Introduction
Fertilization stimulates the eggs of all studied species arrested at different stages of meiosis to resume and complete meiotic cell cycle via intracellular calcium signals (Runft et al., Reference Runft, Jaffe and Mehlmann2002).
Transient increases in the concentration of egg cytosolic Ca2+ are essential for cortical granule release, completion of meiosis and pronuclear formation. (Nuccitelli, Reference Nuccitelli1991; Whitaker & Swann, Reference Whitaker and Swann1993; Schultz & Kopf, Reference Schultz and Kopf1995; Stricker, Reference Stricker1999; Abbott & Ducibella, Reference Abbott and Ducibella2001; Oterino et al., Reference Oterino, Sánchez Toranzo, Zelarayán, Valz-Gianinet and Bühler2001; Horner et al., Reference Horner and Wolfner2008).
Two types of intracellular calcium channels, located in specialized regions of the endoplasmic reticulum (ER), have been identified to control calcium signalling in eggs: the universal inositol trisphosphate receptor (IP3R), and the species-specific ryanodine receptor (RyR), which can be found individually or together depending on the particular egg species (Galione et al., Reference Galione, Lee and Busa1991, Reference Galione, McDougall, Busa, Willmott, Gillot and Whitaker1993a,Reference Galione, White, Willmott, Turner, Potter and Watsonb; McPherson et al., Reference McPherson, McPherson, Mathews, Campbell and Longo1992; Swann, Reference Swann1992; Nuccitelli et al., Reference Nuccitelli, Yim and Smart1993; Fissore & Robl, Reference Fissore and Robl1993; Lee et al., Reference Lee, Aarhus and Walseth1993; Kline & Kline, Reference Kline and Kline1994; Ayabe et al., Reference Ayabe, Kopf and Schultz1995; Yue et al., Reference Yue, White, Reed and Bunch1995, Reference Yue, White, Reed and King1998; Sousa et al., Reference Sousa, Barros and Tesarik1996; Albrieux et al., Reference Albrieux, Sardet and Villaz1997; Herbert et al., Reference Herbert, Gillespie and Murdoch1997; Macháty et al., Reference Macháty, Funahashi, Day and Prather1997; Balakier et al., Reference Balakier, Dziak, Sojecki, Librach, Michalak and Opas2002; Petr et al., Reference Petr, Urbánková, Tománek, Rozinek and Jílek2002; Tesarik, Reference Tesarik2002; Wang et al., Reference Wang, White, Reed and Campbell2005). Our laboratory has previously reported evidence about the existence and functionality of IP3Rs and RyRs of in vitro matured Bufo arenarum oocytes. We observed that IP3Rs were able to trigger oocyte activation by exerting their effect in an independent manner; by contrast, RyRs-induced oocyte activation showed a relationship between both pathways, probably through a calcium-induced calcium-release (CICR) mechanism (Ajmat et al., Reference Ajmat, Bonilla, Zelarayán, Oterino and Bühler2010).
Several regulatory pathways that tailor the response of these channels to the specific cell type have been proposed. Among its many modulatory actions, calcium can serve as an activator of an 85 kDa cytosolic phospholipase A2 (cPLA2) (Clark et al., Reference Clark, Lin, Kriz, Ramesha, Sultzman, Lin, Milona and Knopf1991). It was demonstrated that sea urchin eggs inseminated in the presence of 4-bromophenacyl bromide (BPB), a specific inhibitor of phospholipase A2, failed to undergo fertilization envelope formation in a concentration-dependent manner. In contrast, melittin, known to be an activator of PLA2, strongly induced the envelope formation in sea urchin eggs. These observations led to the assumption that the PLA2 reaction participates in the Ca2+-triggered reaction system to cortical vesicles in sea urchin eggs (Ferguson & Shen, Reference Ferguson and Shen1984; Kamata et al., Reference Kamata, Mita, Fujiwara, Tojo, Takano, Ide and Yasumasu1997).
Once activated, cPLA2 releases arachidonic acid, a poly-cis unsaturated fatty acid (20:4), from phospholipids of the ER as well as from the nuclear envelope. Arachidonic acid acts as a lipophilic messenger and it is the precursor of the synthesis of the eicosanoids.
Previous studies have suggested that arachidonic acid is able to modulate the activity of several ion channels and furthermore, that arachidonic acid and/or its metabolites might be involved in the regulation of the intracellular calcium homeostasis. (Maruyama, Reference Maruyama1993; Rowles & Gallacher, Reference Rowles and Gallacher1996). Striggow & Erlich (Reference Striggow and Erlich1997) have investigated the influence of arachidonic acid and some of the eicosanoids on the single channel properties of the IP3R and the RyR on microsomes of canine cerebellum. They showed that IP3Rs and the RyRs are regulated differently by arachidonic acid and its product leukotriene B4; IP3Rs are inhibited by arachidonic acid, whereas RyRs are activated by arachidonic acid through its metabolite leukotriene B4.
It has been proposed that cyclic ADP ribose could be the endogenous activator of some types of RyRs, but this view remains controversial. Recent observations have suggested that arachidonic acid acts as a physiological activator of ryanodine receptors in pancreatic beta-cells (Woolcott et al., Reference Woolcott, Gustafsson, Dzabic, Pierro, Tedeschi, Sandgren, Bari, Nguyen, Bianchi, Rakonjac, Rådmark, Ostenson and Islam2006).
However, there is no evidence of the effect of arachidonic acid nor of its metabolites on the calcium signalling pathways that led to the intracellular calcium increase during egg activation in frogs. Therefore, we have studied the participation of the PLA2 pathway in the process of Bufo arenarum oocyte activation and the probable interrelation between any of its metabolites and the ion channels involved in the calcium release from the intracellular reservoirs at fertilization.
Materials and methods
Animals
Adult specimens of Bufo arenarum were collected in the northwestern area of Argentina and kept at 15 °C until use, which generally took place 7 days after collection. Experimental manipulation and oocyte culture were conducted at room temperature (22–25 °C) using amphibian Ringer solution (AR): NaCl 6.6 g /l, KCl 0.15 g /l, CaCl2 0.15 g /l, that contains penicillin G-sodium (30 mg/l), streptomycin sulphate (50 mg/l) and 0.005 M Tris–HCl buffer (pH 7.4).
Hormones and reagents
Progesterone (Sigma) was dissolved in ethanol and added directly to the culture medium to give a final concentration of 1 μg/ml. Melittin (Sigma) was dissolved in dimethylsulphoxide (DMSO) and stored in a stock solution of 250 μg/ml and quinacrine (Sigma) was dissolved in DMSO (stock solution 4 mM). Arachidonic acid (Sigma) was dissolved in DMSO in order to obtain a stock solution of 20 mM. Aliquots of 100 μl were purged with gaseous nitrogen and stored at –20 °C until use to avoid oxidation by oxygen, light or heat. All experiments were performed under dim light. Thimerosal (Lilly) was added directly to the culture medium. Ruthenium red (Ted Pella) was dissolved in ddH2O in order to obtain a suspension; then it was heated at 60 °C for 5 min while shaking and centrifuged at 1600 rpm for 10 min. The supernatant resulted in a stock solution < 1%.
All the compounds injected were diluted into calcium-free Tris-buffered saline solution containing NaCl 7.59 g /l, Tris–HCl 2.40 g /l pH 7.4.
Gametes collection
Fully grown ovarian oocytes were obtained from adult female specimens of Bufo arenarum. Oocytes were denuded by manually pulling off the follicle epithelium and theca layer using fine forceps under a stereoscopic microscope. Follicle cells were removed by shaking in AR for 5 min with gentle shaking (100 oscillations/min). Oocytes remained with only the vitelline envelope.
In vitro maturation of oocytes
Hormonal maturation was induced by treatment of denuded oocytes with progesterone 2.5 μM. Oocyte maturation was assessed 18 h after follicle cells removal or after hormone addition. Meiosis reinitiation was scored both by the presence of a transient white spot at the animal pole and by cytological examination. In our working conditions, oocytes reached metaphase (M)II in 16 h after defolliculating or progesterone treatment.
Microinjection procedures
Oocytes were microinjected using a Leitz Wetzlar Micromanipulator. Glass micropipettes HUMAGEN™ FERTILITY DIAGNOSTICS were filled by suction of a microdrop (30 nl) that contained either 2.5–40 μg/ml melittin, 25–200 μM arachidonic acid or 20–200 μM ruthenium red. All injections were carried out at room temperature while oocytes were held in calcium-free Tris-buffered saline solution. Microinjections were performed at approximately 18 to 20 h after progesterone addition.
Cytological preparations
Oocytes were fixed in Ancel and Vintemberger's solution (10% formaldehyde, 0.5% acetic acid and 0.5% NaCl), embedded in paraffin and sliced into 5 μm thick sections. Slides were bleached in 30% hydrogen peroxide solution for 3 days to prevent pigment from interfering with microscopic observations. Then they were stained with Alcian blue at pH 2.5 and counterstained with eosin. This method allowed us to observe cortical granules.
Statistical analysis
Results are expressed as mean ± standard error of the mean (SEM). Comparisons among different treatments were carried out using Student's t-test. A value of p < 0.05 was considered to be statistically significant.
Results
Participation of the PLA2 pathway in oocyte activation
In order to determine the participation of the PLA2 pathway in the activation process we tested the effect of different doses of melittin, a 26 amino acid peptide that represents 50% of the total protein of the bee venom and it is known to able to activate the PLA2.
Denuded ovarian oocytes matured in vitro with progesterone (2.5 μM) were microinjected with different doses of melittin (2.5–40 μg/ml). Egg activation was scored after 20 min of culture.
Results show that melittin is able to induce activation in a dose-dependent manner up to a concentration of 10 μg/ml. We found a decrease in egg activation levels at higher concentrations of melittin (Fig. 1).
Figure 1 Effect of melitin on the activation of in vitro matured oocytes. Dose–response curve for melittin-induced egg activation. Groups of 20 denuded fully grown ovarian oocytes matured in vitro with progesterone were microinjected in amphibian Ringer solution (AR) with different doses of mellitin (2.5–40 μg/ml) and parameters were evaluated after 20 min activation. Values are the mean ± standard error of the mean (SEM) of six experiments. Each experiment was performed on a different animal.
Subsequently, in order to test whether melittin exerted its activator effect specifically through the stimulation of the PLA2 pathway, oocytes were exposed to quinacrine, an inhibitor of PLA2, before treatment with melittin.
Results that showed the specificity of the melittin activation effect are summarized in Fig. 2. Data show that pre-treatment with quinacrine was able to inhibit significantly melittin-induced egg activation, so we could infer that melittin exerts its effect through phospholipase A2 pathway.
Figure 2 Effect of quinacrine on the mellitin-induced egg activation. Groups of 20 denuded fully grown ovarian oocytes matured in vitro with progesterone 2.5 μM were exposed to quinacrine 10 μM before microinjection of 10 μg/ml melitin. Activation was scored after 20 min of insemination. Values are the mean ± standard error of the mean (SEM) of three experiments. Each experiment was performed on a different animal.
The influence of PLA2 pathway on the oocyte activation process was also studied by direct stimulation with one of the products of the enzyme reaction: arachidonic acid.
Results exhibited a bell-shaped curve activation dependence with arachidonic acid levels (Fig. 3), similar to that of melittin. At doses less than 100 μM, we observed a stimulatory effect of arachidonic acid in a dose-dependent manner, whereas at higher concentrations, we noted a decrease in activation levels. Maximum effective concentration of arachidonic acid was 100 μM and at this concentration the activation response was not higher than 50%.
Figure 3 Effect of arachidonic acid on the activation of in vitro matured oocytes. Dose–response curve for arachidonic acid-induced egg activation. Groups of 20 denuded fully grown ovarian oocytes matured in vitro with progesterone 2.5μM were microinjected with different doses of arachidonic acid (25–200 μM) and activation parameters were evaluated after 20 min. Values are the mean ± standard error of the mean (SEM) of five experiments. Each experiment was performed on a different animal.
Interrelation between arachidonic acid and ryanodine receptors
Several effects of arachidonic acid on intracellular ion channels have been described. Striggow & Erlich (Reference Striggow and Erlich1997) showed that inositol 1,4,5-trisphosphate receptors and ryanodine receptors are modulated in an opposing manner by arachidonic acid and its product leukotriene B4. The IP3Rs were inhibited by arachidonic acid, whereas the RyRs were unaffected by this compound. In contrast, LTB4 was able to fully activate the RyRs but did not influence the IP3Rs.
In order to determine whether a similar regulatory mechanism is underlying the experimental findings about arachidonic acid-induced activation mentioned above (Fig. 3), we tested the effect of arachidonic acid in combination with agonists and antagonists of the two types of ion channels.
To test the hypothesis that arachidonic acid-induced activation is mediated by RyRs, we incubated oocytes with ruthenium red, a specific blocker of RyRs before treatment with arachidonic acid.
We found that the pre-injection of 50 μM ruthenium red strongly inhibited 100 μM arachidonic acid-induced egg activation (Fig. 4). These observations allow us to confirm that arachidonic acid-induced egg activation is carried out through RyRs.
Figure 4 Effect of ruthenium red (RR) on arachidonic acid (AA)-induced oocyte activation. Groups of 20 denuded fully grown ovarian oocytes matured in vitro with progesterone were microinjected with RR 50 μM before treatment with AA 100 μM and activation parameters were evaluated after 20 min. Values are the mean ± standard error of the mean (SEM) of three experiments. Each experiment was performed on a different animal.
To determine whether arachidonic acid released calcium through IP3Rs we used heparin (1 μg/ml), a specific blocker of IP3Rs, before treatment with arachidonic acid 100 μM.
As expected, we found that arachidonic acid-induced activation was not affected by pre-treatment with heparin (Fig. 5). These results indicate that IP3Rs do not mediate the release of calcium by arachidonic acid.
Figure 5 Effect of heparin on arachidonic acid (AA)-induced oocyte activation. Groups of 20 denuded fully grown ovarian oocytes matured in vitro with progesterone were microinjected with heparin 1 μg/ml before treatment with AA 100 μM and activation parameters were evaluated after 20 min. Values are the mean ± SEM of three experiments. Each experiment was performed on a different animal.
To examine whether arachidonic acid exerted an inhibitory effect on IP3Rs in Bufo arenarum oocytes, previous injections with arachidonic acid were performed, before exposing oocytes to thimerosal 200 μM, which is a potent activator of IP3Rs.
A significant decrease on the activation induced by thimerosal was observed when mature oocytes were microinjected with arachidonic acid 100 μM, before exposing to high doses of thimerosal (200 μM). This response indicates that arachidonic acid is able to inhibit egg activation through IP3Rs (Fig. 6).
Figure 6 Effect of arachidonic acid (AA)-induced on thimerosal-induced oocyte activation. Groups of 20 denuded fully grown ovarian oocytes matured in vitro with 2.5 μM progesterone were microinjected with 100 μM AA (AA 100) before treatment with 200 μM thimerosal (TMS 200) and after 20 min activation parameters were evaluated. Values are the mean ± standard error of the mean (SEM) of three experiments. Each experiment was performed on a different animal.
Discussion
Participation of the PLA2 pathway in egg activation
Probable participation of PLA2 pathway in the process of fertilization has not been elucidated yet. Many investigations have focused on the involvement of this intracellular mechanism in the sperm acrosomal reaction but only a few have reported its participation in triggering egg activation.
As mentioned earlier, PLA2 inhibition was able to block some activation events such as cortical vesicle exocytosis and subsequent fertilization envelope formation probably through calcium-triggered sequential reactions (Kamata et al., Reference Kamata, Mita, Fujiwara, Tojo, Takano, Ide and Yasumasu1997).
Evidence has suggested the implication of the PLA2 pathway in sperm-zona pellucida interaction. Hydrolysis of phospholipids by PLA2 generates free fatty acids and lysophospholipids that are important either as substrates for the generation of other metabolites (e.g., eicosanoids) or as they have a direct, essential action in the final stages of membrane fusion. (Roldan & Shi, Reference Roldan and Shi2007)
It was suggested that sperm PLA2 and one of its modulators, lysophosphatidylcholine (LPC), may contribute to membrane-fusion events in mammalian fertilization (Riffo & Párraga, Reference Riffo and Párraga1997). Studies in guinea pig spermatozoa suggested that PLA2 plays a fundamental role in agonist-stimulated acrosomal exocytosis (Chen et al., Reference Chen, Ni, Pan, Shi, Yuan, Chen, Mao, Yu and Roldan2005).
Activation of PLA2 at fertilization in Xenopus is a matter of discussion. There were no detectable lipid changes that reflect significant activation of PLA2 during fertilization in Xenopus laevis eggs (Petcoff et al., Reference Petcoff, Holland and Stith2008).
We found that addition of both melittin, a potent PLA2 activator, and arachidonic acid, the main PLA2 reaction metabolite, was able to induce activation events such as cortical granule exocytosis. Moreover, quinacrine treatment before melittin stimulation was proved to be inhibitory. These findings suggest that phospholipase A2 pathway is involved in the calcium-release mechanism that led to Bufo arenarum egg activation.
Results from melittin–induced egg activation as well as arachidonic acid-induced egg activation showed a Gauss curve (Figs. 1 and 3), suggesting the involvement of an underlying regulation mechanism.
Interrelation between arachidonic acid and ryanodine receptors
Arachidonic acid has been shown to interact with intracellular ion channels in many cell types. For instance, it was proved that arachidonic acid inhibits L-type calcium channel currents in cardiac myocytes (Petit-Jacques & Hartzell, Reference Petit-Jacques and Hartzell1996) and in CA1 pyramidal cells of hippocampus (Keyser & Alger, Reference Keyser and Alger1990).
Mobilization of stored calcium from the ER through IP3Rs and RyRs is a crucial step of intracellular calcium signalling at egg activation.
In contrast, it has been proposed that arachidonic acid could be the endogenous activator of ryanodine receptors in pancreatic beta-cells (Woolcott et al., Reference Woolcott, Gustafsson, Dzabic, Pierro, Tedeschi, Sandgren, Bari, Nguyen, Bianchi, Rakonjac, Rådmark, Ostenson and Islam2006).
It is based on these findings that we have approached the study of the effect of arachidonic acid in combination with agonists and antagonists of the two main families of ion channels in mature Bufo arenarum oocytes. The findings presented here at a cellular level are in line with previous studies undertaken on microsomal fractions in other cell models (Striggow & Erlich, Reference Striggow and Erlich1997). Differential regulation of IP3Rs and RyRs by arachidonic acid and its products could explain melittin and arachidonic acid behaviour in Bufo arenarum egg activation shown in Figs. 1 and 3. The concerted action of arachidonic acid and/or its metabolites could provide controlled mobilization of calcium from intracellular reservoirs by terminating IP3-induced calcium release and activating the RyR calcium-release channel.
Which of the products of the arachidonic acid cascade is involved in this complex intracellular calcium signalling? Preliminary reports have shown that LTB4 is the only eicosanoid able to induce intracellular calcium release in oocytes (Silver et al., Reference Silver, Oblak, Jeun, Sung and Dutta1994). Additional experiments would be necessary to identify the specific arachidonic acid-derived compounds that exert its effects on activation events of Bufo arenarum oocytes.
Knowledge of these recently identified agonists of the calcium channels will provide useful tools to understand the calcium homeostasis in eggs expressing both types of receptors and to improve in vitro protocols of egg activation in frogs.
Acknowledgements
This work was supported by a grant from the Science Council of the National University of Tucumán (CIUNT), and the National Agency for Promotion of Science and Technology (FONCYT).
