Using Frozen Sperm

Arrival of Xenopus laevis sperm samples

The frozen sperm samples should arrive at the lab in a sealed polystyrene box covered in dry ice. Samples must remain frozen and should be placed immediately toward the back of a -80oC freezer until use.

  1. Quickly remove the vial containing frozen sperm from the -80oC freezer and thaw (30 seconds) the contents in a 37°C water bath, moving the tube in a circular ‘figure of 8’ motion1.
  2. Once thawed, add 125 µL room temperature 0.1 x MBS to the contents of the vial2.
  3. Using pre-cut tips immediately transfer the entire contents of the vial (approx. 250 µL) to the Petri dish containing eggs (ensuring all eggs are covered with the sperm suspension) and incubate at room temperature for 10 minutes3.
  4. After the 10-minute incubation period, flood the eggs with 0.1 x MBS.
  5. Once the embryos have turned, de-jelly the embryos in 2% cysteine, pH 7.8 – 8.0 and continue as normal.

Notes:

1Before starting ensure that a water bath is preheated to 37oC, 0.1xMBS is at room temperature and that you have large orifice or pre-cut 20-200µL tips available. We usually assess the quality of a small clutch of unfertilised eggs prior to defrosting the sperm. When recovering frozen sperm stored in distant -80oC freezers it is recommended that it is first transported on dry ice to the fertilisation bench before continuing with the protocol.

2frozen sperm sediments, pipette gently up and down to maintain in suspension.

3 If eggs are clustered and layered, gently shake the Petri dish or separate egg layers using pipette tips prior to incubating. An additional wash step  (125 µL 0.1 x MBS) of the vial containing sperm is recommended for maximum efficiency.

Equipment

  • Pipettes
  • 18°C incubator
  • Pre-heated 37°C waterbath

Note: a hot block is not a suitable alternative

  • Optional: Industrial razor blade

Materials

  • 15cm Petri dishes (e.g. Greiner bio-one, 639102)
  • Optional: Large orifice 200 µL filter tips (e.g. ThermoFisher Scientific, TF118-200-Q)
  • Optional: SureOne Beveled pipette tips 200 µL (e.g. Fisherbrand, 11933446)

Note: it is recommended that standard tips, if used, should be cut using a razor blade to enlarge the orifice preventing sperm damage

Reagents

  • Modified Barth’s Saline (MBS – Gurdon, 1977)can be stored at 4°C prior to use for up to 30 days.
  • Cysteine (Sigma, 168149) should be made fresh prior to each experiment and temperature adjusted to 18°C prior to use.
  • Human chorionic gonadotropin (HCG – Sigma, CG10) is reconstituted in sterile water (Sigma, W3500) and stored as aliquots at -20oC.

Modified Barth’s Saline (Gurdon, 1977)

Barth-HEPES Saline (10X): Weigh 51.3 g NaCl (e.g. Sigma, S7653), 0.748 g KCl (e.g. Sigma, P9541), 2.02 g NaHCO3 (e.g. Sigma, S6297), 23.832 g HEPES (e.g. Sigma, H4034) and dissolve in 800 mL de-ionised water. Add 5.6 mL 5 M NaOH (e.g. Sigma, S5881), 2.02 g MgSO (7H20 – e.g. Sigma, M2773), 0.920 g Ca(NO3)2 (4H20 – e.g. Sigma, C1396) and 0.603 g CaCl2 (2H20 – e.g. Sigma, C5080). Make up to a final volume of 1 L using de-ionised water and filter sterilise (or autoclave), the pH of the final solution should be around pH 7.6. Store MBS at 4oC and check for salt precipitation prior to use.

Generate 1 x MBS and 0.1 x MBS using de-ionised water. To 1 L of 1 x MBS or 0.1 x MBS, 1 mL penicillin-streptomycin solution (P/S – e.g. Sigma, p0781) is added to generate a final working concentration of 5 U/mL penicillin and 5 µg/mL streptomycin.

Cysteine

Mix 2 g Cysteine per 100 mL de-ionised water, stir until dissolved, adjust to pH 7.8-8.0 by adding NaOH (e.g. Sigma, S8045) and store in an air-tight container until use.