Calcium plays an important role in the
regulation of meiotic maturation in mammalian oocytes. In the present study,
mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases,
was used to mobilize intracellular calcium deposits in growing pig oocytes,
which had not attained full meiotic competence and in which maturation is thus
spontaneously blocked at the metaphase I stage. CPA treatment significantly
increased the ratio of growing oocytes that are able to overcome the
spontaneously occurring metaphase I block to complete their maturation at the
metaphase II stage. CPA treatment of a least 2 hours’ duration is
necessary to overcome the metaphase I block in growing oocytes. A similar
effect upon release from the spontaneous meiotic block at the metaphase I
stage was observed after treatment of growing pig oocytes with thapsigargin,
another inhibitor of endogenous calcium-dependent ATPases. Numerous calcium
deposits in vacuoles, the mitochondria and on the surface of yolk granules in
growing pig oocytes were observed. CPA treatment is able to mobilize calcium
from the mitochondria, but deposits in vacuoles and deposits on the surface of
yolk granules seem to remain intact after CPA treatment. A microinjection of
heparin, which is known to bind with the inositol trisphosphate receptors,
significantly decreased the ratio of CPA-treated growing oocytes overcoming
the block at the metaphase I stage. This indicates that CPA might mobilize
calcium in growing pig oocytes through inositol trisphosphate receptors. On
the other hand, a microinjection of procaine or a microinjection of ruthenium
red, both inhibitors of ryanodine receptors, did not prevent the overcoming of
the metaphase I block, induced by CPA treatment. The calcium channel blocker,
verapamil, significantly reduces the proportion of CPA-treated growing oocytes
that overcome the metaphase I block. This indicates that the influx of calcium
from extracellular sources is necessary to over-come the metaphase I block.
The calmodulin inhibitors ophiobolin A and W7 also reduce the proportion of
CPA-treated growing oocytes overcoming the metaphase I block.
Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors