/ . Embryol. exp. Morph. Vol. 60, pp. 255-269, 1980
Printed in Great Britain © Company of Biologists Limited 1980
255
Immunosurgical studies on Inner cell
mass development in rat and mouse blastocysts
before and during implantation in vitro
By HORST SPIELMANN1, URSULA JACOB-MULLER, AND
WERNER BECKORD
From the Institut fiir Toxikologie und Embryopharmakologie,
Freie Universitdt, Berlin
SUMMARY
Eighty per cent of rat blastocysts (Wistar, SW72) cultured for 96 h in NCTC-109 supplemented with fetal calf serum (FCS) hatched from the zona pellucida and developed a trophoblast giant cell layer. Thirty seven per cent of the rat blastocysts developed an inner cell mass
(ICM) which, in about 7 %, consisted of two germ layers (ectoderm and endoderm), compared
to 84% in NMRI mice. A significantly better ICM development was obtained with cultured
rat blastocysts that had hatched in vivo. Similar to the in vivo situation LDH-5 was present in
rat blastocysts after implantation in NCTC-109-FCS. Differentiation of C57BL mouse
blastocysts in NCTC-109-FCS proceeded as poorly as in the rat.
ICM development of rat and mouse blastocysts in NCTC-109-FCS was studied in detail.
ICMs of the two species were isolated immunosurgically using complement from different
species, e.g. human, rat and rabbit complement, since guinea-pig complement did not lyse
trophectoderm cells of rat blastocysts. All immunosurgically isolated rat ICMs degenerated
within 48 h, but mouse ICMs isolated with rat or rabbit complement developed significantly
better than mouse ICMs isolated with guinea-pig complement. Determinations of the
blastocyst total cell number (BTCN) and of the cell number of immunosurgically isolated
ICMs were performed in rat and mouse blastocysts to investigate growth kinetics of the ICM
before implantation in vitro. In the mouse an exponential increase in both BTCN and cell
number of the ICM was observed during the 48 h before implantation in NCTC-109-FCS
and also during the 16-24 h before implantation in vivo. In the rat, doubling of the BTCN
was found only during the first 24 h in NCTC-109-FCS and there was hardly any increase in
the cell number of the ICM during the first 48 h in culture. ICM growth of blastocysts in
NCTC-109-FCS is, therefore, stimulated in the mouse before and after implantation and. in
the rat it is inhibited already before implantation.
INTRODUCTION
Mouse embryos can be cultured routinely from the two-cell to the blastocyst
stage in simple media (Biggers, Whitten & Whittingham, 1971). Further
development of the blastocysts will not occur unless they are transferred to a
richer medium, which characteristically is more complex and contains fetal
1
Author's address: Institut fiir Toxikologie und Embryopharmakologie, Freie Universitat
Berlin, Garystrasse 9, D-1000 Berlin 33, West Germany.
17-2
256
H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
calf serum (FCS) (Spindle & Pedersen, 1973; Pienkowski, Solter & Koprowski,
1974; Ansell & Snow, 1975; Sherman, 1975). Since reproducibly simple culture
conditions for rat embryos during cleavage have so far not been established
(Elliott, Maurer&Staples, 1974; Mayer & Fritz, 1974) investigations on differentiation of rat blastocysts in vitro during implantation have not been attempted.
In rat blastocysts cultured in medium NCTC-109 supplemented with 10 %
FCS according to Sherman (1975) differentiation of the inner cell mass (ICM)
was considerably poorer than in mouse blastocysts. To study the difference in
ICM development in the two species in more detail, growth of immunosurgically
isolated ICMs of rat and mouse blastocysts was compared. Since the standard
procedure for isolating ICMs from mouse blastocysts by immunosurgery
(Solter & Knowles, 1975) was unsatisfactory for the rat, ICMs of rat blastocysts
were isolated immunosurgically with complement from different species.
Attempts to culture these rat ICMs in NCTC-109-FCS were unsuccessful
although the medium allowed differentiation of mouse ICMs isolated by the
same procedure.
The different rates of ICM development found in rat and mouse blastocysts
both after implantation in vitro and during culture of the isolated ICMs may
indicate that ICM development in the two species is already quite different
during the 24-48 h incubation period before implantation of the blastocysts in
NCTC-109-FCS. Determinations of the growth kinetics of ICM and trophectoderm cells in mouse and rat blastocysts during development in vivo and also
during culture in NCTC-109-FCS revealed an increase before implantation
in vitro in blastocyst total cell number (BTCN) and in cell number of the ICM
in cultured mouse blastocysts in the same exponential fashion as before implantation in vivo. In rat blastocysts, however, hardly any ICM growth but a
considerable increase in BTCN was observed in NCTC-109-FCS before
implantation.
MATERIALS AND METHODS
Recovery of blastocysts. Medium PB-1 (Whittingham & Wales, 1969) was
used for sampling and handling of blastocysts. The 24 h following the 2 h
mating period from 6 a.m. to 8 a.m. of the NMRI mice and C57BL mice
(Institut fur Tierzucht, Hannover, West Germany) and SW72 Wistar rats
(Winkelmann, Kirchborchen, West Germany) were called day 0 of pregnancy.
Mouse blastocysts were flushed from the uteri of pregnant animals either at
2 p.m. on day 3 or at 8 a.m. on day 4 with medium PB-1 (Whittingham & Wales,
1969) supplemented with 10 % fetal calf serum (FCS). In NMRI mice implantation is occurring between 8 a.m. and 2 p.m. on day 4 of gestation. Rat blastocysts were flushed from the uteri either at 8 a.m., at 2 p.m. or at 10 p.m. on day 4.
In the rats implantation is occurring between 10 p.m. on day 4 and 2 a.m. on
day 5.
In vitro culture of blastocysts. Mouse blastocysts were cultured without oil in
ICM development in rat and mouse blastocysts in NCTC-109-FCS
257
groups of 10-15 in plastic culture dishes (NUNC, Nulcon, Denmark) in 5 ml of
medium NCTC-109 (Microbiological Associates, Frankfurt, West Germany)
supplemented with 10 % FCS according to Sherman (1975) in a humidified 5 %
CO2-in-air atmosphere at 37°C for 96 h. The following time-related success
rates were obtained (Eibs & Spielmann, 1977; Spielmann, Eibs, Jacob-Miiller &
Bischoff, 1978; Spielmann & Eibs, 1978): after 24 h 10 % of the blastocysts had
hatched, after 48 h 85 % had attached to the surface of the culture dish and 32 %
showed trophoblast outgrowth, after 72 h 82 % had an ICM and 52 % an ICM
with two germ layers, and after 96 h the ICM of 84 % of the embryos consisted
of two germ layers (ectoderm and endoderm). Rat blastocysts were cultured
under the same conditions. In a few experiments 0-25 % pronase (Boehringer,
Mannheim, West Germany) in medium PB-1 was used to dissolve the zonae of
rat blastocysts. Photomicrographs of the blastocysts were taken on a Biovert
photomicroscope (Reichert, A.G., Austria) on Ilford Pan F film (Ilford Co.,
England).
Electrophoretic analysis of LDH isozymes. Electrophoresis in 7-5 % polyacrylamide gels at pH 9-0 with a continuous tris-glycine buffer system and the
staining procedure for the detection of LDH activity were carried out as
described previously (Epstein, Kwok & Smith, 1971.; Spielmann, Erickson &
Epstein, 1973; Spielmann et al. 1978). The rat skeletal muscle LDH preparation
was the 20000 g supernatant of a 1:10 homogenate in 0-02 M Tris-HCl, pH 7-4.
Normal rat blastocysts and also blastocysts which had implanted and developed
in culture were washed three times and were then taken up in 20 /*1 of deionized
water. Complete disruption of the embryonic cells was achieved by three times
freezing-thawing before electrophoresis (Spielmann et al. 1978).
Immunosurgery and determination of cell numbers. Immunosurgery was
performed in the rat and mouse after removing the zonae by exposure of the
blastocysts to pronase (0-25 % in Tris-citrate buffer pH 7-0) for 5-10 min at
37 °C and carrying them through several washes in PB-1. The two steps of
incubation for immunosurgical lysis of the trophectoderm cells were carried out
as described by Handyside & Barton (1977) with rabbit antiserum to mouse
embryo homogenate diluted 1 in 5 with PB-1 in the first step and guinea-pig
complement (Behring Werke, Marburg, West Germany) diluted 1 in 5 with PB-1
in the second step. Incubation time was 30 min for each step.
Antibodies against mouse embryo homogenate (day 11) and rat embryo
homogenate (day 13) were prepared by injecting 0-5 ml of the homogenate
subcutaneously into the clavicular region of rabbits. The homogenates were
diluted with Freund's complete adjuvant for the first injection and with Freund's
incomplete adjuvant (Behring Werke, Marburg, West Germany) for the
subsequent injections in weekly intervals for up to 6 weeks, when antibodies
were usually detectable against the embryo homogenates in the Ouchterlony
double diffusion test. Rabbit serum that contained antibodies against embryo
homogenate of one of the two species was incubated for 30 min at 56 °C to
258 H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
destroy complement and stored frozen at — 20°C after filtering through a
millipore unit.
Immunosurgery was performed with rat blastocysts under similar conditions
as described for mouse blastocysts but with either antibody against rat embryo
homogenate or antibody against mouse embryo homogenate in the first step of
the incubation procedure. The second step of incubation was performed with
guinea-pig complement (dilution 1:5 with PB-1), with rabbit complement
(Behring Werke, Marburg, West Germany) and with sera that had not been
heated to 56 °C from the following species: mouse, rat, rabbit and man (dilution
1:5 with PB-1). Peroxidase-labelled goat-anti-rabbit IGG (Miles Laboratories,
Frankfurt, West Germany) was used to study the binding of the antibodies
against embryo homogenate to the surface of the trophoblast cells of mouse and
rat blastocysts after dissolving the zona by pronase. The peroxidase-staining
procedure was performed as described earlier (Spielmann, Eibs, Mentzel &
Nagel, 1977).
To study the developmental potential of immunosurgically isolated ICMs,
mouse blastocysts were flushed from the uterus at 2 p.m. on day 3 and rat
blastocysts at 2 p.m. on day 4. After an overnight incubation in 5 ml of NCTC109-FCS ICMs were immunosurgically isolated between 10 a.m. and noon on
the following day and cultured in NCTC-109-FCS for up to 120 h. Cell counts
were performed on whole blastocysts (BTCN = blastocyst total cell number)
and on immunosurgically isolated ICMs according to Tarkowski's method
(Tarkowski, 1966).
RESULTS
In vitro culture of rat blastocysts in NCTC-109-FCS
Similar to experiments on mouse blastocysts (Spielmann et ah 1978) culture
of rat blastocysts in 5 ml of media without oil was attempted in either Whitten's
medium (Whitten, 1971) or Eagle's minimal essential medium (MEM) both
supplemented with BSA or FCS and also in NCTC-109-FCS. Again, growth
and differentiation of blastocysts were significantly better in NCTC-109-FCS
after 96 h than in any other media tested. The percentage of blastocysts that
reached the characteristic steps of differentiation in vitro was significantly lower
in the rat than in the mouse (Table 1). This table indicates that ICM development was particularly poor in the rat, since only 37 % of the embryos developed
an ICM and in less than 10 % of the cases the ICM consisted of two germ layers
(Fig. 1). Pronase treatment as described by Pienkowski and co-workers (Pienkowski et al. 1974) to improve success rates for cultured mouse blastocysts did
not show any beneficial effect on rat blastocysts. However, blastocysts that had
already hatched from their zona pellucida in vivo, showed a slightly better ICM
development in culture than rat blastocysts that were still surrounded by their
zona (Table 1).
ICM development in rat and mouse blastocysts in NCTC-109-FCS 259
Table 1. Development of mouse and rat blastocysts during implantation in vitro in
medium NCTC-109-FCS (culture period 96 h)
Species
(strain)
Hatching
ICM with
Number of
Trophoblast ICM two germ
and
blastocysts Number of attachment giant cells growth
layers
(100%) experiments
(%)
(%)
(%)
(%)
Mouse (NMRI)
Mouse (C57BL)
Rat (with zona)
Rat (without zona)
540
84
280
68
43
6
24
7
97
84
87
90
95
82
81
87
90*
56**
37
65**
84f
5
7
16i
Blastocysts were obtained on day 3 at 2 p.m. (mouse) and on day 4 between 6 p.m. and
10 p.m. (rat).
ICM growth: * Better than ICM growth of both rat and C57BL mouse blastocysts at
/*< 0-001 (Wilcoxon test). ** Better than rat blastocysts with zona at P < 0 0 1 ; no difference
between C57BL mouse and hatched rat blastocysts (Wilcoxon test).
TCM with two germ layers: t Better than rat and C57BLmouse blastocysts atP<0001
(Wilcoxon test). % Better thanC57BL mouse and rat blastocysts with zonaatP< 0-05 (Wilcoxon
test).
Differentiation of rat blastocysts proceeded time-dependently in the following
manner: After 24 h 34% had hatched, after 48 h 4 8 % showed trophoblast
outgrowth with giant-cell formation and 27 % had an ICM, after 72 h 65 %
showed trophoblast outgrowth, 30 % had an ICM and the data for 96 h are
given in Table 1. Freeze-thaw lysates of hatched and developed rat blastocysts
were run on polyacrylamide gels and assayed for their LDH isozyme pattern.
Similar to development after implantation in vivo an LDH-5 band could be
observed in addition to the LDH-1 band that is characteristic for preimplantation rat embryos up to the blastocyst stage (Fig. \d) (Engel & Petzold, 1973;
Spielmann et at. 1978).
The NMRI mouse strain, which is used for blastocyst culture during implantation in our laboratory, shows a block after the first cleavage division
during culture, like most other mouse strains (Biggers, 1971). Mouse embryos
of the C57BL strain, which can be cultured routinely from the zygote to the
blastocyst stage did not grow any better in NCTC-109-FCS than rat blastocysts.
Comparison of the in vitro development of mouse and rat blastocysts in
NCTC-109-FCS showed that there was less difference in differentiation of the
trophoblast layer than in development of the two layers of the ICM (Table 1).
To study the species difference of ICM development in culture in more detail,
development of immunosurgically isolated ICMs of rat and mouse blastocysts
in NCTC-109-FCS was compared.
Jmmunosurgical isolation of ICMs from rat blastocysts (Fig. 2)
Rat blastocysts that were incubated with either antibody to rat or mouse
embryo homogenate and subsequently with guinea-pig complement did not
260
H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
Vv
, .t
ICM development in rat and mouse blastocysts in NCTC-109-FCS
261
undergo lysis of the trophectoderm cells like mouse blastocysts under identical
conditions (Table 2; Fig. 2d). Studies with peroxidase-labelled goat-anti-rabbit
IGG demonstrated that the antibody against mouse embryo homogenate and
the antibody against rat embryo homogenate that had been produced in
rabbits were tightly bound to the surface of mouse and rat blastocysts in an
identical manner after removal of the zona pellucida with pronase. This indicates
that there is no species-specific difference in the first incubation step of immunosurgery and that guinea-pig complement is unable to induce lysis of the trophectoderm cells of rat blastocysts. It was, therefore, attempted to achieve lysis by
using sera from other species that had not been heated and were still containing
complement. According to Table 2, complete lysis of trophectoderm cells can be
obtained in the rat with rabbit, rat and human sera, the latter being more
effective than any other sera. Rabbit complement from commercial suppliers
was also found to induce trophectoderm cell lysis, It was, therefore, used
routinely to isolate ICMs of rat blastocysts. Furthermore, complement that was
effective in the rat could also be used successfully in immunosurgery of mouse
blastocysts.
Growth of immunosurgically isolated ICMs of rat and mouse blastocysts
Growth and development of ICMs of mouse blastocysts isolated with guineapig complement were comparable to results of earlier investigators (Fig. 2;
Table 3) (Handyside & Barton, 1977; Wiley, Spindle & Pedersen, 1978; Hogan
& Tilly, 1978a, b). However, rat ICMs isolated using either rabbit complement
or fresh sera from rabbit, rat or humans degenerated within 48 h in NCTC-109FCS and never showed any sign of development. To exclude that these sources
of complement were embryotoxic, ICMs were isolated from mouse blastocysts
under the same conditions. Table 3 shows that ICMs obtained with rabbit
complement and with rat serum developed even better than after isolation with
Fig. 1. In vitro development of mouse and rat blastocysts in medium NCTC-109FCS (culture period 96 h)
(a) A single well developed mouse blastocyst consisting of an ICM (centre) with
two germ layers (endoderm and ectoderm) and a trophoblast layer (with giant cell
nuclei)fillingthe rest of the picture (x 90).
(b) Two well developed rat blastocysts, morphological structures are identical to 1 (a),
the ICMs are not as well developed as in the mouse (x 90).
(c) A single well developed rat blastocyst, similar to \{b), the lower magnification
(x 45) shows the total area of the trophoblast giant cell layer.
(cl) LDH-isozyme pattern of in v/7ro-cultured rat blastocysts on polyacrylamide
gels. LDH-1, the fastest migrating enzyme, is at the bottom (anode) and LDH-5,
the slowest migrating enzyme, is at the top (cathode). From left to right: rat kidney
homogenate (reference), LDH-1—LDH-5; 30 well developed rat blastocysts,
LDH-1, LDH-5 and 3 additional bands; 10/*1 medium NCTC-109, no rat LDH
isozyme bands, only bovine LDH isozyme bands, LDH-1—LDH-3, which are the
additional bands on the blastocyst gel.
262
H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
o
t
» *>>
ICM development
in rat and mouse blastocysts
in NCTC-109-FCS
263
Table 2. Imtnunosurgery of rat blastocysts: Effect of different sources of complement on immunosurgery of rat blastocysts
Lysis of trophectoderm cells (time given
in minutes)
Source of complement
(2nd step of incubation)
Guinea-pig complement
Human serum
Mouse serum
Rat serum
Rabbit serum
Rabbit complement
15
30
45
60
./.
+
•/•
(+)
+
./.
+( + )
•/•
+
+
(+)
++
•/•
+( + )
+(+)
•A
The sera had not been heated to 56 °C and were, therefore, still containing complement. In
the first step of incubation antibody against mouse embryo homogenate was slightly
more effective than antibody against rat embryo homogenate.
Lysis of the trophectoderm cells: + + , Complete lysis; + ( + ) , nearly complete lysis; + ,
moderate lysis; ( + ), weak reaction; ./., no reaction.
guinea-pig complement. Human serum was also toxic to mouse ICMs. These
results suggest that rabbit and rat sera and rabbit complement are not toxic to
rat ICM. cells.
The low rates of ICM growth both of whole rat blastocysts and of isolated rat
ICMs in NCTC-109-FCS indicate that this medium does not support growth
and differentiation of ICM cells in the rat in the same manner as in mouse
blastocysts after implantation in culture. In addition, there are at present no
data available on growth of ICM cells of mouse blastocysts before implantation
Fig. 2. Immunosurgery of mouse and rat blastocysts
(a) Two mouse blastocysts during the second step of incubation in rabbit complement, lysis of trophectoderm cells, ICMs remain intact (x 90).
(b) Seven rat blastocysts during the second step of incubation in rabbit complement,
lysis of trophectoderm cells, ICMs remain intact (x 90).
(c) Immunosurgically isolated mouse ICMs, three small ICMs from early blastocysts day 3, 2 p.m., and three big ICMs from blastocysts that had developed in
NCTC-109-FCS for 24 h (x 90).
(d) Effect of different sources of complement on the second step of incubation in
immunosurgery of rat blastocysts (day 4, 2 p.m.). First step of incubation in antirat-embryo antibody from rabbit. Second step of incubation with either rabbit complement, three isolated small ICMs are shown, or with guinea-pig complement,
lysis of the trophectoderm cells did not occur as shown by two intact blastocysts
(x 90).
(e) Tmmunosurgically isolated mouse ICM that was cultured in NCTC-109-FCS
for 48 h. A second layer of cells has grown around the ICM and has attached to the
bottom of the culture dish where solitary outgrowth can be seen (x 90).
(/) Three egg-cylinder-like vesicles that have attached to the bottom of the culture
dish after culture of mouse ICMs for 120 h in NCTC-109-FCS (x 30).
264
H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
Table 3. Immunosurgery of mouse blastocysts: Effect of different sources of
complement on development of ICMs in NCTC-109-FCS
Source of
Number of
Two germ Egg-cylinder-like
structures
complement (2nd blastocysts Number of Degenerated layers
experiments
step of incubation)
(100%)
(%)
(%)
(%)
Guinea-pig compl.
Rabbit compl.
Rat serum
Human serum
148
31
28
29
16
5
5
5
12
23
78
67
14
32*
22
86
68
14t
35**
1
Mouse blastocysts were flushed from the uterus at 2 p.m. on day 3 and cultured in NCTC109-FCS overnight. Immunosurgery was performed between 10 a.m. and noon on the next
morning. Immunosurgically isolated ICMs were cultured for 120 h. Development of two
germ layers of an I CM and egg-cylinder-like structures are shown in Fig. 2(e) and (/).
Development of two egg cylinders (Wilcoxon test): * Significantly better than guinea-pig
complement at P < 0 0 5 ; ** significantly better than guinea-pig complement at P< 0-01.
Development of two germ layers: f Significantly lower than any other group at P < 0-001
(Wilcoxon test).
in vitro, which is occurring after 24-48 h in NCTC-109-FCS (Spielmann et ah
1978). Therefore, blastocyst total cell number (BTCN) and cell number of
immunosurgically isolated ICMs were determined in mouse blastocysts during
the first 48 h in culture and compared with changes of the two parameters in vivo.
BTCN and cell number of the ICM were also determined in rat blastocysts to
see if ICM growth was already inhibited before implantation in NCTC-109-FCS.
Growth kinetics of BTCN and cell number of ICMs of mouse and rat blastocysts
in vivo andin vitro in NCTC-109-FCS
In mouse blastocysts doubling of BTCN and of the cell number per ICM was
observed in vivo during the 18 h period between day 3, 2 p.m., and day 4,
8 a.m., and also in vitro during the first 24 h of culture in NCTC-109-FCS
beginning on day 3, 2 p.m. (Table 4; Fig. 3). Additional doubling of BTCN and
cell number per ICM occurred in vitro in the second 24 h period (Table 4; Fig. 3).
At the beginning of the culture ICM cells accounted for 38 % of the BTCN,
this percentage decreased in vivo and in vitro to 28 % and to 29 % in the first 24 h,
and it increased to 36% in NCTC-109-FCS during the following 24 h. The
medium, therefore, seems to be very favourable for mouse ICM development
before implantation.
Development of rat blastocysts in vivo was characterized by BTCNs that were
considerably smaller than in the mouse (Table 4; Schiffner & Spielmann, 1976;
Surani, 1975, 1977). Furthermore, only a slight increase but no doubling of the
BTCN was observed in rat blastocysts in vivo between 8 a.m. and 10 p.m. on
day 4 (Table 4). Therefore, development of rat blastocysts in NCTC-109-FCS
was compared with mouse blastocysts in the same medium. Doubling of the
ICM development in rat and mouse blastocysts in NCTC-109-FCS 265
Table 4. Developmental changes of blastocyst total cell number (BTCN) and cell
number per ICM in the mouse and rat in vivo and during in vitro culture in
medium NCTC-109-FCS
Time of determination
Day 3,2 p.m.
Day 3,9 p.m.
Day 4, 8 a.m.
BTCN
Cell number per ICM
Mouse in vivo
12-4 ± 4-2(14)
33O± 30(20)
421 ± 9-6(23)
13-5 ± 2-9(13)
72-5 ±14-8 (31)
20-8 ±6-3(41)
Cell number per ICM
as % of BTCN
38
32
29
Mouse in vitro in NCTC-109-FCS (begin on day 3 at 2 p.m.)
75-3±10-0(14)
21-0± 3-1(11)
28
129-6 ±22-3 (12)
46-7 ± 12-8 ( 9)
36
Rat in vivo
Day4,8 a.m.
23-6± 5-0(15)
9-1 ± 20(15)
38
Day 4,2 p.m.
26-4± 3-3(40)
ll-0± 1-8(21)
41
Day4, 10p.m.
351 ± 8-1(56)
130± 0-8(48)
37
24h in vitro
48 h in vitro
Rat in vitro in NCTC-109-FCS (begin on day 4 at 2 p.m.)
24h in vitro
51-4± 81 (23)
15-1 ±3-4(12)
29
48h/«v/7w
64-3± 8-8(19)
18-2± 2-7(9)
28
Blastocysts are implanting in vitro in NCTC-109-FCS after 48-60 h, therefore, cell numbers
can only be determined up to 48 h. Number of determinations is given in parentheses.
BTCN and only a very small increase in cell number per ICM was found in rat
blastocysts during the first 24 h in culture and a slight increase of both growth
parameters was observed during the following 24 h (Table 4, Fig. 3). Figure 3,
therefore, demonstrates that the only growth parameter that increased at a
similar rate in the two species during blastocyst culture in NCTC-109-FCS was
the BTCN during the first 24 h period. ICM growth, however, was already
inhibited in rat blastocysts before implantation in vitro. This, of course, explains
the poor ICM development of whole rat blastocysts after implantation and also
of immunosurgically isolated rat ICMs in NCTC-109-FCS.
DISCUSSION
Medium NCTC-109-FCS does not support post-blastocyst development as
effectively in SW72 rats as in NMRI mice. In particular, the ICM never reaches
its characteristic organization in the rat. In view of these shortcomings it is
notable that even in the rat trophoblast cells develop and express both morphological and biochemical properties which have been associated with their
differentiation in vivo and which have also been found in mouse blastocysts
developing in the same medium. This indicates for both species that cells of the
blastocyst are strongly disposed towards certain characteristic diflferentiative
266
H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
12
f
24
481
Implantation
Implantation
in vivo
in vitro
Time of development (h)
Fig. 3. Development of cell numbers of mouse blastocysts and their ICMs in vivo
and of mouse and rat blastocysts and their ICMs during culture in NCTC-109-FCS
(original data Table 4). Since blastocysts and ICMs implant after 48-72 h in culture,
cell numbers were determined up to 48 h. Cell number of blastocysts: Mouse
in vivo ( •
• ) , mouse in vitro (x
x), rat in vitro (o — o). Cell number
of ICMs: Mouse in vivo (©
©), mouse in vitro (®
(*)), rat in vitro (®
®)
pathways, even under conditions which are less than ideal for cell organization.
ICM development of mouse blastocysts in vitro has usually been compared
to the in vivo situation after implantation. Our data on mouse blastocysts before
implantation in vitro clearly demonstrate that growth kinetics of both BTCN
and ICM cells are identical in NCTC-109-FCS to the in vivo situation of the
NMRI strain up to 20 h, when implantation is occurring in vivo. It is of importance that the size of the ICM in early and late blastocysts in our study is
comparable to the data reported in other immunosurgical investigations on
different strains of mice (Handyside, 1978; Magnuson, Jacobsen & Stackpole,
1978; Rossant & Lis, 1979). The ratio of inside to outside cells is similar to
calculations in a study on serial histological sections of mouse blastocysts
(Copp, 1978). However, as already discussed by Rossant & Lis (1979), the
inside numbers of cells obtained immunosurgically are slightly lower than from
serial reconstruction of embryos of a similar total cell number.
Furthermore, the decrease in the ratio of inside to outside cells that we
observed in early (BTCN 33) and late mouse blastocysts in vivo (BTCN 72) and
ICM development in rat and mouse blastocysts in NCTC-109-FCS 267
in vitro (BTCN 75) is similar to the constant fall of the ratio throughout blastocyst development as calculated by Copp (1978). However, during the second
24 h period in NCTC-109-FCS we found an increase in this ratio in blastocysts
before implantation in vitro (BTCN 129). All previous investigators of blastocyst
differentiation in vitro have observed a delay of implantation of more than 24 h
as compared to development in vivo (Spindle & Pedersen, 1973; Sherman, 1975;
Hsu, 1978). In addition, our study indicates that this delay is inducing significant
differences in BTCN and in the ratio of inside to outside cells at implantation
in vitro as compared to mouse blastocysts at implantation in vivo.
During development in vivo growth kinetics of preimplantation rat and mouse
embryos are quite different at the morula and at the blastocyst stage, as the
BTCN was significantly smaller in the rat during this period in all previous
studies (Surani, 1975, 1977; Schiffner & Spielmann, 1976). Since blastulation
occurs at a lower BTCN in the rat than in the mouse, and since the ratio of ICM
cells to BTCN is similar in the two species, the size of the ICM
is, of course, lower in early rat blastocysts than in early mouse blastocysts
(Table 4). According to earlier studies (Magnuson et al. 1978; Rossant & Lis,
1979) a preimplantation mouse embryo with nine inner cells is a late morula and,
according to our data, a rat embryo with nine inner cells already is a blastocyst.
These observations indicate that blastulation does not seem to be dependent
upon the size of either BTCN or ICM in the two species. Our previous studies
on the effects of cyclophosphamide treatment in vivo on the size of BTCN and
cell number of the ICM in the mouse (Spielmann, Jacob-Miiller, Eibs &
Beckord, 1980) and the present data, therefore, support the results of investigators who suggest that blastulation does not depend upon a certain number of
cells per embryo but rather seems to be a time-related developmental process
(Smith & McLaren, 1977).
Very careful studies on the developmental potential of isolated lCMs from
mouse blastocysts did not show any differences between microsurgically and
immunosurgicaHy isolated ICM cells (Rossant & Lis, 1979). This result proves
the reliability of the method of immunosurgery not only for investigations on
differentiation of isolated cells but also for studies on the growth kinetics of the
two groups of cells of blastocysts before implantation in vivo and in vitro. In
addition, determination of ICM growth before implantation is important in
teratological investigations on the differential sensitivity of ICM and trophectoderm cells (Spielmann et al. 1980) and it allows us to analyse ICM growth of
blastocysts already before implantation in vitro in media that are tested for
their ability to support differentiation of blastocysts in culture.
This work was supported by grants of the Deutsche Forschungsgemeinschaft awarded to
Sonderforschungsbereich 29 'EmbryonaleEntwicklungundDifferenzierung'. We express our
sincere thanks to Dr Colin Liddiard for editorial assistance.
268
H. SPIELMANN, U. JACOB-MULLER AND W. BECKORD
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{Received 28 January 1980, revised 21 February 1980)
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