Development 143: doi:10.1242/dev.129643: Supplementary information
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.
Development • Supplementary information
Fig. S1. Growth of normal testicular fragments and testicular aggregates mixed with SSCs of
Development 143: doi:10.1242/dev.129643: Supplementary information
Fig. S2. Maintenance of spermatogenesis following the subcutaneous transplantation. Testis
fragments recovered from rag1t26683 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
Development • Supplementary information
spermatogonia in large clusters (arrows) incorporate BrdU. Scale bar, 5
Fig. S3. 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.
Development • Supplementary information
Development 143: doi:10.1242/dev.129643: Supplementary information
Development 143: doi:10.1242/dev.129643: Supplementary information
Fig. S4. 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 rag1t26683
mutants to allow SSC maintenance and differentiation. Transplants were collected from hosts
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.
Development • Supplementary information
one month later for histological analysis. (A, B) Aggregates with sox17::egfp SSCs.
Development 143: doi:10.1242/dev.129643: Supplementary information
Fig. S5. 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
Development • Supplementary information
sox17::egfp embryos.
Fig. S6. 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
Development • Supplementary information
Development 143: doi:10.1242/dev.129643: Supplementary information
Development 143: doi:10.1242/dev.129643: Supplementary information
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
Development • Supplementary information
observed at the periphery of SSC clumps (green). Scale bars, 50 µm.
Development 143: doi:10.1242/dev.129643: Supplementary information
Table S1. Survivability of grafted fragments of wild-type testis.
recipient line
# host fish
rearing period
(week)
survived
recipients
grafts recovered
rag1 t26683
10
4
10
8
wild-type
20
4
20
0
grafted
testis
hyperplasia
recipient
line
# host fish
rearing
period
(weeks)
survived
recipients
grafts
recovered
grown
grafts
A
rag1t26683
8
4
8
8
8
B
rag1t26683
2
4
2
2
2
C
rag1t26683
3
4
3
3
3
D
rag1t26683
3
4
3
3
3
D
wild-type
12
4
12
0
0
Development • Supplementary information
Table S2. Survivability and growth of grafted fragments of testis hyperplasia.