Allogeneic Transplantation of Testicular Hyperplasia in rag1 Mutant Zebrafish

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Feb 2016



Allogeneic organ transplantation is a powerful tool for clinical and basic research studies. However, the graft is often rejected by the host organism. Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish. These zebrafish escaped rejection, which made it possible to successfully transplant fragments of an allogeneic testis and testicular hyperplasia. This protocol can be used to amplify and maintain testicular hyperplasia grafts for several years (Kawasaki et al., 2016). The amplified hyperplasias are likely to be a good source of somatic and germ cells such as Sertoli cells and spermatogonial stem cells.


Zebrafish have emerged as a tractable teleost genetic model for the study of vertebrate biology because several thousand mutants have been isolated by various genetic methods (Granato and Nüsslein-Volhard, 1996). Recently, this organism was used to study human diseases such as cancer (White et al., 2013). Although the incidence of spontaneous cancers is low, with many zebrafish eventually surviving cancer, allogeneic organ transplantation is a powerful tool, because many of the cancers are not syngeneic. Unfortunately, this method is not well developed. A previous study reported that zebrafish embryos accept cell grafts prior to the development of a mature immune system (Nicoli et al., 2007). However, it is difficult to successfully transplant grafts into embryos due to their minute size. For transplantation into adult zebrafish, sublethal γ-irradiation or immunosuppression with dexamethasone can block the rejection of the graft (Stoletov et al., 2007; White et al., 2008). However, it can be difficult to maintain cell grafts for long periods of time due to the short lifespans of recipients and the recovery of the immune response by 20 days after irradiation (Smith et al., 2010; Eguiara et al., 2011). Tissue grafts between identical clonal or inbred lines can survive without rejection (Kawasaki et al., 2010; Mizgirev and Revskoy, 2010; Shinya and Sakai, 2011).

T lymphocytes are central to the allograft response (Ingulli, 2010). The Recombination activating gene 1, 2 (rag1, Rag2) are important for immune function, because it creates double-stranded DNA breaks and is essential for V(D)J recombination, as well as for T and B cell function. rag1 mutant mice lack mature T and B cells, and they maintain allogeneic heart grafts for long periods of time (Zhang et al., 2006). By contrast, allogeneic transplantation has failed in rag1 mutant rats, probably due to the insufficient depletion of T and B cells (Ménoret et al., 2013). Hypomorphic rag2E450fs mutant zebrafish has been created, which have reduced V(D)J rearrangement and lymphocytes, and maintains various allogeneic cancer cells (Tang et al., 2014). Although rag1t26683 mutant zebrafish (hereafter rag1 mutant) have been isolated and they lack functional T and B cells (Wienholds et al., 2002; Petrie-Hanson et al., 2009), they were not used for transplantation. Our recent study reported that rag1 mutant zebrafish accept and maintain allogeneic testis organ and testicular hyperplasia grafts for long periods of time (Kawasaki et al., 2016). Here, we describe a protocol that uses immunodeficient rag1 mutant zebrafish for the subcutaneous transplantation of testis and testicular hyperplasia grafts.

Materials and Reagents

  1. 100 mm dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 263991 )
  2. Paper towels
  3. Surgical blades (FEATHER Safety Razor, catalog number: No. 25 )
  4. Aluminum foil
  5. rag1 mutant adult male zebrafish (Wienholds et al., 2002)
  6. L-15 medium (Sigma-Aldrich, catalog number: L5520-500ML )
  7. 25x phosphate-buffered saline (PBS)
  8. Ethyl p-aminobenzoate (Wako Pure Chemical Industries, catalog number: 057-03832 )
  9. Gentamicin (10 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15710064 )
  10. Ethanol
  11. 10% ethyl p-aminobenzoate stock solution (see Recipes)
  12. 0.01% ethyl p-aminobenzoate working solution (see Recipes)
  13. 0.4x PBS containing 10 μg/ml gentamicin (see Recipes)


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


  1. 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

  2. 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.


  1. 10% ethyl p-aminobenzoate stock solution
    Dissolve 5 g of ethyl p-aminobenzoate in 50 ml of ethanol
  2. 0.01% ethyl p-aminobenzoate working solution
    Dissolve 50 μl of 10% ethyl p-aminobenzoate stock solution in 50 ml of rearing water
  3. 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


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)


  1. Eguiara, A., Holgado, O., Beloqui, I., Abalde, L., Sanchez, Y., Callol, C. and Martin, A. G. (2011). Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification. Cell Cycle 10(21): 3751-3757.
  2. Granato, M. and Nüsslein-Volhard, C. (1996). Fishing for genes controlling development. Curr Opin Genet Dev 6(4): 461-468.
  3. Ingulli, E. (2010). Mechanism of cellular rejection in transplantation. Pediatr Nephrol 25(1): 61-74.
  4. Kawasaki, T., Saito, K., Shinya, M., Olsen, L. C. and Sakai, N. (2010). Regeneration of spermatogenesis and production of functional sperm by grafting of testicular cell aggregates in zebrafish (Danio rerio). Biol Reprod 83(4): 533-539.
  5. Kawasaki, T., Siegfried, K. R. and Sakai, N. (2016). Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture. Development 143(4): 566-574.
  6. Ménoret, S., Fontanière, S., Jantz, D., Tesson, L., Thinard, R., Rémy, S., Usal, C., Ouisse, L. H., Fraichard, A. and Anegon, I. (2013). Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J. 27(2): 703-711.
  7. Mizgirev, I. and Revskoy, S. (2010). Generation of clonal zebrafish lines and transplantable hepatic tumors. Nat Protoc 5(3): 383-394.
  8. Nicoli, S., Ribatti, D., Cotelli, F. and Presta, M. (2007). Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res 67(7): 2927-2931.
  9. Petrie-Hanson, L., Hohn, C. and Hanson, L. (2009). Characterization of rag1 mutant zebrafish leukocytes. BMC Immunol 10: 8.
  10. Sakai, N. (2006). In vitro male germ cell cultures of zebrafish. Methods 39(3): 239-245.
  11. Shinya, M. and Sakai, N. (2011). Generation of highly homogeneous strains of zebrafish through full sib-pair mating. G3 (Bethesda) 1(5): 377-386.
  12. Smith, A. C., Raimondi, A. R., Salthouse, C. D., Ignatius, M. S., Blackburn, J. S., Mizgirev, I. V., Storer, N. Y., de Jong, J. L., Chen, A. T., Zhou, Y., Revskoy, S., Zon, L. I. and Langenau, D. M. (2010). High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia. Blood 115(16): 3296-3303.
  13. Stoletov, K., Montel, V., Lester, R. D., Gonias, S. L. and Klemke, R. (2007). High-resolution imaging of the dynamic tumor cell vascular interface in transparent zebrafish. Proc Natl Acad Sci U S A 104(44): 17406-17411.
  14. Tang, Q., Abdelfattah, N. S., Blackburn, J. S., Moore, J. C., Martinez, S. A., Moore, F. E., Lobbardi, R., Tenente, I. M., Ignatius, M. S., Berman, J. N., Liwski, R. S., Houvras, Y. and Langenau, D. M. (2014). Optimized cell transplantation using adult rag2 mutant zebrafish. Nat Methods 11(8): 821-824.
  15. White, R. M., Sessa, A., Burke, C., Bowman, T., LeBlanc, J., Ceol, C., Bourque, C., Dovey, M., Goessling, W., Burns, C. E. and Zon, L. I. (2008). Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2(2): 183-189.
  16. White, R., Rose, K. and Zon, L. (2013). Zebrafish cancer: the state of the art and the path forward. Nat Rev Cancer 13(9): 624-636.
  17. Wienholds, E., Schulte-Merker, S., Walderich, B. and Plasterk, R. H. (2002). Target-selected inactivation of the zebrafish rag1 gene. Science 297(5578): 99-102.
  18. Zhang, Y., Kirken, R. A., Furian, L., Janczewska, S., Qu, X., Hancock, W. W., Wang, M., Tejpal, N., Kerman, R., Kahan, B. D. and Stepkowski, S. M. (2006). Allograft rejection requires STAT5a/b-regulated antiapoptotic activity in T cells but not B cells. J Immunol 176(1): 128-137.


细胞色素(Cyt)b 559 是光合电子传递系统中光系统II的重要和必需的核心组分,是一种血红素桥接异二聚体蛋白,由α亚基 PsbE )和β亚基( PsbE ),其还原形式在559nm的α波段具有吸收最大值。通过用等吸光度点在580nm处的吸光度归一化的减少氧化的差异光谱的分光光度测量可以测定Cyt b 559的量。作者使用的差别消光系数为Cyt b 559 [Δε(559-580nm)= 15.5mM -1 > cm -1 ],这已由Garewal和Wasserman(1974)报道。除了Cyt b559之外,该程序可用于Cyt b 和Cyt 的定量,Cyt > b 6 / f 复合体和P 700 改编自Fujita和Murakami(1987),用于蓝细菌,细长聚球藻PCC 7942,以及其他蓝细菌菌株,包括集胞藻属(Synechocystis)。 PCC 6803。...


  1. 100mm皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:263991)
  2. 纸毛巾
  3. 外科刀片(FEATHER安全剃刀,目录号:25)
  4. 铝箔
  5. rag1 突变成年雄性斑马鱼(Wienholds et al 。,2002)
  6. L-15培养基(Sigma-Aldrich,目录号:L5520-500ML)
  7. 25x磷酸盐缓冲盐水(PBS)
  8. 乙基对氨基苯甲酸酯(Wako Pure Chemical Industries,目录号:057-03832)
  9. 庆大霉素(10mg/ml)(Thermo Fisher Scientific,Gibco TM ,目录号:15710064)
  10. 乙醇
  11. 10%对氨基苯甲酸乙酯储备溶液(见配方)
  12. 0.01%对氨基苯甲酸乙酯工作溶液(参见配方)
  13. 0.4x含有10μg/ml庆大霉素的PBS(见Recipes)


  1. 立体显微镜(OLYMPUS,型号:SZ61)
  2. 钳(样式5)(Dumont,目录号:0108-5-PO)


  1. 皮下移植
    1. 在移植前1天,保持所有成年男性接受者的rag1 突变斑马鱼无食物。
    2. 如(Sakai,2006)所述,从供体鱼中取出正常睾丸或自发性睾丸增生,并在L-15培养基中切成包含睾丸上皮的约2-3mm正方形。
    3. 将纸巾放在100毫米的盘子里,倒入少量的0.4×PBS进入盘子
    4. 用0.01%的对氨基苯甲酸乙酯麻醉受体斑马鱼。
    5. 将麻醉的受体鱼放入100 mm培养皿中
    6. 用湿纸巾覆盖受体鱼的头部和尾部(图1A,视频1)。
    7. 去除移植部位。
    8. 用剃刀刀片切开长约5 mm的腹部切口。
    9. 将一对镊子的尖端插入肌肉和皮肤之间,以准备移植(图1B,视频1)。
    10. 插入睾丸或包含一小部分邻近组织的睾丸增生的片段(图1C,视频1)。
    11. 将受体斑马鱼转移到含有0.4x PBS,补充有10μg/ml庆大霉素的槽中
    12. 用铝箔包裹水箱,以防止光线进入,并将水箱在26°C下孵育4天,以促进伤口愈合。
    13. 手术后4天后移植鱼。

      图1.皮下移植的视图 A.准备受体鱼。通过用湿纸巾覆盖头部和尾部来保护受体鱼免受干燥。 B.皮肤和肌肉的分离。插入的镊子(箭头)将腹部肌肉与受体鱼的皮肤分开。 C.移植的睾丸片段的视图。将睾丸碎片(箭头)插入(B)中所示的处理区域。比例尺= 5mm。

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  2. 系列移植睾丸增生
    1. 在睾丸增生移植物生长之后,用0.01%对氨基苯甲酸乙酯麻醉受体。
    2. 取出睾丸增生并转移到含有L-15培养基的60mm培养皿中
    3. 将移植物切成大约10个相等大小的碎片。
    4. 为了确认睾丸增生移植物的状况,留出几个片段用于组织学
    5. 如程序A:皮下移植中所述,重新移植其余的含有增生表皮的碎片。


我们使用4种睾丸增生进行了总共156例连续移植,并且在149例中观察到移植移植物的生长,包括先前报道的数据(数据呈现在Kawasaki等人,2016 )。我们已经成功地在最长时间内维持了超过3年的睾丸增生(数据呈现在Kawasaki等人,2016)。图2显示通过连续移植超过一年的额外睾丸增生的树图。然而,在睾丸增生的连续移植中非常偶然地出现恶性转化和睾丸-ova(数据呈现在Kawasaki等人,2016)。因此,应当在每一代中进行移植的睾丸增生的组织学观察,以避免不希望的转化。

图2.通过连续移植维持睾丸增生。A.睾丸增生移植片段的生长。箭头指示移植的移植物。比例尺= 5mm。 B.睾丸增生的连续移植的树图。每个框显示移植片段的数目,受体存活的数目超过1个月,以及在每个移植步骤中生长的移植物的数目。注意,在1-1的情况下移植的原始睾丸增生的片段(左侧照片)在第一次移植后保持458天,并且所有移植物在第二次移植中再次生长。比例尺= 5mm。 C.嫁接增生的组织学观察。每个睾丸增生的切片对应于(B)中所示的移植物。虽然每个阶段的精子细胞的比例似乎改变,精子发生通过连续移植维持。箭头,精原细胞PC,精母细胞; SP,精子。比例尺=20μm。


  1. 10%对氨基苯甲酸乙酯储备液
  2. 0.01%对氨基苯甲酸乙酯工作溶液 将50μl10%的对氨基苯甲酸乙酯原液溶解在50ml的饲养水中。
  3. 0.4x含有10μg/ml庆大霉素的PBS 将16ml 25x PBS和1ml庆大霉素储备溶液(10mg/ml)混合在983ml高压灭菌饲养水中




  1. Eguiara,A.,Holgado,O.,Beloqui,I.,Abalde,L.,Sanchez,Y。,Callol,C.and Martin,AG(2011)。  斑马鱼胚胎中的异种移植物作为乳腺癌干细胞样细胞鉴定的快速功能测定。细胞周期 10(21):3751-3757。
  2. Granato,M.和Nüsslein-Volhard,C.(1996)。  钓鱼用于控制发育的基因。 6(4):461-468。
  3. Ingulli,E。(2010)。  细胞排斥机制 Pediatr Nephrol 25(1):61-74。
  4. Kawasaki,T.,Saito,K.,Shinya,M.,Olsen,LCand Sakai,N。(2010)。  通过在斑马鱼中移植睾丸细胞聚集体来重建精子发生和产生功能性精子( Biol Reprod 83(4):533-539。
  5. Kawasaki,T.,Siegfried,KR和Sakai,N。(2016)。  将培养中的斑马鱼精原干细胞分化为功能性精子。 143(4):566-574。
  6. Ménoret,S.,Fontanière,S.,Jantz,D.,Tesson,L.,Thinard,R.,Rémy,S.,Usal,C.,Ouisse,LH,Fraichard,A。和Anegon, )。  生成 Rag1 - 敲除免疫缺陷大鼠和使用改造的大范围核酸酶的小鼠。 FASEB J 。 27(2):703-711。
  7. Mizgirev,I.和Revskoy,S。(2010)。  Generation of clonal zebrafish lines and transplantable hepatic tumours。 Nat Protoc 5(3):383-394。
  8. Nicoli,S.,Ribatti,D.,Cotelli,F.和Presta,M。(2007)。  哺乳动物肿瘤异种移植物诱导斑马鱼胚胎中的新血管形成。癌症研究67(7):2927-2931。
  9. Petrie-Hanson,L.,Hohn,C.和Hanson,L。(2009)。  突变斑马鱼白细胞的表征 BMC Immunol 10:8.
  10. Sakai,N。(2006)。  体外雄性生殖细胞培养物的斑马鱼。方法 39(3):239-245。
  11. Shinya,M。和Sakai,N。(2011)。  通过全同胞交配产生高度同质的斑马鱼。 1(5):377-386。
  12. Smith,AC,Raimondi,AR,Salthouse,CD,Ignatius,MS,Blackburn,JS,Mizgirev,IV,Storer,NY,de Jong,JL,Chen,AT,Zhou,Y.,Revskoy,S.,Zon,LI和Langenau,DM(2010)。  高通量细胞移植确定肿瘤起始细胞在斑马鱼T细胞急性淋巴细胞白血病中丰富。血液 115(16):3296-3303。
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  14. Tang,Q.,Abdelfattah,NS,Blackburn,JS,Moore,JC,Martinez,SA,Moore,FE,Lobbardi,R.,Tenente,IM,Ignatius,MS,Berman,JN,Liwski,RS,Houvras,和Langenau,DM(2014)。  优化的细胞移植使用成年突变斑马鱼。 11 11(8):821-824。
  15. White,RM,Sessa,A.,Burke,C.,Bowman,T.,LeBlanc,J.,Ceol,C.,Bourque,C.,Dovey,M.,Goessling,W.,Burns,CEand Zon, LI(2008)。  透明成人斑马鱼作为工具 移植分析 2(2):183-189。
  16. White,R.,Rose,K.和Zon,L.(2013)。  Zebrafish cancer:the state of the art and the path forward。 Nat Rev Cancer 13(9):624-636。
  17. Wienholds,E.,Schulte-Merker,S.,Walderich,B.and Plasterk,RH(2002)。  目标选择的斑马鱼rag1基因的灭活。 297(5578):99-102。
  18. Zhang,Y.,Kirken,RA,Furian,L.,Janczewska,S.,Qu,X.,Hancock,WW,Wang,M.,Tejpal,N.,Kerman,R.,Kahan,BDand Stepkowski,SM (2006)。  同种异体移植排斥需要STAT5a/b-调节抗凋亡活性在T细胞而不是B细胞中。 J Immunol 176(1):128-137。
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引用:Kawasaki, T. and Sakai, N. (2016). Allogeneic Transplantation of Testicular Hyperplasia in rag1 Mutant Zebrafish. Bio-protocol 6(21): e1992. DOI: 10.21769/BioProtoc.1992.