| dc.description.abstracteng | In sexually reproducing species, life begins with the fusion of egg and sperm to form the one-cell embryo, called the zygote. Maternal and paternal DNA form the female and male pronuclei, and must migrate to each other to allow the unison of genetic information and the first mitotic division. This pronuclear migration relies on microtubules and microtubule-related motors in many studied systems. Interestingly, canonical centrosomes are absent in the mouse zygote, and studies suggest that actin is essential for pronuclear migration in this centriole-free system (Chaigne et al., 2016; Maro et al., 1984). However, the precise temporospatial dynamics of pronuclear migration, as well as the role of specific cytoskeletal players, remained unknown. It had been previously shown by a postdoctoral fellow in the lab that live imaging of zygotes that male and female pronuclei showed different migration dynamics, in that male pronuclei formed closer to the cortex and showed a fast migration inwards. In this study, the mechanisms of this fast migration and the function of different actin cytoskeleton components was investigated using a combination of observational and functional experimental approaches. On an observational level, optimisation of live and fixed cell imaging allowed quantification of Rab11a-positive vesicles, which were present in the cytoplasm from fertilisation until shortly after pronuclear formation. Furthermore, live imaging of Rab11a and Spire2 showed that they transiently accumulate in the cortical region adjacent to the forming male pronucleus. Perturbation of Rab11a using a dominant-negative variant, and of Spire2-Formin-2-interaction with injected FH2, inhibited the fast migration of the male pronucleus, while overexpression of Spire2 elongated the fast migration phase. Interestingly, when the maternal DNA was induced to form a pronucleus close to the cortex, fast migration and transient accumulations of Rab11a and Spire2 were also observed by the forming female pronucleus. Inhibition of actomyosin contractions of the fertilisation cone with ML-7 did not affect fast migration of pronuclei. Lastly, addition of cytochalasin D after pronuclear formation inhibited any further migration towards the centre of the zygote, indicating F-actin to be important for later pronuclear migration through other mechanisms than Rab11a, Spire2 and Formin-2. Overall, this study was able to implicate a functional role of Rab11a and related actin nucleators in early pronuclear migration in mouse zygotes, contributing to our understanding of pronuclear migration mechanisms in the absence of canonical centrosomes. | de |