PIGMENT GRANULES CAN MIGRATE TO THE CORTEX IN
SEA URCHIN EGGS ACTIVATED WITHOUT CORTICAL
GRANULE DISCHARGE
著者
OSANAI KENZI
journal or
publication title
東北大学浅虫臨海實驗所報告
volume
17
number
2
page range
109-116
year
1982-03-30
URL
http://hdl.handle.net/10097/00131452
BULL· MAR. BroL. STN, AsAMUSHI, TOnoKu UNrv., 17(2), 109-116, 1982
PIGMENT GRANULES CAN MIGRATE TO THE CORTEX
IN SEA URCHIN EGGS ACTIVATED WITHOUT
CORTICAL GRANULE DISCHARGE
1l
KENZI 0SANAI2)
Man'ne Biologz'cal Statt'on, T6hok:u Um"ver81:ty, .Asamu.sM,
Aomori, 039-34 Japan
In Temnopleurus hardwio'ki eggs brown pigment granules are scattered throughout the inner cytoplasm before fertilization. Mter fertilization, most of them migrate to the cortex and become embedded. In unfertilized eggs incubated in sea water containing procaine hydrochloride the pigment granule migration occurs without cortical granule discharge. After longer incubation a clear cortex lacking pigment granule.s appears though cleavage does not occur.
Unfertilized eggs of Tewnopleurus hardwicki contain brown pigment granules.
These pigment granules are scattered throughout the irmer cytoplasm. After
fertilization most of them migrate to the egg cortex and become embedded (OSANAI,
1963, 1964, 1969; SAWADA and OsANAI, 1980). Echinochrome granules in
Arbacia
eggs show similar movement after fertilization (ALLEN and RowE, 1958; BELANGER
and RusTAD, 1972: ScHROEDER, 1980). Pigment granules migrate in eggs
exposed to ionophore A23187, which induces the discharge of cortical granules and
the elevation of the fertilization envelope (SAwADA and OsANAI, 1980; ScHROEDER,
1980).
In partially fertilized eggs, in which the cortical granule discharge has been
interrupted and the fertilization envelope has been partially elevated, the pigment
granules migrate ouly to the changed cortex. It was proposed that pigment granule
migration depends on ooplasmic activation relating to cortical granule discharge
(ALLEN and RowE, 1958; 0SANAI, 1963, 1964). The activation of sea urchin
eggs can be induced without cortical granule reaction. Ammonia and procaine
parthenogenetically initiate DNA synthesis and cytoaster formation in spite of
cortical granule remaining in the cortex (MAziA, 197 4; MAzrA et al., 1975; VACQUIER
and BRANDRIFF, 1975; MoY et al., 1977).
These previous works lead me to examine whether the pigment granule
migra-tion is also induced independent of cortical granule discharge.
I) Contribution from the Marine Biological Station, TOhoku University, No. 468. 2) :!i:l'lOOiil
110 K. OSANAI
MATERIAL AND METHODS
Ternnopleurus hardwicki
(JAY) were collected in the vicinity of the Asamushi
Marine Biological
Stat
ion
,
Aomori. Their gametes were obtained by injecting 0.5
M potassium chloride (pH
8)
int
o
the intracoelomic cavity. To remove
the
jelly
coat the eggs were treated with acid
sea
water
(pH
5.1)for
1-2minutes. The
vitelline coat was removed by expos
ing
them to
0.01
% tyrpsi
n
(Sigma) in
sea water
for
10
minutes
.
To induce egg activation with cortical
granule b
r
eakdown, intact
or dejellied eggs were
expsoe
d
to
1
M
urea
solution which was adjusted to pH 8
by the addition of sod
ium
bicarbonate
(
lVIoTOM
URA
1934).Procaine hydrochloride
(Sigma) was dissolved
in filtered
sea water at
5 mM and used to
induce
partheno-ge
neti
c
activation without cortical granule discharge.
Ob
serva
ti
ons
were carried
out with a Nomarski differential interference microscope (Olympus
BHB-N) at
22-24°0.RESULTS
Migration of pigment granules to the egg cm·tex
The distribution of pigment granules in
the
unfertilized and
fertil
i
zed eggs of
Temnopleurus hardwickiwas previously described (OSANAI,
1969).Th
e
pigment
granules
are
di
st
ributed
throughout the inner cytoplasm prior to
fertili
zation
(Figs.
1
and
2) and undergo
randomly-dir
ected
movements. Within 5 minutes
of
fertilization, most of them migrate to the egg periphery, and
become
embedded in
the cortex. Those particles remaining
in
the
inner
cytoplasm move inward and
gather around the aster.
Unfertilized eggs were exposed to
1
M urea for 5 minutes and then incubated
in
normal
sea
water. The
eggs
were parthenogenetically activated
and
developed
the
mona
ste
r
.
How
ever,
they did not
formed
the
fertiliz
at
ion
envelope
and
the
hyaline
lay
e
r,
because
th
e
vitelline
coat
and cortical granules, the precursors of
the
f
ertilizat
ion
e
nv
elope,
had
been removed
b
y
the urea-treatment (MoTOMURA,
1941).In
these activated eggs many pigment granules were embedded in
the
cortex, while a
few
were
di
st
ributed
around the
mona
ster
(Fig.
3).
To induce egg activation without cortical granule discharge, intact or dejellied
unfertilized
eggs
were incubated in
sea water
containing 5 mM procaine. Mter
about 3
hour
s,
the
eggs
underwen
t
nucl
ear
change
an
d
formed monaster as
shown
by
previous workers (VACQUEIR and BRANDRIFF,
1975;MoY
et al., 1977).Most of
the pigment granules become embedded in the cortex
and
the rest of them
gathered around the aster (Fig. 4). The activated
eggs
did not
s
ho
w
any
sign
of
cortical granule
reaction
, such
as fertilization
enve
lo
pe
formation
or
cortical
granule
di
scharge,
but
their
surface
had extended microvilli (Figs. 5 and 6).
PIGMENT GRAI'HJLE 1\HGRATION I l l
4a 4b
-_Figs. 1-4. The distribution of pigment granules in unfertilized eggs a,nd parthenogenetically activated eggs. Figs. l-2: Unfertilized eggs. Pigment granules are distributed throughout the cytoplasm and undergo salta.tory movements. (Fig. I X 330, Fig. 2
x
800). Fig. 3: Urea-activated egg which were exposed to Il\!1 urea for 5 minutes and then incubated in normal sea water for 95 minutes. Optical section (a) and surface view (b). Monaster develops and pigment granules segregate to the cortex (
x
300). Fig. 4: Procaine-activated egg. The dejellied unfertilized egg was incubated in sea water containing 5 mM: procaine for 2 hours and 10 minutes. Optical section (a) and surface (b) (X 260). Bars indicate 10 ,um.112 K. OSANAI
Figs. 5 and 6. Extended microvilli in procaine-treated eggs. The eggs were exposed to 5 mM procaine for 3 hours (X 420). The btu· indicates 10 fiiD.
n~:stribution of cortical pigment granules in ectrly development
Cortical pigment granules homogeneously embedded in the whole cortex after
fertilization are not carried into the boundary of the blastomeres after cleavage, but
7
8
9b
Figs. 7-9. Cortical pigment granules at the 16-cell stage. Figs. 7 and 8: Eggs exposed to 0.01% trypsin for lO minutes and then insemina.ted in normal sea water. 2 hours and 10 minutes after insemination (X 270). Fig. 9: The 16-cell stage of normally fertilized embryo, 2 hours and 20 minutes after insemination. Optical section (a)
and sm·face view (b) (X 270). The bar indicates 10 p;m.
---J?J GMENT URAN ULE .MJGR,ATJ.ON 11:3
remam 111 the periphery of the embryo (MoTOMUR-A, 1935). In several sea urchin species, a clear zone lacking pigment granules appears around the vegetal pole by the
fourth cleavage, resulting in the formation of unpigmented micromeres (TANAKA,
1979; ScHR-OEDEit, 1980). The vegetal clear zone appears also in 1'emnopleums hctrdwicki embryos.
The 1'. hardwicki eggs reached the 16-cell stage and the 32-cell stage about 2 hours and 2.5 hours after .fertilization, respectively. At the fourth cleavage, the peripheral cortex of micromeres contained some pigment granules in the area near macromeres, but was unpigmented near the vegetal pole (Figs. 7-9). At the fifth cleavage, the primary micromere divided into smaller and larger micromeres. The
large micromeres contained small numbers of pigment granules, while the small
micromeres lacked them (Figs. 10 and 11).
10a
10b
Figs. 10 and 11. The 32-cell stage, about 2.5 hours after insemination. Views from the vegetal pole (Fig. 10) and optical sections (Fig. 11). Small micromeres do not contain pigment granules (X 270). The bar indicates 10 pm.
Dist1·ibtttion. of cortical pigment granules in ]Xt1·thenogenetically activated eggs
Eggs activated by the treatment with urea or procaine fmmed the aster, but did not cleave. Thus, I examined whether the vegetal clear zone appeared without
114 K. OSA)iA(
cleavage after the
tim
e
corresponding
to
the 16-cell
stage.
Unfe
r
t
ili
zed
eggs
were exposed to
1
M
u
rea
for
10
seco
nd
s
and then
plac
ed
in
norm
al sea
water.
The
egg
s
elevated the fertilizat
i
on envelope and formed
the
mona
ster
.
Af
ter
about 3
hour
s
.
cortical
pigme
nt
granule
s
aggregated in
s
mall
groups
.
A wide unp
i
gmented area was observed in
t
h
e cortex (F
i
g.
12).
The
aggregation of the pigment granules wa
s
also observed in
the
eggs
incubated
in
p
rocaine
for about 3
h
ours. Some
procaine-t
re
ate
d
eggs
s
howed a concavity
of the
s
urf
ace,
which lacked the cortica
l
pigment
granules.
A part
i
ally elevated
e
n
ve
l
ope
wa
s
ob
se
rved on t
h
e
concave
surface (
Fig
.
13).
I
t was not ascertained
whether these unpigmented
areas
corre
s
pond
ed
to the vegeta
l
clear zone o
f
Arbaciaeggs
shown
by ScmWEDER (1980).
12
13b
-""!~,..
-- -\
..L•'igs. 12 and 13. Clear areas in activated eggs \\'ithout cleavage. Fig. 12: Urea-trea.ted
egg which was exposed to l M urea for 10 seconds and then placed in normal sea
water for 3 hours and 50 minutes. Surface view (X 270). Fig. 13: Procai ne-activated egg which was incubated in sea water containing 5 mlVI procaine for 5 hours.
a-c: Serial optical sections. Concave . surface (an·my) lacking pigment grunules is observed in a. and b (X 270). The bar indicate 10 ttm.
PIGMENT GRANULE MIGRATION 115
DISCUSSION
Pigm
ent
granules randomly
distributed in unfertilized
eggs of
sea
urchins
migrates to the cortex after fert
ili
zation (ALLEN and RowE, 1958
;
0
SAN
AI, 1969;
ScHROEDER
1
980).
Ithad been proposed
that
pigment granule migration depends
o
n
ooplasmic activation
relating
to
cortical
granu
l
e
di
scharge (ALLEN
and RowE,
1958;
OsANA
I
, 1963, 1964).
However,
pigment granule migration occurs without
cortica
l
granule discharge in the procaine-treated
eggs
.
This
shows
that pigment
granule migration
doe
s
not direct
l
y corre
l
ate with cortical granule discharge.
Sea urchin eggs parthenogenet
i
cally activated w
i
th ammonia do not discharge
cortica
l
granules, b
u
t the egg
s
urface
ha
s
man
y
extended
microvilli.
T
he
egg
activation was considered to be
du
e
to
th
e
r
e
l
ease
of a
s
urface
inhibitor that caused
the
extension of surface microvilli (l\'lAz
i
A 197 4; MAZIA
et al.,197
5;
JOHNSON
and
EPEL,
1975). The
extension of mocrovilli without cortical
g
ranule
discharge was
observed
in
procaine-treated eggs. The microvilli and
cor
t
ex
of
sea
urch
i
n eggs
contain actin. The extens
i
on of t
h
e microvilli
i
s
attributed to the po
l
ymerization
of
actin fi
l
aments (BuRGESS and ScHROEDER,
1977: BEGG
and REB
H
UM,
1979;
SPUDICH and SPUDICH,
1979). Pigment
granule migrat
i
on is inhibited by
cyto-cha
l
asin B (BELANGER and RuSTAD
,
1972; SAWADA
and
OsANAI,
19
80).
The
pigment
granules
l
odged in the cortex are
lib
era
ted
again
into
the inner
cytop
la
sm
by
cytochalasin
B
(SAwADA and 0SANAI
,
1980). This
s
uggests that the migration
and
em
bedding
of the
pigme
nt
granules in the cortex relate to actin filaments.
Procaine may activate an actin-related
system,
inducing microvillous
ex
ten
s
ion
and
pigment granu
l
e
migration.
Whether microvillous
extens
ion
is prerequisite to the
pigment granule m
i
gration requires further
exami
nation.
Micromeres
produced
after the fourth cleavage contain
little
or no pigment.
Two
explanations on the appearence of the
unpigm
ented
micromere
s
can be
considered; (
1)
The
s
urface
of the micromeres at the 32
-
cell
stage
is derived from the
boundary
s
u
rface which is not underlain by the cortical pigment
gra
nul
es.
(2) The
clear cortex lacking the pigment granules appears around the vegetal pole
independent of cleavage and t
h
e
inicromere
s
are formed only
of
this clear part of the
cortex.
The
vegetal clear zone appears
prior
to ferti
li
zation in
Paracentrotus lividuseggs.
Fertilized
Arbaciaeggs
incubated in
colchicine form a circular clear
zone though cleavage is b
l
ocked (ScHROEDER,
19
8
0).
Ifthe
clear area
o
b
served
in
the urea-treated egg and
the
concave
sur
face
in the procaine-incubated egg appear
at t
h
e
vegetal pole, the present observation may
sup
port
the second
po
ssibility
proposed
by
ScHROEDER
(1980).
W
h
ether these unp
i
gmented areas corre
s
pond
with the vegeta
l
clear zone remains to
be
examined.
HEFERENCE
S
ALLEN, R.D. and E.C. RowE, 1958 The dependence of pigment granule migration on the
116 K. OSANAI
BEGG, D.A. and L.I. REBlroM, 1979 pH regulates the polymerization of actin in the sea urchin egg cortex. J. Cell Bioi. 83: 241-248.
BELANGER, A.M. and R.C. RusTAD, 1972 Movements of eohinochrome granules during the early development of sea urchin eggs. Nature, New :Sial. 239: 81-83.
BUBGESS, D.R. and T. SoBROEDER, 1977 Polarized bundles on actin filaments within microvilli of fertilized sea urchin egga. J. Cell Bioi. 74: 1032-1037.
JoHNSON, J.D. and D. EPEL, 1975 Relationship between release of surface proteins and metabolic activation of sea urchin eggs at fertilization. Pro c. N atl. A cad. Sci. USA 72: 4474-4478.
MAziA, D., 1974 Chromosome cycles turned on in unfertilized sea urchin eggs exposed. to NR,OH. Proc. Nat!. Acad. Sci. USA 71: 690-693.
MA.zu, D., G. SOHATTEN and R. STEINHARDT, 1975 Turning on of activities in unfertilized sea urchin eggs: Correlation with changes of surface. Proc. Natl. Acad. Sci. USA 72: 4469-4473.
MOTOMUBA, I., 1934 On the mechanism of fertilization and development without membrane formation in the sea urchin egg, with notes on a new method of artificial par-thenogenesis. Sci. Rept. Tohoku Imp. Univ. Ser. IV (Bioi.) 9: 33-45.
MOTOMURA, I., 1935 Determination of the embryonic axis in the eggs of Amphibia and echinoderms. Sci. Rept. Tohoku Imp. Univ. Ser. IV (Bioi.) 10: 211-245. MoTOMU:RA, I., 1941 Materials of the fertilization membrane in the eggs of echinoderms.
Sci. Rept. Tohoku Imp. Univ. Ser. IV (Bioi.) 16: 345-363.
MoY, G.W., B. BRANDRIFF and V.D. VAOQUIER, 1977 Cytoasters from sea urchin eggs parthenogenetically activated by procaine. J. Cell Bioi. 73: 788-793.
OsANAI, K., 1963 Relation between cortical change and endoplasmic activation in the sea urchin eggs. Jap. Jour. Exptl. Morph. 17: 86-94. (In Japanese)
OsANAI, K., 1964 Ecto- and endoplasmic relation in the partially activated egg of the sea urchin. Sci. Rept. Tohoku Univ. Ser. IV (Bioi.) 30: 105-117.
OsANAI, K., 1969 Behavior of pigment granules during early development in the eggs of a sea urchin, Temnopleurus hardwickii. Ann Rept. Fac. Educat. Univ. Iwate 29, Pt. 3: 35-38.
SAWADA, T., and K. OsANAI, 1980 Movements of the pigment granules in the egg of Temnopleurus hardwicki. Bull. Mar. Bioi. Stn. Asamushi, Tohoku Univ. 16:
213-219.
SCHROEDER, T.E., 1980 Expressions of the prefertilization polar axis in sea urchin eggs. Develop. Bioi., 79: 428-443.
SPUDIOH, A. and J.A. SPUDIOH, 1979 Actin in Triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs. J. Cell Bioi. 82: 212-226.
TANAKA, Y., 1979 Effects of the surfactants in the cleavage and further development of the sea urchin embryos. II. Disturbance in the arrangement of cortical vesicles and change in cortical appearance. Develop. Growth Differ. 21: 331-342.
VAOQUIER, V.D. and B. BRANDRmF, 1975 DNA synthesis is unfertilized sea urchin egg can be turned on and turned off by the addition and removal of procaine hydro-chloride. Develop. Bioi. 47: 12-31.
