Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: A potential marker of nuclear reprogramming

Nuclei of differentiated cells can acquire totipotency following transfer i nto the cytoplasm of oocytes. While the molecular basis of this nuclear rep rogramming remains unknown, the developmental potential of nuclear-transfer embryos is influenced by the cell-cycle stage of both donor and recipient. As somatic H1 becomes immunologically undetectable on bovine embryonic nuc lei following transfer into ooplasm and reappears during development of the reconstructed embryo, suggesting that it may act as a marker of nuclear re programming, we investigated the link between cell-cycle state and depletio n of immunoreactive H1 following nuclear transplantation. Blastomere nuclei at M-, G1-, or G2-phase were introduced into ooplasts at metaphase II, tel ophase II, or interphase, and the reconstructed embryos were processed for immunofluorescent detection of somatic histone H1. Immunoreactivity was los t more quickly from donor nuclei at metaphase than at G1 or G2. Regardless of the stage of the donor nucleus, immunoreactivity was lost most rapidly w hen the recipient cytoplast was at metaphase and most slowly when the recip ient was at interphase. When the recipient oocyte was not enucleated, howev er, immunoreactive H1 remained in the donor nucleus. The phosphorylation in hibitors 6-DMAP, roscovitine, and H89 inhibited the depletion of immunoreac tive H1 from G2, but not G1, donor nuclei. In addition, immunoreactive H1 w as depleted from mouse blastomere nuclei following transfer into bovine ooc ytes. Finally, expression of the developmentally regulated gene, eIF-1A, bu t not of Gapdh, was extinguished in metaphase recipients but not in interph ase recipients. These results indicate that evolutionarily conserved cell-c ycle-regulated activities, nuclear elements, and phosphorylation-linked eve nts participate in the depletion of immunoreactive histone H1 from bias to mere nuclei transferred in oocyte cytoplasm and that this is linked to chan ges in gene expression in the transferred nucleus. (C) 2001 Academic Press.