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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 separatio....

Protokół

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
   ....

Wyniki

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 (

Dyskusje

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 .......

Materiały

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
Hostone 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