SSC Niche and SSC In Zebrafish Perinucleolar oocyte from vasa::egfp ovaries immunostained for gfp. Compare similar vasa expression pattern between donor-derived oocyte and perinucleolar oocyte from vasa::egfp ovaries. Scale bar = 25 mm. doi:10.1371/journal.pone.0012808.g006 SSC activity still have to be clarified, the available evidence in fish strongly suggests that, different from mammals, androgens stimulate spermatogonial differentiation [2]. On the other hand, estrogens stimulated SSC self-renewal in Japanese eel [52], so that Leydig cells may be relevant as a source for aromatizeable androgens, thereby contributing directly and/or indirectly to the zebrafish SSC niche. Moreover, Leydig cell paracrine signaling might influence SSC behavior, since colony stimulating factor 1 (Csf1) produced by Leydig and myoid cells is an extrinsic stimulator of SSC self-renewal in mice [10]. Endothelial cells support the expansion of normal and malignant stem cells not only by delivering oxygen and nutrients, but also by the paracrine release of endothelial cell growth factors and trophogens, which are referred to as ‘‘angiocrine factors’’ [53]. Thus, endothelial cells have been pointed out as an important element of the stem cell niche in several systems, including the rodent SSC niche [8,54]. In the zebrafish model, the distribution of SSC candidates (Aund* and Aund) close to blood vessels might indicate an involvement of endothelial cells in regulating SSC function. While there is no information on angiocrine signaling in the zebrafish testis, it has been shown that platelet-derived endothelial cell growth factor (PD-ECGF) induced SSC self- renewal in Japanese eel [55,56]. The functional capacity of the identified SSC candidates in zebrafish was investigated by transplantation assays. In this assay, SSC candidates were transplanted into zebrafish in which endogenous spermatogenesis had been depleted by busulfan, similar to the technique developed for rodents and others species [14–17,19]. It seems that similar to tilapia [16], zebrafish spermatogenesis is more sensitive to busulfan at elevated temperatures (35uC), resulting in 88% of spermatogenic tubules showing Sertoli cells only after 10 days of treatment. Interestingly, spermatogenesis recovered quickly after having passed the nadir of busulfan-induced depletion. It has been shown previously that proliferation of surviving, undifferentiated spermatogonia is greatly enhanced when the number of differentiating spermato- gonia is reduced following busulfan exposure [57,58]. In addition, differentiated spermatogonia can act as ‘‘potential stem cells’’, shifting their nature of transit-amplifying cells to self-renewal in order to rapidly recover spermatogenesis in depleted testes [59]. Analyzing the composition of type A spermatogonia in normal versus busulfan-depleted testes in zebrafish, we have demonstrated a relative increase in the population of Aund, and a relative decrease in the population of Aund* spermatogonia in busulfan- depleted testes. Then, considering Aund as the ‘‘active’’ stem cell, our results suggest that the spermatogenic recovery after busulfan treatment might be associated with a preponderance of the active stem cell population Aund. The treatment with busulfan induced an increase in androgen plasma levels as well as an increase in testicular androgen release. This might reflect a suppression of the negative feedback on gonadotropin release. In rat, the negative feedback on FSH release exerted by germ cells via Sertoli cell-derived inhibin is transiently eliminated after busulfan treatment [60], explaining the increase in circulating FSH levels between 6 and 10 weeks after busulfan injection [61,62]. In mammals, androgen plasma levels were not elevated in busulfan-treated animals [60–62]. However, in fish, also Leydig cells express the receptor for Fsh, which is a strongly steroidogenic gonadotropin in fish (e.g. [33,63,64]). Hence, we attribute the increased androgen levels after busulfan treatment to higher Fsh plasma levels. Consistent with the elevated androgen production, busulfan treatment increased the mRNA levels of transcripts encoding proteins involved in steroidogenesis, such as star and cyp17a1. The down-regulation of amh mRNA expression in busulfan-depleted testes also seems coherent with higher androgen levels, since androgens down-regulated amh expression in juvenile Japanese eel testis and stimulated spermatogonial differentiation towards meiosis [65]. Moreover, since Sertoli cells also proliferate in the adult fish testis, in particular those associated with expanding spermatogonial cysts [23,24], the busulfan treatment may have depleted the proliferating Sertoli cell population as well, thereby contributing to lower levels of amh mRNA, and possibly also to the arrest in spermatogenesis. With regard to the expression of igf gene family members, a strong up-regulation in particular of igf1b mRNA levels has been observed. This is interesting in the context of ongoing work showing that recombinant zebrafish Fsh increases igf1b levels in adult testis tissue culture (unpublished data). Spermatogonial proliferation was stimulated in newt testis in primary tissue culture by exposure to Igf1 [66]. Since Sertoli cells also express a factor (similar to platelet-derived endothelial cell growth factor) that stimulates SSC self-renewal in Japanese eel [55], busulfan-induced changes in the number and/or physiolog- ical state of Sertoli cells might have resulted in a more generalized imbalance of factors released by Sertoli cells and relevant for SSC self-renewal and differentiation. Taken together, our data suggest that busulfan treatment might create a microenvironment in zebrafish testes where the balance of somatic (mainly Sertoli) cell- derived factors has shifted to a reduced level of factors supporting self-renewal and/or an elevated level of factors stimulating differentiation. While this might facilitate the fast recovery after the severe loss of germ cells induced by busulfan, it may also influence the behavior of transplanted cells. Thus, transplanted SSCs may differentiate rather than self-renew, possibly limiting the efficiency of donor-colonization (30%) in the recipient seminifer- ous tubules; however, a relatively low efficiency of donor-derived colonization is not exceptional for zebrafish and has been observed in other vertebrates as well [67]. Future studies will aim at weakening the pro-differentiation or strengthening the self-renewal environment, for example by an estrogen treatment [51] of the recipients. Despite of the limited efficiency, we have taken important steps towards the development and standardization of SSC transplan- tation assay to confirm the ‘‘stemness’’ of SSC candidates in zebrafish. Transplantation of a FACS-enriched fraction of Aund* and Aund into busulfan-depleted testes showed that stem cell candidates were able to colonize, self-renew, and to differentiate in the recipient seminiferous tubules. This confirms the presence of stem cells in the transplanted pool of type A undifferentiated spermatogonia. Moreover, spermatogonial colonization started from regions within the recipient’s seminiferous tubules which are adjacent to the interstitial compartment, the putative SSC niche in zebrafish testes. This might indicate that the SSC niche creates a cellular and molecular environment which is optimal for colonization and development of transplanted SSCs. Since zebrafish have been used as a model for understanding the mechanisms of testicular germ cell tumors [68], and most of these tumors have an unknown etiology, a standard experimental design including reciprocal transplantation of germ cells from affected donors to wild-type testes and vice versa could be applied to evaluate whether the defect is on Sertoli cells or intrinsic to the PLoS ONE | www.plosone.org 12 September 2010 | Volume 5 | Issue 9 | e12808