Materials and Reagents

100 mm dish (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 263991 )
Paper towels
Surgical blades (FEATHER Safety Razor, catalog number: No. 25 )
Aluminum foil
rag1 mutant adult male zebrafish (Wienholds et al., 2002)
L-15 medium (Sigma-Aldrich, catalog number: L5520-500ML )
25x phosphate-buffered saline (PBS)
Ethyl p-aminobenzoate (Wako Pure Chemical Industries, catalog number: 057-03832 )
Gentamicin (10 mg/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15710064 )
Ethanol
10% ethyl p-aminobenzoate stock solution (see Recipes)
0.01% ethyl p-aminobenzoate working solution (see Recipes)
0.4x PBS containing 10 μg/ml gentamicin (see Recipes)

Equipment

Stereoscopic microscope (OLYMPUS, model: SZ61 )
Forceps (Style 5) (Dumont, catalog number: 0108-5-PO )

Procedure

Subcutaneous transplantation
1. Maintain all adult male recipients of rag1 mutant zebrafish without food 1 day prior to transplantation.
2. Remove normal testes or spontaneous testicular hyperplasia from a donor fish as described in (Sakai, 2006), and cut to approximately 2-3 mm square containing the testis epithelium in L-15 medium.
3. Place a paper towel in a 100 mm dish and pour a small amount of 0.4x PBS into the dish.
4. Anesthetize the recipient zebrafish with 0.01% ethyl p-aminobenzoate.
5. Place the anesthetized recipient fish into the 100 mm dish.
6. Cover the head and tail of the recipient fish with wet paper towels (Figure 1A, Video 1).
7. Descale the site of transplantation.
8. Make an abdominal incision of approximately 5 mm in length with a razor blade.
9. Insert the tips of a pair of forceps between the muscle and skin to prepare for the transplantation (Figure 1B, Video 1).
10. Insert a fragment of the testis or testicular hyperplasia containing a small portion of adjacent tissue (Figure 1C, Video 1).
11. Transfer the recipient zebrafish to a tank containing 0.4x PBS supplemented with 10 μg/ml gentamicin.
12. Wrap the tank with aluminum foil to prevent the entry of light and incubate the tank at 26 °C for 4 days to promote wound healing.
13. Rear the transplanted fish 4 days after surgery.
  
  Figure 1. View of subcutaneous transplantation. A. Preparation of the recipient fish. The recipient fish is protected from drying by covering the head and tail with wet paper towels. B. Separation of the skin and muscle. The inserted forceps (arrow) separate the abdominal muscle from the skin of the recipient fish. C. View of the transplanted testis fragment. The testis fragment (arrowhead) is inserted into the treated area shown in (B). Scale bar = 5 mm.
  
  Video 1. Procedures of subcutaneous transplantation
Serial transplantation of testicular hyperplasia
1. After growth of the testicular hyperplasia graft, anesthetize the recipient with 0.01% ethyl p-aminobenzoate.
2. Remove the testicular hyperplasia and transfer to a 60 mm dish containing L-15 medium.
3. Cut the graft into approximately ten fragments of equal size.
4. To confirm the condition of the testicular hyperplasia graft, set aside several fragments for histology.
5. Re-transplant the remaining fragments, which contain epidermis of the hyperplasia, as outlined in Procedure A: Subcutaneous transplantation.

Data analysis

We have performed a total of 156 cases of serial transplantation using 4 testicular hyperplasias, and growth of the transplanted grafts was observed in 149 cases, including data previously reported (Data is presented in Kawasaki et al., 2016). We have succeeded in maintaining a testicular hyperplasia more than 3 years at the longest (Data is presented in Kawasaki et al., 2016). Figure 2 shows the tree diagram of an additional testicular hyperplasia by serial transplantation for more than one year. However, malignant transformation and testis-ova arose very occasionally in the serial transplantation of testicular hyperplasias (Data is presented in Kawasaki et al., 2016). Therefore, histological observation of the grafted testicular hyperplasias should be performed in each generation to avoid undesired transformation.

Figure 2. Maintenance of testicular hyperplasias by serial transplantation. A. Growth of the grafted fragment of a testicular hyperplasia. Arrowheads indicate the transplanted grafts. Scale bar = 5 mm. B. The tree diagram of serial transplantation of a testicular hyperplasia. Each box shows the number of the grafted fragments, the number of recipients survived for more than 1 month, and the number of the grown graft in each transplantation steps. Note that the fragment of the original testicular hyperplasia (the left photograph) grafted in the case of 1-1 was maintained for 458 days after the 1st transplantation, and that all grafts regrown in the 2nd transplantation. Scale bar = 5 mm. C. Histological observation of the grafted hyperplasia. Sections of each testicular hyperplasia correspond to the grafts shown in (B). Although the proportion of each stage of spermatogenic cells seemed to be changed, spermatogenesis was maintained through the serial transplantation. Arrows, spermatogonia; PC, spermatocytes; SP, sperm. Scale bar = 20 µm.

Recipes

10% ethyl p-aminobenzoate stock solution
Dissolve 5 g of ethyl p-aminobenzoate in 50 ml of ethanol
0.01% ethyl p-aminobenzoate working solution
Dissolve 50 μl of 10% ethyl p-aminobenzoate stock solution in 50 ml of rearing water
0.4x PBS containing 10 μg/ml gentamicin
Combine 16 ml of 25x PBS and 1 ml of gentamicin stock solution (10 mg/ml) in 983 ml of autoclaved rearing water

Acknowledgments

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sport, Science and Technology, Japan (Grant Nos. 23570260, 25251034, and 25114003). This protocol was adapted from the previous work (Kawasaki et al., 2016)

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