/. Embryo/, exp. Morph. Vol. 45, pp. 237-247, 1978
Printed in Great Britain © Company of Biologists Limited 1978
237
Isolation and development of the
inner cell mass after exposure of mouse embryos to
calcium ionophore A23187
By M. AZIM H. SURANI, 1 DAVID TORCH1ANA 2
AND SHEILA C. BARTON 1
From the Physiological Laboratory, University of Cambridge
SUMMARY
Compacted morulae and blastocysts were obtained from CBA, BALB/c and CFLP strains
of mice. The embryos were incubated in medium containing 2 x 10~5 M or 2 x 10~6 M ionophore A 23187. With 2 x 10~6 M ionophore, morulae survived for up to 12 h showing slight
decompaction. Normal development resumed when the morulae were explanted to fresh
medium. There was no detectable effect on blastocysts. With 2 x 10~5 M ionophore, morulae
survived for about 20 min and then extensive cell death occurred after this time. With
blastocysts however, selective lysis of trophectoderm cells occurred after approximately
30 min following their swelling and vesiculation but the inner cell mass cells (ICM) remained
apparently intact and viable. Nearly 80 % of the early blastocysts obtained 87 h postovulation and all of the late blastocysts used after 12 h in culture (99 h blastocysts) showed
this response. Individual fluid accumulating cells were detected in a few isolated ICMs after
their overnight culture in vitro, especially in those obtained from early blastocysts, but the
majority of the ICMs did not have these cells. All aggregates of three to five ICMs, except
one which reformed into a blastocyst, developed as embryoid bodies after 2 days in culture
and these survived for up to 10 days; in some cases they developed into cystic embryoid
bodies or attached to the culture dish displaying a variety of cell types. The development of
the isolated ICMs in vivo was judged to be normal after their transfer to intact host blastocysts as these developed as chimaeric embryos to term.
INTRODUCTION
Development of eight-cell preimplantation mouse embryos, consisting of loosely associated blastomeres, is followed by formation of intercellular desmosomes,
gap junctions and tight junctions which result in the formation of compacted
morulae (Ducibella, Albertini, Anderson & Biggers, 1975). The process of compaction is calcium-dependent (Ducibella & Anderson, 1976) and precedes blastulation by about 12 h. Blastocysts consist of 30-40 cells with an outer layer of
trophectoderm cells with complex intercellular tight junctions called 'zonula
1
Authors' address: Physiological Laboratory, University of Cambridge, Cambridge
CB2 3EG, U.K.
2
Author's address: Harvard Medical School, 45, Shattuck Street, Boston Massachusetts
02115, U.S.A.
16
EMB
45
238
M. A. H. SURANI, D. TORCHIANA AND S. C. BARTON
occludens' (Ducibella et al. 1975; McLaren & Smith, 1977), and the inner cell
mass (ICM) at one end of the blastocoelic cavity. We investigated the influence of
the divalent ionophore A23187, which is a monocarboxylic acid antibiotic specific
for divalent cations, on early mammalian development since the ionophore has
a variety of effects on different cells, which are mediated partly by an increase in
intracellular ionized calcium (Reed & Lardy, 1972). These include mitogenic
effects on lymphocytes (Freedman, Raff & Gomperts, 1975; Hesketh et al. 1977),
inhibition of morphological changes in cells induced by dibutyryl cyclic AMP
(Henneberry, Fishman & Freese, 1975), release of histamine from mast cells
(Foreman, Mongar & Gomperts, 1973), prevention of retinal orientation in
developing eyes of Xenopus laevis (Jacobson, 1976), probably due to uncoupling
of permeable junctions (Jacobson, 1976; Rose & Loewenstein, 1975) and parthenogenetic activation of unfertilized eggs (Steinhardt, Epel, Carroll &
Yanagimachi, 1974). We observed that the influence of A23187 on compacted
morulae was reversible under the experimental conditions employed; although
partial decompaction of blastomeres occurred, normal development resumed
when the embryos were explanted to fresh medium. During the course of
experiments to detect the mitogenic action of A23187 on blastocysts we observed
that the ionophore caused selective lysis of trophectoderm cells but the ICM
remained apparently unaffected. The development of the ICM isolated in this
manner was therefore investigated in vitro and in vivo.
MATERIALS AND METHODS
Animals
Two inbred strains of mice, CBA and BALB/c, and one outbred strain, CFLP
(Anglia Laboratories Ltd), were used in these experiments. They were maintained
on a lighting schedule of 05.00 h to 19.00 h. Two or three adult females (approximately 6 weeks old) were caged with each male, checked the following morning
for copulation plugs, and embryos were retrieved on day 3 or day 4 of pregnancy
(day 1 = day of plug). Some mice were superovulated before mating by intraperitoneal injections of 5 i.u. of pregnant mare serum gonadotrophin followed
48 h later by 5 i.u. of human chorionic gonadotrophin.
Recovery of embryos
Compacted morulae were flushed from the oviducts between 15.00 h and
17.00 h in the afternoon of day 3 of pregnancy approximately 60 h after
ovulation. Blastocysts were retrieved from uterine horns between 10.00 h and
12.00 h on day 4 of pregnancy approximately 87 h after ovulation. Some of the
blastocysts were cultured for a period of 12 h (99 h late blastocysts) before being
used in experiments.
Isolation of ICM by ionophore A23187
239
Preparation of media, ionophore and pronase
The medium used for embryo retrieval and culture was that described by
Whittingham (1971), supplemented with amino acids, vitamins and glutamine
as specified for Eagle's Basal Medium, and 4 mg ml" 1 bovine serum albumin
(BSA). Ten per cent heat inactivated foetal calf serum was substituted for BSA
for long-term cultures of isolated ICM. A 2 mM stock solution of the ionophore
A23187 (Eli Lilly) was made in methanol (Analar, BDH) and stored at - 2 0 °C.
A 0-5 % solution of the enzyme pronase (B grade, Calbiochem) was freshly
prepared each time by dissolving in 01 M phosphate buffered saline which
contained 10 mg ml"1 polyvinylpyrrolidone (mol wt. 40000, May and Baker
Ltd).
Culture conditions
Embryos were flushed from the genital tract and cultured in Falcon plastic
Petri dishes under paraffin oil at 37 °C in 5 % CO2 in air for 1 h (except for the
late blastocysts) before transferring to medium containing the ionophore.
Either 10 or 1 JLI\ stock solution of A23187 was added to 1-0 ml culture medium
in an embryological glass dish to give a final concentration of the ionophore
at 2 x 10~5 M or 2 x 10"6 M respectively. The embryological dish was covered in
a glass Petri dish and placed in an incubator at 37 °C in 5 % CO2 in air for at
least 0-5 h to allow equilibration of the medium. The medium with ionophore
was prepared fresh immediately before use. Embryos were washed six times
before transfer to the ionophore medium and incubated for up to 0-75 h in the
higher concentration ionophore ( 2 X 1 0 ~ 5 M ) or for 6-12 h in the lower concentration ionophore (2x 10~ 6 M). The embryos were then removed from the
medium, washed six times in fresh culture medium under paraffin oil and transferred to fresh droplets of medium under paraffin oil for culture at 37 °C in
5 % CO2 in air. Where there was evidence for selective lysis of trophectoderm
cells of blastocysts, the embryos were transferred after 20-30 min in culture to
the solution containing pronase for 3-5 min at 37 °C under paraffin oil to
remove the zona pellucida and the lysed trophectoderm cells. The ICMs were
freed of any remaining trophectoderm cells by repeated pipetting in a finely
drawn Pasteur pipette to yield groups of ICMs. The ICMs were washed in
fresh medium containing 10% foetal calf serum and then cultured in fresh
drops of this medium under paraffin oil in Falcon plastic Petri dishes. Some
were cultured individually, others were placed in groups of three to five and
allowed to aggregate.
Transfer and washing of embryos was carried out under a Wild M5 dissecting microscope and detailed observations and photographs were taken using
a Wild M40 inverted phase microscope.
16-2
240
M. A. H. SURANI, D. TORCHIANA AND S. C. BARTON
Microsurgery
The developmental potential of the ICM isolated from the early and late
blastocysts by the new technique described here was further tested in vivo by
microsurgical transfer to host blastocysts. The mice used to provide the donor
ICMs were selected from our outbred colony of mice to have black non-agouti
coat colour and to be homozygous for the fast moving form of the isozyme
glucose phosphate isomerase (GPI), Gpi-lb/Gpi-lb, detected by electrophoretic
analysis on starch gels (Chapman, Whitten & Ruddle, 1971). Host blastocysts
were obtained from matings of CFLP albino mice selected to be homozygous
for the slow moving form of GPI, Gpi-la\Gpi-la. Isolated ICMs were introduced
into the host blastocysts by microsurgery using a Leitz micromanipulator
(Gardner 1971). The operated blastocysts were cultured for 2-3 h before transfer
to the uteri of day 3 recipient CFLP mothers (also homozygous for Gpi-la),
made pseudopregnant by mating with vasectomized males. Twenty-eight operated
blastocysts were transferred to five pseudopregnant mothers; three females were
killed on day 9 of pregnancy and the embryos and their respective ectoplacental
cones typed for GPI to assess the extent of chimaerism. The other two females
were allowed to reach term and live born mice were examined for coat colour
and blood samples analysed for GPI. These animals were later test bred to
assess the contribution of the donor ICMs to the germ line.
RESULTS
Influence of the ionophore on compacted morulae
One hundred and eighty-nine compacted morulae from the three strains of
mice were cultured for up to 12 h in medium containing 2 x 10~6 M ionophore.
The embryos showed partial decompaction so that individual blastomeres could
be easily identified. After washing and transfer to fresh medium, 181 morulae
continued development to the blastocyst stage. All blastocysts were apparently
normal and out of 120 randomly selected blastocysts transferred to 13 pseudopregnant recipients, 98 developed to term. When incubated in 2 x 10~5 M
ionophore, 71 morulae from the three strains of mice showed partial decompaction after 15-20 min and within 30 min extensive cell death of blastomeres was
observed. These treated embryos showed individual fluid-accumulating cells
when explanted to fresh medium for culture overnight but the majority of the
blastomeres appeared pycnotic with only four poorly developing blastocysts
being observed. Of the 159 morulae cultured in 2 x 10~5 M ionophore for 5 min,
142 developed to the blastocyst stage and out of 96 randomly selected blastocysts
transferred to 10 pseudopregnant recipients, 74 developed to term.
A total of 251 further morulae from the three strains of mice was incubated
as above in 2 x 10~5 and 2 x 10~6 M ionophore in the absence of extracellular
Ca 2+ and Mg 2+ and the results in terms of the response shown and the number
Isolation oflCM by ionophore A23187
241
Table 1. Recovery of ICMs from early and late blastocysts and the
detection offluid-accumulatingcells
Strain of
of mice
CFLP
CBA
BALB/c
Blastocysts
early (E) and
late (L)
No. of
blastocysts
No. of
ICMs
isolated
E
L
E
L
E
L
50
56
33
19
20
21
41
56
27
19
15
21
No. of ICMs Overall total
with fluidof fluidaccumulating accumulating
cells
cells
9
0
2
1
2
1
11
0
2
1
2
1
Isolated ICMs were cultured for 24-48 h and examined for fluid-accumulating cells
(see Fig. 1 c).
of morulae developing to the blastocyst stage were similar to those described
above. Of the 93 blastocysts transferred to seven pseudopregnant recipients,
75 developed to term.
Influence of the ionophore on blastocysts
When the early and late blastocysts were cultured in medium containing
2 x 10~6 M ionophore for 6-12 h, 85 out of 97 blastocysts remained apparently
normal but the remainder showed gross disorganization and failed to recover
on transfer to fresh medium. Seventy-one of these blastocysts developed normally to term on transfer to eight pseudopregnant females.
The response of blastocysts in the presence of 2 x 10~5 M concentration of the
ionophore differed from that described above. After 20-30 min exposure, there
was morphological evidence for swelling, vesiculation and lysis of trophectoderm cells whereas the 1CM remained apparently unaffected. When these
embryos were cultured in fresh medium for a further 20-30 min, all the outer
trophectoderm cells appeared to be uniformly lysed when examined under the
inverted phase microscope (Fig. la). On transfer to the solution containing
pronase, the zona pellucida was lysed, and the lysed trophectoderm cells were
dispersed when the fragments were repeatedly pipetted through a finely drawn
Pasteur pipette, finally releasing the ICM intact (Fig. 1 b).
The response described above was observed in 83 out of 103 early blastocysts;
in the remainder of the embryos, all the cells were affected and no distinct group
of ICM cells was recovered. All the 96 late blastocysts, with and without the
zona pellucida, responded equally to the ionophore and all of these yielded
ICMs (Table 1). The response of blastocysts described above was observed in
a further 113 early and late blastocysts even when extracellular Ca 2+ and Mg 2+
242
M. A. H. SURANI, D. TORCHIANA AND S. C. BARTON
» *
IT
Fig. 1. Mouse blastocysts after exposure to 2x 10~5 M A23187; note swelling and
vesiculation of trophectoderm cells (T) whilst the ICM is apparently unaffected (a).
Group of ICMs after the removal of zonae pellucidae (ZP) with pronase and dispersal of dead trophectoderm cells (b). The ICMs were cultured individually for 2448 h; an occasional ICM showed a single fluid-accumulating cell (FAC) (c). (bar =
Isolation of ICM by ionophore A23187
243
Fig. 2. Groups of three to five isolated lCMs were cultured in Falcon Petri dishes
in medium containing 10% heat inactivated foetal calf serum. Groups of ICMs
aggregated, a property characteristic of these cells (a), and formed into embryoid
bodies 2 days later {b). (bar = 20 /tm.)
salts in the medium were omitted during the exposure of embryos to the
ionophore.
The re-exposure of the isolated ICM to the ionophore apparently destroyed
some of the cells and these appeared pycnotic. The dead cells resembled the
dead blastomeres in morulae and did not display the vesiculation, swelling and
lysis characteristic of trophectoderm cells. Approximately half the total number
of cells survived the treatment as judged after their culture in vitro.
Development of the isolated ICM in vitro
The isolated ICMs were cultured individually for up to 48 h and checked for
cells showing intracellular fluid accumulation. Between 8 and 20% of the
ICMs obtained from the early blastocysts and up to 5 % from the late blastocysts showed evidence of fluid-accumulating cells. However, there was rarely
more than one fluid-accumulating cell detected in an ICM (Fig. 1 c, Table 1).
Between three and five ICMs from blastocysts of CFLP mice were allowed
to aggregate in culture, a property characteristic of ICM cells (Gardner &
Johnson, 1972). The aggregates were cultured for about 10 days in medium
containing 10 % foetal calf serum and the medium was changed every 2-3 days.
Six aggregates each, from early and late blastocysts, were cultured. One ICM
aggregate from the early blastocysts reformed into a blastocyst, albeit with an
exceptionally large ICM. This blastocyst attached to the Petri dish displaying
trophoblast giant cell outgrowth. The remaining 11 aggregates developed into
embryoid bodies after 2 days; by 7-10 days in culture, some had attached to the
Petri dish and a variety of cell types were observed. Similar findings have been
28
5
3
17
21
15
15
23
11
4
6
4
2
No.
chimaeric
11 (day 9)
4 (term)t
8
6(day 9)
2 (term)f
No. embryos
or live young
(day of
pregnancy)
70:30
50:50
20:80
5:95
Contribution from donor ICM*
(%Gpi-la:Gpi-P)
0:100
C/5
n
H
O
z
H
O
* Values represent subjective assessment from GPI analysis based on the time of appearance and relative intensities of the two isozyme bands, o
t Live born.
"
Overall total
(EB + LB)
2
1
11
6
2
11
LB
LB
Total LB
1
1
6
5
EB
EB
Total EB
No.
recipient
mothers
No. host
blastocysts
transferred
Donor ICM from
early (EB) and late
(LB) blastocysts
Table 2. Analysis for chimaerism in day-9 embryos and at term
>
to
Isolation oflCM by ionophore A23187
245
previously reported for ICMs cultured in vitro (Solter & Knowles, 1975; Hogan
& Tilly, 1977).
Development of the ICM in vivo
Development of the ICM isolated by this method was assessed by making
chimaeras to ensure that the treatment did not have any lasting deleterious
effects on their development. Of the 18 embryos examined on day 9 of pregnancy,
16 were normal and chimaeric as judged from GPI typing. Chimaerism was
confined to the embryonic tissues. The ectoplacental cones did not have the
Gpi-lb isozyme band apart from occasional trace levels and this could be due to
incomplete dissection or due to contribution made to this tissue from any
remaining trophectoderm cells. These results, however, suggest that the donor
ICMs were essentially denuded of trophectoderm cells (Gardner &Papaioannou,
1975). Six live born young were apparently normal and all of these were chimaeric as detected by coat colour markings and GPI typing of their blood,
except for one which was wholly of the donor ICM type. Indeed more than half
of all the chimaeric embryos appeared to have developed predominantly from
the donor ICM cells (Table 2). All six chimaeras have themselves had young;
three have shown their germ cells to be entirely of the donor ICM type, one to
have a mixed germ line and the remaining two to have germ cells all of the host
blastocyst type.
DISCUSSION
The aim of these studies on compacted morulae was primarily to determine
if the exposure to the ionophore caused gross anomalies in subsequent development. Under the experimental conditions reported here, only partial decompaction of morulae was observed and development was normal if the morulae were
explanted to fresh medium before extensive cell death occurred. Detailed studies
may show more subtle effects of the ionophore on the timing of blastocoele
formation and even anomalies of development. However, a comprehensive
series of experiments using the ionophore at different concentrations on a
variety of stages and after exposure for various durations would be required.
The first stage at which the outer cells are preferentially lysed also needs to be
identified.
The selective lysis of the trophectoderm cells in the presence of 2 x 10~5 M
ionophore is morphologically analogous to the vesiculation and lysis of the
cells observed after treatment of blastocysts with antibodies and complement
(Solter & Knowles, 1975). The mechanism of trophectoderm cell lysis could in
both cases be due to an osmotic phenomenon. The ionophore induced lysis of
trophectoderm cells may result from the uncontrolled influx of Ca2+ (Reed &
Lardy, 1972) followed by swelling and vesiculation after the entry of water into
the cells. Similarly, lysis of cells following complement damage can be explained
246
M. A. H. SURANI, D. TORCHIANA AND S. C. BARTON
by the suggestion that Na+ flows into the cells more quickly than the outward
flow of K + ; influx of water then causes swelling and lysis of cells (Green, Barrow
& Goldberg, 1959). The observation that similar results were obtained with the
ionophore in the absence of extracellular Ca2+ and Mg 2+ ions is difficult to
reconcile with the explanation proposed here. However, there is some evidence
to show that the ionophore can cause release of intracellular sequestered
calcium (Henneberry et al. 1975) and this may give rise to the same cell response
in the modified medium.
The reasons why ICM in blastocysts survives the treatment with ionophore
may partly be due to the protection offered by the outer trophectoderm cells
and partly because of intrinsic differences in the two cell types. The presence of
'zonula occludens' between the trophectoderm cells (Ducibella et al. 1975;
McLaren & Smith, 1977) may restrict the passage of macromolecules into the
blastocoele and to some extent the entry of A23187, which is a considerably
smaller molecule. Some evidence suggests that the ICM may also possess a
slightly greater tolerance towards the action of the ionophore as shown by
experiments where the isolated lCMs were re-exposed to A23187. In addition
the characteristic swelling and vesiculation is only observed in trophectoderm
cells but not in the blastomeres of morulae or ICM where the cytoplasm was
granular and the cells appeared pycnotic when cell death occurred. The changes
in cell properties such as the increase in the phagocytic activity of trophectoderm cells but not the ICM cells may affect their response, partly by the amount
of ionophore entering the cells.
Fluid-accumulating cells were observed in a few ICMs, especially in those
isolated from early blastocysts when these were cultured in vitro. No direct
evidence was obtained to show if these were trophectodermal or primitive
endodermal cells. However, one aggregate of ICMs from early blastocysts
reformed into a blastocyst and trophoblast giant cell outgrowth was observed
after 72 h in culture. Trophectoderm cells may therefore be present in a few
isolated ICMs either because these escape lysis by the ionophore or there may
be a population of uncommitted cells which subsequently differentiates into
trophectoderm cells (Johnson, Handyside & Braude, 1977). We have no
evidence to distinguish between the two possibilities.
The transfer of the isolated ICMs into intact blastocysts further shows that
these cells are not affected by their treatment with ionophore in that they contribute extensively to the development of the chimaeric embryos to term. The
donor ICM contributes almost exclusively to the embryo and not to the ectoplacental cone as would be expected from previous studies (Gardner &
Papaioannou, 1975). This technique has also recently been used for isolating
ICMs from diploid parthenogenetic blastocysts which on transfer to normal
blastocysts participate in the development of chimaeric embryos to term
(Surani, Barton & Kaufman, 1977). Experiments are in progress to establish
if the ionophore stimulates cell proliferation in the ICM and also to show if
Isolation of ICM by ionophore A23187
fluctuations in intracellular Ca
the ICM.
2+
247
precede proliferation and differentiation of
We thank Andrea Burling for technical assistance and Dr R. L. Hamill of Eli Lilly for a
gift of A23187. The work was supported by a Ford Foundation Grant. M. A.H.S is supported
by an M.R.C. Project Grant and D.T. by a Churchill Foundation Scholarship.
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{Received 10 November 1977, revised 8 January 1978)