phosphorus-radioisotopes and 1-methyladenine

An increased phosphorylation of ribosomal protein S6 has been shown to be correlated with an increase of intracellular pH (pHi) and with stimulation of protein synthesis in many systems. In this research changes in ribosome phosphorylation following hormone-induced meiotic maturation and fertilization or activation by ionophore A23187 were investigated in starfish oocytes. The hormone was found to stimulate, even in the absence of external Na+, the phosphorylation on serine residues of an Mr 31,000 protein identified as S6, as well as that of an acidic Mr 47,000 protein, presumably S1, on threonine residues. Phosphorylation of ribosomes was an early consequence of hormonal stimulation and did not decrease after completion of meiotic maturation. Fertilization or activation by ionophore of prophase-arrested oocytes also stimulated ribosome phosphorylation. Only S6 was labeled in this case, but to a lesser extent than upon hormone-induced meiotic maturation. Changes in pHi were monitored with ion-specific microelectrodes throughout meiotic maturation and following either fertilization or activation. The pHi did not change before germinal vesicle breakdown (GVBD) following hormone addition, but it increased before first polar body emission. It also increased following fertilization or activation by ionophore or the microinjection of Ca-EGTA. In all cases, alkalinization did not depend on activation of an amiloride-sensitive Na+/H+ exchanger. Microinjection of an alkaline Hepes buffer or external application of ammonia, both of which increased pHi, prevented unfertilized oocytes from arresting after formation of the female pronucleus and induced chromosome cycling. Phosphorylation of S6 was still observed following fertilization or induction of maturation when pHi was decreased by external application of acetate, a treatment which suppressed the emission of polar bodies. Protein synthesis increased in prophase-arrested oocytes after fertilization or activation. It also increased after ammonia addition, although this treatment did not stimulate S6 phosphorylation.

Topics: Adenine; Amino Acids; Animals; Calcimycin; Female; Fertilization; Hydrogen-Ion Concentration; Ionophores; Meiosis; Oocytes; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Ribosomal Proteins; Starfish

Maturation-promoting factor (MPF) activity and the protein phosphorylation pattern were monitored throughout the time course of meiotic maturation following hormonal stimulation of prophase-arrested starfish oocytes. MFP activity disappeared or decreased dramatically during the first and second meiotic cleavages. MPF activity came back to a very high level after the first but not the second meiotic cleavage. The state of protein phosphorylation was monitored using both tracer experiments and direct measurements of the absolute amount of phosphate in phosphoproteins. High and low levels of MPF activities were, respectively, associated with high and low levels of protein phosphorylation. It is suggested that the turn over of phosphate already bound to proteins in prophase-blocked oocytes does not change following hormone addition.

Topics: Adenine; Adenine Nucleotides; Animals; Egg Proteins; Female; Growth Substances; Kinetics; Maturation-Promoting Factor; Meiosis; Oocytes; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Starfish