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Embryonální vývoj a transplantace primordiálních zárodečných buněk u candáta obecného Sander lucioperca
GÜRALP, Hilal
It is the purpose of this thesis to implement primordial germ cell (PGC) transplantation, one of the new biotechnological reproductive methods, and for this to explain the details that we have to know about embryo development and PGC migration in pikeperch. We provide several specific useful methods such as GFP labelling and blastodisc surgery which are required for efficiency assessment of the transplantation technique. The main results of the publications in the thesis could be informative and useful for generation of germline chimera by using pikeperch. We described pikeperch embryo development to first feeding at 15°C in detail and demonstrated effects of temperature on the rate of embryogenesis to determine temperature limits for slowing development with minimum negative effects on growth and survival rate. We also developed a technique to soften the pikeperch chorion by enzyme in order to remove it by forceps for in depth observation. Additional groups of eggs were fertilised and incubated at different temperatures to document embryo developmental stages, developmental rate, and survival. The optimum fertilisation and incubation temperature was 15°C, with the highest fertilisation, survival, and hatching rates. Embryo development was drastically slowed down at 10 °C, with 45% of fertilised embryos surviving to hatching. Development was accelerated at 20 °C, with a 56% survival rate of fertilised embryos. After the series of experiments to characterize the embryo development of pikeperch, it could be a valuable model percid for research in which flexible incubation temperatures is required. We described the important early embryonic events, namely, yolk syncytial layer (YSL) formation and midblastula transition (MBT) during the blastula stage in pikeperch embryos. The chorion was removed as we described in the first study. The YSL was formed after the breakdown of marginal cells during the 512- to 1k-cell stage. Cell division analysis by 4'-6-diaminido-2-phenylindole (DAPI) staining revealed that transition from synchronous to asynchronous division occurred after 1k-cell stage. Our results indicate that MBT starts after this stage. Next, we performed blastodisc isolation assay to find the competent stage for embryonic manipulation. Embryos were manipulated by using a microneedle every hour from the 512-cell to the sphere stage, and then developmental rates were evaluated at the hatching stage. The highest survival rate was obtained when we performed this manipulation at the 1k-cell stage. These results clearly showed that the MBT is the best stage for transplantation of PGCs or any cells in pikeperch. We described PGC migration and performed blastomere transplantation in pikeperch. PGCs were visualised by injection of synthesised green fluorescent protein (GFP) within the 3'untranslated region (UTR) mRNA of nanos3. GFP-positive PGCs appeared in all embryos at approximately 100% epiboly. Time-lapse imaging revealed the PGC migration pattern from their initial appearance to the location at the gonadal ridge. We conducted blastomere transplantation at the blastula stage. Donor embryos were labelled with GFP-nos3 3'UTR mRNA and tetramethylrhodamine dextran to label PGCs and somatic cells, respectively. Twelve blastomere transplantation chimeras were produced, with eight surviving to hatching. All exhibited donor-derived somatic cells in the developing body. The PGCs from donor embryos were observed to migrate towards the gonad region of the host embryos. Our results indicated that blastomere transplantation can be successfully applied in pikeperch, and these findings may be useful to produce germline chimeras in percids.
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