 1× PBS from the surfaces of slides without touching the surfaces. Mount the slightly wet slides with an antifade mountant and microscope cover glasses appropriate for available imaging equipment (see Note 15).

3.3. Immunostaining Chromosome Spreads with Antibodies Raised in the Same Species

  1. To prevent the overlap of excitation emission wavelengths, plan experiments with respect to available fluorophores and the components of available imaging equipment (e.g., prisms, beamsplitters, lasers, etc.). Immunostain slides with the first of two primary antibodies raised in the same species.

  2. Wash and incubate slides with an appropriate secondary F(ab’)2 fragment antibody conjugated to a fluorophore.

  3. After incubation, fix each slide by coating its surface in 1 mL of a fresh, ice-cold solution of 4% paraformaldehyde in 1× PBS for 10 min. Perform the fixation without Parafilm covering in a humid chamber in darkness at room temperature.

  4. Briefly wash the fixed slides in PBST before performing a subsequent round of immunostaining with the second of two primary antibodies raised in the same species.

  5. Incubate slides with an appropriate F(ab’)2 secondary antibody conjugated to a fluorophore that is distinct from the initial F(ab’)2 secondary antibody.

  6. Perform immunostaining for additional primary antibodies raised in separate species with appropriate secondary antibodies. Wash, counterstain, and mount slides.

3.4. Staging Spermatocytes in Prophase Via SYCP3 Immunostaining

Precise staging of spermatocytes in meiotic prophase is an essential prerequisite for the analysis of MSCI. Meiotic prophase is divided into four broad stages based on the presentation and pairing of homologous chromosomes: the leptotene, zygotene, pachytene, and diplotene stages. The immunolocalization of SYCP3—a marker of the meiotic synaptonemal complex that begins to localize on chromosomes in the leptotene stage—offers a means for careful, distinct staging of spermatocytes in meiotic prophase (Fig. 2). Here, we provide detailed guidelines to stage mouse meiotic prophase through immunofluorescence microscopy of SYCP3. The text and images in subheading 3.4 are adapted from our 2016 publication in Cell Reports [16].

Fig. 2.

Fig. 2

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Illustrations and images of anti-SYCP3 immunostaining to identify stages of meiotic prophase. (a–h) Stages are indicated above illustrations in the panels. Nuclear regions (a, b) and the sex chromosomes (c–h) are indicated by dashed squares, and these squares are magnified in the bottom images of each panel. Arrows: described in the text; arrowheads: described in the text; X: X chromosome; Y: Y chromosome; PAR: pseudo-autosomal region. Scale bars: 5 μm

  1. In the leptotene stage, chromatids begin to condense and elongate, although they do not synapse yet. To identify leptotene spermatocytes, check chromosome spread slides for staining patterns that resemble short threads of SYCP3 (arrows in Fig. 2a) and small aggregates of SYCP3 staining (arrowheads in Fig. 2a).

  2. In the zygotene stage, homologous chromosomes have begun to synapse, but synapsis is not yet complete.
    1. To identify zygotene spermatocytes, check chromosome spread slides for SYCP3 staining patterns that resemble thick and thin SYCP3 threads: Thick threads indicate synapsis of homologous chromosomes (arrows in Fig. 2b, c), and thin threads that branch from thick threads indicate unsynapsed portions of homologous chromosomes (arrowheads in Fig. 2b, c).
    2. It is useful to categorize zygotene spermatocytes as “early” and “late:” score zygotene spermatocytes with ≤50% of chromatid synapsis as “early” (Fig. 2b) and zygotene spermatocytes with >50% of chromatid synapsis as “late” (Fig. 2c).
    3. It is possible to discern the male sex chromosomes, which are not yet synapsed, in the late zygotene stage (dashed box in Fig. 2c). X appears as a long, thin SYCP3 thread that may overlap autosomes, and Y appears as a short, thin SYCP3 thread that may also overlap autosomes.
  3. The third stage of meiotic prophase, the pachytene stage, lasts the longest and, as such, is subdivided into three stages to account for its many appearances: The “early,” “mid,” and “late” pachytene stages.
    1. MSCI is established at the onset of the early pachytene stage. To identify early pachytene spermatocytes, check for the synapsis of all autosomes (arrows in Fig. 2d), SYCP3 aggregates throughout the nucleus (arrowheads in Fig. 2d), and for the partial synapsis of X and Y at an area of homology known as the pseudoautosomal region (PAR; dashed box in Fig. 2d). As the early pachytene stage progresses, X and Y undergo nonhomologous synapsis beyond the PAR (dashed box in Fig. 2d). Nonhomologous synapsis continues until most of Y appears synapsed to X, after which nonhomologous desynapsis begins and Y gradually “peels off” of X.
    2. Mid pachytene spermatocytes are notable for distinct changes to sex chromosome localization and appearance. X and Y are sequestered away from synapsed autosomes in a large, ovoid nuclear subdomain known as the XY body (also known as the “sex body”), which is established in this stage (dashed box in Fig. 2e). X and Y synapse at only the PAR region (dashed box in Fig. 2e). There are no overt changes to autosome morphology in this stage, and few-to-no SYCP3 aggregates are present.
    3. The late pachytene stage is distinguished by unique changes to sex chromosome and autosome morphology. The axes of X and Y elongate within the XY body (Fig. 2f dashed box), and small SYCP3 extrusions branch off of Y (arrows in Fig. 2f). Further, SYCP3 becomes concentrated at the telocentric end of X and both the telocentric and telomeric ends of autosomes (arrowheads in Fig. 2f).
  4. In the diplotene stage, chromosomes progressively desynapse.
    1. To identify diplotene spermatocytes, check autosomes for progressive desynapsis (arrows in Fig. 2g, h).
    2. It is useful to subdivide diplotene spermatocytes into two stages, “early” and “late,” for analyses: Distinguish early diplotene spermatocytes by the desynapsis of ≤50% of autosomes (Fig. 2g) and the late pachytene-like morphology of the X and Y axes (dashed box in Fig. 2g); distinguish late diplotene spermatocytes by the broad desynapsis of >50% of autosomes (Fig. 2h) and the compacted appearance of the X and Y axes (dashed box in Fig. 2h).

3.5. Immunostaining Against γH2AX to Understand the Dynamics of MSCI

Phosphorylation of histone variant H2AX at Serine 139 (denoted as γH2AX) is an early and essential event in both meiotic recombination and the initiation of MSCI [6]. Because phosphorylation of H2AX is a prominent characteristic of both processes, it is necessary to differentiate the two by examining nucleus-wide γH2AX accumulation patterns in meiotic prophase. We offer guidelines to classify nuclear γH2AX accumulation into three broad categories, termed Patterns I, II, and III (Fig. 3). In the early pachytene stage, distinct γH2AX accumulation patterns on the sex chromosomes reveal DDR signaling events in MSCI. Thus, to analyze MSCI, we describe two γH2AX accumulation patterns specific to the sex chromosomes, termed “semiordered” accumulation and “ordered” accumulation (Fig. 3).

Fig. 3.

Fig. 3

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Illustrations and images of anti-γH2AX immunostaining to identify informative accumulation patterns in meiotic prophase. (a–h) Stages are indicated above illustrations in the panels. Nuclear regions (a) and the sex chromosomes (b–d) are indicated by dashed squares, and these squares are magnified in the bottom images of each panel. Arrows: described in the text; arrowheads: described in the text; X: X chromosome; Y: Y chromosome; PAR: pseudoautosomal region. Scale bars: 5 μm

  1. Nuclear γH2AX accumulation patterns.
    1. Pattern I: In the leptotene and early zygotene stages, γH2AX immunostaining is broad and largely uninterrupted throughout the nuclear chromatin (Fig. 3a). This accumulation pattern coincides with the initial steps of meiotic recombination, in which programmed DNA double strand breaks intersperse the genome.
    2. Pattern II: In the late zygotene and early pachytene stages, γH2AX immunostaining transitions from nucleus-wide accumulation to a pattern of staining distinguished by large, partial regions of accumulation, especially on and around the sex chromosomes (Fig. 3b, c). γH2AX accumulation appears on both the chromatin of synapsed axes (arrows in Fig. 3b, c) and unsynapsed axes (arrowheads in Fig. 3b).
    3. Pattern III: Beginning in the mid pachytene stage (Fig. 3d), γH2AX accumulation occurs on the sex chromosomes, while scant γH2AX accumulation may appear on autosome axes and chromatin as small puncta (arrows in Fig. 3d).
  2. Sex chromosome-specific γH2AX accumulation patterns.
    1. Semiordered accumulation: In early pachytene spermatocytes, γH2AX accumulation on the XY chromatin appears indeterminate and connects with γH2AX accumulation in separate portions of the nucleus (dashed boxes in Fig. 3b, c).
    2. Ordered accumulation: Beginning in the mid pachytene stage, the γH2AX accumulation pattern on the XY chromatin is tightly bounded and does not extend to different portions of the nucleus.

3.6. Extending the Analyses of Prophase Staging and MSCI

By following these guidelines for analysis, it is possible to image and judge the accumulation dynamics of a massive range of MSCI factors with clarity, efficiency, and precision. For examples, we direct you to recent publications from our laboratory [7, 8, 13, 15, 25].

Acknowledgments

We thank past and present members of the Namekawa laboratory for optimizing the experimental conditions. We thank Anna Sosso for reviewing the chapter. This work was supported by the Albert J. Ryan Fellowship to K.G.A. and the NIH Grant GM098605 to S.H.N.

Footnotes

1.

Although it dissolves more slowly than Na2B4O7·10H2O powder, anhydrous Na2B4O7 powder may also be used to make 200 mM Na2B4O7 solution: While stirring 400 mL of water, dissolve 20.1 g of anhydrous Na2B4O7 powder. Add water to 500 mL. Filter-sterilize or autoclave.

2.

Storage at a sub-7 pH value drastically slows the oxidation of 1 M DTT stock solution in contact with air, lengthening its “shelf life” and enabling many freeze-thaws.

3.

Use 60–100 mL of fixation solution per sample to be prepared. From one wild-type mouse testis at 8 weeks of age, one can prepare 24–30 chromosome spread slides. For mouse models with germ cell depletion phenotypes, the number of slides that one can prepare varies with phenotype severity. For example, we could prepare approximately six slides from one testis at 6 weeks of age from models deficient for the Fanconi anemia factors FANCB [14] and FANCD2 [16], both of which undergo marked germ cell loss during and after entry into adulthood. When testing a model deficient for the germline-specific Polycomb factor SCML2 [15], which undergoes a comparatively longer period of germ cell loss, we could prepare approximately 16–20 slides from one testis at 8 weeks of age.

4.

Of potential interest to readers, we have adapted our chromosome spread preparation protocol for fetal ovaries at embryonic day 17.5, which contain oocytes in the midst of meiotic prophase, and we include an abbreviated protocol here. Dissect ovaries from euthanized fetuses and remove the Müllerian ducts; we typically use 4–5 fetal embryos per experiment. Wash 1–5 ovaries once in a single 1.9-cm2 well, part of a 4-well dish, containing 1 mL of ice-cold 0.1% BSA in 1× PBS; use additional well(s) for any ovaries beyond a maximum of 5 per well. Transfer 1–5 ovaries to a single well containing 1 mL of ice-cold HEB; use additional well(s) for any ovaries beyond a maximum of 5 per well. Incubate the ovaries in HEB on ice for 45–60 min with gentle stirring every 15 min. 2–4 min before applying samples to slide surfaces, soak positively charged slides in chilled fixation solution; use 1 slide per 1 ovary. Pipet 15-μL of ice-cold sucrose (100 mM) to a dry, clean uncharged microscope slide. Using fine tweezers, transfer 1 ovary to the sucrose, then gently pull and mash the ovary between the tips of the fine tweezers. After approximately 20–30 mashes, a semitranslucent cell suspension is formed; apply the full volume of suspension to a positively charged slide that has been soaked in chilled fixation solution. Place the slide in a closed humid chamber at room temperature for a minimum incubation period of 4 h to a maximum overnight period of incubation. Repeat the sucrose, cell suspension, slide application, and humid chamber steps for the appropriate number of ovaries/slides. Then, continue from step 11 of the main chromosome spread preparation protocol until completion. Slides can be stored and/or stained as described in the main protocol.

5.

If using a sample with decreased testis size, such as a mutant or juvenile sample, the testis can be torn into two or three chunks, as opposed to four chunks, with tweezers. This is to promote the preparation of slides with sufficient numbers of cells for imaging and to minimize the amounts of buffers/reagents used in the experiment. Alternatively, if using a sample with increased testis size, such as adults ≥10 weeks in age, then approximately 2/3 of the testis can be torn off before division into four pieces. This is to minimize the fixation of broken nuclei, intracellular contents, and extracellular contents to slide surfaces. Broken nuclei and the like can result from the unraveling of an excessive amount of tubules in the small confines of a well.

6.

Again, perform the serial wash steps to prevent the inadvertent fixation of broken nuclei, intracellular contents, and extracellular contents to chromosome spread slides. This, in turn, will minimize background noise when imaging the slides. Furthermore, these serial unraveling steps promote the thorough suffusion of hypotonic extraction buffer, which facilitates the clean, consistent isolation of nuclei and their subsequent spreading into flat, bounded planes on the slide surfaces.

7.

Incubate samples for a shorter or longer time based on the tendency of sample tubules to clump, where an increased incubation time is used for samples that continue to clump despite the unraveling steps. For example, this tendency to clump is often evident in testis samples from mouse models with severe germ cell loss, such as models deficient for various DNA damage response factors [7, 8, 13–16].

8.

The spreading of testicular cells occurs rapidly in the presence of detergent [26]. An extended incubation in hypotonic extraction buffer, combined with the gentle formation of cell suspension, promotes the clean, consistent isolation and spreading of nuclei after exposure to fixation solution.

9.

“Humid chambers” are closed pipet tip boxes filled to approximately 2/3 volume with water at room temperature (Fig. 1). A high-humidity environment is important to prevent overdrying of the slides, which decreases the signal-to-noise ratio when imaging. However, we recommend some slight drying to promote the stable adherence of chromatin to the slide surface: When we have imaged slides that were drenched in suspension/fixation solution before washing in 0.4% Photo-Flo, we have observed nuclear chromatin that appears slightly smudged over small areas.

10.

The application of 0.4% Photo-Flo 200 will minimize the appearance of watermarks and streaks on dried slides, which promotes even immunostaining across the slide surface and increases the signal-to-noise ratio when imaging.

11.

When placing Parafilm on the slide surface, be careful to avoid the formation of bubbles, which can result in uneven staining of the slide. Furthermore, premature drying of the slides will result in a markedly low signal-to-noise ratio in imaging applications. Thus, be careful to avoid solution spilling off of the slides and always incubate slides in a high-humidity environment. To safeguard against spilling, cut the dimensions of the Parafilm cover to slightly less than that of the slide surface and gently place the cover on the surface using tweezers.

12.

Recommended incubation times and temperatures vary with the primary antibodies used. It is imperative that the slides do not dry during the incubation period as this decreases the signal-to-noise ratio in downstream imaging analyses. Thus, for longer incubation times, including overnight incubation, 4 °C in high-humidity conditions is preferable. For example, when imaging slides immunostained against SYCP3 and γH2AX, we get consistently high signal-to-noise ratios, which appear as signals of emission spectra set against deep black backgrounds, after 12–16 h of primary antibody incubation at 4 °C.

13.

A short incubation time of 30–60 min allows for the binding of secondary antibodies to primary antibodies while minimizing background noise, which increases as the incubation period increases.

14.

For example, two approaches are used to apply DAPI. (1) Minimizing exposure to light, dilute 0.2 μL of DAPI (1 mg/mL) in 200 μL of 1× PBS (~1 μg/mL final concentration) per slide to be counterstained. Coat each wet slide surface with 200 μL of DAPI/1× PBS solution. Incubate the slides for 5 min in a humid chamber at room temperature and in darkness. (2) Alternatively, add DAPI (1 mg/mL) to the final 5-min wash with PBST (step 10) to a final concentration of ~1 μg/mL (e.g., 4 μL of DAPI in 40 mL of PBST).

15.

The mountant and cover glasses should be appropriate for available imaging equipment (e.g., with respect to refractive indices, etc.). For example, mount FisherBrand cover glasses with no. 1.5 thickness range (ThermoFisher) after application of 20–30 μL of ProLong gold Antifade Mountant (ThermoFisher), which hardens for permanent mounting, or 10–20 μL of Vectashield Antifade mounting medium (Vector Laboratories H-1000), which remains viscous. If using a permanent mountant, allow sufficient time for curing.

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