ABSTRACT
Intracellular Ca2+ (Ca2+i) transients during fertilization are critical to the activation of eggs in all species studied. Activation of both the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and ryanodine receptor (RYR) are responsible for the calcium oscillations during fertilization in sea urchin eggs. Using in vitro matured bovine oocytes loaded with Fura-2 AM ester as Ca2+i indicator, we addressed whether IP3Rs and RYRs coexist in mammalian eggs. Our results indicate that microinjection of 50-250 nM IP3 or 10-20 mM caffeine, 100-200 μM ryanodine and 4-8 μM cyclic ADP-ribose all induced Ca2+i release. The Ca2+i release induced by 250 nM IP3 could only be inhibited by prior injection of 1 mg/ml heparin which was overcome by continuous injection of IP3 to 1 μM. Prior injection of either 50 μM ruthenium red, 50 μM procaine or 1 % vehicle medium (VM) did not affect the Ca2+i release induced by IP3. Prior injection of heparin or VM did not affect the Ca2+i release induced by 10-20 mM caffeine or 200 μM ryanodine, but prior injection of 50 μM ruthenium red or procaine completely inhibited the effect of 10-20 mM caffeine. In addition, continuous injection of caffeine up to 40 mM overcame the inhibitory effect of ruthenium red or procaine. The same 50 μM concentration of ruthenium red or procaine only partially blocked the effect of 200 μM ryanodine, but 200 μM ruthenium red or procaine completely blocked the effect of 200 μM ryanodine. Oocytes were refractory for 15 minutes to further injections of IP3 after the initiation of Ca2+i release induced by 200 nM IP3; a 10 minute refractory period was observed for 10-20 mM caffeine and 200 μM ryanodine; either caffeine or ryanodine can desensitize RYRs to the other. However, the desensitization of IP3Rs by 200 nM IP3 does not abolish the effect of 200 μM ryanodine, as effectively as the reciprocal treatment. Prior injection of a subthreshold concentration of ryanodine itself only induced a slight increase in Ca2+i level, but it sensitized the RYR to a subsequent injection of a subthreshold concentration of caffeine. Similar results were obtained when ryanodine was first injected followed by injection of caffeine Based on these results, we conclude that independent IP3Rs and RYRs exist in mature bovine oocytes.
INTRODUCTION
Intracellular Ca2+ (Ca2+i) transients and oscillations have been reported to occur during the fertilization in all species studied (Cuthbertson and Cobbold, 1985; Fissore et al., 1992; Fissore and Robl, 1993; Galione et al., 1993; Jeffe, 1983; Kline and Kline, 1992, 1994; Lee et al., 1993; Miyazaki et al., 1986, 1992; Nuccitelli et al., 1993; Poenie et al., 1985; Sun et al., 1992, 1994). The importance of Ca2+i in oocyte activation during fertilization or cell cycle control has been well documented (Whitaker and Patel, 1990; Whitaker and Swann, 1993). In mammalian eggs, mimicking the fertilization Ca2+i transients and oscillations has been widely applied to artificial activation of oocytes in nuclear transplantation (Collas et al., 1993; Rickords and White, 1992, 1993). Although many mechanisms have been proposed to explain Ca2+i transients and oscillations, it still remains unresolved (Berridge, 1993a). The most accepted model involves two pathways that rely on the existence of two well-identified receptors in different species: the universally existing inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) and the species-specific ryanodine receptor (RYR) identified only in sea urchin eggs (Dargie et al., 1990). IP3-induced Ca2+i release (IICR) from IP3-sensitive Ca2+i store is mediated by IP3R; while Ca2+-induced Ca2+i release (CICR) from Ca2+-sensitive Ca2+i store is mediated by RYR (Takeshima et al., 1989). Although the interaction between these two pathways is very complex, they share a common sensitivity to Ca2+i level. It has been suggested that IP3R may also have a CICR mechanism (Berridge, 1993a), and both these two pathways can produce regenerative Ca2+i oscillations (Berridge and Galione, 1988; Missiaen et al., 1991; Taylor and Marshall, 1992). How these two pathways combine in eggs expressing both receptors is unresolved (Berridge, 1993b). Investigation of the existence of dual receptors in mammalian oocytes will facilitate further understanding of their role in fertilization.
IP3 induces repetitive or single Ca2+i release in oocytes of all species studied (Carroll and Swann, 1992; Clapper and Lee, 1985; Fissore et al., 1992; Fissore and Robl, 1993; Galione et al., 1993; Kline and Kline, 1994; Miyazaki et al., 1992; Nuccitelli et al., 1993; Rickords and White, 1993; Swann, 1992; White et al., 1993). A response to IP3 indicates the existence of an IP3R in these species, and IP3Rs have been characterized in both sea urchin and Xenopus eggs (Parys et al., 1992, 1994). There are at least two different types of IP3Rs, which may reflect their different allosteric regulators (Südhof et al., 1991).
RYR agonists include exogenous ryanodine and caffeine, and cyclic ADP-ribose (cADPR), which is the only known endogenous CICR modulator and has been proposed as a new second messenger (Berridge, 1993b; Galione et al., 1993). These agonists have potentiation and desensitization effects (Buck et al., 1992, 1994; Lee, 1993). RYR antagonists include ruthenium red and procaine. Three types of RYRs (Sorrentino and Volpe, 1993) have been identified: skeleton muscle RYR (type I), cardiac muscle RYR (type II) and brain isoform (type III). These dual receptors work in a backup manner during fertilization of sea urchin eggs. The inhibition of either receptor does not abolish normal fertilization Ca2+i transients. Therefore, IP3Rs may not play a major role in sea urchin fertilization (Galione et al., 1993; Lee et al., 1993; Shen and Buck, 1993; Whitaker and Swann, 1993). The search for RYRs in the oocytes of other species has not been productive. Frog eggs do not possess RYRs (Galione et al., 1993). Only IP3Rs exist in hamster eggs and mediate the Ca2+ release at fertilization (Miyazaki et al., 1992). Conflicting results have been obtained in mouse oocytes or eggs (Carroll and Swann, 1992; Kline and Kline, 1994; Swann, 1992). Injection of ryanodine into rabbit oocytes at a pipet concentration of 10 mM did not cause the release of Ca2+i (Fissore and Robl, 1993). In bovine oocytes, sperm-induced Ca2+i oscillations apparently could not be inhibited by prior injection of a high concentration of heparin (Sun et al., 1994). This indicates that RYRs may exist and function in the bovine oocyte. However, direct evidence is lacking. We investigated the existence of these dual receptors in mature bovine oocytes by evaluating (1) the ability of agonists to induce Ca2+i release; (2) the inhibitory effect of antagonists on agonists; (3) the desensitization of IP3Rs and RYRs by agonists and (4) the potentiation of RYRs by caffeine and ryanodine.
MATERIALS AND METHODS
In vitro maturation of bovine oocytes
Bovine ovaries were collected from a local abattoir. Oocytes between 1 and 6 mm were aspirated into 50 ml centrifuge tubes using a 18-gauge needle connected to a vacuum pump. Oocytes with intact layers of cumulus cells and evenly shaded cytoplasm were selected and washed with Hepes-buffered Tyrode’s medium (Fissore et al., 1992) supplemented with 3 mg/ml BSA, then oocytes were transferred into 250 μl TCM 199 (M199: HyClone laboratories) maturation medium containing 10% FBS, 0.5 μg/ml FSH, 5 μg/ml LH, 100 units/ml penicillin, and 100 μg/ml streptomycin into 4-well culture dishes (Nunc, Holland) and cultured at 39°C in a humidified atmosphere of 5% CO2 and air for 24 hours.
Ca2+i indicator Fura-2 AM loading
At 24 hours after the initiation of maturation, oocytes were vortexed in 1 ml PB1+ (phosphate-buffered saline plus 5.55 mM glucose, 0.32 mM sodium pyruvate and 3 mg/ml BSA) at setting 8 for 3.5 minutes to remove cumulus cells completely. Oocytes having an extruded first polar body were selected for use in the experiment. Oocytes were loaded with Ca2+i indicator by incubation in 2 μM Fura-2 AM ester and 0.02% Pluronic F-127 (Molecular Probe Inc) in PB1− solution (Ca2+-and Mg2+-free PB1+ with 100 μM EGTA) at 39°C in darkness for 40-50 minutes. After loading Fura-2 AM, oocytes were washed extensively with PB1+ and maintained in this solution at 39°C in a CO2 incubator until use.
Microinjection procedures
A glass chamber containing 600 μl M2− medium, pH 7.4 (M2; Wood et al., 1987, modified by excluding Ca2+ and Mg2+, adding 100 μM EGTA and replacing BSA with 0.5% polyvinylpyrrolidone to reduce the background) was put on a warm stage on a Nikon Diaphot microscope equipped with 20× fluorescence objective. Fura-2-loaded oocytes were washed in M2− and transferred into the chamber, which was then covered with prewarmed mineral oil. Temperature on the warm stage was maintained at 39°C throughout the experiment. Oocytes held with a holding pipet were injected with a injection pipet. Holding pipettes were pulled from 1.0 mm wall glass capillaries (World Precision Instruments) with KOPF vertical pipet puller (David KOPF Instruments), then bent and fire polished using Narishige microforge (Narishige Scientific Instrument Lab) to O.D. 120 μm, I.D. 20 μm. Injection pipettes were directly pulled to suitable size with long narrow shank without further modification from thin-wall glass capillaries with filament (World Precision Instruments) using a Narishige vertical pipet puller. Microinjection was carried out with a Narishige pneumatic microinjector connected to compressed N2 gas. All the compounds injected except cADPR, were dissolved in the vehicle medium (VM) which consisted of Ca2+- and Mg2+-free PBS and 100 μM EGTA. cADPR was dissolved in Ca2+-and Mg2+-free PBS containing 10 μM EGTA. IP3 was from Molecular Probes; caffeine: sodium benzoate (50:50 w/w mixture), heparin (H-3393), ruthenium red and procaine were from Sigma; ryanodine (high purity) was from Calbiochem; and cADPR was purchased from Amersham.
Estimation of intracellular concentrations of compounds injected
Intracellular concentrations of injected compounds were calculated from the estimation of injection volume and the average volume of a bovine oocyte. Both oocyte volume and injection volume were measured with Image-1 software (Universal Imaging). Oocytes volume was standardized as 800 pl although large variation (from 529 to 914 pl) was observed. Injection volume was estimated by measuring the volume of solution droplets extruded from an injection pipet into mineral oil. The injection volume was controlled by changing the injection pressure and duration, which were usually set at 300 psi and 110 millseconds, respectively. These settings gave an injection volume of approximately 8-9 pl, which is about 1% of the oocyte volume. However, the injection volume ranged from 0.5 to 10%. All the concentrations indicated in the text were the final intracellular concentrations and the injection volume of VM was 1% of the oocyte volume.
Intracellular Ca2+ monitoring and calibration
The Ca2+i was measured with Image-1 software by measuring ratio changes of fluorescence intensity at emission 510 nm wavelength when excited at 340 nm and 380 nm. A camera control switched the filter wheel between the 340 nm and 380 nm wavelengths. Images of whole oocytes were collected with a 20× fluorescence objective, then transmitted via a video camera to a PC 486 computer where images were processed, saved and displayed on the monitor in pseudocolor. The system was calibrated at 39°C using Calibration Buffer Kit I with magnesium according to the protocol provided by Molecular Probes. Fura-2 potassium salt was used as the Ca2+ indicator with a Kd value of 224 nM. However, we present our data in ratio with a value of 3 approximately equal to 717 nM free Ca2+i ion concentration. For measurement of Ca2+i oocytes were washed in M2− three times and transferred into the chamber. After a 20-30 second baseline reading, oocytes were injected. The injection pipettes were then withdrawn to avoid possible leakage. Only those oocytes that survived the first injection without obvious damage to the cytoplasm received a second injection after a 3-3.5 minutes delay. Ca2+i changes were recorded for each individual oocyte.
RESULTS
The effect of IP3, ryanodine, caffeine and cADPR on Ca2+i release
Microinjection of 50-250 nM IP3 immediately induced significant Ca2+i release in bovine oocytes as shown by Fura-2 fluorescence ratio change (n=15, Figs 1A, 2A), and 2 out of 15 oocytes exhibited second peaks with much smaller amplitudes at an interval of 500-1000 seconds (Fig. 1B). Injection of 100-200 μM ryanodine also induced significant Ca2+i release (n=15, Figs 1C, 2B). Oocytes responded to 1.88-20 mM caffeine injection (n=15, Fig. 1D; only the response to 10-20 mM caffeine is shown) in a dose-dependent manner, 1.88 mM caffeine induced a Ca2+i peak of 0.3 (an average increase from the baseline), 7.5 mM caffeine caused Ca2+i peak of 1.5 and 10-20 mM caffeine induced a increase of Ca2+i of 3.22. The response time (expressed as mean+s.d.) to 10-20 mM caffeine injection is significantly longer than that to 200 μM ryanodine (from beginning to full response: 80.4+54.3 seconds for 10-20 mM caffeine versus 23.2+10.2 seconds for 200 μM ryanodine). Injection of 4-8 μM freshly prepared cADPR solution immediately induced significant Ca2+i release (n=15, Fig. 1E). However, we also observed that injection of frozen-thawed cADPR solution failed to induce any significant increase in Ca2+i level (data not shown). Injection of 1% VM did not significantly change Ca2+i level (n=15, Fig. 1F). Thus, neither contaminating Ca2+ in the injection solution nor the injection itself were responsible for the observed increase in Ca2+i concentration.
Intracellular calcium release in mature bovine oocytes reflected by the ratio changes in Fura-2 fluorescence. Compounds were injected at the time indicated by arrows to the following intracellular concentrations. (A) 50-250 nM IP3 (oocytes with single peak); (B) 50-250 nM IP3 (oocytes with 2 peaks); (C) 100-200 μM ryanodine (Rya); (D) 10-20 mM caffeine (Caf); (E) 4-8 μM cADPR; (F) 1% vehicle medium (VM).
Intracellular calcium release in mature bovine oocytes reflected by the ratio changes in Fura-2 fluorescence. Compounds were injected at the time indicated by arrows to the following intracellular concentrations. (A) 50-250 nM IP3 (oocytes with single peak); (B) 50-250 nM IP3 (oocytes with 2 peaks); (C) 100-200 μM ryanodine (Rya); (D) 10-20 mM caffeine (Caf); (E) 4-8 μM cADPR; (F) 1% vehicle medium (VM).
Intracellular calcium changes were measured using the whole-cell imaging technique. Injected compound was deposited in the center of oocytes; no differences in calcium change patterns were observed among areas 1, 2, 3 and 4 within an oocyte. Only whole cell images (labeled area 4 in A and area 1 in B) were subsequently measured and fluorescence expressed as the ratio change with values of 4, 3 and 1 in the color bar approximately equal 1075 nM, 717 nM and 172 nM of free intracellular calcium ion. (A) Intracellular calcium release induced by 50-250 nM IP3. (B) by 100-200 μM Rya. The number above each image indicates the time in seconds after the start of recording. Injections were delivered at 20-40 seconds after initiation of record.
Intracellular calcium changes were measured using the whole-cell imaging technique. Injected compound was deposited in the center of oocytes; no differences in calcium change patterns were observed among areas 1, 2, 3 and 4 within an oocyte. Only whole cell images (labeled area 4 in A and area 1 in B) were subsequently measured and fluorescence expressed as the ratio change with values of 4, 3 and 1 in the color bar approximately equal 1075 nM, 717 nM and 172 nM of free intracellular calcium ion. (A) Intracellular calcium release induced by 50-250 nM IP3. (B) by 100-200 μM Rya. The number above each image indicates the time in seconds after the start of recording. Injections were delivered at 20-40 seconds after initiation of record.
The specific inhibitory effect of heparin, ruthenium red and procaine on IP3-, caffeine- and ryanodineinduced Ca2+i release
To determine whether these different types of agonists are using different mechanisms to mobilize the Ca2+i, we further evaluated the effect of specific antagonists on those agonists. We found that prior injection of 1 mg/ml heparin (n=5, Fig. 3A) completely inhibited 250 nM IP3-induced Ca2+i release, while continuous injection of IP3 to 1 μM in 2 oocytes overcame the inhibitory effect of heparin (Fig. 3B). This also served as a control to determine the viability of oocytes after prior injection of heparin. Prior injection of either 1% VM (n=5, Fig. 3C) or 50 μM ruthenium red (n=5, Fig. 3D) or 50 μM procaine (n=5, Fig. 3E) failed to inhibit Ca2+i release induced by 250 nM IP3. The Ca2+i release induced by 200 μM ryanodine was not affected by the prior injection of either 1 mg/ml heparin (n=5, Fig. 4A) or 1% VM (n=5, Fig. 4B). However, prior injection of either 50 μM ruthenium red (n=5, Fig. 4C) or 50 μM procaine (n=5, Fig. 4E) partially affected the Ca2+i release induced by 200 μM ryanodine. Injection of a higher concentration of ruthenium red (200 μM, n=5, Fig. 4D) or procaine (200 μM, n=5, Fig. 4F) completely abolished the effect of 200 μM ryanodine. Prior injection of 1 mg/ml heparin (n=5, Fig. 5A) or 1% VM (n=5, Fig. 5B) did not inhibit the 20 mM caffeine-induced Ca2+i release. Prior injection of 50 μM ruthenium red or 50 μM procaine (n=5, Fig. 5E) completely blocked 20 mM caffeine-induced Ca2+i release (n=5, Fig. 5C), but continuous injection of caffeine to 30-40 mM overcame their inhibitory effect (for ruthenium red, N=4, Fig. 5D; for procaine, n=3, Fig. 5F), indicating the competitive inhibitory effect of procaine and ruthenium red on RYR.
The inhibition of 250 nM IP3-induced intracellular calcium release by prior injection of (A) 1 mg/ml heparin (Hep); (B) 1 mg/ml Hep but followed by continuous injection of IP3 to 1 μM; (C) 1% VM; (D) 50 μM ruthenium red (RR); (E) 50 μM procaine (Pro). Second injections were usually given 2.5 minutes after the first injections.
The inhibition of 250 nM IP3-induced intracellular calcium release by prior injection of (A) 1 mg/ml heparin (Hep); (B) 1 mg/ml Hep but followed by continuous injection of IP3 to 1 μM; (C) 1% VM; (D) 50 μM ruthenium red (RR); (E) 50 μM procaine (Pro). Second injections were usually given 2.5 minutes after the first injections.
The inhibition of 200 μM Rya-induced intracellular calcium release by prior injection of (A) 1 mg/ml Hep; (B) 1% VM; (C) 50 μM RR; (D) 200 μM RR; (E) 50 μM Pro; (F) 200 μM
The inhibition of 20 mM Caf-induced intracellular calcium release by prior injection of (A) 1 mg/ml Hep; (B) 1% VM; (C) 50 μM RR; (D) 50 μM RR followed by continuous injection of Caf to 40 mM; (E) 50 μM Pro; (F) 50 μM Pro followed by continuous injection of Caf to 40 mM.
The desensitization effect of IP3, caffeine and ryanodine on their respective receptors
Within 15 minutes of a 200 nM IP3-induced-Ca2+i release, another injection of IP3 was given to the same oocyte and no additional significant release of Ca2+i was observed (n=5, Fig. 6A) indicating that IP3 receptor was desensitized and had not recovered its ability to be activated by IP3. A similar desensitization phenomenon was observed for 200 μM ryanodine (n=5, Fig. 6B) and 20 mM caffeine (n=5, Fig. 6C) demonstrating a 10 minute refractory period after the start of Ca2+i release. We found that caffeine and ryanodine can desensitize the oocyte to each other (n=5 for each order, Fig. 6D,E). However, immediately after the initiation of Ca2+i release induced by 200 nM IP3, injection of ryanodine induced further Ca2+i release, but to a lower peak value (n=5, Fig. 6F). Similar results were obtained when ryanodine was injected first followed by IP3 (n=3, Fig. 6G).
(A-E) The desensitization (F,G) and non-desensitization of IP3R and RYR respectively by (A) 200 nM IP3 to further injection of IP3; (B) 200 μM Rya to further injection of Rya; (C) 10 mM Caf to further injection of Caf; (D) 200 μM Rya to 10 mM Caf; (E) 10 mM Caf to 200 μM Rya; (F) 200 nM IP3 to further injection of 200 μM Rya; (G) 200 μM Rya to further injection of 200 nM IP3.
(A-E) The desensitization (F,G) and non-desensitization of IP3R and RYR respectively by (A) 200 nM IP3 to further injection of IP3; (B) 200 μM Rya to further injection of Rya; (C) 10 mM Caf to further injection of Caf; (D) 200 μM Rya to 10 mM Caf; (E) 10 mM Caf to 200 μM Rya; (F) 200 nM IP3 to further injection of 200 μM Rya; (G) 200 μM Rya to further injection of 200 nM IP3.
The potentiation effect of caffeine and ryanodine on ryanodine receptor
Injection of a subthreshold concentration of ryanodine (n=5; Fig. 7C) or caffeine (n=5; Fig. 7A) only induced a slight increase of Ca2+i; however, subsequent injection of low concentration caffeine (n=4, Fig. 7B) or ryanodine (n=5, Fig. 7D) induced significant release of Ca2+i. This indicates that RYR has been potentiated by the prior injections of subthreshold concentrations of either ryanodine or caffeine.
The potentiation of RYR by subthreshold concentrations of Rya and Caf on each other. (A)Control: effect of injection of a subthreshold concentration of Caf; (B)Caf effect potentiated by prior injection of a subthreshold concentration of Rya; (C) control: effect of injection of a subthreshold concentration of Rya; (D) Rya effect potentiated by prior injection of a subthreshold concentration of Caf.
The potentiation of RYR by subthreshold concentrations of Rya and Caf on each other. (A)Control: effect of injection of a subthreshold concentration of Caf; (B)Caf effect potentiated by prior injection of a subthreshold concentration of Rya; (C) control: effect of injection of a subthreshold concentration of Rya; (D) Rya effect potentiated by prior injection of a subthreshold concentration of Caf.
DISCUSSION
Ca2+i release profiles induced by various agonists and the inhibitory action of specific antagonists: evidence for the existence and independence of the IP3 and ryanodine receptors in mature bovine oocytes
The effects of various agonists on the free Ca2+i concentration were very different as clearly shown (Fig. 1) by their different effective concentrations and response kinetics. IP3 appears to be the most potent inducer of Ca2+i release as indicated by its lowest effective concentration (50 nM) and the immediate response of oocytes. Injection of IP3 alone was able to induce periodic release of Ca2+i in some of the oocytes (2/15) as reported in other species. This is in agreement with our previous report using electroporation (White et al., 1993), which strongly argues that the periodic release of Ca2+i was not due to the experimental procedure, rather oocytes have the potential periodically to release Ca2+i when exposed to IP3. It appears that the IP3R alone is responsible for the periodic release of Ca2+i induced by IP3 in bovine oocytes, and CICR is not involved in the formation of a second peak. This conclusion is supported by the observation that the addition of 200 μM thimerosal, which only activates IP3Rs in bovine oocytes (data not shown), to the culturing solution induced Ca2+i oscillations in bovine oocytes preinjected with the RYR antagonist, procaine, at a concentration of 200 μM. It is more likely that the elevation of Ca2+i may either feedback and activate the IP3 production cascade (Harootunian et al., 1991) or it may lower the threshold of IP3Rs, so that a lower level of IP3 is then able to trigger Ca2+i release from IP3-sensitive stores (Berridge, 1993a; Miyazaki et al., 1992). The appearance of periodic Ca2+i release may rely on the height of first peak, which in turn depends on the amount of IP3 injected coupled to the competency of oocytes as indicated by the low proportion (2 of 15) of oocytes showing two peaks in our experiment. This may explain why Fissore et al. (1993) injected bovine oocytes with a pipet concentration of 5 μM, and observed no repetitive release of Ca2+i in a sample of 6 oocytes.
Heparin at a concentration of 1 mg/ml completely inhibited the effect of IP3, and this inhibition is competitive (Ghosh et al., 1988), because 1 μM IP3 overcame the effect of 1 mg/ml heparin. However, this inhibitory effect requires the prior injection of heparin. Simultaneous injection of even higher concentrations of heparin with IP3 could not inhibit the action of IP3. Ruthenium red and procaine are known RYR antagonists and they, like prior injection of VM, do not have a significant effect on IP3R as indicated by their inability to affect IP3-induced Ca2+i release. This observation manifested the independence of IP3Rs and RYRs.
Ca2+i release upon injection of caffeine is regarded as the hallmark for the existence of RYRs. In sea urchin oocytes, the response to caffeine is graded in contrast to that in nongerminal cells (Buck et al., 1994). Our experiment indicated that bovine oocytes responded to 1.88-20 mM caffeine injection in a dose-dependent manner. However, we also found that the response to 10-20 mM caffeine was delayed as compared to that for ryanodine (from beginning to full response: 80.4+54.3 seconds for 10-20 mM caffeine versus 23.2+10.2 seconds for 200 μM ryanodine). This observation is different from the data reported using sea urchin eggs. It was hypothesized that caffeine opens CICR channels, while ryanodine preferentially binds to open channels and locks them in this conformation (Lai and Meissner, 1989). Thus, it was expected that caffeine would induce a more rapid response than ryanodine, as is the case in sea urchin eggs (Galione et al., 1991). Conflicting results may indicate the existence of a different type of RYR in bovine oocytes, supported by the observation that culturing bovine oocytes with 200 μM thimerosal still induced Ca2+i release in 200 μM procaine-preinjected oocytes but not in the oocytes preinjected with 1 mg/ml heparin. Thimerosal, therefore, only activates IP3Rs in mature bovine oocytes. In contrast, thimerosal activates both IP3Rs and RYRs in sea urchin eggs (Galione et al., 1993; Tanaka and Tashjian, 1994). With the availability of the RYR sequence (Takeshima et al., 1989), the potential difference can be identified.
Freshly prepared cADPR (4-8 μM) also induced a significant Ca2+i release (Fig. 1D) while frozen-thawed cADPR solution was unable to induce Ca2+i release. This observation completely excludes the possibility that the Ca2+i release that we observed with fresh cADPR solution was due to contaminating Ca2+ in the cADPR preparation. Injection of 1% VM did not significantly change the Ca2+i level (Fig. 1E), which also argues against the possibility that contaminating Ca2+ in VM or that the injection itself induced an artifact. However, the requirement for such a high concentration of cADPR and the observation that, although frozen-thawed cADPR solution failed to induce Ca2+i release in bovine oocytes, the same cADPR solution was still fully active on sea urchin egg homogenate (H. C. Lee, personal communication) leads to the following possibilities. First, the RYR in bovine oocytes may be different from that in sea urchin eggs and has less affinity for cADPR. Freezing-thawing may decrease the bioactivity of cADPR enough to prevent the activation of the RYR in bovine oocytes, yet not enough to be detectable using the sea urchin egg homogenates assay. Second, it has been reported that, in skeletal muscle, cADPR binds to RYRs but does not elicit the release of Ca2+i and that it may not be an effective endogenous modulator at the concentration of 2 μM (Meszaros et al., 1993). The same phenomenon may be applicable in bovine oocytes.
RYR antagonists such as ruthenium red and procaine are all able to inhibit competitively the effect of ryanodine and caffeine (Figs 3, 4). Ryanodine appears to be more potent than caffeine, because with the same concentrations of ruthenium red and procaine, caffeine effect was completely inhibited, while ryanodine effect was only partially affected. As we expected, injection of heparin or VM did not significantly affect the Ca2+i release induced by ryanodine and caffeine, which indicates the independent relationship between IP3R and RYR.
Desensitization: another indication of the independence of IP3Rs and RYRs and the functional similarity of caffeine and ryanodine
As reported in sea urchin eggs (Clapper and Lee, 1985; Galione et al., 1993; Lee et al., 1993), desensitization is a major characteristic of IP3Rs and RYRs and has been used to distinguish RYRs from IP3Rs. In bovine oocytes, a 15 minute refractory period was observed after the initiation of Ca2+i release induced by 200 nM IP3 or a 10 minute refractory period for 200 μM ryanodine and 10-20 mM caffeine (Fig. 5A-C). The fact that either caffeine or ryanodine desensitized RYRs to the other reinforced the concept that they are using the same receptor to mobilize Ca2+i. The independent relationship between IP3Rs and RYRs was further explored by injecting IP3 (or ryanodine) immediately after the initiation of Ca2+i release induced by ryanodine (or IP3). Because in this period, the IP3R is still desensitized to IP3, the release of Ca2+i by injecting ryanodine must be the result of activating the RYR which is apparently not desensitized by prior injection of IP3. The same is true for the reverse. The low amplitude of Ca2+i peak induced by the second injection (Fig. 5F,G) may be due either to the possible overlapping of two stores as reported in sea urchin eggs (Clapper and Lee, 1985) or to the downregulation of receptor sensitivity by Ca2+i level (Berridge, 1993a).
Potentiation effect of caffeine and ryanodine on RYRs: further indication of their functional similarity
It has been firmly established in sea urchin eggs and egg homogenates that ryanodine, caffeine and cADPR can potentiate RYRs after subthreshold amounts of any of the others. The potentiation effect is illustrated by increased release of Ca2+i and shortened initiation period (for ryanodine) upon injection of a subthreshold concentration of one RYR agonist in the oocytes preinjected with subthreshold concentration of another agonist (Buck et al., 1992, 1994; Lee, 1993). In our experiment, we observed both an increased release of Ca2+i and curtailed initiation period upon the injection of subthreshold concentration of caffeine after the potentiation of RYR by ryanodine in bovine oocytes. Although the mechanism for the potentiation effect is not quite clear, it is believed to be due to different agonists binding to different parts of the receptor or intermediate proteins, which have different effects on the receptor. Caffeine mainly opens channels, while ryanodine binds to the open channels (Lai and Meissner, 1989) and, in skeletal muscle, high and low affinity sites for ryanodine have been reported (Callaway et al., 1994). Therefore, a cooperation between caffeine and ryanodine is possible. For cADPR to induce Ca2+i release in sea urchin egg homogenates, the presence of calmodulin is necessary and calmodulin appears to sensitize CICR (Lee et al., 1994). cADPR inhibited labeling of two proteins with relative molecular mass of 140×103 and 100×103 when microsomes from sea urchin eggs preincubated with 32P8N3-cADPR were photolyzed, while caffeine inhibited the labeling of only the protein with the Mr of 100×103 (Walseth et al., 1993). It is conceivable that cADPR effect could be modulated by other RYR agonists. The potentiation effect is of great advantage to oocytes in modulating the Ca2+i level.
CONCLUSIONS
Using the four commonly used approaches, we evaluated the existence of both IP3Rs and RYRs in mature bovine oocytes. Our data provided the following conclusions: (1) independent IP3Rs and RYRs coexist in mature bovine oocytes, (2) the RYR in bovine oocytes may be different from that in sea urchin eggs and (3) the cause of Ca2+i release induced by fresh cADPR solution is undefined and further studies will be focused on this. The finding of RYRs in bovine oocytes will provide impetus to screen other farm animals for its presence. The effect of these dual receptors in the events associated with bovine fertilization is currently under investigation.
ACKNOWLEDGEMENTS
We thank Dr H. C. Lee for suggestions and critical evaluation of the manuscript. We acknowledge the contribution of E. A. Miller Inc. This project is partially supported by the Utah Department of Community and Economic Development grant Nos 94-1046 and 93-1135 and USDA project No. W-171. Published as Utah Agricultural Experiment Station journal article 4694.