Role of the oocyte nucleus in physiological maturation in Rana pipiens

Abstract

The events of maturation (germinal vesicle dissolution and meiosis to the second metaphase) have been induced with progesterone in vitro in full-grown oocytes dissected from their ovarian follicles. Oocytes from which germinal vesicles (GV) were removed prior to progesterone exposure were subsequently tested for the capacity to be artificially activated, cleave, and synthesize control levels of protein. The enucleated eggs responded to an activation stimulus when tested about 48 hours after a 1-hour exposure to progesterone. This positive response included breakdown of the cortical granules, elevation of the vitelline membrane, rotation within the perivitelline space, increase in egg turgidity, and changes in the appearance of the egg surface. Removal of the egg vitelline membrane and any adherent follicle cells with pronase, prior to progesterone exposure, did not prevent enucleated eggs from undergoing activation; the sole criterion in this case was breakdown of the cortical granules. Many of the enucleated, activated eggs displayed abortive cleavages. Enucleated eggs lacking GV material in their cytoplasm were incapable of genuine cleavage when tested by nuclear transplantation. This restriction was partially removed by injecting GV material back into the enucleated progesterone-stimulated eggs. Donor GV material from non-hormone-treated eggs was essentially as effective in this respect as GV material from progesterone-stimulated eggs. Incubation of oocytes with actinomycin D at concentrations as high as 50 μg/ml had no effect in progesterone-induced maturation. Likewise, injection of actinomycin D directly into oocytes did not prevent progesterone-induced maturation. Puromycin, on the other hand, suppressed all stages of maturation. These results suggest that maturation does not require concomitant DNA-dependent RNA synthesis, but that protein synthesis is obligatory for all stages of maturation. Protein synthesis was measured in eggs enucleated prior to progesterone exposure and at several times after the hormone stimulation. The rate of protein synthesis during the entire period of maturation was essentially identical in enucleated eggs and their nucleated controls. The rate was initially low, increased only after progesterone exposure, reached a maximum level about when nucleated controls gave off their first polar bodies, and the elevated rate was maintained for at least a day after nucleated controls were first capable of being fertilized. Based on these experiments, we conclude that the mixing of GV material with the egg cytoplasm is not a prerequisite to attaining the capacity to undergo artificial activation or to synthesize protein, but is required for the enucleated eggs to cleave. We further suggest that the site of action of progesterone in inducing maturation is extranuclear, possibly at the egg surface.