474
Cellular Interactions
out of metaphase II arrest into anaphase
II and subsequently into telophase II is
promoted by CaM KII. This was demon-
strated in two ways: (1) Living eggs were
activated with calcium ionophore after the
application of membrane-permeant CaM
KII inhibitors, which blocked the transi-
tion from metaphase II into anaphase II;
(2) When permeabilized eggs were equi-
librated in a medium whose free calcium
concentration was adjusted to 1
ยต
M(using
a calcium/BAPTA buffer), calmodulin was
present at a concentration sufFcient to es-
tablish a 4 : 1 ratio of calcium : calmodulin,
and an ATP regenerating system was
present; the permeabilized eggs transited
through anaphase into telophase with the
same timing that would occur in a fer-
tilized living egg. As the ratio of calcium
to calmodulin was sequentially dropped to
0 : 1 in the permeabilized egg, the transit
to telophase was progressively reduced to
zero. When inhibitors to CaM KII were ap-
plied, the transit to telophase was blocked,
but other kinase inhibitors, including in-
hibitors to PKC, could not prevent the
transit through anaphase into telophase.
The
above
results
strongly
indicate
that
the
movement
of
chromosomes
from meiotic metaphase II arrest into
anaphase II and subsequently telophase
II is regulated by activation of CaM KII.
These results also explain investigations
discussed earlier, which reported that
when PKC is activated in mammalian
e
g
g
sinth
ea
b
s
en
c
eo
fane
l
e
v
a
t
i
onin
the level of intracellular-free calcium, an
abnormal transit out of metaphase II
occurs. CaM KII is not activated in the
absence of a rise in the level of [Ca
2
+
]
i
and the chromosomes do not transit
into anaphase, but other features of egg
activation occur.
It is important to remember that John-
son et al. reported that CaM KII remains
on elements of the mammalian meiotic
spindle as the cell transits into anaphase
and at subsequent stages. Interpolar mi-
crotubules form an overlapping assembly
in the midzone region during the latter
part of M-phase and the microtubules
become bundled. Such midzone micro-
tubules contain many spindle-related pro-
teins. It has been proposed that these
proteins have a role in the formation of
the contractile ring during cleavage. John-
son et al. reported, in accordance with this
proposal, that CaM KII appears to tran-
sit along the midzone microtubules to the
contractile ring of the second polar body
and localizes on the contractile ring sug-
gesting it may play an active role in the
cytokinetic process. In addition, a recent
study by Hatch and Capco demonstrated
that the active form of CaM KII associ-
ated with the cytokinetic ring, a structure
that forms perpendicular to the midzone
microtubules. This study conFrms and ex-
tends on work by earlier investigators who
blocked CaM KII activity with drugs. An
inhibitor to calmodulin function was em-
ployed by Xu et al. and showed that this
delayed the formation of the second po-
lar body. Since calmodulin is required to
activate CaM KII, the authors speculated
that CaM KII activity was needed for the
formation of the second polar body. This
idea was further supported by two sepa-
rate studies, which reported that second
polar body formation was delayed after
inhibitors to CaM KII were employed.
The above observations also address an-
other paradox that was reported when
PKC-induced egg activation was investi-
gated. Gallicano et al. reported that after
treatment with PKC agonists, the second
polar body initially appeared, but then
would frequently be absorbed into the egg.
When PKC agonists are applied, there is
no increase in the level of [Ca
2
+
]
i
and
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