/. Embryol. exp. Morph. Vol. 21, 2, pp. 361-8, April 1969
Printed in Great Britain
361
Haemoglobins of the foetal and adult rat: sites of
synthesis and the effects of erythropoietin
By J. A. HUNTER 1 & J. PAUL 1
From the Beatson Institute for Cancer Research, Glasgow
Early studies on rat haemoglobins indicated the existence of several components (Gratzer & Allison, 1960). Seven components were found in the blood
of adult random-bred Wistar rats by CM-cellulose chromatography; their rates
of synthesis appeared unaltered during erythroid cell maturation (Brada &
Tobiska, 1964). A 5-component electrophoretic pattern found in some randombred Wistar rats was considered to be a combination of two 4-component
patterns observed in two inbred strains (Marinkovic, Martinovic & Kanazir,
1967). A 4-component electrophoretic pattern was also reported by Cole,
Hunter & Paul (1968). During gestation the number of components resolved in
this buffer system increased from 1 at 13 days to 4 at 15 days; the pattern
appeared identical to that of the adult from 18 days onward.
Erythropoietin stimulates stem cells to mature to erythrocytes (Filmanowicz
& Gurney, 1961; Orlic, Gordon & Rhodin, 1965; Nakao, Miura & Takaku,
1966). It appears to have no direct effect on haemoglobin synthesis (Erslev, 1962,
1964). In preliminary studies it was found that although erythropoietin increases
the rate of haemoglobin synthesis by foetal rat liver cells in the early stages of
organogenesis, it does not alter the nature of the haemoglobin produced (Cole
et al. 1968). This paper presents a more complete study of haemoglobin synthesis
during rat foetal development and the effects exerted by erythropoietin.
METHODS
Following the technique of Cole et al. (1968), foetuses of known developmental age were obtained from random-bred Wistar rats; haemoglobin samples
were prepared from these and from adult animals by chloroform extraction.
Suspensions of cells were prepared (Cole & Paul, 1966) and grown in replicate
tubes at 1-3 x 106 cells/ml of culture medium (Waymouth's medium MB 752/1
+10 % foetal bovine serum).
Adult marrow cells were obtained by flushing the femur with culture medium
and were disaggregated by pipetting in medium before being dispersed to
1
Authors' address: The Beatson Institute for Cancer Research, Royal Beatson Memorial
Hospital, 132 Hill Street, Glasgow, C.3, Scotland.
362
J. A. HUNTER & J. PAUL
replicate tubes. Haemoglobin synthesized in culture was labelled with 59Fe for
fixed periods and was released from the cells by lysis in distilled water and
extraction with chloroform. The haemoglobins were separated by starch gel
electrophoresis using the discontinuous buffer system of Poulik (1957). The
distribution of radioactivity after electrophoresis was determined as previously
described (Cole et al. 1968). Gels were stained for protein with naphthalene black
(Smithies, 1955) and for haemoglobin with o-dianisidine (O'Brien, 1961).
RESULTS
Five components were separated in this buffer system; the fastest was termed
haemoglobin 'a'. The relative mobilities of the other four components, termed
haemoglobins ' b ' , V , ' d ' and ' e \ were respectively 0-87, 0-68, 0-45 and 0-17
of that of haemoglobin a. In adult blood these were present in the following
ratios, taking the value for haemoglobin a as 1; haemoglobin b, 1; haemo_ 100
T-l
I
J
I
I
I
I
I
I
I
I
I
I
I
I
i
i
1 i
r
L__l
-I
I
I
I
i
i
i
i
I
I
I
I
50
I
l~l
lOrigin
12 13 14 15 16 17 18 19 20 21 Adult
I
Birth
Foetal age (days)
Fig. 1. Haemoglobin components from rat peripheral blood at different developmental ages. The diagram represents positions on starch gel electrophoretograms,
run in the conditions described in the text.
globin c, 0-0-06; haemoglobin d, 0-2; haemoglobin e, 0-17. As shown in Fig. 1,
only haemoglobins b and c were found in 12- and 13-day foetal blood, haemoglobin b being the fainter of the two. Haemoglobin a appeared early on day 14,
haemoglobin d later in the same day; haemoglobin e appeared on day 15. From
day 18 the proportions of haemoglobins a, d and e appeared constant and
similar to the adult pattern; the proportion of haemoglobin b increased before
and after birth while that of haemoglobin c decreased.
Cultures of 10-day yolk-sac cells, prepared from embryos after removal of the
ectoplacental cone, synthesized only haemoglobins b and c, and were unaffected
by erythropoietin treatment (Table 1).
Foetal rat haemoglobins
363
Cultures of 12- and 13-day foetal liver cells synthesized haemoglobins a, b
and c; the relative rates of synthesis of the components were the same during
periods of incubation some 24 h apart (Table 1). A small amount of haemoglobin d was made by 14-day foetal liver cell cultures, in addition to haemoglobins a, b and c. Regardless of erythropoietin treatment, the relative rates of
synthesis of the components were unaltered during incubation (Table I).
Table 1. Rates of synthesis of different haemoglobin components in erythropoietic
tissues during development of the rat. Ages are expressed as times after fertilization
Tissue of origin
Incubation
time
in vitro
(h)
a
b
c
d
e
Increase
of all
components
with Ep
treatment
(%)
Haemoglobin components as % of
total haemoglobin
Untreated cultures
10-day yolk sac
15
—
10-8
89-2
—
—
—
12-day foetal liver
6
30
4-3
5-2
12-5
14-2
831
80-6
—
—
27
36
13-day foetal liver
6
32
10-4
81
13 1
11-7
76-5
80-2
14-day foetal liver
6-5
29-5
91
9-7
11-9
12-7
76-2
72-7
2-8
2-5
15-day foetal liver
6
30
12-4
12-7
121
12-7
68-2
660
41
4-3
3-3
4-2
37
165
18-day foetal liver
6
28
16-4
161
17-2
16-5
53-6
55-2
7-2
7-2
5-5
50
—
20-day foetal liver
5
28
19-8
18-7
210
191
47-4
501
5-9
5-8
5-8
6-3
—
Neonatal liver
(21-5 day)
6
28
26-3
27-7
39-2
40-1
5
29
21-3
22-5
50-3
46-4
6-6
4-7
4-8
5-7
3-9
5-4
20-day foetal spleen
23-8
22-1
17-6
19 7
60
5-7
—
Neonatal spleen
(21-5 day)
6
28
24-6
27-8
250
23-9
38-6
40-3
6-7
6-4
5-1
6-6
—
Adult marrow
(6 months)
4
28
41-8
401
40-4
41-9
2-7
2-5
80
8-6
7-1
6-9
13
78
29
50
30
244
Cultures of 15-day foetal liver cells synthesized all five components and were
sensitive to erythropoietin treatment. The proportion of each component was
unchanged during incubation, irrespective of erythropoietin stimulation (Fig. 2).
Eighteen-day foetal liver cells were unaffected by erythropoietin, quantitatively
as well as qualitatively; the relative amounts of each component were unchanged during incubation (Table 1). Cultures of adult marrow cells also
synthesized all five components, in proportions differing from those observed in
cultures of foetal cells (Fig. 3). These cells responded to erythropoietin with an
24
JEEM
21
364
J. A. HUNTER & J. PAUL
over-all increase in the synthesis but no change in the proportions of the
components; the latter was also unaltered during 30 h incubation (Table 1).
These findings show that treatment with erythropoietin in vitro does not alter
the nature of the haemoglobins made by rat erythroid cells. To determine
whether this were the case in vivo, studies were made of the pattern of haemoglobin synthesis by marrow cell cultures from normal and anaemic adult rats.
A group of animals was made anaemic by bleeding, another by injection of
(a)
D
BB
D
Adult
15-day
foetal
Adult
15-day
foetal
1500
1000
1000
500
500.
10
20
30
40
50
0
10
20
30
40
50
Distance from origin (mm)
Fig. 2. Rates of synthesis of different haemoglobin compounds from 15-day foetal
rat livers. The cells were incubated with 59Fe and the incorporation into haemoglobin determined as described in the text. No erythropoietin added, # — • ;
0-5u/ml step 2 sheep erythropoietin added, O—O. (a) 59Fe added from 0 to 6 h;
(b) 59Fe added from 24 to 30 h.
phenylhydrazine; an untreated third group of animals provided cultures of
normal marrow cells. As shown in Table 2, the patterns of haemoglobin synthesis were virtually identical in cultures of normal and anaemic marrow.
It can be seen that the proportions of the different haemoglobins in the circulating blood is reflected in the proportions synthesized by the livers of different
ages. Erythropoiesis also occurs in the rat spleen during the last third of
gestation; the pattern of haemoglobin synthesis in this organ is similar to that
in livers of the same age (Table 1).
It can be seen from Table 1 that in culture the haemoglobin components were
synthesized in the same ratios during 30 h incubation. To determine whether
this pattern persisted in the last stages of cell maturation in vivo the haemoglobin
synthesized by circulating cells was examined. The results are shown in Table 3.
Foetal rat haemoglobins
365
Haemoglobin synthesis by 12-day foetal blood cells resembled that of 10-day
yolk-sac cells, some 42 h earlier. The pattern observed in cultures of 16-day
blood cells resembled that of 15-day foetal liver cells some 28 h earlier in
(a)
1000
o
U
D
•
•
D
D
DAdult
(fa)
1000
•
• Adult
500
500
10
20
30
40
30
50
0
10
20
Distance from origin (mm)
50
40
Fig. 3. Rates of synthesis of different haemoglobin compounds from adult rat bone
marrow. The cells were incubated with 59Fe and the incorporation into haemoglobin determined as described in the text. No erythropoietin added, • — # ;
0-25u/ml step 1 erythropoietin added, O—O. (a) 59Fe added from 0 to 4 h; (b) 59Fe
added from 22 to 28 h.
Table 2. Effects of anaemia on the synthesis of haemoglobin
components in the marrow of the adult rat
Treatment
Bleeding
Phenylhydrazine
Haemoglobin component as % of
total haemoglobin
Incubation
time
(h)
a
8
9
7
40-2
39-8
40-5
b
e
416
40-2
41-1
d
2-4
3-9
2-7
e
7-9
81
7-6
7-9
80
8-1
Table 3. Synthesis of different haemoglobin components by maturing
cells in the peripheral blood, at different developmental stages
Foetal
age
(days)
12
16
Haemoglobin components as % of total haemoglobin
a
b
c
d
e
12-6
20-2
130
19-6
13 9
65-4
48
861
4-8
6-5
4-1
61
(neonatal)
24-2
366
J. A. HUNTER & J. PAUL
gestation but not that of 14-day foetal liver cells some 50 h earlier. Haemoglobin synthesis by cultures of neonatal blood cells resembled that of 20-day
foetal liver and spleen cells some 36 h earlier. If cells are released into the circulation between 30 and 50 h after maturation begins, as suggested by the pattern
of synthesis in 16-day blood cultures, these results indicate that the proportion
of each component synthesized remains the same during much if not all of cell
maturation. Moreover, the results show that the normal changing pattern of
haemoglobin synthesis does not occur in tissue cultures.
DISCUSSION
The developmental pattern of rat haemoglobins differs from those observed
in other animals. In several mouse strains having one or more adult haemoglobin components three foetal haemoglobins have been found (Craig &
Russell, 1963, 1964; Cole et al. 1968); these are replaced by the adult haemoglobin before birth. Two foetal haemoglobins have been observed in rabbit
foetal blood; again these are replaced by adult haemoglobin before birth
(Hunter, 1968). The mouse foetal haemoglobins are made by yolk-sac cells,
while the adult haemoglobin is made by foetal liver cells (Kovach, Marks,
Russell & Epler, 1967; Hunter, 1968).
In the rat, on the other hand, haemoglobins electrophoretically identical with
two of the adult components persist from yolk-sac to adult marrow erythropoiesis and three further components are added during foetal liver erythropoiesis. The pattern of synthesis is characteristic of the stage of gestation, even
when two sites are simultaneously active. This behaviour suggests an evolving
pattern in a uniform stem cell population rather than several kinds of stem cell,
each responsible for a unique spectrum of components.
It is of interest that the pattern of synthesis remains constant during 30 h
incubation in vitro even in livers from 13-day foetuses although cells explanted
during this period display different patterns. The changes are not brought about
by erythropoietin alone since only a quantitative change follows erythropoietin
treatment in vitro. Although the pattern of haemoglobin synthesis differs in
adult and foetal cells, erythropoietin has the same effect on each: it increases
either the number of maturing cells or their rate of haemoglobin synthesis or
both.
It may be postulated that the stem cells of the foetal rat are initially 'programmed' for a given pattern of haemoglobin synthesis. Evolution of the
programme might depend either on the number of stem cell divisions (and
hence on the age of the foetus) or on changes in the environment. Since anaemia
does not cause reversion to a more immature pattern one would have to postulate that the evolution of the programme in the rat is not as freely reversible as
would be predicted by the hypothesis proposed by Baglioni (1963). Hence,
erythropoietin acts solely to increase the rate of haemoglobin synthesis while
Foetal rat haemoglobins
367
the nature of the haemoglobin produced is determined by another mechanism.
The factors, cellular or humoral, which determine the kinds of haemoglobin
produced remain unknown.
SUMMARY
1. In the foetal rat two haemoglobin components are formed during the
yolk-sac stage of erythropoiesis. The minor component persists into adult life.
The other becomes the major component during the hepatic phase of foetal
erythropoiesis. It diminishes in amount shortly after birth. At the commencement of the hepatic phase (14-15 days gestation) three other components appear;
they persist in the adult.
2. The types of haemoglobin made are not influenced by erythropoietin and
remain unchanged during 24-30 h culture in vitro.
RESUME
Les hemoglobines du rat foetal et adulte: les sites de synthese
et les effets de I'erythropoietine
1. Chez le foetus d'embryon de rat, deux hemoglobines se forment pendant
la phase erythropoietique dans le sac vitelline. L'hemoglobine mineure persiste
jusqu'a la vie adulte. L'autre hemoglobine devient le composant majeur pendant
la phase hepatique d'erythropoiese embryonnaire, mais elle diminue en quantite
peu apres la naissance. Au debut de la phase hepatique d'erythropoiese (14 a
15 jours de gestation) trois autres hemoglobines apparaissent, qui persistent chez
l'adulte.
2. Les types d'hemoglobines qui se forment ne sont pas influences par
l'erythropoietine, et restent inchanges pendant 24 a 30 heures de culture in vitro.
The erythropoietin used in these studies was a gift from the National Blood Resources
Programme of the U.S. National Institutes of Health. This research was supported by grants
from the Medical Research Council and the British Empire Cancer Campaign for Research.
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{Manuscript received 14 October 1968)