RNA and Protein Synthesis during Progesterone-Induced Germinal Vesicle Breakdown in R. pipiens Ovarian Tissue

Abstract

Rana pipiens oocytes with intact follicle cells were induced to undergo meiotic maturation in vitro under the influence of progesterone or related steroids or with pituitary extract. Uptake and incorporation of 3H-uridine and 14C-leucine were measured using pulse-labelling techniques. A transient increase in uridine incorporation (expressed as percent intracellular acid soluble pool) occurred 1–4 h after exposure to inducing levels of progesterone with both amino acid-containing and amino acid-free incubation media; but total incorporation was greatest in amino acid-containing medium. Nanomolar concentrations of a single amino acid such as leucine, tryptophan, or arginine (but not glycine) stimulated uridine incorporation into TCA precipitable material 8–10-fold in non-induced follicles, but had little effect on total uridine uptake. Hydrocortisone or testosterone also induced germinal vesicle breakdown, but did not stimulate uridine incorporation above the control level. A comparison of pulse-labelled RNA recovered in ovulated eggs with that in follicular tissue indicated that progesterone-induced RNA synthesis occurred primarily in the oocyte. Induction in Cal+-free Ringer's solution inhibited uridine incorporation more than 95%, with no effect on total uridine uptake and only 30% inhibition of germinal vesicle breakdown. Twenty μg/ml ethidium bromide inhibited uridine incorporation about 70% in non-induced follicles and only about 30% in progesterone-induced follicles, with no effect on germinal vesicle breakdown. 10–40 μg/ml actinomycin D did not significantly effect either uridine incorporation or germinal vesicle breakdown in non-induced or progesterone-induced follicles. These findings suggest that progesterone (or its metabolites) may act at a transcriptional as well as a post transcriptional level and confirm that the induction of germinal vesicle breakdown is post-transcriptional.