Biochemical and Biophysical Research Communications 290, 1015–1021 (2002)
doi:10.1006/bbrc.2001.6286, available online at http://www.idealibrary.com on
Ca 2⫹ Response to cADPr during Maturation
and Fertilization of Starfish Oocytes
Gilda A. Nusco,* Dmitri Lim,* Pawel Sabala,† and Luigia Santella* ,1
*Laboratory of Cell Biology, Stazione Zoologica “A. Dohrn” Villa Comunale, I-80121, Naples, Italy; and †Department
of Molecular and Cellular Neurobiology, Polish Academy of Sciences, Nencki Institute, 02-093 Warsaw, Poland
Received December 18, 2001
During the reinitiation of the meiotic cycle (maturation) induced by the hormone 1-methyladenine (1-MA),
starfish oocytes undergo structural and biochemical
changes in preparation for successful fertilization.
Previous work has shown that the sensitivity of internal Ca 2ⴙ stores to InsP 3 increases during maturation of
the oocytes. Since Astropecten auranciacus oocytes
also respond to cADPr, we have studied whether the
response to cADPr also changes during maturation.
We have found that the photoactivation of injected
cADPr in immature oocytes immediately induces multiple patches of Ca 2ⴙ release in the cortical region. The
Ca 2ⴙ signal then spreads from these initial points of
increase to the entire cell. In mature oocytes, the uncaging of cADPr induces instead a single (or at most a
dual) initial point of Ca 2ⴙ release, which is immediately followed by the formation of a cortical Ca 2ⴙ flash
and then by the globalization of the wave and by the
elevation of the fertilization envelope. External Ca 2ⴙ
plays a role in the Ca 2ⴙ responses. Inhibition of L-type
Ca 2ⴙ channels does not affect the initial Ca 2ⴙ release, but
abolishes the cortical flash and impairs the elevation of
the fertilization envelope. External Ca 2ⴙ has other effects, as shown by the irregular appearance of the surface of oocytes incubated in Ca 2ⴙ-free sea water. The
sequence of Ca 2ⴙ responses induced by cADPr in mature
oocytes mimics those seen at fertilization, i.e., a first
localized Ca 2ⴙ increase followed by a cortical flash and
by the globalization of the Ca 2ⴙ signal. As in the case of
maturation, L-type Ca 2ⴙ channel blockers abolish the
sperm induced cortical flash. © 2002 Elsevier Science (USA)
Key Words: starfish oocytes; maturation; fertilization; cADPr; Ca 2ⴙ response; nifedipine.
Fully grown starfish oocytes are arrested in the
ovary at the prophase of the first meiotic division. At
this stage of the meiotic cycle (maturation) the oocytes
are characterized by a large nucleus (germinal vesicle).
To whom correspondence should be addressed. Fax: ⫹39 081 764
1355. E-mail: [email protected].
1
The resumption of meiosis can be syncronously induced in vitro by adding the maturing hormone
1-methyladenine (1-MA) to the arrested oocytes suspended in sea water (1). The first morphological sign
that the meiosis reinitiation is going on is indicated by
the breakdown of the germinal vesicle (GVBD). During
the maturation process the oocytes undergo the structural and biochemical changes required for a successful
fertilization. Earlier work in our laboratory has detected increases in both cytoplasmic and nuclear calcium following the addition of 1-MA to the oocytes. In
our hands, the nuclear Ca 2⫹ increase was essential
because the injection of BAPTA into the nucleus completely blocked the continuation of meiosis (2, 3). The
nature of the Ca 2⫹ stores mobilized by 1-MA is still
discussed. Although previous work has shown that the
sensitivity of internal Ca 2⫹ stores to inositol 1,4,5trisphosphate (InsP 3) increases during maturation of
starfish oocytes (4) it has been recently claimed that
InsP 3 dependent calcium release (IICR) is not essential
for meiosis reinitiation (5). In addition to the sensitivity to InsP 3 maturation also increases the sensitivity to
the new Ca 2⫹-messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in Asterina pectinifera
oocytes. We have shown that the response to NAADP is
linked to the external Ca 2⫹ and plays an important role
in initiating the Ca 2⫹ response at fertilization (6, 7).
These oocytes do not respond to the other Ca 2⫹mobilizing second messengers cyclic ADP-ribose
(cADPr). At variance with Asterina pectinifera, Astropecten auranciacus oocytes respond to cADPr (8). In
this contribution we have studied whether the response to cADPr also changed during maturation in
these oocytes. Photoactivation of injected caged cADPr
induced a Ca 2⫹ response which varied substantially at
different times of maturation.
MATERIALS AND METHODS
Starfish (Astropecten auranciacus) were collected during the
breading season in February–June in the gulf of Naples and kept in
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FIG. 1. Ca 2⫹ response induced by the photoactivation of cADPr in immature oocytes. (A) The Ca 2⫹ increase starts simultaneously in
several cortical regions, then spreads to the entire cytoplasm. (B) Light microscopy image of an immature oocyte with the nucleus (n) showing
the region (circle) where the Ca 2⫹ increase is measured. (C) The graph of the relative fluorescence of the Ca 2⫹ increase following the liberation
of cADPr shows the numerical equivalent of the colours in the fluorescent images.
FIG. 2. Ca 2⫹ elevation following the photoliberation of cADPr in a mature oocyte. (A) The Ca 2⫹ response initiates in a circumscribed
cortical site and is followed by the formation of a cortical flash (arrow). (B) Light microscopy image showing the region (circle) where the Ca 2⫹
elevation is measured. Fertilization envelope elevation induced by the uncaging of cADPr (arrow). (C) Graph of the relative fluorescence of
the Ca 2⫹ increase following the photoactivation of cADPr.
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FIG. 3. Ca 2⫹ response induced by the photoactivation of NAADP in a mature oocyte. (A) The Ca 2⫹ increase is limited to the cortical region
of the oocyte and fails to spread throughout the remainder of the cell. (B) Light microscopy image showing the region where the Ca 2⫹ elevation
occurs. The Ca 2⫹ elevation induced by NAADP is not sufficient to trigger the cortical granules exocytosis (arrow). (C) Graph of the relative
fluorescence of the Ca 2⫹ increase measured in the cortex as shown in Fig. 3B.
FIG. 4. Role of the L-type Ca 2⫹ channel inhibitor nifedipine on the cortical Ca 2⫹ flash induced by the uncaging of cADPr. (A) The uncaging
of cADPr in oocytes kept in sea water containing nifedipine fails to generate a cortical flash. (B) Light microscopy image showing the
inhibition of the elevation of the fertilization envelope. (C) Graph showing the relative fluorescence of the Ca 2⫹ increase following the
uncaging of cADPr in oocytes kept in normal sea water (control) and in sea water containing nifedipine.
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running natural sea water (16°C). Immature oocytes (containing the
germinal vesicle, nucleus) were dissected from the ovaries and kept
in artificial sea water (ASW) (ASW; 490 mM NaCl, 8 mM KCl, 10 mM
CaCl 2, 12 mM MgCl 2, 2.5 mM NaHCO 3, pH 8.0) for 30 min before
use. Oocytes in which the breakdown of the germinal vesicle (GVBD)
occurred spontaneously were discarded. Maturation was promoted
by adding the hormone 1-methyladenine (Sigma Chemical Co., St.
Louis, MO) at a final concentration of 5 ␮M. For fertilization experiments, 1 ␮l of dry sperm was suspended in 2 ml of artificial sea
water, and 40 ␮l of this suspension were added to 1 ml of the oocyte
suspension to obtain a final sperm dilution of 1:50000.
Microinjections. The calcium fluorescent dye, Oregon Green 488
BAPTA-1 coupled to a 10 kDa dextran (OGBD, Molecular Probes,
Eugene, OR) was injected into the cytoplasm of immature and mature oocytes. The concentration of the dye in the pipette (diameter of
the tip 1 ␮m) was adjusted to 5 mg/ml with injection buffer (IB, 450
mM potassium chloride, 10 mM HEPES, pH 7.0).
For the experiments with the caged compounds the solution in the
pipette contained 400 ␮M caged cADPr (Calbiochem, La Jolla, CA) or
100 ␮M caged NAADP (Molecular Probes) in IB. The volume of
injected dye corresponded to 1–2% of the total cell volume: thus, the
final concentration of injected substances in the cellular environment was 50 –100 times lower than in the micropipette.
For the experiments with Ca 2⫹-free sea water (CaFSW) injected
with caged compounds oocytes were transferred for 3 min in a solution containing (500 mM NaCl, 8 mM KCl, 12 mM MgCl 2, 2.5 mM
NaHCO 3, 2 mM EGTA, pH 8.0).
Photolysis of caged cADPr and Ca 2⫹ imaging. Caged cADPr was
injected in immature or mature oocytes. Photolysis of the caged
compound was performed using a computer controlled shutter
(Lambda 10-2, Sutter Instruments, Co., Novato, CA) by irradiating 7
times with a wavelenght of 330 nm. Cytosolic Ca 2⫹ changes were
measured every sec using a cooled CCD camera (MicroMax, Princeton Instruments, Inc., Trenton, NJ) mounted on a Zeiss Axiovert 200
microscope with a Plan-Neofluar 20⫻/0.50 objective or a confocal
laser scanning microscope Olympus FVX-ZM-IL (Olympus Optical
Co., Ltd., Japan), an UplanApo 20⫻/0.70 objective, laser power 20%
and confocal aperture No. 2. Fluorescence images were processed
with a MetaMorph Imaging System software (Universal Imaging
Corporation, West Chester, PA). To exclude variations of fluorescent
intensity, the signals were corrected for variations in dye concentration by normalizing fluorescence (F) against baseline fluorescence
(F 0). The antagonists of the L-type sensitive Ca 2⫹ channels (Sigma)
were prepared as a 1 mM stock solution in DMSO and kept frozen.
RESULTS
Spatio-Temporal Pattern of Ca 2⫹ Response to cADPr
during Maturation
Immature oocytes injected with caged cADPr were
irradiated for 3 s 10 min after the injection. Two seconds following the photoactivation of cADPr, a Ca 2⫹
elevation started simultaneously in several cortical regions, spread to the entire cytoplasm and decayed after
about 20 s. Figure 1A shows the fluorescent images
taken at 1 s intervals after the liberation of cADPr. The
cortical region of the oocytes showed a level of fluorescence which was much higher than in the center of the
oocyte. The graph of the relative fluorescence of the
dye, measured in the center of the cell (Fig. 1B) shows
the numerical equivalent of the colors shown in the
fluorescent images of Fig. 1A. The relative fluorescence
of the Ca 2⫹ increase reached an amplitude of 1–1.2 ⫾
0.1 arbitrary units and decayed to the baseline level
20 s later (Fig. 1C).
Ca 2⫹ Response Induced by cADPr in Mature Oocytes
The Ca 2⫹ response to the uncaging of cADPr in mature oocytes 50 min after the addition of 1-MA was
strikingly different from that observed in immature
oocytes. Figure 2A (first fluorescence image) shows
that the response initiated 2 s after the irradiation in
one (or at most a dual) circumscribed cortical site. This
initial Ca 2⫹ release was followed 2 s later by the formation of a cortical Ca 2⫹ flash (second fluorescent image). The cortical flash failed to decay, but spread instead centripetally to the center of the oocyte in about
10 s. The global Ca 2⫹ increase then decayed to the
baseline level in about 1 min. The graph of the relative
fluorescence shows that the Ca 2⫹ increase reached a
peak level of 1.3 ⫾ 0.1 (Fig. 1C), measured in the center
of the oocyte (see Fig. 2B). The cortical Ca 2⫹ flash and
the subsequent centripetal spreading of the wave triggered the elevation of the fertilization envelope, as a
result of the exocytosis of the cortical granules (Fig.
2B). The pre-injection of the specific antagonist of the
cADPr/ryanodine receptors 8NH 2cADPr completely
blocked the Ca 2⫹ response. The latter was also analyzed in immature and mature oocytes using confocal
microscopy, monitoring the center of an equatorial
plane of the oocyte. The confocal images confirmed the
findings with the cooled CCD camera (data not shown).
Ca 2⫹ Response Induced by the Photoactivation
of NAADP
Previous work in our laboratory had shown that the
recently discovered second messenger NAADP induced
a cortical Ca 2⫹ response in the starfish species (Asterina pectinifera) which was linked to the external Ca 2⫹
(6, 7). In this contribution we have investigated
whether Astropecten auranciacus oocytes also responded to NAADP. Photoactivation of the latter indeed induced a strictly cortical Ca 2⫹ liberation in both
immature and mature oocytes. The Ca 2⫹ response was
analyzed using both a cooled CCD and a confocal laser
microscope, monitoring the cortical region and the center of an equatorial plane of the oocyte. We have found
that the response to NAADP was lower in immature
oocytes (not shown) whereas it was very evident, and
strictly limited to the cortical domain in mature oocytes suspended in normal sea water (Fig. 3A). At
variance with the Ca 2⫹ response in Asterina pectinifera
oocytes (6, 7), the Ca 2⫹ increase failed to spread
throughout the oocyte and to trigger the elevation of
the fertilization envelope. The graph of the relative
fluorescence shows that the Ca 2⫹ increase in the cortex
reached a peak of 0.55 ⫾ 0.1 (n ⫽ 9), which decayed to
the baseline in about 10 s. The cortical response measured following the uncaging of NAADP was com-
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pletely blocked when the irradiation was performed on
oocytes kept in Ca 2⫹ free sea water (data not shown).
Role of External Ca 2⫹ in the cADPr-Induced Cortical
Ca 2⫹ Flash
To investigate whether the cortical Ca 2⫹ flash induced by the photoactivation of cADPr was due to the
influx of external Ca 2⫹ and to explore the nature of the
possible Ca 2⫹ entry path, mature oocytes were kept in
normal sea water containing the L-type Ca 2⫹ channel
inhibitor nifedipine. Figure 4A shows that the uncaging of cADPr produced the normal initial circumscribed
cortical site of fluorescence, which was followed by the
globalization of the wave without first generating a
cortical flash. The latter was thus generated by the
influx of external Ca 2⫹. The graph of the relative fluorescence in Fig. 4C shows that in addition to inhibiting
the cortical Ca 2⫹ flash, nifedipine also affected the
overall intracellular Ca 2⫹ increase, measured in the
center of the oocyte (Fig. 4B) which was much faster
than that measured in the controls: The propagation of
the Ca 2⫹ wave to the remainder of the cell occurred in
about 2 s (Fig. 4A). At variance with control oocytes, in
which the Ca 2⫹ increase decayed to the baseline level in
about 50 s, in the presence of nifedipine the increase
failed to reach the baseline during the time of measurement (about 3 min). The Ca 2⫹ channel inhibitor also
inhibited the elevation of the fertilization envelope
(Fig. 4B).
The absence of external Ca 2⫹ also abolished the cortical flash. However, it affected the spatio-temporal
pattern of the Ca 2⫹ response in additional ways. Figure
5A shows that it induced multiple initial points of Ca 2⫹
release throughout the entire oocyte, strikingly resembling the response to the photoactivation of cADPr in
immature oocytes. It also conferred to the surface of
the oocyte an irregular appearance (Fig. 5B). The fertilization envelope elevated simultaneously all over the
surface, but failed to completely detach from its surface.
Ca 2⫹ Response at Fertilization
The Ca 2⫹ response to the addition of spermatozoa
observed in this contribution using a cooled CCD camera strikingly resembled that induced by the photoactivation of cADPr. Figure 6A shows that in all imaged
oocytes (n ⫽ 10) the cortical flash (third fluorescent
image) started 2 s after the first Ca 2⫹ elevation at one
cortical point (first fluorescent image) reaching a relative fluorescence amplitude of about 0.3 ⫾ 0.1, and
decaying afterwards in about 4 s. None of the reports
available in the literature had so far shown a distinct
point-source pattern of initiation of the signal. This
was probably due to the difficulty of detecting a Ca 2⫹
increase restricted to a very limited region using only a
confocal plane of the oocyte instead of imaging the
whole cell in fertilization as well in the response to
cADPr. The wave spread from the initial point of initiation throughout the oocyte reaching an amplitude of
0.9 ⫾ 0.1 (n ⫽ 10) arbitrary units in about 3 min, at a
rate of 1.65 mm/s. The graph of the relative fluorescence in Fig. 6C shows the Ca 2⫹ response to the sperm
measured in different regions of the oocyte. The cortical flash was uniformly distributed in the contour of
the oocyte and particularly well visible at the pole
opposite to the point of sperm fusion (see the arrow in
the graph of Fig. 6C), where the cortical flash was
difficult to detect because of the superposition of the
propagating wave.
Also in the case of fertilization the cortical flash
involved calcium influx from outside. Experiments
with nifedipine (Fig. 6B) showed that the blockade of
the L-type Ca 2⫹ channels had no effect on the circumscribed initial cortical Ca 2⫹ release nor on the globalization of the signal, but it abolished the cortical Ca 2⫹
flash. The graph of the relative fluorescence measured
in the cortex and in the center of the oocyte shows that
the cortical flash is no longer visible (Fig. 6D) (see the
arrow in Fig. 6C for comparison). The pre-injection of
8NH 2cADPr into mature oocytes prior to the addition
of the spermatozoa failed to affect the Ca 2⫹ responses
induced by the sperm, showing that the initial Ca 2⫹
increase and the globalization of the wave were not
linked to a Ca 2⫹-induced Ca 2⫹ release (CICR) mechanism.
DISCUSSION
At fertilization sperm activate eggs by inducing a
calcium wave which starts from the sperm egg point of
interaction and travels throughout the cell. The pattern of calcium change may vary from a single Ca 2⫹
transient (9) to repetitive Ca 2⫹ oscillations (10). In
frogs, mammalians and ascidians Ca 2⫹ increase at fertilization is blocked by the pre-injection of heparin
(11–13). In contrast, the application of heparin or of
antagonists of the ryanodine receptor, such as ruthenium red or 8NH 2cADPr, does not block the sperm
induced Ca 2⫹ wave in sea urchin eggs, but a combination of heparin and ruthenium red or 8NH 2cADPr inhibits its increase (14, 15). This has led to the suggestion that the Ca 2⫹ wave is regulated by two types of
mechanisms: IICR mediated by the InsP 3 receptors
and CICR mediated by the ryanodine receptors (16). In
starfish oocytes of the species Astropecten auranciacus
the pre-injection of heparin into the cytoplasm of mature oocytes induced a delay in the propagation of the
Ca 2⫹ wave at fertilization. By contrast, when oocytes
were injected with 8NH 2cADPr no inhibition in the
propagation of the Ca 2⫹ wave was observed (17) indicating that in these oocytes the globalization of the
wave was not linked to a CICR. The Ca 2⫹ response to
cADPr prior to the spreading of the wave to the entire
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FIG. 5. Role of the external Ca2⫹ on the Ca 2⫹ response induced by the photoactivation of cADPr. (A) The absence of external Ca2⫹ abolishes
the cortical flash and induces multiple initial points of Ca2⫹ release. (B) Light microscopy images of a mature oocyte showing surface irregularity
and the failure to elevate the fertilization envelope (arrow).
FIG. 6. Ca 2⫹ response induced by the sperm. (A) The Ca 2⫹ increase begins at a circumscribed cortical spot and is followed by a cortical
flash and by the globalization of the wave in oocytes fertilized in normal sea water. (B) The blockade of the L-type Ca 2⫹ channels by nifedipine
only abolishes the cortical flash. (C) The graph of the relative fluorescence of the Ca 2⫹ response in normal sea water (control) showing the
cortical flash well visible at the cell pole opposite to the point of sperm fusion (arrow). (D) The graph of the relative fluorescence of the Ca 2⫹
increase at fertilization in the presence of nifedipine shows that the cortical flash is no longer visible (see the arrow in Fig. 6C for comparison).
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cell has been studied here in oocytes of Astropecten
auranciacus. The Ca 2⫹ response induced by the photoliberation of cADPr initiated in the cortical domain in
both immature and mature oocytes identifying a more
sensitive area in the sub-surface region than in the
cytoplasm. Multiple patches of cADPr responsive zones
in this region join to form a circumscribed sensitive site
following maturation from which the uniform cortical
flash is generated. Since the cortical Ca 2⫹ flash induced
by cADPr was strongly affected by L-type Ca 2⫹ channel
inhibitors or by the removal of Ca 2⫹ from the medium,
cADPr receptors located beneath the plasma membrane are evidently linked to external Ca 2⫹. Thus,
cADPr receptors may play an important role in the
initial Ca 2⫹ response at fertilization in Astropecten auranciacus oocytes.
ACKNOWLEDGMENTS
The authors are very grateful to Mr. Gianni Gragnaniello for his
help with the preparation of the figures and to the Marine Resources
Service for maintaining the starfish.
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