Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

Summary

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).

Abstract

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.

Introduction

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 tr....

Protocol

1. 3D Slide Preparation

1. Prepare the following reagents:
   1. CSK buffer: 100 mM NaCl, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl₂. Adjust pH to 6.8 with 1 M NaOH. Store the CSK buffer at 4 °C.
   2. CSK buffer with 0.5% Triton X-100: Add 200 μl of Triton X-100 to 40 ml of CSK buffer. Mix the solution using magnetic stirrer until Triton X-100 is completely dissolved. Store the CSK buffer with 0.5% Triton X-100 at 4 °C.
   3. 4% Paraformaldehyde (PFA)–PBS, pH 7.4: Dissolve 4 g of PFA in approximately 70-80 ml of water. In order to expedite the dissolving process, add approximately 20 μl of 4 N NaOH and keep t....

Results

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 phosphory.......

Discussion

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 embryos^7,8. However, if the fixation step precedes the permeabilizat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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