/. Embryol. exp. Morph. Vol. 31, 3, pp. 707-719, 1974
707
Printed in Great Britain
Serum variants causing the
formation of double hearts and other abnormalities
in explanted rat embryos
By C. E. STEELE 1 AND D. A. T. NEW1
From the Physiological Laboratory, University of Cambridge
SUMMARY
Rat embryos explanted before organogenesis (8£ days gestation) were grown in culture in
homologous serum. When the serum was prepared from blood centrifuged after clotting,
the embryos developed double hearts. In serum prepared from blood centrifuged before
clotting had occurred, and in plasma, the embryos developed normal single hearts. The
delayed-centrifuged (D.C.) serum also supported less growth of older embryos than the
immediately-centrifuged (i.e.) serum. The harmful properties of D.C. serum appeared rapidly
in contact with a normal blood clot but did not develop in contact with separated blood cells
and fibrin clot. Mixtures of D.C. and r.c. sera gave results intermediate between those from
the two sera alone. No significant differences were found between D.C. and i.e. serum in
calcium or complement content but both supported better embryonic development after
pre-heating.
INTRODUCTION
In a study of rat embryos explanted at the egg-cylinder stage and grown in
culture in homologous serum, New & Daniel (1969) found that most of the
explanted embryos developed abnormal double hearts as a result of failure of
fusion of the heart primordia. The serum used in their cultures was prepared
from freshly extracted blood that was allowed to clot and stand for 18-24 h
before centrifuging and decanting. Steele (1972) obtained much improved
embryonic development, with the formation of normal single hearts and a
functional blood circulation, in serum decanted from blood centrifuged immediately after extraction. It is interesting that such an apparently small variation
in the preparation of the serum should have such a large developmental effect
and suggests that some abnormalities in vivo might result from minor variations
in blood composition. At present the factors in the serum responsible for the
single and double hearts are unknown and the experiments described in this
paper have been aimed at obtaining further information about serum variations
and their effects on embryonic development.
1
Authors' Address: Physiological Laboratory, Cambridge CB2 3EG, U.K.
708
C. E. STEELE AND D. A. T. NEW
Fibrin clot
removed
Immediate
YJ
2500 rev min.
Freshly
^
5 min
extracted
blood
Not
cen trifu ned
Standard
blood clot
cen tri fillet
2500 re\ min.
Retracted 5 m i n
standard
ck)t
Fig. 1. Preparation of i.e. and D.C. sera.
MATERIALS AND METHODS
Explanation and culture methods
The methods used for explantation and culture of the embryos were as
described by New (1971) and New, Coppola & Terry (1973).
Rat embryos of the CFHB strain were explanted in Tyrode saline during the
afternoon of the 9th or 10th days of gestation, i.e. the embryos were 8[ or
9\ days post-coitum. Reichert's membrane was torn open but the visceral yolk
sac and ectoplacental cone left intact. In comparing different culture media,
embryos from each litter were divided between the different media to eliminate
any effects of inter-litter variation.
Except where stated otherwise, egg-cylinders (8^-day embryos) were incubated
for 48 h in watchglass cultures with a gas phase containing 40 % O2, 5 % CO2 and
55 % N2. Each watchglass contained 1 ml medium (serum or plasma) and up to
eight egg-cylinders, which attached to the floor of the watchglass by the ectoplacental cone. At the end of the culture period, heart development was classified
as single, double, uncertain/intermediate, or none.
Headfold (9^-day) embryos were cultured in rotating tubes for periods of
48-67 h in medium equilibrated with 5 % CO2 in air. These embryos invariably
formed normal single hearts, and development in culture was assessed by the
number of new somites formed, the condition of the blood circulation, the
adoption by the embryo of the foetal (ventrally curved) position and the expansion of the yolk sac.
Growth was assessed by determining the protein content of the embryos by
the colorimetric method of Lowry, Rosebrough, Farr & Randall (1951).
Serum variants and double hearts
709
i.e. serum, D.C. serum and plasma
The sera (Fig. 1) and plasma used as nutrient media were prepared from blood
withdrawn from the dorsal aorta of rats anaesthetized with ether. Except where
stated otherwise, the media compared in any one experiment were made from
the same blood samples.
Immediately-centrifuged (i.e.) serum. The blood was centrifuged at 2500 rev/
min for 5 min immediately after withdrawal from the rat. This precipitated the
cells and a white fibrin clot rapidly formed in the supernatant. The clot was
carefully removed with forceps, the fluid being allowed to drain from it back
into the centrifuge tube. The contents of the tube were then recentrifuged and
the i.e. serum decanted. In some samples the recentrifuging and decanting were
delayed for 18 h; these are designated 'i.e. serum-(d)'.
Delayed-centrifuged (D.C.) serum. The blood was not centrifuged at the time
of withdrawal but was allowed to clot and stand overnight (approx. 18 h) at
4 °C. The clot was then broken up with a sterile glass rod, the blood centrifuged
at 2500 rev/min for 5-10 min and the D.C. serum decanted.
Plasma. Heparin (0-02%) or sodium oxalate (1-3%) was added to the extracted blood which was immediately centrifuged at 2500 rev/min for 5 min.
No clot formed and the plasma was decanted.
The i.e. serum and plasma were pale yellow fluids but the D.C. serum was
sometimes pink, presumably as the result of slight haemolysis. Streptomycin
was added to all media to a final concentration of 50 /tg/ml and the media were
stored at 4 °C if they were to be used within 24 h or at - 10 °C for longer periods.
Some samples of serum were pre-heated to 56 °C for 30 min before culturing,
and the concentration of complement determined by the method of Demant,
Capkova, Hinzova & Voracova (1973).
Seven series of experiments were made, with media prepared as follows:
(1) Immediately-centrifuged (i.e.) serum, delayed-centrifuged (D.C.) serum,
and plasma (8-]-, 9^-day embryos).
(2) Reduced storage periods of clotted blood for D.C. serum preparation
(8^-day embryos).
(3) Addition of cells or clot to i.e. serum (8^-day embryos).
(4) Delayed decanting of i.e. serum (8£- and 9^-day embryos).
(5) Mixtures of i.e. serum and D.C. serum (8^-day embryos).
(6) Calcium content of i.e. and D.C. sera (8^-day embryos).
(7) Pre-heating and complement assay of i.e. and D.C. sera (8^-day embryos).
710
C. E. STEELE AND D. A. T. NEW
Single heart
Double heart
(Often the hearts
are completely
separated and 6
not touch as in
this diagram.)
Uncertain (heart only)
Fig. 2.'Single','double' and' uncertain' hearts, ys, Yolk sac; a,amnion; /;, headfold;
ht, heart;/, foregut; s, somite.
RESULTS
Series 1. Immediately-centrifuged (i.e.) serum, delayed-centrifuged
(D.C.) serum and plasma (8-j- and 9j-day embryos)
After 24 h in culture, the 8^-day embryos in all three media were at the
headfold stage and were very similar in size and appearance. After 48 h, however, a difference in heart development was clearly visible, the embryos in i.e.
serum and plasma tending to form single hearts and those in D.c. serum, double
hearts (Figs. 2, 3). The average width of the embryo measured across the heart
region was greater in explants with a double heart (0-9 mm) than in those with
a single heart (0-6 mm), but in both the yolk sac was spherical with an average
diameter of 1-9-20mm. Table 1 shows the number of 8^-day embryos developing double or single hearts in each of the three media (data previously
reported by Steele, 1972).
Six experiments were made to examine the growth of 9-^-day embryos in the
three media. Growth was assessed by determining the final protein content of
each embryo, after removal of the membranes. The results are summarized in
Table 2. Each entry in the table gives the mean and standard error of the
5-6 embryos in one culture tube. All the culture tubes in each experiment
D
Rat embryos with single (A, B) and double (C, D) hearts. The embryos were explanted at 8^ days gestation and grown in culture for
48 h. Whole embryos magnified x 30, transverse sections x 150. Abbreviations as in Fig. 2.
FIGURE 3
B
lit
712
C. E. STEELE AND D. A. T. NEW
Table 1. Heart development of 8\-day rat embryos grown in immediatelycentrifuged (i.e.) serum, delayed-centrifuged (D.C.) serum, and plasma
Heart
Medium
No. of embryos
Single
Double
Uncertain
None
29
23
23
0
0
17
5
0
1
6
13
17
6
0
0
4
4
8
3
5
1st experiment
i.e. serum
D.C. serum
2nd experiment
Plasma
D.C. serum
Table 2. Final protein (//g) of9\-day embryos cultured in different sera
{described in the text) and plasma
(Each entry gives the mean and standard error of the 5-6 embryos in one culture tube.
Letters refer to the blood samples from which the sera and plasma were taken. The
embryos in experiments 1-5 were grown for 48-51 h in culture, in experiment 6 for
67 h. Significant differences (P < 001) between i.e. serum and D.C. serum from the
same blood sample are indicated by >.)
Experiment
no.
i.e. serum
1
A 136 ±17
—
2
3
4
C158 + 10
—
5
—
6
—
i.e. serum-(d)
D.C .
serum
Plasma
A 71 ±8
B 175±16\
B 185±15/
—
>
D 181 ±12
F 164 ±15
G 146 ±31
H218±13 >
IB 105 ±9
IB 120 ± 15
C
D
E
F
166 ±9
196 ± 16
69±4
140 ±17
G 93 + 34
H 119 + 25
C 184±5
D 206 ±18
F 142 ±14
G 134 ±17
contained embryos from the same litter or litters. The letters in the Table refer
to the blood samples from which the sera and plasma were taken.
Table 2 shows that mean final protein of the embryos in the eight tubes of
i.e. serum and the four tubes of plasma ranged from 134 to 218 /ig. The values
for D.C. serum were more variable and usually lower, in six out of the nine tubes
being 120 /eg or less. In three experiments (1, 2, 6) the protein of the embryos
in i.e. serum was significantly higher than in the D.C. serum from the same blood
samples (A, B, H). In none of the experiments was embryo protein in D.C. serum
significantly higher than in the corresponding i.e. serum. In blood samples
C, D, F and G there were no significant differences between i.e. serum, D.C.
serum or plasma.
Serum variants and double hearts
713
Table 3. Heart development of 8\-day embryos in D.c. serum prepared from
clotted blood stored for different periods
Heart
Medium
No. of
embryos
Single
Double
Uncertain
None
i.e. serum
I h D.C. serum
3 h D.C. serum
18 h D.C. serum
8
10
10
9
7
5
2
1
0
2
5
5
0
2
1
2
1
1
2
1
The sera were heated at 56 °C for 30 min before culturing.
Table 4. Heart development of 8\-day rat embryos in i.e. serum pre-treated with
added blood cells or protein clot
Heart
Medium
i.e. serum + cells
i.e. serum -t- clot
Mixed i.e. + cells
and i.e. 4- clot
Control i.e. serum
D.C. serum
No. of
embryos
Single
Double
Uncertain
None
7
7
7
4
6
4
1
0
1
2
1
2
0
0
0
7
7
7
0
0
6
0
1
0
0
The amount of development shown by the 9^-day embryos in culture varied
with the amount of protein synthesis. For example, in Expt 3 most of the
embryos in all three media attained 24-26 somites, rotated into the foetal
position and developed a yolk sac 3-5-4-0 mm diameter with a good blood
circulation; similar development was shown by the embryos of Expt. 1 in i.e.
serum, but those in D.C. serum averaged only 19 somites, failed to rotate,
developed smaller yolk sacs and lacked a blood circulation.
Series 2. Reduced storage periods of clotted blood for D.c. serum
preparation (8\-day embryos)
Freshly extracted blood was allowed to clot (without centrifuging) and stored
at 4 °C. The clot was broken up, centrifuged and the serum decanted after shorter
periods (\ h a n ^ 3 h) than normally used for the preparation of D.C. serum. The
effects of these sera on the heart development of 8^-day embryos were examined
and compared with those of standard D.C. serum and i.e. serum. The results,
summarized in Table 3, suggest that serum prepared from blood stored for as
little as y h before centrifuging has already acquired some tendency to cause the
formation of abnormal double hearts, and that by 3 h this effect has become
accentuated almost to that of the standard D.c. serum prepared from blood
stored 18 h.
714
C. E. STEELE AND D. A. T. NEW
Table 5. Heart development of 8^-day rat embryos in i.e. serum-{d) prepared by
delaying decanting for 18 h, compared with controls
Heart
Medium
1st Experiment
i.e. serum
i.e. serum-(d)
D.C. serum
2nd experiment
i.e. serum-(d)*
D.C. serum*
embryos
Single
Double
Uncertain
None
6
15
6
5
10
0
0
0
5
0
0
0
1
5
1
11
11
9
4
0
1
1
0
1
6
Heated at 56 °C for 30 min before culturing.
Series 3. Addition of cells or clot to i.e. serum (SJ2-day embryos)
The cells and clot removed during the preparation of some i.e. serum were
washed in Tyrode saline and then each recombined with the serum. The serum
+ cells and the serum + clot were allowed to stand for about 18 h at 4 °C and
then centrifuged and decanted. The serum removed from the cells was usually
pink, similar to D.C. serum, but the serum removed from the clot remained pale
yellow. Development of 8^-day embryos in each of these two sera alone was
compared with development in a mixture (in equal proportions) of the two
together, and in control i.e. and D.C. sera.
The results are shown in Table 4. All the seven embryos grown in control
i.e. serum formed single hearts. The i.e. serum that had been stored with the
protein clot gave similar results (6 single hearts; 1 uncertain). In each of the
other two i.e. sera, one embryo developed a double heart but in the D.C. serum
six of the embryos formed double hearts.
Evidently storage in contact with blood cells or fibrin clot does not give i.e.
serum the double heart-forming character of D.C. serum.
Series 4. Delayed decanting of i.e. serum (8\- and 9\-day embryos)
Samples of blood, centrifuged immediately after extraction, were left to stand
in the centrifuge tubes at 4 °C for 18 h and then centrifuged again and the
serum decanted. In Tables 2 and 5 this serum is designated 'i.e. serum-(d)'.
Table 5 shows the results of two experiments with 8^-day embryos. The i.e.
serum-(d) in the first experiment was stirred with the cells and clot between the
first and second centrifugations, that in the second experiment was left unstirred.
Both these sera supported development of single hearts in most of the explanted
embryos. In this they resembled control i.e. serum and differed from D.C. serum.
Table 2 shows the effect of i.e. serum-(d) prepared from five different samples
of blood (B, D, F, G, H) on protein synthesis by 9|-day embryos. In two of the
Serum variants and double hearts
715
Table 6. Heart development of 8-\-day embryos grown in i.e. serum and
D.C. serum mixed in various proportions
Medium
i.e. serum
100%
80?/o
50%
20%
Heart
D.C. serum
20%
50%
80%
100%
No. of
embryos
7
9
9
9
7
Single
Double
Uncertain
None
6
8
2
3
2
0
0
5
6
5
0
0
1
0
0
1
1
1
0
0
The sera were pre-heated at 56 °Cfor 30 min.
Table 7. Calcium content of different samples of i.e. serum and D.c. serum; heart
development of 8\-day embryos in these media
(Embryos cultured in i.e. serum 1, 2 or 3 were from the same litter as
those in D.C. serum, 1, 2 or 3, respectively.)
Heart
Medium
i.e.
Serum 1
Serum 2
Serum 3
Ca 2+
(mg%)
No. of
embryos
Single
Double
Uncertain
None
10-9
12-6
11-3
2
3
5
2
3
3
0
0
0
0
0
2
0
0
0
10-6
10 8
11-4
5
4
5
1
0
0
2
4
5
2
0
0
0
0
0
D.C.
Serum 1
Serum 2
Serum 3
samples (B, H) protein synthesis was significantly higher in i.e. serum-(d) than
in D.c. serum. (Of the two tubes of i.e. serum-(d) prepared from blood sample B,
one was stirred with the clot and cells during preparation, the other left unstirred; both gave similar results.) Although no trials were made with i.e.
serum-(d) and control i.e. serum from the same blood sample, growth in these
two sera appeared to be similar.
Series 5. Mixtures of i.e. serum and D.c. serum {8\-day embryos)
Table 6 shows the heart development of 8^-day embryos grown in mixtures of
i.e. serum and D.C. serum, compared with development in whole i.e. serum or
whole D.C. serum. The addition of up to 20% i.e. serum appears to have little
effect on the frequency of double hearts formed in D.c. serum, nor does the
addition of up to 20 % D.c. serum affect the frequency of single hearts in i.e.
serum. In a mixture of i.e. and D.C. serum in equal proportions, five out of
nine embryos formed double hearts and only two single hearts.
716
C. E. STEELE AND D. A. T. NEW
Table 8. Effect ofpre-heating i.e. serum on development of
explanted 8\-day embryos
Medium
i.e. serum
i.e. serum
pre-heated
No. of
embryos
Embryonic
protein (fig)
mean ± S.E.
Mean
yolk-sac
diameter
(mm)
Embryos
with
heartbeat
after 72 h
Embryos with
yolk sac
circulation
after 72 h
12
12
35 ±4
64±4
2-3
30
7
12
1
4
Table 9. The effect of pre-heating the culture serum on heart
development of 8\-day rat embryos
Heart
Medium
i.e. serum
pre-heated
D.C. serum
pre-heated
D.C. serum
No. of
embryos
Single
Double
Uncertain
None
0
0
0
12
0
6
0
12
12
0
0
Series 6. Calcium content of i.e. and D.c. sera (8%-day embryos)
The calcium ion content of three samples of i.e. serum and three samples of
D.C. serum was determined by absorption spectrophotometry. Embryos of
8£ days were grown in each of the six media but the type of heart development
obtained appeared to be unrelated to the small differences in calcium content
(Table 7).
Series 7. Pre-heating and complement assay of i.e. and D.C. sera
{8\-day embryos)
The protein content of embryos explanted at 8^- days and cultured in preheated i.e. serum was significantly higher (P < 0-0005) than in i.e. serum from
the same batch which had not been pre-heated (Table 8). (The embryos in this
experiment were cultured for 24 h in watchglasses and then transferred to
rotating tubes for a further 48 h.)
Pre-heating also improved development in D.c. serum, resulting in more
single hearts and fewer double hearts than in control D.c. serum (Table 9 - and
see also Tables 3, 5 and 6). The pre-heating inactivated most of the complement
in the sera but no significant difference could be detected between the complement content of i.e. and D.C. serum (Table 10).
Serum variants and double hearts
111
Table 10. Complement content of i.e. and D.C. rat sera expressed as percentage
of content in standard guinea-pig serum
(Assay method of Demant et al. 1973.)
Rat no.
1
Serum
i.e.
D.C.
D.C. pre-heated
i.e.
D.C.
i.e. pre-heated
D.C. pre-heated
i.e.
D.C.
i.e.
D.C.
i.e. pre-heated
D.C. pre-heated
Complement
116
86
7
95
106
6
6
71
55
88
65
5
5
DISCUSSION
The results that have been described show that serum (D.C.) as usually prepared from clotted blood causes the formation of double hearts in rat embryos
explanted before organogenesis; but if serum (i.e.) is prepared from blood
centrifuged before clotting has occurred, or if plasma is used instead of serum,
the embryos develop normal single hearts (series 1). D.c. serum also supports
less growth of older embryos than i.e. serum, though different samples of D.C.
serum vary widely in this respect (Table 2). The harmful properties of D.C.
serum develop rapidly in contact with a normal blood clot (series 2) but do not
appear after storage in contact with separated blood cells and fibrin clot
(series 3 and 4). Mixtures of D.C. and i.e. sera give results intermediate between
those from the two sera alone (series 5). No significant differences were found
between D.C. and i.e. serum in calcium (series 6) or complement content
(series 7) but both supported better embryonic development after pre-heating.
The factor(s) in D.C. serum that cause double heart development appear to be
a product of normal blood clotting in which the blood cells are trapped in the
fibrin coagulum; they are not produced by a cell-free clot nor by cells in suspension, even when both are present in the same serum. An example of a difference
between cells held in a fibrin clot as compared with those suspended in plasma
was demonstrated by Bernstein (1953), who found more variability in potassium
exchange by erythrocytes in the clot compared with those in suspension. But
differences in potassium concentration do not seem to be the cause of the heart
abnormalities. Although Steele (1972) found that D.c. serum contains more than
twice as much potassium as either i.e. serum or plasma, the addition of extra
718
C. E. STEELE AND D. A. T. NEW
potassium to i.e. serum did not result in the development of double hearts.
DeHaan (1958a, b) showed that acetylcholine, sodium acetate and EDTA all
produced double hearts in chick embryos and he suggested that these agents
disrupted normal intercellular relationships by binding calcium. But in the
present study, calcium concentrations in i.e. and D.C. sera were found to be
similar and unlikely to be the cause of differences of heart development in the
cultured rat embryos. At present it remains unknown how the formation of a
normal blood clot renders D.C. serum teratogenic.
The pre-heating of the sera in series 7 inactivated most of the complement, as
confirmed by a subsequent assay (Table 10). Pre-heating both D.C. and i.e. sera
gave improved development of the 8^-day embryos and suggests that in untreated sera development might be hindered by a complement-dependent
immune reaction. Some of the embryos grown in pre-heated D.C. serum formed
single hearts, suggesting that the heat treatment may remove some of the harmful factor(s) acquired from the blood clot, but further data are needed to confirm
this.
The double hearts result from a failure of the two heart primordia to fuse and
it is interesting that Severn & Holyoke (1973) have recently described failures
of heart formation in human development which they attribute to a similar
mechanism ('primary cardia bifida'). The cause of these failures remains unknown but in view of the present findings the possibility seems worth examining
that such congenital defects might arise in vivo as a result of any small haemorrhage and clotting of maternal blood in the uterus.
This work was supported by the Medical Research Council.
We are grateful to C. W. S. Howe for performing the complement assay.
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719
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