Cryopreservation of Xenopus Sperm and In Vitro Fertilization Using Frozen Sperm Samples

Freezing Sperm

• 1. Euthanize the male frog(s) painlessly according to local regulations and remove testes as in Protocol: Obtaining Xenopus laevis Embryos (Shaidani et al. 2021) or Protocol: Obtaining Xenopus tropicalis Embryos by In Vitro Fertilization (Lane and Khokha 2021). Use tweezers to carefully remove any blood vessels and excess fat tissue. Roll the testes on paper towels to remove all traces of blood.

  • X. laevis testes can be stored in 1× MBS or in L-15 (unsupplemented) for up to 1 wk at 4°C, but X. tropicalis sperm should be frozen immediately because success of freezing diminishes rapidly over time.

• 2. For each pair of testes, place 1 mL of ice-cold cryoprotectant solution in a 2-mL tube and leave on ice.

  • Testes from 2–6 males can be pooled per batch. If pooling, scale up Steps 2–5 as necessary and use 30-mL tubes.

• 3. Label 16 500-µL thin-walled PCR tubes, and place them in a rack ready for rapid filling.

  • Eight samples of 125 µL per testis will be frozen (16 samples per male).

• 4. Transfer testes to a 6-mm Petri dish placed at a 45° angle with 1 mL of L-15 medium supplemented with FBS and l-glutamine, and carefully macerate the testes with tweezers.

  • X. tropicalis testes can be transferred to a microcentrifuge tube containing 0.5 mL of L-15 supplemented with FBS and l-glutamine and macerated gently using a plastic disposable pestle made for use in Eppendorf tubes. Once the testes are homogenized, add an additional 0.5 mL of ice-cold, supplemented L15 medium. Macerating X. laevis testes with pellet pestle is not recommended. We saw very low sperm recovery compared to maceration with the tweezers.

• 5. Transfer the sperm suspension using a 1-mL tip with a cut end to the 2-mL tube containing 1 mL of cryoprotectant on ice. Gently mix the contents by inversion.

• 6. With a cut-off pipette tip or a large-orifice 200-µL filter tip, transfer 125-µL aliquots of the sperm into the prepared tubes. Immediately place the samples in a polystyrene box and cover with aluminum foil (instead of the normal lid). Place the box in a −80°C freezer.

  • In total, 96 tubes (sperm from six frogs) can be put in a polystyrene box in any one round of freezing. For larger numbers of aliquots, racking the sperm samples in cardboard CryoBoxes within the polystyrene box makes it easier to transfer them to long-term storage and has the advantage of ensuring the sperm suspension is at the bottom of all the tubes.

  • Aim to get the samples into the −80°C freezer as fast as possible. We can get 96 aliquots of sperm into the freezer in <10 min from the maceration. Label all the tubes and organize them in racks at room temperature in advance.

• 7. The next day, move the samples from the polystyrene box into long-term storage at −80°C.

  • Samples can be also be successfully frozen in cryotubes and stored in liquid nitrogen.

In Vitro Fertilization Using Frozen Sperm

• Keep tubes with frozen sperm at −80°C or on dry ice until the eggs are ready to be fertilized.

• 8. Extrude approximately 500 X. laevis or X. tropicalis eggs into a dry 90-mm Petri dish as described in Protocol: Obtaining Xenopus laevis Embryos (Shaidani et al. 2021) or in Protocol: Obtaining Xenopus tropicalis Eggs (Lane et al. 2021). Check egg quality under a dissecting microscope as there is no point wasting frozen sperm on poor-quality eggs.

  • If you are using valuable GA sperm, we recommend testing the first batch of eggs produced on the fertilization day by fertilizing with fresh sperm so that precious frozen samples are not wasted.

• 9. Thaw frozen sperm in a water bath for 30 sec at 37°C by moving it in a figure 8 until most of the frozen sperm has thawed.

• 10. Immediately add 250 µL of 0.1× MBS or 0.1× MMR to the 125-µL sperm sample, and mix gently by pipetting up and down five to10 times using large-orifice or cut-off pipette tips.

• 11. As quickly as possible, using the same tip, apply the sperm suspension to the eggs and mix gently. Spread the eggs into a monolayer with a pair of fresh, uncut, 200-µL pipette tips. Make sure all eggs are covered by the sperm and place the lid on the Petri dish. Incubate the dish at 18°C for X. laevis or at 23°C for X. tropicalis.

• 12. After 5–10 min, flood the eggs with 0.1× MBS or 0.1× MMR. Incubate the dish at 18°C for X. laevis or at 23°C for X. tropicalis.

• 13. Within 2 h, estimate the fertilization rate by comparing divided to undivided eggs.

  • See Troubleshooting.

• 14. Culture the embryos as described in Protocol: Obtaining Xenopus laevis Embryos (Shaidani et al. 2021) or Protocol: Obtaining Xenopus tropicalis Embryos by In vitro Fertilization (Lane and Khokha 2021), changing the medium on a daily basis.