Molecular Reproduction & Development 77:856–867 (2010)
Dynamic Roles of Ion Currents in Early Development
Animal Physiology and Evolution Laboratory, Stazione Zoologica Anton Dohrn, Napoli, Italy
Excitable cells have the capacity to modify their electrical properties in response to different stimuli. This speci？c feature is due to a flux of ion currents that flow via ion [D]evelopment is accompanied channels in the plasma membrane. In all species so far studied, ion channels are by a dramatic modi？cation of proteins expressed in the zygote and in the blastomeres of the developing embryo, and the electrical properties of the their activity is subject to dynamic changes throughout the early cleavage stages. plasma membrane in the newly Although these complex patterns imply that ion currents play a role in signal transduc- developing embryo tion and the control of embryogenesis, a speci？c developmental function for the appearance, loss, and alterations of the channels remains to be elucidated. This review reports several aspects surrounding the involvement of ion currents in early embryo development, from invertebrates to human. It focuses on the occurrence, modulation, This work is dedicated to the memory of and dynamic role of ion fluxes through external, intra- and inter-cellular ion channels my father Giulio. from the zygote up to the blastula and pre-implantation stages. The implications for a * Corresponding author: role of ion currents in development, and their possible clinical and technological Animal Physiology and Evolution applications are discussed. Laboratory Stazione Zoologica Anton Dohrn Villa Comunale Mol. Reprod. Dev. 77: 856–867, 2010. ß 2010 Wiley-Liss, Inc. Naples 80121, Italy. E-mail: firstname.lastname@example.org
Published online 28 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.21215 Received 4 February 2010; Accepted 11 May 2010
INTRODUCTION their electrical properties in response to different stimuli (Tosti
and Boni, 2004). At the time of their interaction, one of the The electrical characteristics of a plasma membrane are earliest events is the change in the ionic permeability of the linked to a differential distribution of electrical charges on the oocyte plasma membrane. Electrophysiological studies have inner and outer surfaces of the cell. This distribution gener- demonstrated that this is due to the gating of plasma mem- ates a gradient known as ‘‘voltage,’’ that in turn gives rise to a brane ion channels that generate a flux of ions across the transmembrane potential which is known as the resting plasma membrane of the fertilized oocyte (Dale, 1994). The potential (RP). Ion channels are speci？c proteins located ion current is accompanied by a change in the oocyte plasma
membrane potential, which transiently depolarizes in some within the lipid bilayer of the membrane that allow the passage
of ions across the plasma membrane, and this results in a flux aquatic animals (Dale and DeFelice, 1984; Chambers, 1989; known as ion current (DeFelice, 1997). Ion currents have De Simone et al., 1998; Glahn and Nuccitelli, 2003), and been found in most excitable membranes, and it has been hyperpolarizes in some mammals (Miyazaki, 1988; Gianaroli suggested that they play a role in triggering some intracellular
processes. In particular, electrical modi？cations of the plasma
membrane are amongst the ？rst events in the processes Abbreviations: FC ? fertilization current; GJ ? gap junction; IP3r? inositol involved in reproduction. Gametes are electrogenic cells, and 1,4,5-triphosphate receptor(s); RP ?resting [membrane] potential; Ryr ? ryano- during their maturation and reciprocal activation they change dine receptor
ß 2010 WILEY-LISS, INC.
ON CURRENT AND DEVELOPMENT I
In this respect, the generation of polarity is a fundamental et al., 1994; Tosti et al., 2002). Thus, the beginning of
development is accompanied by a dramatic modi？cation of event establishing the basic architecture of the embryo. As a the electrical properties of the plasma membrane in the newly general rule, in mammals the zygote divides into equal developing embryo. This review reports the expression and daughter cells and then equal blastomeres; at this stage, activity of plasma membrane ion currents in various stages of apart for some regulatory proteins (Antczak and Van Bler- early embryo development in different animal species. After a kom, 1999), there is no clear evidence of polarity in human brief description of the main steps of development and the (Dale et al., 1995). In the morula, at the 32-cell stage, the signi？cance of the ion currents, I report information on the organization becomes less clear, and the cells change their properties and changes in intracellular ions, ion channels, and shape with a complete remodeling of membrane lipids and currents occurring from zygote up to blastula/pre-implanta- become much more tightly apposed, a process known as tion stages. The correlation between the changes in ion compaction. Shortly afterwards, the embryo starts its ？rst
channel properties and differentiation of the embryonic mem- differentiation process into two cell lineages: a flat layer of branes suggests a dynamic role for ion currents in the estab- epithelial cells termed trophectoderm, and the inner cell lishment and functionality of early embryo development, mass, a small group of cells that are progenitors of the polarity, and interblastomere communication. embryo proper (Wiley, 1987; Biggers et al., 1988); a fluid-
？lled blastocoel cavity also forms during this process. The
acquisition of cell polarity and differentiation of the outer cells EARLY EMBRYO DEVELOPMENT of the early embryo are processes that are spatially and Although a large volume of literature deals with develop- temporally interdependent (Fleming and Johnson, 1988) mental biology, early embryo development is a very complex and these form the basis for the prime differentiation of early process that is dif？cult to summarize step by step. In sexual cell lineages. In this context, intercellular devices such as reproduction, the fusion of sperm and oocyte results in a gap junction (GJ) ion channels play a fundamental role. zygote, the ？rst diploid cell of a new individual. The zygote
divides by mitosis to create a number of smaller cells known ION CURRENTS as blastomeres, and the details of this ？rst mitotic division
The different distribution of electrical charges inside and differ between species. Early cleavages are often synchro-
nous, until an unequal distribution of cytoplasmic compo- outside the cell creates an electrical gradient across the nents and/or induction by neighboring cells cause a loss membrane known as voltage. The ions that are principally synchrony, and the blastomeres become arranged in layers involved in the establishment of voltage are potassium þor groups that mark speci？c differences between the cells. cations (K), the most signi？cant intracellular ion, whereas þ2The overall result is the initiation of a developmental pro- sodium (Na) and calcium (Caþ) cations are more concen-
gram, giving rise to cell lines of the future embryonic tissue trated in the extracellular domain. Chloride anion (Cl ) is (epidermis, muscle, nerve, etc.). present at higher concentration externally, whereas other The newly formed zygote faces critical decisions regard- anions vary extensively inside the cell.
ing cell fate, and in the process of cleavage, each blastomere Although the literature shows that these ions are widely undergoes a commitment to form different tissues. In every distributed on either side of the plasma membrane, it is þspecies, there is a critical embryonic stage at which devel- possible to attribute a principal role to each one. Kions are
opmental fate is segregated into certain blastomeres. Most essential in determining and regulating the RP, whereas the þof the information on cell fate speci？cation comes from the principal role of Naions is to generate an action potential. 2cell-lineage studies performed in ascidians at the turn of the Caþ ions seem to be involved in cell signaling in many animal 2last century (Conklin, 1905). Each blastomere of an eight- species, and Caþcurrents have been shown to be vital in cell ascidian embryo shows a commitment to future tissues, regulating a broad range of physiological processes (Carafoli, easily recognizable by speci？c pigmentation in certain spe- 2002; Berridge et al., 2003; Whitaker, 2006). cies (Satoh, 1994). Similarly, in sea urchin embryos the The flux of these ions across the plasma membrane occurs fourth cleavage division represents a critical stage, with the through ion channels, proteins that are characterized by their appearance of clones that contribute to one lineage or speci？city, gating, conductance, and sensitivity to drugs. another (Cameron and Davidson, 1991). Speci？c ion channel gating is triggered in response to: (i) a Although the details differ among species, the main initial ligand (second messenger-operated channels) (Sutcliffe steps of embryogenesis may be summarized as blastula- et al., 1998), (ii) a change in voltage (voltage-operated tion, gastrulation, and neurulation, whereas later events channels) (Terlau and Stuhmer, 1998), or (iii) a mechanical account for organ formation and the establishment of stimulus (e.g. stretch-activated channels) (Hamill and three-dimensional, multicellular structures. The extent of McBride, 1996; Saitou et al., 2000).
differentiation depends on the species, such as the forma- The passage of ions across membrane ion channels tion of swimming larvae and subsequent metamorphosis in determines an electrical flux that is called ion current. Nor- marine invertebrates versus embryo implantation in the mally, ion current is associated with a change of RP, since the mammalian genital tract (Alberts et al., 1983; Menezo and concentration of ions inside the cell is transiently modi？ed. In
Renard, 1993). Up to the stage of blastula/blastocyst forma- particular, depolarization of the plasma membrane causes a tion, early embryos may be considered a requisite stage for shift of RP towards more positive values, whereas hyperpo- larval development and implantation, and may represent the larization modi？es the RP towards more negative values. major determinant of embryo quality. With the advent of vibrating probe (Jaffe and Nuccitelli, 1974)
Mol Reprod Dev 77:856–867 (2010) 857
Molecular Reproduction &Development OSTI T
and then of patch-clamp techniques (Neher and Sakmann, concurrent cell shrinkage have been identi？ed as indicators
1992), it was possible to study the electrophysiological status of cell death in mouse embryos (Trimarchi et al., 2000). and changes of the plasma membrane by recording the The gametes and embryos of ascidian marine animals steady-state currents, the modi？cation generated by a volt- are very suitable for in vivo electrophysiological analysis of age clamp and/or by the response to different stimuli. channel function, since their extracellular membranes are A large volume of literature supports a role for these easily removed, leaving the nude membrane accessible for currents in cellular process involved in reproduction, such patch clamping. Cleavage-arrested embryos of the ascidian as gamete maturation and fertilization (Hagiwara and Jaffe, Halocynthia roretzi, for example, were analyzed at various 1979; Miyazaki and Igusa, 1982; Darszon et al., 2001; Tosti stages of development in order to examine blastomeres with and Boni, 2004; Cuomo et al., 2005, 2006; Tosti, 2006; different developmental fate and steady-state currents of Silvestre et al., 2009). In post-fertilization events, although plasma membrane (Takahashi and Yoshii, 1981). These a loss of excitability during early development (Arnoult and authors provided a detailed description of blastomere excit- Villaz, 1994) has been reported, voltage-gated ion currents ability and the correlation between certain speci？c ion cur-
undergo considerable modulation during embryogenesis, rent patterns and the presumptive tissue regions. In 2suggesting that they play a clear developmental role (for particular, Caþ spikes were evoked in the presumptive þ2a review see Moody et al., 1991). muscle blastomeres, and Na/Caþ-dependent action po-
tentials were induced in some ectodermal blastomeres. Both þ2Naand Caþ currents decreased during the initial 10 hr ELECTRICAL PROPERTIES OF THE EARLY EMBRYO þafter cleavage arrest, whereas an anomalous (inward) K PLASMA MEMBRANE current increased gradually without changes in potential Embryonic cells cleave with a continuous involvement of dependence throughout development. Similarly, Hirano the cell cycle and renewal of the plasma membrane. These et al. (1984) identi？ed four types of membrane response: processes involve a combination of different chemical and neural, epidermal, muscular, and nonexcitable, on the basis physical forces, including voltage gradients, hence it is clear of the shapes and ionic dependence of action potentials in that ion concentration gradients and activity contribute to the blastomeres from 8- to 32-cell embryos. These authors morphogenetic events during development. This is espe- hypothesized that the membrane differentiation of blasto- 2cially true for Caþ ions, which not only generate a current meres occurred in several critical steps rather than as a but also directly influence the permeability of the plasma result of continuous linear growth.
membrane to other ions, leading to other transcellular cur- In the Boltenia villosa embryo, signi？cant ？ndings
rents that are critical for morphogenesis (Nuccitelli, 1988). showed that at the eight-cell stage the total surface area of Early studies on the role of ion currents in embryo physi- the embryo, measured by capacitance or calculated from ology date back to 1974, when the cleavage furrow mem- cell diameters, increased about 2.5-fold with respect to the þbrane of amphibian embryos was shown to exhibit an unfertilized egg. Furthermore it was shown that high Na þincreased permeability to Kconductance during each suc- current activity in the egg disappeared almost completely cessive division (deLaat and Bluemink, 1974). Similar cyclic from the embryo by the time of ？rst cleavage, and was þbehavior is observed for stretch-activated Kcurrents dur- undetectable in any of the blastomeres at the eight-cell 2þing embryo cleavage in freshwater ？sh, suggesting a poten- stage, whereas Caþ and Kcurrents remained at a con- þtial association between Kcurrents, RP and membrane stant density in all the cells. Although no differences in mean conductance (Medina and Bregestovski, 1988). Therefore, a current densities of blastomeres of different developmental þchange in Kion channel activity occurs in very early fates were detected through the eight-cell stage, these 2þdevelopment. Other studies indicate that this pattern may results suggested that Caþ and Kchannels were added
be quite general, providing evidence that channel modula- along with new membranes during these stages (Block and tion is cell-cycle dependent (Bregestovski et al., 1992). Moody, 1987). However, this situation changed during the þIn early mouse embryos, a voltage-activated Kchannel progression from eight-cell to gastrula. The oscillatory ap- activity is linked to the cell cycle, being active throughout M pearance of ion currents through subsequent developmen- and G1 phases, and switching off during the G1-to-S transi- tal stages leads to profound implications for differentiation of tion (Day et al., 1993). The same authors demonstrated the the cell types. As an example, muscle-lineage blastomeres þexistence of other two ion current patterns: an ERG-like K in these embryos developed a voltage-dependent calcium channel that acts as a timer in early mouse embryo devel- current while surrounding blastomeres of other lineages did opment (Day et al., 1998a, 2001), and a T-type calcium not (Simoncini et al., 1988). Consistent with the cyclical þþcurrent change driven by a similar cell cycle clock (Day et al., behavior of Kcurrents, several types of Kchannels in
1998b); this suggests that these channels exert an autono- Halocynthia embryos were found to co-exist with differenti- mous function that may be of developmental signi？cance. ating muscular blastomeres, starting from the eight-cell þAnother link between Kcurrent activity and early devel- stage up to fully differentiated muscular cells, with develop- opment has been demonstrated recently in establishment of mental changes in either percentage or in kinetics (Shidara left/right patterning in the Xenopus early embryo. These and Okamura, 1991). This study con？rmed previous ？ndings
studies provide the ？rst clues that there is a bioelectrical that the eight-cell stage is critical: single blastomeres with a component to the mechanisms involved in large-scale axis different cell fate express various types of different ion patterning in vertebrate embryogenesis (Morokuma et al., channel, with a possible developmental role (Okado and þ2008). Finally, a current efflux through Kchannels and Takahashi, 1990).
858 Mol Reprod Dev 77:856–867 (2010)
ON CURRENT AND DEVELOPMENT I þ2gradients. The same authors later characterized the electri- Functional expression of Caþ and Nacurrents has
been described during early development of Ciona intesti- cal properties of the whole sea urchin embryo at the 16-cell nalis (Cuomo et al., 2006). An accurate electrophysiological stage, the highly polarized stage in which three populations characterization of each blastomere plasma membrane of blastomere have been formed (macromeres, mesome- 2from two-cell up to eight-cell stage showed a signi？cant res, and micromeres). A dramatic polarization of Caþ 2þdecline of Caþ and Nacurrents from the zygote to the four channels activity was shown along the animal/vegetal axis, -cell stage, indicating that these currents have a minor role in with a cluster at the animal pole decreasing up to disappear the signaling events related to the ？rst embryonic mitotic in the micromeres. In contrast, the micromeres exhibited a cycle. In agreement with the critical role of the eight-cell threefold higher steady-state conductance than the me- þstage embryo, there is a signi？cant increase in all of the someres and macromeres, which may be attributed to K
current activities, measured as total membrane conduc- conductivity. This study provided the ？rst clue for a functional 2tance at this stage, with no difference between the speci？c involvement of Caþ channels in sea urchin embryo devel- blastomeres except for the posterior vegetal blastomere opment, supported by the fact that incubation of the dividing þ22(B4.1), where both Naand Caþ showed lower current zygote with speci？c ion Caþ channel inhibitors caused
values. Although these data might suggest a minor role for serious developmental defects in the later embryos (Dale 2þCaþ and Nain the signaling event related to the ？rst mitotic et al., 1997).
embryonic cycle, the high membrane permeability mea- The functional role of ion channel activity has also been sured as steady-state conductance seems to correspond investigated in the early zebra？sh embryo, which offers
to initial tissue segregation. This suggests that channel particular advantages in embryology studies due to the fact expression during the post-eight-cell stage of embryo de- that its genome has been fully sequenced. Although ion velopment is possibly lineage related. channel activity studies did not report direct ion current þA Nacurrent on the plasma membrane of ascidian MI recording, recent interesting results showed a temporal and þoocytes has been widely described (Block and Moody, spatial dynamic expression pattern of voltage-gated Na
1987; Hice and Moody, 1988; Coombs et al., 1992). These during embryonic stages (Novak et al., 2006). Similar results þ2authors showed that Nacurrents undergo redistribution have been shown for expression of voltage-gated Caþ
after fertilization, decreasing after cleavage of the ？rst channels in early two-cell embryos, whose inhibition re- zygote, attributing a speci？c function to this pattern in sulted in late developmental defects (Sanhueza et al., embryogenesis. 2009). Taken together these ？ndings indicate that voltage
In Ciona, the ？rst event at fertilization is the generation of -gated channel expression occurs suf？ciently early during
the inward ion current, that is, the fertilization current (FC). embryogenesis to play a part in the development of future Electrical characterization of the FC indicated that it is driven embryonic tissues.
by a new population of nonspeci？c channels (Dale and Changes in ion channel properties also occur during early DeFelice, 1984; DeFelice and Kell, 1987). Subsequent mammalian embryo development. Under voltage clamp, the þ2studies demonstrated that Nacurrents play a major role amplitude of inward Caþ currents in mouse and hamster
in the electrical modi？cation of the plasma membrane at decreased with time during early development, and were fertilization (Cuomo et al., 2006) and there is a relationship undetectable by the eight-cell stage. In hamster embryos an between primary electrical events and embryo develop- abrupt increase of the outward current is observed after the þment. Inhibiting Nacurrents at the time of the FC generates two-cell stage, showing an inverse correlation with the 2a rosette, an anomalous embryo previously described in the pattern of Caþcurrents (Mitani, 1985). These data are in 2þ1960s (Reverberi and Ortolani, 1962; Ortolani, 1992). A favor of a possible role for both Caþ and Kcurrents in
rosette is the equivalent of an 8–16 cell stage embryo that membrane differentiation; in mammalian embryos, one of has lost spatial orientation, and it was suggested that the the initial signs of polarization appears at the morula stage resulting partitioning formation is due to the removal of the when all eight blastomeres are compacted. A cyclical be- þvegetative plasms region that is responsible for the correct havior of some Kchannels has been described in the þcleavage pattern. The fact that a lack of Nacurrents at embryo; interestingly several other studies have indicated þfertilization generates this anomalous embryo reinforced an involvement of speci？c ERG-Kchannels during mouse
？ndings by Tosti et al. (2003) who suggested that ions embryo development by showing the expression and loca- generating the FC are involved in correct embryo/larval tion of these protein channels on the plasma membrane. development of C. intestinalis. Inhibition of such currents leads to stage-speci？c develop-
Other animal species provided useful information on a mental defects (Winston et al., 2004), suggesting a true role, role for ion currents in development; the activity of different although the mechanism is not easily explained. functional ion channel populations was also determined in The ability of an early embryo to grow is also due to factors 2sea urchin embryos. A cyclical L-type Caþ channel activity present in the external milieu. During in vitro embryo culture, was determined during a speci？c stage of the mitotic cycle in the deprivation of certain trophic factors results in a marked two-cell embryos (Yazaki et al., 1995). These studies ？rst reduction in embryo survival (O’Neill, 1997). Platelet-acti-
demonstrated that peculiar ion currents are expressed in vating factor (PAF) has been shown to induce an increase in 2sea urchin blastomeres, with a polarized localization to the intracellular Caþ concentration, with an absolute require- 2apical membrane. This behavior supported a functional link ment for external Caþ. Whole-cell patch clamp measure- 2between the embryonic cell cycle and Caþ channels ments in mouse two-cell embryos detected an L-type 2through the generation of localized intracellular calcium voltage-gated Caþ channel, whose inhibition signi？cantly
Mol Reprod Dev 77:856–867 (2010) 859
Molecular Reproduction &Development OSTI T
2blocked the PAF-induced Caþ transient needed for signal nels may have a profound impact on events during devel- transduction during normal embryo growth and survival (Lu opment (Vermassen et al., 2004).
et al., 2003). These observations provided evidence for the An interesting bilateral asymmetry of different families of 222regulation of ligand-induced Caþ channel activation. Like- IP–Caþ channels and related Caþ signaling are reported 3wise, during embryo culture the uptake of the amino acids in the two-cell stage of the ascidian Phallusia mammillata required is mediated by Cl channels in the mouse (Sonoda embryo. However, electrophysiological studies revealed no 2et al., 2003). Cl channels play several roles, including cell change in the density of voltage-dependent Caþ channels
swelling-mediated activation that regulates cell volume in in each blastomere (Albrieux and Villaz, 2000). This asym- somatic cells. In the mouse, these channels have been metry suggests that there may be a one-way communication 2identi？ed in the interaction between a developmental clock in the IP-dependent Caþ signaling between the two blas- 3and the cell cycle of developing early embryo (Kolajova et al., tomeres, with a possible implication for establishment of 2001). embryo polarity.
The role of IPr channels was studied during early em- 3 bryogenesis in Xenopus laevis, revealing a massive in-
crease in these channels at early gastrula stages (Kume IP-MEDIATED ION CURRENTS 3et al., 1997b; Kume, 1999), implying a potential role during 2Caþ is known to be a second messenger involved in gastrulation. In early mouse embryos, a detailed study of many cellular processes (Berridge et al., 2000), including mRNA expression of different isoforms of IPr demonstrated 3reproductive processes from oogenesis to early and late a different regulation during development, and differential embryo development (Stricker, 1995; Jones, 1998; Ducibel- role for the three subtypes between fertilization and early la et al., 2006; Whitaker, 2008; Whitaker and Smith, 2008). In development (Parrington et al., 1998). The presence and 2excitable cells, Caþ entry occurs by means of two principal distribution of the IPr channels in human embryos demon- 3mechanisms: the opening of voltage-gated channels in strated a dynamic redistribution of these channels during response to membrane depolarization (Catteral, 2000) and early cleavage divisions. In particular, a higher receptor 2an efflux from Caþ stores via ligand-gated channels on density appeared at speci？c peripheral sites in blastomeres organelle membranes. Most of the events underlined by the of two-cell and four-cell embryos, suggesting that this may 2latter mechanism are associated with two families of Caþ serve as the sites of origin for future cleavage divisions. ion channels stored in the endoplasmic and/or sarcoplasmic Progressing to the eight-cell stage, receptor clusters were reticulum in all cell types: the ryanodine receptor (Ryr) and observed around the blastomere nuclei. This movement the inositol 1,4,5-trisphosphate receptors (IPr) (Berridge, 3from the periphery to intracellular sites may imply that the 1993). A connection between the two pathways is repre- embryo needs newly synthesized channels, as this timing 2sented by the capacitative/store-operated Caþ entry in also corresponds with embryonic genome activation (Goud 2which the emptying of intracellular Caþ stores activates et al., 1999). In partial agreement with this pattern, IP 32Caþ influx (Parekh, 2003). Although both IPand Ryr receptor channels subtypes 1 and 2 are localized in highly 3channels have been well studied during fertilization organized and developmentally regulated patterns in blas- (Miyazaki et al., 1993), little is still known about their possible tomeres of human pre-implantation embryos (Balakier et al., involvement during subsequent development. 2002). Similar observations in early cleavage stage embryos 2Evidence to support the hypothesis that intracellular Caþ of Xenopus suggested a close correlation between the IP- 32signals may regulate pre-implantation embryogenesis was mediated Caþ signaling system and cell-cycle progression provided in murine models, by showing a calcium-dependent in these embryos (Kubota et al., 1993). improvement in peri- and pre-implantation development More recently, investigations in early zebra？sh develop- (Stachecki et al., 1994a,b). On this basis, the same authors ment showed that three distinct IPreceptor genes are 322showed that Caþ release in mouse morulae occurred pre- expressed. IP-sensitive Caþ stores had previously been 3dominantly through the IPr and that alteration of intracellular shown to be critical for progression from the zygotic to 32Caþ levels caused acceleration or delay of embryonic cleavage stage (Lee et al., 2003). The authors further growth and differentiation (Stachecki and Armant, 1996). demonstrated that early blastula formation was disrupted The relative position and subcellular localization of IPr is 3by pharmacological inhibition of IPreceptors, thus these 3an important factor for the correct initiation and propagation channels are obviously important during early development 2of Caþ signals. The majority of cell types express more than of this species (Ashworth et al., 2007).
one IPr isoform, and their subcellular distribution is cell- 3
speci？c. In particular, the IPr type 1 isoform has been 3reported for human oocytes and embryos (Goud et al., ION CURRENTS AND EARLY EMBRYONIC 1999), mouse (Mehlmann et al., 1996; Pesty et al., 1998; NEURAL INDUCTION Fissore et al., 1999), and Xenopus oocytes (Kume et al.,
Development of the embryonic nervous system is char- 1993, 1997a; Parys et al., 1994), whereas isoform IPr type 2 3has been localized in human maturing oocytes, zygotes, and acterized by a series of signals that often emanate from other 2pre-implantation embryos. pre-existent blastomeres or tissues. Voltage-operated Caþ
The cell is able to express and redistribute ion channels channels, for example, appear to be signi？cant for the
on the basis of physiological need. Therefore, it is likely that control of neuronal differentiation during development in a growing embryo the different localization of such chan- (Spitzer, 1991). The cleavage-arrested eight-cell ascidian
860 Mol Reprod Dev 77:856–867 (2010)
ON CURRENT AND DEVELOPMENT I
velopment (White and Bruzzone, 2000). GJ play a variety of embryo provides the simplest model system: neural induc-
tion is mediated by speci？c cell contact between ectodermal roles in cells, tissue and biological processes, and the nature and vegetal blastomeres. In these embryos, long-lasting and behavior of ion channels have been extensively re- 2Caþ-dependent action potentials develop autonomously viewed (Beyer et al., 1990; Beyer, 1993).
with epidermal differentiation, whereas there is a turnover During the 1980s growing evidence suggested that þof Nachannel subtypes at the time of neural induction, cell–cell communication via GJ was an important feature in þpossibly correlated with suppression of an inward recti？er K early embryo development (see DeHaan, 1994 for a review; channel just after neural induction (Takahashi and Oka- Larsen and Wert, 1988; Lo, 1996). GJ were shown to be mura, 1995; Takahashi and Tanaka-Kunishima, 1998). formed during compaction, playing an important role during Previous studies in X. laevis reported the detailed kinetics the process of cavitation (Ducibella et al., 1975). The in- 2of L-type Caþ channel subunit expression and localization volvement of GJ in development is supported by many lines during the early stages of Xenopus embryogenesis (Drean of experimental evidence, such as: (i) a wide expression of et al., 1995). At the time, these data substantiated the pro- multiple members of the connexin gene family during em- posal that acquisition of neural competence in Xenopus may bryonic developmental stages in Xenopus (Landesman 2be regulated by L-type Caþchannel expression in the plasma et al., 2003) and mouse (Davies et al., 1996; Kidder and membrane. More recently, early nervous system induction Winterhager, 2001) and (ii) a timely related assembly of GJ from the adjacent ectoderm has been observed in early channels during compaction (Kidder et al., 1987). Other vertebrate embryos. In particular, an involvement of mammals, including the human, were found to have similar- 2dihydropyridine-sensitive Caþ channels has been reported ities with the mouse model at the late pre-implantation in the orientation of ectodermal cells toward a neural fate in blastocyst stage, where biochemical studies identi？ed reg-
Xenopus, reinforcing the initial idea that a calcium influx ulation of connexin expression during development (Bloor 2through L-type Caþchannels may play a central regulatory et al., 2004; Flechon et al., 2004). At present, GJ are widely role in neural induction (Moreau and Leclerc, 2004; Leclerc recognized to play a pivotal function in junctional commu- et al., 2006). nications, providing diffusion of ions and cell metabolites
between coupled blastomeres. However, apart from the
proved impact of GJ on embryo viability (Fleming et al.,
2000), the question of whether these channels might regu- ION CURRENTS AND CAVITATION late normal growth of the embryo remains unresolved. Cavitation is one of the polarizing events that accompa- A very detailed review by Houghton (2005) summarizes nies the transformation of the morula into the blastocyst. It the controversial literature on functional requirements for GJ has been proposed that the fluid-？lled cavity, the blastocoel, in pre-implantation development. As an example, in early derives from the expansion of pre-existing intercellular mouse embryos intercellular junctional permeability is very spaces (Calarco and Brown, 1969) via mechanisms that diffuse (Lo and Gilula, 1979) but in terms of their speci？c role, regulate intracellular osmolarity and cell volume through the conflicting data report that: (i) perturbation of GJ communi- controlled release of osmolytes from the cytoplasm. Many cation during the 8–16 cell stage effectively decompacts cells, different types of channel are involved in the generation of either excluding them from further development or inducing blastocoelic fluid, including swelling-activated anion chan- developmental defects (Bevilacqua et al., 1989; Becker and nels (Baltz, 2001), two types of external Cl -dependent Cl Davies, 1995; Becker et al., 1995) while (ii) abolishing dye channels in early medaka ？sh embryos (Shigemoto, 1999), passage through GJ did not affect blastocyst formation or the and via a Na/K-ATPase pump (Watson and Barcroft, 2001). polarized allocation of cell lineages (Vance and Wiley, 1999). Although inhibition of the Na/K-ATPase pump resulted in Since the function of GJ depends on the composition of abnormal blastocoel formation in amphibians (Slack et al., connexin family members in the channel (Bruzzone et al., 1973), at present there is evidence that aquaporins are the 1996), expression studies have remained inconclusive. main ion channels involved in osmoregulation. Aquaporins However, electrophysiological studies appear to be more are integral membrane proteins that serve as channels to reliable for the investigation of GJ function during different transfer water, and in some cases, small solutes across the stages of embryogenesis. In the ascidian C. intestinalis, membrane (Benga, 2009). Members of the aquaporin family electrical coupling between blastomeres occurs through GJ are implicated in numerous physiological processes; they in the two-cell embryo, with functional maternal expression take part in the processes of blastocyst formation as physi- present at the zygote stage (Dale et al., 1991a; Tosti, 1997). ological mediators of fluid movement across the trophecto- No further functional role for these channels has been derm during cavitation and implantation (Cho et al., 2003; investigated in this species, but in 16-cell stage sea urchin Richard et al., 2003; Watson et al., 2004; Huang et al., 2006). embryos a functional GJ cluster is localized opposite to the L 2-type Caþ channels at the vegetal pole (Yazaki et al., 1999). The regionalization of GJ between macromeres and micro- GAP JUNCTIONS meres correlates with the inductive interaction between Communication between blastomeres represents one of these blastomeres and their descendants (Yazaki, 2001). the main processes that allows equivalent cells to express Furthermore, inhibition of GJ electrical communication at the different fates. GJ are specialized transmembrane pores 16-cell stage strongly delays successive gastrulation, induc- formed by the protein connexin, expressed speci？cally in ing developmental defects in archenteron formation. Taken cells with a pattern of expression that changes during de- together, these data are in favor of a coordinated role for both
Mol Reprod Dev 77:856–867 (2010) 861
Molecular Reproduction &Development TOSTI
TABLE 1. Review of the Literature Concerning the Types of Ion Channels/Ion Currents Involved in Various Stages of Early Embryo Development of Different Animal Models Animal models Stage Channels/currents Activity/putative functions Refs.
2Sea urchin þ L-type CaCell cycle related Yazaki et al. (1995)
2 L-type Caþ Cluster at the animal Dale et al. (1997) pole/developmental role Early embryo Gap junctions Yazaki et al. (1999) Cluster at the vegetal pole/developmental role Ascidians Stage related Gap junctions Dale et al. (1991a) 2Possible embryo polarity Albrieux and Villaz (2000) IPr-Caþ 2-cell 3
þ2þStage related Block and Moody (1987), Na–Caþ–K Simoncini et al. (1988), and Shidara and Okamura (1991) þ2Blastomere-related/ Caþ–K Okado and Takahashi (1990) developmental role þ2Early embryo Na–Caþ Cuomo et al. (2006) Stage-related/normal embryo development þ2þ Na–Caþ–K Takahashi and Okamura (1995) Neural induction and Takahashi and Tanaka-Kunishima (1998) Stage related Gap junctions Dale et al. (1991a) and Tosti (1997) 2Sanhueza et al. (2009) Normal embryo Fishes Caþ development 2-cell
þCell cycle related K Medina and Bregestovski (1988) Early embryo þ Stage related Novak et al. (2006) Na 2 Developmental role Lee et al. (2003); IPr-Caþ 3 Ashworth et al. (2007) Generation of cavity Cl Shigemoto (1999) þCell cycle related deLaat and Bluemink (1974) K Amphibians Early embryo Left-right pattern Morokuma et al. (2008) 2 Cell cycle related Kubota et al. (1993) IPr–Caþ 3 2Neural induction Drean et al. (1995) and L-type Caþ Leclerc et al. (2006) 2Lu et al. (2003) Embryo development, L-type Caþ growth and survival 2-cell Mouse þCell cycle related Day et al. (1993) K þCell cycle related Day et al. (1998a, 2001) ERG-K Early embryo 2Cell cycle related Day et al. (1998b) T-type Caþ þCell death Trimarchi et al. (2000) K þStage related Mitani (1985) 2K–Caþ Stage-speci？c Winston et al. (2004) þERG-K developmental effects Sonoda et al. (2003) Cl Amino acid uptake Cell cycle related Kolajova et al. (2001) Cl 2Parrington et al. (1998) Cleavage-related distribution IPr–Caþ 3 Normal embryo development Becker and Davies (1995); Gap junctions Becker et al. (1995); Bevilacqua et al. (1989) 2Normal embryo growth and IPr–Caþ Stachecki and Armant (1996) 3 differentiation Morula Lee et al. (1987) Gap junctions Compaction related Blastocoel formation Richard et al. (2003) Aquaporins Blastocyst (Continued)
Mol Reprod Dev 77:856–867 (2010) 862
ION CURRENT AND DEVELOPMENT TABLE 1. (Continued)
Channels/currents Activity/putative functions Animal models Stage Refs.
Bovine Gap junctions Stage related Boni et al. (1999)
Human Gap junctions Stage related Dale et al. (1991b) 2Balakier et al. (2002) Blastocyst IPr–Caþ Development-related 3organization
2Caþ channels and GJ in induction of critical embryonic lishment and polarizing events (Table 1). Although their processes that occur during the 16-cell stage. functional role is not fully understood, the many patterns Electrical coupling established between blastomeres dur- described argue for a link between the dynamics of ion ing early stage decreases up to the blastocyst stage; this currents and embryogenesis.
pattern, observed in ascidians and sea urchins and con？rmed The characteristic changes displayed by ion currents in in bovine (Boni et al., 1999), reflects the hypothesis that early embryos may account for: (i) a current-related function junctional permeability changes during the early stages. during speci？c periods, and the subsequent lack of a require- Results in bovine, however, showed that in vitro produced ment for them until a new differentiating plasma membrane embryos exhibit a late and reduced expression of intercellular appears and (ii) a pre-requisite for a further differentiation step communication compared with those produced in vivo. or the consequences of the previous one. Indirect evidence These data corroborate with the lower developmental for a role for ion currents is found in the fact that they appear ef？ciency of embryos in vitro, and further con？rm a speci？c during gametogenesis (Hagiwara and Jaffe, 1979; Tosti and role for GJ in regulating normal embryo development (Boni Boni, 2004); their modulation is continuous, and associated et al., 1999). with certain cell populations and lineages in the embryo. The temporal appearance of embryonic GJ varies be- More direct evidence can be found in experimental data tween species. In mouse embryos, electrical and dye cou- showing that perturbation of electrical properties in the em- pling established at the eight-cell stage correlates with bryo induces serious developmental defects that affect even compaction (Lee et al., 1987) similar to human embryos, in ？nal differentiation and morphogenesis. Finally, the regional which dye coupling appears at the blastocyst stage (Dale restrictions of GJ forming communication compartments et al., 1991b). This common situation may be attributed to in the embryo strongly support the role of GJ as regulators maternal junctions being replaced with those resulting from of growth, pattern formation, and tissue differentiation genomic activation, or to a requirement for these channels (Caveney, 1985). Although a clear role for ion currents in during polarizing events of blastulation. In either case, the the physiology of embryo development emerges from the evidence for a different functional role for GJ in speci？c results described, little is known about the molecular basis of developmental stages further supports the proposal that this phenomenon. De？nitive tests of their functional relevance these channels play a part in establishing communication are still lacking, and the mechanisms involved remain compartments during embryogenesis (Lo, 1996). unclear.
The studies described suggest that not only the pres-
ence, but the expression levels and regulation of ion
currents may be of a great importance in a diverse range CONCLUDING REMARKS of scienti？c areas. For example, in the ？eld of reproductive This review reports the presence of ion currents in early biotechnology, aquaporins may be of considerable interest embryos and their modi？cation during early stages of devel- for improving embryo viability after cryopreservation opment. The electrical properties of blastomeres during pre- (Cho et al., 2003). Similarly, improvement of the implan- implantation embryo development have been described in tation rate after in vitro fertilization may be provided by a several species. During embryogenesis, ion currents ap- channel-dependent trophic factor in the culture medium.
Finally, modulation of ion channels may represent a pear, disappear, or simply change their permeability in
relation to certain developmental stages. Their localization, promising ？eld for the discovery of non-hormonal contracep- clustering, and activity are often related to lineage estab- tive tools.
863 Mol Reprod Dev 77:856–867 (2010)
Molecular Reproduction &Development OSTI T
ACKNOWLEDGMENTS Block ML, Moody WJ. 1987. Changes in sodium, calcium and
potassium currents during early embryonic development of the I thank Dr. Kay Elder and Dr. Francesco Silvestre for valuable ascidian Boltenia villosa. J Physiol 393:619–634. comments on the manuscript; Mr. Giuseppe Gargiulo and Mr. Bloor DJ, Wilson Y, Kibschull M, Traub O, Leese HJ, Winterhager E, Giampiero Lanzotti for ？gure preparation. Kimber SJ. 2004. Expression of connexins in human preimplan-
tation embryos in vitro. Reprod Biol Endocrinol 2:25.
Boni R, Tosti E, Roviello S, Dale B. 1999. Intercellular communica- REFERENCES tion in in vivo and in vitro produced bovine embryos. Biol Reprod Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD. 1983. 61:1050–1055.
Cellular mechanisms of development. In: Alberts et al., editors. Bregestovski P, Medina I, Goyda E. 1992. Regulation of potassium Molecular biology of the cell. New York:Garland Publishing, pp conductance in the cellular membrane at early embryogenesis. J 813–890. Physiol Paris 86:109–115.
Albrieux M, Villaz M. 2000. Bilateral asymmetry of the inositol Bruzzone R, White TW, Goodenough DA. 1996. The cellular trisphosphate-mediated calcium signaling in two-cell ascidian Internet: On-line with connexins. Bioessays 18:709–718.
embryos. Biol Cell 92:277–284. Calarco PG, Brown EH. 1969. An ultrastructural and cytological Antczak M, Van Blerkom J. 1999. Temporal and spatial aspects of study of preimplantation development of the mouse. J Exp Zool fragmentation in early human embryos: Possible effects on 171:253–284.
developmental competence and association with the differential Cameron RA, Davidson EH. 1991. Cell type speci？cation during
elimination of regulatory proteins from polarized domains. Hum sea urchin development. Trends Genet 7:212–218.
Reprod 14:429–447. Carafoli E. 2002. Calcium signaling: A tale for all seasons. Proc Natl Arnoult C, Villaz M. 1994. Differential developmental fates of the Acad Sci USA 99:1115–1122. 2two calcium currents in early embryos of the ascidian Ciona Catteral WA. 2000. Structure and regulation of voltage-gated Caþ
intestinalis. J Membr Biol 137:127–135. channels. Ann Rev Cell and Dev Biol 16:521–555.
Ashworth R, Devogelaere B, Fabes J, Tunwell RE, Koh KR, De Caveney S. 1985. The role of gap junctions in development. Annu Smedt H, Patel S. 2007. Molecular and functional characteriza- Rev Physiol 47:319–335.
tion of inositol trisphosphate receptors during early zebra？sh Chambers EL. 1989. Fertilization in voltage clamped sea urchin development. J Biol Chem 282:13984–13993. eggs. In: Nuccitelli R, Cherr G, Clark WH, Jr., editors. Mechanism Balakier H, Dziak E, Sojecki A, Librach C, Michalak M, Opas M. of egg activation. New York:Plenum Press, pp 1–18.
2002. Calcium-binding proteins and calcium-release channels in Cho YS, Svelto M, Calamita G. 2003. Possible functional implica- human maturing oocytes, pronuclear zygotes and early preim- tions of aquaporin water channels in reproductive physiology and plantation embryos. Hum Reprod 17:2938–2947. medically assisted procreation. Cell Mol Biol 49:515–519.
Baltz JM. 2001. Osmoregulation and cell volume regulation in the Conklin EG. 1905. The organization and cell lineage of the ascidian preimplantation embryo. Curr Top Dev Biol 52:55–106. egg. J Acad Natl Sci (Phyladelphia) 13:1–119.
Becker DL, Davies CS. 1995. Role of gap junctions in the develop- Coombs JL, Villaz M, Moody WJ. 1992. Changes in voltage-de- ment of the preimplantation mouse embryo. Microsc Res Tech pendent ion currents during meiosis and ？rst mitosis in eggs of an
31:364–374. ascidian. Dev Biol 153:272–282.
Becker DL, Evans WH, Green CR, Warner AE. 1995. Functional Cuomo A, Di Cristo C, Paolucci M, Di Cosmo A, Tosti E. 2005. analysis of amino acid sequences in connexin 43 involved in Calcium currents correlate with oocyte maturation during intercellular communication through gap junctions. J Cell Sci the reproductive cycle in Octopus vulgaris. J Exp Zool 108:1455–1467. 303A:193–202. 2þBenga G. 2009. Water channel proteins (later called aquaporins) Cuomo A, Silvestre F, De Santis R, Tosti E. 2006. Caþ and Na
and relatives: Past, present, and future. IUBMB Life 61:112–133. current patterns during oocyte maturation, fertilization, and early Berridge MJ. 1993. Inositol trisphosphate and calcium signalling. developmental stages of Ciona intestinalis. Mol Reprod Dev
73:501–511. Nature 361:315–325.
Berridge MJ, Lipp P, Bootman MD. 2000. The versatility and Dale B. 1994. Oocyte activation in invertebrates and humans. universality of calcium signalling. Nat Rev Mol Cell Biol 1: Zygote 2:373–377.
11–21. Dale B, De Felice LJ. 1984. Sperm-activated channels in ascidian Berridge MJ, Bootman MD, Roderick HL. 2003. Calcium signalling: oocytes. Dev Biol 101:235–239.
Dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol Dale B, Santella L, Tosti E. 1991a. Gap-junctional permeability in 4:517–529. early and cleavage- arrested ascidian embryo. Development Bevilacqua A, Loch-Caruso R, Erickson RP. 1989. Abnormal de- 112:153–160.
velopment and dye-coupling produced by antisense RNA to gap Dale B, Gualtieri R, Talevi R, Tosti E, Santella L, Elder K. 1991b. junction protein in mouse preimplantation embryos. Proc Natn Intercellular communication in the early human embryo. Mol Acad Sci USA 86:5444–5448. Reprod Dev 29:22–28.
Beyer EC. 1993. Gap junctions. Int Rev Cytol 137C:1–37. Dale B, Tosti E, Iaccarino M. 1995. Is the plasma membrane of the Beyer EC, Paul DL, Goodenough DA. 1990. Connexin family of gap human oocyte reorganized following fertilisation and early cleav- junction proteins. J Membr Biol 116:187–194. age? Zygote 3:31–36.
Biggers JD, Bell JE, Benos DJ. 1988. Mammalian blastocyst: Dale B, Yazaki I, Tosti E. 1997. Polarized distribution of L-type Transport functions in a developing epithelium. Am J Physiol calcium channels in early sea urchin embryos. Am J Physiol Cell Physiol 255:C419–C432. 273:C822–C825.
864 Mol Reprod Dev 77:856–867 (2010)
ON CURRENT AND DEVELOPMENT I Darszon A, Beltran C, Felix R, Nishigaki T, Trevino CL. 2001. Ion Hagiwara S, Jaffe LA. 1979. Electrical properties of egg cell mem- transport in sperm signalling. Dev Biol 240:1–14. branes. Ann Rev Biophys Bioeng 8:385–416.
Davies TC, Barr KJ, Holstead Jones D, Zhu D, Kidder GM. 1996. Hamill OP, McBride DW, Jr. 1996. The pharmacology of mechano- Multiple members of the connexin gene family participate in gated membrane ion channels. Pharmacol Rev 48:231–252.
preimplantation development of the mouse. Dev Genet Hice RE, Moody WJ. 1988. Fertilization alters the spatial distribu- 18:234–243. tion and the density of voltage-dependent sodium current in the Day ML, Pickering SJ, Johnson MH, Cook DI. 1993. Cell-cycle egg of the ascidian Boltenia villosa. Dev Biol 127:208–420.
control of a large-conductance Kþ channel in mouse early Hirano T, Takahashi K, Yamashita N. 1984. Determination of embryos. Nature 365:560–562. excitability types in blastomeres of the cleavage-arrested but Day ML, Johnson MH, Cook DI. 1998a. A cytoplasmic cell cycle differentiated embryos of an ascidian. J Physiol 347:301–325.
controls the activity of a Kþ channel in pre-implantation mouse Houghton FD. 2005. Role of gap junctions during early embryo embryos. EMBO J 17:1952–1960. development. Reproduction 129:129–135.
Day ML, Johnson MH, Cook DI. 1998b. Cell cycle regulation of a T- Huang HF, He RH, Sun CC, Zhang Y, Meng QX, Ma YY. 2006. type calcium current in early mouse embryos. Pflugers Arch Function of aquaporins in female and male reproductive systems. 436:834–842. Hum Reprod Update 12:785–795.
Day ML, Winston N, McConnell JL, Cook D, Johnson MH. 2001. Jaffe LF, Nuccitelli R. 1974. An ultrasensitive vibrating probe for tiKþ toKþ: An embryonic clock? Reprod Fertil Dev 13:69–79. measuring steady state extracellular currents. J Cell Biol De Simone ML, Grumetto L, Tosti E, Wilding M, Dale B. 1998. Non- 63:614–628. 2speci？c currents at fertilisation in sea urchin oocytes. Zygote Jones KT. 1998. Caþ oscillations in the activation of the egg and 6:11–15. development of the embryo in mammals. Int J Dev Biol 42:1–10.
DeFelice LJ. 1997. Electrical properties of cells, Patch clamp for Kidder GM, Winterhager E. 2001. Intercellular communication in biologists. Chapter 2 New York:Plenum Press, pp 49–122. preimplantation development: The role of gap junctions. Front DeFelice LJ, Kell MJ. 1987. Sperm-activated currents in ascidian Biosci 1:D731–D736.
oocytes. Dev Biol 119:123–128. Kidder GM, Rains J, McKeon J. 1987. Gap junction assembly in the DeHaan R. 1994. Gap Junction communication and cell adhesion in preimplantation mouse conceptus is independent of microtu- development. Zygote 2:183–188. bules, micro？laments, cell flattening and cytokinesis. Proc Natn deLaat S, Bluemink JG. 1974. New membrane formation during Acad Sci USA 84:3718–3722.
cytokinesis in normal and cytochalasin B-treated eggs of Xeno- Kolajova M, Hammer MA, Collins JL, Baltz JM. 2001. Developmen- pus laevis. J Cell Biol 60:529–540. tally regulated cell cycle dependence of swelling-activated Drean G, Leclerc C, Duprat AM, Moreau M. 1995. Expression of L- anion channel activity in the mouse embryo. Development 128: 2type Caþ channel during early embryogenesis in Xenopus 3427–3434.
laevis. Int J Dev Biol 39:1027–1032. Kubota HY, Yoshimoto Y, Hiramoto Y. 1993. Oscillation of intracel- Ducibella T, Albertini DF, Anderson E, Biggers JD. 1975. The lular free calcium in cleaving and cleavage-arrested embryos of preimplantation mammalian embryo: Characterization of inter- Xenopus laevis. Dev Biol 160:512–518.
cellular junctions and their appearance during development. Dev Kume S. 1999. Role of the inositol 1,4,5-trisphosphate receptor Biol 45:231–250. in early embryonic development. Cell Mol Life Sci 56:296–
Ducibella T, Schultz RM, Ozil JP. 2006. Role of calcium signals in 304.
early development. Semin Cell Dev Biol 17:324–332. Kume S, Muto A, Aruga J, Nakagawa T, Michikawa T, Furuichi T, Fissore RA, Longo FJ, Anderson E, Parys JB, Ducibella T. 1999. Nakade S, Okano H, Mikoshiba K. 1993. The Xenopus IP3 Differential distribution of inositol trisphosphate receptor iso- receptor: Structure, function, and localization in oocytes and forms in mouse oocytes. Biol Reprod 60:49–57. eggs. Cell 73:555–570.
Flechon JE, Degrouard J, Flechon B, Lefevre F, Traub O. 2004. Kume S, Yamamoto A, Inoue T, Muto A, Okano H, Mikoshiba K. Gap junction formation and connexin distribution in pig tropho- 1997a. Developmental expression of the inositol 1,4,5-trispho- blast before implantation. Placenta 25:85–94. sphate receptor and structural changes in the endoplasmic Fleming TP, Johnson MH. 1988. From egg to epithelium. Annu Rev reticulum during oogenesis and meiotic maturation of Xenopus Cell Biol 4:459–485. laevis. Dev Biol 182:228–239.
Fleming TP, Ghassemifar MR, Sheth B. 2000. Junctional complexes Kume S, Muto A, Okano H, Mikoshiba K. 1997b. Developmental in the early mammalian embryo. Semin Reprod Med 18:185– expression of the inositol 1,4,5-trisphosphate receptor and local- 193. ization of inositol 1,4,5-trisphosphate during early embryogene- Gianaroli L, Tosti E, Magli C, Iaccarino M, Ferraretti AP, Dale B. sis in Xenopus laevis. Mech Dev 66:157–168.
1994. Fertilization current in the human oocyte. Mol Repr Dev Landesman Y, Postma FR, Goodenough DA, Paul DL. 2003. 38:209–214. Multiple connexins contribute to intercellular communication in Glahn D, Nuccitelli R. 2003. Voltage-clamp study of the activation the Xenopus embryo. J Cell Sci 116:29–38.
currents and fast block to polyspermy in the egg of Xenopus Larsen WJ, Wert SE. 1988. Roles of cell junctions in gametogene- laevis. Dev Growth Diff 45:187–197. sis and in early embryonic development. Tissue Cell 20:809–
Goud PT, Goud AP, Van Oostveldt P, Dhont M. 1999. Presence and 848.
dynamic redistribution of type I inositol 1,4,5-trisphosphate re- Leclerc C, Neant I, Webb SE, Miller AL, Moreau M. 2006. Calcium ceptors in human oocytes and embryos during in-vitro matura- transients and calcium signalling during early neurogenesis in the tion, fertilization and early cleavage divisions. Mol Hum Reprod amphibian embryo Xenopus laevis. Biochim Biophys Acta 5:441–451. 1763:1184–1191.
Mol Reprod Dev 77:856–867 (2010) 865