/. Embryol. exp. Morph. Vol. 67, pp. 51-58, 1982
Printed in Great Britain © Company of Biologists Limited 1982
51
Effect of Li+ on preimplantation mouse embryos
ByL. I Z Q U I E R D C ^ A N D M. 1. BECKER1
From the Department of Biology, University of Chile
SUMMARY
Two-cell mouse embryos were cultured in vitro for different periods in a medium in which
NaCl was partially replaced by LiCl at concentrations ranging from 1 to 30 mM. The relative
cell number diminished according to increasing LiCl concentrations but the onset of blastulation was not affected, thus resulting in blastulae with fewer cells than normal and with
a reduced or absent inner cell mass. Results are discussed in terms of the possible mechanisms
involved and are related with the vegetalization induced by Li+ on early embryos of echinoderms and amphibia.
INTRODUCTION
The vegetalization induced by Li + on early embryos of several species has
been ascribed to its interference with a dorsoventral morphogenetic pattern,
although the underlying molecular mechanism still remains obscure (see
Stanisstreet & Osborn, 1976). No pre-existing morphogenetic pattern has been
convincingly described nor experimentally demonstrated in early mammalian
embryos (see Izquierdo, 1977) and for the differentiation of morula to blastocyst the current explanation is the so called inside-outside model (Tarkowski &
Wroblewska, 1967). It states that peripheral cells of an advanced morula
become trophectoderm and interior cells become inner cell mass as a result of
their different microenvironment. However, the accumulated evidence on
behalf of this model does not exclude the possibility of an inside-outside
gradient being set up during cleavage. Even assuming that all blastomeres of
a late morula are similar, if each one produces an equivalent amount of a substance that diffuses to the space around the embryo, one would expect the
establishment of a concentration gradient (Crick, 1970). Would Li + interfere
with such a gradient ? If this is so, may the effect be compared with the vegetalization induced by Li + in early echinoderm and amphibian embryos? Although
these were the main reasons for this inquiry, we were also interested in knowing
whether Li + might affect fertility or development in humans, in view of its
frequent utilization for the treatment of manic-depressive illness (see Baldessarini & Lipinski, 1975).
1
Authors' address: Department of Biology, University of Chile, Casilla 653, Santiago,
Chile.
L. IZQUIERDO AND M. I. BECKER
52
10
0-8
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3
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-V .
* ^#
0-6
^v^
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o
>
"lJ
9
9
9
9
^s^
0-4
0-2
i
i
i
i
i
i
10
15
20
25
30
i
35
mM-LiCl
Fig. 1. Effect of Li+ on relative cell number after culturing embryos for 48 h in
different media. Abscissa represents LiCl concentration in the culture medium.
The ordinate represents the ratio: mean number of cells in treated embryos over
mean number of cells in control embryos. The line, fitted by non-linear regression
analysis, is given by the equation:
ka)x
+k(2), where y(o) = 0-98, k(\) = 00455 and k(2) = 0.
y = y(0) eMean number of cells: controls = 2 4 1 ; 2 HIM = 22-65; 4mM = 19-52; 5 mM =
17-83; 6 m M = 17-59; 8 mM = 16-68; 10 mM = 14-94; 12 mM = 13-25; 15 mM =
1301; 16 mM = 10-84; 20 mM = 8-23; 25 mM = 7-80; 30 mM = 6-38.
MATERIALS AND METHODS
Two-cell embryos were collected from spontaneously ovulating mice of the
Swiss Rockefeller strain after 40 h of development; counting began at 0 h of the
day in which the vaginal plug was detected. The embryos were cultured in
Falcon plastic Petri dishes in microdrops of Bigger's medium (Biggers, Whitten
& Whittingham, 1971) supplemented with 4 mg/ml Bovine Serum Albumin
(Calbiochem) under mineral oil in an atmosphere of 5 % (v/v) CO2 in humid
air at 37 °C. NaCl in the medium was partially replaced by LiCl in concentrations ranging from 1 to 30 mM which means, at most, reducing NaCl by less
than one third. After different periods in culture, all embryos which had not
developed beyond a 4-cell stage were eliminated. The rest were described under
a dissecting microscope and the cell nuclei were counted by Tarkowski's
method (1966). Some embryos were fixed in 3 % glutaraldehyde in 0-1 M cacodylate buffer pH 7-4 for 1 h, post fixed with OsO4 1 %, dehydrated in graded
acetone and embedded in Spurr's resin (Polyscience). Semithin sections
(0-5 ftm) were stained with Toluidine blue in borax and observed with a light
microscope.
Effect of\A+ on preimplantation mouse embryos
100
100
100
100
5 mM-LiCl
100
Controls
95
105
100
Development (h)
110
115
Fig. 2. Effect of Li+ on timing of blastulation. Abscissa represents hours of development in situ plus hours of development in vitro. The ordinate represents percentage
of blastulae developed in control medium and in media with different LiCl concentrations. The lines, fitted by non-linear regression analysis, is given by the
equation:
y = 100-JC(3) Exp (x(l)Tx(2).
Values: for controls: x(l) = 0175, x(2) = 86-3, x(3) = 100; for LiCl 5mM:
x(\) = 0137, x(2) = 85-8, x(3) = 100; for LiCl 10HIM: X(\) = 0133, x(2) =
860, x(3) = 100; for LiCl 15 HIM: X(\) = 0110, x(2) = 87-3, x(3) = 85; for LiCl
20HIM: X(\) = 0097, x(2) = 86-8, *(3) = 75.
53
54
L. IZQUIERDO AND M. I. BECKER
30
Fig. 3. Effect of Li+ on the number of cells at blastulation. Abscissa represents
LiCl concentrations. The ordinate represents number of cells at the morulablastocyst transition. The line, fitted by non-linear regression analysis, is given by
equation:
y
= y(o)e-m)*+k(2),
where y(o) = 15-81, *(1) = 00815 and k(2) = 719
The experimental series were designed so as to test the effect of different LiCl
concentrations on relative cell number, on timing of blastulation, and on
number of cells at blastulation.
RESULTS
+
Effect ofLi on relative cell number. In each experiment the embryos obtained
from two or more females were pooled and cultured in two microdrops, one
with and the other without LiCl. After 48 h in culture, the cells in each embryo
were counted and the relative cell number expressed as the ratio of the mean
number of cells in treated embryos over the mean number of cells in control
embryos. This procedure takes care of wide variations that might occur in the
timing of ovulation and fertilization. Figure 1 shows that the relative cell
number diminishes as a function of Li + concentration. Each point represents
one experiment with an average of 10 embryos cultured with Li + and 6 control
embryos. The total number of embryos examined in this experimental series
is 566.
The cumulative effect of Li + during a 48 h culture provides no information
on whether early or late cleavage might be preferentially affected; therefore, in
another series of experiments, 170 2-cell embryos were cultured for two
successive 24 h periods separated by a brief rinse. When the embryos were
Effect ofLi+ on preimplantation mouse embryos
55
Fig. 4. Semithin section through a meridian plane of a blastocyst developed in
control medium (a) a blastocyst with a reduced inner cell mass developed in
a medium with 15 mM-LiCl (b) and a trophoblastic vesicle developed in a medium
with 20 mM-LiCl (c).
cultured with 10 mM-LiCl during the first period and without the salt during
the second period, the relative cell number was 0-91 ±0-05 while inverting the
sequence of cultures the ratio was reduced to 0-78 ± 0-06. These results show
that late cleavage is more affected than early cleavage and additionally, that
rinsing removes Li + from its site of action. Shorter culture periods were not
tried because the different phases of the cell cycle, which in this material
normally takes 11 h (Luthardt & Donahue 1975), may introduce some confusion in the interpretation of the results.
Effect of Li+ on timing of blastulation. The onset of blastulation in embryos
which were cultured in different LiCl concentrations is shown in Fig. 2. The
abscissa represents hours of development in situ plus hours of development in
vitro. The points represent the percentage of blastulae in microdrops containing
9 or 10 embryos. Each microdrop was observedtwo or three times during the
30 h long experiments. The total number of embryos observed in control
cultures was 214 and in cultures with 5, 10, 15 and 20 mM-LiCl, the number of
embryos was 121, 87, 88 and 75, respectively. Figure 2 shows that control
embryos, as well as embryos cultured in different Li + concentrations, begin to
blastulate at 87-88 h of development and that all those which blastulate do so
within a 20 h interval. After 108 h of development, 100 % of the embryos have
blastulated in cultures with up to 10 mM-LiCl whereas only 85 % blastulated in
15 mM and 75 % in 20 mM-LiCl, even though these cultures weie observed for
24 additional hours.
Our observations on the timing of compaction are somewhat incomplete but
they suggest that compaction, similarly to blastulation, is not retarded by Li + .
Effect ofLi+ on number of cell at blastulation. Since the precise recognition of
nascent blastulae lends itself to subjective appraisals we took as the number of
56
L. IZQUIERDO AND M. I. BECKER
cells at blastulation the mean number of cells per embryo in populations which
included only late morulae and early blastulae. That is, excluded from these
populations were all morulae with less cells than the evident blastula with less
cells, and all blastulae with more cells than the evident morula with more cells.
For example: 122 embryos cultured during 48 h without Li+ attained different
stages, from a 15-cell morula up to a 30-cell blastocyst; however, since the
latest evident morula had 25 cells and the earliest evident blastocyst had 21 cells,
we calculated the mean number of cells per embryo in a population including
only the 31 embryos which had more than 21 and less than 25 cells. The mean
number was 23-32 + 28 (Fig. 3). Each point in Fig. 3 represents the mean number
of cells per embryo in populations comprising an average of 17-5 embryos. The
total number of embryos considered in these series was 141. Figure 3 shows that
cell number at blastulation decreases as LiCl concentration increases.
Effect of Li+ on embryo morphology. The blastulae obtained were either
normal blastocysts or blastocysts with a reduced or even absent inner cell mass.
The latter, that is trophoblastic vesicles, account for 6, 21 and 43 % of the
embryos which blastulated in cultures containing 10, 15 and 20 mM-LiCl
respectively. Figure 4 shows sections through the meridian plane of a blastocyst
developed in control medium, a blastocyst with reduced inner cell mass developed
in a medium' with 15 mM-LiCl and a trophoblastic vesicle developed in a medium
with 20 mM-LiCl.
DISCUSSION
It has been shown in mammalian nervous tissue that Li + , at concentration
similar to those used in this research, inhibits the synthesis of cyclic nucleotides
probably by interfering with the role of divalent cations on the activity of
adenyl cyclase and guanyl cyclase (Forn & Valdecasas, 1971; Bunney et ah
1979; Zatz, 1979). Since the levels of cyclic nucleotides have been related to cell
proliferation in several differentiated tissues (Berridge, 1975) one might assume
that this is the mechanism by which Li + affects mammalian cleavage. However,
in sea-urchin embryos, no variations in adenyl cyclase activity have been
detected during cleavage nor are variations in cleavage induced by exogenous
cAMP, though its intracellular level increases greatly (Amy & Rebhun, 1977).
As to the onset of blastulation, since it was almost simultaneous in all
experimental series, our results support the idea that its timing in the mouse
depends neither on the total number of cells in the embryo nor on the number
of cell cycles elapsed since fertilization (Smith & McLaren, 1977; Fernandez &
Izquierdo, 1980). This conclusion does not imply that mammalian blastulation
depends on a process which is triggered by the activation of the oocyte and then
proceeds unchecked by embryonic metabolism; actually, blastulation in the
mouse has been supressed by a-amanitin (Braude, 1979), reversibly retarded by
an inhibitor of polyamine biosynthesis (Alexandre, 1979) and by rabbit antiserum to a mouse embryonal carcinoma cell line (Johnson et al. 1979).
Effect of\A+ on preimplantation mouse embryos
57
The increasing percentage of trophoblastic vesicles which developed in
cultures with increasing concentration of LiCl finds a suitable interpretation in
the inside-outside model. Since cleavage is retarded by Li + while blastulation is
not, when the latter occurs all cells may lie outside and therefore, according to
the model, would differentiate into trophectoderm. However convincing this
explanation may be in the case of mammals it may not account for vegetalization induced by Li + in early sea-urchin and amphibian embryos.
Cleavage of amphibian ectoderm is retarded by Li + (Flickinger, Lauth &
Stambrook, 1970; Osborn & Stanisstreet, 1977) although at concentrations
which are about ten times higher than those used in our research. However, if
Li + were to retard cleavage without delaying blastulation in echinoderms and
amphibia as it does in mammals, one might conceive a general interpretation of
vegetalization assuming that cells of each presumptive germ layer must divide
a certain number of times before blastulation (and/or gastrulation) in order to
differentiate normally. This conjecture, in our opinion, deserves a test. Information available at present does not refute nor support the idea that trophoblastic vesicle formation is a vegetalization effect induced by Li + . Therefore,
it would be p emature to suggest that during cleavage of the mouse embryo
emerges a Li+-sensitive gradient.
Now, as to the effect that Li + may have on humans, since the effective concentrations of this ion in our experiments are at least 2 or 3 times higher than
therapeutical doses (Baldessarini & Lipinski, 1975), our results do not support
reservations about unwanted effects on fertility or preimplantation development.
This research was partially financed by Grants from PLAMIRH, PNUD/UNESCO
Regional Program and the Ford Foundation.
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{Received 22 December 1980, revised 2 August 1981)