B. Meinecke et S. Meinecketillmann, EFFECTS OF ALPHA-AMANITIN ON NUCLEAR MATURATION OF PORCINE OOCYTES INVITRO, Journal of Reproduction and Fertility, 98(1), 1993, pp. 195-201
The effects of alpha-amanitin on cumulus enclosed and denuded porcine
oocytes exposed to this heterogeneous nuclear RNA (hnRNA) synthesis in
hibitor 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 dete
rmine the sequence of nuclear progression after removing the inhibitor
. The addition of 10 mug alpha-amanitin ml-1 to a gonadotrophin contai
ning medium (10 iu PMSG ml-1 in TCM 199) at 0, 0.5 or 1 h after the on
set of culture prevented cumulus expansion, and only 4.4, 6.4 and 3.5%
of oocytes underwent germinal vesicle breakdown. This inhibitory effe
ct was considerably reduced by delaying the addition of the drug to th
e 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 cultu
red 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. D
enudation of the oocytes before culture in either medium supplemented
with alpha-amanitin or microinjection of alpha-amanitin into the oopla
sm at concentrations of 1.0 and 10.0 mg ml-1 remained without any effe
ct on nuclear progression. When cumulus-enclosed oocytes were first in
cubated for 24 h in a medium supplemented with alpha-amanitin, and the
n denuded and cultured for a further 8 h in an inhibitor-free medium,
92% (90 of 98) reached the metaphase I stage. This observation indicat
es 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 induc
ed nuclear maturation and cumulus expansion of porcine oocytes in vitr
o. The data further suggest that the transcriptional event that induce
s progression of the cell cycle from the G2- to the M-phase of the ooc
yte occurs within 2 h after explantation.