Somatic cells derived from haploid larvae are feasible as donors for nuclear transplant in zebrafish. Preliminary results

SummarySomatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish.

Definition of three somatic adult cell nuclear transplant methods in zebrafish (Danio rerio): before, during and after egg activation by sperm fertilization

SummaryZebrafish somatic nuclear transplant has only been attempted using preactivated eggs. In this work, three methods to carry out the nuclear transplant using adult cells before, during and after the egg activation/fertilization were developed in zebrafish with the aim to be used in reprogramming studies. The donor nucleus from somatic adult cells was inserted: (method A) in the central region of the egg and subsequently fertilized; (method B) in the incipient animal pole at the same time that the egg was fertilized; and (method C) in the completely defined animal pole after fertilization. Larval and adult specimens were obtained using the three methods. Technical aspects related to temperature conditions, media required, egg activation/fertilization, post-ovulatory time of the transplant, egg aging, place of the donor nucleus injection in each methodology are presented. In conclusion, the technical approach developed in this work can be used in reprogramming studies.

96 TRICHOSTATIN A IMPROVED THE QUALITY OF RABBIT NUCLEAR TRANSFER EMBRYOS

Nuclear reprogramming is dependent upon a number of factors, including chromatin organization and modification. Trychostatin A (TSA), a histone deacetylase inhibitor, was used to increase histone acetylation and to improve reprogrammability in both cattle and mice. The objective of the study was to determine whether TSA could improve the pre-implantational development potential of rabbit nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts of superovulated donors treated with the regime of FSH and hCG. Cumulus cells were then denuded from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in 10% FBS M199 and confirmed under fluorescence microscopy. Cumulus cells were prepared as nuclear donors for NT; a donor cell with the diameter approximately 15–19 µm was transferred into the perivitelline space of an enucleated oocyte, and subsequently fused with the oocyte recipient by application of 3 direct current pulses at 3.2 kV cm−1 for a duration of 20 µs/pulse. Fused embryos were activated by the same electrical stimulation regime described above, and subsequently cultured in M199 + 10% FBS containing 2.0 mM 6-dimethylaminopurine (DMAP) and 5 µg mL−1 cycloheximide for 1 h. Rabbit NT embryos were cultured in 5 nM TSA-2.5% FBS-B2 medium for 10 h before being transferred into regular medium (FBS-B2). The TSA-treated embryos (5 nM vs. 0 nM) were cultured in 400 µL FBS-B2 medium for 5 days in 5% CO2 in a humidified atmosphere at 38.5°C (initiation of activation = Day 0). NT embryo development to cleaved (2 to 4 cell), morula, and blastocyst stages was evaluated on Day 2, Day 3, and Day 5, respectively. The selected NT blastocysts were counted for cell numbers following Hoechst 33342 epifluorescenin staining. The results (Table 1) showed that there was no difference on pre-implantational development of cloned embryos between TSA-added and control groups (P > 0.05). However, a significantly higher cell number per NT blastocyst was found in the TSA-added group (357 vs. 113; P < 0.05). This indicated that the blastocyst quality in NT embryos was improved with the addition of TSA by increasing histone acetylation activity. The developmental potential of TSA-treated NT embryos to term is under investigation. Table 1.Effects of TSA on the pre-implantational development of cloned rabbit embryos This work was supported by NIH/NCRR-SBIR grant: 1R43RR020261-01.