On the origin and persistence of a cytoplasmic state inducing nuclear DNA synthesis in frogs' eggs.

Nucleocytoplasmic interactions are believed to be of great importance in early animal development, since theresponse of genetically identical nuclei to different regions of egg cytoplasm provides the most satisfactory explanation for the initial appearance of cell differences.'-' However, very little is known at present about the mechanism of nucleocytoplasmic interactions. With this in mind, we have selected an apparently simple example of this kind of interaction for detailed study in the frog Xenopus laevis. This is the induction of DNA synthesis by egg cytoplasm, a phenomenon which has been observed in male and female pronuclei shortly after fertilization.4 20 The analysis of this example of a nucleocytoplasmic interaction is much facilitated by the finding that large numbers of nuclei isolated from frog brain and other adult cell types are rapidly induced to synthesize DNA after injection into unfertilized eggs,5 though very few nuclei from these tissues normally synthesize DNA. In all experiments to be reported here, nuclei from adult frog brain have been used. Although these nuclei do not support normal development after their injectioii into unfertilized eggs, they respond to egg cytoplasm by DNA synthesis as do egg and sperm pronuclei or single transplanted embryonic nuclei, which do support normal development.' For this reason the induction of DNA synthesis in adult brain nuclei can be justifiably used to study one aspect of normal nucleocytoplasmic interactions. The experiments reported here contribute to our understanding of this nucleocytoplasmic interaction in three principal ways. First, they show that the state of egg cytoplasm which induces DNA synthesis is totally absent from o6cytes, the cells which mature into eggs. Second, it has been found that this cytoplasmic state arises as an effect of pituitary hormone on mature o6cytes. Last, the experiments demonstrate the persistence of the effective cytoplasmic state which seems to be sufficiently stable to permit eventual identification of its molecular basis. Materials and Methods.-Preparation and incubation of o6cytes: A suspension of nuclei and label was inserted into obcytes of Xenopus laevis laevis by microinjection (50-100 m,4l for full-sized oocytes and 10-15 mA for growing o6cytes of half the full size diameter). Injected o6cytes, still surrounded by follicle cells, were incubated at 210C in modified Barth's medium' (i.e., 0.176 M NaCl, 2.0 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, 10 mg per liter of both streptomycin sulphate and benzylpenicillin sodium salt, and 1.5 mM TrisHCl to bring the pH of the whole solution to 7.6). Odcytes incubated under these conditions retained the same external appearance as they had immediately after removal from the donor female for a period varying from 2 to 3 days. Isolated brain nuclei: These were used as a crude preparation obtained in the way described previously.5 In any 2-hr labeling period, less than 1% of adult frog brain nuclei synthesize DNA in vivo, or as isolated nuclei in vitro.5 Labeling methods: H'-thymidine (H3-TdR) was used as a specific label for DNA synthesis.' It was obtained at 22.1 c/mM, labeled primarily in the 6C position, from the Radiochemical Centre, Amersham, England, and was made up at 2 mc/ml in modified Barth's solution. In most experiments it was mixed with brain nuclei (1:1 ratio by volume) just before use. A utoradiography: O6cytes or eggs, fixed in Perenyi's fixative and sectioned at 6-7 ju, were