EMMA - Mouse sperm cryopreservation protocol

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Cryopreservation of mouse sperm using L-glutamine supplemented CPA: including an IVF recovery procedure incorporting methyl-β cyclodextrin and reduced glutathione

This protocol is based on methods developed by Naomi Nakagata's laboratory, at Kumamoto University (Japan).

A. Sperm collection and freezing

1. For each male to be frozen label ten 250μl plastic semen straws, 1 x Kleenex tissue and 1 x 35mm Falcon 351008 petri dish with the

appropriate sample code. Using a permanent marker pen, mark each straw at a distance approximately 4.5cm and 2.3cm (Fig. 1) from the

end furthest from the cotton/PVA plug. Label the straws with the appropriate sample code near the end of the cotton/PVA plug.

Fig. 1

2. Prepare the cooling chamber e.g. a 47 litre LN2 Dewar (Fig. 2a) filled

with LN2 to a depth of 20-25cm. Also, prepare the floating sperm freezing apparatus. This can be made from a 50ml syringe attached

to a perspex rod. It is important to seal the needle hub (Fig. 2b)

Fig.2a Cooling chamber. Depth of LN2 (h) = 20 to 25 cm.

cotton plug

4.5cm

2.3cm

hLN2

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50cm

Acrylic bar

Styrofoam(30mm thick)

50ml syringe

Heat sealed

Fig.2b Floating sperm freezing apparatus

3. Place a 60µl aliquot of the sperm cryoprotective agent (gCPA containing 100mM L-glutamine - described in Appendix A) into the

base of a 35 mm culture dish and cover with mineral oil.

4. A second 60µl drop of gCPA should be added into the drop (final

volume: 120µl) to make a tall, semi-spherical drop (Fig. 3). Prepare a separate dish for each male to be sperm frozen.

Note: The optimal volume of gCPA is 120µl per two epididymides. If you need to freeze down one epididymis, use a 60µl drop .

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Fig. 3

5. Sacrifice the male and swab the abdomen with 70% ethanol.

6. Dissect the cauda epididymides and clean off all adipose and vascular tissue. This is best achieved by placing the organs on a paper tissue

and examining them under a dissecting microscope lit from above.

7. Transfer the cauda epididymides into a 120µl drop of gCPA.

8. Make five to six cuts across the cauda epididymides using a pair of fine scissors or a similar sharp bladed tool and place the dish on a

37°C hot plate (Fig. 4). Swirl the dish gently every minute for 3 minutes to help disperse the sperm.

Fig. 4

9. While the sperm is equilibrating, prepare the straws (250μl plastic semen straws, Planer; FZA201) for freezing, as follows:

9.1. Attach a 1ml syringe to the labelled end of the straw. Using the syringe, aspirate HTF (high calcium Human Tubal Fluid) (see

Appendix B table 1) solution until the meniscus reaches 4.5cm marker.

9.2. Then aspirate 2.3cm air and lie the syringe and straw assembly on the bench until required.

gCPA

Cauda epididymides

120µl gCPA Mineral oil

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10. After 3 minutes, the sperm suspension should be divided into 10 x

10µl aliquots on a culture dish (Fig. 5). Then aspirate one drop into each of the 10 straws.

Fig. 5

11. Aspirate air until the HTF meniscus reaches the polyvinyl alcohol

section, half way along the cotton plug. This will seal the labelled end of the straw.

12. Seal both ends of the straw (Fig. 6) using a double impulse heat sealer or similar device.

heat sealed aliquots of sperm HTF cotton plug

airair

10ul

heat sealed

Fig. 6

13. Load the sealed straws into a floating sperm freezing device and then place them into the pre-prepared cooling chamber (Fig. 7) for 10

minutes. After 10 minutes plunge the straws directly into liquid nitrogen.

10 x 10µl aliquots of sperm

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LN2

h

Fig. 7

14. Whilst minimising their exposure to air, transfer the straws into their

long term storage locations.

B. Preparation of sperm dispersal dishes

1. Pipette 90µl TYH + 0.75mM MBCD (see Appendix B table 2) into the

centre of a 35mm Petri Dish (Falcon 351008) (Fig.8)

TYH+MBCD

Fig. 8

2. Overlay with mineral oil and equilibrate 10-20min at 37°C, in 5% CO2

in air.

C. Thawing the frozen sperm samples

1. Remove the required straws from the storage tanks and place in a flask of liquid nitrogen.

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2. Quickly transfer a straw into a 37°C water bath and leave for 10

minutes.

Note: To ensure warming of the frozen sperm, completely immerse

the part of the straw containing the sperm in the water bath. Furthermore, frozen-thawed mouse sperm are sensitive to

environmental changes. If the straw is not kept in the water bath long enough (10 minutes), the motility of the cryopreserved sperm will be

reduced.

3. Carefully dry the straw with paper towel. Cut the heat-sealed end of

straw and use a straw pusher to expel only the 10µl sperm suspension into the centre of the drop of 90µl TYH+MBCD pre-incubation medium

(Fig. 9). Do not expel any of the HTF media contained within the straw.

Note: Do not disturb the dishes containing the frozen/thawed sperm

until the sperm are moving rapidly within the medium. If the dishes are disturbed before the sperm start to move they will not recover full

motility

Fig. 9

4. Pre-incubate the frozen/thawed sperm in TYH+MBCD medium for 30min at 37ºC in the CO2 incubator.

D. Preparation of fertilisation medium (CARD MEDIUM) containg 1mM reduced glutathione (GSH – Sigma: G4251)

1. Take 1ml HTF medium and add it to a tube containing 30.7mg reduced glutathione (GSH). Close the lid, mix the medium and the

powder in the tube (Fig. 10).

90µl TYH + MBCD

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GSH1ml HTF mixGSH stock

Fig.10

2. Take 50µl of the GSH solution and add it to 5ml HTF medium and mix

them together gently (Fig. 11).

50µl GSH stock

5ml HTF

filter

Fig. 11

3. Before use, filter the solution using 0.22µm syringe end filter.

4. Make a 90µl drop of the medium in a 35mm Petri Dish (Falcon

351008) (Fig. 12), and then place the dish in an incubator (37⁰C, 5%

CO2 in air) for 10-20minutes.

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Mineral oil

90µl

Fig.12

E. Oocyte harvesting and in vitro fertilisation

1. Dissect the oviducts from three superovulated female mice (for

superovulation method, see Appendix C) and transfer them into the mineral oil overlaying the pre-incubated fertilisation drop.

2. Under a dissecting microscope, hold each oviduct down with forceps and gently tear the swollen ampulla with a second pair of forceps to

release the cumulus masses into the oil. Using the forceps, drag the clutches through the oil and into the fertilisation drop. Then remove

the oviduct from the dish.

3. Once the frozen/thawed sperm has been equilibrated for 30 minutes in

the incubator, take a 10µl aliquot of the sperm suspension from the peripheral part of the pre-incubation drop. This region will contain the

most motile sperm. Try to avoid aspirating any sperm debris (Fig. 13).

Note: This step should be performed gently under a light microscope

and is best achieved using a wedge-shaped 10µl pipette tip (Axygen Inc; T-400). Following this procedure it is easy to collect good quality

motile sperm without picking up dead sperm or cell debris.

Wedge-shaped pipette tip

Non-motile sperm

motile sperm

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Fig. 13

4. Repeat steps 1-3 for each fertilisation dish in succession (i.e. complete all of the steps from collecting the oviducts to returning the fertilisation

dishes to the incubator for one batch of females before starting the next batch). Aim to take no more than 5 minutes from collecting the

oviducts to returning the fertilisation drop (including oocytes) to the incubator.

5. When all oocytes have been added to the fertilisation drops, remove one dish from the incubator and assess the sperm motility and

concentration again. Also observe whether cumulus cells are being removed. If the motility and concentration of sperm is poor and few

cumulus cells are being removed, it may be necessary to add more of the sperm suspension to each fertilisation drop.

6. Incubate the dishes at 37°C, in 5% CO2 in air for approximately 3-4hrs to allow fertilisation to occur.

E. Washing and culturing the fertilized oocytes

1. Prepare the wash drops by placing 4 X 150µl drops of HTF (without GSH) in a 35mm culture dish (Falcon 351008) and cover with mineral

oil (Fig. 14). Place the dishes in an incubator (37°C) for at least 3hrs or overnight.

1

4 3

21

Fig. 14

2. Take the oocytes through three washes (drops 1, 2 & 3) to remove

the cell debris, degenerating oocytes and dead sperm.

3. Move the presumptive zygotes into drop 4 and culture them

overnight.

4. Next morning, separate the 2-cell embryos from the unfertilised or

degenerating oocytes. Place all the 2-cell embryos in drop 4 and the 1-cell or degenerated oocytes/embryos in drop 3.

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5. Either transfer the 2-cell embryos to the oviducts of 0.5 day

pseudopregnant foster mothers, or:

6. Prepare the 2-cell embryos for cryopreservation according to a

standard protocol for in vivo derived embryos, or:

7. Culture the embryos in KSOM, plus amino acids

F. Experimental results

The following results show the fertilisation rates of frozen/thawed sperm

harvested from six C57BL/6NTac males. In each trial the sperm from one of each pair of cauda epididymides harvested from two males was

combined and then frozen using different protocols, namely:

a) the protocol described by Ostermeier et al (2008) (18% raffinose, 3% skimmed milk, supplemented with 447µM Monothioglycerol [MTG]) in

conjunction with the standard MRC-Harwell IVF procedure but in the ‘absence’ of MBCD and GSH.

b) the protocol described above (18% raffinose, 3% skimmed milk, supplemented with 100mM L-Glutamine [gCPA]) in conjunction with the

MBCD+GSH in vitro fertilisation procedure described here.

12 x C57BL/6NTac females were used for each group within each of the 3

individual trials.

Mean fertilisation for C57BL/6NTac USA frozen sperm

Group Trial 1 Trial 2 Trial 3 Mean (%)

MTG 38.5 45.7 29.3 37.8

MBCD+ GSH 82.8 93.2 61.0 79.0

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Appendix A__ Preparation of sperm cryopreservation agent

(gCPA) containing 100mM L-glutamine (FERTIUP®cryoprotectant).

Table 1: Composition of sperm cryopreservation medium

Reagent mg/10ml mg/20ml mg/40ml

Raffinose pentahydrate 1,800 3,600 7,200

Skimmed Milk 300 600 1,200

L-glutamine 146 292 584

1. Add 584mg L-glutamine (Sigma: G8540) to 40ml of embryo tested

water contained in a 50ml disposable tube, vortex for 3 minutes.

2. Put the tube into 60°C water bath.

3. Add 7200mg raffinose pentahydrate (Sigma: R-7630) and 1200mg skimmed milk powder (Becton Dickinson: 232100) to the tube,

vortex for 3 minutes;

4. Incubate the solution in a 60°C water bath for 90 minutes. Vortex for

3 minutes after every 30 minutes.

5. Divide the solution into 1.0ml aliquots and transfer to 1.5ml

centrifuge tubes.

6. Centrifuge the samples at 10,000g for 60 minutes.

7. Carefully collect 0.7ml of the supernatant from the central region of each tube. (Fig. 15).

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Pipette

Surface of supernatant

Supernatant

Pellet

Fig. 15

8. Filter the supernatant using a disposable filter unit (pore size: 0.22µm) discard the pellets.

9. Place 1.0ml aliquots of the filtered solution into 1.5ml microfuge tubes and seal them with parafilm. Store at room temperature (use

within 3 months of preparation).

Appendix B Preparation of IVF media (stored at 4°C in fridge up to 3 months)

Table 1: Composition of high calcium HTF medium

Reagent Name mg/100ml Vendor Cat. number

NaCl 593.8 Sigma S-5886

KCl 35.0 Sigma P-5405

MgSO4·7H2O 4.9 Sigma M-7774

KH2PO4 5.4 Sigma P-5655

CaCl2·2H2O 75.5 Sigma C-7902

NaHCO3 210.0 Sigma S-5761

Glucose 50.0 Sigma G-6152

Na-lactate (ml)* 0.34 Sigma L-7900

Na-Pyruvate 3.7 Sigma P-4562

Penicillin G 7.5 Sigma P-4687

Streptomycin 5.0 Sigma S-1277

BSA (Fraction V, Fatty Acid-Free) 400.0 Merck 126575

Phenol Red (0.5% soln) 0.04(ml) Sigma P-0290

Note: Solubilise the CaCl2.2H2O separately from the other reagents

before combining the solutions.

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Table 2: Composition of sperm pre-incubation medium

(TYH+MBCD: FERTIUP®preincubation medium) (stored at 4°C in fridge up to 3 months)

Reagent Name mg/100ml Vendor Cat. number

NaCl 697.6 Sigma S-5886

KCl 35.6 Sigma P-5405

MgSO4·7H2O 29.3 Sigma M-7774

KH2PO4 16.2 Sigma P-5655

NaHCO3 210.6 Sigma S-5761

Na-Pyruvate 5.5 Sigma P-4562

Glucose 100.0 Sigma G-6152

CaCl2·2H2O 25.1 Sigma C-7902

Methyl-β-cyclodextrin 98.3 Sigma C-4555

Penicillin G 7.5 Sigma P-4687

Streptomycin 5.0 Sigma S-1277

Polyvinylalcohol 100.0 Sigma P-8136

Note: Solubilise the CaCl2.2H2O separately from the other reagents before combining the solutions.

Appendix C Timetable of events for IVF

Day -3

(e.g. Saturday)

Day -1

(e.g. Monday)

Day 0

(e.g. Tuesday)

Day 1

(e.g. Wednesday)

Prepare dishes for

oocyte harvest,

fertilisation/wash/

culture and sperm

dispersal (if using a

freshly harvested

sample).

07:45 Thaw

cryopreserved sperm

sample, or collect

and disperse freshly

harvested sperm.

Morning: score the

IVF success (2-cell

vs others).

Superovulate

between nine

and thirty 3-4

week old females

by injecting

0.1ml (5iu) PMS

at 16.00-17.00.

Induce ovulation in the

females by injecting

0.1ml (2.5 to 5iu) hCG

at 16.00-17.00

08:00-09:00 Harvest

oocytes and place

into diluted sperm

preparation.

Prepare the 2-cell

embryos for

cryopreservation,

embryo transfer or

culture.

12:30 Wash the

presumptive zygotes

and place into culture

drops.

This timetable assumes that the mice are exposed to 12 hours of

darkness between 19:00 and 07:00.

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References

1. Ostermeier CG, Wiles M.V, Farley J.S and Taft R.A. Conserving, Distributing and Managing Genetically Modified Mouse Lines by

Sperm Cryopreservation. PLoS One. 2008 Jul 30;3(7):e2792

2. Takeo T, Nakagata N. Combination medium of cryoprotective agents

containing L-glutamine and methyl-β cyclodextrin in a pre-incubation medium yields a high fertilization rate for cryopreserved C57BL/6J

mouse sperm. Lab Anim. 2010 Apr; 44(2):132-7.

3. Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, Yamamura K, Irie

T, Nakagata N. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating

cholesterol efflux from the cells. Biol Reprod. 2008 Mar; 78(3):546-51.

4. Takeo T, Nakagata N. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-Beta-

cyclodextrin. Biol Reprod. 2011 Nov;85(5):1066-72.

5. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in

vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985; 44: 493-498.