Chapter 11 Ultrastructural and Immunofluorescent Methods for the Study of the XY Body as a Biomarker Roberta B. Sciurano and Alberto J. Solari Abstract Structural and immunohistochemical methods have been extremely useful for the characterization of the XY body (the structure formed by the XY pair during meiotic prophase) in Man and in other mammals. These methods are widely used at the present time for the detection of abnormalities leading to human infertility. The basic ultrastructural methods are spreading of pachytene spermatocytes, thin-sectioning techniques with or without 3-D reconstructions, and the monitoring of all specimens with semi-thin sections. Immunofluorescent techniques also use spreading of meiotic cells for the analysis of the XY body, and they can be combined with the fluorescence in situ hybridization (FISH) technique, in the so-called immuno-FISH. Epitope retrieval techniques are also used. Key words XY body, Human spermatocytes, Meiosis, Immuno-FISH
1
Introduction The term “XY body” was coined by Solari in 1974 [1] to label a special chromatin domain formed by the sex chromosomes during meiotic prophase, which is seen only in eutherian and metatherian mammals [1–4]. The functional importance of this subnuclear domain—or XY body—began to be noticed with the demonstration of its transcriptional inactivity [5] and it has been greatly increasing, to become a model of epigenetic processes [6, 7]. Furthermore, the XY body of meiotic spermatocytes has been shown to have special features in rare or abnormal processes that affect fertility in man and other mammals [8–11]. Thus, this subnuclear domain can be used as a biological marker, that is, a chromatin domain that may indicate a physiological state of the nucleus, through its variations in structure, in protein content, in repression of transcription, and in other aspects that are being analyzed in several laboratories. The XY body can be studied by several techniques that include light microscopy of routine, paraffin sections and semi-thin sections
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prepared with ultramicrotomes; it can be studied also with the “spreading” techniques using whole spermatogenic cells, freshly obtained from non-fixed testicular biopsies. Specific features of the XY body, like its proteins and DNA content, can be studied by immunofluorescence (IF) and special FISH techniques, respectively. This chapter is aimed to describe the presently applied microscopic techniques that are useful for the demonstration of the XY body as a biological marker in the spermatogenesis of man and other eutherian and metatherian mammals.
2
Materials
2.1 Testicular Biopsies
1. Human testicular biopsy—fresh tissue (preferentially) or cryopreserved at −80 °C (see Note 1). Each testicular biopsy— generally bilateral—is about 5–10 mm3 in volume (see Note 2). 2. Two clean vials with safety caps of 20 mL capacity and 3 cm diameter. 3. Hanks’ balanced salt solution (HBSS, 14170-120 Gibco® Life Technologies Corp., USA).
2.2 Semi-Thin Sections of Testicular Tissue and Staining with Toluidine Blue
1. Plain microscope slides 75 × 25 mm, 0.9–1.1 mm thick (294775 × 25, Corning Inc., USA) (see Note 3). 2. Two 10 mL glass vials with caps. 3. Cacodylate buffer: 0.1 M sodium cacodylate in distilled water. Adjust to pH 7.3 with 0.1 N HCl. Store at 4 °C (for 2–4 weeks). 4. Fixative solution A: 2.5 % glutaraldehyde (EM grade, 8 %, 10 mL ampoule, Polysciences Inc., USA) in cacodylate buffer. 5. 2 % osmium tetroxide (ampoule of 1 g OsO4, crystalline, Polysciencies) in distilled water. Prepare this solution carefully following the manufacturer’s instructions. 6. Post-fixative solution: 1:1, 2 % osmium tetroxide: 0.1 M cacodylate buffer. 7. Ethanol dilution series 40°, 70°, 96°, 100°. 8. Propylene oxide 100°. 9. Embedding medium: 1:1, propylene oxide 100°: Araldite 502 kit (Sigma-Aldrich). Prepare the epoxy-resin embedding medium following the manufacturer’s instructions. 10. Pelco’s flat embedding mold (#105, Ted Pella Inc., USA). 11. Toluidine blue staining: 1 % alkaline solution of toluidine blue. Add 1 g of toluidine blue in 100 mL distilled water, and heat to 60–80 °C using a magnetic stirrer until the toluidine blue has been dissolved. Adjust to pH 10–11 by adding traces of sodium borate, cool, and filter.
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2.3 Spermatocyte Spreads from Testicular Biopsies (Modified from Ref. 12)
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1. Hypotonic-shock solution: 200 μL of 0.1 M sucrose that contains a cocktail of protease inhibitors (final concentration 0.1 μg/mL each for pepstatin A, chymostatin, and leupeptin, and 1 μg/mL for aprotinin) or a general-use protease inhibitor cocktail (used following manufacturer’s instructions P27141BTL, Sigma-Aldrich) to give a final total volume of 1,200 μL per biopsy. 2. Fixative solution B (for electron microscopy, EM): 4 % paraformaldehyde, pH 8.5–9 (see Note 4), that contains 0.15 % Triton X100. 3. Fixative solution C (for immunofluorescence technique): 2 % paraformaldehyde, pH 8.5–9, containing 0.15 % Triton X100. 4. 0.4 % (v/v) of Photo-Flo 200 (Eastman Kodak) in distilled water with traces of phenol red as pH indicator. Adjust to pH 8 with 0.05 % tetrasodium borate. Store at 4 °C for 2–4 weeks, checking the pH before use.
2.4 Immunofluorescence (IF) Technique on Spermatocyte Spreads
1. Pretreatment solutions: (a) Epitope retrieval solution: 0.01 M sodium citrate buffer, pH 6, adjusted with 1 N HCl (see Note 5). (b) Deoxyribonuclease I (DNAse I): final concentration of 250 μg/mL DNAse I enzyme (DN25, Sigma-Aldrich) in distilled water. 2. Phosphate-buffered saline (PBS), pH 7.4: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, 2 mM KH2PO4 (see Note 6). 3. Blocking buffer A: 5 % bovine serum albumin (BSA, SigmaAldrich) and 0.1 % Tween 20 in phosphate-buffered saline (PBS). 4. Primary and secondary antibodies of interest (see Table 1). 5. DAPI staining: 0.1 μg/mL 4,6-diamidino-2-phenylindol in distilled water (DAPI, Sigma-Aldrich). 6. Mounting-and-antifading medium: 2 mM 1,4-diazabicyclo [2.2.2]octane (DABCO, Sigma-Aldrich) in 0.1 M Tris–HCl/ 90 % glycerol.
2.5
Immuno-FISH
1. Stringency wash solution A: 4× SSC (Saline-Sodium Citrate), 0.1 % Tween 20, pH 7.2–7.4. 2. Ethanol series: 70°, 80°, 100°. 3. Pretreatment with proteinase K (if necessary, see Note 7): 0.4 μg/mL proteinase K (P6556, Sigma-Aldrich) in 20 mM Tris–HCl, 2 mM CaCl2, pH 8. 4. Invitrogen’s SPoT-Light® Chromosome X/Y Probe Cocktail (Digoxigenin-labeled chromosome X centromere probe; and biotinylated chromosome Y band q12 probe, 84-1800 Invitrogen Corp.) or the DNA probe of interest.
Chromatin remodeling – – – – –
1:1,000
Chromatin remodeling
m γ-H2AX (Pser139) rb γ-H2AX (Pser139)
g α rb-FITC g α rb-TRICT g α mouse-FITC g α mouse-TRICT α human-FITC
1:1,000
Lateral elements of SC
rb SYCP3
1:50 1:50 1:25 1:25 1:200
1:400
1:200 1:200
Transversal filaments of SC Transversal filaments of SC
rb SYCP1 m SYCP1
1:50 1:30 1:10 1:30 1:500 1:100
Differential XY-axes Kinetocores Late recombination nodules Early recombination nodules Single axes of cohesins Central element of SC
Dilution in PBS
rb BRCA 1 (C 20) h CREST m MLH1 rb RAD51 (H92) rb SMC3 rb SYCE3
Labelling
R.T./37 R.T./37 R.T.37 R.T./37 R.T./37
37
37
4
4 4
4/37 4/37 4 37 4/37 4
Incubation (T °C)
F0382, Sigma, USA 111-025-045, Jackson ImmunoResearch, USA F0257, Sigma, USA 115-025-146, Jackson ImmunoResearch, USA IFI Fluor Multitest, Lab. IFI, Arg.
ab-2893, Abcam Ltd., UK
sc-642, Santa Cruz Biotech, USA IF I Fluor Multitest, Lab. IF I, Arg 550838, BD Pharmingen, USA sc-8349, Santa Cruz Biotech, USA ab3914, Millipore Corp., USA Courtesy from Prof. Alsheimer (University of Würzburg Germany) ab15090, Abcam Ltd., UK Courtesy from Moens-Spyropoulus (York University, Canada) ab15093, Abcam Ltd., UK/Courtesy from Moens-Spyropoulus 05-636, Millipore Corp., USA
Suppliers
rb rabbit, m mouse, h human, g goat, T °C temperature in degree Celsius, R.T. room temperature, SC synaptonemal complex
Secondary antibodies
Primary antibodies
Antibodies
Table 1 List of some useful primary and secondary antibodies to analyze the human XY body by immunofluorescence (IF) technique
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5. Stringency wash solution B: 0.5× SSC buffer, pH 7.2–7.4. 6. Blocking buffer B: 2.5 % BSA in PBS. 7. Fluorescein (FITC)-conjugated IgG fraction monoclonal mouse anti-digoxin (200-092-156, Jackson ImmunoResearch Inc.). Immediately before use, dilute 1:100 in PBS, mix softly, and short spin. 8. Rhodamine (TRICT)-conjugated streptavidin (016-020-084, Jackson ImmunoResearch Inc.). Immediately before use, dilute 1:500 in PBS, mix softly, and short spin. 9. DAPI staining (see above). 10. Mounting and antifading medium (see above).
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Methods
3.1 Testicular Biopsies
1. Transfer the fresh biopsies (generally, bilateral) in each vial containing 5–10 mL of HBSS (depends on the volume of each biopsy). 2. Place the testicular tissue on a dental wax plate with a drop of HBSS and subdivide the testicular tissue into small pieces using a new, clean razor blade to perform different techniques. Based on the aim of the analysis, the biopsies could be subdivided into four or more pieces for (a) histopathological diagnosis with routine methods (fixation in Bouin fixative, embedding in paraffin and staining with hematoxylin-eosin, Fig. 1a) and for
Fig. 1 (a–c). Identification of the human XY body in normal spermatocytes using different routine techniques. The XY body is a clearly distinct, oblong chromatin domain within the pachytene nuclei (arrows). (a) Paraffin section stained by hematoxylin-eosin. (b) Semi-thin section stained by toluidine blue. (c) Electron micrograph of a thin section stained with lead citrate [13]. n nucleolus, f and g fibrillar and granular regions of the nucleolus, respectively, arrowhead heterochromatin of an acrocentric chromosome, lines differential axes of X and Y chromosomes. Bars: (a) 5 μm, (b) 5 μm, (c) 1 μm
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Fig. 2 (a–d). Immunofluorescence (IF) localization of meiotic proteins and FISH in human spermatocyte spreads from control (a, b) and infertile (c, d) men. (a) A triple IF of SYCP3 (red axes), BRCA1 (green axes), and γ-H2AX (red chromatin) shows the autosomal bivalents and a normal XY body (arrow). The BRCA1 and γ-H2AX proteins are normally absent in the pseudoautosomal region (PAR, arrowhead). DAPI counterstaining, blue. (b) A double IF of SYCP3 (red axes) and MLH1 (green foci) marks the only recombination nodule at the distal end of the XY pair. (c) Immuno-FISH technique (SYCP3, red axes; CREST, green spots; X centromere probe, purple) allows identifying euploid, 46, XY spermatocytes in a non-mosaic Klinefelter patients. (d) A double IF of BRCA1 (green axes) and γ-H2AX (red chromatin-patches) shows the lack of a normal XY body in an azoospermic man with a double-strand DNA break-processing deficiency. Bars: (a) 5 μm, (b) 10 μm, (c) 5 μm, (d) 10 μm
immunohistochemistry in paraffin sections (see [14]); (b) histological analysis of semi-thin sections by light microscopy (fixation in glutaraldehyde, postfixation in osmium tetroxide, embedding in araldite, and staining with toluidine blue) (Fig. 1b, see Subheading 3.2 in this chapter); (c) ultrastructural analysis of the XY body by electron microscopy (Fig. 1c); (d) air-drying technique for meiotic metaphases [15]; and (e) immunolocalization of XY-body proteins (combined with FISH, if interesting) on spermatocyte spreads (Fig. 2, see Subheading 3.3 in this chapter).
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3.2 Semi-Thin Sections of Testicular Biopsies
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1. Place 1 mm3 of the testicular tissue (removing the tunica albuginea, vasculature, and fat) in a 10 mL glass vial with 2–3 mL of fixative solution A for 1 h at room temperature (RT). 2. Remove the fixative solution A and rinse in 2–3 mL of cacodylate buffer into the same glass vial (see Note 8). 3. Remove the cacodylate buffer, add 2 mL of post-fixative solution, and leave on for 1 h at RT. 4. Wash twice in cacodylate buffer for 5 min each. 5. Dehydrate through an ethanol series 40°, 70°, 96° for 10 min, twice each, and ethanol 100° for 5 min, three times each. 6. Remove the ethanol 100° and wash twice in propylene oxide 100° for 5 min each. 7. Add 2 mL of embedding medium, cap tightly the glass vial, and leave on overnight (16–24 h) at 4 °C. 8. Remove the glass vial from the refrigerator, leave it at RT to raise the temperature, and remove the solvent from the embedding medium. 9. Evaporate the remnant of propylene oxide under a lamp until the epoxy resin retrieves the initial viscosity. 10. Add the embedding medium in a flat embedding mold and immerse the piece of tissue. 11. Place in a laboratory oven overnight at 58 °C to allow the polymerization of the epoxy resin. 12. Section the semi-thin slices (0.5–1 μm) using a Porter-Blumtype ultramicrotome fitted with a glass knife, place on a clean slide, and dry at 70–80 °C on a hot plate until water evaporates. 13. Add a drop of toluidine blue staining solution using a plastic pipette and heat at 70–80 °C on a hot plate until it begins to give off vapor (around 1–3 min). 14. Rinse in distilled water, air-dry, and observe under the light microscope (see Note 9).
3.3 Spermatocyte Spreads from Testicular Biopsies
1. Remove the tunica albuginea, vasculature, and fat. 2. Put the mass of seminiferous tubules on a depression in the glass or plastic plate filled with hypotonic-shock solution and chop this mass in equally smaller pieces with a clean razor blade. 3. Take one small piece of tissue, add one drop of hypotonicshock solution using a plastic pipette, and, softly, squeeze the fragments with two clean frosted-one-side slides to extract the germinal cells from the seminiferous tubules. 4. Using a handmade aspirator (see Note 10), place one drop of the cell suspension on a clean slide.
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5. Monitor the number of spermatocytes per field by phase contrast microscopy (the correct dilution should be around 5–15 spermatocytes per field observed by 10× objective and 10× ocular lens; see Note 11). 6. After determining the correct dilution, incubate the testicular tissue in the hypotonic-shock solution for 20–30 min at RT. 7. Label the slide with a carbide-tip pen. 8. Drop 80 μL of fixative solution B or C (see Note 12) on a clean slide and spread the solution holding the tip of the pipette parallel to the slide. 9. Carefully, place a drop of cell suspension (according to the dilution determined in Subheading 3.3, step 5) on the slide, avoiding any bubble. 10. Place them horizontally (to prevent leakage of fluid) in a humid chamber and sediment for 2 h at RT. 11. After incubation, rinse the slides in 0.4 % Photo-Flo 200 for 5 min and air-dry. 12. Check and classify the quality of the cellular spreads—under a phase contrast microscopy—based on the number of spermatocytes per 10× field, the extraction of the chromatin, and the optical contrast between synaptonemal complexes and chromatin (see Note 13). These spreads could be kept at −80 °C until used (see Note 14). 3.4 Immunofluorescence (IF) Localization of XY-Body Proteins Using Spermatocyte Spreads
1. Defrost the spreads from −80 °C (or use them directly after air-drying). 2. Place them in a dark humidity chamber for maintaining a wet atmosphere and protecting from the light (from this step until the end, the technique should be performed in this dark humidity chamber). 3. For protein components of synaptonemal complexes (like SYCP1, SYCP3, SYCE3) or recombination nodules (like RAD51, MLH1), it is recommended to choose one of the following pretreatments to unmask the corresponding epitope (see Note 15): (a) Citrate-buffer epitope retrieval: Place the slides in 200 mL of epitope retrieval solution and heat them in two rounds of heating using a microwave: (1) at 720 W for 1–2.5 min, (2) at 240 W for 2 min (see Note 16). At the end of the microwave heating, the temperature should have increased to a maximum of 83–85 °C. Cool the slides placing them in a Coplin jar with the citrate buffer at RT for 5 min and wash three times in PBS for 3 min each. (b) Chromatin digestion using DNAse I (see Note 17): Add 100 μL of 250 μg/mL DNAse I (final concentration),
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apply a plastic or a glass coverslip, and incubate for 8–10 min at RT in a dark humidity chamber. Quickly wash with distilled water. 4. Blocking step: Add 100 μL of blocking buffer A, apply a plastic coverslip, and incubate for 20–30 min at RT in a dark humidity chamber. 5. After blocking, remove the coverslip and the blocking buffer, add the primary antibodies of interest (100 μL), apply a plastic coverslip, and incubate overnight (see Table 1 and Note 18). 6. Remove the coverslip and wash three times in PBS for 3 min. 7. Add 100 μL of secondary antibodies, apply a plastic coverslip, and incubate for 2 h (Table 1). 8. Remove the coverslip, wash three times in PBS for 3 min and three times in distilled water for 2 min each. 9. Counterstain by adding 100 μL of 0.1 μg/mL DAPI, add coverslip, and incubate for 5 min. 10. Rinse twice in distilled water for 1 min each. 11. Air-dry, mount in antifading mounting medium, and apply a glass coverslip. 12. Observe, photograph, and annotate the position of the spermatocytes of choice (see Note 19). 3.5 Combination of Immunofluorescence Staining and FISH (Immuno-FISH) to Study the Behavior of the XY Body
After examining and photographing the spermatocyte spreads, it is possible to perform a fluorescence in situ hybridization (FISH)— on the same spermatocytes which were previously immunolabeled—using DNA probes that bind specifically to the X chromosome or the Y chromosome or both (see Subheading 2.5, Invitrogen’s SPoT-Light® Chromosome X/Y Probe Cocktail). 1. Carefully, remove the glass coverslip. 2. Rinse the slides three times for 10 min each in a Coplin jar (protected from light) with the stringency wash solution A, at RT. 3. Dehydrate the slides through an ethanol series 70°, 80°, 100° for 2 min each and air-dry until no traces of ethanol are left on them. 4. If necessary, incubate with 20 mg/mL of proteinase K in buffer Tris-CaCl2 for 20 min at 37 °C. This step should not be performed if the slides were preheated in epitope retrieval solution during the immunofluorescence technique. 5. Add 25 μL of probe to the center of the sample, apply a 24 × 40 mm coverslip (use more probe for bigger and larger coverslips), and place in a dark humidity chamber. 6. Denature the DNA of spermatocyte nuclei and the DNA probe on a hot plate at 80 °C for 3 min.
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7. Incubate overnight (16–24 h) at 37 °C in a dark humidity chamber. 8. Remove coverslip and immerse the slides in a Coplin jar with stringency wash solution B at 72 °C for 5 min. 9. Rinse three times in stringency wash solution A for 2 min each at RT. 10. Add 50 μL of blocking buffer B, apply a plastic coverslip, and incubate for 15 min at RT in a dark humidity chamber. 11. Add 50 μL (total volume) of diluted FITC-conjugated mouse anti-digoxin and/or TRICT-conjugated streptavidin. 12. Repeat Subheading 3.5, step 9. 13. Counterstain by adding 50 μL of 0.1 μg/mL DAPI, add coverslip, and incubate for 5 min. 14. Rinse twice in distilled water for 1 min each. 15. Air-dry, mount in antifading mounting medium, and apply a glass coverslip. 16. Observe, localize again the spermatocytes of choice, and photograph (see Note 20).
4
Notes 1. Cryogenic preservation is based on Sudman [16]. 2. Surgeons should avoid damaging the testicular tissue (do not place it on a piece of gauze or cotton). The testicular tissue should be put immediately in a culture medium or in a fixative solution at room temperature and transferred to the laboratory as soon as possible. It is recommended to use the testicular tissue up to 6 h after surgery to obtain good spreads. 3. The slides should be perfectly clean, devoid of irregularities and fat (particularly, for electron microscopy). They could be examined by phase contrast microscopy at 400× magnification. If they are not enough clean, put in Coplin jars with 96° and 100° ethanol for 15 min each, and dry using a lint-free paper. 4. A 4 % paraformaldehyde solution is made with 4 g in 100 mL of distilled water, plus traces of phenol red as pH indicator; it is heated slowly at 50–55 °C while stirring; the pH of the solution is brought alkaline (purple-red, pH about 8) by slowly adding drops of 0.1 N NaOH until the paraformaldehyde is solubilized. Allow to cool, filter, and adjust pH to 8.5–9 with 0.05 M tetrasodium borate. Store at 4 °C (up to several months), check the pH, and adjust it if necessary. 5. A stock solution of 0.1 M sodium citrate buffer (pH 6) can be prepared and stored at room temperature. The citrate epitope
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retrieval unmasks epitopes in paraformaldehyde-fixed spreads, thus enhancing staining intensity of antibodies. 6. A 10×-concentrated stock solution could be prepared and stored at RT. If the 1×-diluted PBS solution is not sterile, it is recommended to store it at 4 °C to prevent bacterial growth. 7. If the slides were previously preheated in epitope retrieval solution, they should not be treated with proteinase K. The proteinase K can be used as an epitope retrieval solution: firstly, by breaking the protein cross-links and, secondly, by unmasking the epitopes in paraformaldehyde-fixed cells. 8. The testicular tissue can be stored in cacodylate buffer for weeks by adding 2–3 drops of fixative solution A using a plastic pipette. 9. If the semi-thin section is overstained with toluidine blue, it could be rinsed in ethanol 96°. 10. To make the handmade aspirator, take a scalp vein set 21G, cut the end which has a needle, remove the needle, and put a small piece of cotton in the opposite side (in order to prevent the aspiration of any liquid, including a cell suspension). Then, hold an 8 mm capillary tube by the ends, place the center of the capillary tube under the flame of a Bunsen burner, and heat it until the glass softens; quickly extend the capillary tubes from the ends until both sides are separated. Cut the tip of the thinner side of the capillary tube and insert it to the scalp vein set. 11. The amount of tissue should be optimal (5–15 spermatocytes per field observed by a 10× objective) to allow the correct spreading of each spermatocyte, and avoid crowding of the nuclei on the slide or a low number of nuclei per field. Testicular biopsies from infertile men show a great variability depending on the type of spermatogenesis failure, which ranges from almost normal spermatogenesis to a complete lack of germ cells (Sertoli-cell-only syndrome and/or hyalinization of seminiferous tubules). 12. It is recommended to fix the cell suspension with fixative solution B for electron microscopy, and fixative solution C for immunofluorescence technique/FISH. Once the spermatocyte spreads for EM are ready, they could be silver stained [17], coated by a plastic solution, and placed in center-marked grids for EM (see details in ref. 18). 13. The “quality” of the slides is widely variable: the attachment of the nuclei on the slides depends on the state of the tissue, dilution, and the senescence of the cells. 14. It is possible to keep the cellular spreads at −80 °C for months (or even, for years). In the latter case, it is suggested to pretreat them with citrate-buffer epitope retrieval.
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15. These pretreatments should not be performed if the protein of interest is a chromatin-related protein. 16. It is important that the temperature of the citrate buffer does not rise to 90 °C. It should be controlled in such a way that the temperature at the first round of heating ranges between 80 and 82 °C. 17. This pretreatment should not be done if a FISH technique will be performed. 18. The antibodies of interest should be incubated at concentrations and temperatures previously determined for each specific antibody (Table 1). 19. For double immunodetection using two primary antibodies on the same host, two successive rounds of incubations could be performed. For example, if the antibodies against BRCA1 and SYCP3 were inoculated in rabbit, perform a first round of immunolabelling using BRCA1 antibody— from Subheading 3.4, steps 1–12. To carry out the second round of immunolabelling using SYCP3 antibody, remove the coverslip, rinse the slides three times for 10 min each in a Coplin jar (protected from light) with 4× SSC plus 0.1 % Tween 20, and repeat Subheading 3.4, steps 4–11. Based on the annotated position of the spermatocytes of choice, localize again, observe, photograph, and superimpose the separated images using Adobe Photoshop CS software (Adobe Systems Inc., San Jose, CA, USA). 20. Using Adobe Photoshop CS, the separated images can be superimposed. Furthermore, the color of the FISH signal could be artificially modified in order to distinguish this label from the one/s resulting from the immunofluorescence technique (Fig. 2c).
Acknowledgments We thank M.Sc. I.M. Rahn for the useful comments on the manuscript and for experimental help. Thanks to Mrs C. Deparci for the able technical help. We acknowledge Prof. R. Ponzio for the histopathological diagnosis of the biopsies, and R. Coco, Ph.D.; G. Rey Valzacchi, M.D.; and S. Brugo Olmedo, M.D., for the clinical and surgical procedures. We also thank the generous gift of antibodies from P. J. Moens, B. Spyropoulus (York University, Toronto, Ontario, Canada), and M. Alsheimer (University of Würzburg, Würzburg, Germany). This work was supported by UBACYT 20020100100030 (AJS) and FONCYT PICT 2010-2718 (RBS).
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References 1. Solari AJ (1974) The behaviour of the XY pair in mammals. Int Rev Cytol 38:273–317 2. Solari AJ (1994) Sex chromosomes and sex determination in vertebrates. CRC Press, Boca Raton, FL 3. Franco MJ, Sciurano RB, Solari AJ (2007) Protein immunolocalization supports the presence of identical mechanisms of XY body formation in eutherians and marsupials. Chromosome Res 15:815–824 4. Sciurano RB, Rahn MI, Rossi L et al (2012) Synapsis, recombination, and chromatin remodeling in the XY body of armadillos. Chromosome Res 20:293–302 5. Monesi V (1965) Differential rate of ribonucleic acid synthesis in the autosomes and sex chromosomes during male meiosis in the mouse. Chromosoma 17:11–21 6. Handel MA (2004) The XY body: a specialized meiotic chromatin domain. Exp Cell Res 296:57–63 7. Ichijima Y, Sin HS, Namekawa SH (2012) Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways. Cell Mol Life Sci 69:2559–2572 8. Coco R, Rahn MI, Estanga PG et al (2004) A constitutional complex chromosome rearrangement involving meiotic arrest in an azoospermic male: case report. Hum Reprod 19:2784–2790 9. Sciurano RB, Rahn MI, Pigozzi MI et al (2006) An azoospermic man with a doublestrand DNA break-processing deficiency in the spermatocyte nuclei: case report. Hum Reprod 21:1194–1203
10. Burgoyne PS, Mahadevaiah SK, Turner JM (2009) The consequences of asynapsis for mammalian meiosis. Nat Rev Genet 10:207–216 11. Sciurano RB, Rahn MI, Rey-Valzacchi G et al (2012) The role of asynapsis in human spermatocyte failure. Int J Androl 35:541–549 12. Anderson LK, Reeves A, Webb LM et al (1999) Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics 151:1569–1579 13. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212 14. Sciurano RB, Luna Hisano CV, Rahn MI et al (2009) Focal spermatogenesis originates in euploid germ cells in classical Klinefelter patients. Hum Reprod 24:2353–2360 15. Evans EP, Breckon G, Ford CE (1964) An air-drying method for meiotic preparations from mammalian testes. Cytogenetics 15:289–294 16. Sudman PD (1989) Cryogenic preservation of mammalian testicular material for synaptonemal complex analysis. Cytogenet Cell Genet 52:88–89 17. Howell WM, Black DA (1980) Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1 step method. Experientia 36:1014–1015 18. Solari AJ (1998) Structural analysis of meiotic chromosomes and synaptonemal complexes in higher vertebrates. Methods Cell Biol 53: 236–254
1
Introduction The term “XY body” was coined by Solari in 1974 [1] to label a special chromatin domain formed by the sex chromosomes during meiotic prophase, which is seen only in eutherian and metatherian mammals [1–4]. The functional importance of this subnuclear domain—or XY body—began to be noticed with the demonstration of its transcriptional inactivity [5] and it has been greatly increasing, to become a model of epigenetic processes [6, 7]. Furthermore, the XY body of meiotic spermatocytes has been shown to have special features in rare or abnormal processes that affect fertility in man and other mammals [8–11]. Thus, this subnuclear domain can be used as a biological marker, that is, a chromatin domain that may indicate a physiological state of the nucleus, through its variations in structure, in protein content, in repression of transcription, and in other aspects that are being analyzed in several laboratories. The XY body can be studied by several techniques that include light microscopy of routine, paraffin sections and semi-thin sections
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prepared with ultramicrotomes; it can be studied also with the “spreading” techniques using whole spermatogenic cells, freshly obtained from non-fixed testicular biopsies. Specific features of the XY body, like its proteins and DNA content, can be studied by immunofluorescence (IF) and special FISH techniques, respectively. This chapter is aimed to describe the presently applied microscopic techniques that are useful for the demonstration of the XY body as a biological marker in the spermatogenesis of man and other eutherian and metatherian mammals.
2
Materials
2.1 Testicular Biopsies
1. Human testicular biopsy—fresh tissue (preferentially) or cryopreserved at −80 °C (see Note 1). Each testicular biopsy— generally bilateral—is about 5–10 mm3 in volume (see Note 2). 2. Two clean vials with safety caps of 20 mL capacity and 3 cm diameter. 3. Hanks’ balanced salt solution (HBSS, 14170-120 Gibco® Life Technologies Corp., USA).
2.2 Semi-Thin Sections of Testicular Tissue and Staining with Toluidine Blue
1. Plain microscope slides 75 × 25 mm, 0.9–1.1 mm thick (294775 × 25, Corning Inc., USA) (see Note 3). 2. Two 10 mL glass vials with caps. 3. Cacodylate buffer: 0.1 M sodium cacodylate in distilled water. Adjust to pH 7.3 with 0.1 N HCl. Store at 4 °C (for 2–4 weeks). 4. Fixative solution A: 2.5 % glutaraldehyde (EM grade, 8 %, 10 mL ampoule, Polysciences Inc., USA) in cacodylate buffer. 5. 2 % osmium tetroxide (ampoule of 1 g OsO4, crystalline, Polysciencies) in distilled water. Prepare this solution carefully following the manufacturer’s instructions. 6. Post-fixative solution: 1:1, 2 % osmium tetroxide: 0.1 M cacodylate buffer. 7. Ethanol dilution series 40°, 70°, 96°, 100°. 8. Propylene oxide 100°. 9. Embedding medium: 1:1, propylene oxide 100°: Araldite 502 kit (Sigma-Aldrich). Prepare the epoxy-resin embedding medium following the manufacturer’s instructions. 10. Pelco’s flat embedding mold (#105, Ted Pella Inc., USA). 11. Toluidine blue staining: 1 % alkaline solution of toluidine blue. Add 1 g of toluidine blue in 100 mL distilled water, and heat to 60–80 °C using a magnetic stirrer until the toluidine blue has been dissolved. Adjust to pH 10–11 by adding traces of sodium borate, cool, and filter.
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1. Hypotonic-shock solution: 200 μL of 0.1 M sucrose that contains a cocktail of protease inhibitors (final concentration 0.1 μg/mL each for pepstatin A, chymostatin, and leupeptin, and 1 μg/mL for aprotinin) or a general-use protease inhibitor cocktail (used following manufacturer’s instructions P27141BTL, Sigma-Aldrich) to give a final total volume of 1,200 μL per biopsy. 2. Fixative solution B (for electron microscopy, EM): 4 % paraformaldehyde, pH 8.5–9 (see Note 4), that contains 0.15 % Triton X100. 3. Fixative solution C (for immunofluorescence technique): 2 % paraformaldehyde, pH 8.5–9, containing 0.15 % Triton X100. 4. 0.4 % (v/v) of Photo-Flo 200 (Eastman Kodak) in distilled water with traces of phenol red as pH indicator. Adjust to pH 8 with 0.05 % tetrasodium borate. Store at 4 °C for 2–4 weeks, checking the pH before use.
2.4 Immunofluorescence (IF) Technique on Spermatocyte Spreads
1. Pretreatment solutions: (a) Epitope retrieval solution: 0.01 M sodium citrate buffer, pH 6, adjusted with 1 N HCl (see Note 5). (b) Deoxyribonuclease I (DNAse I): final concentration of 250 μg/mL DNAse I enzyme (DN25, Sigma-Aldrich) in distilled water. 2. Phosphate-buffered saline (PBS), pH 7.4: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, 2 mM KH2PO4 (see Note 6). 3. Blocking buffer A: 5 % bovine serum albumin (BSA, SigmaAldrich) and 0.1 % Tween 20 in phosphate-buffered saline (PBS). 4. Primary and secondary antibodies of interest (see Table 1). 5. DAPI staining: 0.1 μg/mL 4,6-diamidino-2-phenylindol in distilled water (DAPI, Sigma-Aldrich). 6. Mounting-and-antifading medium: 2 mM 1,4-diazabicyclo [2.2.2]octane (DABCO, Sigma-Aldrich) in 0.1 M Tris–HCl/ 90 % glycerol.
2.5
Immuno-FISH
1. Stringency wash solution A: 4× SSC (Saline-Sodium Citrate), 0.1 % Tween 20, pH 7.2–7.4. 2. Ethanol series: 70°, 80°, 100°. 3. Pretreatment with proteinase K (if necessary, see Note 7): 0.4 μg/mL proteinase K (P6556, Sigma-Aldrich) in 20 mM Tris–HCl, 2 mM CaCl2, pH 8. 4. Invitrogen’s SPoT-Light® Chromosome X/Y Probe Cocktail (Digoxigenin-labeled chromosome X centromere probe; and biotinylated chromosome Y band q12 probe, 84-1800 Invitrogen Corp.) or the DNA probe of interest.
Chromatin remodeling – – – – –
1:1,000
Chromatin remodeling
m γ-H2AX (Pser139) rb γ-H2AX (Pser139)
g α rb-FITC g α rb-TRICT g α mouse-FITC g α mouse-TRICT α human-FITC
1:1,000
Lateral elements of SC
rb SYCP3
1:50 1:50 1:25 1:25 1:200
1:400
1:200 1:200
Transversal filaments of SC Transversal filaments of SC
rb SYCP1 m SYCP1
1:50 1:30 1:10 1:30 1:500 1:100
Differential XY-axes Kinetocores Late recombination nodules Early recombination nodules Single axes of cohesins Central element of SC
Dilution in PBS
rb BRCA 1 (C 20) h CREST m MLH1 rb RAD51 (H92) rb SMC3 rb SYCE3
Labelling
R.T./37 R.T./37 R.T.37 R.T./37 R.T./37
37
37
4
4 4
4/37 4/37 4 37 4/37 4
Incubation (T °C)
F0382, Sigma, USA 111-025-045, Jackson ImmunoResearch, USA F0257, Sigma, USA 115-025-146, Jackson ImmunoResearch, USA IFI Fluor Multitest, Lab. IFI, Arg.
ab-2893, Abcam Ltd., UK
sc-642, Santa Cruz Biotech, USA IF I Fluor Multitest, Lab. IF I, Arg 550838, BD Pharmingen, USA sc-8349, Santa Cruz Biotech, USA ab3914, Millipore Corp., USA Courtesy from Prof. Alsheimer (University of Würzburg Germany) ab15090, Abcam Ltd., UK Courtesy from Moens-Spyropoulus (York University, Canada) ab15093, Abcam Ltd., UK/Courtesy from Moens-Spyropoulus 05-636, Millipore Corp., USA
Suppliers
rb rabbit, m mouse, h human, g goat, T °C temperature in degree Celsius, R.T. room temperature, SC synaptonemal complex
Secondary antibodies
Primary antibodies
Antibodies
Table 1 List of some useful primary and secondary antibodies to analyze the human XY body by immunofluorescence (IF) technique
140 Roberta B. Sciurano and Alberto J. Solari
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5. Stringency wash solution B: 0.5× SSC buffer, pH 7.2–7.4. 6. Blocking buffer B: 2.5 % BSA in PBS. 7. Fluorescein (FITC)-conjugated IgG fraction monoclonal mouse anti-digoxin (200-092-156, Jackson ImmunoResearch Inc.). Immediately before use, dilute 1:100 in PBS, mix softly, and short spin. 8. Rhodamine (TRICT)-conjugated streptavidin (016-020-084, Jackson ImmunoResearch Inc.). Immediately before use, dilute 1:500 in PBS, mix softly, and short spin. 9. DAPI staining (see above). 10. Mounting and antifading medium (see above).
3
Methods
3.1 Testicular Biopsies
1. Transfer the fresh biopsies (generally, bilateral) in each vial containing 5–10 mL of HBSS (depends on the volume of each biopsy). 2. Place the testicular tissue on a dental wax plate with a drop of HBSS and subdivide the testicular tissue into small pieces using a new, clean razor blade to perform different techniques. Based on the aim of the analysis, the biopsies could be subdivided into four or more pieces for (a) histopathological diagnosis with routine methods (fixation in Bouin fixative, embedding in paraffin and staining with hematoxylin-eosin, Fig. 1a) and for
Fig. 1 (a–c). Identification of the human XY body in normal spermatocytes using different routine techniques. The XY body is a clearly distinct, oblong chromatin domain within the pachytene nuclei (arrows). (a) Paraffin section stained by hematoxylin-eosin. (b) Semi-thin section stained by toluidine blue. (c) Electron micrograph of a thin section stained with lead citrate [13]. n nucleolus, f and g fibrillar and granular regions of the nucleolus, respectively, arrowhead heterochromatin of an acrocentric chromosome, lines differential axes of X and Y chromosomes. Bars: (a) 5 μm, (b) 5 μm, (c) 1 μm
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Fig. 2 (a–d). Immunofluorescence (IF) localization of meiotic proteins and FISH in human spermatocyte spreads from control (a, b) and infertile (c, d) men. (a) A triple IF of SYCP3 (red axes), BRCA1 (green axes), and γ-H2AX (red chromatin) shows the autosomal bivalents and a normal XY body (arrow). The BRCA1 and γ-H2AX proteins are normally absent in the pseudoautosomal region (PAR, arrowhead). DAPI counterstaining, blue. (b) A double IF of SYCP3 (red axes) and MLH1 (green foci) marks the only recombination nodule at the distal end of the XY pair. (c) Immuno-FISH technique (SYCP3, red axes; CREST, green spots; X centromere probe, purple) allows identifying euploid, 46, XY spermatocytes in a non-mosaic Klinefelter patients. (d) A double IF of BRCA1 (green axes) and γ-H2AX (red chromatin-patches) shows the lack of a normal XY body in an azoospermic man with a double-strand DNA break-processing deficiency. Bars: (a) 5 μm, (b) 10 μm, (c) 5 μm, (d) 10 μm
immunohistochemistry in paraffin sections (see [14]); (b) histological analysis of semi-thin sections by light microscopy (fixation in glutaraldehyde, postfixation in osmium tetroxide, embedding in araldite, and staining with toluidine blue) (Fig. 1b, see Subheading 3.2 in this chapter); (c) ultrastructural analysis of the XY body by electron microscopy (Fig. 1c); (d) air-drying technique for meiotic metaphases [15]; and (e) immunolocalization of XY-body proteins (combined with FISH, if interesting) on spermatocyte spreads (Fig. 2, see Subheading 3.3 in this chapter).
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1. Place 1 mm3 of the testicular tissue (removing the tunica albuginea, vasculature, and fat) in a 10 mL glass vial with 2–3 mL of fixative solution A for 1 h at room temperature (RT). 2. Remove the fixative solution A and rinse in 2–3 mL of cacodylate buffer into the same glass vial (see Note 8). 3. Remove the cacodylate buffer, add 2 mL of post-fixative solution, and leave on for 1 h at RT. 4. Wash twice in cacodylate buffer for 5 min each. 5. Dehydrate through an ethanol series 40°, 70°, 96° for 10 min, twice each, and ethanol 100° for 5 min, three times each. 6. Remove the ethanol 100° and wash twice in propylene oxide 100° for 5 min each. 7. Add 2 mL of embedding medium, cap tightly the glass vial, and leave on overnight (16–24 h) at 4 °C. 8. Remove the glass vial from the refrigerator, leave it at RT to raise the temperature, and remove the solvent from the embedding medium. 9. Evaporate the remnant of propylene oxide under a lamp until the epoxy resin retrieves the initial viscosity. 10. Add the embedding medium in a flat embedding mold and immerse the piece of tissue. 11. Place in a laboratory oven overnight at 58 °C to allow the polymerization of the epoxy resin. 12. Section the semi-thin slices (0.5–1 μm) using a Porter-Blumtype ultramicrotome fitted with a glass knife, place on a clean slide, and dry at 70–80 °C on a hot plate until water evaporates. 13. Add a drop of toluidine blue staining solution using a plastic pipette and heat at 70–80 °C on a hot plate until it begins to give off vapor (around 1–3 min). 14. Rinse in distilled water, air-dry, and observe under the light microscope (see Note 9).
3.3 Spermatocyte Spreads from Testicular Biopsies
1. Remove the tunica albuginea, vasculature, and fat. 2. Put the mass of seminiferous tubules on a depression in the glass or plastic plate filled with hypotonic-shock solution and chop this mass in equally smaller pieces with a clean razor blade. 3. Take one small piece of tissue, add one drop of hypotonicshock solution using a plastic pipette, and, softly, squeeze the fragments with two clean frosted-one-side slides to extract the germinal cells from the seminiferous tubules. 4. Using a handmade aspirator (see Note 10), place one drop of the cell suspension on a clean slide.
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5. Monitor the number of spermatocytes per field by phase contrast microscopy (the correct dilution should be around 5–15 spermatocytes per field observed by 10× objective and 10× ocular lens; see Note 11). 6. After determining the correct dilution, incubate the testicular tissue in the hypotonic-shock solution for 20–30 min at RT. 7. Label the slide with a carbide-tip pen. 8. Drop 80 μL of fixative solution B or C (see Note 12) on a clean slide and spread the solution holding the tip of the pipette parallel to the slide. 9. Carefully, place a drop of cell suspension (according to the dilution determined in Subheading 3.3, step 5) on the slide, avoiding any bubble. 10. Place them horizontally (to prevent leakage of fluid) in a humid chamber and sediment for 2 h at RT. 11. After incubation, rinse the slides in 0.4 % Photo-Flo 200 for 5 min and air-dry. 12. Check and classify the quality of the cellular spreads—under a phase contrast microscopy—based on the number of spermatocytes per 10× field, the extraction of the chromatin, and the optical contrast between synaptonemal complexes and chromatin (see Note 13). These spreads could be kept at −80 °C until used (see Note 14). 3.4 Immunofluorescence (IF) Localization of XY-Body Proteins Using Spermatocyte Spreads
1. Defrost the spreads from −80 °C (or use them directly after air-drying). 2. Place them in a dark humidity chamber for maintaining a wet atmosphere and protecting from the light (from this step until the end, the technique should be performed in this dark humidity chamber). 3. For protein components of synaptonemal complexes (like SYCP1, SYCP3, SYCE3) or recombination nodules (like RAD51, MLH1), it is recommended to choose one of the following pretreatments to unmask the corresponding epitope (see Note 15): (a) Citrate-buffer epitope retrieval: Place the slides in 200 mL of epitope retrieval solution and heat them in two rounds of heating using a microwave: (1) at 720 W for 1–2.5 min, (2) at 240 W for 2 min (see Note 16). At the end of the microwave heating, the temperature should have increased to a maximum of 83–85 °C. Cool the slides placing them in a Coplin jar with the citrate buffer at RT for 5 min and wash three times in PBS for 3 min each. (b) Chromatin digestion using DNAse I (see Note 17): Add 100 μL of 250 μg/mL DNAse I (final concentration),
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apply a plastic or a glass coverslip, and incubate for 8–10 min at RT in a dark humidity chamber. Quickly wash with distilled water. 4. Blocking step: Add 100 μL of blocking buffer A, apply a plastic coverslip, and incubate for 20–30 min at RT in a dark humidity chamber. 5. After blocking, remove the coverslip and the blocking buffer, add the primary antibodies of interest (100 μL), apply a plastic coverslip, and incubate overnight (see Table 1 and Note 18). 6. Remove the coverslip and wash three times in PBS for 3 min. 7. Add 100 μL of secondary antibodies, apply a plastic coverslip, and incubate for 2 h (Table 1). 8. Remove the coverslip, wash three times in PBS for 3 min and three times in distilled water for 2 min each. 9. Counterstain by adding 100 μL of 0.1 μg/mL DAPI, add coverslip, and incubate for 5 min. 10. Rinse twice in distilled water for 1 min each. 11. Air-dry, mount in antifading mounting medium, and apply a glass coverslip. 12. Observe, photograph, and annotate the position of the spermatocytes of choice (see Note 19). 3.5 Combination of Immunofluorescence Staining and FISH (Immuno-FISH) to Study the Behavior of the XY Body
After examining and photographing the spermatocyte spreads, it is possible to perform a fluorescence in situ hybridization (FISH)— on the same spermatocytes which were previously immunolabeled—using DNA probes that bind specifically to the X chromosome or the Y chromosome or both (see Subheading 2.5, Invitrogen’s SPoT-Light® Chromosome X/Y Probe Cocktail). 1. Carefully, remove the glass coverslip. 2. Rinse the slides three times for 10 min each in a Coplin jar (protected from light) with the stringency wash solution A, at RT. 3. Dehydrate the slides through an ethanol series 70°, 80°, 100° for 2 min each and air-dry until no traces of ethanol are left on them. 4. If necessary, incubate with 20 mg/mL of proteinase K in buffer Tris-CaCl2 for 20 min at 37 °C. This step should not be performed if the slides were preheated in epitope retrieval solution during the immunofluorescence technique. 5. Add 25 μL of probe to the center of the sample, apply a 24 × 40 mm coverslip (use more probe for bigger and larger coverslips), and place in a dark humidity chamber. 6. Denature the DNA of spermatocyte nuclei and the DNA probe on a hot plate at 80 °C for 3 min.
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7. Incubate overnight (16–24 h) at 37 °C in a dark humidity chamber. 8. Remove coverslip and immerse the slides in a Coplin jar with stringency wash solution B at 72 °C for 5 min. 9. Rinse three times in stringency wash solution A for 2 min each at RT. 10. Add 50 μL of blocking buffer B, apply a plastic coverslip, and incubate for 15 min at RT in a dark humidity chamber. 11. Add 50 μL (total volume) of diluted FITC-conjugated mouse anti-digoxin and/or TRICT-conjugated streptavidin. 12. Repeat Subheading 3.5, step 9. 13. Counterstain by adding 50 μL of 0.1 μg/mL DAPI, add coverslip, and incubate for 5 min. 14. Rinse twice in distilled water for 1 min each. 15. Air-dry, mount in antifading mounting medium, and apply a glass coverslip. 16. Observe, localize again the spermatocytes of choice, and photograph (see Note 20).
4
Notes 1. Cryogenic preservation is based on Sudman [16]. 2. Surgeons should avoid damaging the testicular tissue (do not place it on a piece of gauze or cotton). The testicular tissue should be put immediately in a culture medium or in a fixative solution at room temperature and transferred to the laboratory as soon as possible. It is recommended to use the testicular tissue up to 6 h after surgery to obtain good spreads. 3. The slides should be perfectly clean, devoid of irregularities and fat (particularly, for electron microscopy). They could be examined by phase contrast microscopy at 400× magnification. If they are not enough clean, put in Coplin jars with 96° and 100° ethanol for 15 min each, and dry using a lint-free paper. 4. A 4 % paraformaldehyde solution is made with 4 g in 100 mL of distilled water, plus traces of phenol red as pH indicator; it is heated slowly at 50–55 °C while stirring; the pH of the solution is brought alkaline (purple-red, pH about 8) by slowly adding drops of 0.1 N NaOH until the paraformaldehyde is solubilized. Allow to cool, filter, and adjust pH to 8.5–9 with 0.05 M tetrasodium borate. Store at 4 °C (up to several months), check the pH, and adjust it if necessary. 5. A stock solution of 0.1 M sodium citrate buffer (pH 6) can be prepared and stored at room temperature. The citrate epitope
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retrieval unmasks epitopes in paraformaldehyde-fixed spreads, thus enhancing staining intensity of antibodies. 6. A 10×-concentrated stock solution could be prepared and stored at RT. If the 1×-diluted PBS solution is not sterile, it is recommended to store it at 4 °C to prevent bacterial growth. 7. If the slides were previously preheated in epitope retrieval solution, they should not be treated with proteinase K. The proteinase K can be used as an epitope retrieval solution: firstly, by breaking the protein cross-links and, secondly, by unmasking the epitopes in paraformaldehyde-fixed cells. 8. The testicular tissue can be stored in cacodylate buffer for weeks by adding 2–3 drops of fixative solution A using a plastic pipette. 9. If the semi-thin section is overstained with toluidine blue, it could be rinsed in ethanol 96°. 10. To make the handmade aspirator, take a scalp vein set 21G, cut the end which has a needle, remove the needle, and put a small piece of cotton in the opposite side (in order to prevent the aspiration of any liquid, including a cell suspension). Then, hold an 8 mm capillary tube by the ends, place the center of the capillary tube under the flame of a Bunsen burner, and heat it until the glass softens; quickly extend the capillary tubes from the ends until both sides are separated. Cut the tip of the thinner side of the capillary tube and insert it to the scalp vein set. 11. The amount of tissue should be optimal (5–15 spermatocytes per field observed by a 10× objective) to allow the correct spreading of each spermatocyte, and avoid crowding of the nuclei on the slide or a low number of nuclei per field. Testicular biopsies from infertile men show a great variability depending on the type of spermatogenesis failure, which ranges from almost normal spermatogenesis to a complete lack of germ cells (Sertoli-cell-only syndrome and/or hyalinization of seminiferous tubules). 12. It is recommended to fix the cell suspension with fixative solution B for electron microscopy, and fixative solution C for immunofluorescence technique/FISH. Once the spermatocyte spreads for EM are ready, they could be silver stained [17], coated by a plastic solution, and placed in center-marked grids for EM (see details in ref. 18). 13. The “quality” of the slides is widely variable: the attachment of the nuclei on the slides depends on the state of the tissue, dilution, and the senescence of the cells. 14. It is possible to keep the cellular spreads at −80 °C for months (or even, for years). In the latter case, it is suggested to pretreat them with citrate-buffer epitope retrieval.
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15. These pretreatments should not be performed if the protein of interest is a chromatin-related protein. 16. It is important that the temperature of the citrate buffer does not rise to 90 °C. It should be controlled in such a way that the temperature at the first round of heating ranges between 80 and 82 °C. 17. This pretreatment should not be done if a FISH technique will be performed. 18. The antibodies of interest should be incubated at concentrations and temperatures previously determined for each specific antibody (Table 1). 19. For double immunodetection using two primary antibodies on the same host, two successive rounds of incubations could be performed. For example, if the antibodies against BRCA1 and SYCP3 were inoculated in rabbit, perform a first round of immunolabelling using BRCA1 antibody— from Subheading 3.4, steps 1–12. To carry out the second round of immunolabelling using SYCP3 antibody, remove the coverslip, rinse the slides three times for 10 min each in a Coplin jar (protected from light) with 4× SSC plus 0.1 % Tween 20, and repeat Subheading 3.4, steps 4–11. Based on the annotated position of the spermatocytes of choice, localize again, observe, photograph, and superimpose the separated images using Adobe Photoshop CS software (Adobe Systems Inc., San Jose, CA, USA). 20. Using Adobe Photoshop CS, the separated images can be superimposed. Furthermore, the color of the FISH signal could be artificially modified in order to distinguish this label from the one/s resulting from the immunofluorescence technique (Fig. 2c).
Acknowledgments We thank M.Sc. I.M. Rahn for the useful comments on the manuscript and for experimental help. Thanks to Mrs C. Deparci for the able technical help. We acknowledge Prof. R. Ponzio for the histopathological diagnosis of the biopsies, and R. Coco, Ph.D.; G. Rey Valzacchi, M.D.; and S. Brugo Olmedo, M.D., for the clinical and surgical procedures. We also thank the generous gift of antibodies from P. J. Moens, B. Spyropoulus (York University, Toronto, Ontario, Canada), and M. Alsheimer (University of Würzburg, Würzburg, Germany). This work was supported by UBACYT 20020100100030 (AJS) and FONCYT PICT 2010-2718 (RBS).
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References 1. Solari AJ (1974) The behaviour of the XY pair in mammals. Int Rev Cytol 38:273–317 2. Solari AJ (1994) Sex chromosomes and sex determination in vertebrates. CRC Press, Boca Raton, FL 3. Franco MJ, Sciurano RB, Solari AJ (2007) Protein immunolocalization supports the presence of identical mechanisms of XY body formation in eutherians and marsupials. Chromosome Res 15:815–824 4. Sciurano RB, Rahn MI, Rossi L et al (2012) Synapsis, recombination, and chromatin remodeling in the XY body of armadillos. Chromosome Res 20:293–302 5. Monesi V (1965) Differential rate of ribonucleic acid synthesis in the autosomes and sex chromosomes during male meiosis in the mouse. Chromosoma 17:11–21 6. Handel MA (2004) The XY body: a specialized meiotic chromatin domain. Exp Cell Res 296:57–63 7. Ichijima Y, Sin HS, Namekawa SH (2012) Sex chromosome inactivation in germ cells: emerging roles of DNA damage response pathways. Cell Mol Life Sci 69:2559–2572 8. Coco R, Rahn MI, Estanga PG et al (2004) A constitutional complex chromosome rearrangement involving meiotic arrest in an azoospermic male: case report. Hum Reprod 19:2784–2790 9. Sciurano RB, Rahn MI, Pigozzi MI et al (2006) An azoospermic man with a doublestrand DNA break-processing deficiency in the spermatocyte nuclei: case report. Hum Reprod 21:1194–1203
10. Burgoyne PS, Mahadevaiah SK, Turner JM (2009) The consequences of asynapsis for mammalian meiosis. Nat Rev Genet 10:207–216 11. Sciurano RB, Rahn MI, Rey-Valzacchi G et al (2012) The role of asynapsis in human spermatocyte failure. Int J Androl 35:541–549 12. Anderson LK, Reeves A, Webb LM et al (1999) Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein. Genetics 151:1569–1579 13. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212 14. Sciurano RB, Luna Hisano CV, Rahn MI et al (2009) Focal spermatogenesis originates in euploid germ cells in classical Klinefelter patients. Hum Reprod 24:2353–2360 15. Evans EP, Breckon G, Ford CE (1964) An air-drying method for meiotic preparations from mammalian testes. Cytogenetics 15:289–294 16. Sudman PD (1989) Cryogenic preservation of mammalian testicular material for synaptonemal complex analysis. Cytogenet Cell Genet 52:88–89 17. Howell WM, Black DA (1980) Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a 1 step method. Experientia 36:1014–1015 18. Solari AJ (1998) Structural analysis of meiotic chromosomes and synaptonemal complexes in higher vertebrates. Methods Cell Biol 53: 236–254
