18 Characterisation of early embryonic cellular defects after somatic cell nuclear transfer in fish

Abstract

Cryopreservation of genetic resources is of major interest for the security of biodiversity and the sustainability of the agronomic industry. Cryopreservation of somatic cells is one means of preserving the genome of both parents, but regeneration of functional breeders requires the mastering of somatic cell NT (SCNT). In mammals, SCNT consists of injecting the somatic nuclei into enucleated recipient oocytes to obtain clones that bear the genome of the donor. Cloning in fish presents some advantages over cloning in mammals: oocytes are produced by the hundreds, and development is external. Moreover, embryonic genome activation takes place after 10 mitoses only. These features are favourable to a better understanding of cellular reprogramming of the somatic cell. Nonenucleated oocytes are often used for SCNT, which allows the production of 2n clones bearing only the donor genome when the maternal chromatin is spontaneously removed by an unknown mechanism (25% of cases). This ability can help us understand the fate of the maternal chromatin and the role of its surrounding factors on the cellular behaviour of the somatic cell during the first developmental steps of embryonic development: meiosis resumption (MR) and mitosis. In this study, fin somatic cells and mature oocytes were obtained from a 2-year-old goldfish (Carassius auratus). For SCNT, the whole fin cell was injected into the sperm entry site, and the oocyte was activated by water contact after 30 min to begin the development. Somatic and maternal chromatin behaviour during MR and early mitosis were characterised by immunofluorescence (Hoechst/Vybrant labelling for chromatin identification and α-tubulin for spindle organisation). Variability in chromatin condensation was observed after the somatic cell injection before the activation. Clone analysis (n = 16) revealed that some of them presented a condensed somatic nuclei (71%) and others presented a decondensed nuclei (29%). After activation, most clones underwent normal polar body extrusion of the maternal chromatin (66%, n = 69/95 clones v. 96%, n = 96/100 controls oocytes) without extrusion of a somatic polar body. Afterward, clones that presented a first symmetric division (n = 14/35) were analysed at the 2-cells stage and compared with fertilized embryos (n = 7). The maternal chromatin was observed as fragmented on the cleavage furrow (79%), where it cannot contribute to the development. Spindle defects were observed in 50% of clone cells (v. 0% in controls), such as multicentrosomal spindles, chromosome misalignment, abnormal segregation, and DNA fragmentation. To conclude, those clone (2n or 3n) defects are probably due to the chromatin condensation observed before activation. The fish oocyte volume did not allow the MR of the condensed somatic chromatin, and that may induce an abnormal anaphase and the following clone developmental defects.