EFFECTS OF ALPHA-AMANITIN ON NUCLEAR MATURATION OF PORCINE OOCYTES INVITRO

The effects of alpha-amanitin on cumulus enclosed and denuded porcine oocytes exposed to this heterogeneous nuclear RNA (hnRNA) synthesis inhibitor at different times (0, 0.5, 1, 2, 4, 6, 8, 12 and 20 h) after the start of culture was investigated. A further objective was to determine the sequence of nuclear progression after removing the inhibitor. The addition of 10 mug alpha-amanitin ml-1 to a gonadotrophin containing medium (10 iu PMSG ml-1 in TCM 199) at 0, 0.5 or 1 h after the onset of culture prevented cumulus expansion, and only 4.4, 6.4 and 3.5% of oocytes underwent germinal vesicle breakdown. This inhibitory effect was considerably reduced by delaying the addition of the drug to the culture medium for 2-8 h (2 h: 34.9%,4 h: 53.5%,6 h: 46.9%,8 h: 59.2% germinal vesicle breakdown), and no inhibition of nuclear maturation was observed when alpha-amanitin was added after 12 or 20 h following explantation of the oocytes. When cumulus-oocyte complexes were cultured for greater-than-or-equal-to 2 h in inhibitor-free medium and then transferred to medium supplemented with alpha-amanitin, full cumulus expansion was observed in all cases, at the end of the 44 h culture. Denudation of the oocytes before culture in either medium supplemented with alpha-amanitin or microinjection of alpha-amanitin into the ooplasm at concentrations of 1.0 and 10.0 mg ml-1 remained without any effect on nuclear progression. When cumulus-enclosed oocytes were first incubated for 24 h in a medium supplemented with alpha-amanitin, and then denuded and cultured for a further 8 h in an inhibitor-free medium, 92% (90 of 98) reached the metaphase I stage. This observation indicates that porcine oocytes released from a block of hnRNA synthesis enter the M-phase of the cell cycle in an interval three times shorter than that required for control oocytes. It is concluded that de novo mRNA synthesis within the cumulus cells is required for gonadotrophin induced nuclear maturation and cumulus expansion of porcine oocytes in vitro. The data further suggest that the transcriptional event that induces progression of the cell cycle from the G2- to the M-phase of the oocyte occurs within 2 h after explantation.