9.00 -11.00 Testicular spermatozoa
(isolation, cryopreservation and IVF)
Anna Noble and Alan Jafkins, Lab 6.12, Old
St. Michael’s Building
Sperm freezing
This protocol is modified by Harland from the protocol developed by Michael Sargent,
NIMR: Sargent, MG and Mohun, TJ (2005) Cryopreservation of sperm of Xenopus laevis
and Xenopus tropicalis Genesis 41(1):41-6
http://tropicalis.berkeley.edu/home/obtaining_embryos/sperm-freezing/sperm-freeze.html
1. Remove testes. All traces of blood or blood vessels are removed effectively from the
testes by rolling them on paper towels.
2. Testes are transferred to an E ppendorf tube in 0.5ml L15 supplemented to 10% calf
serum and dissociated by gentle application of an Eppendorf pestle.
3. Sperm preps are diluted 1:1 with ice cold cryoprotectant and frozen immediately. The
capacity to be frozen successfully starts to be lost quite soon after macerating the testis
although their actual motility does not change.
4. To prepare cryoprotectant disperse one egg yolk (about 15ml) in an equal volume of
distilled water. Then dilute it to 20% v/v in sucrose (0.4M) in sodium bicarbonate
(10mM) containing 2mM pentoxyfylline. Samples are then centrifuged for 20 min at
13,000 rpm in an SS24 rotor or equivalent. Aliquots of the supernatant are then stored
frozen at -20C and the pellets discarded. Cryoprotectant should be discarded after
~1month. For freezing sperm, ice cold cryoprotectant were added to ice cold sperm in
L-15 in a 1:1 ratio immediately before processing.
5. Freeze 4 samples of X. tropicalis sperm per testis: 250µl aliquots in 500µl thin walled
eppendorfs.
NOTE: We have tested three different freezing techniques (slow, medium, and fast) and
found the slow freeze with a Styrofoam box to work the best. This is in contrast to the
original published protocol of Sargent et al. which uses a slow freeze using either a dry
ice/ethanol bath or thermoregulator for freezing.
6. Place 4 aliquots into a polystyrene box closed with aluminium foil instead of the normal
lid and place directly into the –80C freezer overnight.
7. The next day move the tubes into a normal box in the –80C.
Cryprotectant
0.4M sucrose………………………..136.92g
10mM NaHCO3…………………….…0.84g
2mM pentoxyfilline……………………0.56g
1000ml
40ml
+
17.5ml yolk
+ 17.5ml Sigma H20
35ml
10ml
____________
50ml
Divide 50ml into 2 x 25ml ultracentrifuge tubes. Spin 20’ at 10000 g @ 10ºC.
5ml aliquots can be stored for up to one month at -20°C.
L-15+calf serum
L-15…………………….….….……9ml
10% calf serum….…………..……..1ml - FBS (Fetal Bovine Serum)
L-glutamine 2mM…………..……..10µl
10ml
X. laevis
Macerate ½ of one testis in 500µl L-15 and add 500µl ice cold cryoprotectant
(⅛ of one testis, Mohun 2005)
divide into 4 tubes (thin walls 0.5ml) - 250µl each and place quickly at -80ºC in a polystyrene
box with one layer of aluminium foil on top
X. tropicalis
Macerate both testis in 500µl L-15 and add 500µl ice cold cryoprotectant
divide into 4 tubes (0.5ml) - 250µl each and place quickly at -80ºC in a polystyrene box with one
layer of aluminium foil on top
In vitro fertilisation
1. Keep tubes on dry ice untill the moment of thawing
2. Place roughly 200 eggs into a Petri dish
3. Thaw sperm 30s by hand, add 250µl 0.1x MBS (X. laevis) or sterile nuclease free water
(X. tropicalis)
4. Add sperm to the eggs and mix
5. After 5 min flood with 0.1x MBS (X. laevis) or sterile nuclease free water (X. tropicalis)