©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ionomycin, Thapsigargin, Ryanodine, and Sperm Induced Ca Release Increase during Meiotic Maturation of Mouse Oocytes (*)

(Received for publication, October 12, 1994; and in revised form, January 9, 1995)

Keith T. Jones (§) John Carroll David G. Whittingham

From the Medical Research Council Experimental Embryology and Teratology Unit, St. George's Hospital Medical School, Cranmer Terrace, London, SW17 0RE, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Fertilization of mature mouse oocytes triggered highly repetitive Ca oscillations lasting 2-3 h. However, immature oocytes generated only two or three oscillations, which ceased within 1 h. Development of repetitive Ca transients to sperm occurred late in oocyte maturation and was dependent on cytoplasmic modifications that were independent of cell cycle progression from metaphase I to metaphase II. Immature oocytes released significantly less Ca from stores than mature oocytes in response to ionomycin and thapsigargin. Ryanodine had no effect on intracellular Ca in maturing oocytes but stimulated an increase in Ca in mature oocytes. The ability of ryanodine to increase Ca levels was, however, strain-dependent.

Preincubation of oocytes with thapsigargin or ryanodine significantly attenuated the normal fertilization Ca response, causing a decrease in the number and the rate of rise of the transients. The inhibition of sperm-induced Ca transients by ryanodine was independent of its ability to cause an immediate Ca increase. Low concentrations of ryanodine had no effect on resting Ca levels but inhibited Ca oscillations at fertilization. Similarly Ca oscillations were blocked in oocytes from a strain of mouse that showed no immediate Ca increase with ryanodine. These results suggest that modifications in Ca stores and ryanodine-sensitive Ca release mechanisms during oocyte maturation play an important role in Ca oscillations at fertilization.


INTRODUCTION

In mammalian oocytes a series of highly repetitive Ca transients lasting several hours are triggered at fertilization(1, 2, 3) . The Ca transients act both to initiate completion of meiosis and to block polyspermy by stimulating cortical granule exocytosis(4, 5) . Precisely how these Ca oscillations are generated is not clear.

Ca oscillations, like those observed at fertilization, occur in a wide variety of cell types (6, 7, 8) and are mediated by two families of Ca channels on the endoplasmic reticulum(9) . The generation of Ca oscillations is normally stimulated by an agonist-induced increase in inositol trisphosphate (InsP(3)). (^1)Ca released in response to InsP(3) acts, by positive feedback, to stimulate further Ca release through the InsP(3) receptor (10) or the ryanodine receptor(11) . The ability of Ca to stimulate further Ca release from intracellular stores is known as Ca-induced Ca release (CICR). InsP(3)-induced Ca release is present in both mouse and hamster oocytes since microinjection of InsP(3) triggers Ca oscillations(12, 13) . Additionally an antibody to the InsP(3) receptor inhibits Ca oscillations induced by InsP(3), Ca injection, and sperm (14) . This suggests that both InsP(3)- and Ca-induced Ca release in oocytes are mediated through the InsP(3) receptor(15) .

In mouse oocytes there is also evidence for the presence of a ryanodine receptor. Ryanodine binds to the ryanodine receptor and blocks it in an open state that has low conductance(16) . Its effects are complex, with activation and inhibition of CICR through the ryanodine receptor being reported depending on experimental conditions(17) . The ability of ryanodine to raise Ca levels in mouse oocytes is controversial, with Swann finding that ryanodine triggers Ca release, inhibits agonist-induced oscillations, and enhances CICR (18) whereas Kline and Kline find no effect(19) .

The ability to undergo normal fertilization is acquired during oocyte maturation(20) . Immature mouse oocytes arrested at prophase of the first meiotic division are morphologically identified by a germinal vesicle (GV)(20) . In vivo, maturation is stimulated by gonadotropins but can occur in vitro by removal of the immature oocyte from the follicle into culture medium(21) . GV breakdown (GVBD) occurs about 2 h after the resumption of meiosis. At 8-10 h the first polar body is extruded, and then the oocyte arrests at metaphase II until fertilization stimulates the completion of meiosis and entry into the first mitotic cell cycle. In addition to the events of meiotic maturation, cytoplasmic modifications such as the ability to release cortical granules (22, 23) and decondense the sperm nucleus (24) are necessary for normal fertilization.

Recent observations suggest that an important aspect of cytoplasmic maturation is modifications in Ca homeostasis. GV stage oocytes show spontaneous Ca oscillations, which decrease following GVBD(25) . Mature oocytes, but not immature oocytes, show Ca waves in response to a Ca-releasing factor from sperm(26) . Additionally, during maturation there is an increase in sensitivity to InsP(3)-induced Ca release (27) and an increase in the amount of Ca released by ionomycin(28) , suggesting that Ca stores increase in preparation for fertilization.

The aim of the present study was to examine how Ca homeostasis is modified during oocyte maturation by examining the responses to ionomycin, thapsigargin, ryanodine, and sperm. We find that immature oocytes do not show highly repetitive Ca transients during fertilization, and the development of such transients occurs late in oocyte maturation. This modification in the mechanism of sperm-induced Ca release is correlated with an increase in sensitivity to ionomycin, thapsigargin, and ryanodine.


EXPERIMENTAL PROCEDURES

Materials

Compounds were from Sigma unless otherwise stated and tissue culture grade where appropriate. Ryanodine (Calbiochem) contained >80% ryanodine + dihydroryanodine, and identical effects were seen using high purity ryanodine (data not shown).

Preparation of Gametes

Unless otherwise stated, the mice used in this study were 21-23-day-old female F1 hybrids of B6CB (C57Bl/6JLac times CBA/CaLac). For one set of experiments 6-8-week-old mice from an outbred MF1 strain were used. Oocytes were collected in medium M2 plus bovine serum albumin (BSA) (4 mg/ml) (29) and further cultured in medium M16 plus BSA (4 mg/ml) at 37 °C and 5% CO(2)(30) . Immature GV stage oocytes were collected 48 h after peritoneal injection of 7.5 IU of pregnant mares' serum gonadotropin. The antral follicles were punctured with a sterile needle, and cumulus-intact oocytes were collected. For in vitro maturation studies, the GV stage cumulus-oocyte complexes were cultured in Waymouth's medium MB752/1 (Life Technologies, Inc.) supplemented with 0.23 mM pyruvate and 5% fetal calf serum. For Ca recording the cumulus cells were removed by pipetting through a fine bore pipette. Oocytes matured in vivo were collected from pregnant mares' serum gonadotropin-primed mice 14 h after injection of 5 IU of human chorionic gonadotropin. The cumulus cells were removed using hyaluronidase (0.3 mg/ml). Sperm were collected from known fertile male mice of the same strain and capacitated at a concentration of 1-2 times 10^6 sperm/ml for 90 min in T6 medium containing 15 mg/ml fraction V BSA(31, 32) .

Ca Measurement

Measurements were made using oocytes incubated with either 50 µM Fluo-3 AM or 50 µM Indo-1 AM for 30 min as described previously(25) . The zona pellucida was removed by a brief treatment in acid Tyrode's solution. The temperature of the chamber containing the oocyte was kept at 33-36 °C. Zona-free oocytes were attached to chamber coverslips in M2 without BSA. For fertilization M2 plus fraction V BSA (final concentration, 7 mg/ml) and 10 µl of capacitated sperm suspension were added to the 1-ml chamber.


RESULTS

Ca Oscillations at Fertilization of Mature and Immature Oocytes

Fertilization of mature oocytes typically generated a series of 20-40 Ca oscillations that continued over a period of 2-3 h (Fig. 1a) (n = 7). Transients were initiated by a pacemaker rise in basal Ca followed by a rapid upstroke. There were a number of clear differences between the first Ca transient and those that occurred subsequently. The first transient was longer in duration, had a distinct shoulder during its rising phase, and at its peak had a series of superimposed high frequency Ca spikes (Fig. 1b). The frequency of the transients was constant in any one oocyte but showed significant variation (5-15 min) between oocytes.


Figure 1: Comparison of sperm-induced Ca oscillations in mature and immature oocytes. a, representative Ca recording from a mature oocyte loaded with Indo-1. The oocyte continued to show Ca oscillations for 3.5 h. Arrows indicate the pacemaker Ca triggering further Ca release. b, the first transient in a mature oocyte. Arrow indicates a shoulder during the rise phase. Note the superimposed spikes during the peak. c, representative Ca recording in an immature oocyte showing that the Ca transients do not continue. Inset, a magnified trace of the second and third transient and subsequent pacemaker Ca that fails to initiate a further transient. d, the first transient in an immature oocyte. Arrow indicates a shoulder during the rise phase. Note the absence of spikes during the peak. The timebar is 5 min.



The pattern of Ca oscillations in response to sperm was markedly different in immature oocytes where there were a maximum of three much smaller transients, which ceased after 1 h (Fig. 1c) (n = 15). Similar to mature oocytes the first transient showed a shoulder in its rising phase but was shorter in duration and had no superimposed spikes (Fig. 1d). A common feature in oocytes in which oscillations ceased was that the pacemaker increase in basal Ca occurred but failed to initiate a complete transient (Fig. 1c, inset). To determine the state of filling of the Ca stores once oscillations had ceased, 10 µM ionomycin was added to the oocytes after chelation of extracellular Ca with 10 mM EGTA. Ionomycin did not trigger a Ca transient in these oocytes (Fig. 2, n = 4), suggesting that fertilization had depleted intracellular Ca stores.


Figure 2: Depletion of Ca stores in an immature fertilized oocyte. Once sperm-induced Ca transients had ceased, 10 mM EGTA (openbar) and 10 µM ionomycin (solidbar) were added sequentially. No Ca increase was recorded.



The Development of Continuous Ca Oscillations to Sperm

Maturing oocytes were fertilized at different times after release from the follicle. In response to sperm only one to three Ca transients were seen in oocytes during the first 10 h after release (n = 10). A similar attenuated response was seen in oocytes that had extruded the first polar body and fertilized between 10 and 11 h (n = 5) (Fig. 3a). It was not until 12 h after release from the follicle that sperm-induced Ca transients typical of mature oocytes were first observed (n = 3) (Fig. 3b). This suggests that the ability to generate mature oocyte-like Ca oscillations occurs 1-2 h after first polar body formation.


Figure 3: The development of continuous oscillations in response to sperm during maturation. a, at 10.5 h after release from the follicle, oocytes that had extruded the first polar body showed an attenuated response to sperm. Continuous Ca oscillations following fertilization were observed both in b, oocytes at 12 h, and c, metaphase I-arrested oocytes at 14 h after meiotic resumption.



During in vitro maturation some oocytes arrest spontaneously at metaphase I. These arrested oocytes when fertilized 14 h after the resumption of meiosis generated continuous Ca oscillations similar to mature ovulated oocytes (Fig. 3c). Therefore, the mature oocyte-like response to sperm does not require progression from metaphase I to metaphase II but is dependent on cytoplasmic modifications, which occur about 12 h after release from the follicle.

Ca Stores in Immature and Mature Oocytes

Recently Tombes et al.(28) demonstrated that the amount of Ca released in response to the Ca ionophore, ionomycin, increased between metaphase I and metaphase II. We have confirmed these findings and extended the study to examine the amount of Ca released by the endoplasmic reticulum Ca-ATPase inhibitor thapsigargin (10 µM)(33) . In Ca-free medium only one Ca transient was observed for either agent (Fig. 4a). The peak ratio and area under the transient (Fig. 4, b and c) were consistently greater for ionomycin than thapsigargin (p < 0.05, Student's unpaired t test), suggesting that the ionomycin-sensitive Ca store is larger. In mature oocytes there was an 11-fold increase in the area under the thapsigargin-induced transient and a 3-fold increase in response to ionomycin compared with GV stage oocytes. After GVBD, the area under the transient increased for both agonists but remained significantly less than that seen in mature oocytes (Fig. 4, b and c).


Figure 4: Comparison of thapsigargin and ionomycin-induced Ca transients in immature and mature oocytes. a, GV stage (a, b) and mature (c, d) oocytes were placed in Ca-free medium containing 3 mM EGTA. The Ca increase was recorded following addition of 10 µM thapsigargin (a, c) or 10 µM ionomycin (b, d). The responses of mature oocytes were greater than immature oocytes. The timebar represents 5 min. b, the area under the transient. c, peak ratio when thapsigargin (openbars) or ionomycin (hatchedbars) were added to oocytes at the GV stage (GV), 2-4 h after GVBD (GVBD), or to mature oocytes (mature). * (p < 0.05, Student's unpaired t test) and** (p < 0.01), significantly different from corresponding GV stage oocyte. ++ (p < 0.01), significantly different from GVBD stage oocytes.



Sensitivity to Ryanodine in Mature and Immature Oocytes

Ryanodine added to the extracellular medium causes a rise in intracellular Ca or modulation of agonist-induced Ca rises in many cell types(34, 35, 36, 37, 38) , although its effects on oocytes are controversial(18, 19) . The ability of oocytes to respond to ryanodine was therefore examined. The majority (88%, n = 17) of mature oocytes showed a series of Ca rises in response to 1 mM extracellular ryanodine, which returned to resting levels within 20 min (Fig. 5a). In contrast, ryanodine caused little or no response in 94% of immature oocytes (Fig. 5b) (n = 32). Lower concentrations of 100-500 µM ryanodine did not affect intracellular Ca levels in mature oocytes (data not shown).


Figure 5: The effects of ryanodine on intracellular Ca in mature and immature oocytes. Ryanodine (1 mM) was added extracellularly at the indicated time (solidbar) to mature (a) and immature (b) oocytes. Ryanodine caused an increase in Ca in mature (but not immature) oocytes. Oocytes were loaded with Fluo-3 AM, and therefore fluorescence intensity is arbitrary.



The Effects of Thapsigargin and Ryanodine on Sperm-induced Ca Transients in Mature and Immature Oocytes

Since the thapsigargin- and ryanodine-sensitive Ca stores are modified during maturation we examined their contribution to sperm-induced Ca oscillations at fertilization. Mature oocytes were preincubated with thapsigargin or ryanodine during loading with Indo-1. Preincubation with 10 µM thapsigargin blocked continuous sperm-induced oscillations in three of seven oocytes (Fig. 6, a and b). 1 mM ryanodine inhibited oscillations in all oocytes studied (n = 10) (Fig. 6c). A lower concentration of ryanodine (100 µM), which did not increase Ca directly, also inhibited sperm-induced Ca transients (four of five oocytes).


Figure 6: Fertilization-induced Ca changes in mature oocytes treated with thapsigargin or ryanodine. Preincubation of oocytes with 10 µM thapsigargin had a variable effect on Ca transients at fertilization. In some oocytes, the first transient was reduced but oscillations continued (a) while in others, continuous oscillations were inhibited (b). c, preincubation with 1 mM ryanodine inhibited repetitive Ca oscillations in all oocytes studied.



The first four sperm-induced Ca transients in mature oocytes treated with thapsigargin and ryanodine were compared with controls with respect to the rate of rise (Fig. 7a), duration (Fig. 7b), and peak ratio (Fig. 7c). The first transient showed a distinct shoulder (see Fig. 1, b and d); therefore, the rise time before and after this shoulder was calculated. In mature oocytes the duration of the first transient was longer and the peak ratio greater than subsequent transients (Fig. 7, b and c). However, the rise time of the transients became progressively faster (Fig. 7a). In mature oocytes preincubated with ryanodine or thapsigargin the rate of rise and peak ratio were reduced for all transients.


Figure 7: Analysis of the fertilization-induced Ca transients in mature and immature oocytes. The rise time (a), the duration (b), and the peak ratio (c) of the first four transients following fertilization in both mature and immature oocytes in controls or after treatment with 10 µM thapsigargin (Tg) or 1 mM ryanodine (Ry) are shown. Transients are in sequential order: box, first transient; &cjs2106;, second transient; &cjs2110;, third transient, ,fourth transient. The first transient had a distinct shoulder during its rising phase; therefore, the rate of rise is measured before and after this shoulder.



In immature oocytes the rise time, duration, and peak ratio of the sperm-induced Ca transients were found to be less than in mature oocytes (Fig. 7). Thapsigargin preincubation abolished the shoulder of the first transient and greatly shortened the duration of all the oscillations. Only one sperm-induced transient was observed in immature oocytes preincubated with 1 mM ryanodine, and this was of shorter duration and smaller peak ratio. This suggests that ryanodine had an inhibitory effect on the immature oocyte response to sperm without inducing an immediate rise in intracellular Ca. The possibility that immature oocytes have a ryanodine receptor was further examined in spontaneously oscillating GV stage oocytes since ryanodine binding is influenced by the open state of the channel and Ca concentration(17) . 1 mM ryanodine induced an immediate Ca rise superimposed on the spontaneous oscillations in 71% (n = 7) of oocytes studied (Fig. 8).


Figure 8: Ryanodine induces a Ca rise in GV stage oocytes undergoing spontaneous oscillations. 1 mM ryanodine (solidbar) added to GV stage oocytes undergoing spontaneous oscillations induced a rise in Ca. Spontaneous oscillations occurred superimposed on the ryanodine-induced Ca transient.



The Effect of Strain Differences in the Response of Mature Oocytes to Ryanodine

In all studies described so far oocytes from an F1 hybrid mouse have been used. However, Kline and Kline(19) , using mature oocytes from an outbred CF1 strain, reported no Ca rise in response to ryanodine in contrast to the F1 hybrid here (Fig. 5). We examined the possibility that oocytes may have a ryanodine receptor, but its presence cannot be successfully assayed by ryanodine addition. In oocytes from an outbred MF1 strain ryanodine added for up to 50 min did not raise Ca (Fig. 9a) (n = 5) in agreement with Kline and Kline(19) . Fertilization of MF1 oocytes produced a typical series of Ca oscillations (Fig. 9b) that were inhibited by preincubation with 1 mM ryanodine (Fig. 9c). Therefore, oocytes from the MF1 strain, although not responding with a Ca rise to ryanodine addition, do possess a ryanodine-sensitive Ca release channel that is involved at fertilization.


Figure 9: The effect of ryanodine on Ca levels in MF1 outbred mice. a, 1 mM ryanodine did not raise Ca levels when added to mature MF1 oocytes; b, normal sperm-induced Ca transients at fertilization in MF1 oocytes; c, 1 mM ryanodine preincubation during dye loading blocked the normal sperm-induced Ca transients.




DISCUSSION

We have investigated the ability of oocytes to generate Ca oscillations in response to sperm at different times during oocyte maturation. In agreement with previous studies(1, 2, 3) , we have found that fertilization of mature mouse oocytes triggered a series of 20-40 Ca transients continuing for 2-3 h. In immature oocytes the response to sperm was attenuated, with the generation of only 2-3 transients that ceased within 1 h. The ability to generate multiple Ca transients occurred late during oocyte maturation, after first polar body formation. This transition was associated with an increase in the amount of Ca released in response to ionomycin and thapsigargin in addition to an increase in the sensitivity to ryanodine. These results show that modifications in Ca homeostasis are an integral part of oocyte maturation.

The Development of the Mature Oocyte Ca Response to Sperm Late in Maturation

Ca responses typical of mature oocytes occurred late in oocyte maturation at about 12 h after meiotic resumption. Prior to this time only two to three Ca oscillations were generated by sperm. Ca responses similar to those of mature oocytes were also seen in metaphase I-arrested oocytes 14 h after meiotic resumption. This suggests that the ability to generate repetitive Ca transients to sperm is dependent on cytoplasmic changes that occur about 12 h after meiotic resumption and is independent of progression from metaphase I to metaphase II. These findings are supported by recent observations showing metaphase I-arrested oocytes at 14 h undergo normal fertilization but not metaphase I oocytes at 6-8 h, as assessed by second polar body extrusion, pronucleus formation, and cleavage(39) . Metaphase I-arrested oocytes have a similar protein synthesis profile to oocytes at metaphase II showing some aspects of cytoplasmic maturation occur independently of cell cycle progression (39) . Our results suggest that one important aspect is the ability to generate repetitive Ca transients in response to sperm.

An increase in intracellular Ca at fertilization is known to stimulate cell cycle re-entry and cortical granule exocytosis. The attenuated Ca response of oocytes 10-12 h after meiotic resumption may explain the low rate of oocyte activation induced by sperm and parthenogenetic stimuli in these oocytes(40) . Similarly the ability to undergo global cortical granule release does not occur until late in maturation between metaphase I and metaphase II(41) , suggesting that this response may require the maturation of intracellular Ca stores. Together these data suggest that the changes in Ca homeostasis during oocyte maturation are an important mechanism for normal fertilization to occur. Further, the late onset of these changes would prevent premature oocyte activation and cortical granule exocytosis.

Fertilization-induced Ca Oscillations in Mature and Immature Oocytes

Ca release in response to fertilization was characterized by an initial long lasting Ca transient followed by a series of Ca oscillations that were shorter in duration and smaller in magnitude. Preceding each Ca transient was a small gradual elevation in intracellular Ca, which acted as a trigger for the transient. This pacemaker Ca, which has been observed in human oocytes undergoing fertilization(42) , may be due to a decrease in the Ca buffering capacity of the cytoplasm as the Ca stores are replenished(9) .

A common feature of the first transient was a shoulder during its rising phase suggesting there are two Ca release mechanisms or stores, triggered at differing levels of cytoplasmic Ca. This shoulder has been observed using the Ca-sensitive protein aequorin in both mouse and human oocytes at fertilization (2, 42) but not previously using conventional dyes. The subsequent transients had a faster rate of rise and did not show a distinct shoulder during the rising phase, suggesting that Ca release from the second store had undergone sensitization. This is consistent with previous findings that demonstrate that fertilization leads to an increased sensitivity of CICR induced by Ca injection(43) . In immature oocytes, similar to mature oocytes, the first transient was larger and longer lasting than subsequent oscillations. However, oscillations did not continue and had a smaller peak ratio, a shorter duration, and a slower rate of rise compared with mature oocytes. The possible reasons for this attenuated response are discussed in the following sections.

Ca Stores Increase during Oocyte Maturation

The amount of Ca released by ionomycin increased during oocyte maturation in agreement with the results of Tombes et al.(28) . In that study Ca stores increased late in maturation so that a 75% maximal response was seen 12 h after meiotic resumption, similar to when repetitive Ca oscillations to sperm develop. An increase in Ca stores may be associated with an increased Ca-sequestering capacity, and this was examined using thapsigargin. Our data showed an 11-fold increase in thapsigargin-induced Ca release during maturation, suggesting that an increased number or activity of the thapsigargin-sensitive Ca pumps contributes to the increased Ca stores during maturation. The decreased ability of immature oocytes to sequester Ca may explain the rapid run down in oscillations seen at fertilization. After Ca oscillations had ceased in immature oocytes, ionomycin did not elicit further release, suggesting that Ca stores had depleted. This run down in stores occurs in response to fertilization but not InsP(3) or thimerosal(25) .

The increased thapsigargin-sensitive Ca store appears to be important for fertilization since preincubation with thapsigargin decreased the rate of rise, the duration, and the number of oscillations generated by sperm. The ability of thapsigargin to inhibit repetitive Ca oscillations was correlated with its effect on the first Ca transient, which was normal in thapsigargin-treated oocytes that continued to oscillate but was not present in oocytes where oscillations ceased (Fig. 6). A similar observation that thapsigargin is able to block continuous oscillations in only some oocytes has previously been made (44) and suggested to be due to the time between thapsigargin addition and fertilization, which in the present study was kept constant. We therefore suggest that sensitivity to thapsigargin varies among oocytes.

The sensitivity of InsP(3)-induced Ca release is directly correlated with an increase in Ca stores (45) . Therefore, the observed increase in Ca stores seen during oocyte maturation may explain the increased sensitivity to InsP(3) during the maturation of hamster oocytes(27) . Ca store size is also known to regulate the sensitivity of Ca release from ryanodine- and caffeine-sensitive Ca stores(46, 47) . While there is no evidence for caffeine-sensitive stores in mouse oocytes, ryanodine-sensitive release mechanisms have been reported(18) .

Sensitivity to Ryanodine Increases during Oocyte Maturation

In mature (but not in immature) oocytes 1 mM ryanodine consistently increased intracellular Ca, suggesting a maturation-associated modification in the ryanodine-sensitive Ca release mechanism. A similar conclusion has been made in the sea urchin where there is an increase in protein recognized by a ryanodine receptor antibody during oocyte maturation(48) . There does appear to be a ryanodine-sensitive Ca release mechanism in immature oocytes because ryanodine raised Ca in GV stage oocytes that were undergoing InsP(3)-dependent spontaneous oscillations. The relationship between ryanodine and its receptor is complex, depending on many factors(17, 47, 49, 50) , and we suggest that the ryanodine receptor is present in immature oocytes but that it is regulated differently.

Further support for a ryanodine-sensitive Ca release mechanism in both mature and immature oocytes is provided by the observation that ryanodine blocks sperm-induced Ca oscillations. This is independent of the ability of ryanodine to induce an immediate increase in Ca. The addition of 100 µM ryanodine to mature oocytes and 1 mM ryanodine to immature oocytes has no immediate effect on Ca but inhibits sperm-induced oscillations. The presence of a ryanodine receptor in oocytes is controversial since Kline and Kline (19) report no Ca increases following ryanodine addition, in contrast to the present findings and those of Swann(18) . Results presented here may explain the discrepancy between these studies and those of Kline and Kline (19) because we have shown that the ability of ryanodine to cause an immediate Ca rise is strain-dependent. Ryanodine stimulates an immediate Ca rise in oocytes from an F1 hybrid as used here and by Swann (18) and not in oocytes from an outbred MF1 strain, similar to the outbred CF1 strain used by Kline and Kline(19) . However, in both strains ryanodine inhibited sperm-induced Ca oscillations. Therefore, results presented here with ryanodine and those of Miyazaki et al.(14, 51) suggest that both ryanodine and InsP(3) receptors are essential for the generation of Ca oscillations at fertilization in the mouse.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-181-725-2824; Fax: 44-181-767-9109.

(^1)
The abbreviations used are: InsP(3), inositol trisphosphate; CICR, Ca-induced Ca release; GV, germinal vesicle; GVBD, GV breakdown; BSA, bovine serum albumin.


ACKNOWLEDGEMENTS

We thank Dr. Karl Swann for critical reading of this manuscript.


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