Pronuclear transfer has been successfully used in human-assisted reproduction to suppress the adverse effects of a defective oocyte cytoplasm or to bypass an idiopathic developmental arrest. However, the effects of the initial parental genome remodelling in a defective cytoplasm on the subsequent development after pronucleus transfer have not been systematically studied. By performing pronuclear transfer in pre-replication and post-replication mouse embryos, we show that the timing of the procedure plays a critical role. Although apparently morphologically normal blastocysts were obtained in both pre- and post-replication pronuclear transfer groups, post-replication pronuclear transfer led to a decrease in developmental competence and profound changes in embryonic gene expression. By inhibiting the replication in the abnormal cytoplasm before pronuclear transfer into a healthy cytoplasm, the developmental potential of embryos could be largely restored. This shows that the conditions under which the first embryonic replication occurs strongly influence developmental potential. Although pronuclear transfer is the method of choice for mitigating the impact of a faulty oocyte cytoplasm on early development, our results show that the timing of this intervention should be restricted to the pre-replication phase.

• Keywords
• DNA damage, developmental rate, embryo, pronuclear transfer, replication,
• MeSH
• Blastocyst * metabolism   cytology   MeSH
• Cell Nucleus metabolism   MeSH
• Time Factors MeSH
• Cytoplasm metabolism   MeSH
• Embryo, Mammalian MeSH
• Embryonic Development * MeSH
• Mice MeSH
• Oocytes metabolism   cytology   MeSH
• Nuclear Transfer Techniques * MeSH
• Gene Expression Regulation, Developmental MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

It is now approximately 25 years since the sheep Dolly, the first cloned mammal where the somatic cell nucleus from an adult donor was used for transfer, was born. So far, somatic cell nucleus transfer, where G1-phase nuclei are transferred into cytoplasts obtained by enucleation of mature metaphase II (MII) oocytes followed by the activation of the reconstructed cells, is the most efficient approach to reprogram/remodel the differentiated nucleus. In general, in an enucleated oocyte (cytoplast), the nuclear envelope (NE, membrane) of an injected somatic cell nucleus breaks down and chromosomes condense. This condensation phase is followed, after subsequent activation, by chromatin decondensation and formation of a pseudo-pronucleus (i) whose morphology should resemble the natural postfertilization pronuclei (PNs). Thus, the volume of the transferred nuclei increases considerably by incorporating the content released from the germinal vesicles (GVs). In parallel, the transferred nucleus genes must be reset and function similarly as the relevant genes in normal embryo reprogramming. This, among others, covers the relevant epigenetic modifications and the appropriate organization of chromatin in pseudo-pronuclei. While reprogramming in SCNT is often discussed, the remodeling of transferred nuclei is much less studied, particularly in the context of the developmental potential of SCNT embryos. It is now evident that correct reprogramming mirrors appropriate remodeling. At the same time, it is widely accepted that the process of rebuilding the nucleus following SCNT is instrumental to the overall success of this procedure. Thus, in our contribution, we will mostly focus on the remodeling of transferred nuclei. In particular, we discuss the oocyte organelles that are essential for the development of SCNT embryos.

• Keywords
• Nucleus, Remodeling, Reprogramming, Selective enucleation,
• MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin metabolism   MeSH
• Oocytes MeSH
• Sheep genetics   MeSH
• Mammals genetics   MeSH
• Nuclear Transfer Techniques * veterinary   MeSH
• Animals MeSH
• Zygote * metabolism   MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin MeSH

Differentiated nuclei can be reprogrammed/remodelled to totipotency after their transfer to enucleated metaphase II (MII) oocytes. The process of reprogramming/remodelling is, however, only partially characterized. It has been shown that the oocyte nucleus (germinal vesicle - GV) components are essential for a successful remodelling of the transferred nucleus by providing the materials for pseudo-nucleus formation. However, the nucleus is a complex structure and exactly what nuclear components are required for a successful nucleus remodelling and reprogramming is unknown. Till date, the only nuclear sub-structure experimentally demonstrated to be essential is the oocyte nucleolus (nucleolus-like body, NLB). In this study, we investigated what other GV components might be necessary for the formation of normal-sized pseudo-pronuclei (PNs). Our results showed that the removal of the GV nuclear envelope with attached chromatin and chromatin-bound factors does not substantially influence the size of the remodelled nuclei in reconstructed cells and that their nuclear envelopes seem to have normal parameters. Rather than the insoluble nuclear lamina, the GV content, which is dissolved in the cytoplasm with the onset of oocyte maturation, influences the characteristics and size of transferred nuclei.

• Keywords
• Nucleus transfer, Oocyte, Selective enucleation,
• MeSH
• Cell Nucleolus metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin metabolism   MeSH
• Cytoplasm metabolism   MeSH
• Nuclear Lamina metabolism   MeSH
• Nuclear Envelope metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mice MeSH
• Oocytes cytology   metabolism   MeSH
• Oogenesis MeSH
• Ovarian Follicle metabolism   MeSH
• Cellular Reprogramming * MeSH
• Nuclear Transfer Techniques * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin MeSH
• RNA, Messenger MeSH