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Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.

Nuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid offspring which bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fish, because enucleation is difficult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explores which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fish oocyte enucleation. The fate of the somatic and maternal DNA during oocyte activation and first cell cycle was studied using DNA labeling and immunofluorescence in goldfish clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally affected after oocyte activation. During the first cell cycle, only 40% of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80% of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed.

Nuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid offspring who bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fish, because enucleation is difficult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explore which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fish oocyte enucleation. The fate of the somatic and maternal DNA during meiosis resumption and first cell cycle was studied using DNA labeling and immunofluorescence in goldfish clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally affected after meiosis resumption. During the first cell cycle, only 40 % of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80 % of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed.

Le transfert nucléaire chez les poissons. Une technologie de régénération de poissons précieux. Un modèle de développement perturbé. Journées d'Animation Scientifique du département Phase (JAS Phase 2018)

Import nucléaire de facteurs ovocytaires dans les cellules somatiques en culture : une avancée prometteuse pour leur reprogrammation en vue du transfert nucléaire. Journées d'Animation Scientifique du département Phase (JAS Phase 2018)

Le transfert nucléaire chez les poissons. Une technologie de régénération de poissons précieux. Un modèle de développement perturbé. Journées d'Animation Scientifique du département Phase (JAS Phase 2018)

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