Maintenance and growth of testicular hyperplasias following subcutaneous transplantation. (A,B) Histological observation of a testicular hyperplasia (A) and normal testis (B). Arrows indicate a single spermatogonium surrounded by Sertoli cells. DG, differentiating spermatogonia in large clusters surrounded by Sertoli cells; PC, primary spermatocytes; SP, sperm. Note that the hyperplasia contains a large number of single spermatogonia. Scale bar: 50µm. (C-G) Views of the rag1^t26683 mutant just after transplantation beneath the abdominal skin (C) and after 3months (D). A testicular hyperplasia (E) was cut down to transplantable size (F). After 3 months of transplantation, the grafted fragment was removed (G). Note that the grafted region swelled (arrows in C,D) and the transplanted fragment grew to almost original size (E,G). Scale bar: 1cm in C,D and 3.3mm in E-G.

Serial transplantation of the testicular hyperplasia A. (A) Each box shows the number of the grafted fragments, the number of recipients surviving for more than 1month, and the number of the grown graft in each transplantation steps. The number in parentheses indicates day/month/year of the transplantation. (B) Histological observation of a grafted hyperplasia. Sections of each testis correspond to the grafts described in A. Many single spermatogonia (arrows) were observed in grafts and sperm (arrowhead) were present. Testis-ova (the presence of oocytes in testis) were observed in A3-8 (fish no. 8 of the third serial transplantation of hyperplasia A; double arrowheads). Scale bar: 50µm. Serial transplantation of other testicular hyperplasia lines are shown in Fig. S2.

Propagation of SSCs in culture. (A,B) Effects of basal medium (A) and heparin at 25-50U/ml (B) on proliferation of early spermatogonia. Grafts of the sox17::egfp testicular hyperplasias recovered from rag1^t26683 mutant host were dissociated and cultured under various conditions. The gas condition was adjusted to the basal culture medium; in air for L-15 and in 5% CO₂ for DMEM. The culture medium in B was DMEM with 5% CO₂ and 20% O₂. Bars indicate the mean (n=4). (C) Dissociated sox17::egfp testicular hyperplasias were cultured for 1, 31 and 48days under SSC propagation conditions. Inset shows the morphology of the early stage of spermatogonia. Top panels: phase contrast; bottom panels: fluorescence. Arrows indicate GFP-negative differentiated spermatogonia. Scale bar: 50µm.

Differentiation of SSCs to sperm in culture. (A) SSCs that underwent propagation culture for 31days were transferred onto Sertoli ZtA6-12 feeders and cultured under the differentiation condition. After 1day of transfer, SSCs attached tightly on Sertoli feeders. Spermatocytes (inset) and sperm (inset) appeared around 10 and 17days after the transfer, respectively. Top panels: phase contrast; bottom panels: fluorescence. (B) Sycp3 expression in cells under differentiation culture. SSCs that underwent propagation culture for 31days were transferred onto ZtA6-12 feeders and cultured for 9days. Sycp3 signal is observed as a small particle on one side of the nucleus and extends into the nucleus (inset), suggesting normal meiotic initiation. (C) Sycp3 immunostaining of SSC clumps cultured under SSC propagation conditions for 40 days. Cells expressing Sycp3 were observed only rarely, at the periphery of SSC clumps (arrows). Scale bars: 50µm.

Growth of normal testicular fragments and testicular aggregates mixed with SSCs of testicular hyperplasia after transplantation. (A, B) The normal testicular fragment prior to the transplantation into rag1t²⁶⁶⁸³ mutant hosts (A), and the grafted testis fragment (B, enclosed by a dotted line) uncovered by peeling of host’s skin after 2 months of transplantation. (C, D) The aggregate of the inbred IM line that was mixed with spermatogonia of normal vas::egfp testes after 3 months of transplantion in the IM line. Arrows indicate GFP-positive cells (D). (E, F) The aggregate of the inbred IM line that was mixed with cultured SSCs of vas::egfp hyperplasias after 3 months of transplantation in the IM line. The SSCs were cultured for 4 weeks in the previous culture condition (Kawasaki et al., 2012). Arrowheads indicate GFP-positive cells (F). Sperm obtained from this aggregate generated fertilized embryos. Scale bar, 1mm.

Maintenance of spermatogenesis following the subcutaneous transplantation. Testis fragments recovered from rag1^t26683 mutant hosts after treatmemt with BrdU for 3 hours. (A, B) Serial sections of the grafted testis fragment stained with hematoxylin and eosin (A) and anti-BrdU antibodies (B). Note that the single spermatogonium (arrowheads) and differentiating spermatogonia in large clusters (arrows) incorporate BrdU. Scale bar, 50 µm.

Sequential transplantations of testicular hyperplasias B-D. (A, B) B series from hyperplasia B; (C, D) C series from hyperplasia C; (E, F) D series from hyperplasia D. In A, C, and E, graft information is provided in the same way as in Figure 2. (B, D, F) Histological observation of grafted testicular hyperplasias. Sections of each testis correspond to grafts of the same serial number shown in panels A, C and E, respectively. The single spermatogonium is indicated by arrows, sperm with arrowheads. Note that spermatogonia were rarely found in B4-1 (fish #1 of the fourth serial transplantation of hyperplasia B). Testis-ova were observed in the B3-1 and B4-1 (double arrowheads). Scale bar, 50µm.

Stemness test of SSCs propagated in culture. sox17::egfp SSCs that had been cultured under the propagating condition for 31 days were aggregated with dissociated testis of moto mutant, which lacks late stage germ cells. The aggregate was transplanted into rag1^t26683 mutants to allow SSC maintenance and differentiation. Transplants were collected from hosts one month later for histological analysis. (A, B) Aggregates with sox17::egfp SSCs. Self-renewal of GFP-positive spermatogonia (single spermatogonium: red arrowhead; spermatogonia in small clusters: red arrow), spermatocytes (PC) and sperm (SP) were observed. (C, D) Control experiment in which dissociated moto testis was self-aggregated. Black arrowheads and arrows indicate moto-derived GFP-negative single spermatogonia or those in small clusters, respectively. (A, C) hematoxylin-eaosin staining; (B, D) GFP immunostaining. Scale bar, 20µm.

Functional test of sperm generated in culture from SSCs. Unfertilized eggs from wild type females were artificially inseminated with sperm generated in culture from sox17::egfp SSCs. (A, B) Embryos at 24 hr-post-fertilization (A) expressed GFP (B) as in normal sox17::egfp embryos.

Propagation and differentiation culture of SSCs of normal testis. (A) Effects of basal medium and heparin at 50 U/ml on proliferation of dissociated testicular cells of the sox17::egfp transgenics. The gas condition was adjusted to the basal culture medium; in air for L-15 and in 5% CO2 and 10% O2 for DMEM. Bars indicate the average (n=4). (B) Dissociated sox17::egfp testicular cells were cultured for 1, 20, and 31 days under the SSC propagation condition. Upper panels: phase contrast; bottom panels: fluorescent. Arrows indicate GFP-negative differentiated spermatogonia. (C) Sycp3 expression in cells under differentiation culture. SSCs of normal testis that underwent propagation culture for 31 days were transferred onto ZtA6-12 feeders and cultured for 9 days. Sycp3 signals are observed (green). (D) Sycp3 immunostaining of SSC clumps cultured under the SSC propagation condition for 40 days. Cells expressing Sycp3 were observed at the periphery of SSC clumps (green). Scale bars, 50 µm.