Collection ofmouse spermatogenic cells
Bioresource Research Center
Bioresource Engineering Division
protocol #2

2020/05/12

1Graphical Abstract

TestisSpermatogenic cells

Microinsemination

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Contents• Introduction• Reagents and Equipment• Methods
1.
2.
3.
4.

Preparation of Reagents
Collection of mouse testes
Mechanical collection of spermatogenic cells
Chemical collection of spermatogenic cells• Appendix• Contact3

Introduction
PurposeSpermatogenic cells for microinjection can be collected from thetestes by a mechanical or chemical (enzyme) treatment.

Introduction

In the mechanical collection method, seminiferous tubules are
cut into small pieces and pipetted to allow spermatogenic cells
released into medium. This method enables collection ofspermatocytes, round spermatids, elongated spermatids andspermatozoa, which are located at the luminal side of theseminiferous tubules.
The enzyme-based chemical collection method enables
collection of all the testicular cells including spermatogonia andSertoli cells attached to the basement membrane of the tubules.
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Reagents and Equipment
Anatomy Props
35 mm petri dish

IWAKI

Erythrocyte-lysing buffer

Home-madeHome-madeGL-PBS

Home-madeHome-made

Pasteur pipette

Shorten the tip and round thecut surface with a burner

Home-madeNylon mesh

NIPPON RIKAGAKU KIKAI

I.D. 37-50 μm

15 ml tube

IWAKI

1000-035

Cold pack (4C)

Centrifuges(Chemical methods)
Collagenase

SIGMA

C-2674

DNase

SIGMA

DN-25

Trypsin

SIGMA

T-9201

Shaker5

Methods
1. Preparation of reagents• Erythrocyte-lysing buffer
NH4Cl 155 mM 0.829 g/ml
KHCO3 10mM 0.1 g/ml
EDTA
2 mM 0.744 g/ml
(pH 7.4)• GL-PBS
Dulbecco-PBS(+) with 0.1 mg/ml polyvinyl alcohol 500 ml
Glucose
5.6 mM 5.045 mg
Sodium lactate
5.4 mM 6.051 mg
BSA
2.5 g
6Methods
2. Collection of mouse testes
All operations should be performed on a refrigerated (not frozen) cold-pack , taking
care not to decrease the temperature below 4oC① Testes removed from male mice are placed in erythrocyte-lysingbuffer.
② Remove Tunica albuginea using tweezers, and transfer theseminiferous tubules into GL-PBS.
③ Loosen the seminiferous tubules with fine tweezers (be careful
not to break the tubules).
①②③7

Methods
3. Mechanical collection of spermatogenic cells① Cut the seminiferous tubules into small pieces
using fine scissors (5-6 cuts/testis) and pipettethem gently to allow spermatogenic cells
released into the medium.
② Filter the cell suspension through a nylon mesh,then centrifuge it at 800 rpm, 4 min at 8oC in a
15 ml tube.
③ Resuspend cells in 3-4 ml GL-PBS and pipettegently. Repeat centrifugation and washing
three times.
④ Finally, add 0.5-1 ml GL-PBS to the sedimentand store at 4oC until the experiment.

①②④Methods
4. Chemical collection of spermatogenic cells① After removal of Tunica albuginea as above and put the seminiferous
tubules into a 15 ml tube containing 1.5 ml GL-PBS +150 μl 1%collagenase +50 μl 0.5% DNase.
② Shake gently in a 37oC shaker until the seminiferous tubules are
dispersed (about 30 min).
③ After centrifugation at 800 rpm, 4 min at RT, resuspend cells in 100 μl
GL-PBS (BSA free)+200 μl 1% trypsin+20 μl 0.5% DNase and pipettegently.
④ Shake gently in a 37oC shaker (about 5-10 min).
⑤ Add 700 μl GL-PBS (with BSA), pipette gently and centrifuge at 800rpm for 4 min at RT. Repeat centrifugation and washing three times.
⑥ Finally, add 0.5-1 ml GL-PBS to the sediment and store at 4oC until theexperiment.

Appendix
Isolated spermatogenic cells (by mechanical collection )Spermatozoon

Spermatocyte

Elongated
spermatid

Round spermatid

Appendix• The isolated cells can be stored for several days at 4-10oC.
• For the cell freezing, the pellet of cell are resuspended in GL-PBS containing
7.5% glycerol + 7.5% fetal bovine serum and frozen at -80oC (Ogura et al., 1996).
• Spermatogenic cells can be collected from testes that have been refrigerated (410oC) for 1 to 2 days.
• Sperm or elongated spermatids collected from frozen whole testes (at -80oC)
can produce normal offspring after microinsemination (Ogonuki et al., 2006).
• For collection of only round cells, cell suspension is treated with 0.1-0.2 mg/ml
pronase in GL-PBS and centrifused/washed. This treatment removes mostelongating spermatids and spermatozoa from cell suspension as agglutinatedsticky masses (Figure below) (Ogura et al. 1993).

11References• Ogura et al., 1993, Biol. Reprod. 48, 219-225.
• Ogura et al., 1996, J.Assist. Reprod. Genet. 13, 431-434.
• Ogura et al., 2005, Int. Rev. Cytol., 246, 189-229.
• Ogonuki et al., 2006, PNAS 103, 13098-13103.

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Contact• Bioresource Engineering Division• Question
Narumi Ogonuki: ogonuki@rtc.riken.jp

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