Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Summary

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

Abstract

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Introduction

Drosophila testes are a valuable model for the study of nuclear architecture. In mitotic spermatogonial cells, the nuclear volume is largely occupied by chromatin. These cells undergo four rounds of division, producing a cyst of 16 primary spermatocytes. Over the next several days, the spermatocytes pass through 6 developmental stages (S1-S6), greatly increasing in volume, approximatively 20-fold^1,2. They also change their nuclear morphology. The outline of the nucleus, revealed by the lamin Dm0, becomes irregular and shows deep invaginations^1,3. This is accompanied by extensive gene expression changes and modifications in chromatin organization^1,4,5,6,7. The interphase chromosomes are well-defined in stage S4-S5, forming three distinct masses corresponding to the 2 major bivalent autosomes and the X-Y pair docked with the nucleolus.

Mad1 was originally isolated as a key component of the mitotic checkpoint (reviewed in⁸). In interphase, it is described as located primarily at the nuclear envelope but also in the nucleoplasm^9,10,11. In Drosophila testis, we reported that Mad1 is prominent in the nucleoplasm, intimately associated with the two major autosomal chromatin masses and to a lesser extent with the XY chromatin. We defined this chromatin-associated structure as MINT (Mad1-containing IntraNuclear Territory)¹². Four other proteins were associated with MINTs in spermatocytes: the nucleoporin Mtor/Tpr, the SUMO peptidase Ulp1, the mitotic checkpoint protein Mad2 and a subunit of a Polycomb-like complex Raf2¹².

To complete the description of MINTs, we needed to use antibodies and were confronted with the technical problems generally encountered with immunostaining in testis: a poor permeability resulting in a significant non-uniformity of staining in the depth of the testis, particularly in the large spermatocytes. Squashed preparations increased antibody access but led to compressed images, restricting the 3D analyses, and preventing measurement of quantitative fluorescence, including colocalization. The problem of antibody penetration could also be addressed by permeabilizing agents such as 0.3% Na deoxycholate¹³, but this procedure did not in our hands yield reproducible results. Because we performed many co-labeling experiments, we looked to improve the permeabilization protocol. The combination of 1% NP40 plus heptane resulted in more reproducibility, particularly in studying the large spermatocytes.

This protocol performs fixation and immunostaining of testes in suspension, improving the sample quality as well as enhancing reproducibility, and rendering it suitable for confocal microscopy. We used the protocol to better define the spatial relationships of certain nuclear envelope proteins with the nucleoplasmic structures of large spermatocytes. This method will permit better investigations of the changes that accompany spermatocyte development, for example the dynamic structural changes of the nucleus including chromatin, nucleoplasm and the nuclear pores.

Protocol

1. Drosophila testes collection

NOTE: Young males (0-15 h post-eclosion) are necessary for examining diploid germ cells (i.e., spermatogonia and spermatocytes).

1. Select young flies as much as possible to minimize interference from developing spermatids.
2. Anesthetize flies by brief exposure to carbon dioxide and transfer to a fly pad^14,15.
3. Under a dissecting microscope (10x magnification), use forceps to remove the head.
4. Transfer 5 to 10 decapitated flies to 100 µL of Ringer’s buffer (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, complete protein inhibitor, pH 6.8) on a silicone-coated glass slide on a black background support.
5. With dissecting microscope at a 40x magnification, use one pair of number 5 forceps to hold a fly between the thorax and the abdomen, and, with other forceps, pull the abdomen away from the thorax. Rapidly isolate the testes and adjacent tissues from the fly carcass¹⁵ and place in a new drop of Ringer’s buffer on the same slide.
6. Once all the testes are ready, clean the testes from the remaining tissues (accessory gland and external genitalia).
7. Remove excess buffer from the drop containing the dissected testes. Typically, use a p200 pipette with a 10 μL tip fitted on top of a 200 μL tip. This allows the removal of the liquid excess through a small aperture. Place a drop of 4% paraformaldehyde fixative on top of the drop containing the dissected testes, and then rapidly transfer the testes to a tube containing fresh fixative solution.

2. Paraformaldehyde fixation

1. Prepare 2 mL of fixative solution just before use: 600 µL of 4% paraformaldehyde diluted in phosphate buffered saline (PBS) with 30 µL of NP40 20% plus 1200 µL of heptane in a 2 mL siliconized microcentrifuge tube. Use 4% paraformaldehyde in PBS prepared in 600 μL aliquots and stored at -20°C.
   Caution: Heptane and paraformaldehyde are toxic and should be handled in a fume hood. Dispose of them according to the host institute’s regulations.
2. Transfer the testes using a p200 micropipette with a wide aperture tip to the PFA/NP40/heptane solution. Shake the tube up and down for 30 s by hand. Continue fixing the testes on a rotary mixer at room temperature for 30 min.
3. Stop the rotary mixer and place the tube in a tube rack to allow phase separation and for the testes to settle to the bottom of the tube.
4. Remove all the heptane and the fixative solution and rinse the testes quickly three times with 1x PBS.
5. Transfer to a clean 200 µL individual tube. Perform all incubations in these small tubes to minimize the amounts of antibodies employed.
6. Remove excess buffer using a 200 µL micropipette with both a P200 and a P10 tip attached. Take care not to aspirate testes.
7. Keep testes in 1x PBS on ice until all samples are ready.

3. Antibody staining in solution

1. Discard the PBS and leave testes in 0.3% PBS-T for 30 min at room temperature to permeabilize cell membranes.
2. Pre-incubate in the same buffer plus 5% normal goat serum (NGS) for 1 h.
3. Add 200 µL of the primary antibody diluted in 0.3% PBS-T plus 5% NGS (Table of Materials).
4. Incubate the primary antibody overnight at 4 °C (or room temperature) with gentle agitation on a rocker.
5. Wash 3 times in 0.3% PBS-T for 15 min on a rocker at room temperature. Allow the testes to settle to the bottom of the tube by gravity.
6. Add 200 µL of secondary antibody diluted 1:500 from the DyLight conjugated series.
7. Incubate the secondary antibody in a dark chamber for 4 h at room temperature.
8. Wash in 0.3% PBS-T for 15 min on a rocker at room temperature. Then repeat with 0.2% PBS-T and 0.1% PBS-T, each time for 30 min on a rocker at room temperature. Do a final wash in 1x PBS for 30 min to remove the detergent.
9. Add 180 µL of DAPI solution at 1 µg/mL in 1x PBS for 15 min. The stock solution is 10 mg/mL in H₂O. Avoid making stock solution in PBS, which can reduce the DAPI staining signal on diffuse chromatin in larger spermatocytes.
10. Wash 3x for 5 min each.
11. Use a hydrophobic barrier pen to draw a circle on a clean slide.
12. Aspirate testes and put them on the slide. Pre-rinsing the wide aperture tip with 0.1% PBS-T prevents the testes from sticking to the tip. Remove excess PBS.
13. Add a drop (approximately 100 µL) of Citifluor AF1.
14. Gently place a glass coverslip over the tissue, taking care to avoid trapping air bubbles.
15. Seal the coverslip to the slide using clear nail polish.
16. Observe slides on a microscope.

4. Colocalization analyses

1. Acquire confocal images. We used a Zeiss LSM 710 confocal microscope (63x Plan Apochromatic oil) with a z-resolution of 0.5 or 1 µm.
2. Select different cells from independent testes.
3. Import images to processing software (e.g., Imaris).
4. Define the nucleus by manual segmentation in order to exclude the neighboring nuclei. Use the Coloc software; it collects the Pearson’s correlation coefficients (PCCs) inside the entire volume between 2 fluorophores.

Results

The main obstacle to obtaining adequate staining of Drosophila testes is the limited penetrability of antibodies, which has been partially resolved by using squashed preparations but at the cost of inducing a restriction of the 3D analyses (for example 3D representation or measure of colocalization). The procedure permits uniform labeling and preserves the volume of all the cells of the testis. Here we have focused the illustration of the method on the 3D representation of S4-S5 spermatocytes, as they are the largest cel...

Discussion

This protocol provides a method for successful immunolabeling of whole-mount adult Drosophila testes. As reported in other procedures young males must be used (we even shortened the age to less than 20 hours after eclosion) to minimize the presence of developing sperm, which risks obstructing the spermatocytes. The dissection should be rapid and the antibody dilution must be adjusted to optimize the signal-to-noise ratio^21,22,23...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drosophila srains
ketelGFP y; ketelGFP/y+CyO			Gift from A. Debec (Institut Jacques Monod, UMR 7592, Paris, France)
Mad1-GFP			J Cell Sci. 2011 May 15;124(Pt 10):1664-71.
Mtor RNAi	VDRC	ID 110218	Vienna Drosophila RNAi Center
Materials
DAPI	Thermo	D1306	Stock solution must be prepared in H2O
Dumont # 5 Fine Forseps	Fine Science Tools
MBP Wide Bore Pipette Tips	Fisherbrand	11917744	Wide aperture tip
Thermo Scientific 0.2 mL Individual Tube	Thermo	AB-0620
2 ml micro centrifuge tubes	Sigma	T3531-200EA	Siliconized 2ml tube
Citifluor AF1	Citifluor	AF1
Dakopen	Sigma	Z377821-1EA
Normal Goat Serum (NGS)	Sigma	NS02L-1ML
Paraformaldehyde 4%	Sigma	P6148-500G	Paraformaldehyde (4%) dissolved in PBS 1X ; aliquot by 600 ml
PBS 1X	Thermo	14190094	DPBS, no calcium, no magnesium
0.1% (0.2 or 0.3%) PBS-T			PBS 1X  containing 0.1% (0.2 % or 0.3%) Triton X-100
Triton X-100, for molecular biology,	Sigma	T8787
Antibodies
GFP booster	Chromotek	gba488	1/200
Mouse anti-Mtor			1/40 gift from J. Johansen, Iowa State University
Rat anti-Nup62			1/200 gift from H. Ohkura (University of Edinburgh, UK
Guinea-pig anti-Ulp1			1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam
Rabbit anti-Raf2			1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam
DyLight conjugated series	Thermo		1/500 Donkey anti-(guinea-pig, mouse,  rabbit or rat) as second antibodies