Calcium Homeostasis in Early Hamster Preimplantation Embryos¹

^a Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The development in culture of 1-cell hamster embryos prior to the completion of fertilization is not well understood. In this study it was observed that culture for only 6 h of these early 1-cell embryos collected before pronuclei formation (3 h post-egg activation; PEA) significantly increased intracellular free calcium levels (194.3 ± 3.1 nM) compared to levels in similarly aged 1-cell embryos collected from the oviduct at 9 h PEA, after pronuclei formation is complete (134.2 ± 6.8 nM). Not only was the developmental competence of cultured 3-h PEA embryos with elevated intracellular free calcium levels compromised as compared with that of embryos collected from the oviduct at 9 h PEA; these embryos also had impaired cytoplasmic mitochondrial distribution (ratio of 0.62 ± 0.06 for cultured embryos compared to 0.44 ± 0.04 for in vivo-developed embryos) and decreased lactate metabolism (2.93 ± 0.22 pmol/embryo per 3 h for cultured embryos compared to 5.37 ± 0.36 for in vivo-developed embryos). This impairment in mitochondrial distribution and function and reduced development in culture by 3-h PEA embryos appears related to the ability to regulate intracellular calcium homeostasis. Intracellular free calcium levels were reduced by culture with increased medium magnesium concentrations, calcium channel inhibitors nifedipine or verapamil, or an intracellular calcium chelator. All of these treatments also stimulated development of 3-h PEA embryos to the morula/blastocyst stages and prevented impairment in mitochondrial organization and function. Conversely, culture with low medium magnesium and high calcium concentrations that increased intracellular free calcium levels resulted in low development and reduced mitochondrial function. Therefore, it appears that removal of the early embryo from the oviduct results in an inability to regulate intracellular calcium levels. As increased magnesium concentrations, nifedipine, and verapamil inhibit L-gated calcium channels, it may be a loss of regulation of these channels that alters calcium homeostasis resulting in impaired developmental competence.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The early events of fertilization of mouse and hamster oocytes involve recurring transient hyperpolarizations of the membrane [1] that are a result of an increase in K⁺ conductance [2]. These hyperpolarizations are followed by a series of calcium oscillations that last for several hours until the pronuclei are formed [3–5]. In the hamster embryo, pronuclei are formed during the first 5–6 h post-egg activation (PEA) by the sperm. The continued calcium oscillations during the first cleavage of hamster embryo development are important for the stimulation of later development [5]. The calcium oscillations regulate many developmentally important processes during fertilization such as release of cortical granules, protein synthesis, and gene transcription [6–8].

Intracellular calcium levels also regulate many important cellular functions such as cell division, membrane fusion, exocytosis, cell-cell communication, and metabolism as well playing a role in second messenger systems [9]. Cells utilize several mechanisms such as membrane channels, transporters, sequestration by organelles, and binding to proteins to maintain intracellular calcium at low and constant levels [9]. However, cellular stress or trauma results in disturbances in cellular calcium homeostasis [10].

Development in culture of early hamster embryos collected 3 h PEA (when they are still completing pronuclei formation) is significantly reduced compared to that of embryos collected at 9 h PEA (after pronuclei formation is completed) [11, 12]. During this critical 6-h period of development, the 1-cell hamster embryo undergoes morphological changes such as pronuclei formation and second polar body extrusion, as well as considerable organelle redistribution [13, 14], as the process of fertilization is being completed. It was recently demonstrated that hamster 1-cell embryos that developed poorly in culture had elevated intracellular free calcium levels at the 2-cell stage [12]. It is possible that stress induced by removal of the early embryo from the reproductive tract while fertilization is still occurring results in loss of calcium homeostasis by depletion of physiological regulators present in the oviduct. The aim of this study therefore was to determine whether the loss in development of 3-h PEA embryos (before pronuclei formation is completed) as compared to later-stage embryos (9-h PEA, after pronuclei formation is completed) is associated with perturbations in intracellular calcium levels and to investigate whether conditions that reduce intracellular calcium levels can maintain developmental competence in culture. The effects of altered magnesium concentrations in the medium, calcium transport inhibitors, or chelating agents on intracellular free calcium levels, development in culture, total cell number, inner cell mass (ICM) and trophectoderm (TE) development, embryo metabolism, and mitochondrial reorganization were determined.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Culture Media

Embryos were cultured in a protein-free, chemically defined hamster embryo culture medium, HECM-9 [15]. The medium was prepared the day before use from stock solutions with Milli-Q water and stored at 4°C. The formulation of HECM-9 is as follows: NaCl, 113.8 mM; KCl, 3.0 mM; CaCl₂·2H₂O, 2.0 mM; MgCl₂·6H₂O, 0.5 mM; NaHCO₃, 25.0 mM; DL-sodium lactate, 4.50 mM; asparagine, 0.01 mM; aspartate, 0.01 mM; cysteine, 0.01 mM; glutamate, 0.01 mM; glutamine, 0.20 mM; glycine, 0.01 mM; histidine, 0.01 mM; lysine, 0.01 mM; proline, 0.01 mM; serine, 0.01 mM; taurine, 0.5 mM; pantothenate, 3 µM; and polyvinyl alcohol, 0.1 mg/ml.

All salts, lactate, amino acids, EGTA, verapamil, nifedipine, and ruthenium red were purchased from Sigma Chemical Co. (St. Louis, MO). N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-,bis[(acetyloxy)methyl] ester (BAPTA-AM) was purchased from Molecular Probes (Eugene, OR).

Animals and Embryo Collection

Embryos were collected from 3- to 6-mo-old cycling golden hamsters. Hamsters were housed as previously described [16]. Females were superovulated by an i.p. injection with 10–20 IU eCG (Pregnyl; Diosythn, Chicago, IL) on the morning of postestrus discharge (Day 1 of cycle). Females were mated to fertile males on the evening of Day 4. One-cell embryos were collected at either 3 h, 6 h, or 9 h PEA (at 0700 h, 1000 h, and 1300 h the following day) [17]. Embryos were flushed from the oviduct with warmed and gas-equilibrated (10% CO₂:5% O₂:85% N₂) HECM-9 medium. One-cell embryos collected at either 3 h or 6 h PEA were denuded by a 1-min incubation with 0.5 mg/ml hyaluronidase (Sigma). Denuded embryos were washed twice in HECM-9 and once in the appropriate culture medium modification before being placed in culture. Exposure to hyaluronidase for 2 min was found not to affect 1-cell embryo development (data not shown).

Embryo Culture

All embryos were cultured in 35-µl drops of medium under silicone oil (Aldrich Chemical Company, Milwaukee, WI) for 72 h at 37.5°C in a humidified atmosphere of 10% CO₂:5% O₂:85% N₂. Embryos from individual females were cultured separately and evenly distributed between treatment groups. Any embryos remaining after the even distribution were discarded. Embryos collected at 3 h PEA were cultured in either HECM-9 (control); HECM-9 with altered magnesium (1.0, 2.0, or 4.0 mM) and calcium (1.0 or 2.0 mM) concentrations; HECM-9 supplemented with 50 µM verapamil, 10 µM nifedipine, 10 µM ruthenium red, 5 µM BAPTA-AM, or 100 µM EGTA [18]; or HECM-9 without calcium (with 1 mM EGTA). Embryos were cultured for 6 h in each treatment, with the exception of BAPTA, and then were either returned to culture in HECM-9 for a further 72 h or used for assessment of intracellular free calcium levels, lactate oxidation, or mitochondrial distribution. For BAPTA treatment, embryos were cultured with BAPTA for only 30 min to enable loading of the acetyloxymethyl ester form and then returned to culture in HECM-9. In vivo control embryos were collected at 9 h PEA without hyaluronidase treatment and cultured in medium HECM-9 for 72 h. An additional control for development of 3-h PEA embryos to the morula/blastocyst stage was medium HECM-10 (HECM-9 modified to contain a magnesium concentration of 2.0 mM and calcium concentration of 1.0 mM), which increases hamster 1-cell development [12].

Determination of Intracellular Calcium Levels

Intracellular calcium concentrations were determined by ratiometric analysis using the calcium-sensitive probe Fura-2-AM (Molecular Probes) [19]. One-cell embryos (either 3 h, 6 h, or 9 h PEA) were loaded with 5 µM of Fura-2-AM in HECM-9 for 30 min at 37°C. Embryos were washed twice in medium not containing dye and placed inside a temperature-controlled tissue culture chamber (Biophysica, Baltimore, MD) containing HECM-9 and were immediately analyzed. Each individual embryo was viewed, and fluorescent intensities were recorded using a Nikon Diaphot inverted microscope connected with a Nikon Dual Optical Path Tube to a PXL cooled camera (Photometrics, Huntington Beach, CA) for high-resolution recording of epifluorescent images. Image analysis of fluorescent images was performed using Metamorph/Metafluor hardware and software (Universal Imaging Corp., West Chester, PA). Emission wavelength was set to 535 nm, and the ratio of fluorescence intensities of excitation wavelengths 340–380 nm was obtained for each embryo. The ratio of fluorescence for each embryo was determined by averaging 5 readings taken at 3-min intervals. Intracellular free calcium levels were found to be consistent and not oscillating at this stage (9 h PEA) in development (data not shown). Relative intracellular calcium levels were quantitated from these ratios using a two-point in situ standard [19, 20] run on each day of experiment using the ionophore Br-A23187 (20 µM; Sigma).

Assessment of Embryo Morphology

Development to the 4- to 8-cell stage was assessed after 48 h of culture, and morula/blastocyst development was determined after 72 h of culture. As the cultured hamster blastocyst in vitro undergoes extensive cycles of expansion and collapse [21], morulae and blastocysts (observed at 72 h of culture) were grouped together as an additional endpoint to blastocyst formation.

Differential Nuclear Staining of ICM and TE Cells

Embryo cell number and allocation of cells to the ICM and TE were determined using a modification of the technique described by Hardy et al. [22]. Embryos were incubated in 0.5% pronase for 30 sec to dissolve the zona pellucida. Embryos were incubated in picrylsulfonic acid (Sigma) for 10 min at 4°C before incubation in 0.1 mg/ml of anti-DNP BSA (ICN Technologies, Costa Mesa, CA) at 37°C. Embryos were then incubated in a 1:5 dilution of guinea pig serum containing 25 µg/ml of propidium iodide for 5 min and subsequently placed in 25 µg/ml bisbenzimide (Hoechst 33258; Sigma) in ethanol overnight at 4°C. The following morning, embryos were washed in absolute ethanol and mounted in glycerol on siliconized slides overlaid with a coverslip. Under a mercury lamp and a UV dichroic mirror, the nuclei of the TE appear pink while the nuclei of the ICM appear blue. Using a blue/green dichroic mirror, only the TE cells are visible. Numbers of ICM and TE cells and the percentage of ICM cells to total cell number were determined for each blastocyst.

Assessment of Embryo Metabolism

Embryo metabolism was assessed by incubation with the radioisotope [U-¹⁴C]lactate in a microcentrifuge tube as described by O'Fallon and Wright and Rieger et al. [23, 24]. Individual embryos were incubated in a 3-µl drop of medium HECM-9, containing [U-¹⁴C]lactate (0.02 µCi/µl), on the lid of a microcentrifuge tube over 1.5 ml of 25 mM NaHCO₃. The tubes were gassed with 10% CO₂:5% O₂:85% N₂ into around 0.2 ml of air space, sealed, and placed in an incubator at 37°C for 3 h. Control tubes of medium without embryos were included to control for nonspecific breakdown of the label, and total radioactivity was determined by adding 3 µl of label directly to the NaHCO₃ trap. After the 3-h incubation period, the tubes were opened; 1 ml of the NaHCO₃ trap was placed into a scintillation vial containing 200 µl of 0.1 M NaOH, and the vials were stored at 4°C. The following day, 10 ml of scintillation fluid (Ultima Gold; Packard, Meriden, CT) was added to each vial, the vials were vortexed, and disintegrations were counted for 4 min. Lactate metabolism was calculated using the recovery efficiency of the radioisotope as previously described [24].

Determination of Mitochondria Distribution in 1-Cell Embryos

Zygotes were incubated in HECM-9 containing 100 nM Mitotracker-rosamine (Molecular Probes) for 15 min. Embryos were then washed twice and fixed by a series of steps through increasing ratios of formalin in PBS. After fixation, embryos were washed twice in PBS and mounted in PBS on a siliconized slide for imaging [13]. Images were obtained using laser scanning confocal microscopy (Bio-Rad MRC-600; Richmond, CA) with a krypton-argon mixed gas laser. Mitotracker was excited at 534 nm. Images were obtained in the center of the embryo using the pronuclei as reference points. Image analysis was performed using Metamorph software. Quantification of mitochondrial distribution was adapted from the method for 2-cell embryos described by Barnett et al. [14]. Boxes 20 x 20 pixels in size were placed in 6 areas adjacent to the pronuclei and in 6 areas 15 pixels from the cell membrane 90° to the pronuclei (intermediate area), giving 12 regions per embryo (Fig. 1). The average intensity of each 20 x 20-pixel box was determined. The mean pixel intensity of the 6 boxes in the intermediate area and the 6 boxes in the perinuclear region was determined, and a ratio of intermediate to perinuclear areas was obtained for each embryo.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Schematic diagram illustrating areas for analysis of pixel intensity for quantification of mitochondria distribution within a 1-cell embryo. Solid boxes indicate areas measured within the intermediate area of the embryo and were placed 15 pixels from the cell membrane. Open boxes indicate areas measured within the perinuclear area of the embryo and were placed adjacent to the pronuclei.

Statistical Analysis

Data for embryo development in culture and cell numbers were initially corrected for day of experiment and donor female variation. Embryo development was analyzed using log-logistic regression assuming a binomial distribution [25]. The null hypothesis of no treatment effect against a treatment effect was tested using the log-likelihood ratio statistic, which is approximately chi-square distributed. Embryo cell number, ICM and TE cell number, lactate oxidation, intracellular free calcium levels, and ratio of mitochondrial distribution by embryos were initially analyzed using ANOVA. Differences from the values in the control medium HECM-9 were determined using Dunnett's test. Between-treatment differences were examined using the Bonferroni procedure for multiple comparisons [26].

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Determination of Intracellular Calcium Levels of 1-Cell Embryos

The intracellular free calcium concentration of 3-h PEA embryos developed in vivo was 256.0 ± 7.4 nM, which decreased to 161.2 ± 5.7 nM for embryos collected at 6 h PEA and to 134.1 ± 6.8 nM for embryos collected at 9 h PEA. Culture of 3-h PEA embryos for 6 h in control medium HECM-9 significantly increased intracellular free calcium levels compared to those of in vivo-developed 1-cell embryos collected at 9 h PEA (p < 0.05; Table 1). Culture of embryos for 6 h in medium HECM-9 with magnesium and calcium concentrations adjusted to 0.5 mM further increased the intracellular free calcium levels, while increasing the magnesium concentration to 2.0 mM or 4.0 mM resulted in significantly decreased intracellular free calcium levels compared to those in 3-h PEA embryos cultured for 6 h in medium HECM-9. Addition of nifedipine or verapamil to HECM-9 also significantly reduced intracellular free calcium levels to 122.6 ± 7.4 nM (p < 0.01) and 135.2 ± 5.6 nM (p < 0.01), respectively, in 3-h PEA embryos cultured for 6 h as compared to embryos cultured in HECM-9 alone (Table 1). In contrast, culture of 3-h PEA embryos with either ruthenium red or EGTA for 6 h did not affect intracellular free calcium levels. However, culture of embryos with BAPTA or in medium with no external calcium significantly reduced intracellular free calcium levels compared to those of embryos cultured in medium HECM-9 (Table 1).

Embryo Development in Culture

Altering both the magnesium and calcium concentrations in the culture medium to 0.5 mM for the first 6 h of culture for embryos collected at 3 h PEA significantly reduced subsequent development compared to that in the control medium (Tables 2 and 3). In contrast, increasing the magnesium concentration to either 2.0 mM or 4.0 mM for the first 6 h of development significantly increased development to the 8-cell stage (Table 2), mean cell number at 48 h (Table 2), and morula/blastocyst and blastocyst development (Table 3) compared to values in the control medium. Furthermore, resultant blastocysts had significantly increased cell numbers and ICM development (Table 3). Culture of 3-h PEA embryos for 6 h with the calcium channel inhibitor nifedipine significantly increased development to the 8-cell stage, while both channel inhibitors nifedipine and verapamil increased mean cell number of embryos after 48 h of culture (Table 2). Furthermore, nifedipine significantly increased both morula/blastocyst and blastocyst development as well as elevating blastocyst cell number and ICM development (Table 3). Culture with verapamil did not affect development to the morula/blastocyst or blastocyst stages compared to that in the control medium, although resultant blastocysts had a significantly higher cell number and ICM development compared to embryos cultured in the control medium (Table 3). Culture for 6 h with ruthenium red significantly reduced subsequent development to the 8-cell stage, morula/blastocyst, and blastocyst stages and blastocyst cell number compared to values in embryos cultured in the control medium (Table 3). Culture with the chelator EGTA or the absence of external calcium did not affect development after either 48 h (Table 2) or 72 h (Table 3) of culture. Similarly, culture with the internal calcium chelator BAPTA for 30 min prior to continued culture in the control medium did not affect development to the 8-cell stage but did increase development to the blastocyst stages, blastocyst cell number, and ICM cell number (Table 3). Culture with an increased magnesium concentration for the entire culture period (HECM-10) significantly increased development to the 8-cell stage (Table 2) as well as increasing development to the morula/blastocyst and blastocyst stages, blastocyst cell number, and ICM development compared to values in all other treatment groups. Development of embryos collected at 9 h PEA to the 8-cell stage after 48 h (Table 2), and morula and blastocyst development after 72 h (Table 3), were significantly increased compared to values in all 6-h treatments for the culture of 3-h PEA embryos.

Assessment of Metabolism of 1-Cell Embryos

Initially, the levels of lactate oxidation by in vivo-developed 1-cell embryos was determined at 3, 6, and 9 h PEA. Lactate oxidation was highest in 1-cell embryos collected at 3 h PEA (8.3 ± 1.0 pmol/embryo per 3 h). Lactate oxidation was similar at 6 h PEA (7.7 ± 0.5 pmol/embryo per 3 h) but was significantly lower in 1-cell embryos collected at 9 h PEA (5.4 ± 0.4; pmol/embryo per 3 h; p < 0.05).

Culture of 3-h PEA 1-cell embryos for 6 h in medium containing increased magnesium levels, 2.0 mM or 4.0 mM, significantly increased lactate oxidation by 1-cell embryos compared to that in embryos cultured in the control medium, HECM-9 (Table 4). In contrast, when the magnesium and calcium concentrations in the medium were adjusted to 0.5 mM for the 6-h culture period, resultant 1-cell embryos had significantly reduced rates of lactate oxidation compared to the control. Culture with either nifedipine, verapamil, BAPTA, or no calcium in the medium significantly increased lactate oxidation by 3-h PEA embryos compared to embryos cultured in HECM-9 (Table 4). Furthermore, culture with nifedipine, BAPTA, or no calcium in the medium increased lactate oxidation to levels that were not different from those for in vivo-developed 9-h PEA embryos. Culture with EGTA did not alter the levels of lactate oxidation compared to those in medium HECM-9 (Table 4); however, culture with ruthenium red significantly reduced the levels of lactate oxidation by more than half compared to the level in the control medium (Table 4).

Examination of Mitochondrial Distribution in 1-Cell Embryos

The ratios of intermediate:perinuclear staining intensities, a marker of mitochondrial distribution, were determined for in vivo-developed 1-cell embryos (Fig. 2). As the 1-cell embryo developed, mitochondria normally became distributed in a perinuclear pattern, and the ratio of intermediate to perinuclear pixel intensities significantly decreased from 0.91 ± 0.09 in 3-h PEA embryos to 0.84 ± 0.11 for embryos collected at 6 h PEA and 0.44 ± 0.04 for embryos collected at 9 h PEA.

View larger version (46K): [in this window] [in a new window]   FIG. 2. Micrographs illustrating the changes in mitochondrial distribution in in vivo-developed 1-cell embryos. Micrographs represent a) a 3-h PEA embryo: the mitochondrial distribution is initially dispersed throughout the cytoplasm; b) a 6-h PEA embryo: the first signs of perinuclear clustering are evident at 6 h PEA; c) a 9-h PEA embryo: the mitochondria have a distinct perinuclear clustering, and the sperm tail is also evident (arrow).

Culture of 3-h PEA 1-cell embryos for 6 h in the control medium HECM-9 resulted in an intermediate:perinuclear ratio of 0.63 ± 0.03, significantly higher than that for similarly aged (9 h PEA) in vivo embryos (p < 0.05). Culture in medium with a magnesium and calcium concentration of 0.5 mM significantly increased the intermediate:perinuclear mitochondrial ratio compared to culture in the control medium (Fig. 3). In contrast, culture with increased magnesium levels of 2.0 mM or 4.0 mM, nifedipine, or BAPTA significantly reduced the ratio of intermediate:perinuclear mitochondrial intensity compared to that in the control medium (Fig. 3). Culture with ruthenium red or EGTA did not affect the mitochondrial distribution (Fig. 3).

View larger version (17K): [in this window] [in a new window]   FIG. 3. Ratios of intermediate:perinuclear pixel intensity of mitochondria for 3-h PEA 1-cell embryos cultured for 6 h; n = at least 15 embryos imaged per treatment group (3 replicates). *Significantly different from embryos cultured in control medium HECM-9 (p < 0.05). Numbers (0.5/0.5, 2.0/1.0, and 4.0/1.0) indicate magnesium/calcium concentration in the medium. Nif, nifedipine; Verap, verapamil; RR, ruthenium red.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Culture for 6 h of early 1-cell embryos collected at 3 h PEA (before completion of pronuclei formation) resulted in elevated levels of intracellular free calcium compared to values in embryos flushed from the oviduct at 9 h PEA. Development in culture was also reduced, as was metabolic activity, and organelle redistribution was delayed compared to that in in vivo-developed counterparts. Culture conditions that resulted in reduced levels of intracellular free calcium, such as increased magnesium concentration in the medium from 0.5 mM to 2.0 mM or 4.0 mM, the addition of calcium channel blockers nifedipine or verapamil, and addition of the intracellular free calcium chelator BAPTA, all increased subsequent development of 3-h PEA 1-cell embryos to the morula/blastocyst stage and increased oxidative metabolism and organelle distribution associated with fertilization. It is therefore apparent that the loss of developmental competence of early 1-cell embryos collected at 3 h PEA is at least partially attributable to a loss in the regulation of calcium homeostasis. The resultant increase in intracellular free calcium levels impairs cellular functions, for example disturbing mitochondrial metabolism and organelle redistribution, which results in reduced developmental competence.

Developmental competence of 3-h PEA embryos was significantly increased by culture with increased medium magnesium concentration. Culture in the presence of increased levels of magnesium resulted in embryos with elevated levels of lactate oxidation, the carbohydrate preferentially utilized for energy production by the preimplantation hamster embryo [27]. The maintenance of adequate energy production is important to the developing embryo, as correct pathway utilization reflects embryo viability [28, 29]. Increased magnesium levels in the medium also maintained the normal timing of redistribution of mitochondria, which is essential for normal development of hamster 1-cell embryos [13].

Interestingly, the abnormal increase in free intracellular calcium levels of cultured embryos was prevented by culture with increased medium magnesium levels. A similar effect—a reduction of intracellular free calcium levels by increasing magnesium levels in the medium—has recently been reported for cultured 2-cell hamster embryos [12]. Similarly, for cardiac cells, increasing external magnesium reduces free intracellular calcium levels by inhibiting either L-gated calcium channels on the cell membrane or the activity of the Ca²⁺/Mg²⁺ ATPase, thereby reducing the movement of calcium into the cell [30]. It therefore appears that the increased magnesium concentration in the culture medium enhances early embryo development by preventing any detrimental increase in intracellular free calcium levels.

As culture for 6 h with nifedipine or verapamil significantly reduced free intracellular calcium levels, we predicted that culture with nifedipine or verapamil should also increase the development of early 3-h PEA embryos. This was found experimentally to be true up to the 8-cell stage. However, only culture with nifedipine increased subsequent development to the morula and blastocyst stages, although both inhibitors increased resultant blastocyst cell numbers. Culture with either nifedipine or verapamil also increased lactate metabolism and mitochondrial redistribution of cultured 1-cell embryos. Both verapamil [31] and nifedipine [32, 33] affect calcium levels by inhibiting slow inward potential dependent channels (L-gated channels) in a dose-dependent manner. However, the reduced effects of verapamil are likely due to its selective interaction with channels in the depolarized inactivated state [34] and nonspecific actions on potassium and sodium channels [31, 35].

As both increased extracellular magnesium levels and nifedipine and verapamil act on L-gated calcium channels, it appears that early embryos are unable to regulate these channels in culture. Further evidence for this hypothesis is that removal of calcium from the external medium for the initial 6 h of culture also reduced intracellular free calcium levels compared to that in embryos cultured in the control medium. Removal of extracellular calcium from the medium would prevent the influx of external calcium that was inhibited by the channel blockers verapamil and nifedipine and therefore enable the early embryo to maintain calcium homeostasis. L-gated calcium channels are present in both mouse and hamster embryos up to the 8-cell stage of development [36, 37]. Furthermore, there is a decrease in calcium channel currents during development of the hamster 1-cell embryo between 3 h and 9 h PEA [37], which may account for the increased susceptibility of the 3-h PEA 1-cell embryo to the external culture medium. Furthermore, loss of regulation of calcium channels in 3-h PEA embryos may disturb the calcium oscillations during fertilization that control developmentally important processes such as protein synthesis [6, 7] and gene transcription [8], as these calcium oscillations during the first cleavage stimulate subsequent development [5]. This observation that the culture conditions can adversely affect fertilization and subsequent development has significant implications for assisted reproductive technologies, such as clinical in vitro fertilization. Many of these technologies have poor success rates caused by a loss of developmental competence by early embryos. It is possible that embryos from other species such as the human may also lose the ability to regulate ionic homeostasis during early development, contributing to the observed losses in developmental competence.

In many cell types, the stimulation and regulation of both L- and T-gated calcium channels can be due to either calcium- or voltage-dependent protein kinases [38]. In mouse spermatozoa, T-gated calcium channels are present in two states, as either a low- or a high-conductance channel, and transitions between the states are also due to modulation by protein kinases [39]; in cardiac cells, calcium influx through these channels can be regulated by intracellular pH [40, 41]. It is, however, currently unclear why the early embryo is unable to regulate its calcium channels in culture. It is possible that removing the embryo from the oviduct results in loss of physiologically important regulators.

Further evidence that reduced development in culture is associated with an increase in free intracellular calcium is provided by the finding that culture with the chelator BAPTA also stimulated development. In contrast, EGTA, also a chelator of divalent cations with a high selectivity for calcium, did not affect embryo development, lactate oxidation, or mitochondrial distribution. However, EGTA did not alter free intracellular calcium levels. Use of the acetyloxymethyl ester form of BAPTA would enable this chelator to enter the cell and chelate intracellular calcium [42], resulting in the stimulation of embryo development in culture. In contrast, EGTA is an external chelator of calcium and appears to be unable to cross the cell membrane of 1-cell hamster embryos.

One of the mechanisms that cells utilize to maintain low and constant intracellular calcium levels is the sequestration of calcium by organelles. The endoplasmic/sarcoplasmic reticulum [43, 44], mitochondria [45], and nucleus [46] sequester around 90% of the cellular calcium, equivalent to around 2 mM [9]. Small changes in the activity of the calcium influx and efflux mechanisms of these organelles can elicit large changes in free intracellular calcium levels. However, high levels of calcium influx into mitochondria in response to increases in intracellular free calcium levels have detrimental effects on mitochondrial activity [45]. To determine whether the decreased oxidation of lactate by cultured embryos was due to an increase in mitochondria calcium levels, ruthenium red was used to inhibit calcium influx into mitochondria. In this study, the presence of ruthenium red did not increase mitochondrial activity. Rather, incubation of 1-cell embryos with ruthenium red reduced mitochondrial lactate metabolism and inhibited subsequent development. Therefore the reduction of mitochondrial function does not appear to be due to an increase in calcium influx into mitochondria. Rather, the appropriate influx of calcium through this channel is necessary for adequate energy production and therefore developmental competence of early 1-cell-stage embryos.

The loss of developmental competence of 3-h PEA 1-cell hamster embryos appears related to the inability of these embryos to regulate intracellular calcium homeostasis. Development was stimulated by culture conditions that reduced free intracellular calcium levels such as increased medium magnesium concentrations, calcium channel inhibitors, or BAPTA. As the increase in free intracellular calcium levels of embryos was prevented by removal of extracellular calcium, it appears that the increase in intracellular free calcium levels is due to an influx of external calcium. Moreover, increased magnesium, verapamil, and nifedipine all act by regulating calcium entry into cells by inhibiting voltage L-gated calcium channels, indicating that these channels are also associated with the embryo's inability to regulate calcium homeostasis in culture. Removal of embryos from the oviduct and exposure to culture media appear to result in loss of regulation of these calcium channels, causing an elevation in free intracellular calcium levels that impairs cellular functions, thereby resulting in reduced developmental competence in culture. It is currently unclear why the early embryo loses control of these L-gated calcium channels in culture.

	ACKNOWLEDGMENTS

 
The authors wish to thank Dr. Ralph Albrecht for use of the equipment for determination of intracellular calcium levels and Jayne Squirrell for technical assistance with the confocal microscopy. We would also like to thank Drs. Baltz, Boatman, Eppig, Gardner, Prather, and Tasca for their critique of the manuscript.

	FOOTNOTES

 
¹ This research was supported by National Cooperative Program on Non-Human In Vitro Fertilization and Preimplantation Embryo Development by the National Institute of Child Health and Human Development (NICHD) through Grant HD22023.

² Correspondence: Michelle Lane, Department of Animal Health and Biomedical Sciences, University of Wisconsin, 1655 Linden Drive, Madison, WI 53706. FAX: 608 262 7420; lane{at}ahabs.wisc.edu

Accepted: June 8, 1998.

Received: March 9, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Miyazaki S, Igusa Y. Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations. Nature 1981; 290:706–707.[CrossRef][Medline]
2. Miyazaki S, Igusa Y. Ca-mediated activation of a K current at fertilization of golden hamster eggs. Proc Natl Acad Sci USA 1982; 79:931–935.[Abstract/Free Full Text]
3. Cuthbertson KSR, Cobbold PH. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca²⁺. Nature 1985; 316:541–542.[CrossRef][Medline]
4. Jones KT, Carroll J, Merriman JA, Whittingham DG, Kono T. Repetitive sperm-induced Ca²⁺ transients in mouse oocytes are cell cycle dependent. Development 1995; 121:3259–3266.[Abstract]
5. Bos-Mikich A, Whittingham DG, Jones KT. Meiotic and mitotic Ca²⁺ oscillations affect cell composition in resulting blastocysts. Dev Biol 1997; 182:172–179.[CrossRef][Medline]
6. Prostko CR, Brostom MA, Brostom CO. Reversible phosphorylation of eukaryotic initiation factor-2 alpha in response to endoplasmic reticular signaling. Mol Cell Biochem 1993; 100:117–127.
7. Sirvastava SP, Davies MB, Kaufman RJ. Calcium depletion from the endoplasmic reticulum activates the double-stranded RNA-dependant protein kinase (PKR) to inhibit protein synthesis. Biol Chem 1995; 270:16619–16624.[Abstract/Free Full Text]
8. Pribnow D, Muldoon LL, Fajardo M, Theodorf L, Chen L-YS, Magun BE. Endothelin induces transcription of fos-jun family genes: a prominent role for calcium ions. Mol Endocrinol 1992; 6:1003–1012.[Abstract]
9. Campbell AK. Intracellular calcium: its universal role as a regulator. Chichester: John Wiley and Sons; 1983: 206–361.
10. Dormer RL, Hallet MB, Campbell AK. Measurement of intracellular free Ca²⁺: importance during activation and injury of small cells. In: Parratt JR (ed.), Control and Manipulation of Calcium Movements. New York: Raven Press; 1985: 1–27.
11. Barnett DK, Bavister BD. Hypotaurine requirement for in vitro development of golden hamster one-cell embryos into morulae and blastocysts, and production of term offspring from in vitro-fertilized ova. Biol Reprod 1992; 47:297–304.[Abstract]
12. Lane M, Boatman DE, Albrecht RM, Bavister BD. Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo. Mol Reprod Dev 1998; 50:443–450.[CrossRef][Medline]
13. Barnett DK, Kimura J, Bavister BD. Translocation of active mitochondria during hamster preimplantation embryo development studies by confocal laser scanning microscopy. Dev Dyn 1996; 205:64–72.[CrossRef][Medline]
14. Barnett DK, Clayton MK, Kimura J, Bavister BD. Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol Reprod Dev 1997; 48:227–237.[CrossRef][Medline]
15. McKiernan SH, Bavister BD. Pantothenate stimulates development of hamster embryos in culture. Theriogenology 1998; 49:209 (abstract).
16. Schini SA, Bavister BD. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol Reprod 1988; 39:1183–1192.[Abstract]
17. Bavister BD, Leibfried ML, Lieberman G. Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 1983; 28:235–247.[Abstract]
18. Abramczuk J, Solter D, Koprowski H. The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium. Dev Biol 1977; 61:378–383.[CrossRef][Medline]
19. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440–3450.[Abstract/Free Full Text]
20. Tsien RY. New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis and properties of prototype structures. Biochemistry 1980: 19:2396–2404.
21. Gonzales DS, Bavister BD. Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo. Biol Reprod 1995; 52:470–480.[Abstract]
22. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development 1989; 107:597–604.[Abstract]
23. O'Fallon JV, Wright RW Jr. Quantitative determination of pentose phosphate pathway in preimplantation mouse embryos. Biol Reprod 1986; 34:58–64.[Abstract]
24. Rieger D, Loskutoff NM, Betteridge KJ. Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro. J Reprod Fertil 1992; 95:585–595.[Abstract/Free Full Text]
25. Nelder JA, Wedderburn RWM. Generalized linear models. J R Statist A 1972; 135:370–380.
26. Ludbrook J. On making multiple comparison in clinical and experimental pharmacology and physiology. Clin Exp Pharmacol Physiol 1991; 18:379–392.[Medline]
27. McKiernan SH, Tasca RJ, Bavister BD. Energy substrate requirements for in-vitro development of hamster 1- and 2-cell embryos to the blastocyst stage. Hum Reprod 1991; 6:64–75.[Abstract/Free Full Text]
28. Barnett DK, Bavister BD. What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol Reprod Dev 1996; 43:105–133.[CrossRef][Medline]
29. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod 1996; 11:1975–1978.[Abstract/Free Full Text]
30. Herman B. Regulation of calcium metabolism in cells. In: Anderson JJB, Garner SC (eds.), Calcium and Phosphorus in Health and Disease. Boca Raton, FL: CRC Press; 1995: 83–93.
31. Ehara T, Kaufmann R. The voltage- and time dependent effects of(-) verapamil on the slow inward current in isolated cat ventricular myocardium. J Pharmacol Exp Ther 1978; 207:49–55.[Abstract/Free Full Text]
32. Kohlhardt M, Fleckenstein A. Inhibition of the slow inward current by nifedipine in mammalian ventricular myocardium. Naunyn-Schmiedebergs Arch Pharmacol 1977; 298:267–272.[CrossRef][Medline]
33. Bayer R, Ehara T. Comparative studies on calcium antagonists. In: van Zwieter PA, Schonbaum E (eds.), The Action of Drugs on Calcium Metabolism. New York: Fischer Verlag; 1978: 31–37.
34. Triggle DJ. Calcium antagonists: some basic chemical and pharmacological aspects. In: Weiss GB (ed.), New Perspectives on Calcium Antagonists. Bethesda: American Physiological Society; 1981: 1–18.
35. Kass RS, Tsien RW. Multiple effects of calcium antagonists on plateau currents in cardiac Purkinje fibers. J Gen Physiol 1978; 66:169–192.[Abstract/Free Full Text]
36. Yamashita N. Enhancement of ionic currents through voltage-gated channels in the mouse oocyte after fertilization. J Physiol 1982; 339:631–642.[Abstract/Free Full Text]
37. Mitani S. The reduction of calcium current associated with early differentiation of the murine embryo. J Physiol 1985; 363:71–86.[Abstract/Free Full Text]
38. Hille B. Modulation of ion-channel function by G-protein coupled receptors. Trends Pharmol Sci 1994; 17:531–536.
39. Arnoult C, Lemos JR, Florman HM. Voltage dependent modulation of T-type calcium channels by protein tyrosine and phosphorylation. EMBO J 1997; 16:1593–1599.[CrossRef][Medline]
40. Irisawa H, Sato R. Intra- and extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells. Circ Res 1986; 59:348–355.[Abstract/Free Full Text]
41. Kaibara M, Mitarai S, Yano KA, Kameyama M. Involvement of Na⁺-H⁺ antiporter in regulation of L-type Ca²⁺ channel current by angiotensin II in rabbit ventricular myocytes. Circ Res 1977; 75:1121–1125.[Abstract/Free Full Text]
42. Dieter P, Fitzke E, Duyster J. BAPTA induces a decrease of intracellular free calcium and a translocation and inactivation of protein-kinase-C in macrophages. Biol Chem 1993; 374:171–174.
43. Ebashi S. Calcium binding and relaxation in actomyosin system. J Biochem 1960: 48:150–151.
44. Hales CN, Luzio JP, Chandler JA, Herman L. Localization of calcium in the smooth endoplasmic reticulum of rat isolated fat cells. J Cell Sci 1974; 15:1–15.[Abstract/Free Full Text]
45. McCormack JG, Crompton M. The role and study of mammalian mitochondrial Ca²⁺-transport and matrix Ca²⁺. In: McCormack JG, Cobbold PH (eds.), Cellular Calcium: A Practical Approach. New York: Oxford University Press; 991: 345–382.
46. Maunder CA, Yarom R, Dubowitz V. Electron-microscopic x-ray microanalysis of normal and diseased human muscle. J Neurol Sci 1977; 33:323–334.[CrossRef][Medline]