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/. Embryol. exp. Morph. Vol. 26, 3, pp. 475-480, 1971
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475
Stimulation of haem synthesis by erythropoietin
in mouse yolk-sac-stage embryonic cells
By A. E. BATEMAN 1 AND R. J. COLE 1
From the School of Biological Sciences, Sussex University
SUMMARY
Cultures of disaggregated cells from mouse embryos at the stage of yolk-sac erythropoiesis
have been used to test sensitivity of primary erythroid cells to erythropoietin, the hormone
which controls adult red cell production. Synthesis of haem was stimulated by 89% in cells
of 8-day embryos, 23 % in cells of 9-day embryos and by 19% in peripheral nucleated blood
cells of 12-day embryos. Differentiation of primary embryonic erythroid cells may therefore
be controlled by erythropoietin in situ. The haem precursor #-aminolaevulinic acid (ALA)
did not stimulate haem synthesis under similar conditions.
INTRODUCTION
The production of red blood cells (erythropoiesis) in adult mammals is controlled by the hormone erythropoietin (Ep) which is known to stimulate the
differentiation of recognizable erythroid cells from unrecognizable precursors
(Jacobson, Goldwasser, Plzak & Fried, 1957; Hanna, 1967), to effect the release
of increased numbers of new red cells into the circulation after 24 h in vivo
(reviewed by Hodgson, 1970) and also to reduce the cell cycle time of maturing
erythroid cells (Hanna, Tarbutt & Lamerton, 1969) and to increase haem
synthesis in marrow cells in vitro (Krantz, Gallien-Lartigue & Goldwasser,
1963).
Ep may also control erythropoiesis in the embryo. Mammalian erythropoiesis occurs in four successive organs throughout development. The first
erythroid cells (the primary series producing large nucleated erythrocytes) occur
in the yolk sac; then foetal liver, spleen and bone marrow become successive
sites of erythropoiesis. Foetal liver and foetal spleen cells synthesize haem in
vitro in response to Ep (Cole, Hunter & Paul, 1968) but haem synthesis in
cultured whole yolk-sac-stage embryos is independent of added Ep (Cole &
Paul, 1966; Hunter & Paul, 1969).
This study has used cultures of disaggregated cells from whole embryos and
yolk-sac tissues, and also cultures of nucleated blood cells which result from
yolk-sac erythropoiesis, to investigate the sensitivity of these cells to Ep.
1
Authors' address: School of Biological Sciences, University of Sussex, Falmer, Brighton,
Sussex, BN1 9QG, U.K.
476
A. E. BATEMAN AND R. J. COLE
In a comparison with erythropoiesis in chick embryos, the haem precursor
molecule £-aminolaevulinic acid (ALA), which stimulates haemoglobin
accumulation in cultured chick blastoderms (Levere & Granick, 1965;
Wainwright & Wainwright, 1970), was added to cultures of mouse embryonic
cells, then haem synthesis measured.
MATERIALS AND METHODS
Random-bred Swiss mice of the Porton strain were used. Females were
primed with Folligon and Chorulon (Organon Ltd.) before mating: vaginal
plugs were seen on the morning of day 0 gestation. Eight- and 9-day embryos
were dissected as previously described (Cole & Paul, 1966). Embryos and yolk
sacs were then disaggregated to give a suspension of single cells, after trypsin
treatment similar to that used for foetal liver tissue (Cole & Paul, 1966). Foetal
blood cells from 12- and 13-day embryos were collected in Hanks's saline (Paul,
1970). Twelve-day blood contains negligible numbers of enucleate liverproduced erythrocytes, 13-day blood contains nucleated cells with more condensed nuclei, and ca. 20 % enucleate cells.
Waymouth's medium MB 752/1 (Waymouth, 1959), supplemented with 5 %
foetal calf serum and 2-5 % Porton mouse serum was used for all cell cultures.
Suspensions of 1 x 106 embryonic cells or blood cells in this medium were
cultured in 1 ml aliquots in 15 ml glass culture tubes at 37 °C in an atmosphere
of 5 % CO2 in air.
Erythropoietin (Ep: National Institute of Health, step IV preparation) was
added at the start of the culture period at concentrations of 01-0-3 units per ml.
The haem precursor £-aminolaevulinic acid (ALA; as the hydrochloride,
Sigma) was added to a series of cultures at concentrations of 0-01-10 HIM.
59
FeCl3 in HC1 (Radiochemical Centre, Amersham, Bucks) was equilibrated
with 50 % mouse serum for several hours at 37 °C before addition to cultures
at 1 /tCi/ml. Labelled haem was extracted from lysed cells by the acid ketone
technique (Teale, 1959; Krantz et ai 1963) and counted in a Nuclear Chicago
gas-flow counter. Results are expressed as counts per min/h/106 cells.
RESULTS
No stimulation of incorporation of 59Fe into haem was observed in cells
incubated with ALA. Concentrations greater than 1 HIM inhibited haem synthesis.
Lower concentrations had neither a short-term nor long-term effect on haem
synthesis.
Erythropoietin did result in significantly raised levels of haem synthesis by
the primary erythroid cells tested. 59Fe was added to cultures for periods of 4 h
or longer after an initial p re-incubation period of at least 30 min. When haem
synthesis at all times of incubation in all experiments is considered, pooled data
Erythropoietin and embryonic blood cell production
Ml
59
show that Fe incorporation into haem is stimulated 89 % in cells from 8-day
embryos, 23 % in cells of 9-day embryos and 19 % in 12-day blood cells. Table 1
presents the pooled data.
Table 1. Stimulation of haem synthesis in blood cells of the primary series by
erythropoietin at 0-15 to 0-3 units per ml.
Source of cells
Mean c.p.m. 59Fe
in haem/h
No. of
1 ml
cultures
8-day embryos
(0-12 somites)
9-day embryos
(13-20 somites)
12-day blood
13-day blood
Control
64-2
20
+ Ep
% increase
above control
±S.E.
Probability that
+ Ep — control
(Wilcoxon test)
121-4
89 ±24
0031
92
200
246
23 ±12
0003
65
12
684
278
778
297
19 + 7
6.3
0003
Not sig.
-
20 r
B
E 10 -_--
5 -
1
1
10
20
i
i
0
10
30
Time of exposure to Ep in vitro
i
i
20
30
Fig. 1. The effect of erythropoietin on synthesis of haem by cells from Porton mouse
embryos and yolk sacs in vitro. Haem synthesis was measured by incorporation of
59
Fe into the acid-ketone-soluble fraction, and plotted as c.p.m./h throughout the
time of labelling with 59Fe. The histograms show synthesis in cells from (A) 8-day
embryos and (B) 9-day embryos.
, Control;
, +Ep.
There was no significant increase in haem labelling by peripheral blood cells
from 13-day embryos, perhaps because the erythrocytes are more mature than
those in 12-day blood. Significance is assessed by the matched-pair technique of
Wilcoxon (Siegel, 1956). This non-parametric test was more sensitive than
matched-pair /-tests applied to these data (because numbers are not from a
Normal distribution).
The pattern of response to Ep at different times is shown in Fig. 1. An
478
A. E. BATEMAN AND R. J. COLE
experiment on 8-day-embryo cells is shown in Fig. 1 A; Fig. IB shows the time
course of response of cells from 9-day embryos.
Experiments on cells of disaggregated 8- or 9-day embryos were limited
because of shortage of material. Blood cells from 12-day embryos were more
plentiful, and the time course of response was studied. Fig. 2 shows results of
three experiments using 12-day blood cells. The percentage increase in haem
synthesis in the presence of Ep, above control levels, varies with time, with a
probable maximum increase after 24-36 h incubation with Ep in vitro. Thus the
kinetics of response of primary series blood cells to Ep in vitro may be similar to
those reported for foetal liver cells in vitro (Cole & Paul, 1966).
100 i—
80
60
40
20
2 4 3 2
40
Time of exposure to Ep. (h)
Fig. 2. The in vitro response to erythropoietin of nucleated erythrocytes from the
blood of Porton embryos of 12 days' gestation. The percentage increase of haem
synthesis in Ep-stimulated cells above control levels is calculated for each period of
labelling with 59Fe, and is plotted at the midpoint of that period. Data of three
experiments, A, B and C are shown.
DISCUSSION
#-aminolaevulinic acid (ALA) has been used in several studies of differentiation of erythroid cells in chick embryo blood islands developing in vitro. Levere
& Granick (1965) and Wainwright & Wainwright (1966, 1967) showed that
ALA increased the haemoglobin content of young blastoderms, and induced
haemoglobin synthesis prior to the normal onset of synthesis at the 7-somite
stage. But Wilt (1968) showed that ALA stimulation of the haemoglobin content
was only detectable on non-rich media. A further analysis by Wainwright &
Wainwright (1970) based on a new very sensitive assay for haem (Wainwright,
1970) showed that ALA causes an increase in haemoglobin, the pattern of which
is specific to the stage of the embryo at which ALA is added.
Erythropoietin and embryonic blood cell production
479
It seems therefore that the production of ALA is the ultimate control factor
of haem and haemoglobin synthesis in chick embryos. However, added ALA
does not stimulate haem synthesis in cells of the mouse yolk-sac-stage embryo,
nor in foetal liver cells of 12- to 14-day embryos (Bateman, 1971).
The hormone erythropoietin (Ep) does stimulate haem synthesis in cells of
young embryos, and in blood cells of the primary generation. Previous experiments using whole embryo cultures found no stimulation of yolk-sac-stage haem
synthesis in the mouse (Cole & Paul, 1966) and the rat (Hunter & Paul, 1969).
These negative results could arise if the external endodermal layer of the yolk
sac is impermeable to Ep, or if the cells within the intact embryo are maximally
stimulated by a factor produced in situ.
Haem synthesized in response to Ep in this study has not been characterized
as haemoglobin, and other haem proteins will be synthesized in embryonic cells
but Cole et al. reported (1968) that synthesis of haem in hepatocytes was not
stimulated by Ep and Ep-dependent haem synthesis probably occurs only in
erythroid cells.
Table 1 shows a decrease in response to Ep with increasing embryonic age.
Similar trends in response are found in cells of chick embryos at equivalent
developmental stages (Malpoix, 1967) and in liver cells from mouse foetuses of
increasing age (Cole & Paul, 1966; Bateman, Cole, Regan & Tarbutt, 1972). As
both absolute and proportional amounts of Ep-induced haem synthesis in vitro
are greater in cells of 8-day embryos than in cells at later stages of primary
erythropoiesis the detected response is probably not due to small numbers of
cells of the intermediate (hepatic) erythroid series which might exist prior to the
development of the liver, as these cells would be more numerous at 9 days of
gestation.
The ability of yolk-sac erythroid cells to divide in the circulation decreases
progressively between the 11th and 13th days of development, and is paralleled
by a decline in RNA content and RNA synthesis. During the early stages of the
phase of yolk-sac erythropoiesis haemoglobin synthesis depends on newly
synthesized RNA (Fantoni, de la Chapelle, Rifkind & Marks, 1968). The
erythropoietin sensitivity of circulating yolk-sac erythroid cells is therefore
limited to a period when there is active DNA synthesis and cell division, and
this is consistent with the known effects of erythropoietin on both foetal liver
and bone marrow erythroid cells.
The conclusion to this work is that Ep can stimulate haem synthesis in cells
from 8- and 9-day embryos and nucleated erythrocytes of 12-day embryos in
vitro. Electrophoretic studies of 59Fe labelled haemoglobins on polyacrylamide
gels suggest that the pattern of synthesis of foetal haemoglobins by 12-day blood
cells is not altered significantly by Ep. Ep therefore stimulates the synthesis of
haemoglobins which the erythroid cell is already programmed to produce, as
previously described in foetal liver cells (Cole et al. 1968).
480
A. E. BATEMAN AND R. J. COLE
This work was supported by the S.R.C. and the M.R.C. We are grateful to Mrs M. Reid
for care of the animals used, and to Mrs J. Garlick for technical assistance. Erythropoietin
was a gift from the National Heart and Lung Institute, U.S. Dept. of Health, Education and
Welfare.
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{Manuscript received 25 June 1971)