NXR Slow-Freezing Protocol

(Testes Extraction)

1. Euthanize a ♂Xenopus following IACUC or institutional standards.

2. Once euthanasia is complete, proceed with the ventral side of the animal exposed on a surface suitable for dissection.

3. Using delicate dissecting scissors, cut an opening through each layer of skin, and pull back with forceps to expose the body cavity.

   As testes extraction is a terminal procedure, care does not need to be taken to minimize damage from dissection.

4. Testes are located centrally in the abdominal cavity connected to the mesorchium.
5. Using delicate dissecting scissors and forceps, isolate testes from surrounding tissue.

6. Remove testes from the abdominal cavity and trim off any excess tissue still connected to the testes.

7. Roll testes across a paper towel to remove any remaining connective tissue or blood before storing testes. Optionally, using the flat side of dissecting forceps, apply light pressure to each testis and run forceps down each side to break apart surface level blood vessels.

   X. laevis: Place testes in a Petri dish filled with pre-chilled 1.2x MMR on ice.
   X. tropicalis: Place testes in a Petri dish filled with 26°C 1x MMR.
    

8. Store testes until ready to use.

   X. laevis: Store testes in 1.2x MMR at 4°C for up to 2 weeks. Some degradation in sperm quality has been observed during the storage period.

   X. tropicalis: Store testes in 1x MMR at 26°C for 1-2 days. Best results are obtained when homogenizing and freezing samples shortly after extracting.

X. tropicalis (Steps 9-16)

9. For best results, plan the procedure to be efficient by ensuring all components are thawed and aliquoted before extracting testes.

• L-15: Aliquot and store in a tube on ice.
• FBS: Thaw at ambient temperature (~21°C) and store on ice.
• Cryoprotectant: Thaw at ambient temperature (~21°C) and store on ice.
• L-glutamine: Thaw in a water bath (~37°C) and vortex to dissolve particulate until clear. Store on ice once liquified.

10. Combine L-15 (900μL), FBS (100μL), and L-glutamine (10μL) in a conical-bottom tube and store the mixture on ice.

    This volume produces 16 vials. For X. tropicalis, scaling down the volume to produce fewer vials (10-14) may result in higher quality samples.

11. Place both testes in the L-15 mix and crush to homogenize with a micro pestle.

    Avoid overworking the tissue, use as few motions as possible to homogenize. Optionally, forceps can be used to tear testes into small pieces rather than crushing which may reduce damage to sperm from the mechanical action.

12. Transfer the mixture to a larger tube (>2mL) and add 1000μL of cryoprotectant.
13. Mix the contents and aliquot 125μL to each of the labelled vials.
    Use appropriately threaded cryovials when storing samples in LN₂ to ensure sample security.    
14. Place tubes upright in a polystyrene box (~8”x6”x5”) covered on top with aluminum foil (no polystyrene lid).
15. Place the polystyrene box in a -80°C freezer.

16. Remove after 3+ hours and store samples in a LN₂ dewar.
    Alternatively, without access to LN₂, store samples in a -80°C freezer until ready for use. Longevity of samples is decreased when stored without LN₂.

X. laevis (Steps 17-24)

17. Due to the longevity of X. laevis testes stored at 4°C, timing is less crucial than with      X. tropicalis, so components can be prepared before or after testes extraction.

• L-15: Aliquot and store in a tube on ice.
• FBS: Thaw aliquot at ambient temperature (~21°C) and store on ice.
• Cryoprotectant: Thaw at ambient temperature (~21°C) and store on ice.
• L-glutamine: Thaw in a water bath (~37°C) and vortex to dissolve particulate until clear. Store on ice once liquified.

18. Combine L-15 (900μL), FBS (100μL), and L-glutamine (10μL) in a conical-bottom tube and store the mixture on ice.

19. Place both testes in the L-15 mix and crush to homogenize with a micro pestle.
    Avoid overworking the tissue, use as few motions as possible to homogenize. Optionally, forceps can be used to tear testes into small pieces rather than crushing which may reduce damage to sperm from the mechanical action.
20. Transfer the mixture to a larger tube (>2mL) and add 1000μL of cryoprotectant.
21. Mix contents and aliquot 125μL to each of the 16 labelled cryovials.
    Use appropriately threaded cryovials when storing samples in LN₂ to ensure sample security.

If LN₂ is available for sample freezing, proceed to Step 25.

22. Place tubes upright in a polystyrene box (~8”x6”x5”) covered on top with aluminum foil (no polystyrene lid).

23. Place the polystyrene box in a -80°C freezer where it won’t be disturbed.

24. Remove after 3+ hours and store samples in a LN₂ dewar.
    Alternatively, without access to LN₂, store samples in a -80°C freezer until ready for use. Longevity of samples is decreased when stored without LN₂.

X. laevis Fast Freeze (Steps 25-38)

• To freeze samples with a controlled-rate freezer proceed to Step 25.
• To freeze samples without a controlled-rate freezer proceed to Step 33.
  • Benefits have been observed when freezing X. laevis samples at a faster rate than can be produced with a -80°C freezer. Faster cooling rates can be obtained with a controlled-rate freezer or by using LN₂ to expose samples to LN₂ vapor.

Controlled-Rate Freezer

25. Fill a polystyrene container or LN₂ transport vessel with enough LN₂ to submerge samples once freezing concludes.

26. Connect the controlled-rate freezer to a LN₂ supply tank.

27. Turn on and ensure proper operation of the controlled-rate freezer. Program or select the following freeze cycle to run:

• 10 minutes hold at 4°C
• -20°C/min ramp to -80°C
• 5 minutes hold at -80°C

28. Begin the freeze cycle with the controlled-rate freezer.

29. Load samples onto a tube rack suitable for -80°C temperatures.

30. When the controlled-rate freezer has reached the starting temperature, load the samples, and start the freeze cycle.

31. When the freeze cycle has concluded, quickly move the samples from the freezer into the waiting LN₂.

32. Load the samples into a freezer box and store in a dewar.

Without a Controlled-Rate Freezer

Without the use of a controlled-rate freezer to produce an optimized cooling rate, it is still possible to cryopreserve samples with a high degree of success. Using Positional Cooling Platform Devices (PCPDs), it is possible to obtain standardized rates of cooling adjusted by increasing or decreasing the distance of samples from LN₂ or LN₂ vapor. Two low-cost methods designed by the Aquatic Germplasm and Genetic Resources Center (AGGRC) are detailed. The CryoKit and CryoArk hold samples at variable heights above LN₂ to produce a range of cooling rates.

https://aggrc.com/CryoKit.php

https://aggrc.com/CryoArk.php

33. Add ~10cm of LN₂ to a polystyrene box of sufficient size to fit the CryoKit or CryoArk.

34. Load samples onto the CryoKit or CryoArk rack.

35. Float the CryoKit or CryoArk in the polystyrene container with LN₂.

36. Begin a timer for the duration of time it will take the samples to reach -80°C and hold for an additional 5-10 minutes.

37. Once samples have been frozen, quickly move the samples from the rack into the waiting LN₂.

38. Load samples into a freezer box and store in a dewar.