POU5F1 (OCT4) is a crucial transcription factor for induction and maintenance of cellular pluripotency, as well as survival of germ cells in mammals. However, the homologues of POU5F1 in teleost fish, including zebrafish and medaka, now named Pou5f3, exhibit considerable differences in expression pattern and pluripotency-maintaining activity. To what extent the POU5F1 homologues are conserved in vertebrates has been unclear. In this study, we report that the POU5F1 homologue from the Nile tilapia (Oreochromis niloticus), OnPou5f3, displays an expression pattern and biological activity somewhat different from those in zebrafish or medaka. The expression of Onpou5f3 at both mRNA and protein levels was abundant in early development embryos until blastula stages, barely detectable as proceeding, and then displayed a transiently strong expression domain in the brain region during neurula stages similar to zebrafish but not medaka. Afterward, OnPou5f3 appeared as germline-restricted (including primordial germ cells and female and male gonad germ cells) expression just like medaka. Notably, OnPou5f3 depletion through morpholino oligos caused blastula blockage or lethality and failure of survival and proliferation of blastula cell-derived cells. These findings indicate that equivalent POU5F1-like expression and activity of Pou5f3 might be conserved accompanying with species-specific expression pattern during evolution. Our study provides insight into the evolutionary conservation of the POU5F1 homologues across vertebrates.

Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations in vitro and functional analysis of target genes in vivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world-wide commercial farmed fish. These cell lines, designated as TES1-3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder-free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26-labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential in vitro and in vivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non-protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES-based biotechnology in commercial fish.

The origin and evolution of molecular mechanisms underlying the self-renewal and differentiation of spermatogonial stem cells (SSCs) are fundamental questions in stem cell biology as well as reproduction medicine. In mammals, glial cell line-derived neurotrophic factor (GDNF) is crucial for SSC self-renewal and maintenance. However, in nonmammals, the role of Gdnf in SSCs still remains unknown. In this study, we report that the two GDNF homologs from medaka fish (Oryzias latipes), namely OlGdnfa and OlGdnfb, can promote proliferation activity and retain the spermatogonial property of SG3, a spermatogonial cell line derived from adult medaka showing the intrinsic property of SSCs by self-renewal and differentiation potential during 2 years of culture. Cloning and sequencing led to the identification of two cDNA sequences as Olgdnfa and Olgdnfb, which are 780-nt and 744-nt in length for 253 and 245 amino acid residues, respectively. Both are homologs of mammalian GDNF and share over 45% identity with the other known vertebrate homologs. Importantly, in a well-defined condition, the recombinant proteins, OlGdnfa and OlGdnfb, can significantly promote the proliferative activity of SG3 cells and retain the spermatogonial gene expression pattern and alkaline phosphatase activity. Meanwhile, both of the two recombinant proteins can upregulate the mRNA expression level of bcl6b, one of the prominent GDNF-regulated genes involved in SSC self-renewal and maintenance in mammals. Taken together, our findings suggest that just like the mammalian counterpart, the nonmammalian Gdnfs might mediate the self-renewal and maintenance of SSCs; moreover, Bcl6b might be a conserved regulator in SSC self-renewal across vertebrate taxa. This study extends our knowledge of GDNF functions in SSC biology during evolution.

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