/. Embryo!. exp. Morph. Vol. 58, pp. 217-229, 1980
Printed in Great Britain © Company of Biologists Limited 1980
217
The role of divalent cations in the metabolic
response of mouse blastocysts to serum
By S. B. FISHEL 1
From the Marshall Laboratory, University of Cambridge
Beit Memorial Research Fellow
SUMMARY
3
Uptake and incorporation of [ H]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 (< 20/4M-Ca2+) cell metabolism was
also inhibited and increasing [Ca2+] resulted in increased uptake and incorporation. Maximum stimulation required an extracellular [Ca2+] of 025 mM. The effects of low Ca2+ were
reversible and could also be alleviated by 15 and 20 mM-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 /*M respectively. Magnesium concentration of 15 mM alleviated the inhibitory effects
of 50 /AM 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.
1
Author's address: Institute of Animal Physiology, Animal Research Station, 307
Huntingdon Road, Cambridge, CB3 OJQ, U.K.
218
S. B. FISHEL
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 & Wigglesworth, 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 Mg 2+ 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 Mg 2+ 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 Mg 2+ 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)7V,Ar-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
Metabolic response of mouse blastocysts to serum
219
calf serum (Flow Laboratories) was extensively dialysed against physiological
saline free of Ca 2+ and Mg 2+ . A^-2-hydroxyethylpiperazine-7V'-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 4mgml~ 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 [A experimental
medium that contained 10 % dialysed foetal calf serum (optimal medium) with
radioactive uridine, 10/^Ci/100/*l medium, and 10/AM unlabelled uridine to
saturate the endogenous UTP pool (Daentl & Epstein, 1971). Depending on the
experiment the following stock solutions were used: 10 and 1 IDM D600 dissolved in dimethyl sulphoxide, 10 HIM papaverine dissolved in 95 % ethanol the medium was warmed at 45 °C for 15 min to remove the alcohol, Na 2 HPO 4
2H2O at 0-1 M, 0-1 M-CaCl2 and 0-1 M-MgCl2. EGTA was used at a concentration of 1 mM, to reduce the ionic concentration of Ca 2+ to < 0-017 mM, and
10 mM Hepes was used as a buffer. In experiments requiring high Ca 2+
concentrations KH 2 PO 4 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 /*M 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 /AM unlabelled uridine
and PVP. The embryos were pipetted into a solubilizing buffer, with a final
volume of 100 /A 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 [A aliquots were withdrawn from each sample, each
pipetted on to a glass-fibre disc (GF/C 2-5 cm, 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 com15
EMB 58
220
S. B. FISHEL
Table 1. The effect of Ca2J-deficient medium on the responsiveness
of blastocysts to serum
[Ca 2+ ] (mM)
Control
BSA (10)
< 0017
0020
0100
0170
Av. acid- Av. acidprecipitable soluble
dpm/blasto- dpm/blastocyst/h
cyst/h
±S.E.M.
±S.E.M.
3135 ±70
1554 ±72
1097±29
1898 ±104
2508 ±107
2815 ±94
9947 ±278
4 855 ±249
35O9±183
7 607 ±449
8981 ±565
9303 ±493
Av.
I/U±S.E.M.
0-3162±00087*, t
0-3212 ±00079
0-3159 ±00129
0-2498 ±00030*,**
0-2805 + 0-0066**, ***, t
0-3132±00161***
0-250
3342±147 10111±360 0-3317 ±00182
Ca2+-deficient\
Optimal
/ 3053 ±123 9657 ±395 0-3165 ±00099
No. of
experiments
Total
no. of
embryos
7
7
7
4
4
76
80
76
39
46
4
44
4
37
3
39
* P < 0001, ** P < 001, *** P < 0-20, t P < 001.
pletely 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
Metabolic response of mouse blastocysts to serum
221
Table 2. The effect of Mg24-deficient medium on the responsiveness
of blastocysts to serum
[Mg2+] (mM)
Av. acid- Av. acidprecipitable soluble
dpm/blasto- dpm/blastocyst/h
cyst/h
±S.E.M.
Control
3544±159
1744 ±104
0025 + 5mM Pi 1578 ±24
0025 + 1 HIM Pi 1924+140
0025
1921 ±92
0050
2 682 ±70
0100
3 361 ±148
0-200
3271 ±68
BSA
±S.E.M.
Av. //£/± S.E.M.
11293 ±613
5071 ±806
4397 ±757
4141 + 536
6343 ±301
7 975 ±620
8 302 ±522
9405 ±578
No. of
experiments
0-3155±00093***, t, t t , ttt
7
O-3115±OO158
0-3776 ±00544**,***
7
3
0-4708 ± 0-0243*, * * , t
3
0-3043 ±00167*
0-3404 ± 00177ft
0-4079 ± 00231 f t t
0-3511 ±00191
4
4
4
4
Total
no. of
embryos
101
75
39
35
40
46
37
39
* P < 001, ** P < 0-20, *** P < 0001, f P < 0001, f t P < 0-30, f t t P < 002.
Table 3. Reversal of inhibition of responsiveness by supranormal levels of
Mg2+ or Ca2+
Supranormal Mg2+ irl Ca2+-deficient medium
Av. acidAv. acidprecipitable
soluble
No. of
dpm/blasto- dpm/blastoexperi[Mg 2+ ] (mM) cyst/h±s.E.M. cyst/h± S.E.M.
Av. I/U± S.E.M.
ments
Control
10
50
100
150
200
250
500
3 071 ±98
1659 ±126
1706 ±55
1786 ±53
3025 + 50
3194 ±99
2179 ±65
1982 ±63
10091 ±425
6 894 ±389
6 040 ±405
5987±253
9688 + 348
10422 ±452
8 206 ±446
7270 ±350
0-3048 ±0-0051*, **K
0-2811 ±00079
0-2857 ±00180
0-3005 ±00188
0-3136 + 0-0129
0-3084 ±00170
0-2673 ±00129*
0-2557±00068**
4
4
4
4
4
4
4
4
Total
no. of
embryos
44
39
41
43
45
29
45
43
* P < 005,** P < 0001.
Supranormal Ca2+ in Mg2+-deficient medium
Av. acidAv. acidprecipitable
soluble
No. of
dpm/blastodpm/blastoexperi[Caa+] (m M ) cyst/h ± S.E.M. cyst/h± S.E.M.
Av. I/U± S.E.M.
ments
Control
50
100
150
200
250
22 36 ±42
1290 ±74
1912 ±26
2 280 ±63
2076 ±85
1420 ±58
6 964 ±252
5 654 ±362
6 355 ±290
7 220 ±282
6 736 ±208
5228 ±321
0-3223 ±00126*
0-2333 ±00268
0-3032 ±00173
0-3170± 00134
0-3081 ±00052
0-2750 ±00205*
4
4
4
4
4
4
Total
no. of
embryos
70
72
62
57
44
60
* P < 010.
15-2
222
S. B. FISHEL
Table 4. Effects of D600 on uptake and incorporation of
[3H]uridine into blastocysts
Av. acidAv. acidprecipitable
soluble
dpm/blasto[D600;I dpm/blasto- % o f
OM) cyst/h ± S.E.M. control cyst/h ± S.E.M.
Control 3 060 ±49
10
3037±lll
50
2 705 ±47
100
2 368 ±41
500
1868 ±71
982 ±22
1000
938 ±20
1250
2500
783 ±5
10000
99-25
88-41
77-41
6707
3210
30-67
25-60
Control 3031 ±102 10000
10
2 733 ±95
9017
50
2 694 ±93
88-88
100
1965 ±63
64-83
500
1738 ±94
57-34
944 ±40
1000
31-14
748 ±49
1250
24-68
654 ±28
2500
21-58
%of
control
Av. I/U
± S.E.M.
No. of
experiments
Short culture (6h)
9611±341 10000 0-3191 ±00119*** 3
3
9832±617 102-29 0-3122 ±00270
3
99-76 0-2878 ±00286
9 588 ±963
3
8 661 ±847
9011 0-2788 ±00275
3
6184±626
64-34 0-3126 + 0-0448
3
30-97 0-3418 ±00449
2977 + 404
3
2 033 ±486
2115 O-5136±O1O93
1530±188
15-92 0-5282 ±00670*** 3
Long culture (overnight)
9 539 ±436 10000 0-3184±00103*, **: 3
3
10081 ±515 105-68 0-2723 ±00149
3
8 894 ±563
93-25 0-3043 ±00129
7381 ±553
77-38 0-2690 ±00193** 3
3
6173±117
64-72 0-2814±0-0119
3
3 699 ±684
38-78 0-2704 ±00417
3
2633 ±451
27-61 0-2953 ±00341
3
1505 ±249
15-78 0-4509 ±00499*
Total
no. of
embryos
41
32
32
33
29
35
30
37
43
34
34
30
36
29
28
33
* P < 005, ** P < 010., *** P < 005.
Table 5. Effects of papaverine on uptake and incorporation of
[3H]uridine into blastocysts
Av. acid[papa- precipitable
verine] dpm/blastoOM) cyst/h ± S.E.M.
%of
Av. acidsoluble
dpm/blasto-
control cyst/h ± S.E.M.
%of
Av. I/U
control
± S.E.M.
Control 2 856 ±49
10000
9264±186
10000
2819±80
2 775 ±71
1580 ±95
1455 ±93
1576±113
1493 ± 83
1483 ±10
98-72
9715
55-33
50-95
55-18
52-29
51-92
9 368 ±395
9924±513
8 619 ±244
6111±215
5964±132
5 480 ±442
3 632 ±239
10112
10712
93 03
65-96
64-37
5915
39-20
50
100
200
500
700
1000
1500
No. of Total
experi- no. of
ments embryos
0-3085 ±00079*, **,3
T
3
0-3105 ±00124
3
0-2817 ±00202
3
01833 ±00098*
O-238O±O-O117*<* 3
3
0-2677 ±00249
3
0-2743 ±0-1553
3
0-4117 ±00255f
* P < 0001, ** P < 001, t P < 002.
33
30
26
36
27
29
31
34
Metabolic response of mouse blastocysts to serum
223
Table 6. Reversal of the inhibitory effects of D600 by
supranormal levels of Mg2+
Av. acidprecipitable
dpm/blasto-
%of
Av. acidsoluble
dpm/blasto-
Experiment cyst/h± S.E.M. control cyst/h± S.E.M.
Control
50 /*M D600
2403 ±61
1432 ±129
5 0 / « M D 6 0 0 +• 2440 ±82
15 mM Mg2+
10000
59-61
101-55
7 806 ±286
4153 ±1227
7 950 ±450
%of
control
Av. I/U
± S.E.M.
10000 0-3083 ±00059*
53-21 0-2724 ±00170*
101-84 0-3090 ±00149
No. of
experiments
Total
no. of
embryos
4
4
4
48
43
40
*P<0- 10.
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
0-1 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 (Pj) was used to ch elate
free Mg 2+ and maximize the effects of low Mg 2+ . The overall results shown in
Table 2 show that uptake and incorporation were inhibited by low Mg 2+ but
this effect was reversed with increasing [Mg2+].
Although incorporation was similar between 1 mM-Pi 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 Ca2Ar or Mg2+ deficient medium by Mg2+ or Ca2+ respectively. A t
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 Ca 2+ 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 Mg 2+ levels in Ca2+-deficient
medium. However, 10 mM-Ca2+ had a significant effect, inducing stimulation to
224
S. B. FISHEL
Table 7. Effects on responsiveness of blastocysts of replacement of
Ca2+ with Sr2+
Av. acidAv. acidprecipitable
soluble
dpm/blasto- % o f
[Sr2+] dp m/blast o- % o f
(mM) cyst/h±s.E.M. control cyst/h ± S.E.M. control
Control
10
50
100
150
2271 ±61
2346±91
2366+146
2 738 ±275
2656±155
10000
103-30
10416
120-55
116-93
7 370 ±294
7665 ±231
7 557 ±220
8 204 ±395
7 939 ±392
10000
10401
102-54
111-32
107-73
Av. I/U
± S.E.M.
No. of
experiments
0-3093 ±00063*, **
0-3067 ±00116
0-3144 ±00216
0-3262 ±00248*
0-3366 ±00202**
5
5
5
5
5
Total
no. of
embryos
112
106
74
79
89
* P < 0-30, ** P < 0-30.
70% of the control (P < 0-001). The rise between 10 and 15 mM Ca 2+ 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 I/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 I/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
I/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 I/U ratio.
Table 7, Fig. 2, shows that Sr2+ was able to replace Ca 2+ 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
Metabolic response of mouse blastocysts to serum
225
incorporation of uridine is stimulated (Fishel & Surani, 1978). The aim of this
study was to determine the requirement for Ca2+ and Mg 2+ in the stimulation of
uridine uptake and incorporation by mouse blastocysts.
The results show that at Ca 2+ 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 Ca 2+ are reversible.
The results in Table 4 and 6 support the view that Ca 2+ 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 Ca 2+ 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 Ca 2+ . Similar findings
have been reported elsewhere (Rubin, 1977).
The inhibitory effect of low Mg 2+ (Table 2) on embryo metabolism is not as
potent as low Ca2+, and a lower concentration of Mg 2+ is required to maintain
optimal stimulation. At 1 and 5 mM-Pj some mechanism of uptake is inhibited,
but at 5 mM-Pj inhibition of transcription is greater than 1 mM-Pi5 hence the
overall reduction in the I/U ratio. This effect could be due to the increased Pj
binding free Ca 2+ , or some other secondary effect on cell metabolism.
Although concentrations of Ca 2+ between 15 and 20 mM alleviate the inhibitory effects of Mg 2+ (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 Mg 2+ deprivation. However, these studies used cells cultured in Eagles
medium and the bicarbonate precipitates out with concentrations of Ca 2+ at or
above 5 mM (unpublished observations).
Previous studies on other cells have shown that a marked reduction in Ca 2+
226
S. B. FISHEL
concentration in medium containing the physiological concentration of Mg 2+
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 Ca 2+ greater
than 0-01 mM for stimulation, but for optimal response 0-5 mM-Ca2+ was necessary: and the omission of Ca 2+ 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 Ca 2+ is caused by an
increased flux of extracellular Ca2+, while others may mobilize intracellular
located Ca 2+ (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 Ca 2+ influx.
Papaverine interferes with Ca 2+ 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 Ca 2+ flux
(Moran & Rice, 1976).
The effects of Mg 2+ on the stimulation of various cell types in response to
mitogens has been less well studied than Ca 2+ . 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 Mg 2+ 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 Ca 2+ and
Mg 2+ 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 Mg 2+ and Ca 2+ are required
above a critical concentration of about 0-2 mM to permit optimal blastocyst
response. Influx of Ca 2+ into embryonic cells is necessary, but, in the absence of
Metabolic response of mouse blastocysts to serum
2+
227
2+
Ca , supranormal levels of Mg induce optimal stimulation; possibly by a
rise in internal Ca 2+ displaced from bound internal sites.
In the presence of D600 and normal levels of Ca 2+ , stimulation is inhibited
although physiological Mg 2+ is maintained and its influx presumably not
affected by the drug. Low Mg 2+ in the presence of physiological Ca 2+ 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 Mg 2+ and
Ca 2+ 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.
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.
REFERENCES
J. F., SPOONER, B. S. & WESSELLS, N. K. (1973). Effects of papaverine and Ca++-free
medium on salivary gland morphogenesis. Devi Biol. 33, 463-469.
BALK, S. D. (1971). Calcium as a regulator of the proliferation of normal but not of transformed chicken fibroblasts in a plasma containing medium. Proc. natn. Acad. Sci. U.S.A.
70, 675-679.
BALK, S. D., POLIMENI, P. I., HOON, B. S., LESTOURGEON, D. N. & MITCHELL, R. S. (1979).
Proliferation of Rous sarcoma virus-infected but not of normal, chicken fibroblasts in a
medium of reduced calcium and magnesium concentrations. Proc. natn. Acad. Sci. U.S.A.
76, 3913-3916.
BAYER, R., KAUFMAN, R. & MANNHOLD, R. (1975). Inotropic and electrophysiological actions
of verapamil and D600 in mammalian myocardium. Archs Pharmacol. 290, 69-80.
BERRIDGE, M. J. (1976). Calcium, cyclic nucleotides and cell division. In Calcium in Biological
Systems (ed. C. J. Duncan), pp. 219-231. New York: Cambridge University Press.
BOWEN-POPE, D. F. & RUBIN, H. (1977). Magnesium and calcium effects on uptake of hexoses
and uridine by chick embryo fibroblasts. Proc. natn. Acad. Sci. U.S.A. 74, 1585-1589.
BOWEN-POPE, D. F., VIDAIR, C , SANUI, H. & RUBIN, A. H. (1979). Separate roles for calcium
and magnesium in their synergistic effect on uridine uptake by cultured cells: significance
for growth control. Proc. natn. Acad. Sci. U.S.A. 76, 1308-1312.
BOYNTON, A. L. & WHITFIELD, J. F. (1976). Different calcium requirements for proliferation
of conditionally and unconditionally tumorigeneic mouse cells. Proc. natn. Acad. Sci.
U.S.A. 73, 1651-1654.
ASH,
BOYNTON,A. L.,WHITFIELD, J. F., ISAACS, R. J. & MORTON, H. J. (1974). Control of 313 pro-
liferation by calcium. In Vitro 10, 12-17.
D. L. & EPSTEIN, C. J. (1971). Development interrelationships of uridine uptake,
nucleotide formation and incorporation into RNA by early mammalian embryos. Devi
Biol. 24, 428-442.
DAENTL,
228
S. B. FISHEL
FISHEL, S.
B. (1979). Analysis of mouse uterine proteins at pro-oestrus, during early pregnancy
and after administration of exogenous steroids. / . Reprod. Fert. 55, 91-100.
FISHEL, S. B. & SURANI, M. A. H. (1978). Changes in responsiveness of pre-implantation
mouse embryos to serum. / . Embryol. exp. Morph. 45, 295-301.
FISHEL, S. B. & SURANI, M. A. H. (1980). Evidence for the synthesis and release of a glycoprotein by mouse blastocysts. J. Reprod. Fert. (in press.)
GULYAS, B. J. & DANIEL, J. C. Jr. (1969). Incorporation of labelled nucleic acid and protein
precursors by diapausing and non-diapausing blastocysts. Biol. Reprod. 1, 11-20.
HAZELTON, B. J. & TUPPER, J. T. (1979). Calcium transport and exchange in mouse 3T3 and
SV4O-3T3 cells. / . Cell Biol. 81, 538-542.
HERSHKO, A., MAMONT, P., SHIELDS, R. & TOMKINS, G. (1971). Pleiotypic response. Nature
New Biol. 232,206-211.
IMAI, S. & TAKEDA, K. (1967). Effects of vasodilators on the isolated taenia coli of the guineapig. Nature, Lond. 213, 509-511.
KOREN, R. & SHOHAMI, E. (1979). Magnesium ions and serum activation of uridine uptake
by quiescent hamster fibroblasts. FEBS Lett. 106, 149-152.
KROEGER, F. A., MARSHALL, J. M. & BIANCHI, C. P. (1975). Effects of isoproterenol and
D600 on calcium movements in rat myometrium. / . Pharmacol, exp. Ther. 193, 309-316.
MCKEEHAN, W. L. & HAM, R. G. (1978). Calcium and magnesium ions and the regulation of
multiplication in normal and transformed cells. Nature, Lond. 275, 756-758.
MCLAREN, A. (1968). A study of blastocysts during delay and subsequent implantation in
lactating mice. / . Endocr. 42, 453-463.
MCLAREN, A. (1973). Blastocyst activation. In The Regulation of Mammalian Reproduction
(ed. S. J. Segal, R. Crozier, P. A. Corfman & D. G. Condliffe). pp. 321-328. Springfield,
Illinois: Charles C. Thomas.
MILNER, S. M. (1979). Activation of lymphocytes by concanavalin A requires calcium ions.
Cell Biol. Int. Rep. 3, 35-43.
MORAN, D. J. & RICE, R. W. (1976). Action of papaverine and ionophore A23187 on neurulation. Nature Lond. 261, 497-499.
++
PAUL, D. & RISTOW, H. J. (1979). Cell cycle control by Ca -ions in mouse 3T3 cells and in
transformed 3T3 cells. / . Cell. Physiol. 98, 31-40.
PRASAD, M. R. N., DASS, C. M. S. & MOHOLA, S. (1968). Action of oestrogen on blastocyst
and uterus in delayed implantation: an autoradiography study. / . Reprod. Fer. 16, 97-104.
RASMUSSEN, H. & GOODMAN, D. B. P. (1977). Relationship between calcium and cyclic nucleotides in cell activation. Physiol. Rev. 57, 421-409.
ROZENGURT, E., MIERZEJEWSKI, K. & WIGGLESWORTH, N. (1978). Uridine transport and
phosphorylation in mouse cells in culture: effect of growth-promoting factors, cell cycle
. transit and oncogenic transformation. / . Cell. Physiol. 97, 241-252.
ROZENGURT, E., STEIN, W. D. & WIGGLESWORTH, N. M. (1977). Uptake of nucleosides in
density inhibited cultures of 3T3 cells. Nature, Lond. 267, 442-444.
RUBIN, H. (1975). Central role of magnesium in coordinate control of metabolism and
growth in animal cells. Proc. natn. Acad. Sci. U.S.A. 72, 3551-3555.
RUBIN, H. (1977). Specificity of the requirements for magnesium and calcium in the growth
and metabolism of chick embryo fibroblasts. / . cell. Physiol. 91, 449-458.
RUBIN, A. H. & CHU, B. (1978). Reversible regulation by magnesium of chick embryo
fibroblast proliferation. / . cell. Physiol. 94, 13-20.
RUBIN, H. & FODGE, D. (1974). Interrelationships of glycolysis, sugar transport and initiation
of DNA synthesis in chick embryo cells. In Control of Proliferation of Animal Cells, vol. 1
(ed. B. Clarkson & R. Baserga), pp. 801-816. Cold Spring Harbor, New York.
RUBIN, H. & KOIDE, T. (1975). Early cellular responses to diverse growth stimuli independent
of protein and RNA synthesis. / . cell. Physiol. 86, 47-58.
RUBIN, H. & KOIDE, T. (1976). Mutual potentiation by magnesium and calcium of growth in
animal cells. Proc. natn. Acad. Sci. U.S.A. 73,. 168-172.
RUBIN, A. H., TERASAKI, M. & SANUI, H. (1978). Magnesium reverses inhibitory effects of
calcium deprivation on coordinate response of 3T3 cells to serum. Proc. natn. Acad. Sci.
U.S.A. 75, 4379-4383.
Metabolic response of mouse blastocysts to serum
229
H., SWIERENGA, H., WHITFIELD, J. F. & KARASAKI, S. (1978). Loss of proliferative
calcium dependence: simple in vitro indicator of tumorigeneity. Proc. natn. Acad. Sci.
U.S.A. 75, 6069-6072.
SURANI, M. A. H. (1977). Cellular and molecular approaches to blastocyst uterine interactions
at implantation. In Development in Mammals, vol. 1 (ed. M. H. Johnson), pp. 245-305.
Amsterdam: North Holland.
TASHIRO, N. & TOMITA, T. (1970). Effects of papaverine on the electrical and mechanical
properties of the guinea pig. Taenia Coli. Br. J. Pharmac. 39, 608-618.
TZARTOS, S. J. & SURANI, M. A. H. (1979). Affinity of uterine luminal proteins for rat
blastocysts. /. Reprod. Fert. 56, 579-586.
VAN BLERKOM, J. & MANES, C. (1977). The molecular biology of the preimplantation embryo.
In Concepts in Mammalian Embryogenesis (ed. M.I.Sherman), pp. 37-94. London:
MIT Press.
WEITLAUF, H. M. (1971). Effect of progesterone on survival of blastocyst in the uteri of
ovariectomized mice. /. Endocr. 51, 375-380.
SABINE,
WHITFIELD, J. F., MACMANUS, J. P., RIXON, R. H., BOYNTON, A. L., YARDALE, T. & SWIERENGA, S. H. M. (1976). The positive control of cell proliferation by the interplay of calcium
ions and cyclic nucleotides - a review. In Vitro 12, 1-18.
D. G. (1971). Culture of mouse ova. /. Reprod. Fert. (Suppl.) 14, 7-21.
WHITTINGHAM,
{Received 10 December 1979, revised 13 February 1980)