Chicken blastodermal cells (stage X) were fused to mouse enucleated oocytes with either no or high maturation promoting factor (MPF) activity. High MPF levels always induced premature chromosome condensation (PCC) irrespective of the number of nuclei fused to a single oocyte. When a single blastodermal cell was fused to a single oocyte without MPF activity the nucleus remained intact for up to 3 h and thereafter underwent PCC. A quite different situation was observed after multiple fusion of several blastodermal cells to a single oocyte without MPF activity. Here, the transplanted nuclei remained intact even after prolonged culture but underwent extensive swelling. DNA synthesis was detected in almost all unfused blastodermal cells. However, after the fusion of several blastodermal cells to a single oocyte no DNA synthesis could be detected. These results provide further evidence that MPF is the universal cell-cycle regulator in the animal kingdom. Its activity is blocked (or neutralised) after fusion to several S-phase cells. Interestingly, our results further suggest that DNA synthesis is suppressed in meiotic cytoplasm even in the presence of an intact nuclear envelope.

Cell fusion experiments were undertaken to determine both whether oocytes possess, in a stage-specific manner, suppressors of pronuclear development and whether these factors could be used to modulate female pronuclear development. The fusion of telophase II eggs obtained 2 hr after activation (A2 eggs) to germinal vesicle (GV)-staged oocytes (GV x A2) suppresses female pronuclear development. The cytoplasm of GV x A2 heterokaryons contains, at 10 hr postfusion, a micropronucleus and a GV which is significantly larger (approximately x1.6) than normal. Fusion of early pronucleate eggs (3 hr postactivation, A3) to GV-staged oocytes (GV x A3) results in the formation of a retarded half-sized pronucleus and a slightly enlarged (approximately x1.2) GV at 10 hr postactivation. Normal pronuclear formation occurs when eggs at 4 hr postactivation (A4) are fused to GV oocytes (GV x A4). Although pronuclear size is suppressed in heterokaryons formed when GV-staged oocytes are fused to eggs activated 2-3 hr earlier (A2 or A3 stage), entry into S-phase and DNA synthesis is not inhibited even in the micropronuclei. Meiotic progression in the oocyte germinal vesicle is arrested after fusion to activated eggs throughout the period during which the pronucleus is undergoing G1 and S-phase. However, at the end of S-phase in the pronucleate partner, germinal vesicle breakdown occurs, cell cycle progression is accelerated, and both nuclei reach M-phase by 12 hr postactivation. That suppressors of pronuclear development are found only in GV (G2)-staged oocytes, and not in mitotic cells at the same cell cycle stage, was demonstrated by fusing G2-staged blastomeres to A2-staged eggs (G2 mitotic x A2). By contrast to the micropronuclei formed in GV x A2 heterokaryons, normal pronuclear development occurred in G2 mitotic x A2 heterokaryons. Our results show that suppressor activity, present only in GV oocytes, restricts female pronuclear development, but only in the first 3 hr after activation. We propose to use the ability to modulate either female (this study) or male pronuclear formation in studies (i) on imprinting and (ii) on the developmental consequences of early asynchrony between male and female pronuclear development.

Summary ― The aim of this study was to characterize embryonic nucleologenesis by determining the appearance and localization of acid argyrophilic, basic lysine-rich and histone proteins in 8-cell bovine embryos. Two silver staining techniques, ethanolic phosphotungstic acid (PTA) and immunocytochemical methods using specific antibodies, were applied at the ultrastructural level. The silverstained proteins were detected at the onset of nucleologenesis on the periphery of the dense nucleolus precursor bodies (NPBs). The amounts of these proteins increased during the transformation of the NPBs into the fibrillogranular nucleolus. At this stage the well-developed dense fibrillar components encircling fibrillar centres showed intense staining. PTA-positive (basic lysine-rich) proteins were present within most nucleolar structures during nucleologenesis as well as in the chromatin. Histones H2B, H3 and H4 were concentrated throughout the chromatin including the nucleolusassociated chromatin. At the onset of nucleologenesis, histones were absent in the NPBs. The first weak histone labelling was detected in the multivacuolated NPBs, both