During oocyte growth in the ovary, the nucleolus is mainly responsible for ribosome biogenesis. However, in the fully-grown oocyte, all transcription ceases, including ribosomal RNA synthesis, and the nucleolus adopts a specific monotonous fibrillar morphology without chromatin. The function of this inactive nucleolus in oocytes and embryos is still unknown. We previously reported that the embryo lacking an inactive nucleolus failed to develop past the first few cleavages, indicating the requirement of a nucleolus for preimplantation development. Here, we reinjected the nucleolus into oocytes and zygotes without nucleoli at various time points to examine the timing of the nucleolus requirement during meiosis and early embryonic development. When we put the nucleolus back into oocytes lacking a nucleolus at the germinal vesicle (GV) stage and at second metaphase (MII), these oocytes were fertilized, formed pronuclei with nucleoli and developed to full term. When the nucleolus was reinjected at the pronucleus (PN) stage, most of the reconstructed zygotes cleaved and formed nuclei with nucleoli at the 2-cell stage, but the rate of blastocyst formation and the numbers of surviving pups were profoundly reduced. Moreover, the zygotes without nucleoli showed a disorder of higher chromatin organization not only in the female pronucleus but also, interestingly, in the male pronucleus. Thus, the critical time point when the nucleolus is required for progression of early embryonic development appears to be at the point of the early step of pronucleus organization.