Optimization of somatic cell injection in the perspective of nuclear transfer in goldfish

<p>Abstract</p> <p>Background</p> <p>Nuclear transfer has the potential to become one strategy for fish genetic resources management, by allowing fish reconstruction from cryopreserved somatic cells. Survival rates after nuclear transfer are still low however. The part played by unsuitable handling conditions is often questioned, but the different steps in the procedure are difficult to address separately. In this work led on goldfish (<it>Carassius auratus</it>), the step of somatic cells injection was explored. Non-enucleated metaphase II oocytes were used as a template to explore the toxicity of the injection medium, to estimate the best location where the cell should be injected, and to assess the delay necessary between cell injection and oocyte activation.</p> <p>Results</p> <p>Trout coelomic fluid was the most suitable medium to maintain freshly spawned oocytes at the metaphase II stage during oocyte manipulation. Oocytes were then injected with several media to test their toxicity on embryo development after fertilization. Trout coelomic fluid was the least toxic medium after injection, and the smallest injected volume (10 pL) allowed the same hatching rates as the non injected controls (84.8% ± 23). In somatic cell transfer experiments using non enucleated metaphase II oocytes as recipient, cell plasma membrane was ruptured within one minute after injection. Cell injection at the top of the animal pole in the oocyte allowed higher development rates than cell injection deeper within the oocyte (respectively 59% and 23% at mid-blastula stage). Embryo development rates were also higher when oocyte activation was delayed for 30 min after cell injection than when activation was induced without delay (respectively 72% and 48% at mid-blastula stage).</p> <p>Conclusions</p> <p>The best ability of goldfish oocytes to sustain embryo development was obtained when the carrier medium was trout coelomic fluid, when the cell was injected close to the animal pole, and when oocyte activation was induced 30 min after somatic cell injection. Although the experiments were not designed to produce characterized clones, application of these parameters to somatic cell nuclear transfer experiments in enucleated metaphase II oocytes is expected to improve the quality of the reconstructed embryos.</p

Epigenetic stability in goldfish sperm upon hormonal induction and over the breeding season

Epigenetic stability in goldfish sperm upon hormonal induction and over the breeding season. 6. International Workshop on the Biology of Fish Gamete

Comparative analysis of global DNA methylation in spermatozoa from fish, molluscs, bird and mammals: effect of cryopreservation

Comparative analysis of global DNA methylation in spermatozoa from fish, molluscs, bird and mammals: effect of cryopreservation. 7. International Workshop on the Biology of Fish Gamete

Défauts de reprogrammation épigénétique des embryons issus du transfert nucléaire somatique chez les poissons

Défauts de reprogrammation épigénétique des embryons issus du transfert nucléaire somatique chez les poissons. Journées d'Animation Scientifique du département Phase (JAS Phase 2018

Epigenetic reprogramming after nuclear transfer in fish

Epigenetic reprogramming after nuclear transfer in fish. 6. International Workshop on the Biology of Fish Gamete

Compréhension des perturbations induites par le transfert nucléaire chez le carassin: Caractérisation de la reprise de la méiose et des premières mitoses

Compréhension des perturbations induites par le transfert nucléaire chez le carassin. Caractérisation de la reprise de la méiose et des premières mitoses. Journées d'Animation Scientifique du département Phase (JAS Phase 2018

Epigenetic and cellular reprogramming after nuclear transfer in fish : characterization of clone defects in relation to the donor cell origin

Epigenetic and cellular reprogramming after nuclear transfer in fish : characterization of clone defects in relation to the donor cell origin. 2. Journées scientifiques GDR 3606 REPRO : ReproSciences 201

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish

Meiosis resumption and early mitosis pattern after somatic cell nuclear transfer in goldfish. 6. International Workshop on the Biology of Fish Gamete