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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.

Introduction: The cryopreservation of genetic resources is of major interest for the security of biodiversity and for the sustainability of agronomic industry. Cryopreservation of somatic cells, which carry the genome of both parents, is one mean to regenerate functional breeders, but this requires the mastering of nuclear transfer. In mammals, somatic cell nuclear transfer consists in injecting the somatic nuclei into an enucleated mature recipient oocyte, in order to obtain clone which bear the genome of interest from the donor animal. However, in fish species, the oocyte structure makes the enucleation step difficult to achieve (presence of the chorion and bulk nutritive reserve), so a non-enucleated oocyte is often used for nuclear transfer. To obtain a real clone and not hybrid embryos, the oocyte chromatin must therefore be spontaneously excluded from the developing embryo. This phenomenon has been described in several studies but the mechanism involved is still unknown. Deciphering this phenomenon requires a high understanding of the first developmental steps that are meiosis resumption and early mitosis. The aim of the study is to understand the interplay between the maternal chromatin and the injected one upon nuclear transfer during these entire critical steps, in order to identify and use means to improve the success rate of nuclear transfer in fish. Methods: Fin somatic cell and mature oocytes were obtained from 2 years old goldfish (Carassius auratus) females. For nuclear transfer, the whole fin cell was injected under the micropyle. The egg activation was induced 30min after cell injection. Somatic and maternal chromatin during meiosis resumption and early mitosis was characterized by immunofluorescence (hoechst and vybrant labelling for chromatin identification and α-tubulin for spindle organization). Results and Discussion: We observed that a high proportion of clone underwent a normal polar body extrusion after activation (60% vs 96 % for the controls). We hypothesize that in most cases, maternal chromatin achieved correctly its meiosis resumption and that the maternal chromatin behavior was not disturbed by nuclear transfer. However, these observations do not augur early mitosis fitness in clone. Indeed, we observed several spindle defects in clone cells: metaphase misalignment, abnormal segregation, and multicentrosomal spindle, associated with DNA fragmentation. Conclusion: Oocyte non-enucleation for SCNT procedure did not drastically alter the meiosis resumption process, but it could be involved in the perturbation of mitosis during clone early development.

National audience; Les milieux aquatiques constituent le réceptacle d’un grand nombre de contaminants, anthropiques et naturels, pouvant être dangereux pour les organismes qui y vivent.

Somatic cell nuclear transfer in rainbow trout (Onrohynchus mykiss): What makes it different from fertilization with a spermatozoa ?. 11. International Symposium on Reproductive Physiology of Fish

International audience

International audience