/. Embryol. exp. Morph. 76, 1-8 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Growth of 9 5-day rat embryos in human serum
By M. GUPTA AND F. BECK 1
From the Department of Anatomy, Leicester University
SUMMARY
Rat embryos were explanted at 9-5 days and cultured for 48 h in human serum supplemented
with glucose. The embryos were anaemic and frequently malformed. The haemoglobin and
DNA content of these embryos was less than those grown in pure rat serum. Addition of 10 %
rat serum improved the embryonic growth as well as the haemoglobin and DNA content. This
suggests that rat serum provides essential growth factors required by the embryos which are
not present in human serum.
INTRODUCTION
Whole-rat-embryo culture based on the New Technique (New, Coppola &
Cockroft, 1976) but using human serum as culture medium has been described
by Shepard and his co-workers (Shepard, Tanimura & Robkin, 1970; Tanimura
& Shepard, 1970; Fantel, Greenaway, Juchau & Shepard, 1979) and more
recently by Chatot, Klein, Piatek & Pierro (1980). The latter supplemented
human serum with glucose to bring the level of sugar to that normally present in
rat serum. Using this technique it was suggested that pure human serum could
be used to evaluate the true teratogenic activity of bioactive materials in man
(Chatot et al. 1980). This approach has great potential because, if it proved
reliable, human serum in a variety of situations could be monitored directly for
potential embryotoxicity. However, Reti, Beck & Bulman (1982) showed that
although human glucose-supplemented serum supported growth of the earlyhead-fold rat embryo, the level of abnormalities was unacceptably high compared to controls. In addition embryos cultured in human serum were smaller
than those cultured in pure rat serum and although a vitelline circulation was
established the embryos were grossly anaemic and on naked eye inspection the
corpuscular element of the blood was quite colourless. The gross morphological
effects were overcome when human serum was supplemented with 10 % rat
serum. This also had the effect of producing red-coloured blood corpuscles. 10 %
rat serum in buffered saline is unable by itself to support embryonic growth (AlAlousi, Ph.D. Thesis in preparation) and it was concluded that supplementation
of human serum with 10% rat serum might provide essential specific
1
Author's address: Department of Anatomy, Medical Sciences Building, Leicester University, University Road, Leicester, LEI 7RH, U.K.
Z
M. GUPTA AND F. BECK
macromolecules required in only trace amounts for normal rat embryo growth,
absent in human serum but present in rat serum.
We now present the results of biochemical studies on rat embryos grown
between 9-5 and 11-5 days in pure human serum compared to those obtained
from embryos grown in rat-serum-supplemented human serum and with embryos of an equivalent age cultured for 48 h in normal rat serum or removed at
11-5 days from a normal pregnancy. We have measured protein, DNA and
haemoglobin levels in each of these.
MATERIALS AND METHODS
Wistar rats were mated overnight and pregnancy was timed from midnight
preceding the morning on which vaginal plugs were observed. Conceptuses were
explanted at 9-5 days according to the standard method of rodent embryo culture
described by New (1978). The embryos were divided into three groups:
Group A Cultured in 100 % human serum
Group B Cultured in 90 % human serum and 10 % rat serum
Group C Cultured in 100 % rat serum.
In addition, a further group of rats was killed at 11-5 days and the conceptuses
removed for comparison with the experimental groups.
Human serum from several donors was prepared in an identical manner to that
used for rat serum except that it was always supplemented with glucose
(1 mg/ml of human serum). The human serum was not pooled but each donor's
blood was used in both Group A and Group B. 0-02 ml of antibiotics (Streptomycin/Penicillin cone. 5000/ig/5000i.u.) were added per ml of rat and human
sera. The embryos were cultured for 48 h. They were gassed initially with a gas
mixture of 5 % O 2 , 5 % CO 2 , 90 % N 2 . After 24 h this was replaced with 20 %
O 2 , 5 % CO 2 , 75 % N2 and after 42h with 40 % O 2 , 5 % CO 2 , 55 % N 2 .
At the end of the culture period the embryos were examined under a dissecting
microscope for any abnormalities. A scoring system (Brown & Fabro, 1981) was
used to determine the morphological characteristics of the embryos. The embryos were then divided into three sub-groups for the estimation of protein
(Lowry, Rosenbrough, Farr & Randall, 1951), DNA (by methods based on Le
Pecq & Paoletti, 1966; Karsten & Wollenberger, 1972) and haemoglobin (based
on Williamson, 1916). For the haemoglobin estimation the yolk sac and the
embryos were frozen intact whereas the protein and DNA content were
estimated on embryos which had been removed from their membranes.
RESULTS
The results of the biochemical investigations are given in Table 1.
All the embryos grown in 100 % human serum looked anaemic while embryos
cultured in human-supplemented rat serum had pink blood cells similar to those
4-68 ±0-38
n = 25
P<0-05
6-24 ±0-56
n = 27
P>0-l
216-32 ±10-31
n = 25
P>0-l
249-25 ±11-27
n = 27
P<0-01
291-14 ±11-31
n = 24
P< 0-001
100% human serum
90% human serum +
10% rat serum
11-5 days in vivo
39-94
28-54
0-43
0-55
0-82
19-94
21-93
23-49
93-32 ±4-89
n = 25
P< 0-001
136-88 ±6-33
n = 26
P>0-l
239-61 ± 13-08
n = 21
P< 0-001
46-22
34-08
0-63
21-53
132-89 ± 6-50
n = 29
Protein/DNA
Ratio
Haemoglobin/
Protein Ratio
Haemoglobin/
DNA Ratio
Haemoglobin
(in /ug)
n = number of embryos. Each value is the mean of the number of embryos + S.E. (Standard Error).
P was calculated using Student's T test to indicate the difference from culture in 100 % normal rat serum.
10-20 ±0-50
n = 23
P< 0-001
6-17 ±0-52
n = 26
210-32 ±6-15
n = 26
100% normal rat serum
DNA
(in j"g)
Protein
(in Pg)
Culture medium
Table 1. Protein, DNA and haemoglobin canteur of the embryos in fig cultured from 9-5 to 11-5 days in 100% human serum
90% human serum +10% rat serum, 100% rat serum and 11-5 days in vivo
S'
8
Iture of rat embry
uman s erum
M. GUPTA AND F. BECK
V--
Fig. 1. 11-5-day rat embryo after 48 h of culture in glucose-supplemented 100%
human serum. Note the presence of an open neural tube (arrow).
Fig. 2. 11-5-day normal rat embryo cultured in 100 % rat serum for 48 h.
Bar= lmm.
cultured in 100% rat serum or to 11-5 days in vivo embryos. 30-66% of the
embryos grown in 100% human serum were malformed (see Table 2). The
malformations included open neural tubes and abnormal turning of the embryo
(see Fig. 1). The presence of an open posterior neuropore, deficiency in somite
numbers and delayed completion of turning were regarded as growth retardations
0%
n = 68
—
25-85 ±0-20
n = 47
P< 0-001
43-10 ±0-04
n = 47
P<001
5%
n = 80
3-60 ± 0-06
n = 54
24-07 ±0-17
n = 54
P< 0-001
11-5 days
n = number of embryos. Each value is the mean of the number of embryos ± standard error.
P values indicate differences from culture in 100% normal rat serum.
90% human serum +
10% rat serum
100% human serum
30-66%
n = 75
0%
n = 81
3-53 ± 0-03
n = 52
3-44 ±0-07
n = 50
24.80 ±0-11
n = 52
23-22 ±0-27
n = 50
P< 0-001
43-0 ±0-00
n = 52
40-14 ±0-51
n = 50
P< 0-001
42-77 ±0-14
n = 54
100% normal rat serum
% growth retarded and
abnormal embryos
Yolk-sac diameter
Somite number
Morphological score
(Brown and Fabro, 1981)
Culture medium
Table 2. Mean morphological score, somite number, yolk-sac diameter and the percentage of growth-retarded and normal
embryos
3
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9
M. GUPTA AND F. BECK
rather than malformations. The majority of the 5 % of deviant embryos grown
in human supplemented rat serum fell into this group. The embryos grown in
100 % rat serum were all normal (see Fig. 2).
DISCUSSION
Our findings do not support a conclusion that rat embryos can be reliably
grown in 100 % glucose-supplemented human serum although the protein content of the embryos grown in 100 % human serum was not significantly different
from the embryos grown in 100 % pure rat serum. A significant difference at a
0-05 level of probability was found for DNA levels and an even greater difference
(P < 0-001) for haemoglobin levels. The protein values reported by Chatot et al.
(1980) are much lower than in the present findings. This may reflect different
methods used for protein estimations; its significance should not be exaggerated
since each set of results is internally consistent.
The protein values when compared with 11-5-days in vivo embryos showed
that the embryos cultured in rat or human serum contained significantly less
protein than the in vivo group. This is consistent with some of the findings
reported by New, Coppola & Cockroft (1976). The protein content improved
significantly (by 15 %) when 90 % human serum was supplemented with 10 % rat
serum.
Our biochemical findings confirm the morphological observations reported by
Reti et al. (1982). Embryonic growth in human serum can be improved when
human serum containing glucose is supplemented with 10 % rat serum and the
advantages of growing mammalian embryos on human serum can thus be
retained by the addition of an aliquot of rat serum which in itself would not be
sufficient to sustain growth.
The haemoglobin content of embryos grown in human serum was significantly
less than in those grown in rat serum, but when the human serum was supplemented with 10 % rat serum haemoglobin synthesis matched that of embryos grown
in pure rat serum. Nevertheless the haemoglobin content in the in vivo group was
much higher than in the other three groups.
It is interesting to observe that the haemoglobin/protein ratio is highest in the
in vivo group of animals and lowest in the group cultured in 100 % human serum.
Addition of 10 % rat serum or culture in 100 % rat serum increased the protein
content of the embryos but it increased the haemoglobin content even further.
This is in keeping with the naked eye appearances of the cultured embryos. The
picture is similar but even clearer when haemoglobin/DNA ratios are considered.
The mammalian embryo has great difficulty in accumulating iron. The uptake
of this element in the rat is thought to be largely achieved through the binding
of a maternal iron-bearing serum /3-globulin known as transferrin to the surface
of the visceral yolk-sac epithelial cells. The possible presence of specific surface
Culture of rat embryos in human serum
1
receptor sites for transferrin has been demonstrated (Huxham, 1982). Many of
the transferrin molecules are interiorized and passed unaltered to the embryo but
the significance of this finding is speculative.
It may be that at this stage of development the embryo is unable to synthesize
its own transferrin in adequate quantities and relies upon transmission of the
maternal form (cf IgG).
Rat serum levels of transferrin are in the region of 6 /Ug/ml while human levels
are 3/ig/ml. This means that human serum contains only about a half of the
circulating iron present in the rat. This cannot alone account for the anaemia
following culture in human serum because culture in 50 % rat serum in Hank's
solution results in fairly normal development. It may be, therefore, that the
specificity of the yolk-sac receptors is such that rat transferrin is much more easily
bound than is the human form.
The protein: DNA ratio (see Table 1) indicates that the embryos grown in
100 % human serum contain fewer cells than those grown in rat-supplemented
human serum, pure rat serum or the 11-5 days in vivo group.
We thank the National Fund for Research into Crippling Diseases for a grant in aid of
research.
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BROWN,
{Accepted 28 March 1983)