Vol. 8, No. 1
REVIEW
Cytoplasmic maturation of mammalian oocytes:
development of a mechanism responsible for
sperm-induced Ca2+ oscillations
Anna Ajduk1, Antoni Małagocki, Marek Maleszewski
Department of Embryology, Institute of Zoology, University of Warsaw,
Warsaw, Poland
Received: 5 January 2008; accepted: 20 February 2008
SUMMARY
Oocytes of most mammalian species, including mouse and human,
are fertilized in metaphase of the second meiotic division. A fertilizing
spermatozoon introduces an oocyte-activating factor, phospholipase C zeta,
triggering oscillations of the cytoplasmic concentration of free calcium ions
([Ca2+]i) in the oocyte. [Ca2+]i oscillations are essential for the activation
of the embryonic development. They trigger processes such as resumption
and completion of meiosis, establishment of the block to polyspermy and
recruitment of maternal mRNAs necessary for the activation of the embryo
genome. Moreover, it has been recently shown that [Ca2+]i oscillations may
also influence the development of the embryo. The ability to generate [Ca2+]i
oscillations develops in mammalian oocytes during meiotic maturation
and requires several cytoplasmic changes, including: 1/ reorganization
of endoplasmic reticulum, the main stockpile of calcium in the oocyte,
2/ increase in the number of 1,4,5-inositol triphosphate (IP3) receptors,
Corresponding author: Department of Embryology, Institute of Zoology, University of Warsaw, ul.
Miecznikowa 1, 02-096 Warsaw, Poland; e-mail: [email protected]
1
Copyright © 2008 by the Society for Biology of Reproduction
Sperm-induced Ca2+ oscillations in mammalian oocytes
3/ changes in their biochemical properties (e.g.: sensitivity to IP3), and
possibly both 4/ an increase in the concentration of Ca2+ ions stored in
endoplasmic reticulum (ER) and 5/ redistribution of Ca2+-binding ER
proteins. The aim of this review is to present the state of current knowledge
about these processes. Reproductive Biology 2008 8 1:3-22.
Key words: oocyte, maturation, mammal, mouse, calcium, calcium
oscillations, endoplasmic reticulum, IP3 receptor, calreticulin, calnexin
INTRODUCTION
Oocytes of majority of mammals, including mouse and human, are fertilized
in metaphase of the second meiotic division. A fertilizing spermatozoon
introduces an oocyte-activating factor, phospholipase C zeta (PLC zeta),
a sperm-specific isoform of phospholipase C [50]. PLC zeta cleaves
phosphatidylinositol 4,5-bisphosphate (PIP2) to 1,4,5-inositol triphosphate
(IP3) and diacylglycerol. IP3 binds to its receptor located in the membranes
of endoplasmic reticulum (ER), inducing the opening of the Ca2+ channels
and release of Ca2+ ions from ER lumen into the cytoplasm. This initiates
the oscillations in the concentration of free Ca2+ ions ([Ca2+]i) in the oocyte
cytoplasm. [Ca2+]i oscillations cease after a few hours, at the time of the
pronuclei formation (reviewed in [29]).
[Ca2+]i oscillations are essential for activation of the embryo development.
They trigger processes such as: resumption and completion of meiosis,
establishment of the block to polyspermy and recruitment of maternal
mRNAs necessary for the activation of the embryo genome (reviewed
in [11]). Recently it has been suggested that [Ca2+]i must be elevated for
different times to properly induce each of these processes [44, 45, 55]. In
other words, the above events are induced by a different number of [Ca2+]i
transients and therefore occur in a certain order, i.e. cortical granules
exocytosis, a main mechanism of the mammalian block to polyspermy,
is initiated before the resumption of meiosis and recruitment of maternal
mRNAs [10]. In addition, [Ca2+]i oscillations influence the development of
the embryo. If the number of [Ca2+]i transients is too low (i.e. total duration
Ajduk et al.
of elevated [Ca2+]i is too short), the embryo implantation is impaired [45,
55]. On the other hand, an excessive number of [Ca2+]i transients (i.e.
[Ca2+]i is elevated for too long) affects postimplantation development of
the embryo [45]. The processes involved in sperm-induced activation of
embryonic development are summarized in Figure 1.
Figure 1. Activation of the embryonic development. Sperm derived oocyte-activating
factor leads to IP3R1-mediated Ca2+ release. Increase in [Ca2+]i triggers several
processes, e.g.: resumption of meiosis, establishment of a block to polyspermy or
recruitment of maternal mRNAs. It also influences further stages of the embryo
development. PLC zeta – phospholipase C zeta, PIP2 – phosphatidylinositol 4,5bisphosphate, IP3 – 1,4,5-inositol triphosphate, DAG – diacylglycerol, IP3R – 1,4,5inositol triphosphate receptor, ER – endoplasmic reticulum
The ability to generate [Ca2+]i oscillations develops in mammalian
oocytes during meiotic maturation, i.e. between prophase of the first
meiotic division (prophase I) and metaphase of the second meiotic division
(metaphase II, MII). Oocytes fertilized in prophase I generate fewer [Ca2+]i
transients than oocytes fertilized in metaphase II [6, 22]. Moreover, the
first [Ca2+]i peak has higher amplitude and lasts longer in mature than in
immature oocytes [5, 22, 37]. Interestingly, the development of a mature
oocyte-like response to sperm does not require transition to metaphase
II. Oocytes spontaneously arrested in metaphase I, unable to achieve
metaphase II even after a prolonged culture, produce continuous [Ca2+]i
oscillations similar to these generated in fertilized MII oocytes [22]. This
Sperm-induced Ca2+ oscillations in mammalian oocytes
suggests that development of the oocyte ability to generate long-lasting
[Ca2+]i oscillations depends rather on cytoplasmic changes than on the
progression of meiosis. Cytoplasmic changes occurring during oocyte
maturation include: 1/ reorganization of endoplasmic reticulum, the main
calcium storage in the oocyte, 2/ increase in the number of IP3 receptors,
3/ changes in their biochemical properties (e.g. sensitivity to IP3), and
possibly 4/ increase in the concentration of Ca2+ ions stored in ER, and 5/
redistribution of Ca2+-binding ER proteins (fig. 2). The goal of this review
is to summarize the state of current knowledge about these processes.
Figure 2. Cytoplasmic maturation of mammalian oocytes. An oocyte ability to
generate sperm-induced long-lasting [Ca2+]i oscillations develops during meiotic
maturation and requires several cytoplasmic changes, such as: reorganization
of endoplasmic reticulum, the main calcium store in the oocyte, increase in the
number of IP3 receptors, changes in their biochemical properties (e.g.: sensitivity
to IP3), and possibly increase in the concentration of Ca2+ ions stored in ER and
redistribution of ER Ca2+ –binding proteins. ER – endoplasmic reticulum, IP3R1
– 1,4,5-inositol triphosphate receptor type 1, [Ca2+]i – cytoplasmic concentration
of free Ca2+ ions, GV – germinal vesicle, MII – metaphase II
Ajduk et al.
MECHANISM OF GENERATION OF [CA2+]i OSCILLATIONS IN
MAMMALIAN OOCYTES
Increased [Ca2+]i can be caused by a Ca2+ release from the ER lumen or
by an influx of Ca2+ ions from the external environment. The Ca2+ release
from ER lumen occurs through the IP3 receptors (IP3R). In mammalian
cells, three different IP3R genes have been identified. They encode three
closely related IP3R proteins with a different expression pattern [48].
The majority of somatic cells express all three IP3R isoforms in various
combinations, but oocytes predominantly express one isoform – type 1
IP3 receptor (IP3R1; [13, 47]). The IP3R1 assembles and functions as a
tetramer with each subunit sharing a common triple-domain organization
(fig. 3). The cytosolic N-terminal portion of the IP3R1 protein contains the
Figure 3. Structure of type 1 IP3 receptor. IR3R1 consists of three domains: an
IP3 binding domain, a regulatory domain and a Ca2+ channel domain. IP3 - 1,4,5inositol triphosphate, ER – endoplasmic reticulum
IP3 binding site. The Ca2+ channel domain is located at the C- terminal end
and consists of six membrane-spanning helices with C-terminus projecting
into the cytoplasm. The IP3-binding domain and the channel pore are
linked by a regulatory domain (over 1700 amino acids) which contains
multiple phosphorylation sites recognized by calmodulin/Ca2+-regulated
kinases, protein kinases C, A and G, M-phase kinases, as well as sites
binding calcium and other modulatory molecules and enzymes [48].
Sperm-induced Ca2+ oscillations in mammalian oocytes
Among all the molecules that regulate IP3R1, IP3 and Ca2+ ions are the
most important. At physiological cytoplasmic calcium concentrations (~
100-300 nM), the Ca2+ channel opens and Ca2+ ions are released into the
cytoplasm (following the gradient of Ca2+ ions). When the [Ca2+]i increases
above a certain level (low micromolar concentrations), the Ca2+ channel
closes, release of Ca2+ stops and the uptake of Ca2+ ions to the ER lumen
is facilitated [48]. The uptake of Ca2+ ions is mediated by ATP-dependent
Ca2+ pumps (the sarco/endoplasmic reticulum Ca2+-ATPases, SERCA). The
restoration of Ca2+ level in the ER is required for the next Ca2+ release [3].
Experiments performed on Xenopus oocytes suggest that IP3R1
sensitivity to [Ca2+]i closely depends on the cytoplasmic level of IP3 [31].
The opening of the Ca2+ channel requires a relatively low level of IP3.
However, when IP3 concentration is low, the Ca2+ channel closes when
[Ca2+]i is only slightly higher than necessary for the opening. Consequently,
Ca2+ release is short lasting, and change in the [Ca2+]i is small and locally
confined but sufficient to cause a closure of the Ca2+ channel. As IP3
concentration rises, the Ca2+ channel of IP3R1 closes on higher [Ca2+]i and
Ca2+ release lasts longer, causing more pronounced change in [Ca2+]i.
In addition, research on Xenopus oocytes indicates that both SERCA
and IP3R1 can be regulated by interactions with ER proteins (fig. 4). One
of these proteins, calreticulin, is a soluble lumenal Ca2+-binding chaperon
regulated primarily by the ER environment such as a concentration of free
Ca2+ ions in the ER lumen ([Ca2+]ER; [7, 8]). Calreticulin forms a complex
with ERp57, an ubiquitous ER thiol-dependent oxidoreductase, that
promotes formation of protein disulfide bonds [7, 30]. Calreticulin can also
interact with SERCA, recruiting ERp57 in close proximity to the lumenal
loops of the pump [21, 30]. When Ca2+ stores are full (~ 300 µM), ERp57
catalyzes formation of a disulfide bond in one of the SERCA loops facing
the ER lumen. This modification inhibits activity of the pump. When Ca2+
stores become depleted (~ 10 µM) by IP3-mediated Ca2+ release, the ERp57
dissociates from SERCA, which results in the formation of a reduced and
more active form of SERCA [30].
Another ER chaperone protein, calnexin, is a transmembrane protein
with a cytosolic domain containing two conserved phosphorylation motifs
Ajduk et al.
Figure 4. Regulation of IP3R1 and SERCA by ER proteins. When Ca2+ stores
are full, complex calreticulin-ERp57 binds to SERCA, inhibiting activity of the
pump. When Ca2+ stores become depleted by IP3-induced Ca2+ release, complex
calreticulin-ERp57 dissociates from SERCA, what leads to the activation of
SERCA. The cytosolic domain of calnexin, another ER protein regulating SERCA,
is phosphorylated, when a cell is in a resting state and Ca2+ stores are full. This
phosphorylation promotes interaction of calnexin with SERCA, keeping the pump
in an inhibited state. Ca2+ release leads to dephosphorylation of calnexin and its
dissociation from SERCA. This in turn activates the pump. IP3R1 is regulated by
ERp44. Low concentration of Ca2+ ions in the ER lumen and free cysteine residues
in the lumenal part of IP3R1 enhance binding of ERp44 to IP3R1 and hinder Ca2+
release via IP3R1. ER – endoplasmic reticulum, IP3R1 – 1,4,5-inositol triphosphate
receptor type 1, SERCA - sarco/endoplasmic reticulum Ca2+-ATPase.
10
Sperm-induced Ca2+ oscillations in mammalian oocytes
(located around seronine 562 and seronine 485) recognized by protein
kinase C [56]. The cytosolic domain of calnexin becomes phosphorylated
when a cell is in a resting state and Ca2+ stores are full. This phosphorylation
promotes interaction of calnexin with SERCA, and inactivates the pump.
When Ca2+ ions are released from the internal stores by IP3, the [Ca2+]i
increases and activates a phosphatase that dephosphorylates calnexin.
Dephosphorylation of calnexin causes its dissociation from SERCA,
allowing the pump to refill the Ca2+ stores [49]. It is very probable that this
dephosphorylation is catalized by calcineurin, the only Ca2+/calmodulindependent seronine/threonine phosphatase identified so far in mammalian
cells [23, 24]. Recently, calcineurin has been reported to play a crucial role
in activation of Xenopus oocytes [41, 42].
Another ER protein participating in regulation of Ca2+ release is ERp44,
an ER lumenal protein of thioredoxin family. Higo et al. [17] showed that
in somatic cells, ERp44 interacts with one of the lumenal loops of IP3R1
in [Ca2+]ER- and redox state-dependent way and inhibits activity of the
receptor. Their results suggest that low [Ca2+]ER and free cysteine residues
in the lumenal part of IP3R1 enhance binding of ERp44 to IP3R1, that in
turn hinders Ca2+ release via IP3R1 and prevents a complete depletion of
the ER calcium stores. It is very plausible that similar mechanism operates
in mammalian oocytes.
Maintaining long-lasting [Ca2+]i oscillations requires also an influx of
2+
Ca ions from the external environment into the oocyte. In mouse and
hamster oocytes cultured in medium without Ca2+ ions, oscillatory changes
of [Ca2+]i cease prematurely or their frequency is greatly reduced [18, 26].
In oocytes, as in other non-excitable cells, the primary Ca2+ influx pathway
consists of store-operated calcium entry (SOCE; [46]), which is activated
by depletion of the intracellular Ca2+ stores. Entry of external Ca2+ into
a mouse oocyte is probably caused by the depletion of intracellular Ca2+
stores (mainly ER) caused by fertilization-induced Ca2+ release. Ca2+
influx is most prominent during the rise of the sperm-induced Ca2+ spike
(i.e. when Ca2+ stores become depleted). It slows down following the
decline of the Ca2+ transient (i.e. during refilling of Ca2+ stores) but usually
remains higher than the basal rate of influx measured in the latent period
Ajduk et al.
11
before the first spike [36]. The nature of the signaling pathway that conveys
the depleted state of the intracellular Ca2+ stores in cells to the Ca2+-influx
channels in the plasma membrane still remains unknown. In the past few
years, several general models have been proposed for the activation of
store-operated influx. One hypothesis is that upon store depletion, a soluble
Ca2+ influx factor diffuses from the empty store to Ca2+-influx channels in
the plasma membrane and activates them. Another possibility is that IP3
receptors located in ER membranes may interact with Ca2+-influx channels
in the plasma membrane and regulate Ca2+ entry [46].
REORGANIZATION OF ENDOPLASMIC RETICULUM DURING
MEIOTIC MATURATION OF MAMMALIAN OOCYTES
Endoplasmic reticulum is the main Ca2+ store in the oocytes. During oocyte
maturation, ER undergoes profound reorganization and this process seems to
play an important role in the establishment of the oocyte ability to generate
long-lasting [Ca2+]i oscillations. Since ER structure can be easily and
reliably visualized by dicarbocyanine dyes (DiI) and confocal microscopy
[25], the reorganization of the ER network has been well documented. In
oocytes in prophase I (germinal vesicle oocytes; GV oocytes) ER forms
a fine network with patch-like accumulations within the inner cytoplasm
(mouse oocytes; [15, 38]) or in the cortex area (hamster oocytes; [51]).
Following the breakdown of a germinal vesicle nuclear envelope (germinal
vesicle breakdown, GVBD), which is a sign of the meiosis resumption, ER
accumulates in a form of a dense ring in the centre of the oocyte around
the meiotic apparatus [15, 38, 51]. Confocal microscopy revealed that some
ER cisterns lie between the individual bivalents [15]. In later stages of
maturation, the ER ring moves together with meiotic spindle towards the
oocyte cortex. Formation of the ER ring is mediated by microtubules, since
in oocytes treated with nocodazole, a microtubule depolymerising agent, the
ER ring around the meiotic apparatus is absent [15]. Transport of organelles
along microtubules occurs via kinesins (towards plus-ends located at cell
periphery) and via dyneins (towards minus-end, docked in microtubule
12
Sperm-induced Ca2+ oscillations in mammalian oocytes
organizing centers, MTOC; [34]). Since the meiotic apparatus in a maturing
oocyte is surrounded by several MTOCs [40], dynein is considered to be
the main candidate for a motor protein responsible for the redistribution of
ER cisterns towards the meiotic spindle. This was proved by experiments in
which oocytes were injected with an anti-dynein antibody or incubated in the
presence of sodium orthovanadate, a phosphatase inhibitor with a high level
of selectivity for dynein over kinesins. Both of these treatments prevented
the formation of the prominent ER ring around meiotic spindle [15].
At the time of transition from metaphase I to metaphase II, the ER ring
and patch-like ER structures disappear and a layer of fine, distinctive ER
clusters (1–2 μm in diameter) forms in the cortical region of the oocyte. At
the same time ER in deeper layers of cytoplasm has a relatively uniform
reticular structure [14, 15, 38, 51]. The ER clusters are absent in the region
above the MII spindle. Accumulation of ER clusters in the oocyte cortex
depends on actin cytoskeleton: latrunculin A, an actin depolymerising
agent, inhibits ER reorganization in the cortical zone. Interestingly, both
phases of ER reorganization, i.e. formation of the ER ring and cortical
ER clusters, occur independently. Nocodazole treatment inhibits ER
ring formation, but not accumulation of cortical ER clusters. Similarly,
latrunculin A prevents the formation of ER clusters in the cortex, but not
the formation of the ER ring around the meiotic spindle [15]. Fitzharris
and co-workers [15] showed that ER clusters in the oocyte cortex form
independently of meiotic progression towards metaphase II. Oocytes
spontaneously arrested in metaphase I, unable to extrude first polar body,
display cortical ER clusters similar to those present in MII eggs.
INCREASE IN IP3R1 LEVEL AND CHANGES IN IP3R1
REGULATION AND DISTRIBUTION DURING MATURATION
OF MAMMALIAN OOCYTES
The number of IP3R1s increases during oocyte maturation. Western
blot analysis indicates that MII oocytes contain almost two-times more
IP3R1s than GV oocytes [13, 39, 47]. This increase plays a crucial role
Ajduk et al.
13
in the development of an oocyte ability to generate long-lasting [Ca2+]i
oscillations. Brind et al. [4] inhibited the maturation-related increase in the
amount of IP3R1 using adenophostin A (an IP3 analog) that activates IP3R1s
and leads to their degradation in a proteosome-dependent way. Mature
mouse oocytes with experimentally reduced number of IP3R1s did not
exhibit [Ca2+]i oscillations following sperm penetration or parthenogenetic
activation with Sr2+ ions. They were able to generate at most a single
[Ca2+]i transient. Similar results were obtained by our group [19], when we
fertilized maturing oocytes by intracytoplasmic sperm injection (ICSI) and
cultured them for several hours until they achieved MII stage. We showed
that MII oocytes fertilized during maturation had a lower amount of IP3R1
and, when fertilized again, they generated less [Ca2+]i transients than control
oocytes, which were not fertilized during maturation. These observations
are in agreement with results of experiments in which IP3R1 was depleted
by the injection of IP3R1 RNAi into maturing mouse oocytes [57]. This
procedure resulted in the complete inhibition of maturation-related increase
in the number of IP3R1s. The number of sperm-induced [Ca2+]i transients
registered in MII oocytes injected during maturation with IP3R1 RNAi
was lower than in control oocytes injected with EGFP RNAi. Interestingly,
while duration of the first [Ca2+]i transient was also significantly lower in
oocytes injected with IP3R1 RNAi than in the control group, the amplitude
of the first [Ca2+]i spike was not affected. This suggests that the amount
of IP3R1 is not the only factor influencing the oocyte ability to produce
proper [Ca2+]i oscillations.
Development of the oocyte ability to generate long-lasting [Ca2+]i
oscillations in response to the fertilization also depends on posttranslational
modifications of IP3R1. IP3R1 has two strongly conserved motifs recognized
by mitogen-activated protein kinase (MAPK), localized near the IP3 binding
site (serine 436) and in the regulatory domain (threonine 945; [28]). In mouse
oocytes the IP3R1 is phosphorylated gradually during meiotic maturation
until the MII stage [28]. A high level of phosphorylated IP3R1 is maintained
through the whole MII stage [20, 28]. Lee et al. [28] performed a series of
experiments proving that the MAPK signaling pathway was involved in
phosphorylation of IP3R1. They matured mouse oocytes in vitro in medium
14
Sperm-induced Ca2+ oscillations in mammalian oocytes
containing U0126, an inhibitor of MEK, a kinase activating MAPK. Since
a significant fraction of oocytes maturing in the absence of MAPK activity
is not able to maintain MII arrest [1], colcemid, a microtubule disruptor,
was added to the culture medium together with U0126 to prevent the
oocyte from exiting metaphase. In MII oocytes treated with U0126 during
maturation the level of phosphorylated IP3R1 was significantly lower than
in control, untreated oocytes [28]. Interestingly, MII oocytes incubated
with U0126 during maturation are not able to generate long-lasting [Ca2+]i
oscillations following fertilization, microinjection of complementary RNA
(cRNA) for PLC zeta or parthenogenetic activation with Sr2+ [28, 35]. In
such oocytes [Ca2+]i oscillations are terminated prematurely or absent
altogether. In addition, [Ca2+]i transients in U0126-treated oocytes are
of lower amplitude and shorter duration compared to those observed in
control, untreated oocytes [28, 35]. In U0126-treated oocytes completion
of meiosis and cell cycle progression were compromised as well, probably
because of impaired [Ca2+]i oscillations [35].
IP3R1 also contains consensus sites for CDK1/MPF. Two of the sites
recognized by CDK1, serine 421 and threonine 799, are located in the IP3
binding domain and in the regulatory domain respectively [32]. Recent
studies have indicated that in somatic cells these sites are phosphorylated
in vitro and in vivo by CDK1/MPF. Therefore phosphorylation by CDK1
may play a significant role in regulating IP3R1 function: it already has been
shown that this phosphorylation increases IP3 binding and IP3-gated Ca2+
release [32, 33]. This observation is in agreement with results obtained by
Deng and Shen [9] in experiments involving mouse oocytes. These authors
showed that the inhibition of MPF activity by roscovitine, a specific
inhibitor of CDK1, suppressed fertilization-induced [Ca2+]i oscillations.
Therefore, although changes in mouse oocytes in the phosphorylation
status of IP3R1 correlate with changes in MAPK activity rather than CDK1/
MPF activity (e.g. phosphorylated IP3R1 is abundant and CDK1 activity is
low at MI/MII transition; [28]), a regulatory role of CDK1/MPF-mediated
phosphorylation of IP3R1 cannot be excluded.
The IP3R1 also contains motifs recognized by other kinases, such as
calmodulin/Ca2+- dependent kinase II and protein kinases C, A and G
Ajduk et al.
15
[48]. Although their role in regulation of IP3R1-mediated Ca2+ release was
widely examined in somatic cells [48], there is little data about their role
in Ca2+-releasing system in mammalian oocytes. There is some evidence
that inhibition of protein kinase C and calmodulin/Ca2+- dependent kinase
II in mature mouse or hamster oocytes does not alter IP3R1-mediated Ca2+
release [52, 53]. However it is possible that phosphorylation of IP3R1
catalyzed by these kinases is required during meiotic maturation rather
than in MII stage. Therefore, the inhibition of their activities in metaphase
II may not be sufficient to reveal a potential function of these kinases in
regulation of IP3R1. When MAPK activity is inhibited in ovulated MII
oocytes, IP3R1-mediated [Ca2+]i oscillations are much less affected than
in oocytes incubated with MAPK inhibitor through the entire maturation
period [35]. Unfortunately, as far as we know, there are no studies
concerning the role of the abovementioned kinases in maturation-related
change in IP3R1 activity.
Distribution of IP3R1s changes during oocyte maturation as well. In
rodent GV oocytes, the IP3R1s are present throughout the cytoplasm, but
are preferentially located in the cortex, where they form a thin layer (in
mouse oocytes; [13, 39]) or patch-like accumulations, resembling those of
ER (in hamster oocytes; [51]). In human oocytes, distribution of IP3R1s is
different: they form patch-like structures in the inner cytoplasm, and are
less abundant in the peripheral zone [16]. In mouse, hamster and human
MII oocytes the IP3 receptors are ubiquitous in the cytoplasm, but they
accumulate in the cortex, where they form well-defined clusters, about the
same size as the ER clusters [16, 27, 39, 51]. Immunofluorescent labeling
of ER cortical preparations isolated from mouse oocytes confirmed that
IP3R1s are localized predominantly in ER clusters [27]. It was also shown
in mouse and human oocytes that the IP3R1s were less abundant in the
region around MII spindle than in the vegetal hemisphere [16, 39].
Since the IP3 and PLC zeta, a sperm factor producing IP3, are likely to be
present at high concentrations in the cortical region after sperm-egg fusion,
the clustering of ER and IP3R1 in the cortex of MII oocytes may facilitate
the initiation of [Ca2+]i oscillations. Dumollard et al. [12] and Kline et al.
[27] showed that waves of increased [Ca2+]i start at the hemisphere opposite
16
Sperm-induced Ca2+ oscillations in mammalian oocytes
the MII spindle, where ER and IP3R1 clusters were the most dense. In
addition, the clustering of IP3R1 may be required, at least partially, for
the enhanced receptor sensitivity observed in mouse MII oocytes. Oda et
al. [43] showed that the concentration of sperm protein extract required
to induce [Ca2+]i rise was significantly lower when the sperm extract was
injected into the periphery of the egg than into its interior. This indicates
that sensitivity to the sperm extract is higher in the cortex. Accumulation
of IP3R1 in cortical clusters may also participate in the regulation of Ca2+
influx, since IP3R1s are postulated to interact with Ca2+ channels in the
plasma membrane [46].
INCREASE IN CA2+ CONCENTRATION IN INTRACELLULAR
STORES IN MATURING MOUSE OOCYTES
There is some evidence that the concentration of Ca2+ ions in the intracellular
stores increases during maturation of the mouse oocytes [22, 37, 54].
This concentration can be estimated by a measurement of the amount of
Ca2+ released in the oocyte by an ionophore treatment. Ionophores, such
as ionomycin or A23187, are lipid-soluble substances that insert into the
membranes of intracellular organelles and allow the release of Ca2+ ions.
They are believed to completely deplete the intracellular Ca2+ stores.
Because ionophores also insert into the plasma membrane, the ionophoretreated oocytes are cultured in medium without Ca2+, often enriched with
Ca2+ chelators, in order to prevent Ca2+ influx. Several groups showed
that MII oocytes treated with ionomycin generated [Ca2+]i transient that
was approximately 4-times bigger than [Ca2+]i transient in GV oocytes
treated in the same way [22, 37, 54]. The greatest increase in the amount
of released Ca2+ occurs between MI and MII stages [54].
However, data obtained by Mehlmann and Kline [37] are equivocal. In
some experiments the authors used thimerosal, a sulfydryl reagent which
in low concentrations sensitizes IP3-dependent Ca2+ release. They showed
that immature oocytes incubated in thimerosal released significantly
more Ca2+ upon IP3 injection than untreated oocytes that also received
Ajduk et al.
17
IP3 injections. Moreover, when thimerosal was present in the medium,
microinjection of IP3 released a similar amount of Ca2+ both in GV and MII
oocytes. These results clearly contrast the data obtained with ionophores
and suggest that the GV oocyte contains a Ca2+ store similar in size to that
of the MII oocyte, but just less responsive to IP3. Interestingly, GV oocytes
incubated with thimerosal and then injected with IP3 released significantly
more Ca2+ than GV oocytes treated with ionophore. Mehlmann and Kline
[37] explained these discrepancies as a different accessibility of ER to the
ionophore in GV and MII oocytes. They assumed that the ionophores might
more effectively insert into the membranes of clustered ER in MII stage
than into the membranes of more diffused ER structures in GV oocytes.
Therefore, the ionophores were able to release all or almost all Ca2+ from
intracellular stores in MII oocytes, but only a fraction of Ca2+ stored in GV
oocytes. However, there is also another explanation that since experiments
with thimerosal and IP3 were conducted in the medium containing Ca2+,
the [Ca2+]i rise possibly consisted not only of Ca2+ released from the
intracellular stores, but also of Ca2+ entering the cell from the exterior.
REDISTRIBUTION OF ER CA2+-BINDING PROTEINS DURING
MATURATION OF MAMMALIAN OOCYTES
Distribution of Ca2+-binding ER proteins, such as calreticulin and calnexin,
changes during maturation of mammalian oocytes. Although there is no
direct evidence indicating that this redistribution participates in development
of the oocyte ability to generate long-lasting [Ca2+]i oscillations, it is very
plausible that maturation-related translocation of these proteins facilitates
Ca2+ release. Immunofluorescent staining showed that the calreticulin, one
of Ca2+-binding proteins regulating SERCA, is localized in a thin layer
(~ 3 µm) in the cortical zone of human GV oocytes. The intensity of staining
decreased gradually towards the cell centre. The distribution of calreticulin
in MI and MII oocytes is similar to that observed in GV oocytes. However,
in MI and MII oocytes, but not in GV oocytes, calreticulin forms patchlike accumulations in the oocyte cortex [2]. Another Ca2+-binding protein
18
Sperm-induced Ca2+ oscillations in mammalian oocytes
that regulates Ca2+ release from ER, calnexin, is also predominantly
localized in the cortex of both immature and mature human oocytes.
Experiments using immunofluorescence showed that in GV oocytes
calnexin formed three distinct cortical zones: two (2-3 µm thick) zones
of stronger fluorescence, separated by an area of less intense staining
(1-2 µm thick). The fluorescent signal in the central cytoplasm was much
weaker. Localization of calnexin changes during meiotic maturation. In
MI and MII oocytes only one thick (10-13 µm) layer of distinctly patchy
labeling is present in the peripheral ooplasm [2]. Formation of cortical
accumulations of calreticulin and calnexin observed in mature MII oocytes
clearly resembled ER clustering at this stage [14, 15, 38, 51]. This suggests
that in MII oocytes both calreticulin and calnexin may be predominantly
present in ER clusters.
CONCLUSIONS
Sperm-induced [Ca2+]i oscillations are crucial for activation and progress
of the embryonic development. Mammalian oocytes can properly respond
to fertilization only when the mechanism responsible for generation of
[Ca2+]i oscillations is well developed. During last 15 years scientists have
managed to learn much about the development of the oocyte ability to
produce [Ca2+]i transients. We know that this ability develops in mammalian
oocytes during meiotic maturation. Several lines of evidence indicate that
it requires reorganization of ER cisterns, increase in the number of IP3R1s,
change in IP3R1 activity, and probably redistribution of ER proteins and
increase in the amount of Ca2+ ions stored in the cell. However many
questions remain unanswered. Involvement of ER proteins in regulation of
IP3R1 and SERCA in mammalian oocytes is still poorly understood. The
possible function of protein kinases C and A or calmodulin/Ca2+-dependent
kinase II in maturation-related activation of IP3R1 as well as regulation of
Ca2+ influx and its role in maintaining [Ca2+]i oscillations also need further
examination. Research focusing on these subjects may help to discover
new factors responsible for female infertility.
Ajduk et al.
19
ACKNOWLEDGEMENTS
The authors would like to thank Prof. M. Kloc for her excellent help during
preparation of the manuscript. AA was supported by a grant 2P04C 047 30
from the Ministry of Science and Higher Education of Poland.
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