Tolerance to paternal genotoxic damage promotes survival during embryo development in zebrafish (Danio rerio).

Spermatozoa carry DNA damage that must be repaired by the oocyte machinery upon fertilization. Different strategies could be adopted by different vertebrates to face the paternal genotoxic damage. Mammals have strong sperm selection mechanisms and activate a zygotic DNA damage response (DDR) (including cell cycle arrest, DNA repair and alternative apoptosis) in order to guarantee the genomic conformity of the reduced progeny. However, external fertilizers, with different reproductive strategies, seem to proceed distinctively. Previous results from our group showed a downregulation of apoptotic activity in trout embryos with a defective DNA repairing ability, suggesting that mechanisms of tolerance to damaged DNA could be activated in fish to maintain cell survival and to progress with development. In this work, zebrafish embryos were obtained from control or UV-irradiated sperm (carrying more than 10% of fragmented DNA but still preserving fertilization ability). DNA repair (γH2AX and 53BP1 foci), apoptotic activity, expression of genes related to DDR and malformation rates were analyzed throughout development. Results showed in the progeny from damaged sperm, an enhanced repairing activity at the mid-blastula transition stage that returned to its basal level at later stages, rendering at hatching a very high rate of multimalformed larvae. The study of transcriptional and post-translational activity of tp53 (ZDF-GENE-990415-270) revealed the activation of an intense DDR in those progenies. However, the downstream pro-apoptotic factor noxa (ZDF-GENE-070119-3) showed a significant downregulation, whereas the anti-apoptotic gene bcl2 (ZDF-GENE-051015-1) was upregulated, triggering a repressive apoptotic scenario in spite of a clear genomic instability. This repression can be explained by the observed upregulation of p53 isoform Δ113p53, which is known to inhibit bcl2 transcription. Our results showed that tp53 is involved in DNA damage tolerance (DDT) pathways, allowing the embryo survival regardless of the paternal DNA damage. DDT could be an evolutionary mechanism in fish: tolerance to unrepaired sperm DNA could introduce new mutations, some of them potentially advantageous to face a changing environment.

In vitro generation of zebrafish PGC-like cells.

The possibility of generating primordial germ cells (PGCs) in vitro from noncommitted embryonic cells represents an extremely useful tool in current research. Primordial germ cell in vitro differentiation has been successfully reported in mammals. However, contrary to fish, PGC specification in mammals is an inductive mechanism. This study is the first to date to describe a rapid method for PGC in vitro differentiation in teleosts. Primordial germ cell-like cells were characterized by several lines of evidence, including gene expression, cell complexity, size, and image analysis for the quantification of fluorescence under vasa promoter. Moreover, differentiated cells were able to colonize the genital ridge after transplantation. Differentiation treatments increased the number of PGCs in culture, causing differentiation of cells rather than inducing their proliferation. These results open up the possibility of differentiating genetically modified embryonic cells to PGC-like cells to ensure their transmission to the progeny and could be crucial for an in-depth understanding of germline differentiation in teleosts.

Germplasm cryobanking in zebrafish and other aquarium model species.

During the last few years zebrafish and other aquarium fish have become more important model system species. In zebrafish valuable biotechnological lines are constantly emerging, and the creation of cryobanks of viable fish sperm, oocytes, and embryos from those lines is a constant and important demand. Cryopreservation is an essential tool for storing specific genetic material and would significantly help in the conservation of such lines, facilitating daily procedures in labs and companies working with this species. In this review, all advances achieved in this field are presented. Protocols for sperm cryopreservation are described, and the new directions of approaches in embryo and oocyte cryopreservation and also in blastomeres, and primordial germ cell cryopreservation are discussed.