The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).
During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.
In testes, germ cells differentiate from diploid spermatogonia through meiosis into mature, haploid spermatozoa. During this process, the nuclear chromatin structure of germ cells is continuously and dynamically remodeled. Surface spreads are commonly used for the cytological examination of individual spermatogenic cells. A prevailing method of surface spreads employs hypotonic treatment by which individual spermatogenic cells are spread and flattened1. These conditions are optimal for detailed analysis of meiotic chromosomes. Chromosomal features such as synaptic status and recombination foci are easily observed using this method. However, the hypotonic treatment disrupts subnuclear chromatin architecture, thus this technique is not suitable for structural analysis of nuclei. Consequently, an improved method has been designed to preserve the three-dimensional chromatin structure of testicular germ cells. This method has been termed as the three-dimensional (3D) slide method (Figure 1). The 3D slide method has enabled the detection of nascent RNA localization in nuclei because it was initially optimized to examine gene expression and chromatin states during testicular germ cell differentiation by RNA fluorescence in situ hybridization (FISH)2,3. This 3D method is also applicable to the combination of immunofluorescence, DNA, and RNA FISH. Additionally, this method has aided in the discovery of postmeiotic sex chromatin (PMSC), a silent compartment of the sex chromosomes found in postmeiotic spermatids2.
The 3D slide method was originally optimized through the combination of two essential steps of slide preparation commonly used for nuclear staining: the fixation step designed to fix nuclear materials and the permeabilization step intended to remove cytoplasmic materials in order to improve the accessibility of staining reagents, such as antibodies and FISH probes. As previously described in another publication3, it has been determined that the permeabilization step must precede the fixation step in order to obtain optimal results with low cytoplasmic background. In this 3D slide method, the permeabilization step and subsequent fixation step are performed directly on seminiferous tubules and are followed by the mechanical dissociation of germ cells with forceps prior to cytospinning onto slides. An alternative method for RNA FISH of spermatogenic cells was developed in another laboratory4. In this method, consistent with the 3D method, the permeabilization step must precede the fixation step in order to obtain optimal results of RNA FISH.
The following protocol describes the 3D slide method and provides an example of a possible application, Cot-1 RNA FISH for the detection of nuclear nascent RNAs. Cot-1 DNA consists of repetitive elements in the genome. Here, a Cot-1 DNA probe is hybridized to an intron and UTR of the nascent transcripts, thereby detecting the transcriptionally active regions in nucleus5,6. 3D slides are versatile and can be applied to a combination of immunofluorescence, DNA and RNA FISH techniques in order to obtain detailed examination of spatial relationships between chromatin architecture.
1. 3D Slide Preparation
2. Cot-1 DNA Probe Preparation by Random Priming
Component | Amount (μl) | Final concentration |
Formamide | 500 | 50% (v/v) |
50% Dextran | 200 | 10% (w/v) |
20x SSC | 100 | 2x |
1% BSA | 100 | 0.1% |
Component | Amount per reaction (μl) | Final |
Cot-1 DNA: 1 mg/ml | 1 | 1 μg |
Random 9mer primer (from kit) | 10 | |
Water | 27 |
Component | Amount per reaction (μl) | Final concentration |
Mixture prepared from step 2.2 | 38 | |
5x nucleotide buffer (from kit) | 9.2 | |
Cy3-dUTP (1 mM) | 0.8 | 16 μM |
Klenow (from kit) | 2 |
3. Cot-1 RNA FISH
Component | Amount per reaction (μl) | Final |
Cot-1 DNA probe (50 ng/μl) | 2 | 100 ng |
Ribonucleoside Vanadyl Complex (200 mM) | 2 | 20 mM |
Hybridization buffer | added to 20 |
A representative result of a 3D slide is shown in Figure 2. In this experiment, Cot-1 RNA FISH was performed to detect nascent transcription together with immunostaining using anti CBX1 and γH2AX antibodies. To combine RNA FISH and immunostaining, immunostaining was performed first, followed by RNA FISH as described in3. CBX1 is a heterochromatin protein that localizes to pericentromeric heterochromatin and silent sex chromosomes in meiosis called an XY body (or sex body). γH2AX is a phosphorylated form of histone variant H2AX and localizes on the XY body. In this picture, Cot-1 signals accumulate on euchromatic regions, but are excluded from pericentromeric heterochromatin and the XY body.
During surface spread preparation, treatment with hypotonic solution swells nuclei and physically extends subnuclear structures. This prevailing method for meiotic study is best suited for capturing all meiotic chromosomes in a single z-plane1. To clarify the difference between the 3D slide method and surface spreads after hypotonic treatment, representative pictures of pachytene spermatocytes of each experiment are shown using the same magnification using an epifluorescent microscope (Figures 2A and 2B). In surface spreads treated with hypotonic solution, the three-dimensional chromatin structure is disrupted and all meiotic chromosome axes can be captured within a single z-plane. 3D slide preparation, however, preserves the intact chromatin structure.
Figure 1. Schematic of 3D slide method. The 3D slide method can preserve the three-dimensional chromatin structure. Due to the three-dimensional nature of the slide, data acquisition with multiple z-section is required to cover a nucleus.
Figure 2. Comparison between the 3D slide method and surface spreads after hypotonic treatment at the same magnification. (A) Using the 3D slide, Cot-1 RNA FISH (cy3) and immunostaining with anti CBX1 (fitc) and γH2AX (cy5) antibodies are performed. A pachytene spermatocyte with a single z-section is shown. (B) Using the surface spreads, immunostaining with anti SCP3 and γH2AX antibodies are performed. A pachytene spermatocyte is shown. Circles: XY body. Bars: 10 μm.
In 3D slide preparation, the permeabilization step precedes the fixation step. These steps are performed directly on seminiferous tubules, thereby preserving the three-dimensional chromatin structure. An alternate option to preserving chromatin structure for RNA/DNA FISH is to perform the permeabilization step and the fixation step simultaneously. This alternate technique has been performed in marsupial germ cells and in mouse preimplantation embryos7,8. However, if the fixation step precedes the permeabilization step, it may give rise to high cytoplasmic background. Therefore, the critical determinant of slide optimization for RNA and DNA FISH is dependent upon the order of the permeabilization and the fixation steps. The duration of the permeabilization step is also an important factor for the optimization of slide preparation. Six minutes is the general condition for this method. However, it can be adjusted depending on variation of the materials used.
The preservation of the three-dimensional chromatin structure is a feature of the 3D slide that distinctly separates it from surface spread slides. This advantage differs greatly from that of chromosome spread preparation. The 3D slide method preserves three-dimensional chromatin structure, but requires data acquisition with multiple z-sections to encompass an entire nucleus. A single z-section cannot capture all signals of a nucleus. Thus, the necessity for multiple z-sections is a major limitation to this method. To capture a nucleus in a single z-section, surface spreads have an advantage. Therefore, the 3D method and the surface spread preparation have distinct advantages and mutually compensate the features of each other for the study of meiosis and spermatogenesis.
The 3D slide method can be applied to all cell types found within the testicles, including spermatogonia, round spermatids, and Sertoli cells. This method has also been applied to examine the chromatin compaction of the sex chromosomes at the onset of meiotic sex chromosome inactivation, and to examine epigenetic modifications on PMSC10. In terms of future applications, this method may be applied to other tissues or cells. If so, it is recommended that the permeabilization step precede the fixation step. Alternatively, the permeabilization step and the fixation step can be performed simultaneously.
The authors have nothing to disclose.
I thank Jeannie T. Lee for supervision of this project when I was in the Lee laboratory and Tyler Broering for editing the manuscript. This work was supported by the Basil O’Connor Starter Scholar Award from the March of Dimes Foundation and the NIH Grant GM098605.
Cot-1 DNA | Invitrogen | 18440-016 |
Stratagene Prime-It Fluor Fluorescence Labeling Kit | Agilent |
300380 |
Herring sperm DNA Solution | Invitrogen | 15634-017 |
Ethanol, 200 proof (absolute) | Pharmco-AAPER | 111000200 |
Sodium acetate | Sigma-Aldrich | S2889 |
Formamide deionized | American Bioanalytical | AB00600-00500 |
Sodium citrate tribasic dihydrate | Sigma-Aldrich | C8532 |
Bovine serum albumin | Lifeblood Medical | 77110-100 |
Dextran sulfate sodium salt from Leuconostoc spp. | Sigma-Aldrich | D6001 |
RPMI 1640 | Invitrogen | A10491-01 |
Sucrose | Sigma-Aldrich | S0389 |
PIPES | Sigma-Aldrich | P6757 |
Magnesium chloride hexahydrate | Sigma-Aldrich | M0250 |
Triton X-100 | Sigma-Aldrich | T8787 |
Paraformaldehyde | Sigma-Aldrich | 76240 |
Sodium hydroxide | Sigma-Aldrich | 221465 |
Phosphate buffered saline | Sigma-Aldrich | P4417 |
VECTASHIELD Mounting Medium with DAPI | Vector Laboratories | H1200 |
Ribonucleoside-vanadyl complex | New England Biolabs | S1402S |
Illustra MicroSpin G-50 Columns | GE Healthcare | 27-5330-01 |
Microcentrifuge | Eppendorf | 5424 |
Forceps | VWR | 300-050 |
Microcentrifuge tubes | Bioexpress | C-3262-1 |
Cytospin 3 | Shandon | |
Coplin jar | Fisher | 08-815 |
Superfrost /Plus Microscope Slides | Fisher | 12-550-15 |
4-well dish | Fisher | 12-566-301 |
Cover glass | Fisher | 12-544-G |
Air incubator | Revolutionary Science | RS-IF-203 |