ABSTRACT
Uptake and incorporation of [3H]uridine by mouse blastocysts was measured in response to serum under various conditions of Ca2+ and Mg2+ availability. Previous studies (Fishel & Surani, 1978) showed that serum stimulated uptake and incorporation of uridine in blastocysts. Here it is shown that in Ca2+-deficient medium ( < 20 μM-Ca2+) cell metabolism was also inhibited and increasing [Ca2+] resulted in increased uptake and incorporation. Maximum stimulation required an extracellular [Ca2+] of 0·25 mM. The effects of low Ca2+ were reversible and could also be alleviated by 15 and 20mM-Mg2+. Magnesium greater than 20 mM was deleterious. Inorganic phosphate (Pi) was used to complex free Mg2+ in order to maximize the effects of Mg2+ deficiency. Inhibition was reversed by increasing [Mg2+] or by 15–20 mM-Ca2+. Calcium concentrations greater than 20 mM inhibited maximum stimulation. Inhibitors of Ca2+ influx D600 and papaverine, inhibited stimulation at concentrations above 5 and 20 μM respectively. Magnesium concentration of 15 mM alleviated the inhibitory effects of 50 μM D600. The effect of Ca2+-deficient medium was also alleviated by Sr2+. The results suggest that both Ca2+ and Mg2+ are required for blastocysts to respond maximally to serum; their initial role appeared to involve the binding of stimulatory serum molecules to the cells of the blastocyst followed by an influx of these cations.
INTRODUCTION
Previous studies have shown that the metabolism of mouse blastocysts is affected by changes in the environment in vivo (McLaren, 1968; Prasad, Dass & Mohola, 1968; Weitlauf, 1971) and in vitro (Gulyas & Daniel, 1969; see Van Blerkom & Manes, 1977; Fishel & Surani, 1978). If, at blastulation, the uterine lumen is not fully conditioned for implantation; such as asynchronous transfer of day-4 embryos to a day-3 uterine horn, or as in the state of ‘lactational delayed embryos’, embryonic metabolism is reduced to a basal level and mitosis ceases. When such blastocysts, in a state of quiescence, are transferred to culture conditions in vitro the rate of cell metabolism increases and mitosis is initiated (see McLaren, 1973). Fishel & Surani (1978) showed that RNA metabolism of blastocysts, cultured in medium containing bovine serum albumin, is enhanced when the embryos are transferred to medium containing 10 % foetal calf serum.
Serum is known to have stimulatory effects on many cell types in culture, it rapidly affects several events which include acceleration in transport of macromolecular precursors (Rubin & Koide, 1975), the rate of nucleoside phosphorylation, which is now thought to be the rate-limiting step rather than uptake (Rozengurt, Stein & Wigglesworth, 1977: Rozengurt, Mierzejewski & Wiggles-worth, 1978) and an array of processes associated with intermediary metabolism and macromolecular synthesis (Rubin & Fodge, 1974; Rozengurt et al. 1977), referred to as the pleiotypic response (Hershko, Mamont, Shields & Tomkins, 1971). Pleiotypic response is postulated to occur in blastocysts (Surani, 1977). Both Ca2+ and Mg2+ have been shown to be involved in the regulation of such diverse cellular functions in many cell types (Balk, 1971; Whitfield et al. 1976; Rubin & Chu, 1978; Balk et al. 1979).
The aim of this work was to determine the role of Ca2+ and Mg2+ in the response of blastocysts to serum, at what concentration of the ion the response was initiated and whether there was a requirement for the influx of the ions into the cell. For the latter no successful Mg2+-influx inhibitor is known, but for inhibition of Ca2+ influx the drug D600 and papaverine were used. D600 is a methoxyl derivative of verapamil originally found to block calcium uptake in cardiac and smooth muscle (Kroeger, Marshall & Bianchi, 1975), the (—) optical isomer was used, having a high specificity for blocking the slow Ca2+ channel (Bayer, Kaufman & Mannhold, 1975); papaverine (6,7-dimethoxy-l veratryl-isoquinoline) has been used by previous workers as an inhibitor of Ca2+ uptake (Imai & Takeda, 1967; Tashiro & Tomita, 1970; Ash, Spooner & Wessells, 1973). This study demonstrates that Ca2+ and Mg2+ are essential for the metabolic response of blastocysts to serum. Calcium can also be replaced by Sr2+.
MATERIALS AND METHODS
Animals
Random bred CFLP mice (Anglia Laboratory Animals) were kept under standard animal house conditions with the lights on between 05.00 and 19.00 h. Immature female mice, aged between 21 and 23 days, were superovulated with an i.p. injection of 5 i.u. pregnant mares serum gonadotrophin followed 45 h later by 2·5 i.u. human chorionic gonadotrophin, and immediately placed with males aged between 3–5 months. The morning when a vaginal plug was detected was designated day-1 p.c. (post coitum).
Materials
Pregnant mares serum (Folliogn) and human chorionic gonadotrophin (Chorulon) were obtained from Intervet (Bar Hill, Cambridge), [5,6-3H]-uridine (sp.act. 58 and 43 Ci/mmol) from the Radiochemical Centre (Amersham) and the general chemicals including ethylene glycol bis (B-amino-ethylether)-N,N’-tetracetic acid (EGTA) from Sigma Chemical Co. (U.K.) or BDH (U.K.). Culture medium was based on Whittingham’s medium (Whittingham, 1971) supplemented with vitamins and amino acids as specified for Eagle’s Minimum Essential Medium (Flow Laboratories). Heat-inactivated virus-screened foetal calf serum (Flow Laboratories) was extensively dialysed against physiological saline free of Ca2+ and Mg2+. N′ -2-hydroxyethylpiperazine-N′ -2-ethanesulphonic acid (Hepes: Flow Laboratories) was used in certain experiments involving supranormal levels of Ca2+. D600 and papaverine were kindly supplied by Dr T. Rink, Physiological Laboratory, Cambridge.
Collection and culture of embryos
Blastocycsts were obtained at 12.00–14.00 h on day-4 p.c. The mice were killed by cervical dislocation, the uterine horns were excised and flushed through with pre-warmed phosphate-buffered saline (PBS) containing 10 mg ml−1 polyvinylpyrrolidine (PVP : May & Baker, U.K.) at 37 °C. Embryos were washed four times in supplemented Whittingham’s medium containing 4 mg ml−1 bovine serum albumin (BSA) (sub-optimal medium) and cultured in the same medium overnight under light liquid paraffin at 37 °C in an atmosphere of 5 % CO2 in air.
The following morning the embryos were transferred to 30 μl experimental medium that contained 10 % dialysed foetal calf serum (optimal medium) with radioactive uridine, 10 μCi/100 μl medium, and 10μM unlabelled uridine to saturate the endogenous UTP pool (Daentl & Epstein, 1971). Depending on the experiment the following stock solutions were used: 10 and 1 mM D600 dissolved in dimethyl sulphoxide, 10 mM papaverine dissolved in 95 % ethanol - the medium was warmed at 45 °C for 15 min to remove the alcohol, Na2HPO4 2H2O at 0·1 M, 0·1 M-CaCl2 and 0T M-MgCl2. EGTA was used at a concentration of 1 mM, to reduce the ionic concentration of Ca2+ to < 0·017 mM, and 10 mM Hepes was used as a buffer. In experiments requiring high Ca2+ concentrations KH2PO4 was omitted to prevent precipitation with Ca2+. The osmolarity of all media was checked on a Precision Systems Osmettes Automatic Osmometer and adjusted to between 290 and 310 mosmole. Calcium and Mg2+-deficient media, prepared with 10% dialysed serum, gave a residual reading of 20 and 25 μM respectively on the Pye Unicam SP90B Series 2 Atomic Absorption Spectrophotometer when measured before each experiment.
Analysis of radioactivity
After a 2 h incubation in labelled medium the blastocysts were washed six times in PBS containing 20 μM unlabelled uridine and PVP. The embryos were pipetted into a solubilizing buffer, with a final volume of 100 μl consisting of 0·14 M 2-mercaptoethanol and 0·1% sodium dodecyl sulphate in a 0·01 M sodium phosphate buffer, pH 7·2, and frozen for up to 1 week.
Samples were frozen and thawed twice and then heated at 65 °C for 1 h in a water bath. Eight 10 μl aliquots were withdrawn from each sample, each pipetted on to a glass-fibre disc (GF/C2-5cm, Whatman) and dried in air. To four of the discs 30 ml ice-cold 10 % trichloracetic acid was run slowly through the discs, under reduced pressure, followed by an equal volume of ethanol. The discs were ether-dried before addition to scintillation vials. The remaining four discs were added directly to scintillation vials. When completely dry 10 ml scintillation fluid, containing 5·5 g Packard-Permablend 1112−1 toluene, was added to each vial. The vials were left overnight in the dark at room temperature and counted in a Tracerlab Scintillation Spectrophotometer with an efficiency of counting computed at 38 %. The soluble counts were determined by subtracting the acid-precipitable counts from the total.
RESULTS
For the purpose of this study the responsiveness of blastocysts was measured by the increased uptake or incorporation of labelled uridine into the acidsoluble pool or acid-precipitable material, respectively. The stimulated uptake and/or incorporation was due to incubating the embryos in medium containing 10% foetal calf serum (control) subsequent to an overnight culture in medium containing bovine serum albumin. This is referred to as the optimal response. The uptake and incorporated values during culture in BSA only were provided as a comparison to the control. However, incorporation values alone do not indicate whether a reduction or stimulation of incorporation is due to a direct effect on transcription per se or a consequence of reduced or enhanced uptake of the precursor, respectively. Hence the calculation of the incorporation : uptake ratio (I/U) for each experiment.
The effect of varying Ca2+ concentration on response
The results shown in Table 1 indicate that incorporation was inhibited to 35 % of the control when embryos were cultured in Ca2+-deficient medium, i.e. 15 % below the BSA value (P < 0·001). In 0·02mM-Ca2+ the stimulation of blastocysts was only 60% of the control (P < 0·05), but significantly different from Ca2+-deficient medium (P < 0·001). Stimulation by 10% dialysed serum was 80% and 90% of the control in 0·1 and 0·12 mM-Ca2+, respectively. However, these two values were not significantly different (P < 0·01). At 0·25 mM-Ca2+ incorporation was significantly higher than at 0·17 mM-Ca2+ (P < 0·02) and similar to the control. There was a significant reduction from the control in the I/U ratio at 0·02 and 04 mM-Ca2+.
Some embryos were maintained in Ca2+-deficient medium for 4 h before transfer to optimal medium. After 3 h in optimal medium the blastocysts were incubated in labelled optimal medium for a further hour. After this period the incorporation of label was similar to the control values also obtained with this experiment (P < 0·60).
The effects of varying Mg2+concentration on response
As in previous work (Rubin, Terasaki & Sanui, 1978), inorganic phosphate (Pi) was used to chelate free Mg2+ and maximize the effects of low Mg2+. The overall results shown in Table 2 show that uptake and incorporation were inhibited by low Mg2+ but this effect was reversed with increasing [Mg2+].
Although incorporation was similar between 1 mM-P, and 0·025 mM-Mg2+ the I/U ratio was significantly higher in the former. Using 5 mM-Pi, incorporation declined, but due to the large sample variation it was not significant (P>0·05 < 0·10). At 0·05 mM-Mg2+stimulation was about 76% of the control, and incorporation in 0·1 and 0·2mM-Mg2+ was similar to the control, but in 0·1 mM the 1/U ratio was significantly higher.
Reversal of Ca2+ or Mg2+ deficient medium by Mg2+ or Ca2+ respectively
At 10mM-Mg2+ inhibition by Ca2+-deficient medium was unaffected, but 15 and 20 mM-Mg2+ alleviated the inhibitory effects. At 25 and 50 mM-Mg2+ incorporation was 71 % and 65 % of the control respectively, and the I/U ratios decreased significantly ; however, the values were not significant from each other (P < 0·10).
In the supranormal Ca2+ experiments the medium was buffered with Hepes to prevent precipitation of high Ca2+ with bicarbonate. Pilot studies indicated no difference between the control experiments and bicarbonate-buffered culture. Similar results were observed to the supranormal Mg2+ levels in Ca2+-deficient medium. However, 10 mM-Ca2+ had a significant effect, inducing stimulation to 70 % of the control (P < 0·001). The rise between 10 and 15 mM Ca2+ was significant (P < 0·01) but between 15 and 20 mM there was no significant difference (P < 0·10).
The effects of D600 and papaverine on response
Table 4 shows the results of culturing blastocysts in various concentrations of D600 for either a short culture of 6 h or an overnight culture. The drug had a dose-dependent inhibitory effect on the uptake and incorporation of [3H]uridine which was enhanced by the longer culture. In the short culture 0·005 mM D600 slightly inhibited incorporation (P < 0·01) and at 0·05 mM D600 stimulation was inhibited by up to 66 %. At 0·1 and 0·25 mM, D600 had deletrious effects on cell metabolism, with an increase in the 1/U ratio. The reduced incorporation at 0·25 mM was significantly lower than 0·1 mM (P < 0·001).
In the long culture, compared to the control, 0·01 mM D600 had an enhanced inhibitory effect on incorporation (P < 0·01). There was a significant enhanced inhibition between 0·125 mM (P < 0·02) and 0·25 mM (P < 0·01) of the short and long cultures and the 1/U ratio of the latter was higher than the control.
Papaverine, Table 5, was not as potent as D600. The drug had a critical effect at 0·02 mM only. Both 0·02 and 0·05 mM induced a significant reduction in the 1/U ratio. At 0·15 mM papaverine, the I/U ratio increased above the control; similar to that observed for D600.
Table 6 shows that the inhibitory effects of D600 were reversed by the addition of 15 mM-Mg2+ and there was no significant difference in the 1/ U ratio.
Table 7, Fig. 2, shows that Sr2+ was able to replace Ca2+ in Ca2+-deficient medium. At 15mM-Sr2+ there was a significant increase in the incorporation value when compared to the control (P < 0·01).
DISCUSSION
When mouse blastocysts are cultured in the absence of serum and then transferred to identical culture conditions with the addition of serum, uptake and incorporation of uridine is stimulated (Fishel & Surani, 1978). The aim of this study was to determine the requirement for Ca2+ and Mg2+ in the stimulation of uridine uptake and incorporation by mouse blastocysts.
The results show that at Ca2+ concentrations below 0·017 mM blastocyst cell metabolism is inhibited. A minimum of 0·25 mM-Ca2+ is required for optimal levels of uptake and incorporation of uridine. Similar I/U ratios indicate that both uptake and, possibly as a consequence of reduced uptake, incorporation are affected. At 0·02 and 0·1 mM-Ca2+ the I/U ratio suggests that some mechanism of transcription is affected. Further experiments show that these effects of low Ca2+ are reversible.
The results in Table 4 and 6 support the view that Ca2+ influx is necessary for stimulation by serum. From the I/U data high [D600] reduce incorporation by affecting uptake, but the critical dose of papaverine seems to act via transcription. As little is known about Ca2+ channels and their blockage by these drugs it is difficult to suggest differences in their mode of action.
Supranormal [Mg2+], 15 and 20 mM, alleviate the effects of Ca2+-deficient medium, but concentrations greater than 25 mM have deleterious effects. Similar results on BALB/c 3T3 cells have been reported (Rubin et al. 1978). The embryo data suggests an inhibition of transcription and is consistent with previous studies on the BALB/c 3T3 cell line (Bowen-Pope, Vidair, Sanui & Rubin, 1979), which showed that a concentration of 40 mM-Mg2+ inhibited the onset of DNA synthesis but did not inhibit uridine uptake.
The inhibitory effect of D600 (Table 5) is also reversed by increasing the [Mg2+], suggesting that D600 inhibition may be due to prevention of Ca2+ flux and not other secondary effects.
The effects of Ca2+-deficient medium are also reversed by replacing the cation with Sr2+ (Table 7), which may displace serum-bound Ca2+. Similar findings have been reported elsewhere (Rubin, 1977).
The inhibitory effect of low Mg2+ (Table 2) on embryo metabolism is not as potent as low Ca2+, and a lower concentration of Mg2+ is required to maintain optimal stimulation. At 1 and 5 mM-P, some mechanism of uptake is inhibited, but at 5 mM-Pi inhibition of transcription is greater than 1 mM-Pi, hence the overall reduction in the I/U ratio. This effect could be due to the increased P, binding free Ca2+, or some other secondary effect on cell metabolism.
Although concentrations of Ca2+ between 15 and 20 mM alleviate the inhibitory effects of Mg2+ (Table 3), at 25 mM the mechanism of uptake is inhibited. This alleviation by supranormal levels of Ca2+ is in contrast to work done on BALB/c 3T3 cells (Rubin et al. 1978), which showed that supranormal Ca2+ (15 mM) has no effect on the stimulation of DNA synthesis in culture inhibited by Mg2+ deprivation. However, these studies used cells cultured in Eagles medium and the bicarbonate precipitates out with concentrations of Ca2+ at or above 5 mM (unpublished observations).
Previous studies on other cells have shown that a marked reduction in Ca2+ concentration in medium containing the physiological concentration of Mg2+ inhibits DNA synthesis and thus the proliferation of cells (Boynton, Whitfield, Isaacs & Morton, 1974; Sabine, Swierenga, Whitfield & Karasaki, 1978; Balk et al. 1979). Milner (1979) showed that mouse spleen cells exposed to the mitogen concanavalin A required an extracellular concentration of Ca2+ greater than 0·01 mM for stimulation, but for optimal response 0·5 mM-Ca2+ was necessary : and the omission of Ca2+ from BALB/c 3T3 cells slightly inhibited uridine uptake after 3 h (Bowen-Pope et al. 1979).
Calcium transport is complex: in 3T3 mouse cells at least two exchangeable cellular compartments are involved : a rapidly exchanging compartment, possibly surface-membrane-localized Ca2+, and a more slowly exchanging intracellular compartment (Hazelton & Tupper, 1979).
In some secretory systems an increase in intracellular Ca2+ is caused by an increased flux of extracellular Ca2+, while others may mobilize intracellular located Ca2+ (see Rasmussen & Goodman, 1977). It has recently been shown that actively metabolizing mouse blastocysts release glycoproteins into their environment in vitro (Fishel & Surani, 1980) and this process, if a secretory mechanism is involved, may be regulated by Ca2+ influx.
Papaverine interferes with Ca2+ flux and may inhibit release of bound Ca2+ (Imai & Takeda, 1967). It has been shown to inhibit characteristic morphogenesis of the salivary gland (Ash et al. 1973), and the neurolation of the amphibian, Ambystoma maculatum, by putative interference with Ca2+ flux (Moran & Rice, 1976).
The effects of Mg2+on the stimulation of various cell types in response to mitogens has been less well studied than Ca2+. Workers on chick embryo fibroblasts showed that uridine incorporation into RNA (Rubin, 1975) and uridine uptake and incorporation (Bowen-Pope & Rubin, 1977) was greatly reduced by Mg2+-deficient media. But similar studies on BALB/c 3T3 cells (Bowen-Pope et al. 1979) showed that omission of Mg2+ from the medium had no effect on uridine uptake. In each of these studies however, only a limiting 1 % serum was used. Magnesium affects the rate and extent of activation of quiescent NIL 8 hamster fibroblasts by serum as judged by uridine uptake (Koren & Shohami, 1979).
There is substantial experimental evidence supporting the role of Ca2+ and Mg2+ as growth-regulating factors in vitro (Berridge, 1976; Rubin & Koide, 1976), and their importance in the co-ordination of cell metabolism has recently indicated that homeostasis of these divalent cations may have a function in initiating tumouriegenicity of different cell types (Boynton & Whitfield, 1976; McKeehan & Ham, 1978; Sabine et al. 1978; Paul & Ristow, 1979; Balk et al. 1979).
The results of this study indicate that extracellular Mg2+ and Ca2+ are required above a critical concentration of about 0·2 mM to permit optimal blastocyst response. Influx of Ca2+ into embryonic cells is necessary, but, in the absence of Ca2+, supranormal levels of Mg2+ induce optimal stimulation; possibly by a rise in internal Ca2+ displaced from bound internal sites.
In the presence of D600 and normal levels of Ca2+, stimulation is inhibited although physiological Mg2+ is maintained and its influx presumably not affected by the drug. Low Mg2+ in the presence of physiological Ca2+ also prevents optimal response. Although it is difficult to resolve the role of these cations definitively in blastocyst response, it is clear that they play an important role by affecting uptake and/or incorporation of uridine.
These findings suggest three possible areas of control of embryonic metabolism in vivo : (a) extracellular components, probably of uterine origin (Fishel, 1979), may interact with the blastocyst cell surface (Tzartos & Surani, 1979) to regulate the permeability of divalent cations or (b) function to lower cytosol divalent cation concentrations – cytosol concentrations of ionized Mg2+ and Ca2+ are known to be regulated by a balance between net passive influx and active extrusion and isolation (Balk et al. 1979) or (c) changes in the luminal content of these cations.
Acknowledgement
I wish to thank Dr M. A. H. Surani for his advice and encouragement with this work, Dr S. Kimber and Mr K. K. Ahuja for helpful suggestions and Dr P. Flatman for help with his atomic absorption spectrophotometer. This work was supported in part by a Medical Research Project Grant to M.A.H.S. and a Ford Foundation Grant. I am grateful to the Medical Research Council for support.