Ion currents modulating oocyte maturation in animals

Abstract

Growing oocytes are arrested at the first prophase of meiosis which is morphologically identified by the presence of a large and vesicular nucleus, called the germinal vesicle. The dissolution of the germinal vesicle marks the resumption of meiosis during which the oocyte undergoes massive modifications up to the second meiotic block, which is removed at fertilization. The interval between the first and the second meiotic block is defined as maturation and the events occurring during this period are crucial for ovulation, fertilization, and embryo development. Oocytes are excitable cells that react to stimuli by modifying their electrical properties as a consequence of ion currents flowing through ion channels on the plasma membrane. These electrical changes have been largely described at fertilization whereas little information is available during oocyte maturation. The aim of this review is to give an overview on the involvement of ion channels and ion currents during oocyte maturation in species from invertebrates to mammals. The results summarized here point to the possible functional role of ion channels underlying oocyte growth and maturation.

Keywords:

• ion channels
• ion currents
• meiosis
• oocyte maturation

Introduction

Oocyte maturation

Oocyte maturation is a complex process occurring at the end of oogenesis during which the oocyte completes its growth and undergoes a series of changes that are necessary for ovulation, fertilization, and early embryo development. Oocyte maturation involves two different but interlinked processes involving nuclear and cytoplasmic events [Tsafriri 1979; Yanagimachi 1994; Eppig 1996].

Nuclear maturation is underpinned by the meiotic process that is arrested in most species, during neonatal life, at the diplotene of the first meiotic prophase (PI) and marked by the large nucleus known as germinal vesicle (GV) in which the chromosomes are in a decondensed state. The oocyte persists in this GV stage up to the time of a stimulus that resumes meiosis inducing the breakdown of the GV (GVBD), chromosome condensation, and spindle formation. A second meiotic arrest then occurs at different stages depending on the species such as the first meiotic metaphase (MI) in ascidians, bivalves, and gastropods, whereas mammalian oocytes complete the first round of meiosis with extrusion of the first polar body and without an interphase progress to the second meiotic metaphase (MII). Apart from some exceptions, this process is completed in almost all the species studied, upon fertilization, by the extrusion of the second polar body (Fig. 1).

Figure 1.  A schematic illustration of the resumption of meiosis in different animal models. The immature oocyte is arrested in the prophase I (PI) marked by the germinal vesicle (GV). Depending on the species, oocytes may be fertilized in PI (anellids and starfish). In response to a stimulus, the first meiotic block is removed up to a second meiotic block at the metaphase I (MI; anellids, starfish, molluscs, and ascidians) or at metaphase II (MII; amphibians, fishes, mammals). MII is marked by the presence of one polar body (yellow). Resumption from the second meiotic block occurs upon sperm penetration.

Cytoplasmic maturation is a less clear process that starts at the time of oocyte growth and can be evaluated by: i) ultrastructural organelle re-organization, ii) molecular modifications, and iii) differentiation of the calcium signalling machinery. Ultrastructural re-organization involves the redistribution of microfilaments and microtubules that in turn support the relocation of cytoplasmic organelles such as cortical granules and mitochondria [Schorderet-Slatkine 1972; Masui 1985; Iwamatsu et al. 1993; Voronina and Wessel 2003; Sun and Schatten 2006; Stricker 2006; Ferreira et al. 2009; Liu et al. 2010; De los Reyes et al. 2011].

Transient changes in cytoplasmic content of some molecules, such as cyclic adenosine mono-phosphate (cAMP) and some analogues, act either by preventing spontaneous maturation and/or blocking GVBD in vitro [Yoshimura et al. 1992; Stricker and Smythe 2001; Conti et al. 2002; Yi et al. 2002; Silvestre et al. 2011a; Tsafriri and Dekel 2011; Stricker et al. 2010]. Contrasting data, however, show that high levels of cAMP may transiently block GVBD [Kishimoto 1998] or even accompany GVBD [Lambert 2008; 2011; Yamashita et al. 2000]. A relevant role in promoting meiosis progression has been also attributed to M-phase promoting factor and cytostatic factors, shown for the first time in amphibian and, subsequently, in mammalian and invertebrate oocytes (for review see [Masui 2001; Russo et al. 2011]). Regulation of these molecular dynamics involves the integration of systemic and local inputs [Menezo and Elder 2011].

The rise in intracellular calcium at fertilization has been widely analyzed and assessed as the main signal involved in the oocyte activation [Malcuit and Fissore 2011]. To date, little information is available on the function of this ion during oocyte maturation; however, an integral attribute of oocyte maturation is the remodeling of calcium signaling pathways endowing the oocyte with the capacity to produce a specialized calcium transient at fertilization [Homa 1995; Carroll et al. 1996; Tosti 2006; Machaca 2007; Boni et al. 2007]. Recent studies have shown that cytoplasmic maturation is also accompanied by a modification of plasma membrane permeability due to ion currents flowing through ion channels located on the plasma membrane.

Ion currents

In the cell, the plasma membrane marks the borderline between the internal and external compartments and this separation is essential for cell functionality. The plasma membrane represents a barrier to ions whose passage occurs through the ion channels, specific proteins located in the lipid bilayer. Ion channels are characterized by their specificity, gating, conductance, and sensitivity to drugs, and their gating is triggered mainly in response to ligand-operated channels [Sutcliffe et al. 1998] or to a change in the voltage-operated channels [Terlau and Stuhmer 1998]. The passage of ions across the ion channels determines an electrical flux, which is called the ion current, that is associated with a change in the resting potential (RP). This is also due to the different distribution of electrical charges inside and outside the cell that creates an electrical gradient across the membrane that, in almost all cells studied, is negative, ranging from –10 mV to –100 mV [De Felice 1997]. Transient modification of the ionic concentration inside the cell shifts RP towards more positive values (depolarization) or toward more negative values (hyperpolarization). The main ion inside the cell are potassium cations whereas those externally are sodium and calcium cations. Due to its elective storage inside the cell, potassium is the ion that mainly contributes to determining and regulating the RP. This is an important parameter that, determining the steady state distribution of actively charged substances, plays a key role as a signal for many cellular processes controlled by voltage-sensitive ion channels. In comparison, sodium channels play a major role in supporting the action potentials [Stephan and Agnew 1991] and calcium channels are voltage-gated channels. These open in response to plasma membrane depolarization and have been shown to regulate a broad range of physiological processes (for a review see [Moosmang et al. 2005]).

The patch-clamp is the most popular technique used for the study of cell currents as first described by Neher and Sakmann [1992]. At present, whole-cell and single channel recordings are the most basic configurations of the patch clamp technique allowing the measurement of the activity and patterns of ion currents flowing throughout the plasma membrane.

Ion currents in invertebrate oocyte maturation

The role of ion currents in the physiology of oocytes has been described in many animal species. The oocyte is an excitable cell that during the crucial periods of maturation and subsequent fertilization and development shows transient modifications of the electrical asset due to ion currents throughout the plasma membrane [Hagiwara and Jaffe 1979; Tosti and Boni 2004; Tosti 2010]. Invertebrate oocytes have been extensively used for electrophysiological studies since the 1940s when Edward Chambers started to investigate sodium and potassium exchange during fertilization in the sea urchin (for review see [Chambers 1989]).

The mechanisms of reproduction of echinoderms have been extensively studied. In particular, starfish and sea urchin have been used to study the electrophysiological properties of the oocyte plasma membrane. In starfish, immature oocytes exhibit three types of voltage-dependent currents: an inward calcium, a fast transient potassium, and an inwardly rectifying potassium current. During the hormone-induced in vitro maturation, a gradual change in the amplitude of all three currents is observed; the calcium current becomes larger whereas both potassium currents become smaller [Moody and Lansman 1983; Moody and Bosma 1985]. These changes are also associated with a decrease in membrane conductance and a depolarization of RP due to changes in sodium and potassium conductance [Miyazaki et al. 1975a; 1975b; Moreau and Cheval 1976].

Interesting differences and similarities in the electrophysiological changes which occur during maturation in two different starfish (Henricia leviuscula and Asterina miniata) were described by Simoncini and Moody [1990] who found that oocytes of both species have the three major voltage-dependent currents. Apart from differences in the amplitudes, during maturation both potassium currents decreased in amplitude whereas the inward calcium currents remained constant in both species.

In the Leptasterias hexactis, two voltage-dependent potassium currents were found early in oogenesis, before the rapid growth phase, and remained stable throughout oogenesis at the same current density and kinetics. As soon as the oocyte reached its full size, the inward current increased in amplitude by about fivefold, and underwent major changes in kinetics. These changes were closely associated with the migration of the GV to the cell periphery that represents the major morphological event at maturation [Moody 1985]. Electrical modifications of the oocyte plasma membrane have been previously described in sea urchin by Dale and De Santis [1981]. These authors described how the high potassium permeability of immature oocytes progressively decreases along with maturation. In both starfish and sea urchin, however, the functional role of these currents has not been elucidated.

Ascidians are invertebrates largely used for studying gamete biology. Their gametes are, in fact, very suitable for electrophysiological analysis of channel function, since their extracellular membranes are easily removed leaving the nude membrane accessible for the patch clamp. In the ascidian Ciona intestinalis, ion currents show typical features characterized by inward and outward currents that follow each other during oocyte maturation. This suggests that each current may play an important role during either nuclear or cytoplasmic maturation.

By using the whole-cell voltage clamp technique, we have studied ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis [Tosti et al. 2011]. First, Cuomo et al. [2006] showed that immature oocytes underwent a spontaneous GVBD in the presence of calcium. The fact that calcium plays a role in prophase/metaphase transition was also supported by a higher calcium current activity found at the GV stage in comparison to the following meiotic stages where sodium currents start to appear (Fig. 2). Subsequently, we identified three stages of immature oocytes with different electrical features. The smallest oocyte class exhibited the highest L-type calcium current activity and was incompetent for meiosis resumption whereas the larger oocyte classes showed the first occurrence of sodium currents that remained high throughout maturation up to the post-vitellogenic stage [Silvestre et al. 2009]. L-type calcium channels, however, do not represent the unique type of calcium channels found in ascidian oocytes that may play a role during oogenesis. Recently, in other ascidian species, we observed, for the first time in oocytes, T-type calcium channels [Gallo et al. 2012], which are typically involved in sperm physiology [Jagannathan et al. 2002]. This novel finding suggests new insights for the mechanism of calcium currents during oocyte maturation.

Figure 2.  Ion current patterns during oocyte maturation of Ciona intestinalis. L-type calcium current activity is high at the GV stage and decreases during maturation. Sodium currents first appear in mature oocytes and remain high up to the zygote.

In mollusks, the influence of calcium entry through the plasma membrane channels has been related to nuclear maturation. In Spisula, nuclear maturation requires external calcium influx through voltage-gated channels in order to undergo GVBD. This is also the case for the oocytes of the surf clam, Ruditapes and Crassostrea [Dubè 1992; Leclerc et al. 2000]. The present hypothesis is that an early calcium influx is a prerequisite to allow nuclear maturation in either PI- [Colas and Dubè 1998] or MI-arrested oocytes of bivalves [Moreau et al. 1996]. This current seems to be mediated by L-type calcium channels [Guerrier et al. 1993; Tomkowiak et al. 1997].

Consistent with these findings, in Octopus vulgaris the accurate electrical characterization of the plasma membrane electrical proprieties of immature oocytes at GV stage showed that L-type calcium currents are higher in small pre-vitellogenic oocytes and significantly lower in early-vitellogenic oocytes. Similarly, a significant decrease of the steady-state conductance occurred from the pre- to early-vitellogenic oocytes indicating that ion and L-type calcium currents play a role in oocyte growth and cytoplasmic maturation, and may be involved in preparing the plasma membrane for the interaction with the spermatozoon [Cuomo et al. 2005].

Ion currents in vertebrate oocyte maturation

Amphibians

Amphibian oocytes exhibit peculiar electrical properties, such as the involvement of chloride channels and sensitivity to neurotransmitters [Jaffe and Cross 1984]. In Xenopus laevis, the ability to respond to neurotransmitters is related to chloride channels which generate a steady current in the early immature oocyte. Earlier studies by Robinson [1979] suggested that the electric field generated by chloride channels may serve to localize components in the growing oocyte and, more generally, these electrical currents might play a role in the establishment of cell polarity during oogenesis. In Xenopus, voltage-sensitive ion channels, selective for sodium and capable of generating action potentials, were shown to first appear during oocyte growth [Baud 1983]. Recently, the ability of chloride channels to induce GVBD was shown using molecular biology techniques [Reyes et al. 2009].

The coordinated action of chloride and sodium channels appears to modulate oocyte maturation in Rana pipiens in which chloride channels disappear throughout maturation; in contrast, the sodium channels remain active up to the full maturity of the oocyte [Baud 1983]. The precise timing of the appearance and disappearance of chloride channels and the increasing activity of sodium channels during oocyte growth suggested that they might play a regulatory role in maturation [Schlichter 1983]. It is well established that progesterone releases amphibian oocytes from the meiotic block and stimulates meiosis progression up to the mature stage [Maller 1985]. In Ambystoma, an interplay between the fluctuation of sodium currents and decreased hydrogen ion conductance was described during the progesterone-induced oocyte maturation [Baud and Barish 1984]. These findings further support the hypothesis that progesterone induced GVBD via a down-regulation of the membrane sodium-potassium ATPase and, hence, influence the ion permeability and transport properties of both nuclear and plasma membranes [Weinstein et al. 1982].

In the European frog Rana esculenta, together with chloride and sodium activity, voltage-dependent potassium current changes occur during oocyte maturation. These changes are considered relevant for successful fertilization [Taglietti et al. 1984]. By accurate electrophysiological recording in Rana perezi, three different types of chloride currents have been characterized during oocyte growth [Ivorra and Morales 1997]. These authors definitively suggested that the presence of most of these infrequent currents in immature oocytes could not be merely the result of random genomic expression but a programmed decision, probably related to a definite functional role in the maturation process.

In Xenopus, there is substantial support for a role for calcium, and its downstream effectors, i.e., calmodulin and calcium-calmodulin-dependent protein kinase II, in mitosis initiation and progression [Means 1994; Whitaker 1995]. Calcium signals are required for GVBD and chromosome condensation during mitosis [Steinhardt and Alderton 1988; Tombes et al. 1992; Ciapa et al. 1994]. In contrast to the well-defined roles for calcium signals in mitosis and after fertilization [Stricker 2000], the role of intracellular calcium signals during oocyte maturation remains contentious. On one hand, early reports suggested that the rise in calcium is sufficient to induce oocyte maturation [Wasserman and Masui 1975; Moreau et al. 1976]. On the other hand, oocytes treated with calcium buffers were unable to mature [Duesbery and Masui 1996] and injection of inositol 1,4,5-trisphosphate (IP₃), which induces calcium release, did not stimulate meiotic maturation [Picard et al. 1985].

Distinct sources of calcium signals can be generated either due to calcium release from intracellular calcium stores or calcium influx from the extracellular compartment. These two calcium sources are mechanistically linked through the store-operated calcium channels (SOCs), which are activated in response to the depletion of calcium stores. The role of SOCs and their interaction with stromal interaction molecule 1 (STIM1), a protein involved in intracellular calcium dynamics from intracellular stores, is still controversial [Worley et al. 2007; DeHaven et al. 2009]. Some of the molecular components of SOCs have been defined. It is known that STIM1 works as the endoplasmic calcium sensor [Liou et al. 2005; Roos et al. 2005], and Orai family proteins work as the pore-forming subunits of SOCs. These channels are known as the calcium release-activated calcium channels [Feske et al. 2006; Vig et al. 2006; Zhang et al. 2006]. A significant percentage of total Orai1 is intracellular at steady state. The depletion of stores completely shifts endosomal Orai1 to the cell membrane [Yu et al. 2010]. In Xenopus oocytes, it has been shown that calcium entry through SOCs, operated by STIM1/Orai1, was responsible for filling the endoplasmic reticulum during maturation [Machaca and Haun 2000]. However, by manipulating intracellular calcium store load and the extent of calcium influx through SOCs, Sun and Machaca [2004] showed that calcium signals were not required for entry into meiosis. On the contrary, high intracellular calcium delays meiosis entry. Moreover, in the absence of calcium signals oocytes arrest in MI, form abnormal spindles, and do not extrude the polar body.

Mammals

In mammals, oocyte maturation may be defined as a process occurring between the first and the second meiotic block that renders the oocyte able to fertilize and to support the early stages of embryonic development. During this time, a number of events occur in which the oocyte plasma membrane plays a crucial role. Before reaching maturity, the mammalian oocyte does not operate as an independent cell. A close cooperation between the oocyte and the surrounding cumulus cells assures the passage of metabolites allowing and/or driving oocyte growth and, finally, maturation. The cumulus cells transmit stimuli to the oocyte, such as those involved in maintaining or removing meiotic arrest (for review see [Silvestre et al. 2011b]). In the former, the oocyte RP is maintained under hyperpolarized conditions, whereas in the latter, depolarization occurs as a consequence of the action of gonadotropins on the cumulus cells [Mattioli et al. 1998], leading to the progression of meiosis. Notwithstanding this interaction, cumulus cells and oocytes do maintain different membrane potentials in spite of the existence of low resistance channels, such as gap junctions [Emery et al. 2001]. Depolarization of granulosa cells was addressed as an essential feature of follicular maturation; this hypothesis is supported by the finding that differential granulosa cell proliferation, steroidogenic capability, and apoptosis can be influenced by selective antagonism of granulosa cell potassium channels [Manikkam et al. 2002]. The interaction between cumulus cells and the oocyte decreases at the end of maturation, together with cumulus expansion and the breaking of heterologous gap junction communications. However, this is not accompanied by a simultaneous decrease in electrical coupling [Racowsky and Setterlie 1987]. In bovine oocytes, we found RP variations during the progression of meiosis [Tosti et al. 2000]. In particular, after a preliminary hyperpolarization at the GVBD stage, RP was depolarized. The opposite pattern has also been recorded in relation to plasma membrane permeability, measured as steady-state conductance [Tosti et al. 2000; Murnane and De Felice 1993]. This parameter is high at the GV stage and decreases during meiosis with little restoration at the mature (MII) stage. Hence, the highest membrane permeability corresponds to the two meiotic arrest phases. It is feasible that the large ion exchange is related to the large metabolic activity of the GV stage or to the preparation of the plasma membrane for fertilization at the MII stage. During maturation, relevant ion dynamics occur through the oocyte plasma membrane due to either an exchange between intra- and extra-cellular compartments or intercellular communications.

Two-pore domain potassium (K_2P) channels that help to set the resting membrane potential have been localized on the plasma membrane of bovine oocytes and may contribute to the electrical activities of the gamete plasma membrane during maturation [Hur et al. 2009]. ATP-sensitive potassium (K⁺_ATP) channels have been found in human, bovine, and porcine oocytes at various maturational stages [Du et al. 2010]. In any tissues where they are expressed, K⁺_ATP channels regulate membrane potential and, at the same time, affect intracellular calcium levels (reviewed by [Nichols 2006]). The link established by these channels between intracellular metabolism, membrane excitability and intracellular calcium signaling suggests a potential role in infertility due to stress injuries or metabolic diseases.

Voltage-dependent anion channels, which are reputed to play a key role in mitochondrial metabolism and ion signalling, have been recently found on the ooplasma membrane of porcine oocytes [Cassarà et al. 2009]. They are arranged as ring-like clusters of structures distributed on the cortical area at GV, but not at MII, stage oocytes. Either their localization or their absence at the end of maturation suggests a functional role during maturation and fertilization of mammalian oocytes.

Another relevant propriety of the plasma membrane is to allow the movement and the proper regulation of fluids between the internal and external side of the cell. The transport of water primarily occurs by simple diffusion through the lipid bilayer membrane [Verkman et al. 1996]. However, in somatic cells high water permeability cannot be explained by simple diffusion because the lipid bilayer is hydrophobic [Preston et al. 1993]. Aquaporins (AQPs) are a family of small membrane spanning monomer and hydrophobic integral membrane channel proteins that are expressed in plasma membranes of many cell types requiring fluid transport [Carbrey and Agre 2009]. In mouse oocytes, the Aqp3 mRNA is expressed in immature oocytes and decreases after either in vitro or in vivo maturation [Jo et al. 2011] suggesting a potential role of these channels in underlying correct maturation progress. In addition, Yamaji et al. [2011] improved the tolerance of the in vitro matured oocytes to vitrification with a glycerol-based solution by injecting AQP3 RNA in immature oocytes.

The role of calcium in oocyte maturation is still a matter of debate [Boni et al. 2007]. Based on the observation that cytoplasmic IP₃ receptors increase during meiotic progression, IP₃-induced calcium release has been suggested as a primary mechanism for maturation of bovine oocytes. A minor role has been attributed to calcium release through ryanodine receptors, which are poorly expressed in bovine oocytes [He et al. 1997]. In the hamster and mouse, a series of spontaneous calcium oscillations occur in the oocyte after isolation from the follicle up to the GVBD stage [Fujiwara et al. 1993; Carroll et al. 1995]. After these oscillations have subsided, calcium does not affect further meiotic progression. In bovine and pig, no calcium oscillations occur during meiotic progression; however, calcium is necessary for meiotic progression since BAPTA, a calcium chelator, causes a delay or block of meiosis [Fujiwara et al. 1993; He et al. 1997]. In addition, the calcium ionophore, A-23187, induces resumption of meiosis in oocytes blocked either at the GV [Wasserman and Masui 1975] or MII stage [Boni et al. 2002]. Because extracellular calcium is required for in vitro GVBD [De Felici and Siracusa 1982] and for the first meiotic division [Paleos and Powers 1981], calcium ion transport through the plasma membrane seems to play a functional role in maturation. Extracellular calcium plays a role in the progression of meiosis. In bovine, we have shown that: i) calcium depletion in the maturation medium severely affects oocyte developmental competence, ii) the activity of plasma membrane L-type calcium channel decreases during maturation, and iii) the exposure to specific inhibitors decreases in vitro maturation efficiency [Tosti et al. 2002]. In addition: i) BAPTA delays kinase activity and inhibits maturation [He et al. 1997], ii) calcium may influence protein synthesis that is essential for maturation [Levesque and Sirard 1996], and iii) calcium modulates gap junction functionality, allowing the drop in cumulus-mediated [cAMP]i levels [Yoshimura et al. 1992]. As a consequence of a net uptake of calcium through the plasma membrane, at the end of maturation, the oocyte achieves an adequate store of calcium. This storage may occur via either gap junctions or specific channels. Electrophysiological and pharmacological studies strongly suggest that ion currents underlying bovine oocyte maturation are L-type calcium channels [Tosti et al. 2000] whose activity decreases throughout meiotic progression. In mouse oocytes, the activity of these channels increases after puberty [Murnane and De Felice 1993], suggesting a role for these channels in meiotic competence.

The STIM1 and Orai1 proteins are components of the plasma membrane-resident calcium channel that are responsible for mediating calcium entry after the mobilization of intracellular calcium. Recently, they have been identified in pig and mouse oocytes and their involvement in store-operated calcium entry has also been demonstrated [Gomez-Fernandez et al. 2009; Koh et al. 2009; Yu et al. 2009]. In porcine, Orai1 expression decreased during oocyte maturation along with the increasing ability to generate a large calcium influx after store depletion [Wang et al. 2012]. In addition, down-regulation of Orai1 expression by siRNA microinjection blocked calcium influx after store depletion and subsequent calcium add-back. Over-expression of Orai1 in the oocytes modified store-operated calcium entry and had an inhibitory effect on the calcium fertilization signal. Co-over-expression of Orai1 and STIM1 led to a dramatic increase in calcium entry after store depletion [Wang et al. 2012].

As above reviewed, the role played by ion currents in oocyte maturation is still unclear. However, the change in plasma membrane electrical properties appear to underlie oocyte growth, meiosis progression, and the preparation of the oocyte for fertilization in all the animals studied. This information may help to develop new culture systems more adequate to the oocyte's physiological needs for proper maturation.

Declaration of interest: The authors have declared that no competing interests exist.

Abbreviations

PI:	=	first meiotic prophase
GV:	=	germinal vesicle
GVBD:	=	breakdown of the GV
MI:	=	first meiotic metaphase
MII:	=	second meiotic metaphase
cAMP:	=	cyclic adenosine mono-phosphate
RP:	=	resting potential
IP3:	=	inositol 1,4,5-trisphosphate
SOCs:	=	store-operated calcium channels
STIM1:	=	stromal interaction molecule 1
K+ATP:	=	ATP-sensitive potassium
AQPs:	=	aquaporins
BAPTA:	=	bis-(o-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid.