Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

Abstract

G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with M r = 38,000 and 39,000 ( α 38 and α 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of α 38–39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of α 38–39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12- O-tetradecanoyl phorbol 13-acetate, TPA), but not an inactive one (4α-phorbol 12,13-didecanoate, 4α-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of α 38–39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo ( Poueymirou and Schultz, 1987. Dev. Biol. 121, 489–498). In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or α-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or much smaller inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of α 38–39. Results of immunoblotting experiments using an antibody that is highly specific for α i1–3 reveal the presence of a cross-reactive species of M r = 38,000 ( α 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos. Relative to these stages, a reduced amount of this species is present in the eight-cell, morula, and blastocyst stages. Treatment of oocytes with PT results in a small but significant acceleration in the rate of GVBD, but has no effect on the extent of polar body emission. Treatment of one-cell embryos with PT has no effect on the extent of cleavage, onset of transcriptional activation at the two-cell stage, or development of two-cell embryos to the hatching blastocyst stage.