Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes.

Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.

Oocyte maturation in starfish is mediated by the beta gamma-subunit complex of a G-protein.

The stimulation of meiotic maturation of starfish oocytes by the hormone 1-methyladenine is mimicked by injection of beta gamma subunits of G-proteins from either retina or brain. Conversely, the hormone response is inhibited by injection of the GDP-bound forms of alpha i1 or alpha t subunits, or by injection of phosducin; all of these proteins should bind free beta gamma. alpha-subunit forms with reduced affinity for beta gamma (alpha i1 or alpha t bound to hydrolysis-resistant GTP analogs, or alpha i1-GMPPCP treated with trypsin to remove the amino terminus of the protein) are less effective inhibitors of 1-methyladenine action. These results indicate that the beta gamma subunit of a G-protein mediates 1-methyladenine stimulation of oocyte maturation.

Polyamines and HeLa-cell DNA replication.

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.

Increased expression of alphaq family G-proteins during oocyte maturation and early development of Xenopus laevis.

G-proteins of the alphaq family link extracellular stimulation of plasma membrane receptors to phospholipase C and consequently to intracellular Ca2+ release. Because they might function in initiating Ca2+ release at fertilization, we examined Galphaq family proteins in oocytes and eggs of Xenopus laevis. Three members of this protein family were identified by immunoblotting and antisense depletion. These proteins are barely detectable in the immature oocyte, but undergo a 6-fold increase in amount during oocyte maturation. This increase in Galphaq family protein expression correlates with the acquisition, during oocyte maturation, of the ability to release Ca2+ at fertilization (Schlichter and Elinson, 1981, Dev. Biol. 83, 33-41). In contrast, amounts of Galphas and Galphai3 are constant during maturation. We also examined the amounts of Galphaq, Galphas, and Galphai3 proteins during early development. While amounts of Galphas and Galphai3 show little or no change, Galphaq family protein expression increases 27-fold between the egg and neurula stages, suggesting that these proteins may be important in initiating Ca2+ release during early development.