Figure 2.

Translational awakening of tPA mRNA in oocytes. (A) Induction of tPA synthesis in primary oocytes. The indicated 3′-UTR transcripts were injected in primary oocytes (GV) at 1 or 3 μg/μl (lanes 4–15); transcript injected at 1 μg/μl, are present in oocytes at a ∼10⁴-fold molar excess over endogenous tPA mRNA. After 6 hr of culture in the presence of dB-cAMP, arrested primary oocytes were lysed in groups of five and assayed in triplicate by zymography (Huarte et al. 1985). Groups of five control noninjected arrested primary oocytes (n.i. GV, lanes 1–3) or maturing oocytes lysed 6 hr after GVBD (n.i. BD, lanes 16–18) were analyzed in parallel. The zymogram was incubated for 40 hr at 37°C. (B) Time course of tPA accumulation in arrested primary and in maturing oocytes. Noninjected primary oocytes and primary oocytes injected with the ACE transcript at 2 μg/μl were incubated in the presence or absence of dB-cAMP. After the indicated periods of time, arrested primary oocytes (GV) or maturing oocytes (BD) were lysed in groups of five, and the samples were assayed in duplicate on a zymogram that was incubated for 40 hr at 37°C; tPA activity was quantitated by measuring the surface of the proteolytic zones [n.i., noninjected; inj., injected (Stutz et al. 1997)].