Spermatogonial stem cell quest: nanos2, marker of a sub-population of undifferentiated A spermatogonia in trout testis

Supplementary DataContinuous or cyclic production of spermatozoa throughout life in adult male vertebrate depends on a sub-population of undifferentiated germ cells acting as spermatogonial stem cells (SSCs). What makes these cells self-renew or differentiate is barely understood, in particular in non-mammalian species including fish. In the highly seasonal rainbow trout, at the end of the annual spermatogenetic cycle, tubules of the spawning testis contain only spermatozoa with the exception of scarce undifferentiated spermatogonia that remain on the tubular wall, which will support the next round of spermatogenesis. Taking advantage of this model, we identified putative SSCs in fish testis using morphological, molecular and functional approaches. In all stages, large spermatogonia with ultrastructural characteristics of germinal stem cells were found, isolated or in doublet. Trout homologs of SSC and/or immature progenitor markers in mammals, nanos2 and nanos3, pou2, plzf and piwil2, were preferentially expressed in the prepubertal testis and in the undifferentiated A spermatogonia populations purified by centrifugal elutriation. This expression profile strongly suggests that these genes are functionally conserved between fish and mammals. Moreover, transplantation into embryonic recipients of the undifferentiated spermatogonial cells demonstrated their high 'stemness' efficiency in terms of migration into gonads and ability to give functional gametes. Interestingly, we show that nanos2 expression was restricted to a sub-population of undifferentiated spermatogonia (less than 20%) present as isolated cells or in doublet in the juvenile and in the maturing trout testis. In contrast, nanos2 transcript was detected in all the undifferentiated spermatogonia remaining in the spawning testis. Plzf was also immuno-detected in A-Spg from spawning testis, which reinforces the idea that these cells are stem cells. From those results, we hypothesize that the subset of undifferentiated A spermatogonia expressing nanos2 transcript are putative SSC in trout