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A cell-free preparation of the cytoplasm from activated eggs of Rana pipiens induces, in demembranated sperm nuclei of Xenopus laevis, formation of a nuclear envelope, chromatin decondensation, initiation of DNA synthesis, and chromosome condensation. Both soluble and particulate cytoplasmic constituents are required to initiate these processes in vitro. The observed changes resemble processes occurring during fertilization and the mitotic cycle in early amphibian embryos. Therefore, this cell-free system may be useful in biochemical analysis of the interactions of nucleus and cytoplasm that control nuclear behavior.

Maturation-promoting factor causes germinal vesicle breakdown when injected into Xenopus oocytes and can induce metaphase in a cell-free system. The cell-free assay was used to monitor maturation-promoting factor during its purification from unfertilized Xenopus eggs. Ammonium sulfate precipitation and six chromatographic procedures resulted in a preparation purified >3000-fold that could induce germinal vesicle breakdown within 2 hr when injected into cycloheximide-treated oocytes. Proteins of 45 kDa and 32 kDa were correlated with fractions of highest activity in both assays. These fractions contained a protein kinase activity able to phosphorylate the endogenous 45-kDa protein, as well as histone Hi, phosphatase inhibitor 1, and casein. The highly purified preparations described here should help to identify the mechanism of action of maturation-promoting factor and to elucidate the role of protein kinases in the induction of metaphase.

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca 2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.Fully grown Xenopus oocytes are physiologically arrested in first meiotic prophase. Upon exposure to progesterone or insulin, oocytes synchronously complete meiotic maturation, undergoing breakdown of the nuclear envelope (germinal vesicle breakdown), chromosome condensation, spindle formation, and extrusion of the first polar body. Oocytes then proceed through meiosis II, arresting at metaphase as an unfertilized egg (see references 1 and 2 for review). After fertilization or parthenogenetic activation, the egg chromosomes decondense, a nuclear envelope reforms, and DNA replication occurs. This series of nuclear changes forms a simple cell cycle, comprising a synchronous G2/M transition as immature oocytes undergo meiotic maturation and then an M/S transition after fertilization. Studies in several laboratories have shown that nuclei from somatic cells or spermatozoa injected into maturing oocytes or eggs cease their previous activities and begin to carry out the same processes as the oocyte nucleus (3-5). These findings suggest that the cytoplasmic components that control nuclear events through the oocyte cell cycle can act on exogenous nuclei.The cytoplasmic control of nuclear behavior in amphibian oocytes has also been demonstrated by cytoplasmic transfer. Injection of the cytoplasm from maturing oocytes (6, 7) or of a partially purified fraction from eggs (8) into immature 518 oocytes induces germinal vesicle breakdown, as well as all of the other events of meiotic maturation. As a result, the activity responsible for inducing these changes has been called maturation-promoting factor (MPF).~ A similar activity has been found in starfish (9) and mouse (10) oocytes undergoing meiotic maturation, as well as duri...