476
Cellular Interactions
preventing pronuclei from forming too
early in the zygote.
CaM KII could have two potential roles
in regulating the conversion of the egg
into the zygote based on the above studies:
(1) to mediate the chromosomal transition
from metaphase II arrest into anaphase
II and subsequently through telophase II
in the fertilization-competent egg and (2)
to interact with MAP kinase to potentiate
its
activity and
prevent formation
of
pronuclei. CaM KII is localized at an
optimal site to be involved with both the
above-stated roles because it is colocalized
with MAP kinase. It subsequently interacts
with, and it is localized on, the meiotic
spindle, which is a mechanical scaffold in
eggs that drives chromosomal movement.
4.2
Remodeling through Changes in Activity of
Protein Kinase C
When eggs are treated with PKC agonists,
fertilization-competent hamster eggs re-
lease from arrest at meiotic metaphase
II, initiate second polar body formation,
and several hours later pronuclei as well
as the Golgi apparatus form (all compo-
nents that are absent in M-phase cells). A
number of the events associated with egg
activation are initiated after PKC is acti-
vated in eggs using a phorbol ester (i.e.
a pharmacologic agent known to activate
PKC) or a diacylglycerol, a natural activa-
tor of PKC in cells. Even after inducing
activation of PKC with agonists while the
level of [Ca
2
+
]
i
was clamped low with a
calcium chelator, these activation events
still occurred. However, in this case the
meiotic spindle did not undergo a normal
metaphase to anaphase transition when
the level of [Ca
2
+
]
i
was clamped low. In-
stead, the entire meiotic spindle attempted
to translocate into the forming second po-
lar body. When the reverse situation was
tested (the level of [Ca
2
+
]
i
was elevated
by experimental manipulation, and the
activity of PKC was clamped low using
antagonists) the eggs did not change and
remained arrested at meiotic metaphase
II. This indicates that PKC acts down-
stream of the calcium signal, although
the speciFc isotypes of PKC that act ap-
pear to differ depending on the study and
species. Even though PKC induced a num-
ber of changes that are associated with egg
activation, some other calcium-dependent
feature was needed to cause the normal
metaphase to anaphase transition. To con-
Frm that the fertilization-induced event
involved PKC at an immunocytochemical
level, the investigators monitored the spa-
tial position of several isotypes of PKC by
using a PKC reporter dye. In this study,
the investigators demonstrated in ham-
ster metaphase II eggs that as a result
of the rise in intracellular-free calcium,
PKCs translocated from a relatively uni-
form distribution in the cytoplasm to the
cell periphery near the plasma membrane
of the egg. Translocation of the reporter
dye to the membrane indicated that the ki-
nasewasactivated.Inthisstudy,aDAG,a
natural simulator of PKC activity produced
in vivo
, was employed. A DAG was used
because the cell itself makes DAGs and
contains enzymes that normally metabo-
lize DAG into other compounds. Thus,
any DAG when transiently applied is more
likely to mimic the normal activation of
PKC. Phorbol esters also activate PKC but,
in contrast, they are foreign to cells and
could likely not be metabolized because
they are not normally present in cells. Use
of phorbol esters as PKC agonists could
lead to abnormal results due to an ex-
tended activation of PKC as well as other
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