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Biology of Reproduction 62, 873-878 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

A Specific Inhibitor of p34cdc2/Cyclin B Suppresses Fertilization-Induced Calcium Oscillations in Mouse Eggs1

Man-Qi Deng2,a, and Sheldon S. Shena

a Department of Zoology & Genetics, Iowa State University, Ames, Iowa 50011-3223


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fertilization-induced Ca2+ oscillations in mouse eggs cease at the time of pronuclear formation when maturation-promoting factor (MPF) is inactivated, but the Ca2+ oscillations are ceaseless if eggs are arrested at metaphase by colcemid, which maintains the activity of MPF. To determine the possible role of MPF in regulation of cytoplasmic Ca2+ excitability, roscovitine, a specific inhibitor of p34cdc2/cyclin B kinase, was used to inactivate MPF, and its effect on fertilization-induced Ca2+ oscillations was investigated. Our results showed that roscovitine at >= 50 µM suppressed fertilization-induced Ca2+ oscillations in normal and colcemid-treated metaphase II (MII) eggs after the first 1–2 Ca2+ spikes. Roscovitine inhibition of fertilization-induced Ca2+ oscillations could be reversed by extensive washing of the eggs. Histone H1 kinase activity in colcemid-treated MII eggs was similarly inhibited by roscovitine, which suggested that the cessation of fertilization-induced Ca2+ oscillations is due to the inactivation of MPF. Thimerosal-induced Ca2+ oscillations in Ca2+-, Mg2+-free medium was also suppressed by roscovitine, suggesting a general inhibitory effect of roscovitine on Ca2+ oscillations. The inhibition may be achieved by disruption of Ca2+ release and refilling of the calcium store. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, induced significantly less Ca2+ release in roscovitine-treated eggs than in the non-drug-treated eggs. Taken together, our results suggest that MPF plays an important role in regulation of the cytoplasmic Ca2+ excitability in mouse eggs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that mammalian eggs produce repetitive calcium oscillations at fertilization, which play an important role in egg activation and early embryonic development [1–5]. Fertilization-induced Ca2+ oscillations last for several hours and cease at the time of pronuclear formation, but they are ceaseless if eggs are arrested at metaphase by colcemid [6]. Therefore, it has been suggested that fertilization-induced Ca2+ oscillations are cell cycle dependent [6]. But at present little is known about the mechanisms controlling the persistence or cessation of fertilization-induced Ca2+ oscillations in the fertilized eggs.

At least two factors appear to trigger and maintain these calcium oscillations. Sperm trigger the oscillations, but they are maintained by the metaphase-like cytoplasm of the eggs. The metaphase state is established by the kinase activity of maturation-promoting factor (MPF) and is characterized by nuclear envelope breakdown, chromatin condensation, and the formation of a mitotic spindle [7]. MPF is a complex of p34cdc2 and cyclin B [8–11] and displays cyclic histone H1 kinase activity peaking at metaphase [11]. Metaphase ends when several proteins, including cyclin B, are targeted via ubiquitination for proteolytic destruction by the anaphase-promoting complex [12–14]. It is interesting to note that the ceaselessness of fertilization-induced Ca2+ oscillations in metaphase II (MII) eggs treated with the microtubule inhibitor (colcemid or nocodazole) corresponds with the maintenance of the MPF activity [15–17]. The temporal association between Ca2+ excitability and MPF activity raises the possibility that the inactivation of MPF may contribute to the cessation of the fertilization-induced Ca2+ oscillations in fertilized eggs. To address this question, we used roscovitine, a potent specific inhibitor of p34cdc2/cyclin B kinase [18, 19], to inactivate MPF and investigate its effect on fertilization-induced Ca2+ oscillations. We found that fertilization-induced ceaseless Ca2+ oscillations in colcemid-treated eggs were suppressed by inactivation of MPF. The inhibition mechanisms of roscovitine were investigated, and its physiological implications are discussed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Roscovitine (BIOMOL, Plymouth Meeting, PA) was dissolved in dimethyl sulfoxide (DMSO) and stored in 5 mM stock solution at -20°C. Roscovitine was freshly prepared using M2 (94.66 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl2·2H2O, 1.19 mM KH2PO4, 1.19 mM MgSO4·7H2O, 4.15 mM NaHCO3, 20.85 mM Hepes, 23.28 mM sodium lactate, 0.33 mM sodium pyruvate, 5.56 mM glucose, 4 mg/ml BSA, pH 7.4 [20]) or in vitro fertilization (IVF) medium [20] prior to its use. The final concentration of DMSO in medium was no more than 2%. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise.

Oocyte Collection and IVF

Ovulated MII-arrested eggs were collected from 4- to 5-wk-old C57BL/6J female mice, which were superovulated by i.p. injection of 5 IU eCG and 10 IU hCG 48 h apart. Cumulus masses were collected from the oviducts 15–16 h after hCG injection in M2 [20]. Cumulus cells surrounding oocytes were removed by a brief incubation in 0.5 mg/ml hyaluronidase.

Sperm were collected from 7- to 8-wk-old C57BL/6J male mice and capacitated in a 5% CO2 incubator for 2 h in IVF medium containing 30 mg/ml BSA (fraction V; Sigma) [20]. For IVF, zona pellucida of eggs was removed with acidic Tyrode's solution (pH 2.3 [20]) before insemination. Zona-free eggs were used for IVF because of the advantage of their synchronization in fertilization and convenience in observation of the fertilization-induced Ca2+ oscillations. Colcemid at 1 µg/ml was included in IVF medium to arrest the cell cycle at metaphase and to maintain the exhibition of ceaseless fertilization-induced Ca2+ oscillations in eggs [6].

Histone H1 Kinase Assay

MII eggs were cultured in M2 containing 0, 20, and 100 µM roscovitine for 40 min. Three eggs from each treatment were transferred by micropipette to 5 µl sample buffer solution (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 mM p-nitrophenyl phosphate, 20 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 5 mM EGTA) in Eppendorf (Hamburg, Germany) tubes at 4°C and stored at -80°C. Histone H1 kinase activity was determined as described by Moos et al. [16].

Ca2+ Measurement

Prior to Ca2+ measurement, eggs were loaded by incubation in 5 µM fura-2/AM (Molecular Probes, Eugene, OR) in M2 at 37°C for 30–40 min and transferred to a thin coverslip chamber. To prevent the possible movement of eggs by sperm during Ca2+ measurement, zona-free eggs were transferred to IVF medium without BSA and allowed to sediment and attach tightly to the coverslip. For intracellular Ca2+ measurement, fura-2-loaded eggs in the chamber were observed with an epifluorescence Olympus (Tokyo, Japan) IMT-2 microscope adapted to an ImageMaster Ratio Fluorescence Imaging System (Photon Technology International, Monmouth Junction, NJ) equipped with a CCD video camera (IC-200). Fura-2 fluorescence was imaged, and the concentration of intracellular Ca2+ was determined by calculating the ratio of fluorescence at 510 nm, excited by UV light alternately at 340 nm and 380 nm from a xenon arc lamp. The Ca2+ images of a field covering eggs were recorded every 2–10 sec. Ca2+ changes in individual eggs were analyzed by ImageMaster software (version 1.48). Eggs in the chamber were maintained at 37°C using a heat stage during Ca2+ measurement. To prevent pH change during the Ca2+ measurement, Hepes (20.85 mM) was included in the IVF medium and equilibrated in a 5% CO2 incubator for several hours. Ca2+ measurement was conducted at room air condition for 2–3 h.

Statistical Analysis

Each group of experiments was repeated at least 3 times. Data are presented as mean ± SEM. Multiple comparisons of the number of Ca2+ spikes between treatments with different dosages of roscovitine were analyzed using Scheffe's test (Statistical Analysis System, General Linear Models Procedure; Cary, NC). Comparison of thapsigargin-induced Ca2+ release between control and roscovitine treatment was conducted using Student's t-test. Differences of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Roscovitine Suppressed Fertilization-Induced Ca2+ Oscillations in a Dose-Dependent Manner

We tried different doses (0, 20, 50, 100, 200 µM) and different application methods for roscovitine treatment of mouse eggs, based on its reported inhibition of p34cdc2/cyclin B and the mammalian cell cycle [18, 19, 21–23]. For one set of experiments, eggs were cultured in M2 containing roscovitine for at least 40 min and inseminated in IVF medium with inclusion of roscovitine. For another set of experiments, roscovitine was added to the IVF medium after fertilization-induced Ca2+ oscillations were observed. The purpose of latter experiments was to determine the effect of roscovitine on the ongoing Ca2+ oscillations. The fertilization-induced Ca2+ spikes were counted and compared between different doses before and after roscovitine addition. In the first set of experiments, the number of fertilization-induced Ca2+ spikes was significantly reduced when the concentration of roscovitine was >= 50 µM (Table 1). Roscovitine had no effect on timing, occurrence, and amplitude of the first 1–2 Ca2+ spikes, but it abolished the subsequent Ca2+ oscillations (Fig. 1). Increasing roscovitine to 200 µM caused eggs to become distorted and fragmented, but intact eggs still produced the first 1–2 Ca2+ spikes after fertilization (data not shown). Even at high concentrations, it does not appear that roscovitine prevents sperm penetration and fusion to eggs.


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  		TABLE 1. Effect of roscovitine on fertilization-induced Ca2+ oscillations



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  		FIG. 1. Fertilization-induced Ca2+ responses in MII eggs at various concentrations of roscovitine. MII eggs were pretreated with various doses of roscovitine (0*, 20, 50, 100 µM) for 1 h and inseminated in IVF medium containing (A) 0* µM, (B) 20 µM, (C) 50 µM, and (D) 100 µM roscovitine, respectively. The arrows indicate the time of sperm addition. This result was typical of 4 observations, each with > 10 eggs in the chamber. *2% DMSO was included in the IVF medium

In the second set of experiments, roscovitine was added after fertilization-induced Ca2+ oscillations were observed. Fertilization-induced Ca2+ oscillations ceased abruptly after addition of >= 50 µM roscovitine even in the colcemid-treated eggs, suggesting again that roscovitine suppresses the regenerative Ca2+ oscillations. Similar to roscovitine pretreatment of eggs, the addition of 20 µM roscovitine had no effect on the ongoing fertilization-induced Ca2+ oscillations. However, the addition of 50 or 100 µM (Fig. 2A) roscovitine caused a nearly immediate cessation of ongoing fertilization-induced Ca2+ oscillations. Subsequent addition of thimerosal, a universal trigger for Ca2+ oscillations [24, 25], could induce new series of Ca2+ oscillations in eggs previously inhibited with >= 50 µM roscovitine (Fig. 2B), suggesting that these eggs were still capable of Ca2+ oscillations. These observations were based on at least 3 repetitive experiments.



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  		FIG. 2. Effect of roscovitine on fertilization-induced ceaseless Ca2+ oscillations in colcemid-treated eggs. Addition of roscovitine (100 µM) blocked the ongoing Ca2+ oscillations in the fertilized egg (A). After cessation of the fertilization-induced Ca2+ oscillations for 1.5 h, addition of thimerosal (200 µM) induced additional Ca2+ oscillations (B). Colcemid (1 µg/ml) was included in the medium during the described manipulation and Ca2+ measurement. The result was typical of 3 observations, each with > 10 eggs in the chamber

Inhibition of Roscovitine on Fertilization-Induced Ca2+ Oscillations Was Reversible

To be certain that the inhibition of roscovitine on fertilization-induced Ca2+ oscillations was not a cytotoxic effect, we examined the reversibility of inhibition by removing the eggs from roscovitine and washing them extensively. After the suppression of fertilization-induced Ca2+ oscillations by 100 µM roscovitine (Fig. 3A), the roscovitine-treated eggs were removed from roscovitine and washed extensively with M2. The washed eggs resumed Ca2+ oscillations in IVF medium without roscovitine (Fig. 3B), suggesting that the inhibitory effect of roscovitine on the Ca2+ oscillations is reversible.



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  		FIG. 3. Reversibility of roscovitine-induced cessation of Ca2+ oscillations in fertilized eggs. Fertilization-induced Ca2+ oscillations in colcemid-treated eggs were suppressed by addition of 100 µM roscovitine (A). After extensive washing and transferral to roscovitine-free IVF medium with inclusion of colcemid, the egg resumed Ca2+ oscillations (B). This result was typical of 3 observations, each with > 10 eggs in the chamber

Roscovitine Inactivated Histone H1 Kinase Activity in Colcemid-Treated Eggs

To be sure that roscovitine used in suppressing Ca2+ oscillations in our experiment is able to inactivate MPF in intact mouse eggs as was observed in an in vitro system [18, 19, 21], we cultured mouse MII eggs in 0, 20, and 100 µM roscovitine for 40 min and measured the histone H1 kinase activity. Our results showed that 100 µM roscovitine, which suppressed the fertilization-induced Ca2+ oscillations, strongly inhibited the activity of histone H1 kinase in colcemid-treated eggs; in contrast, 20 µM roscovitine could not induce the inactivation of histone H1 kinase (Fig. 4). This clearly showed that the concentration of roscovitine used to suppress fertilization-induced Ca2+ oscillations corresponded to that required to inactivate MPF in intact mouse eggs. This suggests that the cessation of fertilization-induced Ca2+ oscillations in colcemid-treated MII eggs is due to the inactivation of MPF.



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  		FIG. 4. Effect of roscovitine on histone H1 kinase activity in mouse eggs. Lane 1 shows the histone H1 kinase activity of untreated MII eggs. Lanes 2 and lane 3 show the activity of histone H1 kinase after culture in M2 (with inclusion of colcemid) containing 20 µM and 100 µM roscovitine for 40 min, respectively. Each band represents the result of 3 eggs

Effect of Roscovitine on Ca2+ Release from Intracellular Calcium Stores

Since roscovitine did not suppress the first 1–2 Ca2+ spikes but only the subsequent Ca2+ oscillations, we speculated that roscovitine might affect the Ca2+ release and refilling of calcium stores. To test this possibility, we examined the possible alteration of Ca2+ release system by roscovitine treatment.

Effect of roscovitine on thimerosal-induced Ca2+ oscillations Since thimerosal can induce Ca2+ oscillations both in Ca2+-containing and in Ca2+-free medium [24–26], we tested the effect of 100 µM roscovitine on thimerosal-induced Ca2+ oscillations in Ca2+-, Mg2+-free medium. Our results showed that thimerosal-induced Ca2+ oscillations ceased quickly in Ca2+-, Mg2+-free M2 after addition of roscovitine (Fig. 5). This suggests that roscovitine affected Ca2+ release and the refilling of internal stores.



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  		FIG. 5. Effect of roscovitine on thimerosal-induced Ca2+ oscillations in unfertilized MII eggs in Ca2+-, Mg2+-free medium. Addition of roscovitine (100 µM) suppressed the thimerosal-induced Ca2+ oscillations in Ca2+-, Mg2+-free M2 with inclusion of 1 mM EGTA. The concentration of thimerosal used was 100 µM. This result was typical of 3 observations, each with > 10 eggs in the chamber

Thapsigargin-induced Ca2+ release in roscovitine-treated eggs To determine whether the suppression of fertilization-induced Ca2+ oscillations by roscovitine was due to the exhaustion of calcium stores, we induced Ca2+ release from intracellular stores by using thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, to deplete calcium stores. The thapsigargin-induced Ca2+ response in Ca2+-, Mg2+-free medium was used as an estimate of the size of intracellular Ca2+ stores [27]. Our results showed that thapsigargin (50 µM) induced much less Ca2+ release in the roscovitine-treated eggs in which fertilization-induced Ca2+ oscillations in colcemid were suppressed than in the fertilized colcemid-treated eggs (control). The peak ratio (340/380 nm) and the duration time of the Ca2+ increase were significantly less in roscovitine-treated eggs than in the control eggs (P < 0.01, Table 2). In these roscovitine-treated eggs, thapsigargin caused no (n = 18) or a minor (n = 5) Ca2+ increase (Fig. 6). The peak ratio and the duration of Ca2+ increase in the 5 roscovitine-treated eggs (with peak ratio of 0.95 ± 0.06 and duration of 142.80 ± 10.58 sec) were also significantly less than in the control eggs (P < 0.05). This suggests that the Ca2+ stores in the roscovitine-treated eggs were exhausted after the first 1–2 spikes after fertilization.


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  		TABLE 2. Comparison of Ca2+ release induced by thapsigargin between roscovitine-treated eggs and MII eggs (mean ± SEM).a



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  		FIG. 6. Comparison of Ca2+ release from intracellular stores between roscovitine-treated and untreated eggs. Thapsigargin (50 µM) induced significantly less (A, n = 5) or no (B, n = 18) Ca2+ release in those eggs in which fertilization-induced Ca2+ oscillations were suppressed by 100 µM roscovitine in comparison with fertilized eggs without roscovitine treatment (C, n = 25). The arrows indicate the time of thapsigargin addition


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Inactivation of MPF Suppresses the Fertilization-Induced Ceaseless Ca2+ Oscillations in Colcemid-Treated MII Eggs

It is known that the microtubule inhibitors, colcemid and nocodazole, maintain MPF activity by preventing the degradation of cyclin B in mouse eggs [15], which normally occurs after fertilization [16, 17]. Therefore we speculated that the persistence of MPF activity in colcemid-treated eggs might be responsible for the maintenance of long-lasting fertilization-induced Ca2+ oscillations. Recent research in the ascidian shows that fluctuation of MPF activity correlates temporally with the exhibition and cessation of fertilization-induced Ca2+ oscillations in MI oocytes [28], suggesting a possible role of MPF in regulation of the Ca2+ excitability in eggs. But there is no direct evidence that artificial inactivation of MPF can result in the cessation of fertilization-induced Ca2+ oscillations in fertilized eggs. In this paper we offer the first evidence that inactivation of MPF by roscovitine, a specific inhibitor of p34cdc2/cyclin B kinase, suppresses the fertilization-induced Ca2+ oscillations in mouse eggs.

Roscovitine and its analogue, olomoucine, are paurine derivatives, and they inhibit specifically the activity of p34cdc2/cyclin B kinase both in vitro and in vivo [18, 19, 21–23], with roscovitine 10 times more potent than olomoucine [18, 19]. They inhibit the activity of p34cdc2/cyclin B by preventing its binding to ATP but without affecting its activation [18, 19]. The inhibitory effect of roscovitine on fertilization-induced Ca2+ oscillations showed a sharp increase between 20 to 50 µM. This feature is similar to the sharp dose-dependent inhibition curve of roscovitine on p34cdc2/cyclin B and cell cycle progression in invertebrate embryos and human cell lines [18, 21, 22]. The necessary concentration of roscovitine used for suppressing fertilization-induced Ca2+ oscillations in mouse eggs (~50 µM) was about 5 times higher than that used for arresting cell cycles in sea urchin and starfish embryos [18], but it was comparable with that of olomoucine used in activation of pig oocytes [23]. The reversibility of roscovitine inhibition on fertilization-induced Ca2+ oscillations in this study, and on cell cycle arrest during oocyte maturation [18], demonstrates that the inhibitory effect of roscovitine is not cytotoxic. Histone H1 kinase assay in our research clearly shows that roscovitine can effectively inactivate MPF in intact mouse eggs, indicating that the suppression of fertilization-induced Ca2+ oscillations by roscovitine is due to the inactivation of MPF. In addition, the resumption of fertilization-induced Ca2+ oscillations after extensive washing of roscovitine-treated eggs suggests that the inactivation of MPF can be reversed and that the recovery of MPF activity could rescue the fertilization-induced Ca2+ oscillations in roscovitine-treated eggs. Taken together, our research suggests that MPF plays an important role in the regulation of the cytoplasmic Ca2+ excitability.

Possible Mechanisms of Roscovitine-Induced Suppression of Ca2+ Oscillations in Fertilized Eggs

We observed that roscovitine suppression of fertilization-induced Ca2+ oscillations in colcemid-treated eggs occurred after the first 1–2 Ca2+ spikes. This suggests that roscovitine does not affect the initial Ca2+ release from intracellular calcium stores but blocks the subsequent periodic refilling and depleting of internal calcium stores, which sustain the regenerative Ca2+ oscillations in living cells [29, 30]. Suppression of thimerosal-induced Ca2+ oscillations by roscovitine in Ca2+-, Mg2+-free medium also supports the notion. But it should be noted that roscovitine could not suppress the thimerosal-induced Ca2+ oscillations in Ca2+-, Mg2+-containing medium, indicating that different mechanisms are employed in fertilization and thimerosal stimulation [26]. The differences between the two kinds of Ca2+ oscillations can also be seen in their different responses to heparin [24–27]. Furthermore, in contrast to the lack of an observed increase in basal level of Ca2+ during roscovitine suppression of fertilized eggs, an increase in basal level was observed in some eggs during roscovitine suppression of the thimerosal-induced Ca2+ oscillations in Ca2+-, Mg2+-free medium (Fig. 5). This suggests that roscovitine may affect pumping of Ca2+ back into the calcium stores or out of the eggs, thus leaving the basal level high.

Even though we observed significantly less Ca2+ release in roscovitine-treated eggs in response to thapsigargin compared with that in untreated eggs, we cannot preclude the possible simultaneous desensitization of inositol 1,4,5-triphosphate (IP3) receptor at this point. Because IP3-induced Ca2+ release also decreases with reduction of the size of calcium stores, we could not determine whether sensitivity of IP3 receptor is also decreased. It has been reported that the sensitivity of IP3 receptor in mouse eggs is reduced dramatically after fertilization but the size of the calcium store remains unchanged, suggesting that the desensitization of the IP3 receptor may be responsible for the cessation of fertilization-induced Ca2+ oscillations in pronuclear stage zygotes [6, 31]. However, in ascidian oocytes, the cessation of fertilization-induced Ca2+ oscillations does not seem to be caused by desensitization of IP3 receptor, since the Ca2+ signaling system remains equally sensitive during the period in which the fertilization-induced Ca2+ oscillations pause [28]. In mouse and hamster immature oocytes, both spontaneous and fertilization-induced Ca2+ oscillations are observed even though they have lower IP3 sensitivity [32–34]. All these findings indicate that much more redundant and complex mechanisms may be involved in controlling the Ca2+ excitability in eggs.

In pig oocytes, olomoucine at 1 mM inactivates MPF by inhibiting p34cdc2 or cdc25 and induces the parthenogenetic activation and formation of an interphase nucleus but without chromosome segregation [23]. In mouse MII eggs, however, we did not observe either the second polar body extrusion or pronucleus formation after culture in 100 µM roscovitine for over 10 h (data not shown). This is consistent with the previous report in the mouse showing that olomoucine cannot activate but only facilitate the parthenogenetic activation process of MII-arrested eggs [21]. It seems that the inactivation of MPF by p34cdc2/cyclin B inhibitors shows a species difference in the induction of egg activation.

It should be pointed out that there is a difference in the mode of MPF inactivation between fertilization and roscovitine. The former induces the inactivation of MPF by Ca2+-induced degradation of cyclin B [15, 35, 36], but the latter inhibits the activity of cyclin B by preventing its binding to ATP [18]. These differences may account for some of the different cellular effects such as egg activation, change of size of calcium stores, and the sensitivity of IP3 receptor.

Regulation of Cytoplasmic Ca2+ Excitability in Mouse Eggs

It is known that mature ovulated eggs are arrested at the metaphase of second meiotic division due to action of MPF and cytostatic factor [7] unless activated by fertilization, which initiates Ca2+ oscillations. The fertilization-induced Ca2+ increases drive the resumption of the cell cycle by inducing the degradation of cyclin B and hence MPF inactivation [35, 36]. Although the biological significance of the long-lasting Ca2+ oscillations in fertilized eggs is still unclear, complete inactivation of MPF seems a possible explanation because the robust synthesis of new cyclin B in MII eggs may lead to the reactivation of MPF [37, 38]. From the standpoint of cell signaling, fertilization-induced Ca2+ oscillations must be terminated after fulfilling their functions (inactivation of MPF, resumption of cell cycle, etc.), as can be seen in the most common form of negative feedback regulation. It is most likely that the inactivation of MPF and the successful completion of egg activation will generate signal(s) to stop the fertilization-induced Ca2+ oscillations at the proper stage. Otherwise, the fertilization-induced Ca2+ oscillations will become ceaseless as observed in colcemid-treated MII eggs in which the inactivation of MPF is blocked. Although the current research has shed some light on the mechanisms controlling fertilization-induced Ca2+ oscillations in eggs, the detailed signaling pathways leading to the cessation of fertilization-induced Ca2+ oscillations remain elusive.


   FOOTNOTES
 
First decision: 17 August 1999.

1 This study was supported by NSF (IBN96-31982). Back

2 Correspondence: Man-qi Deng, University of Connecticut, Department of Animal Science/Biotech Center, 3636 Horsebarn Road, Extension U-40, Storrs, CT 06269-4040. FAX: 860 486 4375; mdeng{at}ansc1.cag.uconn.edu Back

Accepted: November 9, 1999.

Received: July 22, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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