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Optimization of the nuclear transfer procedure in fish

• Our research is developed on the goldfish as a model fish because of the large availability of oocytes bearing optimal development ability. Freshly spawned oocytes are stored in coelomic fluid, in order to prevent egg activation and meiosis resumption. A thawed fin cell obtained from culture of fin explant is injected by the micropyle into the oocyte, by mean of a glass microcapillary. Oocyte enucleation is a complicated matter in fish, and most of our experiments are performed without preliminary enucleation.
• We could observe that most media usually injected with the somatic cell (culture medium, saline) could be toxic for embryo development, and that trout coelomic fluid is the best vector. The cell should be injected at the animal pole close to the egg plasma membrane, where the somatic cell nucleus is rapidly released into the egg cytoplasm. A recovery period prior to egg activation is necessary to obtain significant development rates. We believe that this incubation in metaphase II oocytes is favorable to chromatin reprogramming.
• We demonstrated that half the produced embryos are diploids, and half these diploid embryos bear only the genetic material of the injected cell. This show that a complete erasure of the maternal chromatin occurred in some reconstructed embryos. We observed that the quality of the first mitosis can predict the embryo development outcome, although most defects will be expressed only after embryonic genome activation.

Reprogramming of the donor cell to improve nuclear transfer outcome

• In the normal process after fertilization, sperm and oocyte chromatin undergoes the structural and biochemical changes which will allow embryo development. Ultimately, such reprogramming is responsible for the proper expression of the genes involved in early development. When the injected chromatin comes from a somatic cell, its profile is not adapted to this task. Therefore, the quality of the somatic chromatin reprogramming is a key factor in the success of embryo development after nuclear transfer.
• We study how this reprogramming can be improved by modification of the donor cell features. Culture of fin cells has been optimized so that fin cells can be treated by reprogramming agents prior to nuclear transfer. Our latest strategy is to permeabilize the donor cell and to incubate it with xenopus oocyte extracts at metaphase II stage, in order to allow some embryonic-like pre-reprogramming of the somatic nucleus.

Reprogramming of the embryo after nuclear transfer in fish

• The epigenetic profile of the somatic cell is very different from that of the spermatozoa or of the early embryo. We are studying to what extent the recipient oocyte is able to reprogram the epigenetic profile of this somatic cell. We are studying the DNA methylation pattern of some marker genes that are differentially methylated between somatic cells and spermatozoa or embryonic cell, and we follow this DNA methylation profile in the embryos sired by nuclear transfer.
• Resuming meiosis and triggering embryo development are functional in the oocyte after fertilization. In the case of nuclear transfer, we are wondering to what extent these steps are set up normally. We are studying meiosis resumption and first mitosis after nuclear transfer, in order to understand the extent of the damages induced by nuclear transfer at these early developmental steps.