SSC Niche and SSC In Zebrafish day of injection (day 0), the arrowhead shows that the lowest GSI is observed 10 days after injection. Dots represent mean6SE (n = 6). C. Testes from control (27uC, left) and busulfan-depleted animals. Inset shows a high magnification of the depleted testis after in vivo injection via the urogenital pore of a solution containing trypan blue. D. Frequency of spermatogenic cysts after a single dose of 30 or 40 mg/Kg busulfan. Type A spermatogonia (SGA), type B spermatogonia (SGB), spermatocytes (SC), apoptosis and abnormal metaphase I figures (A+AMF), Sertoli cell only (SCO). Bars represent means expressed as percentage. E–I. Histological sections of testes collected at 0 (E), 2 (F), 4 (G), 6 (H), and 10 (I) days after a single injection of 40 mg/Kg busulfan at 35uC. Note that busulfan induced spermatogonial apoptosis (arrowheads) after 4 days of injection. Scale bars = 10 mm. J. Testicular cell suspensions were obtained from vasa::egfp testes and subjected to FACS. Dot plot shows the total testicular suspension from which a population of large cells (forward scatter, FSC; abscissa), showing an intense fluorescence (FL1 FITC; ordinate) was sorted (black square). K. Histogram shows an enrichment of the sorted cells after FACS. Blue line (sorted cells), black line (total testicular cell suspension). FL1 FITC (x axis) means intensity of fluorescence, and counts (y axis), the number of events. L,M. Fraction of sorted cells under normal light (L) and under fluorescence (M). Arrow indicates a large cell carrying high fluorescence. Scale bars = 10 mm. N. Histogram showing the percentage of germ cells in the unsorted and sorted fractions. Despite of the contamination with other germ cell types (cell clumping before FACS), there is an enrichment of type A undifferentiated spermatogonia (Aund*/Aund) population, which might contain SSC candidates. Undifferentiated type A spermatogonia (Aund* and Aund), differentiating type A spermatogonia (Adiff), type B spermatogonia, spermatocytes (SC), spermatids (ST), spermatozoa (SZ), apoptosis (A) and others (O). Bars represent means expressed as percentage. O. Germ cell transplantation into zebrafish genital pore using a glass capillary needle. doi:10.1371/journal.pone.0012808.g004 found in the recipient seminiferous epithelium (Figure 5E,F). The vasa expression pattern in donor-derived germ cells was the same as observed in vasa::egfp testes immunostained for GFP, or in wild-type testes immunostained for Vasa (Figure S6). The transplantation efficiency was considered not high (30%) as colonization and donor- derived spermatogenesis was observed in about 3 out of 10 recipients. With regard to SSC transplantation into females, cell clusters derived from transplanted SSC were found in recipient ovaries after three weeks of transplantation (Figure 6A, Video S4). Small GFP-positive oocytes at an early stage of oocyte development were also found in recipient females (Figure 6B,C). Surprisingly, these male-derived germ cells progressed into oocyte development, and gave rise to advanced oocytes after one month of transplantation (Figure 6D–F; Video S5). Clusters of GFP-positive cells, possibly clones of oogonia, were also found near male-derived oocytes (Figure 6G,H). The presence of GFP-positive cells in both transplanted males (three weeks after transplantation) and females (one month after transplantation) was also confirmed by PCR analysis (Figure S7). Hormonal characterization and gene expression in busulfan-depleted zebrafish The relatively low efficiency of colonization after transplantation triggered studies on the recipient’s testicular microenvironment prior to transplantation. The plasma levels of 11-KT were ,3 times higher in fish kept at 35uC (with or without busulfan injection) than in non-treated zebrafish kept at 27uC (Figure 7A). The temperature- induced elevation of 11-KT plasma levels was similar to the stimulatory effect of hCG (10 IU/g) or recombinant zebrafish Fsh (100 ng/g) [33]. Also, testicular 11-KT release in primary culture was higher from tissue of males exposed to 35uC and busulfan than from testis tissue of control males kept at 27uC, in particular with regard to basal release (7-fold higher), while the difference in forskolin-stimulated androgen did not reach statistical significance (Figure 7B). Quantifying the expression of selected genes revealed that transcript levels of steroidogenesis and androgen signaling genes (star, ar, cyp17a1) had increased 3-fold in busulfan-depleted testes (Figure 7C). In most cases, exposure to 35uC alone did not change gene expression. There are two interesting exceptions. The transcript levels of amh were significantly down-regulated at 35uC and in busulfan-depleted testes, while igf1b transcript levels were strongly up-regulated. Expression of germ cell-specific genes, such as, piwil1 and sycp3l significantly decreased only on busulfan-treated animals (Figure 7C), while gsdf and insl3 mRNA levels did not change among the groups (Figure 7C). Discussion Using a BrdU pulse-chase approach, we identified the LRC population in zebrafish testis. Within this category of testicular cells, somatic cells were found (e.g. Sertoli and interstitial cells), but type A undifferentiated spermatogonia (Aund* and Aund) were the only germ cells able to retain the label after 18 and 25 days of chase, constituting therefore the putative SSCs. Two distinct patterns of BrdU dilution were found among the two subtypes of type A spermatogonia in zebrafish. The BrdU dilution kinetics for type Aund showed a rapid decline that stabilized at the 18th day after chase, while the percentage of BrdU-positive Aund* spermatogonia remained constant. Applying mathematical mod- eling analysis to hematopoietic stem cells (HSCs) [39], a similar biphasic profile of BrdU dilution was described. The initial, rapid decline was attributed to the faster turnover of activated cells, while the subsequent, decelerated decline resulted from the slow turnover of quiescent cells. This suggested the presence of a heterogeneous population of HSCs which can be activated or deactivated according to the systemic needs [39]. Likewise, the biphasic profile found in zebrafish testes might be attributed to functional differences between the two subtypes of spermatogonia, in which Aund would be the more rapidly dividing, ‘‘active’’ population (related to differentiation and rapid proliferation, as indicated by the more rapid loss of BrdU), and Aund*, the slow- cycling, ‘‘reserve’’ population (related to slow self-renewal proliferation, as indicated by the relatively stable BrdU labeling index). Also in the human testis, two types of single A spermatogonia (pale and dark) are present, and might play distinct roles as ‘‘reserve’’ versus ‘‘active’’ stem cell, respectively [40,41]. In another teleost fish, in female medaka (Oryzias latipes), a BrdU pulse-chase experiment also revealed two distinct populations of oogonial stem cell, one being fast and the other a slow-dividing cell population [42]. Across the vertebrates, it seems that tissues with constant demand for stem cells to proliferate and generate differentiated cells, two populations of stem cells are required; one to rapidly expand and produce differentiated cells, and another which rarely divides and functions as ‘‘reserve’’ in case of insults. According to the ‘‘immortal strand hypothesis’’ [43], stem cells divide asymmetrically, since a hypothetical mechanism would sort chromatids containing the original template DNA strands in one daughter cell, passing therefore the new strands to the other daughter cell [44]. Hence, in a stem cell self-renewal division, the new stem cell would inherit the original templates, the other daughter cell, destined to differentiate, would inherit the new strands. Recently, it has been demonstrated that the ‘‘immortal strand hypothesis’’ is not exclusive of self-renewing cells, but also of divisions in which daughter cells adopt divergent fates [45]. In this case, daughter cells inheriting the original templates would also retain the more immature phenotype, whereas daughter cells inheriting the newer templates acquired a more differentiated phenotype [45]. The BrdU dilution for Aund suggests the existence PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12808