Dreyer C (1987), Differential accumulation of oocyte nuclear pro...

XB-ART-27848

Development 1987 Dec 01;1014:829-46.

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Differential accumulation of oocyte nuclear proteins by embryonic nuclei of Xenopus.

Dreyer C .

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Oocyte nuclear proteins of Xenopus are distributed into the cytoplasm of the maturing egg after germinal vesicle breakdown. Later they are found in all cell nuclei of the embryo. At early stages of development, different nuclear proteins behave differently. A class of 'early shifting' antigens is accumulated by pronuclei and cleavage nuclei, whereas others appear to be excluded from the nuclei at early stages but are shifted into the nuclei at blastula or during and after gastrulation. Accumulation of 'late-shifting' nuclear antigens is a gradual process and occurs during a period characteristic of each protein. Multiple artificial pronuclei can be formed after injection of sperm nuclei, erythrocyte nuclei or pure lambda-DNA into unfertilized eggs. The artificial pronuclei accumulate early- but not late-shifting proteins. Early-migrating proteins rapidly accumulate into the germinal vesicle after de novo synthesis in the oocyte, indicating that the efficiency of translocation into nuclei is an intrinsic property of each protein. Artificial extension of the length of the cell cycle before midblastula transition does not lead to accumulation of the late-shifting nuclear antigens investigated.

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Species referenced: Xenopus
Genes referenced: actl6a myh3 npm1 tbx2 xnf7
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	Fig. 1. Early and late shifting nuclear antigens in embryos of X. laevis. Embryos at late blastula stage 9 (A,B) and gastrula stage 12 (C,D) were fixed with formaldehyde and embedded with wax. Sections were stained with mABs b2-2B10 (A), and 32-5B6 (B,C). The same section shown in C was counterstained with DAPI to locate all cell nuclei (D). Bar 100um.
	Fig. 2. Nucleocytoplasmic distribution of nucleoplasmin in embryos of X. laevis. Embryos after the 7th (A,B), 11th (C,D), and about the 13th cleavage division (E, F) were fixed according to Romeis and embedded in wax. Sections were stained with mAB b7-lA9 that binds to nucleoplasmin. Details of the sections shown in the left panels (A,C,E) are shown in the right panels (B,D,F). Bar 100um.
	Fig. 3. Kinetics of pronucleus formation after injection of sperm nuclei into unfertilized eggs of X. laevis. Sperm nuclei were isolated and injected into eggs as detailed in Materials and methods. Eggs were fixed with TCA 15min (A,B), 30min (C,D), and 60min after injected, embedded in wax, and sectioned. Sections were stained with mAB b2-2B10, which binds to N, (A,C,E) and counterstaind with DAPI (B,D,F). Nuclei that do not swell do not take up the antigen to a significant degree (E,F). Bar 100um.
	Fig. 4. Multiple pronuclei formed after injection of sperm nuclei into unfertilized eggs. X. laevis eggs were fixed with TCA 2h after injection of sperm nuclei. Wax sections were stained with mABs b2-2B10 (A) and L6-8A7, which binds to lamin Lm (B). Counterstaining with DAPI showed that all nuclei had taken up both antigens (not shown). Bar 100um.
	Fig. 5. Selective uptake of early-shifting nuclear antigens into injected sperm nuclei. Eggs were fixed with TCA 2h after injection of sperm nuclei. Wax sections were stained with mABs 32-4A1 (A), 37-1B2 (C), P3 control antibody IgGl secreted by mouse cell line P3x63 Ag8 (E). Counterstaining of each of these sections with DAPI is shown on the right panels (B,D,F). Bar 50um.
	Fig. 6. Selective uptake of early-shifting antigens into injected erythrocyte nuclei. Eggs of X. laevis were fixed with TCA 2h after injection of erythrocyte nuclei. Wax sections were stained with mABs L6-8A7 (A), b2-2B10 (C), and 37-1B2 (E). Counterstaining of the same sections with DAPI is shown in the right panels (B,D,F). Bar 50m.
	Fig. 7. Artificial pronuclei formed after injection of A-DNA. Unfertilized eggs of X. borealis were injected with 5-10ng of A-DNA each. 2h after injection, eggs were fixed with TCA. Wax sections were stained with mABs b2-2B10 (A) and 32-5B6 (C). Counterstaining of each section with DAPI is shown on the right panels (B,D). Bar 50um.
	Fig. 8. Nucleocytoplasmic distribution of oocyte proteins synthesized de novo. Oocytes of X. laevis were labelled with [35S]methionine for 5 h (A,B) or 15 h (C,D), manually enucleated and protein preparations of either four total oocytes (A,C) or of 30 nuclei were subjected to two-dimensional gel analysis and fluorography as detailed in Materials and methods. After fluorography, the areas at the position of the prominent nuclear and cytoplasmic proteins indicated in the figure were excised from each of the four gels and the amount of radioactivity in each area determined by liquid scintillation counting. The results are given in Table 2. A, actin; T, /J-tubulin; N,, antigen b2-2B10; 5, antigen 35-1A7; 7, antigen 37-1A9; 21, antigen 32-5B6 (Dreyer & Hausen, 1983; Dreyer et al. 1985). Nucleoplasmin is not labelled under these conditions.
	Fig. 9. Phosphorylation of germinal vesicle proteins in oocytes and maturing eggs of X. laevis. Oocytes in the absence (A,B) or presence (C) of progesterone were labelled with [32P]phosphate for 4h as detailed in Materials and methods. Protein preparations were subjected to twodimensional gel analysis. The gels were stained with silver and analysed by autoradiography. (A) Oocyte proteins stained with silver. Isoelectric focusing was from right to left, SDS-polyacrylamide gel electrophoresis from top to bottom. (B) Autoradiograph of the gel shown in A. (C) Autoradiograph of gel showing 32P-labelled proteins from maturing egg. Protein patterns of oocytes and of maturing eggs were not significantly different after silver staining (not shown). Germinal vesicle proteins are numbered as previously. Nu, nucleoplasmin.
	Fig. 10. Cell cycle arrest with cycloheximide. Embryos of X. laevis were transferred to lOO^gml"' of cycloheximide in MBSH at the 64-cell stage and incubated therein for 4h, when controls had reached stage 9+. Experimental and control embryos were fixed by freeze substitution, and wax sections were stained with mAB 32-5B6 (A,C) and b2-2B10 (B,D). (A,B) Controls; (C,D) embryos treated with cycloheximide. Bar 100um.