Cell Biology International (2009) 33, 337-343 - S.K. Chaube and others - Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs

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Cell Biology International (2009) 33, 337–343 (Printed in Great Britain)

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Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs

S.K. Chaube^a^*, Anima Tripathi^a, Sabana Khatun^b, S.K. Mishra^b, P.V. Prasad^b and T.G. Shrivastav^b

^aCell Physiology Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India
^bDepartment of Reproductive Biomedicine, National Institute of Health and Family Welfare, Baba Gang Nath Marg, New Delhi 110067, India

Abstract

Non-specific L-type calcium channel blockers, such as verapamil (≥50μM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca²⁺-deficient medium. However, the effects of extracellular Ca²⁺ on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca²⁺-deficient medium. The initiation of apoptotic features was observed at 50μM of verapamil. Extracellular Ca²⁺ (1.80mM) reduced intracellular H2O2 level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50μM, but not higher concentrations of ≥100μM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca²⁺ ions have a role during SEA and protect against verapamil-induced apoptosis in aged rat eggs.

Keywords: Apoptosis, Calcium, Caspase-3 activation, DNA fragmentation, Hydrogen peroxide, Metaphase-II arrest, Verapamil.

^*Corresponding author. Tel.: +91 542 2307149; fax: +91 542 2368174.

1 Introduction

Intracellular calcium homeostasis is very important in maintaining the normal functions of a cell (Whitaker and Patel, 1990). The transition from one meiotic phase to the next is regulated by cell cycle control checkpoints, which are in turn modulated by a transient increase of intracellular calcium ion [Ca²⁺]i (Tosti, 2006; Boni et al., 2007). A transient increase of [Ca²⁺]i triggers several events that activate eggs, including cortical granule exocytosis, pronuclear formation, exit from metaphase-II (M-II) arrest and extrusion of a second polar body at the time of fertilization (Kline and Kline, 1992; Xu et al., 1997; Raz et al., 1998; Whitaker, 2006). In the absence of fertilization, postovulatory aging mimics the action of fertilizing spermatozoa, increases [Ca²⁺]i (Vincent et al., 1992; Xu et al., 1997; Raz et al., 1998) and induces spontaneous egg activation (SEA) only in a few mammalian species such as mouse (Xu et al., 1997; Vincent et al., 1992), hamster (Austin, 1956), and rat (Zernika-Goetz, 1991; Raz et al., 1998; Ross et al., 2006; Galat et al., 2007; Chaube et al., 2007; Tamura et al., 2008). The SEA limits somatic cell nuclear transfer during cloning in rat (Ross et al., 2006).

Changes in intracellular calcium [Ca²⁺]i level modulate various cell functions such as meiotic cell cycle, apoptosis and/or cell death (Homa et al., 1993; Homa, 1995; McConkey and Orrenius, 1997; Tosti, 2006; Whitaker, 2006). For instance, a transient increase of [Ca²⁺]i induces egg activation (Vincent et al., 1992), while high sustained Ca²⁺ level leads to apoptosis (McConkey and Orrenius, 1997; Berridge et al., 1998; Gordo et al., 2002). In contrast, abnormally high [Ca²⁺]i level results in cell death (Gordo et al., 2000). The calcium rise in an egg occurs by means of two principal mechanisms: the efflux from the stores via ligand-gated channels or organelle membrane and entry through ion channels in the plasma membrane (Tosti, 2006).

A significant change in L-type calcium channel activity from diplotene arrest to M-II stage has been identified in mammalian oocytes (Tosti et al., 2000; Tosti, 2006). The L-type calcium channels have a role in resumption of meiosis (Tosti, 2006) since verapamil, a known non-specific L-type calcium channel blocker, inhibits calcium current activity (Tosti et al., 2000), and the resumption of meiosis in rat, pig and bovine eggs cultured in vitro (Paleos and Powers, 1981; Bae and Channing, 1985; Kaufman and Homa, 1993; Tosti et al., 2000). Recently, we reported that verapamil (≥50μM) inhibits resumption of meiosis and induces apoptosis in aged rat eggs cultured in Ca²⁺-free medium (Chaube et al., 2007). Furthermore, the calcium ionophore A23187, which is known to increase [Ca²⁺]i generates hydrogen peroxide (H2O2) and thereby induces apoptosis in aged rat eggs cultured in vitro (Chaube et al., 2008). It is possible that the release of Ca²⁺, mainly from endoplasmic reticulum (Boni et al., 2007), and inhibition of calcium channels by verapamil may induce the accumulation of [Ca²⁺]i in aged eggs (Rozinek et al., 2003), which may generate intracellular hydrogen peroxide (H2O2). This in turn may inhibit the meiotic cell cycle and induce apoptosis in aged rat eggs cultured in vitro. Hence, the objectives of the present study examined whether the generation of intracellular H2O2, changes in bax/bcl2 expression and caspase-3 activation are associated with verapamil-induced egg apoptosis in Ca²⁺-deficient medium. If so, we tested whether extracellular Ca²⁺ protects against verapamil-induced initiation of apoptosis in aged eggs of rat cultured in vitro.

2 Materials and methods

2.1 Chemicals

Unless otherwise stated, all reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

2.2 Preparation of verapamil working concentrations

The culture media used in the present study were either serum-free and Ca²⁺-free medium-199 or serum-free and Ca²⁺-free medium-199 containing 1.80mM Ca²⁺ (HiMedia Laboratories, Mumbai, India). This concentration of calcium ion is generally used to prepare medium-199 (Ca²⁺-supplemented medium) for the culture of various cell types. Working concentrations of verapamil (25, 50, 100, 200 and 300μM) were prepared separately in Ca²⁺-deficient or Ca²⁺-supplemented medium from its stock solution (10mg/ml) and kept at 37°C for 5min before use. Addition of verapamil at its final concentration did not alter the osmolarity (295±5mOsmol) or pH (7.2±0.1) of the culture medium used in the present study. Since absolute alcohol was used as a solvent in the verapamil stock solution, an equivalent dilution of the highest concentration (0.01% absolute alcohol) was used in the control group.

2.3 Rats and the collection of their eggs

Holtzman rats were housed in air-conditioned, light controlled room, with food and water, ad libitum. Twenty-three to 25-day-old female rats were primed with a single subcutaneous injection of (20IU) pregnant mare serum gonadotropin (PMSG) followed 48h later by (20IU) hCG. Eggs were collected 17h after hCG injection. Ovulated cumulus-enclosed eggs were isolated in Ca²⁺-deficient medium under a dissecting microscope (Carl Zeiss, Oberkochen, Germany) and denuded using 0.01% hyaluronidase at 37°C. Denuded eggs were washed 3 times with Ca²⁺-deficient medium. The eggs used in this study were collected 17h post-hCG treatment to obtain aged eggs susceptible for partial SEA (Ross et al., 2006). All procedures conformed to the regulations of the University Animal Ethical Committee of Banaras Hindu University, Varanasi and were in keeping with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication).

2.4 Effect of verapamil on morphological changes in apoptosis

Fifteen to 20 eggs were cultured in a 35mm petri dish containing 2ml of either Ca²⁺-deficient medium, or Ca²⁺-supplemented medium, or either one of these media containing various concentrations (25, 50, 100, 200 and 300μM) of verapamil. Petri dishes were maintained at 37°C for 3h. At the end of incubation period, oocytes were removed, washed 3 times with Ca²⁺-deficient medium, transferred onto a grooved slide with 100μL of medium and examined for morphological changes characteristic of SEA, apoptosis and degeneration using a phase-contrast microscope (Nikon, Eclipse; E600, Japan) at 400×magnification.

Since extracellular calcium protected against 50μM verapamil-induced initiation of morphological apoptotic features such as shrinkage, membrane blebbing and cytoplasmic granulation, 50μM verapamil was selected for further biochemical analysis.

2.5 Quantitative estimation of intracellular H2O2 level

The intracellular H2O2 level was measured using an H2O2 assay kit purchased from R&D Systems, Inc. (MN, USA). Seventy-five eggs collected from 2 animals after superovulation induction were randomly divided into 3 groups of 25. The first was cultured in Ca²⁺-deficient medium (control group). The second and third were cultured separately in Ca²⁺-deficient or Ca²⁺-supplemented media for 3h with both media containing 50μM verapamil. Thereafter, eggs were washed 3 times with 10mM phosphate buffer saline (PBS) and lysed in 100μL hypotonic lysis buffer (5mM Tris, 20mM ethylenediamine tetraacitic acid (EDTA), 0.5% Triton X-100; pH 6.0). After 1h of incubation on ice, lysates were centrifuged at 10,000rpm at 4°C for 15min and the clear supernatant was immediately stored at −30°C until use. Hydrogen peroxide concentrations of the egg lysates were measured by calorimetric assay as described in our previous publication (Chaube et al., 2008). All samples were run in triplicate in one assay to avoid inter-assay variations. Intra-assay variation was &007E;3%.

2.6 Detection of bax and bcl2 proteins by Western blotting

The expression of pro-apoptotic and anti-apoptotic proteins such as bax and bcl2 in egg lysate was analyzed by Western blotting. Approximately 600 eggs were collected from 10 to 12 animals after the induction of superovulation. These cumulus-enclosed eggs were denuded using 0.01% hyaluronidase and then washed twice with Ca²⁺-deficient medium. The denuded eggs were divided into 3 groups of 200. The first was cultured in Ca²⁺-deficient medium (control group). The second and third were cultured separately in Ca²⁺-deficient or Ca²⁺-supplemented media for 3h with both media containing 50μM verapamil. Thereafter, eggs were washed 3 times with 10mM PBS and transferred into 0.6ml microcentrifuge tubes containing 100μL STKM buffer (0.25M sucrose, 50mM Tris–HCl, pH 7.5, 25mM KCl, 5mM MgCl2 and 0.25%Triton X-100) for lysis at 4°C overnight. Bax and bcl2 proteins in the lysates were analyzed using the protocol published earlier (Chaube et al., 2005b). The blots were probed separately with anti-bax and anti-bcl2 rabbit antibodies (1:2500 dilution) (Polyclonal; Santa Cruz Biotechnology Inc., CA, USA). Another set of the same samples was run in parallel and probed with an anti-β-actin rabbit antibody (1:2500 dilutions; Polyclonal; Santa Cruz Biotechnology Inc., CA, USA) to confirm that equivalent amounts of protein were analyzed. The immunodetection of bax and bcl2 proteins were repeated at least 3 times.

2.7 Detection of caspase-3 activity by colorimetric assay

Caspase-3 activity was analyzed using a colorimetric assay kit (R&D Systems, Inc., MN, USA). Seventy-five eggs were divided into 3 groups of 25. The first was cultured in Ca²⁺-deficient medium (control group). The second and third were cultured separately in Ca²⁺-deficient or Ca²⁺-supplemented media for 3h with both media containing 50μM verapamil. Thereafter, the eggs were washed 3 times with PBS and lysed separately in 100μl of chilled hypotonic lysis buffer (5mM Tris, 20mM ethylenediamine tetraacitic acid (EDTA), 0.5% Triton X-100, pH 6.0) at 4°C for 10min. The egg lysates were centrifuged at 10,000g for 1min and 50μl of supernatant was transferred to a fresh tube and frozen at −30°C until assay. The experiment was repeated 3 times and triplicate samples from all the groups were used for the detection of caspase-3 activity by colorimetric assay following the protocol published earlier (Chaube et al., 2005a).

2.8 DNA fragmentation analysis by TUNEL assay

DNA fragmentation was detected using a TUNEL kit (R&D Systems, Inc., MN, USA). Approximately 45 eggs were divided into 3 groups of 15. The first was cultured in Ca²⁺-deficient medium (control group). The second and third were cultured separately in Ca²⁺-deficient or Ca²⁺-supplemented media for 3h with both media containing 50μM verapamil. Thereafter, eggs were washed 3 times with 10mM PBS and then transferred onto slides. These eggs were quickly fixed in 3.7% formaldehyde in PBS for 15min. Slides were washed 3 times with PBS and air dried. All procedures were carried out at 18–20°C unless stated otherwise. The terminal dUTP nick-end labeling (TUNEL) assay was performed as described in our previous publication (Chaube et al., 2006).

2.9 Statistical analysis

Data are expressed as mean±S.E. of mean (SEM) of triplicate samples. All percentage data were subjected to arcsine square-root transformation before statistical analysis. Data were analyzed by either Student's t-test or one-way ANOVA using SPSS software, version 11.5 (SPSS, Inc., Chicago, IL, USA). A probability of P<0.05 was considered significant.

3 Results

3.1 Effect of various concentrations of verapamil on SEA

Eggs collected after 14h post-hCG injection were at M-II stage and exhibited a first polar body without any sign of SEA after 3h of culture (Fig. 1A). Further culture of eggs collected after 17h post-hCG injection when they were at M-II stage and exhibited a first polar body with no sign of SEA at the time of isolation, induced extrusion of a second polar body, a morphological sign of SEA in both Ca²⁺-supplemented and Ca²⁺-deficient media. (Fig. 1B), Nevertheless, eggs collected after 17h post-hCG injection and cultured in Ca²⁺-supplemented medium had significantly (P<0.05) higher rate of partial SEA (92.0±0.72%; as compared to eggs cultured in Ca²⁺-deficient medium (66.8±1.8%), see Table 1) Conversely, verapamil (25, 50, 100, 200 and 300μM) inhibited SEA in a dose-dependent manner in either medium (Fig. 1C) (one-way ANOVA, F=307.5, P<0.001, Ca²⁺-supplemented medium; one-way ANOVA, F=303.6, P<0.001; Ca²⁺-deficient medium). A lower dose of verapamil (25μM) not only induced maintenance of M-II arrest but also egg survival since cytoplasm of treated eggs (Fig. 1C) was morphologically better as compared to respective control eggs that underwent SEA (Fig. 1B).

Fig. 1

Representative photograph showing verapamil-induced inhibition of partial SEA in eggs cultured in vitro for 3h. (1A) Egg at 0h of culture exhibits first polar body with no sign of spontaneous activation (▶). (1B) Eggs showing partial SEA, as extrusion of second polar body is seen after 3h of culture (▶). (1C) Verapamil (25μM) induced inhibition of partial SEA as no second polar body is seen in treated egg (▶).

Table 1.

Effect of various concentrations of verapamil on partial SEA and morphological apoptotic changes in aged rat eggs cultured in Ca²⁺-deficient or Ca²⁺-supplemented medium for 3 h in vitro.

Treatment			Morphological features (in %)
Verapamil	Culture medium	Spontaneous egg activation	Cell shrinkage	Membrane blebbing	Cytoplasmic granulation	Degeneration
Control	(+)	92.0 ± 0.72*	Nil	Nil	Nil	Nil
Control	(−)	66.8 ± 1.84	Nil	Nil	Nil	Nil

25 μM	(+)	88.3 ± 2.49*	Nil	Nil	Nil	Nil
25 μM	(−)	45.3 ± 2.91	Nil	Nil	Nil	Nil

50 μM	(+)	84.7 ± 3.23*	Nil	Nil	Nil	Nil
50 μM	(−)	38.4 ± 1.0	7.4 ± 0.50	5.9 ± 0.97	7.5 ± 0.39	Nil

100 μM	(+)	68.6 ± 2.71*	18.2 ± 1.0⁺	Nil	10.4 ± 1.50⁺	Nil
100 μM	(−)	17.8 ± 0.56	21.7 ± 1.67	16.7 ± 1.93	35.0 ± 1.02	7.4 ± 0.50

200 μM	(+)	26.3 ± 1.59*	24.9 ± 1.24⁺	Nil	40.9 ± 2.79⁺	4.9 ± 0.89⁺
200 μM	(−)	Nil	28.3 ± 0.41	Nil	56.5 ± 1.62	13.3 ± 1.93

300 μM	(+)	Nil	Nil	Nil	Nil	93.9 ± 3.07⁺
300 μM	(−)	Nil	Nil	Nil	Nil	100

3.2 Effect of verapamil on morphological apoptotic changes

A shift from an inhibition of SEA to apoptosis inducing ability occurred as the concentration of verapamil increased. Apoptotic features such as shrinkage, membrane blebbing and cytoplasmic granulation were observed, if the eggs were cultured in Ca²⁺-deficient medium supplemented with 50μM of verapamil for 3h. The 100μM of verapamil induced morphological apoptotic features such as shrinkage (21.7±1.7%), membrane blebbing (16.7±1.9%) and cytoplasmic granulation (35.0±1.0%) prior to degeneration (7.4±0.5%; Table 1). The 200μM dose of verapamil induced maximum apoptotic features such as shrinkage (28.3±0.4%) and cytoplasmic granulation (56.5±1.6%) but no membrane blebbing. Conversely, extracellular Ca²⁺ protected against 50μM verapamil-induced morphological apoptotic changes, but its protective effect was reduced when eggs were treated with higher concentrations (≥100μM) of verapamil (Table 1). In the absence of Ca²⁺ in culture medium, eggs became more susceptible to apoptotic cell death at higher concentrations of verapamil (50–200μM). The verapamil-induced shrinkage (Fig. 2A, a first morphological apoptotic feature) and membrane blebbing (Fig. 2B, a second morphological apoptotic feature) that occurred only when eggs were cultured in Ca²⁺-deficient medium supplemented with 50 or 100μM of verapamil. The last verapamil-induced morphological apoptotic feature was cytoplasmic granulation (Fig. 2C) prior to degeneration of eggs (Fig. 2D).

Fig. 2

Representative photograph showing higher concentrations of verapamil-induced morphological apoptotic features such as shrinkage (2A), membrane blebbing (2B), cytoplasmic granulation (2C) prior to degeneration (2D) after 3h of in vitro culture (▶).

3.3 Effect of verapamil on intracellular H2O2 level

In Ca²⁺-deficient medium, 50μM of verapamil significantly (P<0.05) increased intracellular H2O2 (89.6±1.5ng/egg) when compared to control eggs (77.0±3.0ng/egg). Conversely, extracellular Ca²⁺ significantly (P<0.05) reduced the verapamil-induced generation of intracellular H2O2 (81.7±1.2ng/ml; Fig. 3A).

Fig. 3

(A) Effect of verapamil (50μM) on intracellular H2O2 level. C, Control egg. V, Eggs cultured in Ca²⁺-deficient medium containing 50μM of verapamil. V+Ca, eggs cultured in Ca²⁺-supplemented medium containing 50μM of verapamil. (B) Representative photograph showing 50μM verapamil-induced changes in bax and bcl2 expression. Lane 1, control eggs lysate; Lane 2, lysate of eggs cultured in Ca²⁺-deficient medium containing 200μM of verapamil; Lane 3, lysate of eggs cultured in Ca²⁺-supplemented medium containing 50μM of verapamil. The lower portion shows a control assay for β-actin protein (corresponding to 45kDa) to confirm that the equivalent amounts of protein were analyzed. (C) Effect of verapamil on induction of caspase-3 activity. C, Control eggs. V, Eggs cultured in Ca²⁺-deficient medium containing 50μM of verapamil. V+Ca, eggs cultured in Ca²⁺-supplemented medium containing 50μM of verapamil. Data are means (OD units)±SEM of three replicates. “*” Denotes significantly (P<0.05) higher as compared to control egg. “+”Denotes significantly (P<0.05) lower as compared to verapamil-treated egg (Student's t-test).

3.4 Effect of verapamil on bax and bcl2 protein expression

In Ca²⁺-deficient medium, 50μM of verapamil increased bax protein expression but reduced bcl2 protein expression in treated eggs (lane 2) as compared to control eggs (lane 1; Fig. 3B). Conversely, extracellular Ca²⁺ significantly protected verapamil-induced over-expression of bax protein and the amount of bax protein was comparable to that in the control lane, while bcl2 protein expression was increased (lane 3). The immunodetection of protein was repeated at least 3 times.

3.5 Effect of verapamil on caspase-3 activity

In Ca²⁺-deficient medium, caspase-3 activity of 50μM verapamil-treated eggs was significantly (P<0.05) higher (1.6 times) as compared to control eggs (Fig. 3C). Conversely, extracellular Ca²⁺ significantly (P<0.05) reduced caspase-3 activity and enzyme activity was comparable to control eggs.

3.6 Effect of verapamil on DNA fragmentation

Verapamil-induced DNA fragmentation was confirmed by TUNEL analysis. Control egg showed TUNEL negative staining (Fig. 4A). On the other hand, verapamil (50μM)-treated eggs that had morphological apoptotic features, such as shrinkage, were positively stained (Fig. 4B). Further, extracellular Ca²⁺ protected against 50μM verapamil-induced DNA fragmentation in 26% of eggs shown by negative staining (Fig. 4C). TUNEL analysis was repeated at least 3 times.

Fig. 4

Representative photograph showing 50μM of verapamil-induced DNA fragmentation in eggs cultured in vitro for 3h. (4A, ▶) Control egg cytoplasm showing TUNEL negative staining. (4B, ▶) Verapamil-treated egg showing showing DNA fragmentation as evidenced by TUNEL positive staining. (4C, ▶) Extracellular Ca²⁺ protected against verapamil-induced DNA fragmentation as evidenced by TUNEL negative staining. The irregular shape of zona pellucida is due to the proteinase K treatment during TUNEL assay.

4 Discussion

In rats of Holtzman strain, eggs collected from oviduct after 14h post-hCG surge are arrested at the M-II stage of meiotic cell cycle. Culture of these eggs under in vitro conditions for 3h does not induce exit from M-II arrest (Chaube et al., 2005a,b, 2006). The postovulatory aging increases exit from M-II arrest by extruding a second polar body, a morphological feature characteristic of SEA (Ross et al., 2006). In the present study, eggs collected from oviduct after 17h post-hCG surge were at M-II stage with a first polar body. Culture of these eggs in the Ca²⁺-deficient or -supplemented culture medium induced SEA but the presence of Ca²⁺ induced a higher rate of SEA than that seen in eggs cultured in Ca²⁺-deficient medium. This observation confirms our previous findings that postovulatory aging and extracellular Ca²⁺ induce SEA in rat cells (Chaube et al., 2007; Yoo and Smith, 2007); together with our previous findings suggest that postovulatory aging induces SEA possibly by increasing [Ca²⁺]i. Extracellular Ca²⁺ might have contributed, at least in a part, to the induction of SEA.

L-type calcium channels are distributed on the plasma membrane of mammalian egg, being involved in meiotic cell cycle progression (Tosti et al., 2000; Tosti, 2006). We have shown that verapamil (50–200μM) inhibited SEA and induced morphological feature characteristics of apoptosis in a dose-dependent manner. Verapamil-induced M-II arrest and apoptosis in aged eggs might be associated with increased [Ca²⁺]i due to the release from internal calcium stores. Alternatively aged eggs may be unable to handle increased [Ca²⁺]i that should be redirected to calcium stores or to the extracellular medium. Although we have not analyzed [Ca²⁺]i level to support this hypothesis, previous studies have demonstrated that verapamil induces accumulation of [Ca²⁺]i thereby inducing meiotic cell cycle arrest (Rozinek et al., 2003) and apoptosis (Gordo et al., 2002; Chaube et al., 2007). The extracellular Ca²⁺ protected against 50μM verapamil-induced apoptotic cell death. The protective effect of extracellular Ca²⁺ was reduced when eggs were treated with verapamil at ≥100μM, suggesting that these levels of verapamil might have affected membrane transit of cations other than calcium to induce egg apoptosis.

The mechanisms by which verapamil induces cell cycle arrest and apoptosis remain obscure. Verapamil increases [Ca²⁺]i thereby inducing meiotic cell cycle arrest (Rozinek et al., 2003) and apoptosis (Gordo et al., 2002; Chaube et al., 2007), possibly by stimulating the generation of H2O2 in aged eggs. This is further strengthened by our results showing that verapamil (50μM) induced M-II arrest and apoptosis was associated with increased intracellular H2O2 level in aged rat eggs. Conversely, extracellular Ca²⁺ significantly reduced intracellular H2O2 levels which were comparable to levels in the control eggs. The data suggests that an endogenous burst of Ca²⁺ store, inhibition of calcium channels by verapamil and inability of aged eggs to handle such high sustained [Ca²⁺]i level might have induced generation of H2O2 and thereby apoptosis.

The increase of intracellular H2O2 may generate a pro-apoptotic protein such as bax and reduce anti-apoptotic protein expression, such as bcl2, and thereby induce egg apoptosis (Chaube et al., 2005a). Western blot analysis in the present study suggests that 50μM verapamil-induced bax expression and reduced bcl2 expression in treated eggs compared to control eggs. Conversely, extracellular Ca²⁺ reversed the bax/bcl2 expression pattern and protected against verapamil-induced egg apoptosis. Taken together, these results indicate that increased intracellular H2O2, over-expression of bax protein and reduced expression of bcl2 might shift the balance of intracellular anti-apoptotic signals to apoptotic ones, pushing verapamil-treated aged eggs towards apoptosis, while extracellular Ca²⁺ prevents this shift and thereby egg apoptosis.

Caspases are a family of cysteine-dependent aspartate-directed proteases. Caspase-3 is a group II caspase that destroys structural and specific proteins damaging DNA and leading to apoptotic cell death (Jurisicova and Acton, 2004). Our data indicates that verapamil (50μM) significantly increased caspase-3 activity and thereby egg apoptosis. Interestingly, extracellular Ca²⁺ significantly reduced caspase-3 activity and egg apoptosis (P<0.05). These results indicate that caspase-3 activation is involved during 50μM verapamil-induced apoptosis in aged rat eggs.

A unique biochemical event in apoptosis that precedes morphological changes is fragmentation of genomic DNA into 180–200 base-pair fragments (Jurisicova and Acton, 2004). These DNA fragments can be detected in a single cell using an in situ technique such as the TUNEL assay (Chaube et al., 2005a,b, 2006, 2007, 2008). We have shown that there was no DNA fragmentation in control egg as shown by negative staining in the TUNEL assay. However, verapamil (50μM) induced DNA fragmentation in treated egg as evidenced by TUNEL positive staining. On the other hand, extracellular Ca²⁺ protected against 50μM verapamil-induced DNA fragmentation as evidenced by TUNEL negative staining. The data confirms that extracellular Ca²⁺ protects eggs against 50μM verapamil-induced apoptosis in aged rat eggs cultured in vitro. Verapamil-induced DNA fragmentation has already been reported in aged rat eggs (Chaube et al., 2007).

In conclusion, our results suggest that 50μM verapamil induces egg apoptosis by increasing intracellular H2O2, expression of bax protein and caspase-3 activity. Further, extracellular Ca²⁺ reduces intracellular H2O2, expression of bax protein and caspase-3 activity and thus protects against 50μM but not higher concentrations (≥100μM) of verapamil-induced egg apoptosis in aged rat eggs cultured in vitro.

Acknowledgments

The authors are very grateful to Mr. Vinay K. Dubey and Mr. Ravi S. Chaubey, Biotech India, Nandigram, Lanka, Varanasi, UP, India, for their generous gift of Caspase-3 and H2O2 kits.

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Received 22 May 2008/5 November 2008; accepted 9 January 2009

doi:10.1016/j.cellbi.2009.01.001

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