Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Summary

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Abstract

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Introduction

Spermatogenesis is a complex process wherein a small population of spermatogonial stem cells sustain continuous production of a large number of sperm throughout adult life^1,2. During spermatogenesis, dynamic chromatin remodeling takes place when spermatogenic cells undergo meiosis to produce haploid spermatids^3,4,5. Isolation of meiotic spermatocytes is essential for molecular investigation, and several different approaches to isolate meiotic spermatocytes have been established, including sedimentation-based separation^6,7 and fluorescence-activated cell sorting (FACS)^{8,9,10,11,12,13,14,15,16,17}. However, these methods have technical limitations. While sedimentation-based separation yields a large number of cells^5,6,7, it is labor intensive. The established FACS-based method uses Hoechst 33342 (Ho342) to separate meiotic spermatocytes based on DNA content and light scattering properties and requires FACS cell sorters equipped with an ultraviolet (UV) laser^8,9,10,11. Alternative FACS-based methods require transgenic mouse lines that express florescent proteins, synchronization of spermatogenesis¹², or cell fixation and antibody labeling that is not compatible with isolation of live cells¹³. While there is another alternative method using a cell-permeable DNA binding dye, DyeCycle Green stain^14,15,16,17, this method is recommended for the isolation of spermatogenic cells from juvenile testis. Therefore, there is a critical need to develop a simple and robust isolation method for live meiotic spermatocytes that can be applied to any mouse strain of any age and that can be performed using any FACS cell sorter.

Here we describe such a long-sought cell isolation protocol using the DyeCycle Violet (DCV) stain. DCV is a low cytotoxicity, cell-permeable DNA binding dye structurally similar to Ho342 but with an excitation spectrum shifted toward the violet range¹⁸. In addition, DCV has a broader emission spectrum compared to DCG. Thus, it can be excited by both UV and violet lasers, which improves the flexibility of equipment, allowing the use of an FACS cell sorter not equipped with a UV laser. The DCV protocol presented here uses two-dimensional separation with DCV blue and DCV red, mimicking the advantage of the Ho342 protocol. With this advantage, our DCV protocol allows us to isolate highly enriched germ cells from the adult testis. We provide a detailed gating protocol to isolate live spermatogenic cells from adult mouse testes of one mouse (from two testes). We also describe an efficient and quick protocol to prepare single-cell suspension from mouse testes that can be used for this cell isolation. The procedure requires a short time to complete (preparation of single cell suspension - 1 hour, dye staining - 30 min, and cell sorting - 2-3 hours: total - 4-5 hours depending on the number of needed cells; Figure 1). Following cell isolation, a wide range of downstream applications including RNA-seq, ATAC-seq, ChIP-seq, and cell culture can be completed.

Protocol

This protocol follows the guidelines of the Institutional Animal Care and Use Committee (protocol no. IACUC2018-0040) at Cincinnati Children’s Hospital Medical Center.

1. Equipment and reagent setup for the preparation of testicular cell suspension

1. Prepare each enzyme stock in 1x Hanks’ Balanced Salt Solution (HBSS) and store at -20 °C. (Table1).
   NOTE: Prepare any time before the experiment.
2. One day before the experiment: Coat collecting tubes (1.5 mL tubes) with Fetal bovine serum (FBS) at 4 °C overnight.
3. On the day of the experiment: Set the water bath to 37 °C.
4. Pre-warm Dulbecco's Modified Eagle Medium (DMEM) at 37 °C and prepare 2 mL of 1x dissociation buffer for each sample right before use (Table 1) in 15 mL centrifuge tube.
   NOTE: 2 mL dissociation buffer is prepared for two testes. For the dissociation buffer recipe, please refer to Table 1.

2. Animal dissection and preparation of testicular cell suspension

1. Sacrifice an 8-week-old male mouse by leaving in a carbon dioxide chamber for at least 10 min.
2. Remove both testes and place on a 60 mm Petri dish containing 2 mL of ice-cold phosphate-buffered saline (PBS).
3. Remove the tunica albuginea from the testes. Slightly disperse the seminiferous tubules by gently separating with forceps.
4. Transfer the seminiferous tubules to a 100 mm Petri dish with a new drop of ice-cold PBS and untangle seminiferous tubules gently with forceps. Repeat this wash 3 times to remove interstitial cells as much as possible.
5. Incubate untangled seminiferous tubules in a 15 mL tube containing 2 mL of dissociation buffer at 37 °C for 20 min.
6. Gently pipette the tubules 20 times using a 1000 µL micropipette.
7. Incubate for 6 min. Repeat gentle pipetting 20 times.
8. Incubate for 3 min. Repeat gentle pipetting 10 times until no visible chunks remain.
9. Add 10 mL of FACS buffer to the suspension. Centrifuge at 300 x g for 5 min at room temperature and discard the supernatant.
10. Repeat step 2.9 twice to remove the spermatozoa and as much debris as possible.

3. Cell staining

1. Resuspend the cell pellet with 3 mL of FACS buffer (Table 1) and filter the cell suspension through a 70 µm nylon cell strainer into a 50 mL tube.
   NOTE: The expected cell yield is approximately 100 million cells per two testicles (from an 8-week-old B6 wild-type mouse).
2. Count the cell number and split 10% of the cells into a new tube as the unstained negative control and leave on ice.
3. Add 6 µL of DCV stain (the original concentration is 5 mM) to the remaining cell suspension and mix well. The final concentration is 10 µM, and the capacity is approximately 100 million cells.
4. Incubate at 37 °C for 30 min in the dark. Gently shake the cell suspension every 10 min.
5. After incubation, without a wash step, directly add 5 µL of DNase I (the stock concentration is 10 mg/mL) to the cell suspension and filter the cells into a 5 mL FACS tube through the 35 µm nylon mesh cap. Keep the samples on ice until sorting.

4. Flow cytometry and experimental gates

1. Prepare the FACS cell sorter. Ensure that FACS cell sorter is equipped with Excitation optics: Violet (405-nm) laser; Detection optics: filter combination of 450/50 bandpass [same filter set of 4′,6-diamidino-2-phenylindole (DAPI) for DCV-blue detection] and 665/30 bandpass [same filter set of Allophycocyanin (APC) for DCV-red detection]. Here we use Sony SH800S cell sorter as an example.
2. Create a new experiment and set up the following working plots displaying parameters to be optimized: Click “New Density” on the “Worksheet Tools” menu bar to create a forward scatter - area (FSC-A) vs. back scatter – area (BSC-A) density plot on a linear scale. Click “New Histogram” to create a DCV-blue histogram plot on logarithmic scale.
3. Briefly vortex the unstained negative control (from step 3.2) and load the sample.
4. Click “Start” and “Record” to begin processing the unstained sample. While the sample is running, click “Detector & Threshold Settings” to optimize FSC and BSC voltages by adjusting both the photomultiplier tube (PMT) voltages up or down to place the unstained cells on the scale of the FSC/BSC plot.
5. Adjust the PMT voltage up and down on “FL1: DAPI” while the sample is running to locate the position of the DCV-negative population in the first decade of the DCV-blue histogram logarithmic plot (Figure 2A). After completing the PMT voltage adjustment, click “Stop” to unload the unstained sample.
6. Briefly vortex the sample (from step 3.5) and load the sample.
7. Click “Next Tube” to create a new worksheet for the DCV-stained sample; and click “Start” and “Record” to acquire the DCV-stained sample, record ≥ 1 x 10⁶ events. Add the following working plots: Click “New Density” on the “Worksheet Tools” menu bar to create an FSC-H (height) vs. FSC-W (width) density plot on a linear scale; Click “New Density” to create a DCV-blue vs. DCV-red density plot on a linear scale. After recording ≥ 1 x 10⁶ events, click “Stop” and unload the sample.
8. On the FSC-A vs. BSC-A density plot, click “Polygon” on the “Plot Tools” menu bar to draw a big gate “Cells” to include most cells and exclude small debris (Figure 2B). Apply the gate “Cells” to FSC-H vs. FSC-W density plot. Click “Rectangle” to draw a “Single Cells” gate to exclude non-single cells (Figure 2C).
9. Apply “Single Cells” gate to DCV-blue vs. DCV-red density plot and adjust the scale to capture an extended profile as shown in Figure 2D. Click “Polygon” on the “Plot Tools” menu bar to draw a “DCV” gate to exclude the unstained cells and side population (Figure 2D).
10. Apply “DCV” gate to DCV-blue histogram plot on a linear scale. The three major peaks refer to different DNA content: 1C, 2C, and 4C (Figure 2E).
11. Click “New Density” to create a DCV-blue vs. DCV-red density plot on a linear scale and apply “DCV” gate to perform back gating from Figure 2E to locate the 1C and 4C populations (Figure 2F). Click “Ellipse” to draw a gate on the 1C population, which is within a condensed area (gate 1C). Click “Polygon” to draw a gate on the 4C population which is a continuous curve (gate 4C).
12. Click “New Density” to create a DCV-blue vs. DCV-red density plot on a linear scale and apply “4C” gate; adjust the scale to zoom in and click “Polygon” to draw a “4C_1” gate for more precise selection (Figure 2G).
13. Click “New Density” to create a new FSC-A vs. BSC-A density plot on a linear scale and apply “4C_1” gate; there will be three enriched populations separated by size corresponding to leptotene (L) /zygotene (Z), pachytene (P) and diplotene (D) spermatocytes. Click “Polygon” or “Ellipse” to draw 3 gates: “L/Z”, “P”, and “D” based on the growing size (Figure 2H).
14. Click “New Dot Plot” to create a DCV-blue vs. DCV-red color dot plot on a linear scale to apply gate and ensure the three populations are in a continuous order within the “4C_1” gate (Figure 2I).
15. Similarly, for the 1C population, click “New Density” to create a new FSC-A vs. BSC-A density plot on a linear scale and apply “1C” gate, select the unified size of cells as pure round spermatid population, and click “Ellipse” to draw an “RS” gate (Figure 2J).

5. Sort male germ cell subpopulations

1. Prepare 1.5 mL tubes containing 500 μL 50% FBS for cell collection and load the collection tube into the collector and click “Load Collection”.
2. Click “Next Tube”, “Start”, “Record” and “Sort Start”. For using a two-way system that allows two populations of interest to be sorted at the same time into the collection device, follow pairwise combinations: leptotene (L) /zygotene (Z) and pachytene (P) spermatocytes based on L/Z and P back-gates; Round spermatids (RS) and diplotene (D) spermatocytes based on RS and D back-gates.
3. While the sample is running, adjust the flow rate to ~3000 events/s to get the most efficient sorting.

6. Purity analysis of sorted cells

1. Collect ≥ 10,000 cells/each population. Perform cell immunostaining to confirm the substage.
2. Centrifuge at 300 x g for 5 min at 4 °C and carefully discard the supernatant, keeping around 110 µL of the liquid in the bottom of the tube.
3. Take a 10 µL drop of cell suspension to observe under microscope and evaluate the overview cell morphology and number.
4. Apply 100 µL of cell suspension to each of the sample chamber slides (see Table of Materials), and load the chambers to the Cytospin.
5. Spin the samples at 30 x g for 5 min at room temperature. Draw a circle around the cell with a hydrophobic pen. Dry slides on lab bench for a few minutes at room temperature.
6. Drop 50 µL of PBS in the circle of the slide and tap off.
7. Add primary antibody solution (Dilute primary antibodies with 5% Donkey Serum in PBS with 0.02% Polysorbate 20) in the circle of the slide and incubate at 4 °C overnight.
   NOTE: To judge the substage of meiotic spermatocytes, SYCP3 was detected, which is a marker of meiotic chromosome axes, and γH2AX, which is a marker of DNA damage response. (For the antibody working concentration, please refer to the Table of Materials).
8. Tap off the primary antibody solution from the slides.
9. Drop 50 µL of PBS in the circle of the slide and tap off. Repeat one time.
10. Add secondary antibody solution (dilute secondary antibodies in PBS with 0.02% Polysorbate 20) in the circle of the slide and incubate at room temperature for 1 h in dark.
11. Drop 50 µL of PBS in the circle of the slide and tap off. Repeat one time.
12. Add 50 µL of DAPI (stock concentration is 0.1 μg/mL) stain for 5 min and tap off.
13. Add 1-2 drops of mounting media on the slides. Carefully cover the mounting media with a cover glass and gently press the cover glass to remove extra mounting media and air bubble. The slides are ready for microscopy evaluation.

Results

A representative result of this sorting protocol is shown in Figure 3. The total sorting time of two testes (one mouse) is usually around 3 hours, which is dependent on the concentration of cell suspension and the sorting speed. After sorting, the purity of spermatocytes is confirmed by immunostaining of SYCP3 and γH2AX (Figure 3A). The representative purity of sorted L/Z, P, D spermatocyte fractions are around 80.4%, 90.6%, and 87.6%, respectively (

Discussion

Here we present a practical and simple protocol to isolate subpopulations of spermatocytes and spermatids from a single adult male mouse. To ensure the reproducibility of this protocol, there are some critical steps that need attention. Before enzyme digestion, wash step aims to remove interstitial cells; after digestion, this step helps to remove spermatozoa and debris. Washing/centrifuging 3 times is important. In our dissociation buffer recipe, the combination of several different enzymes facilitates the dissociation ...

Materials

Name	Company	Catalog Number	Comments
1.5 ml tube	Watson	131-7155C
100 mm Petri dish	Corning, Falcon	351029
15 mL Centrifuge tube	Watson	1332-015S
5 ml polystyrene tube with cell strainer snap cap (35 µm nylon mesh)	Corning, Falcon	352235
50 mL Centrifuge tube	Watson	1342-050S
60 mm Petri dish	Corning, Falcon	351007
70 µm nylon mesh	Corning, Falcon	352350
Cell sorter	Sony	SH800S
Centrifuge
Collagenase, recombinant, Animal-derived-free	FUJIFILM Wako Pure Chemical Corporation	036-23141
Collagenase, Type 1	Worthington	LS004196
Cover glass	Fisher	12-544-G
Cytospin 3	Shandon
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Fisher	D1306	working concentration: 0.1 μg/mL
Dnase I	Sigma	D5025-150KU
Donkey serum	Sigma	S30-M
Dulbecco’s phosphate-buffered saline (DPBS)	Gibco	14190144
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	11885076
Fetal bovine serum (FBS)	Gibco	16000044
Histone H1t antibody	gift from Mary Ann Handel		1/2000 diluted
Hank’s balanced salt solution (HBSS)	Gibco	14175095
Hyaluronidase from bovine testes	Sigma	H3506-1G
Phosphate buffered saline (PBS)	Sigma	P5493-4L
Pipettemen
ProLong Gold Antifade Mountant	Fisher	P36930
rH2AX antibody	Millipore	05-635	working concentration: 2 μg/mL
Sperm Fertilization Protein 56 (Sp56) antibody	QED Bioscience	55101	working concentration: 0.5 μg/mL
Sterilized forceps and scissors
Superfrost /Plus Microscope Slides	Fisher	12-550-15
SYCP3 antibody	Abcam	ab205846	working concentration: 5 μg/mL
TWEEN 20 (Polysorbate 20)	Sigma	P9416
Vybrant DyeCycle Violet Stain (DCV)	Invitrogen	V35003
Water bath