Strontium promotes calcium oscillations in mouse meiotic oocytes and early embryos through InsP3 receptors, and requires activation of phospholipase and the synergistic action of InsP3

Di Zhang1,2,*, Lei Pan1,2,*, Ling-Hai Yang1,2, Xiao-Ke He1,2, Xiu-Ying Huang1 and Fang-Zhen Sun1,3

1 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100080, China, 2 Graduate School of the Chinese Academy of Sciences, Beijing 100080, China

3 To whom correspondence should be addressed. E-mail: fzsun{at}genetics.ac.cn


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Sr2+ is the most efficient agent for mouse oocyte activation and functions by inducing Ca2+ oscillations. However, its specific mechanism of action remains unknown. Here we investigated the specificity and possible mechanism of Sr2+-induced Ca2+ oscillations in mouse oocytes and early embryos. METHODS: Ca2+ oscillations in oocytes and embryos were measured by ratiometric fluorescence imaging using fura-2AM. The role of phospholipase C (PLC) and inositol trisphosphate (InsP3) receptors in Sr2+-induced Ca2+ oscillations was examined by selective inhibitors. RESULTS: Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes, and in early embryos. A cell cycle stage-dependent phenomenon to Sr2+ stimulation was observed in 1-cell embryos. By using a low molecular weight heparin to antagonize the function of InsP3 receptors, we were able to show that InsP3 receptors are essential for Sr2+-induced Ca2+ oscillations. Treating metaphase II (MII) oocytes with the PLC inhibitor, U73122, abolished Sr2+-induced increases in Ca2+. This inhibitory effect of U73122 could be rescued by microinjection of InsP3, indicating that Sr2+-induced Ca2+ oscillations require the synergistic action of InsP3. CONCLUSIONS: Sr2+-induced calcium oscillations in mouse oocytes and early embryos are mediated through InsP3 receptors, and require PLC activation and the synergistic action of InsP3.

Key words: calcium oscillations/embryos/InsP3R/oocytes/Sr2+


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In mammals, the sperm activates the egg by triggering long-lasting Ca2+ oscillations in egg cytoplasm (Cuthbertson and Cobbold, 1985Go; Kline and Kline, 1992Go; Sun et al., 1992Go; Miyazaki et al., 1993Go; Schultz and Kopf, 1995Go; Swann and Lai, 1997Go). Accumulated evidence suggests that the sperm-induced Ca2+ oscillations are triggered by a sperm-derived protein factor which diffuses into the egg cytoplasm after gamete membrane fusion (Swann, 1990Go; Swann and Lai, 1997Go). The activity of this sperm factor in triggering Ca2+ oscillations in mammalian eggs appears to be highly conserved during evolution (Dong et al., 2000Go; Li et al., 2001Go). Although the sperm factor has not yet been biochemically purified, recent studies have shown that phospholipase C (PLC){zeta} appears to be a strong candidate for the mammalian sperm factor (Saunders et al., 2002Go; Kouchi et al., 2004Go).

The sperm factor mobilizes Ca2+ release from the egg’s intracellular stores (Swann, 1994Go) mainly through inositol trisphosphate (InsP3) receptors because blocking the function of the InsP3 receptors completely inhibited sperm-induced Ca2+ release (Miyazaki et al., 1992Go, 1993Go). Maintenance of the sperm factor-induced Ca2+ oscillations at fertilization requires Ca2+ influx (Swann, 1994Go), and is critically dependent upon the presence of a functional maternal machinery (Tang et al., 2000Go). The sperm-induced Ca2+ oscillations are spatially organized as propagating waves (Miyazaki et al., 1992Go), last for several hours and cease at about the time of pronuclear formation (Jones et al., 1996; Kono et al., 1996Go). The first Ca2+ transient, and some subsequent increases in Ca2+, are essential for egg activation (Kline and Kline, 1992Go), while later long-lasting Ca2+ oscillations facilitate early embryonic development such as pronuclear formation (Swann and Ozil, 1994Go; Ducibella et al., 2002Go).

Mammalian egg activation can also be achieved by a variety of artificial stimuli (Swann and Ozil, 1994Go). Sr2+ has been shown as the most efficient parthenogenetic agent for activating mouse ovulated eggs (Kline and Kline, 1992Go; Bos-Mikich et al., 1997Go) and nuclear-transplanted eggs (Wakayama et al., 1998Go). In addition, Sr2+-induced Ca2+ transients were also observed in activated eggs (Kono et al., 1996Go). Extending the exposure time to Sr2+-containing medium during egg activation and the first mitosis promoted preimplantation development (Bos-Mikich et al., 1997Go). However, so far, the mechanism by which Sr2+ induces Ca2+ oscillations in mammalian oocytes and early embryos remains unknown. In the present study, we examined the specificity and possible mechanism of Sr2+ -induced Ca2+ oscillations in mouse meiotic oocytes and early embryos. Our results show that Sr2+-induced Ca2+ oscillations in mouse meiotic oocytes and early embryos are mediated through the InsP3 receptors, and require PLC activation and a synergistic action of InsP3.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Unless otherwise stated, all chemicals used in this study were purchased from Sigma chemicals (St. Louis, MO, USA). U73122 and U73343 were from CALBIOCHEM (EMD Biosciences Inc. La Jolla, USA).

Preparation of mouse oocytes, zygotes and parthenogenetic (PG) embryos
For collection of germinal vesicle (GV) oocytes, the ovaries were removed from Kunming Abiro female mice (bred in the Institute of Genetics and Developmental Biology) 46–48 h after injection of pregnant mare serum gonadotrophin (PMSG). Antral follicles were punctured by 30 gauge needles, and cumulus-enclosed GV oocytes were released into M2 medium with 4 mg/ml bovine serum albumin (BSA fraction V, Sigma) (Fulton and Whittingham, 1978Go) containing 0.2 mM 3-isobutyl-1-methylxanthine (IBMX) to inhibit germinal vesicle breakdown (GVBD). Metaphase I (MI) oocytes were collected from oocytes cultured for 6–7 h from GV oocytes. MII eggs were collected from 4- to 6-week-old female Kunming Abiro mice as described previously (Tang et al., 2000Go). Female Kunming Abiro mice were superovulated by serial injection of PMSG and HCG 48 h apart. MII oocytes were collected from the oviducts at 14–16 h post-HCG into pre-warmed M2 medium supplemented with 4 mg/ml BSA. The cumulus cells were removed by a brief incubation at 37°C in hyaluronidase (0.3 mg/ml, Sigma) in M2 + BSA. The MII oocytes were then washed three times in M2 + BSA and transferred to microdrops of M2 + BSA under paraffin oil pending further treatments.

To obtain fertilized eggs (zygotes) at different cell cycle stages, female mice were paired with males immediately following HCG injection. S- and M-phase zygotes were subsequently collected at 23–24 and 30 h post-HCG, respectively. It should be noted here that, in our study, the M-phase zygotes refer to those fertilized eggs that had already undergone complete nuclear membrane breakdown.

PG embryos were produced by exposure of MII oocytes at 17–18 h post-HCG to 7% ethanol in M2 medium for 7 min at room temperature. The oocytes were then washed three times with M2 + BSA, and cultured in KSOM medium (Lawitts and Biggers, 1993Go) at 37°C, 5% CO2 in air. To produce diploid PG embryos, the MII oocytes were treated with cytochalasin B (5 µg/ml) for ~6 h immediately after the activation treatment. S-phase PG 1-cell embryos were obtained at 10–12 h after ethanol activation, while M-phase PG embryos were subsequently obtained at 18–20 h for the ethanol-activated group or at 16–18 h for the Sr2+-activated group.

Microinjection of MII oocytes, zygotes and PG 1-cell embryos
Micromanipulation of mouse MII oocytes and 1-cell embryos was conducted following the procedures described by Tang et al. (2000)Go. All injections were carried out using a pair of manually operated pressure microinjectors (IM-6, Narishige, Japan) filled with paraffin oil. Microinjection pipettes (Clark Electromedical Instruments, UK) were pulled by a micropipette puller (Sutter Instrument company, Model P-87, USA) to give an open tip (inner diameter 1–2 µm) and a long taper of ~10 mm in length. The microinjection pipette was connected to the microinjector and then filled with paraffin oil. Microinjection of heparin and injection volume were measured as described by Tang et al. (2000)Go. In addition, the procedures for bovine sperm extract preparation and injection were the same as described previously (Tang et al., 2000Go).

Intracellular Ca2+ measurements
Before intracellular Ca2+ concentration ([Ca2+]i) measurement, mouse oocytes or zygotes were loaded with 2 µM Fura-2 AM (Molecular Probes) in M2 medium containing 4 mg/ml BSA at 37°C for 30 min. After loading, the cells were washed three times in M2 and then transferred to a chamber containing M2 medium covered by light paraffin oil. The procedures and equipment used for calcium measurement were the same as described by Dong et al. (2000)Go. The emitted fluorescence intensities at 510 nm were recorded at 340 and 380 nm excitation wavelengths by a Mira-1000TE low light level CCD camera. The fluorescence signal is displayed as the ratio of fluorescence intensities for the 340 nm/380 nm excitation wavelengths. [Ca2+]i was estimated from the ratio equation described by Grynkiewicz et al. (1985)Go, which is calculated by computer simultaneously. The intensity of excitation UV light was reduced by neutral density filters. Parameters required for the ratio equation were obtained according to the method of Poenie et al. (1985)Go. [Ca2+]i were recorded immediately after the initial exposure of the oocytes to Sr2+. The [Ca2+]i image was recorded every 2 s for up to 2–4 h.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sr2+ induces Ca2+ oscillations in immature and mature oocytes
The Ca2+ responses to Sr2+ in mouse immature and mature oocytes are illustrated in Figure 1. GV oocytes can exhibit spontaneous Ca2+ oscillations (Carroll and Swann, 1992Go); we therefore used IBMX (50 µg/ml for 3 h) to block (GVBD), and treated GV oocytes with Sr2+ only after their spontaneous Ca2+ oscillations had completely ceased. Table I shows that addition of 20 mM Sr2+ induces GV, MI and MII oocytes to produce a series of Ca2+ oscillations. In comparison with mature oocytes, immature oocytes (GV and MI) exhibited a lower amplitude and a shorter spiking interval. In general, the first Ca2+ transient in MII oocytes induced by Sr2+ was broader and had a larger amplitude than the subsequent spikes. In both immature and mature oocytes, the characteristics of the spikes in the same oocytes induced by 20 mM Sr2+ were remarkably similar. In all cases, the rate of increase of Ca2+ was always faster than the rate of recovery.



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Figure 1. Patterns of Ca2+ oscillations induced by 20 mM Sr2+ in GV (a), MI (b) and MII (c) oocytes.

 

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Table I. Characteristics of Ca2+ oscillations induced by Sr2+ in mouse meiotic oocytes and 1-cell embryosa

 

Sr2+-induced Ca2+ oscillations in 1-cell embryos are cell cycle stage dependent
To investigate whether Sr2+ can induce Ca2+ oscillations at any cell cycle stages in mouse early embryos, and whether Sr2+-induced Ca2+ oscillations in embryos are fertilization status dependent, we treated embryos derived from both fertilized and PG embryos with 20 mM Sr2+ at different cell cycle stages and then examined their responses to the treatment. Figure 2 shows the patterns of Ca2+ signalling in embryos. Characteristics of individual increases induced by Sr2+ in 1-cell embryos are presented in Table I. Sr2+ can induce Ca2+ oscillations in interphase 1-cell embryos in a manner independent of their fertilization status. However, at metaphase, none of the embryos derived from the fertilized or the PG eggs was sensitive to Sr2+, indicating that the ability of Sr2+ to trigger intracellular Ca2+ release in embryos is cell cycle stage dependent. In addition, when interphase 2-cell embryos were treated with 20 mM Sr2+, Ca2+ oscillations also occurred in the embryos in a manner independent of their fertilization status. Clearly, our finding is not consistent with that reported previously (Kono et al., 1996Go; Bos-Mikich et al., 1997Go). To examine whether the lack of calcium oscillations is due to embryo quality or a possible toxic effect of Sr2+ at 20 mM on the metaphase 1-cell embryos, we first tested whether reducing the Sr2+ concentration to 10 mM could induce an increase in Ca2+ in the PG embryos. It is found that all metaphase PG embryos (n = 27) treated with 10 mM Sr2+ failed to generate any Ca2+ oscillations during 2 h of detection. We then examined whether the metaphase PG embryos that failed to respond to 20 mM Sr2+ could generate sperm factor-induced Ca2+ oscillations. Figure 3 shows that injection of a physiological dose of the sperm extract had caused the Sr2+-stimulated PG embryos to generate Ca2+ oscillations (nine out of 12 embryos examined) in M2 medium containing Ca2+, in a manner similar to our previous finding (Tang et al., 2000Go). Moreover, it was found that low frequency oscillatory increases of Ca2+ even occurred in the sperm extract-injected metaphase embryos (eight out of 11) in M2 medium with 20 mM Sr2+. Our results therefore suggest that there is indeed a striking difference in the ability of Sr2+ and sperm protein factor in triggering Ca2+ oscillations in the M-phase 1-cell PG embryos.



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Figure 2. Patterns of Ca2+ oscillations induced by 20 mM Sr2+ in PG embryos at 1-cell interphase (a), 1-cell metaphase (b), 2-cell interphase (c), and fertilized embryos at 1-cell interphase (d), 1-cell metaphase (e) and 2-cell interphase (f). The embryos were prepared as described in Materials and Methods.

 


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Figure 3. Effect of sperm extract injection on Ca2+ oscillations in Sr2+-treated metaphase 1-cell embryos. The embryos were firstly incubated in M2 medium with 20 mM Sr2+ for 30 min, and were thereafter injected with a physiological dosage (2 pl) of the bovine sperm extract. The embryos were transferred to M2 medium containing Ca2+ or Sr2+ for imaging analysis immediately after the injection. (a) and (b) Typical patterns of Ca2+ oscillations observed in the embryos in M2 medium with 2.0 mM Ca2+ and in M2 medium with 20 mM Sr2+, respectively. The arrows indicate the time of sperm extract injection.

 

Sr2+-induced Ca2+ oscillations in MII oocytes and embryos are mediated through the InsP3 receptors
InsP3 receptors play an essential role in determining Ca2+ oscillations at fertilization (Miyazaki et al., 1992Go, 1993Go). Heparin, as an effective InsP3 receptor antagonist, has been widely used to determine the role of InsP3 receptors in regulating intracellular Ca2+ release (Rakow and Shen, 1990Go; Carroll and Swann, 1992Go). Here, we used a low molecular weight heparin to examine whether InsP3 receptors are critical in regulating Sr2+-induced Ca2+ oscillations in mouse oocytes and interphase embryos. In doing so, we firstly injected various concentrations of InsP3 into MII oocytes and established the minimal intracellular dosage of InsP3 required for inducing calcium oscillations. As shown in Figure 4, when the intracellular concentration of injected InsP3 is 100 nM, oscillatory increases in Ca2+ occurred in the oocytes, and the amplitude of the Ca2+ increases appears to be InsP3 dosage dependent. This is consistent with the finding of a previous report (Jones and Whittingham, 1996Go). We then tested the effect of heparin at different concentrations on InsP3-induced Ca2+ oscillations in the oocytes. It is found that when the intracellular concentration of heparin is 120 µM, it completely inhibited the increases in Ca2+ induced by 100 nM InsP3. The effect of various concentrations of heparin on Sr2+-induced Ca2+ oscillations in MII oocytes and 1-cell PG embryos is shown in Table II. In 1-cell PG embryos, Sr2+-induced intracellular increases in Ca2+ were effectively inhibited even at an intracellular concentration of 10–60 µM heparin (n = 13). To confirm further the inhibitory effect of heparin on Sr2+-induced Ca2+ release, we then tested whether injecting MII oocytes with 120 µM heparin could block Sr2+-induced egg activation. It is found that none of the heparin-injected oocytes (n = 39) following 2 h treatment with 20 mM Sr2+ had formed a pronucleus at 8 h after the treatment. However, when the injected oocytes were stimulated with 10 µg/ml cycloheximide, >50% of the them (18 out of 33 examined) had been activated, evidenced by the presence of a female pronucleus following 8 h of incubation. This is consistent with the finding that cycloheximide as a protein synthesis inhibitor could activate mouse eggs in a manner independent of their intracellular Ca2+ increase (Bos-Mikich et al., 1995Go). In conclusion, our results therefore suggest that InsP3 receptors are essential in mediating the Sr2+-induced increase in intracellular Ca2+ in mouse MII oocytes and 1-cell embryos.



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Figure 4. Effect of heparin on Sr2+-induced Ca2+ oscillations in mouse eggs. (a) Ca2+ spikes caused by 100 nM InsP3; (b) effect of heparin on InsP3-induced Ca2+ release; (c) effect of 120 µM heparin on Sr2+-induced Ca2+ oscillations.

 

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Table II. Effect of heparin on Sr2+-induced Ca2+ oscillations in mouse eggs and 1-cell embryos

 

Sr2+-induced Ca2+ oscillations in mouse oocytes require activation of PLC
U73122 is a membrane-permeable inhibitor of PLC (Bleasdale et al., 1990Go; Yule and Williams, 1992Go), and has been used to determine PLC activation of Ca2+ oscillations induced by sperm or sperm protein factor (Dupont et al., 1996Go). Here, U73122 was used to examine whether Sr2+-induced Ca2+ oscillations in mouse oocytes require activation of PLC. As a control experiment, we first tested the effect of U73122 and U73343 on acetylcholine (Ach)-induced increases in Ca2+ in mouse oocytes. Ach-induced increases in Ca2+ in mouse oocytes were mediated by activating PLC (Dupont et al., 1996Go). We found that 40 µM U73122 could effectively block the increase in Ca2+ induced by 50 µM Ach in the oocytes (data no shown here). Pre-incubating MII oocytes with U73122 at an intracellular concentration of 1–30 µM for 30 min before treatment with Sr2+ caused a dose-dependent inhibition of Ca2+ oscillations induced by Sr2+ (Figure 5). When U73122 was at a concentration of 10 µM, <40% of treated oocytes (eight out of 21 examined) showed Sr2+-induced Ca2+ oscillations. However, when increasing the U73122 concentration to 20 µM, Sr2+-induced Ca2+ oscillations were completely inhibited in all oocytes treated (n = 13). On the contrary, pre-incubating oocytes with U73343, an inactive analogue of the PLC inhibitor U73122, had no effect on Sr2+-induced Ca2+ oscillations even at a concentration of 30 µM (n = 12). These results indicate that activation of PLC to generate InsP3 is required for Sr2+-induced Ca2+ oscillation in mouse oocytes.



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Figure 5. Effect of the PLC inhibitor U73122 on Sr2+-induced Ca2+ oscillations. (a) Inhibition by various concentrations of U73122 of Sr2+-induced Ca2+ oscillations in mouse eggs; U73343 is used as the control. (b) Pre-incubating eggs for 30 min with 20 µM U73122 completely stopped Ca2+ oscillations caused by Sr2+. (C) No inhibitory effect is observed following an identical pre-incubation period with U733343 at a concentration of 20 µM.

 



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Figure 6. The synergistic effect of InsP3 with Sr2+ on Sr2+-induced Ca2+ oscillations. (a) Ca2+ spikes caused by 100 nM InsP3. (b) Effect of 20 µM U73122 on Ca2+ spikes induced by 100 nM InsP3 in the absence of Sr2+. (c) U73122 at 20 µM completely inhibited Sr2+-induced Ca2+ oscillations. (d) Injection of 100 nM InsP3 can retrieve the inhibition by 20 µM U73122 of Sr2+-induced Ca2+ oscillations.

 
Sr2+-induced Ca2+ oscillations in mouse oocytes require a synergistic action of InsP3
To determine the relationship between InsP3 and Sr2+ on Sr2+ -induced Ca2+ oscillations in mouse eggs, we tested whether injection of a physiological concentration of InsP3 could rescue the inhibitory effect of U73122 on Sr2+-induced Ca2+ oscillations. Figure 6 shows that in the presence of 20 µM U73122, injection of 100 nM InsP3 induced only 1–2 spikes (seven out of seven), while addition of 20 mM Sr2+ caused the InsP3-injected eggs to generate Ca2+ oscillations (seven out of 10). These observations suggest that there is asynergistic action of InsP3 in determining Sr2+-induced Ca2+ oscillations in mouse oocytes.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The present study investigated the possible mechanism by which Sr2+ promotes intracellular Ca2+ release in mouse oocytes and early embryos. Our major findings are: (i) Sr2+ can induce Ca2+ oscillations in both immature and mature oocytes as well as in early embryos, and its effect on 1-cell embryos is cell cycle stage dependent; (ii) Sr2+-induced calcium oscillations in meiotic oocytes and embryos are mediated through the InsP3 receptors; and (iii) Sr2+-induced Ca2+ oscillations in mouse oocytes require activation of PLC and the synergistic action of InsP3. To our knowledge, this is the first report showing how Sr2+ promotes intracellular Ca2+ release in mouse oocytes and early embryos.

The mechanism underlying the maintenance of Ca2+ oscillations induced by Sr2+ and sperm is different
It has been demonstrated in the mouse that sperm-induced calcium oscillations at fertilization are mediated by the InsP3 receptors (Miyazaki et al., 1992Go, 1993Go). The results of the present study show that Sr2+ can induce calcium oscillations in mouse oocytes and embryos in calcium-free medium and that antagonizing the function of InsP3 receptors by low molecular weight heparin blocked Sr2+-induced calcium oscillations, indicating that calcium oscillations induced by Sr2+ are also regulated through the InsP3 receptors. In an early study, it had been demonstrated that the persistence of the sperm factor-induced calcium oscillations requires a putative maternal machinery, which functions only once and is abolished in the zygotes (Tang et al., 2000Go). The results of the present study show that Sr2+ can induce Ca2+ oscillations not only in meiotic oocytes but also in early embryos derived from fertilized eggs, suggesting that the persistence of the long-lasting calcium oscillations induced by Sr2+ in both mouse oocytes and embryos does not require this putative maternal machinery. We therefore conclude that although sperm protein factor- and Sr2+-induced Ca2+ oscillations are both mediated through the InsP3 receptors, different mechanisms exist with regard to their requirement for a functional maternal machinery in determining the persistence of Ca2+ oscillations.

Action of Sr2+ in promoting intracellular Ca2+ release
Ca2+ signalling in mouse oocytes can be affected by the amount of releasable Ca2+ in the intracellular stores (Jones and Whittingham, 1996Go), the number of intact InsP3-1 receptors (InsP3R-1s) and the sensitivity status of InsP3R-1 to InsP3 (Jellerette et al., 2004Go). Although it is difficult to measure the precise amount of intracellular InsP3 required for its effect on Sr2+-induced Ca2+ oscillations in mouse eggs, the results in this study show that activation of PLC to generate InsP3 is required for Sr2+-induced Ca2+ signalling. Mouse eggs will generate Ca2+ oscillations in Ca2+-containing medium either when stimulated with agonists that activate G-protein or PLC to generate InsP3 (Jaffe, 1990Go) or when injected with InsP3 (Jones et al., 1996; Tang et al., 2000Go). The findings that treatment with U73122 blocked Sr2+-induced Ca2+ oscillations while microinjection of InsP3 to increase intracellular InsP3 promoted long-lasting Ca2+ oscillations imply a synergistic action of InsP3 on Sr2+-induced Ca2+ oscillation.

It has been suggested that Sr2+ can improve the sensitivity of InsP3R. Hamada et al. (2003)Go have shown, by applying electron microscopy and proteolytic analyses, the conformational changes of InsP3R-1 bound by bivalent ions (Ca2+, Sr2+, Ba2+ and Mg2+). It was shown that the Ca2+-specific conformational change can structurally affect the InsP3-triggered channel opening within InsP3R-1. It has also been shown that Sr2+ can trigger fast exocytosis by binding to the Ca2+-binding site of the synaptotagmin 1 C2B domain of InsP3R-1 (Shin et al., 2003Go). In addition, it is reported that at least two Ca2+ interaction sites should exist in InsP3R-1: a stimulatory site sensitive to Sr2+ and an inhibitory site insensitive to Sr2+ (Marshall and Taylor, 1994Go; Xu et al., 2003Go). In another study using sheep cerebellum microsomes (Worley et al., 1987Go), measurements of [3H]InsP3 binding at a low concentration showed that Sr2+ and Ca2+ did not enhance the amount of bound [3H]InsP3, implying that the activating effect of Sr2+ and Ca2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by increasing the receptor’s affinity for InsP3. Based on these findings, together with our observation (Figure 5), we speculate that Sr2+ promotes Ca2+ oscillations in mouse oocytes and embryos probably by sensitizing the InsP3 receptors (the Ca2+ release channels) to release Ca2+. However, we still do not know how Sr2+ affects PLC activation to generate InsP3 and how in Ca2+-free medium the Sr2+-induced Ca2+ oscillations can persist for several hours. One possibility is that Sr2+ acts as a surrogate for Ca2+, such that Sr2+-induced Ca2+ oscillations in Ca2+-free medium reflect changes in the fura ratio in response to changing [Sr2+]i, but direct evidence to support this possibility is at present still lacking.

Sr2+-induced Ca2+ oscillations in early embryos are cell cycle stage dependent
The present study reveals two striking differences between the sperm protein factor and Sr2+ in regulating Ca2+ release in early embryos. First, at interphase of the first mitosis, neither fertilized nor PG embryos were able to generate Ca2+ oscillations upon sperm factor injection (Tang et al., 2000Go); however, both types of embryos at interphase exhibit Ca2+ oscillations when treated by Sr2+. Secondly, at metaphase of the first mitosis, the sperm protein factor can cause PG embryos to undergo Ca2+ oscillations, while Sr2+ cannot. These findings indicate that although at the same cell cycle stage and with theoretically an identical cellular environment, the response of the cells to the stimulation of these two oscillatory Ca2+ stimuli is strikingly different. In theory, since the embryos were generated by the same approach and at the same cell cycle stage, it is most likely that the difference is attributed to the stimulation of Sr2+ and sperm protein factor. There is evidence showing that phosphorylation can modify the function or conductivity of InsP3R (Tang et al., 2003Go; Krizanova and Ondrias, 2004Go) and that phosphorylation of InsP3R-1 occurs at interphase of the first mitosis (Jellerette et al., 2004Go). We consider that the phenomenon of cell cycle stage dependency of Sr2+ and sperm protein factors in triggering Ca2+ oscillations in embryos reflects the basic difference in the mechanisms underlying how Ca2+ release is triggered by Sr2+ and the sperm factor. In the present study, we show that Sr2+ can induce the 1-cell embryos to undergo Ca2+ oscillations, in a manner independent of their fertilization status, in interphase but not M phase. This finding is clearly contrary to two previous reports that Sr2+ triggers Ca2+ oscillations in M phase, but not interphase (Kono et al., 1996Go; Bos-Mikich et al., 1997Go). We have shown that this difference is not due to the concentrations of Sr2+ used, because even using the same concentration (10 mM) as that described in the two previous reports, we still failed to detect any Ca2+ oscillations in the M-phase embryos. In addition, we show that the M-phase embryos that failed to generate Sr2+-induced Ca2+ oscillations were able to do so when injected with a physiological dosage of the sperm extract (Figure 3), suggesting a striking difference between the sperm factor and Sr2+ in inducing Ca2+ oscillations in M-phase 1-cell PG embryos. It should be noted here that the strain of mouse used in our study is different from that used in the previous reports (Kono et al., 1996Go; Bos-Mikich et al., 1997Go). This may be the reason for the striking difference between our finding and that of the two previous reports, but the molecular basis for this variation is still a mystery. The present study has shown that PLC activation is required for Sr2+-induced Ca2+ oscillations, and that Sr2+ need a synergistic activation of InsP3 to generate Ca2+ oscillations. Our findings imply that the activation of PLC may be important for maintaining a basal intracellular InsP3 concentration required for the synergistic action. This possibility is supported by our finding (Figure 6) that injecting InsP3 into U73122-treated eggs reinitiated Ca2+ oscillations in Sr2+-containing medium, while without Sr2+ the same group of eggs generated only 1–2 spikes. The combined findings point to the possibility that the dominant action of Sr2+ in triggering intracellular Ca2+ release is mediated by stimulating intracellular Ca2+ channels, probably by inducing a conformational change in InsP3 receptors (Hamada et al., 2003Go).

Impact of Ca2+ signalling on embryonic development
Sr2+ has been proved to be the most efficient agent for activating mouse eggs (Kline and Kline, 1992Go; Bos-Mikich et al., 1997Go). This is probably due to its ability to trigger fertilization-like Ca2+ oscillations in the egg cytoplasm. In all mammals studied so far, the sperm activates the egg by causing long-lasting Ca2+ oscillations (see the review by Stricker, 1999Go). Similarly, human eggs at fertilization also exhibited Ca2+ oscillations (Taylor et al., 1992Go).

Ca2+ signalling at fertilization is a vital event for switching the oocytes from meiosis to mitosis (Swann and Ozil, 1994Go; Ducibella et al., 2002Go). Ca2+ transients can affect morphological, biochemical and developmental events of egg activation. Moreover, extending the exposure time to Sr2+-containing medium during egg activation and the first mitosis promoted preimplantation development in the mouse (Bos-Mikich et al., 1997Go). Manipulating Ca2+ signalling patterns during egg activation can severely alter mammalian preimplantation development and implantation outcome (Ozil and Huneau, 2001Go). In addition, it has been shown that the Ca2+ signal controls chromosome disjunction in early sea urchin embryos (Groigno and Whitaker, 1998Go). Altering Ca2+ signalling in mammalian cells affects the metaphase–anaphase transition by interfering with the spindle checkpoint (Xu et al, 2003Go). Our findings that Sr2+ can promote Ca2+ oscillations in immature and mature oocytes, and early embryos, and that the action of Sr2+ involves activation of PLC and a synergetic action of InsP3 illustrates that Sr2+ can be used as a physiological agent to dissect the effect of Ca2+ oscillatory signals on nuclear and cytoplasmic events of mammalian oocyte maturation, gene expression and developmental events associated with preimplantation and implantation. It will be extremely interesting and worthwhile to find out in future studies how Ca2+ signals upon egg activation could alter preimplantation development and implantation outcome, and whether improved embryo quality and implantation outcome can be achieved by providing the human eggs with adequate Ca2+ signals at fertilization.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported partly by grants from the State key Basic Research Program of China (Grant No. TG1999055902), and the National Natural Science Foundation of China (Grant No.30370699).


    Notes
 
* Zhang and Pan contributed equally to this work Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 19, 2005; resubmitted on June 13, 2005; accepted on June 24, 2005.