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BOR - Papers in Press, published online ahead of print November 14, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.064527
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BIOLOGY OF REPRODUCTION 78, 307–315 (2008)
DOI: 10.1095/biolreprod.107.064527
© 2008 by the Society for the Study of Reproduction, Inc.

Gamete Biology; 

Store-Operated Calcium Entry in Human Oocytes and Sensitivity to Oxidative Stress1

Francisco Javier Martín-Romero 2 3, Jose Ramón Ortíz-de-Galisteo 6, Javier Lara-Laranjeira 6, Jose Antonio Domínguez-Arroyo 6, Ernesto González-Carrera 5 6, and Ignacio S Álvarez 4 6

Departamento de Bioquímicay Biología Molecular,3 Departamento de Biología Celular,4 and Departamento de Terapéutica Medico-Quirúrgica,5Reproduction and Development Group (REDES), Universidad de Extremadura, Badajoz-06071, Spain Instituto Extremeño de Reproducción Asistida (IERA),6 Badajoz-06004, Spain

ABSTRACT

Calcium signaling is a cellular event that plays a key role at many steps of fertilization and early development. However, little is known regarding the contribution of extracellular Ca2+ influx into the cell to this signaling in gametes and early embryos. To better know the significance of calcium entry on oocyte physiology, we have evaluated the mechanism of store-operated calcium entry (SOCE) in human metaphase II (MII) oocytes and its sensitivity to oxidative stress, one of the major factors implicated in the outcome of in vitro fertilization (IVF) techniques. We show that depletion of intracellular Ca2+ stores through inhibition of sarco(endo)plasmic Ca2+-ATPase with thapsigargin triggers Ca2+ entry in resting human oocytes. Ba2+ and Mn2+ influx was also stimulated following inhibition, and Ca2+ entry was sensitive to pharmacological inhibition because the SOCE blocker 2-aminoethoxydiphenylborate (2-APB) reduced calcium and barium entry. These results support the conclusion that there is a plasma membrane mechanism responsible for the capacitative divalent cation entry in human oocytes. Moreover, the Ca2+ entry mechanism described in MII oocytes was found to be highly sensitive to oxidative stress. Hydrogen peroxide, at micromolar concentrations that could mimic culture conditions in IVF, elicited an increase of [Ca2+]i that was dependent on the presence of extracellular Ca2+. This rise was preventable by 2-APB, indicating that it was mainly due to the enhanced influx through store-operated calcium channels. In sum, our results demonstrate the occurrence of SOCE in human MII oocytes and the modification of this pathway due to oxidative stress, with possible consequences in IVF.

2-APB, calcium, calcium channels, hydrogen peroxide, oocyte, oxidative stress, store-operated calcium entry, thapsigargin

INTRODUCTION

Repetitive and transient oscillations of the intracellular calcium concentration ([Ca2+]i) in oocytes are essential during fertilization [1]. Among other processes, the rise in [Ca2+]i is required for the exocytosis of cortical granules and resumption of the meiotic cell cycle [2, 3]. There is a consensus that the mechanism of increase of [Ca2+]i within the oocyte is initiated by the sperm-specific phospholipase C zeta, which activates the phosphoinositide pathway in the egg, resulting in the production of inositol 1,4,5-trisphosphate (InsP3) and 1,2-diacylglycerol via the hydrolysis of phosphatidylinositol 4,5-bisphosphate (reviewed in [4–9]). InsP3 binds to its receptor (InsP3R) located on the endoplasmic reticulum (ER), leading to the initial Ca2+ release from this intracellular store. The increase of [Ca2+]i induces the subsequent activation of calcium pumps, both plasma membrane Ca2+-ATPase (PMCA) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), that restore basal levels of cytosolic calcium by Ca2+ extrusion or Ca2+ uptake into the ER, respectively. Each of the subsequent calcium discharges starts from stores located in the oocyte periphery, likely mediated by a calcium-induced calcium release (CICR) mechanism, when [Ca2+]i, which shows a slow but significant increase between two sequential spikes, reaches the threshold of CICR activation [10].

The molecular mechanism underlying these sperm-triggered calcium oscillations in mammalian oocytes has been extensively studied considering mainly intracellular stores of calcium. Nevertheless, it has been suggested that a continuous Ca2+ influx is needed to preserve Ca2+ spiking [11], as this signaling ceases by chelating extracellular Ca2+ [12]. However, little is known about the precise molecular mechanism of this Ca2+ influx at fertilization and the nature of the Ca2+ channels or transporters involved in this event. In this regard, a recent review highlights the involvement of voltage-operated calcium channels (VOCs) during oocyte maturation from germinal vesicle to metaphase II (MII) oocytes [13], although the relative contribution of a broad range of plasma membrane calcium channels during early signaling in mammalian fertilized oocytes remains unclear. In other somatic cell types, such as neurons, smooth muscle cells, and a variety of endocrine cells, it is well established that calcium waves triggered by diverse stimuli are dependent on the process of calcium entry through plasma membrane in order to replenish calcium levels in Ca2+ stores to maintain the rapid rate of cytosolic Ca2+ spiking. This calcium entry was originally termed "capacitative calcium entry," although the expression "store-operated calcium entry" (SOCE) is now the most commonly used and indicates the retrograde signaling by which plasma membrane calcium channels are controlled by calcium levels within the ER [14, 15]. SOCE has been described in Xenopus oocytes [16], although in these cells SOCE uncouples from Ca2+-store content following meiotic maturation, and there is a down-regulation during meiosis leading to SOCE inactivation at the germinal vesicle breakdown stage. In mammals, there are few reported works describing SOCE in oocytes, and these are restricted to porcine [17] and mouse oocytes [18, 19], while the function of this Ca2+ entry pathway has yet to be addressed.

It has been shown that oxidative stress significantly alters intracellular calcium homeostasis in other cell types, since Ca2+ transport systems like SERCA [20], PMCA [21], or VOCs [22] have been found to be targets of a variety of reactive oxygen species (ROS). Ex vivo manipulation and in vitro culture of cells at an atmospheric oxygen level, which is approximately 20%, expose these cells to a nonphysiological high oxygen tension, leading to deregulation of the cell redox state. Accordingly, ex vivo culture of mammalian gametes, in particular human gametes during assisted reproductive techniques, may result in alterations in Ca2+ signaling with unknown consequences in fertilization and early development. Consequently, the study of the sensitivity of oocyte Ca2+ homeostasis to oxidative stress is essential to better understand the consequences of manipulating gametes and embryos. In this work, we addressed the question of extracellular calcium entry mechanisms in human MII oocytes and found that mature human oocytes (arrested in MII) display a Ca2+ influx pathway that is highly sensitive to pharmacological SOCE inhibition. Moreover, we confirmed that SOCE is highly sensitive to oxidative stress, and that exposure of oocytes to micromolar hydrogen peroxide severely impairs intracellular calcium homeostasis.

MATERIALS AND METHODS

Materials

SynVitro Flush, Universal in vitro fertilization (IVF) medium, ISM-1, and hyaluronidase (SynVitro Hyadase) from Medicult (Jyllinge, Denmark) were used for the recovery and manipulation of oocytes. Ovarian stimulation was carried out in all cases with a long agonists protocol using rFSH (Gonal-F) from Serono Europe Ltd. (London, U.K.) and GnRH agonist (Leuprolide acetate-Procrin) from Abbot Laboratories S.A. (Madrid, Spain), adding in some cases (when LH levels were under 1.5 IU/ml) HMG (Menopur) from Ferring. Ovulation was induced 36 h before oocyte recovery using CG (Profasi HP or Ovitrelle) from Serono Europe.

Fura 2 acetoxymethyl ester (fura 2-AM) was purchased from Molecular Probes (Eugene, OR). Hydrogen peroxide, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), rhodamine 123 (Rhod123), and diethylstilbestrol were purchased from Sigma Chemical Co. (St. Louis, MO). 2-aminoethoxydiphenylborate (2-APB) and thapsigargin (TG) were from Calbiochem. Other chemicals were of the highest quality available.

Methods

Only spare oocytes were used, and patients gave their written consent prior to participation. Approval for the study was granted by the University of Extremadura, Spain (Universidad de Extremadura, Spain). All the patients involved yielded more than 12 cumulus-oocyte complexes (COC) after aspiration and were not older than 35 yr. Aspiration was performed by ultrasound, and the COC were collected as per routine in flushing medium. The oocytes obtained from each patient were processed as a normal IVF cycle, and only when it was confirmed that we could put aside some oocytes for research purposes did we begin the experiments with spare oocytes. Denudation was carried out with 80 IU/ml of hyaluronidase for 1 min, and afterward the oocytes were maintained in ISM-1 medium at 37°C in a 95% air/5% CO2 controlled atmosphere until processing for the experiments. The experiments presented in this work were carried out with oocytes arrested in MII and performed within a time window of 5–10 h after oocyte recovery, except for the long-time cultured oocytes, which was extended to 24 h.

During the period of the study, no differences were found in the pregnancy rate or other parameters such as fertilization rate, embryo quality, implantation, etc., meaning that the use of some of the oocytes did not produce any detriment in the outcome of the IVF center including the egg donor program. Overall, about 180 women participated in the study, mostly donors.

Measurement of Cytosolic Ca2+ Concentration

[Ca2+]i was measured basically as indicated in previous papers [22, 23]. It is important to highlight that the MII oocytes were not processed by removal of the zona pellucida before fura 2-AM loading. Oocytes were loaded with the dye by incubation in ISM-1 medium for 45 min with 5 µM fura 2-AM and 0.025% Pluronic-F127 (Sigma Chemical Co., St. Louis, MO) rinsed thoroughly with Hanks balanced salt solution (HBSS; Invitrogen cat. no. 14025050) containing Ca2+ and Mg2+, and placed in a 20-µl drop of assay medium covered with mineral oil. Intracellular calcium concentration was measured with a Nikon Diaphot 300 inverted fluorescence microscope equipped with thermostatic controlled plate. Ratio fluorescence images were obtained with excitation filters of 340 and 380 nm and a DM510 dichroic mirror and absorption filter (emission side) of 510 nm (Nikon Instruments Europe B.V., The Netherlands). Digital images were taken with a Hamamatsu Hisca CCD camera (Hamamatsu Photonics, France) and subsequently analyzed with the Argus/Hisca software. [Ca2+]i was calculated as indicated in [24] with the equation


Formula 78

where R is the measured fluorescence ratio (340/380), and Rmax and Rmin are the ratio values (340/380) for Ca2+-bound and Ca2+-free in fura 2-loaded oocytes. Rmax and Rmin were determined experimentally from steady-state fluorescence ratio (340/380) measurements after sequential addition of (1) BrA23187 (5 µg/ml) and (2) 10 mM EGTA (Sigma Chemical Co., St. Louis, MO). The average values obtained for Rmax and Rmin were 3.56 ± 0.11 and 0.14 ± 0.02, respectively, and the average value for the ratio of fluorescence values for Ca2+-free/Ca2+-bound indicator at 380 nm (β) obtained with our instrument setup was 8.4 ± 0.6. A value of 224 nM was used for Kd, the dissociation constant of the complex fura 2:Ca2+ [24].

This protocol was followed for the oocytes throughout the experiment and under the conditions described in the results that include the addition of H2O2, cations, and drugs.

Activation of SOCE

In order to induce the opening of store-operated calcium channels (SOCs) in oocytes, we followed a classical method using TG, a well-known inhibitor of the SERCA [25, 26], as we reported previously for other cells [27]. Briefly, Ca2+ within the ER was depleted by incubating cells with 2 µM TG in EGTA-buffered Ca2+-free HBSS with the following composition: 138 mM NaCl; 5.3 mM KCl; 0.34 mM Na2HPO4; 0.44 mM KH2PO4; 4.17 mM NaHCO3; 4 mM Mg2+; EGTA 0.1 mM (pH 7.4). SOCE and subsequent increase of [Ca2+]i was confirmed with the addition of 2.5 mM Ca2+ to this TG-containing medium. The free Ca2+ concentration in the EGTA-buffered Ca2+-free HBSS was measured experimentally with fura 2 (sodium salt) and found to be 25–30 nM.

Mitochondrial Inner Membrane Potential

To measure the mitochondrial membrane potential, we used Rhod123 as described previously [28], with modifications. Briefly, MII oocytes were incubated with 5 µM Rhod123 for 10 min at 37°C and then washed in HBSS. Rhod123 was excited with a 465–495 nm excitation filter, and emitted light was detected using a long-pass 515- to 555-nm barrier filter, using a Nikon DS-Fi1 CCD camera attached to a Nikon Eclipse TE2000-U inverted microscope (Nikon Instruments Europe B.V., The Netherlands). Control experiments were carried out in the presence of the uncoupler FCCP (10 µM) to follow the relocalization of the dye that is dependent on the mitochondrial inner membrane potential.

RESULTS

SOCE in Human MII Oocytes

SOCE is a mechanism by which the influx of extracellular calcium through plasma membrane SOCs is controlled by the filling state of intracellular Ca2+ stores. It has been previously shown that inhibition of the SERCA with TG, which leads to the depletion of Ca2+ stores [25, 26] or the use of Ca2+ ionophores such as A23187 and ionomycin, can trigger SOCE in a variety of cell types [29]. In order to elucidate whether this mechanism of calcium entry is functioning in mature human MII oocytes, we used 2 µM TG, which evoked a rapid and transient increase in [Ca2+]i (Fig. 1A). This transient peak in fura 2-loaded oocytes reached a maximum ratio F340/F380 of 0.48 ± 0.2, i.e., increasing the resting levels of free intracellular Ca2+ from 68 ± 5 nM to 208 ± 14 nM. This peak was found after 6.8 ± 0.6 min of incubation with TG in Ca2+-free medium. The subsequent rapid decrease of [Ca2+]i to basal levels can be explained by the rapid extrusion of Ca2+ to the extracellular space by the TG-insensitive PMCA. Emptying of intracellular Ca2+ stores by TG led to the activation of SOCE by the opening of SOCs, as revealed by the rapid rise in [Ca2+]i when extracellular Ca2+ was added to TG-containing assay medium. However, the extent of this increase in [Ca2+]i might be explained by the combined activities of SOCE together with intracellular calcium channels, pumps, exchangers, and buffering. For this reason, to better measure the activity of calcium channels involved in SOCE, after depleting Ca2+ stores with TG in Ca2+-free medium, we replaced the addition of extracellular Ca2+ by the addition of 2.5 mM Ba2+. Barium has been shown to enter the cell specifically via SOCs in rat basophilic leukemia cells and mast cells [30, 31], but cannot be extruded by plasma membrane calcium pumps. In this way, after depleting the intracellular stores with TG, we consistently found a rapid increase of the ratio F340/F380 up to 0.98 ± 0.05 after Ba2+ addition to the assay medium (Fig. 1B). We further determined that this influx is mediated by SOCs because this cation entry through the plasma membrane can be blocked by 2-APB in a dose-dependent manner. 2-APB is a potent SOC blocker that has been used extensively to differentiate SOCE from other transport mechanisms (Ki, concentration giving half maximal inhibition; 30 µM; [29, 32]). Short preincubation of oocytes (10 min) with 100 µM 2-APB inhibited 70%–75% of the Ba2+ entry after Ca2+-store depletion, while the inhibition reached 90%–95% for 250 µM 2-APB (Fig. 1C).


Figure 1
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  		FIG. 1 SOCE triggered by TG in MII human oocytes. A) [Ca2+]i was measured with fura 2-AM as indicated in Materials and Methods. After fura 2-AM loading, oocytes were rinsed in HBSS and the ratio F340/F380 was measured for 10 min in Ca2+-free HBSS. Then, 2 µM TG (closed symbols) or the vehicle (1% DMSO, open symbols) were added to the oocytes, and the ratio F340/F380 was measured for the following 20–30 min until [Ca2+]i reached baseline levels in the TG-treated oocytes. At this time, 2.5 mM Ca2+ (A) or 2.5 mM Ba2+ (B, C) was added, and the ratio F340/F380 measured for an additional 20 min. In (C), 2-APB was added 10 min before addition of Ba2+ to the sample (indicated by the arrow). D) Oocytes were loaded with fura 2-AM, and the [Ca2+]-independent emission of fluorescence (Excitation wavelength = 360 nm, Emission wavelength = 510 nm) was measured for 10 min in Ca2+-free HBSS medium. Then, 1 mM Mn2+ and 2 µM TG (closed circles) were added to the sample (indicated in the figure by the arrow), and the quenching of fluorescence was recorded for an additional 20 min. In parallel experiments, 1 mM Mn2+, 2 µM TG, and 100 µM 2-APB were added to the sample, and fluorescence was recorded during the same time (closed up triangles). Control experiments were carried out with the addition of Mn2+ in the absence of TG (open circles). The number of measurements (1 oocyte per trial) was n = 5 for A and B, and n = 4 for each condition in C and D. The figure shows representative traces for each experimental condition for the sake of clarity.

An alternative method to characterize divalent cation entry through the plasma membrane induced by TG is the use of the fluorescence quencher Mn2+ [18, 33]. The influx of Mn2+ has been described to take place through SOCs in muscle cells [27] as well as in porcine oocytes [17]. Fura 2 fluorescence (excitation wavelength 360 nm) in dye-loaded oocytes decreased remarkably in the presence of 1 mM Mn2+ after addition of 2 µM TG, indicating a rapid increase of Mn2+ influx stimulated by TG without a lag phase (Fig. 1D). As in the case of Ba2+ entry, 2-APB prevented TG-stimulated Mn2+ influx, demonstrating its mode of action as an SOCE blocker. This experiment is further confirmation of the conclusion that there exists an influx of divalent cations through the plasma membrane, which is regulated by intracellular Ca2+ stores, in mature MII human oocytes.

Calcium Deregulation in Human Oocytes Induced by H2O2

Given the importance of calcium signaling in oocytes and the sensitivity of Ca2+-transport systems to redox modifications (see below), in this work we addressed the study of the sensitivity of SOCE to oxidative stress in human oocytes. ROS have been used extensively to study the effects of oxidative stress on different calcium transport systems in diverse cell types (reviewed in [34]), and we have reported alterations of the function of VOCs, PMCA, and SERCA induced by oxidative and/or nitrosative stress in other model cell types in vitro [20–22]. In that previous work, we found that hydrogen peroxide-mediated oxidative stress triggered irreversible opening of L-type VOCs and inhibition of Ca2+-pumps that can lead to the impairment of intracellular Ca2+ homeostasis. Figure 2A shows how single additions of micromolar H2O2 to MII oocytes elicited a rapid dose-dependent Ca2+ deregulation. Indeed, this loss of regulation occurred for the range of H2O2 concentrations 10–100 µM and was biphasic, with an initial rise that could be buffered temporarily by the cell, followed by an absolute loss of the [Ca2+]i regulation. Concentrations of H2O2 less than 10 µM were also studied, but did not increase [Ca2+]i significantly during the 3 h following the addition of H2O2 (results not shown). In sum, extracellular H2O2 triggers an increase of [Ca2+]i in human oocytes that leads to the impairment of intracellular calcium homeostasis.


Figure 2
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  		FIG. 2 [Ca2+]i increase after exposure to H2O2 in human oocytes: requirement of extracellular calcium. A) [Ca2+]i was measured with fura 2-AM as indicated in Materials and Methods. After fura 2-AM loading, oocytes were rinsed in HBSS, and the ratio F340/F380 was measured for 10 min. Then, a single pulse of micromolar H2O2 (10, 20, 50, or 100 µM) was added to the sample (indicated by the arrow), and the ratio F340/F380 was monitored until this signal indicated saturation. The number of measurements (1 oocyte per trial) was n = 5 for each H2O2 concentration. Open circles mean the ratio F340/F380 under the same experimental conditions, i.e., in HBSS, without H2O2 addition (n = 5). The figure shows a single representative trace for each concentration for the sake of clarity. B) After fura 2-AM loading, the oocytes were rinsed in Ca2+-free HBSS and the ratio F340/F380 was measured for 10 min. Then, a single pulse of 100 µM H2O2 was added to the sample (indicated by the arrow), and the ratio F340/F380 was recorded (closed symbols). Parallel experiments were carried out without addition of H2O2 under the same experimental conditions, i.e., incubating oocytes in Ca2+-free HBSS (open symbols). The number of measurements (1 oocyte per trial) was n = 5 for H2O2 -treated oocytes and n = 5 for control untreated oocytes. The figure shows a single representative trace for each experimental condition.

To better characterize the alterations of [Ca2+]i induced by H2O2 and to evaluate the transport systems involved in this deregulation, we exposed oocytes to 100 µM H2O2 in Ca2+-free medium, and detected no significant increase in [Ca2+]i for the following 80 min (Fig. 2B). The requirement of extracellular Ca2+ strongly suggested that plasma membrane transport systems could be involved in the H2O2-induced calcium deregulation in oocytes, as [Ca2+]i rise was independent of intracellular stores.

It is important to note that H2O2 may induce a significant loss of mitochondrial membrane potential that would partially explain the mechanism of the Ca2+ deregulation. The mitochondrial inner membrane potential is considered a key regulator of cell death induced by many types of insults, including oxidative stress in human follicles [35]. Mitochondrial membrane potential was measured with Rhod123, which has been previously used to investigate mitochondrial activity in single mouse eggs [28]. Our results show that the addition of 100 µM H2O2, the highest H2O2 concentration tested, did not alter the mitochondrial inner membrane potential during the first 45 min of exposure to this ROS (Fig. 3). Parallel experiments were performed with the mitochondrial uncoupler FCCP to evaluate localization and fluorescence intensity under depolarization conditions, showing a pattern of distribution that did not correspond to that observed during treatment with H2O2. In addition, 100 µM H2O2 did not significantly decrease emission of fluorescence when F360 was used as the excitation wavelength in fura 2-loaded oocytes (results not shown). This result indicates that no significant leakage of the dye takes place during the exposure to H2O2, allowing us to exclude any extensive cell death as responsible for the increase in [Ca2+]i in our experimental conditions.


Figure 3
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  		FIG. 3 Mitochondrial inner membrane potential upon exposure to H2O2. The mitochondrial inner membrane potential was measured with Rhod123 as indicated in Materials and Methods. Oocytes were rinsed in HBSS after incubation with the dye, and emission of fluorescence was measured for 45 min in HBSS or HBSS supplemented with 100 µM H2O2 (labeled H2O2). Images recorded after 2 min and 45 min in these experimental conditions are shown in the figure. Parallel positive control experiments were carried out with oocytes incubated in HBSS with 10 µM FCCP (labeled FCCP) for 15 min after loading with Rhod123 in order to record emission of fluorescence and localization of the dye in depolarizing conditions. Figure shows representative oocytes for each experimental condition (n = 4 oocytes/trial).

Thus, our results indicated that Ca2+ homeostasis was a primary target of oxidative stress in human oocytes, possibly by modifying plasma membrane Ca2+ transport systems.

SOCE Underlies Ca2+ Deregulation Induced by H2O2 in Human Oocytes

We believe that, among the channels that are operating in oocytes in the regulation of the Ca2+ influx, the SOCs described in this paper could be one of the major targets of H2O2 damage. To evaluate the relationship between oxidative stress and SOCE in detail, we first confirmed this extracellular calcium dependence by studying the Mn2+ influx rate through the plasma membrane in fura 2-loaded oocytes treated with H2O2 (Fig. 4A). It has been demonstrated previously that capacitative entry is permeable to Mn2+ [36] and is, therefore, a good tool for measuring divalent cation entry. The rate of Mn2+ influx increased notably after H2O2 addition and was dose dependent. Moreover, the t1/2 of the fluorescence quenching by Mn2+ fits well with the onset of the Ca2+ rise induced by H2O2 shown in Figure 2, suggesting that opening of Ca2+-channels could be involved in calcium overload during the oxidative stress induced by extracellular H2O2. In addition, we measured Ba2+ entry through the plasma membrane and found that the kinetics of this transport had a similar dependence on the intensity of the H2O2 insult (Fig. 4B). It has been reported that Ba2+ entry is mediated by SOCs in other cell types [30, 31], and here we showed that SOCs are permeable to Ba2+ in oocytes as well (Fig. 1). Therefore, our results support the hypothesis that oxidative stress modulates Ca2+ influx through SOCs located at the plasma membrane.


Figure 4
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  		FIG. 4 Divalent cation transport across plasma membrane is enhanced by H2O2. A) After fura 2-AM loading, the oocytes were rinsed in HBSS, and the fluorescence was recorded for 10 min in Ca2+-free HBSS at the excitation wavelength 360 nm (F360). After recording the baseline, 1 mM Mn2+ and H2O2 at the same concentrations as in Figure 2 were added to the sample, and the fluorescence was monitored until the completion of quenching. The number of measurements (1 oocyte per trial) was n = 3 for each concentration of H2O2 and for control oocytes without H2O2. B) The ratio F340/F380 was measured in fura 2-loaded oocytes incubated in Ca2+-free HBSS containing 2.5 mM Ba2+. After recording the baseline for 10 min, a single pulse of H2O2 was added to the sample (indicated by the arrow), and the ratio F340/F380 was measured until reaching a steady signal. As in (A), the number of measurements (1 oocyte per trial) was n = 3 for each H2O2 concentration and the control. The figure shows representative traces from these experiments.

Although these results strongly indicated that calcium channels at the plasma membrane of human oocytes could be a primary target of oxidative stress, we studied the pharmacology of this divalent cation entry to confirm our hypothesis. Accordingly, we analyzed the ability of calcium channel blockers to inhibit calcium deregulation induced by H2O2.

The simplest inhibitors are Ca2+ mimics, such as other divalent cations, which have been reported to block VOCs [37], voltage-independent Ca2+ channels [38], and ICRAC in mast cells [39]. In the human oocytes, we found that 5 mM Ni2+ strongly prevented [Ca2+]i increase in H2O2-treated oocytes (Fig. 5A). More specific inhibitors of the TG-induced capacitative calcium entry are diethylstilbestrol [40] and 2-APB, as we noted above. These two blockers prevented Ca2+ deregulation stimulated by H2O2, the effect of 100 µM 2-APB being more intense and longer-lasting, because [Ca2+]i did not rise above resting levels during the time of the experiment, i.e., 35 min following the addition of 100 µM H2O2.


Figure 5
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  		FIG. 5 Attenuation of calcium deregulation by calcium channel blockers. A) The intracellular free calcium concentration was measured in fura 2-loaded oocytes in Ca2+-containing HBSS medium. At the time indicated by the arrow, 100 µM H2O2 was added to the sample, and the ratio F340/F380 was recorded (closed circles). In parallel experiments, 5 mM Ni2+ (diamonds), 10 µM diethylstilbestrol (Des, triangles), or 100 µM 2-APB (squares) was added 10 min before the addition of H2O2 (time = 0 in the figure). The number of measurements (1 oocyte per trial) was n = 5 per experimental condition. B) Following the same experimental design as described in (A), 100 µM H2O2 was added to oocytes treated with 100 µM 2-APB (squares) or with the vehicle (DMSO, closed circles) in Ca2+-free medium supplemented with 2.5 mM Ba2+. In parallel, oocytes in Ca2+-free medium without Ba2+ were treated with H2O2 (open circles). The number of measurements (1 oocyte per trial) was n = 5 per experimental condition. The figure shows representative traces for the sake of clarity.

In addition, we analyzed barium permeability through the plasma membrane, which is enhanced after exposure to H2O2 (Fig. 4B) in the presence of the SOCE-specific blocker 2-APB. Figure 5B shows the large blocking effect of 2-APB because the Ba2+ influx was completely abolished after treatment with H2O2 in the presence of this drug.

We have previously shown that serum-supplemented media used in the manipulation and culture of oocytes, as in assisted reproductive techniques, can generate fluxes of H2O2 within the low micromolar range [41]. Therefore, the possibility that long periods of culture may alter Ca2+ signaling by altering SOCE in human oocytes deserves to be studied. For that purpose, we have cultured oocytes for 24 h in ISM-1 medium, mimicking conditions used for regular human IVF. Thereafter oocytes were loaded with fura 2-AM, and TG-stimulated Mn2+ influx was monitored with the fluorescence quenching technique (Fig. 6). The results show that Mn2+ influx is faster in cultured oocytes compared with uncultured oocytes, i.e., oocytes used in the same day of the ovary punction, indicating an increased SOCE in aged oocytes. This result supports the hypothesis that the oxidative stress associated with the culture conditions used for IVF can interfere with intracellular Ca2+ signaling by impairing the molecular machinery that regulates SOCE in oocytes.


Figure 6
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  		FIG. 6 Divalent cation transport across plasma membrane in fresh and aged oocytes. MII oocytes were incubated with fura 2-AM and assessed for TG-stimulated Mn2+ influx experiment in the same day of ovary punction, i.e., 5–6 h after retrieval and denudation of oocytes (open symbols, control oocytes, n = 7). Using cells from the same donors, spare oocytes were incubated in ISM-1 medium, and the Mn2+ influx experiment was performed after 24 h of in vitro culture at 37°C in CO2:air (5.5%:94.5%) atmosphere (closed symbols, cultured oocytes, n = 7). The figure shows mean ± SD.

In sum, our results demonstrated the existence of SOCE in resting human MII oocytes and indicated that SOCs play a key role in H2O2-induced Ca2+ deregulation in human oocytes, as they were found to be a primary target of extracellular oxidative stress.

DISCUSSION

Calcium signaling in oocytes has been extensively studied because of its importance in oocyte maturation and the early steps of fertilization. At fertilization, oocytes generate calcium oscillations that arise at least in part from the release of InsP3-sensitive intracellular calcium stores, mainly the ER. Nevertheless, much less is known regarding the role of plasma membrane calcium transport systems in fertilization, although it is known that extracellular calcium is required for maintaining Ca2+ oscillations, because they are abolished in Ca2+-free medium [11, 12]. The involvement of extracellular Ca2+ and the role of Ca2+ influx vary between species. For example, extracellular Ca2+ influx through VOCs seems to be required at least for the initial calcium transient in oocytes of marine bivalves [42], but not in hamster oocytes [11].

In excitable cells, Ca2+ entry through VOCs is assumed to be the most important influx pathway, whereas in non-excitable cells this entry takes place mainly through SOCs [43]. In oocytes, a TG-induced influx pathway for Ca2+ was reported by Kline and Kline in the mouse [12], and increased divalent cation entry simultaneous with periodic release of Ca2+ from internal stores of fertilized mouse oocytes has been described, suggesting the probable existence of a capacitative Ca2+ pathway [18].

Here we have shown for the first time the SOCE in human mature oocytes. In human oocytes, TG was found to elicit a rapid and transient increase in [Ca2+]i because of the specific inhibition of sarco(endo)plasmic Ca2+ pumps and the concomitant leak of Ca2+ from internal stores to the cytosol. This increase in [Ca2+]i occurs without a lag phase, indicating the presence of high Ca2+-sequestering pump activity in ER, sensitive to this drug. This rapid action of TG is similar to the inhibition found for other cell types [26, 27, 44]. The concomitant decrease in the Ca2+ filling state of intracellular stores leads to the opening of plasma membrane SOCs, as was revealed by the rapid Ca2+ entry when extracellular Ca2+ was added to TG-treated oocytes. The discrete rise of [Ca2+]i is indeed the average result of a sum of events, including Ca2+ extrusion by Ca2+ pumps, buffering in TG-insensitive intracellular stores (mitochondria), and cytosolic buffering. For that reason, tracing unidirectional Ba2+ or Mn2+ entry is a reliable method with which to better characterize the permeability to divalent cations. We showed in this work that Ca2+/Mn2+/Ba2+ entry is enhanced after depleting intracellular Ca2+ stores with TG, indicating the existence of a Ca2+-transport system in the plasma membrane that is functionally linked to the Ca2+-filling state of these stores. In early reports, there was some controversy about the analysis of the effects and the mechanism of action of 2-APB because it was used as an InsP3-induced Ca2+ release blocker (reported half maximal inhibition at 36–42 µM [45, 46]); however, it is now accepted that SOCE is a primary target for micromolar 2-APB in a variety of cell types [29, 32, 47]. Here we demonstrated that 2-APB blocks TG-stimulated divalent cation entry in human oocytes, confirming the existence of store-operated channels in these cells.

Calcium transport systems have been shown to be regulated by the redox state, and ROS such as hydrogen peroxide and superoxide can mediate oxidative pathology through changes in channel function [34]. Although ample information is available on the alterations induced by oxidative stress on VOCs [22, 48, 49], InsP3R and RyR [50], PMCA [21, 51], and SERCA [20, 52], much less is known about these modifications on SOCE.

There is currently great interest in oxidative stress and its pathophysiological implications for human health because oxidative stress is believed to be fundamentally involved in the development of a wide range of diseases, and its influence on calcium signaling may potentially be relevant. Because precise control of Ca2+ oscillations is essential for the initial phase of fertilization and for the appropriate resumption of the cell cycle, the study of oxidative stress and its influence on Ca2+-transport systems in oocytes becomes of major interest for reproductive biology. We therefore investigated the sensitivity of human oocytes to hydrogen peroxide, one type of ROS generated as a downstream side-product of many oxidative insults, as well as being formed in normal physiological conditions [53, 54]. Although exogenous addition of H2O2 will not precisely mimic the physiological situations in which H2O2 is involved, its use in studying the role of H2O2 in signaling has become ever more frequent [55]. In the present work, using single additions of micromolar H2O2, we demonstrated that [Ca2+]i in human oocytes increases significantly in a dose-dependent manner, leading to the rapid loss of intracellular calcium homeostasis. Similarly, Takahashi et al. reported H2O2-induced Ca2+-deregulation in mouse oocytes that leads to a low developmental rate to blastocyst formation after fertilization of these H2O2-treated oocytes [56]. Although those authors suggested that deregulation could be due to a probable inhibition of ER Ca2+-ATPases, this hypothesis was not proved, and here we have demonstrated that the rise in [Ca2+]i and subsequent deregulation depends on the presence of Ca2+ in the external medium, because no increase of [Ca2+]i was found in the absence of external Ca2+, ruling out the involvement of internal stores like ER. On the contrary, our data strongly suggest that plasma membrane Ca2+ channels may be involved in Ca2+ overloading. In addition, Ca2+ channel blockers, and in particular store-operated channel blockers, notably reduce Ca2+ entry during H2O2-induced stress, proving that the activity of these channels is a target of oxidative stress. Moreover, these effects occur without a lag phase after the addition of H2O2, as was shown with the Ba2+/Mn2+ unidirectional influx experiments, indicating that divalent cation entry is not a late consequence from a retrograde signaling originating in the ER.

Of particular interest was that the effect of exogenously added H2O2 on Ca2+ homeostasis in human oocytes developed within a certain time window in the absence of significant alteration of mitochondrial membrane polarization, supporting the conclusion that 2-APB-sensitive divalent cation entry is an early and major target of oxidative insult induced by H2O2.

Measurements of steady-state [Ca2+]i show a biphasic effect (Fig. 2A), with an early rise in [Ca2+]i followed by a rapid drop and a final Ca2+ overloading induced by all H2O2 concentrations tested. Since the steady-state [Ca2+]i measured with fura 2 is the result of different activities, i.e., Ca2+ pumps, buffers, and Ca2+ channels, the results obtained with this assay cannot be explained only in terms of Ca2+ entry. However, the results from replacing Ca2+ by Mn2+ or Ba2+ in the extracellular medium during H2O2 insult (Fig. 4) allow one to conclude that the increase of [Ca2+]i in oocytes treated with micromolar H2O2 is accounted for by a continuous calcium entry. The results shown in Figure 4 are not in contradiction with those of Figure 2A. The secondary drop in [Ca2+]i after the initial rise of [Ca2+]i induced by the addition of H2O2 could be due to a probable activation of plasma membrane and ER Ca2+ pumps after the increase of [Ca2+]i, because KM for Ca2+ varies among different isoforms, but it is well below 1 µM for all of them [57]. This Ca2+-pump activation leads to a temporary decrease of cytosolic calcium that cannot, however, be maintained if a continuous Ca2+-influx persists, as suggested by Ba2+/Mn2+ influx studies, leading to an irreversible loss of the intracellular calcium homeostasis. It is important to highlight at this point that the increased Ca2+ entry through SOCs induced by oxidative stress does not inevitably mean an increase in the content of Ca2+ within intracellular stores, but merely a dysfunction of SOCs that could be opened without a functional connection to ER.

The increased divalent cation influx was found even for the lowest H2O2 concentration tested (10 µM). This is within the estimated concentration range that can be generated in vivo under physiological conditions [55] and in vitro when serum-supplemented media are used in the manipulation and culture of oocytes, as in assisted reproductive techniques [41]. Indeed, when unfertilized oocytes are incubated for 24 h in ISM-1, a culture medium commonly used for human IVF, a slight increase of the rate of Mn2+ influx is monitored after TG stimulation when compared to uncultured oocytes. The consequences of oxidative stress on SOCE could explain, at least in part, the previously reported detrimental effects observed in Ca2+ signaling during aging of oocytes [58]. However, the aim of our study is to define a scale of sensitivity of calcium transport systems to oxidative stress in human oocytes, and here we demonstrate that SOCE is a major and early system deregulated by oxidative stress. The molecular mechanism underlying aging in culture is out of the scope of this work, and long-term changes or mechanisms of adaptation of oocytes to culture conditions remain keys questions in reproductive biology that require further investigation. In this regard, modulation of Ca2+ signaling by kinases must be considered, because Ca2+ spiking at fertilization triggers translocation of protein kinase C (PKC) to the egg plasma membrane, and this activates SOCE [19]. The substrate of PKC in this pathway is still unknown, but it has been concluded that positive feedback of Ca2+ influx mediated by PKC ensures the long-lasting Ca2+ oscillations required for early development [19]. An alternative explanation for the observed increase of SOCE induced by H2O2 could be the activation of this PKC activity by oxidative stress, a hypothesis that should be addressed in future studies.

Although our present data have shed some light on the question of calcium homeostasis and its relationship with oxidative stress in human oocytes, the study of the precise molecular nature of SOCs in these cells is essential for the future because this is a wide family of channels with diverse permeability properties and pharmacology [43, 59]. Knowledge of the specific channels expressed in mature human oocytes could improve the description of calcium signaling during the maturation and fertilization of human oocytes and could help with considerations of the pharmacology of these channels during oocyte manipulation for assisted reproductive techniques such as IVF.

ACKNOWLEDGMENTS

We are very grateful to Dr. Carlos Gutierrez-Merino for the use of the Nikon-Diaphot epifluorescence inverted microscope and to Dr. Eva Miguel-Lasobras for support and assistance.

FOOTNOTES

1Supported by grants BFU2004-04500 from the Spanish Ministerio de Educación y Ciencia, SCSS-0510 and SCSS-0676 from the Consejeria de Sanidad y Consumo (Junta de Extremadura), and PRIB06B300 from the Consejeria de Infraestructuras y Desarrollo Tecnológico (Junta de Extremadura). Back

Correspondence: 2Francisco Javier Martín-Romero, Department of Biochemistry and Molecular Biology, Science Faculty, University of Extremadura, Avenida de Elvas s/n, 06071-Badajoz, Spain. FAX: 34 924 289 419; e-mail: fjmartin{at}unex.es

Received: 30 July 2007.

First decision: 25 August 2007.

Accepted: 3 November 2007.

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