J. Embryol. exp. Morph. Vol. 64, pp. 275-293, 1981
Printed in Great Britain © Company of Biologists Limited 1981
275
Early erythropoiesis in foetal rat bone marrow:
evidence for a liver-to-bone marrow relay
ByM. D. NAGEL1, J. NAGEL AND R. JACQUOT1
From the Laboratoire de Physiologie animale - ERA 401 - UER Sciences
Exactes et Naturelles - Universite de REIMS
SUMMARY
Erythropoietic activity of foetal rat femoral marrow was examined during the last four
days of intra-uterine life. Insignificant at day 18, it develops slowly thereafter until birth. In
the non-suckled neonate (not older than two hours), it appears notably enhanced. In order
to test the potential of the foetal marrow to develop precocious or increased erythropoiesis,
the activity of the erythropoietic organ predominant at this time, the liver, was altered by
modifying the level of circulating corticosteroids which govern its function. Maturation and
involution of the hepatic erythron were prevented by corticosteroid deprivation of the
foetus (maternal adrenalectomy and foetal hypophysectomy). Precocious maturation and
exhaustion of the hepatic erythron was induced by submitting foetuses to corticosteroids
excess from day 14. Both corticosteroid deprivation and excess increase the erythropoietic
activity of the femoral marrow. This activity can reach and even exceed by day 20 of intrauterine life that in neonatal marrow. Foetal hepatic erythron misfunction can therefore
initiate and stimulate bone marrow erythropoiesis. The study of circulating red blood cells
demonstrates that: (1) anaemia initiates medullary erythropoietic activity; (2) this anaemia
is largely corrected by the bone marrow. The regulatory mechanism is presumably erythropoietin mediated.
INTRODUCTION
Jt is well known that in most mammalian species, the foetal liver assumes a
major role in haematopoiesis, especially erythropoiesis (see for instance Ackerman, Grasso, & Knouff, 19*61; Grasso, Swift & Ackerman, 1962; Rifkind, Chui
& Epler, 1969). In the rat foetus, the cellularity of the hepatic erythron decreases
sharply after day 18 of gestation (Nagel, 1968). This decrease is governed by
the circulating corticosteroids: in foetuses largely deprived of these hormones
by maternal adrenalectomy and foetal hypophysectomy, further evolution of
liver erythropoietic tissue is prevented, but can be restored by hydrocortisone
administration (Nagel & Jacquot, 1969). Conversely, stressing the pregnant
rat by laparotomy results in an accelerated evolution and a precocious
regression of the foetal hepatic erythron, which is evident 24 h after the
stress, whereas previous maternal adrenalectomy suppresses the effects of the
laparotomy (Nagel & Jacquot, 1968). The foetal hepatic erythron responds to
1
Authors' address: Laboratoire de Physiologie animale, UER Sciences Exactes et Naturelles, 51062 Reims Cedex France.
276
M. D. NAGEL, J. NAGEL AND R. JACQUOT
corticosteroids as early as day 14 of gestation (Billat, Nagel, Nagel & Jacquot,
1980). It is therefore experimentally possible to prematurely deprive the foetus
of a large part of its hepatic erythropoietic tissue by repeated stress inflicted
on the pregnant mother during and after day 14.
Little information on foetal marrow erythropoiesis in the rat is available.
Lucarelli et al. (1968) assert that there is no detectable erythropoietic activity
in the rat foetal femur at day 18 of gestation; this activity appears at birth and
increases strikingly towards the seventh day of post-natal life. Therefore, we
studied the evolution of haematopoietic activity in the foetal rat femur from
day 18 of gestation until birth. Then, we looked for an eventual modification
of femoral erythropoietic activity in foetuses: (1) where normal regression of the
hepatic erythron was prevented by maternal adrenalectomy and foetal hypophysectomy; (2) where evolution of the hepatic erythron was precociously
initiated and accelerated by repeated maternal laparotomies at days 14, 16 and
18 of gestation.
In parallel, the cellular content of the blood was investigated in each experimental situation.
MATERIALS AND METHODS
Animals
Wistar rats (CF strain of the C.N.R.S.) were used. They were housed in a
constant temperature room with 12 h day/12 h night. They had free access to
water and food (UAR rat commercial food). Coitus was assessed by the presence
of spermatozoa in the morning vaginal smear. In this strain of rats, delivery
generally occurs during the night between days 21 and 22 of pregnancy or at
day 22 in the morning.
Surgery
Surgery was performed under ether anaesthesia. Laparotomy consists of an
incision of skin, muscular wall and peritoneum followed by suture, without
touching the viscera. Adrenalectomy of the mother was performed classically,
by the dorsal approach, on day 14 of gestation. Foetal hypophysectomy was
performed by decapitation according to the technique of Jost (1951) on day
18 of gestation.
Cortisol was administered through the maternal uterine wall under the skin
of the foetuses (0-6 mg of hydrocortisone acetate Roussel).
Evaluation of liver erythropoiesis
The evolution of the liver erythropoietic function was evaluated according to
Nagel's methodology (1968). In brief, livers were homogenized in ice-cold
0-25 M-sucrose (Potter-Elvehjem, standardized number of strokes and of
revolutions per minute), the nuclear suspension obtained was enumerated in a
haemocytometer and the number of nuclei per gram liver calculated. In previous
Erythropoiesis regulation in foetal rat
277
studies (Nagel, 1968; Jacquot & Nagel, 1976) the number of hepatic cells per
liver volume has been shown to be roughly constant at all gestational stages
and in all experimental groups; thus, any change in the number of nuclei per
g liver reflects a change in the number of nuclei of haematopoietic cells per g
liver. Since erythropoietic cells are considerably more numerous than other
blood cells in the liver haematopoietic tissue, experimentally induced differences
in the number of nuclei in homogenates reflect experimentally induced differences in the importance of the liver erythron.
Cytological determinations on bone marrow
To study medullary erythropoiesis, femoral marrow imprints were used.
The bone was rapidly dissected, split longitudinally and stuck on a slide. The
imprints were fixed and stained with May-Gruenwald-Giemsa.
Four different cell groups will be considered.
(i) 'Precursor' cells, i.e. morphologically undifferentiated cells, the progeny
of which cannot be predicted on mere cytological observation.
(ii) Unhaemoglobinized erythroid cells (proerythroblasts + basophilic erythroblasts).
(iii) Haemoglobinized cells (polychromato and acidophilic erythroblasts).
(iv) The white cell lineage (myeloid and lymphoid cells, megakaryocytes and
mature granulocytes), called for convenience 'other cells'.
The number of each cellular type per arbitrary surface area of imprint was
determined. Medullary activity was assessed by classifying and numbering the
different cells present in this arbitrary surface area of the imprint. For each gestational age, control or experimental, at least eight foetuses belonging to four
different mothers were used and 3000 to 5000 cells were enumerated and
classified. Numbers of cells per surface area are presented as means (±S.E.M.;
significance of differences between means was evaluated according to Fisher's t
test. Qualitative composition of imprints is depicted by the percentage of each
cellular type versus the whole cellular population of the imprint.
Histological study of bone marrow
The bones, fixed in formol, were treated for paraffin embedding. Sections
were stained with haemalum-eosin.
Blood assays
Foetal blood was collected directly from the axillary artery for the determination of: the total number of circulating cells (10 /d), the number of nucleated
cells (25 fi\), the haemoglobin concentration (20 /A), the hematocrit (12/d) and
the composition of the cellular population (two smears). The simultaneous
determination of these parameters required too much blood to be performed on
single foetuses at day 18. Therefore, cellular enumerations and blood smears
were carried out on some foetuses, haemoglobin and hematocrit being
278
M. D. NAGEL, J. NAGEL AND R. JACQUOT
determined on their littermate brothers. For older stages, all the determinations
were performed simultaneously on each foetus.
For each experimental point, 9 to 25 foetuses were studied, belonging to at
least four different mothers.
(a) Cellular enumerations
(i) Total number of cells (= a) was determined on 10 /d blood samples using
the Unopette 585IF (Becton Dickinson) commercial kit for manual methods.
(ii) Number of nucleated cells (= b) was determined on 25 /d blood samples
using the Unopette 5856F (Becton Dickinson) commercial kit for manual
methods.
(iii) Number of anucleated red cells is by definition (a-b).
(iv) Number of nucleated red cells was determined as follows. Two smears of
each blood were fixed and stained with May-Gruenwald-Giemsa. They allowed
the determination of the ratio of the nucleated red cells to the total population
of nucleated blood cells (more than 1000 nucleated cells were enumerated on
smears for each blood sample). The number of circulating nucleated red cells is
obtained by multiplying (b) by this ratio.
(b) Haemoglobin concentration
Unopette test 5857F (Becton Dickinson) commercial kit was used on 20 /d
blood samples. Cyanmethaemoglobin, oxyhaemoglobin, carboxyhaemoglobin
and methaemoglobin are measured. The coloured reaction developed after
addition of the Uno-heme reagent was read at 530 nm and the haemoglobin
concentration was deduced from a standard calibration curve.
(c) Hematocrit
A standardized micromethod was used for hematocrit determination: 12/d
blood in 15/4 Drummond microcaps, 1650Og centrifugation for 15 sec in a
Janetzki TH 11 centrifuge.
(d) Presentation of the results
Experimental values are presented in the tables, as means ± S.E.M. : anucleated
red cells per ju\ blood, nucleated red cells per /d blood (assuming, for simplification, that the ratio of the nucleated red cells to the whole nucleated population
is determined with a negligible error), haemoglobin content (g/100 ml blood)
and hematocrit (volume of the red cells as percentage of the total volume of
the blood). Fisher's t significance test was used for differences between means.
Mean corpuscular haemoglobin (MCH) was calculated as:
haemoglobin (g/100 ml) x 10
inpg(10- ] 2 g).
number (millions//*!) red cells (nucleated + anucleated)
Erythropoiesis regulation in foetal rat
279
Mean corpuscular volume (MCV) was calculated as:
hematocrit (%) x 10
number (millions//d) red cells (nucleated+ anucleated)
.^
As both MCH and MCV were obtained, for each experimental situation,
from the mean values of haemoglobin content, hematocrit and cell numbers,
they are reported without statistical information.
RESULTS
(1) Control foetuses (normal mothers)
(a) Marrow haemopoietic activity (Table 1)
The total number of cells per arbitrary surface area of imprint increases
regularly from day 18 of gestation until the first hours following birth. The
cells belonging to the white myeloid lineage constitute the major part of the
marrow population. The number of 'precursors' per surface area remains
roughly constant at days 19, 20 and 21. The number of immature and mature
erythroid cells per surface area increases between days 18 and 21 of gestation
but their proportion remains quite low when compared to that of other cell
lines (only 4 % of marrow cells at day 21 are differentiated erythroid cells).
At birth the number of precursors per surface area sharply decreases. In
contrast, the number per surface area of the erythroid cells and, more particularly, of the haemoglobinized ones, increases (in the newborn, differentiated
erythroid cells represent 9-2% of the whole population). Cells of the white
lineage continue to increase after birth. Amongst these cells, the evolution of
granulocytes is spectacular: they represent 003, 1-1, 6-9, 15-4 and 29-5% of the
total population of the imprints at 18, 19,20, 21 foetal days and at birth respectively (not shown on Table 1).
(b) Blood data (Table 2).
The number of circulating anucleated red cells increases between days 18 and
21 of gestation. During the first hours following birth, this increase is very
rapid. On the contrary, the number of nucleated red cells falls sharply between
days 18 and 19, and then decreases more slowly during the last two days of
gestation and after birth. At day 18, the percentage of red cells from yolk-sac
origin as determined on blood smears is very high: 49-8% of total nucleated
red cells. This percentage considerably decreases thereafter so that at birth,
cells from yolk-sac origin have almost disappeared. The percentage of acidophilic erythroblasts increases between day 18 and birth, and that of younger
erythroblasts (polychromatophilic and basophilic), roughly stable until day 20,
falls after this stage (Fig. 1).
Haemoglobin concentration slightly increases between day 18 and birth and
MCH decreases slightly.
Neonates
(< 2h)
21
21
20
19
18
Gestational
age
(days)
Foetuses from normal
mothers
Foetuses from normal
mothers
Foetuses from adrenalectomized mothers
Corticosteroid deprived
foetuses (decapitated)
Foetuses from normal
mothers
Foetuses from adrenalectomized mothers
Corticosteroid deprived
foetuses (decapitated)
Foetuses from normal
mothers
Foetuses from adrenalectomized mothers
Corticosteroid deprived
foetuses (decapitated)
Neonates from normal
mothers
19-4 ±1-3
560%
16-3 ±0-9
31-3%
17-3 + 1-3
31-8%
14-5±l-3
32-1 %
14-2 + 0-7
21-8%
14-1+2-2
20-5%
91+0-4
15-3%
15 9 ± 1 6
21-2%
15-7±2-3
15-9%
9-4+1-2
12-7%
5-4 + 0-4
5-8%
' Precursors'
Haemoglobinized
erythroblasts
0-05 + 00-3
0-14%
015 ±005
0-3%
0-24 + 0-05
0-4%
0-24 + 0-06
0-5%
0-4 ± 0 1
0-6%
0-8+0-3
1-2%
3-3 ±0-3
5-6%
0-9 ±0-3
1-2%
l-6±0-3
1-6%
4 0 ±0-6
5-4%
3-6 + 0-6
3-9%
Unhaemoglobinized
erythroid cells
0-5 + 0-2
1-4%
0-8 ± 0 1
1-5%
1-1+0-2
2-0%
1-0 + 0-1
2-2%
1-5 ±0-2
2-3%
1-3 + 0-2
1-9%
4-4 + 0-3
7-4%
2-1+0-5
2-8%
2-6 + 0-5
2-6%
7-6 ±0-8
10-3%
4-9 + 0-6
5-3%
•
14-7 ±1-9
42-4%
34-8 ±1-1
66-9%
35-7±l-5
65-6%
29-4+1-6
65-2%
48-9 ± 3 0
75-3%
52-5±2-7
76-4%
43-6±ll
71-7%
561 ± 2 0
74-8%
78-6 ±1-9
79-9%
52-8 ±2-9
71-6%
78-9 ±2-5
85-0%
Other cells
(Results (number of cells per surface area of marrow imprint) are means ±S.E.M. The percentages refer to whole of
marrow nucleated cells.)
92-8
73-8
o
98-5
H
c
oo
>
>
cw
1>
z
z
>
o
M
S
K
oo
O
750
59-4
68-7
65 0
451
54-3
521
34-7
Total
Table 1. Marrow haematopoietic activity in control foetuses from normal or adrenalectomized mothers and in decapitated
foetuses from adrenalectomized mothers
Neonates
(< 2h)
21
20
19
18
Gestational
age (days)
Foetuses from normal
mothers
Foetuses from normal
mothers
Foetuses from adrenalectomized mothers
Corticosteroid deprived
foetuses (decapitated)
Foetuses from normal
mothers
Foetuses from adrenalectomized mothers
Corticosteroid deprived
foetuses (decapitated)
Foetuses from normal
mothers
Foetuses from adrenalectomized mothers
Corticosteroid deprived
foetuses (decapitated)
Neonates from normal
mothers
40-9
46-2
6-7 ±0-6
9-9 ±0-3
ll-2±0-6
8-6 ±0-5
10-9 ±0-4
13-3 + 0-5
60-7 ±6-9
10-6 ±2-9
19-3±3-5
34-8 ±3-7
5-6 ±0-9
10-7 ±1-4
6-6 ±0-9
31 ±0-5
1432-3 ±82-4
2147-9±30-3
22411 ±119-8
2450-4 ± 107-1
2241 -9 ± 93-1
2370-6 ±49-7
2657-6 ±109-3
2875-4+118-6
12-2 ±0-2
111 ±0-4
102 ±0-3
42-1 ±5-7
1802-4 ±114-5
51-2
49-4
34-6
49-5
45-9
44-9
55-3
54-3
9-8 ±0-5
200 ±3-5
1784-5 ±165-9
550
9 0 ±0-4
MCH
(pg)
58-5±4-3
Nucleated red
blood cells x 10~3 Haemoglobin
(gm/lOOcc)
1576-8 ±89-7
Anucleated red
blood cells x 10~3
40-2+1-5
441 ±0-9
42-5 ± 1 1
39-4 ±1-4
401 ±1-1
41-7±l-4
39-9 ±2-3
290 ±0-9
39-5 ±1-9
39-6 ±1-9
40-9 ±0-9
Hematocrit
139-7
165-5
178-5
175-3
161-4
184-5
184-9
194-2
214-2
219-5
250-2
MCV
Om3)
Table 2. Blood data in control foetuses from normal or adrenalectomized mothers and in decapitated foetuses from
adrenalectomized mothers
oo
a
1
"1
hropt
282
M. D. NAGEL, J. NAGEL AND R. JACQUOT
90 -
y*
70 50
30
10
- K\
\
o—T
18
I
19
I
T
20
21
Foetal age (days)
r
Neonate
Fig. 1. Evolution of nucleated red cells from yolk-sac origin ( # ) , acidophilic
erythroblasts (T) and younger (polychromatophilic and basophilic) erythroblasts
(O) as percentages of total nucleated red cells.
Between day 18 and birth, hematocrit values stay quite stable. MCV diminishes between day 18 and birth, especially after day 21.
(II) Corticosteroid deprivation (decapitated foetuses)
Maternal adrenalectomy (on day 14) and foetal hypophysectomy (on day
18) reduce the level of foetal circulating corticoids (cf. for instance Arishima
etal. 1977).
(a) Marrow erythropoietic activity (Table 1)
At all foetal ages there is no significant difference between intact foetuses
from normal and from adrenalectomized mothers. At day 19, the values obtained
from decapitated foetuses do not differ significantly from those of intact
foetuses. In contrast, at days 20 and 21, in corticosteroid-deprived foetuses, a
striking increase in the number of unhaemoglobinized and haemoglobinized
cells per surface area occurs. These differences between decapitated foetuses and
controls are statistically significant (P < 0-01). The same conclusions can be
drawn when the results are expressed as percentages of the whole marrow
population.
(b) Blood data (Table 2)
The number of anucleated red cells in intact foetuses from adrenalectomized
mothers is not significantly different (P > 0-05) from that found in foetuses
from normal mothers at any gestational age.
In contrast, the number of nucleated red cells presents a significant difference
(P < 0-01) between foetuses from adrenalectomized mothers and their normal
controls at days 19, 20 and 21.
Haemoglobin concentration, although slightly higher in foetuses from
adrenalectomized mothers, is not really significantly different from that of normal
Erythropoiesis regulation in foetal rat
283
controls and MCH is roughly identical at every gestational age. Hematocrit
and MCV present no difference at any gestational age between foetuses from
adrenalectomized mothers and normal controls.
In decapitated foetuses, the number of anucleated red cells at day 19 is
significantly lower (P < 0-01) than in intact foetuses from normal or adrenalectomized mothers. On the contrary at days 20 and 21, this number is higher
(P < 0-01). The number of nucleated red cells is more elevated (P < 001) in
decapitated foetuses, except for day 21.
Haemoglobin concentration is significantly lowered in decapitated foetuses
at day 19 (P < 001); this difference disappears thereafter. MCH in decapitated
foetuses is below the control values at all gestational stages. Hematocrit at
day 19, is significantly lower in decapitated foetuses than in their controls but
is no longer different thereafter. MCV in decapitated foetuses is always below
the controls.
(Ill) Corticosteroid excess: repeated maternal stress
Repeated laparotomies were performed on pregnant rats from day 14. At this
early stage the foetal liver erythron is already responsive to corticosteroids
(Billat et al. 1980). Mothers were stressed at days 14 and 16 (sampling at day 18),
14-16 and 18 (sampling at days 19 or 20), 14-16-18 and 20 (sampling at day 21).
The results are compared to those of foetuses from normal mothers.
(a) Marrow erythropoietic activity (Table 3)
In 18-day-old foetuses from mothers laparotomized at days 14 and 16, the
number of erythroid cells per surface area of marrow imprint is not significantly
different from that observed in foetuses from normal mothers at the same
gestational age (P > 0-05). On the contrary, this value is statistically much
higher (P < 001) in 19, 20 and 21-day-old foetuses borne by stressed mothers,
than in foetuses from normal mothers at the same gestational ages. At day 19,
the increase of unhaemoglobinized cells, and, at day 20, that of haemoglobinized
erythroblasts, are particularly noticeable.
When percentages referring to the whole marrow population are considered
there is, again, after day 18 a difference between experimental foetuses and their
controls, especially at day 19 for unhaemoglobinized erythroid cells and at
day 20 for haemoglobinized erythroblasts.
It is noteworthy that 21-day-old foetuses from four-fold-stressed mothers do
not differ significantly from 20-day-old foetuses from three-fold-laparotomized
mothers (P > 005). In both cases, cell numbers and percentages are higher
than in 21-day-old control foetuses borne by normal mothers.
(b) Blood data (Table 4)
The number of anucleated red cells is significantly lowered at days 18 and 19
in experimental foetuses (P < 0-01); this difference is no longer observed
21
20
19
18
Gestational
age (days)
Foetuses from normal
mothers
Foetuses from mothers
laparotomized at days
14 and 16
Foetuses from normal
mothers
Foetuses from mothers
laparotomized at days
14, 16 and 18
Foetuses from normal
mothers
Foetuses from mothers
laparotomized at days
14, 16 and 18
Foetuses from normal
mothers
Foetuses from mothers
laparotomized at days
14, 16, 18 and 20
561 ± 2 0
74-8%
69-2 + 3-5
84-5%
0-9±0-3
1-2%
2-2 + 0-5
2-7%
2-1+0-5
2-8%
3-4 + 0-4
4-1 %
15 9±1 6
21-2%
71 ±1-4
8-7%
48-9 + 3-0
75-3%
37-8 ±1-4
68-7%
0-4 + 0-1
0-6%
1-9 + 0-3
3-4%
1-5 ±0-2
2-3%
3-2 + 0-3
5-8%
14-2 + 0-7
21-8%
12-2 ±0-9
21-1%
34-8±ll
66-9%
31-9 + 0-8
66-7%
015 + 005
0-3%
0-6 + 0-1
1-3%
0-8 + 0-1
1-5%
2-7 ±0-3
5-6%
16-3 ±0-9
31-3%
12-6 + 0-9
26-4%
14-7 ±1-9
42-4%
26-9 ±1-6
56-5%
005 ±003
0-14%
019±007
0-4%
0-5 + 0-2
1-4%
0-6 + 0-3
1-3%
Other cells
Haemoglobinized
erythroblasts
19-4±l-3
560%
19-9±2-7
41-8%
' Precursors'
Unhaemoglobinized
erythroid cells
81-9
750
551
o
cCJ
"
/H"\
o
<_i
r
>
47-8
650
>
m
m
•
47-6
521
o
2
34-7
Total
(Results (number of cells per surface area of marrow imprint) are means±S.E.M. The percentages refer to the whole of marrow nucleated cells.
Table 3. Marrow haematopoietic activity in control foetuses from normal mothers and in foetuses from mothers
submitted to repeated stress
to
oo
*.
Foetuses from normal
mothers
Foetuses from mothers
laparotimized at days
14 and 16
Foetuses from normal
mothers
Foetuses from mothers
laparotomized at days
14, 16 and 18
Foetuses from normal
mothers
Foetuses from mothers
laparotomized at days
14, 16 and 18
Foetuses from normal
mothers
Foetuses from mothers
laparotimized at days
14, 16, 18 and 20
34-2 ±4-8
10-6 ±2-9
10 0± 11
5-6 ±0-9
5-3±0-5
1551-7±86-7
2147-9 ±30-3
2135O± 103-1
2241-9 ± 93 1
2239-4 ±68-8
48-5
49-4
ll-l±0-4
10-9 ±0-5
461
45-9
9-9 ±0-7
9-9 ±0-3
61-8
54-3
9-8 ±0-5
200 ±3-5
1784-5 ±165-9
9-8 ±0-4
63-3
9-3 ±0-3
65-2 ±4-1
550
14041 ±341
90 ±0-4
(Pg)
MCH
58-5 ±4-3
Nucleated red
blood cells x 10~3 Haemoglobin
/fi\
(gm/lOOcc)
1576-8 ±89-7
Anucleated red
blood cells xlO~3
39-9 ±1-4
39-4 ±1-4
40-7 ±1-5
39-9 ±2-3
41-4±l-6
39-6 ±1-9
371 ±0-9
40-9 ±0-9
Hematocrit
177-7
175-3
189-7
184-9
2610
219-5
252-5
250-2
MCV
1'
55'
s*
to
oo
foetal
ts
s
21
20
19
18
Gestational
age (days)
Table 4. Blood data in control foetuses from normal mothers and in foetuses from mothers submitted to repeated stress
*ulatio,
286
M. D. NAGEL, J. NAGEL AND R. JACQUOT
N, foetuses from normal mothers
A, foetuses from adrenalectomized mothers
D, decapitated foetuses
ID, cortisol injected decapitated foetuses
2OX5-1(TS
19
18
17
16
15
14
13
-
:
->-—
IX
T
1
T
N
A
D
ID
Fig. 2. Cortisol injection at day 18 to decapitated rat foetuses: effect on the number
of anucleated red cells at day 19.
thereafter. The numbers of nucleated red cells, slightly higher in experimental
foetuses at days 18 and 19 (005 > P > 001), are thereafter analogous to control
values.
Haemoglobin concentration is unchanged in experimental foetuses, and MCH,
slightly increased at days 18 and 19, is the same as in controls at days 20 and 21.
Hematocrit, lower at day 18 in experimental foetuses (0-5 > P > 0-01) is not
different from controls at other gestational stages, and MCV, increased at day
19 in experimental foetuses, is not different from controls at other ages.
(IV) Control experiments
(a) Foetuses (adrenalectomized mothers) were decapitated at 18 days and
simultaneously injected with cortisol. Blood measurements were performed at
19 days. As shown in Fig. 2, the number (per /A) of anucleated red cells was
higher than in non-injected decapitated foetuses (P < 0-01) and equal to the
values observed in intact foetuses from normal or adrenalectomized mothers.
(b) Foetuses, decapitated at 18 days (adrenalectomized mothers), were
cortisol-injected at 20 days and sampled at 21 days. As shown in Table 5, they
presented a significant (P < 0-01, versus non-injected decapitated foetuses)
decrease in the number of unhaemoglobinized erythroblasts per surface area
of marrow imprint, and a concomitant increase in the number of more mature
erythroblasts. The number of their circulating red cells (anucleated and nucleated)
was significantly increased (P < 0-01) (Table 5).
(c) Liver cellularity (number of nuclei per volume unit of liver) was evaluated,
between 18 and 21 days, in intact foetuses from normal and adrenalectomized
20 day-old corticosteroid deprived
foetuses (decapitated)
21 day-old corticosteroid deprived
foetuses (decapitated)
21 day-old corticosteroid deprived
foetuses (decpitated) cortisol injected
at day 20
4-4±0-3
7-4%
7-6±0-8
10-3%
2-3±0-3
2-3%
3-3 ±0-3
5-6%
40±0-6
5-4%
7-1 ±1-3
7-1%
UnhaemogloHaemoglobinized erythroid binized erythrocells
blasts
Marrow erythropoietic activity
34-8 + 3-7
6-6 + 0-9
18-5±31
2657-6±109-3
3043-4±291-4
//a
Nucleated red
blood cells x 10"
2450-4±1071
A nucleated red
blood cells x 10~3
Red blood cells
Table 5. Effects of hydrocortisone administration at day 20 to decapitated foetuses on femoral marrow erythropoietic
activity and number of anucleated and nucleated red blood cells at day 21
(Marrow results (number of cells per surface area of femoral imprint) are means ±S.E.M. Percentages refer to the whole of marrow nucleated cells.)
00
to
S'
o*
<«••.
^5
s:
288
M. D. NAGEL, J. NAGEL AND R. JACQUOT
1300
-
1100
900
700
\
I
500
300
1
18
1
1
1
21
19
20
Foetal age (days)
Fig. 3. Evolution of the hepatic haematopoietic tissue in foetuses from normal ( • )
and adrenalectomized (T) mothers.
1300 73 <u
3 .>
C —
5-
1100
T
T
Os%
o
i
1
1
19
20
21
n> O
3x
900
-
Foetal age (days)
Fig. 4. Evolution of the hepatic haematopoietic tissue in corticosteroid deprived
foetuses (decapitated: O). Effect of cortisol administration at day 20 (#).
mothers (Fig. 3), in decapitated foetuses from adrenalectomized mothers (Fig.
4) and in foetuses borne by stressed mothers (Fig. 5). The results previously
described (Nagel & Jacquot, 1968; Nagel & Jacquot, 1969; Jacquot & Nagel
1976) are confirmed. Corticosteroid deprivation prevents the decrease of liver
cellularity normally observed as pregnancy progresses; corticosteroid excess, on
the contrary, induces an early decrease. Cortisol administration at day 20 to
foetuses decapitated at day 18 induces a rapid decrease of liver cellularity
(Fig. 4).
Erythropoiesis regulation in foetal rat
289
1300 _
1100
-
500
-
300
-
Foetal age (days)
Fig. 5. Anticipated and accelerated evolution of the hepatic haematopoietic tissue
in foetuses submitted to corticosteroid excess (A) by maternal repeated stress as
compared to normal controls ( # ) .
DISCUSSION
In the foetal rat the adrenals become significantly functional at day 17-18
(Holt & Oliver, 1968; Kamoun, 1970; Cohen, 1973; Milkovic, Milkovic &
Paunovic, 1973; Dupouy, Coffigny & Magre, 1975; Martin, Cake, Hartmann &
Cook, 1977). In intact foetuses borne by adrenalectomized mothers, they are
particularly active (Milkovic, Paunovic, Kniewald & Milkovic, 1973a; Cohen &
Brault, 1974), but remain largely inactive in decapitated foetuses (Arishima
et al. 1977; Cohen & Brault, 1974; Dupouy et al. 1975; Milkovic et al 1973a).
(I) Normal evolution of prenatal erythropoiesis
At the end of intrauterine life in the rat, the number of anucleated red cells
increases with age while MCV decreases; this is quite obvious during the
first hours after birth. The anucleated red cells produced in the last two days of
gestation and after birth are thereafter smaller than the ones produced earlier.
The functioning of the hepatic erythron has direct repercussions on the
composition of foetal blood: around day 18, a new population of red cells,
differing in size and morphology from those of yolk-sac origin, appears in the
blood (Nagel, 1972). Similar conclusions were reported in the foetal mouse
(Craig & Russel, 1964: Barker, 1968; Brotherton, Chui, Gauldie & Patterson,
1979). Accordingly, we observed, particularly between days 18 and 19, a rapid
290
M. D. NAGEL, J. NAGEL AND R. JACQUOT
decrease in both the number and the percentage of nucleated red cells of yolk-sac
origin.
Around birth, two other erythropoietic organs may be implicated: the spleen
and the bone marrow. The role of the spleen has not been considered in the
present paper: preliminary results suggest that the supply of erythrocytes by
the spleen is not important before birth in the rat (cf. also Lucarelli et al. 1968).
Under normal conditions, the bone marrow of the foetal rat contains only
few differentiated erythroid elements and the increase of its cell population
during the last three days of foetal life concerns essentially other lineages. At
day 21, only 4 % of the whole population are differentiated erythroid cells, and
at birth, 9-2 %. This last value is in good agreement with the one published by
Lucarelli et al. (1968) for the neonatal rat.
(II) Experimentally induced modifications of prenatal erythropoiesis
The corticosteroid environment of the foetus was modified in two opposite
ways: deprivation (maternal adrenalectomy and foetal decapitation) or early
excess of these hormones (repeated maternal stress). Paradoxically both modifications produced a similar effect: an increase of the erythropoietic tissue in the
bone marrow. The first conclusion which can be drawn is that the foetal bone
marrow is able, as early as day 20 of gestation, to adjust its erythropoietic
activity. The similarity of the effects produced by two apparently opposed
procedures suggests that, in fact, these procedures lead to a common physiological situation, as far as foetal erythropoiesis is concerned.
In corticosteroid-deprived foetuses, the stability of the hepatic haematopoietic
tissue was established by Nagel & Jacquot (1969) and Jacquot & Nagel (1976)
(and here, Fig. 4). Such a 'frozen' situation strongly suggests a block in the
delivery of circulating red cells by the liver, at a time when the foetal growth
is rapid. According to this hypothesis, the precocious increase of the erythroid
line in the bone marrow observed in decapitated foetuses occurs in compensation for precocious cessation of liver erythron functioning.
In fact, at day 19, an anaemia is detectable in decapitated foetuses: the number of anucleated red cells and haemoglobin concentration are lowered. By
day 20, the number of anucleated red cells is more than restored. Accordingly,
haemoglobin concentration increases between days 19 and 20, but MCH falls.
These observations suggest that these new red cells are supplied by the stimulated bone marrow and not by the liver. These red blood cells from medullary
origin appear to be smaller than those from hepatic origin.
The anaemia observed at day 19 in decapitated foetuses is really due to a
hepatic misfunction and not to a surgical haemorrhage for it is lacking in
foetuses decapitated at day 18 and simultaneously injected with cortisol.
Cortisol administration to decapitated foetuses at day 20 restores the efficiency of their liver erythropoiesis. Twenth-four hours later, part of their liver
erythroid line has been discharged as suggested by the decrease in nuclear count
Erylhropoiesis regulation in foetal rat
291
of liver homogenates and by the fact that the number of erythroid blood cells
is significantly higher than in non-treated decapitated controls. This suggests
that both anucleated and nucleated red cells are delivered by the liver.
In these cortisol-injected decapitated foetuses, the suddenly resumed supply
of blood cells from the hepatic erythron appears to block erythropoiesis in the
marrow (this medullary erythropoiesis is initially stimulated by the failure of
hepatic erythropoiesis). The number (by imprint surface area) and the percentage of unhaemoglobinized erythroid cells, which were both increasing rapidly
decrease sharply after cortisol injection, while the more mature haemoglobinized cells accumulate in the marrow, and are not liberated as circulated reticulocytes.
Repeated maternal laparotomies at 14, 16 and 18 days reduce the number of
nuclei in liver homogenates from 18- and 19-day-old foetuses. Here again, it is
tempting to speculate that the liver, prematurely drained of the major part of its
erythropoietic cells is unable to deliver enough red cells to the blood.
In agreement with this hypothesis, red blood cells, and especially anucleated
ones, are less numerous at 18 and 19 days (in fact this situation may well be
present already at 17 days, stress having been inflicted at 14 and 16 days).
Afterwards, the numbers of anucleated red cells reach the control values, and
an increased number of reticulocytes is observed at days 19 and 20. It is therefore logical to think that, here again, alterations in red blood cells number due
to hepatic deficiency brought about a medullary response. Nevertheless,
foetuses from stressed mothers differ in their behaviour from decapitated
foetuses: their liver erythron, although already depleted by the two first
maternal laparotomies at days 14 and 16, is still able to react to a third one at
day 18. As a consequence, the decrease in the number of their circulating red
cells, although already present by day 18, is not so drastic (haemoglobin
concentration is not affected) and their medullary response, although occurring
earlier (at day 19), is less pronounced.
It is also possible that the glucocorticoids act directly on marrow erythroid
cells. The status of effects, tested with in vitro systems, is controversial (Golde,
Bersh & Cline, 1976), Singer, Samuels & Adamson (1976), Gidari & Levere
(1979), Urabe, Hamilton & Shigeru (1979) and Zalman, Maloney & Pratt
(1979)). In our study, erythropoiesis in the marrow is not stimulated at day 21
by the fourth maternal laparotomy, but is considerably increased in corticoiddeprived foetuses. Such results do not sustain the hypothesis of corticosteroids
acting directly on the marrow.
The case of intact foetuses borne by adrenalectomized mothers (littermate
brothers of decapitated foetuses) deserves some comment. Their hepatic erythron presents an accelerated evolution (Nagel & Jacquot, 1969) and seems to
deliver to the blood an increased supply of nucleated and perhaps also anucleated red cells.
Misfunction of the hepatic erythron, whether induced by glucocorticoid
292
M. D. NAGEL, J. NAGEL AND R. JACQUOT
deficiency or by glucocorticoid excess, and leading to transitory anaemia, is
associated with premature initiation of medullary erythropoiesis. It is tempting
to believe that the bone marrow activity is triggered by an erythropoietinmediated mechanism of adult type, as suggested by Matoth & Zaizov (1971) and
reported by Meberg (1980) in the case of foetal hypoxia. Preliminary data
indicate that plasma erythropoietin-like activity is higher in both types of
experimental foetuses.
This work was supported in part by the DGRST Grant no. 77-7-0673.
REFERENCES
ACKERMAN, G. A., GRASSO, J. A. & KNOUFF, R. A. (1961). Erythropoiesis in the mammalian
embryonic liver as revealed by electron microscopy. Lab. Invest. 10, 787-796.
ARISHIMA, K., NAKAMA, S., MORIKAWA, Y., HASHIMOTO, Y. & EGUSHI, U. (1977). Maternalfoetal interrelations of plasma corticosterone concentrations at the end of gestation in the
rat. J. Endocr. 72, 239-240.
BARKER, J. E. (1968). Development of the mouse hematopoietic system. I. Types of hemoglobin produced in embryonic yolk sac and liver. Devi Biol. 18, 14-29.
BILLAT, C , NAGEL, M. D., NAGEL, J. & JACQUOT, R. (1980). Early reactivity of liver erythropoietic tissue of the rat foetus towards glucocorticoids. Biol. Cell. 38, 3, 187-194.
BROTHERTON, T. W., CHUI, D. H. K., GAULDIE, J. & PATTERSON, M. (1979). Hemoglobin
ontogeny during normal mouse fetal development. Proc. natn. Acad. Sci., U.S.A. 76, 6,
2853-2857
COHEN, A. (1973). Plasma corticosterone concentration in the foetal rat. Horm. Metab. Res. 5,
1,66.
COHEN, A. & BRAULT, S. (1974). Modifications de la concentration en corticosterone des
surrenales et du plasma chez le foetus de rat apres surrenalectomie maternelle. C.r. hebd.
Seanc. Acad. Sci. Paris. 278, 1755-1758.
CRAIG, M. L. & RUSSEL, E. S. (1964). A developmental change in hemoglobins correlated
with an embryonic red cell population in the mouse. Devi Biol. 10, 191-201.
DUPOUY, J. P., COFFIGNY, H. & MAGRE, S. (1975). Maternal and foetal corticosterone levels
during late pregnancy in rats. / . Endocr. 65, 347-352.
GIDARI, A. S. & LEVERE, R. D. (1979). Glucocorticoid mediated inhibition of erythroid
colony formation by mouse bone marrow cells. J. Lab. Clin. Med. 93, 5, 872-878.
GOLDE, D. W., BERSH, N. & CLINE, M. J. (1976). Potentiation of erythropoiesis in vitro by
dexamethasone. / . Clin. Invest. 57, 57-62.
GRASSO, J. A., SWIFT, H. & ACKERMAN, G. A. (1962). Observations on the development of
erythrocytes in mammalian fetal liver. / . Cell. Biol. 14, 235-254.
HOLT, P. G. & OLIVER, I. T. (1968). Plasma corticosterone concentrations in the perinatal
rat. Biochem. J. 108, 339-341.
JACQUOT, R. & NAGEL, J. (1976). Glucocorticoids and evolution of haemopoietic tissue in
the liver of the rat foetus. Ann. Immunol. (Inst. Pasteur) 126 C, 895-903.
JOST, A. (1951). La physiologie de l'hypophyse foetale. Biol. Med. 40, 205-229.
KAMOUN, A. (1970). Activite cortico-surrenale au cours de la gestation, de la lactation et du
development pre et post natal chez le rat. / . Physiol. Paris 62, 5-32.
LUCARELLI, G., PORCELLINI, A., CARNEVALI, C , CARMENA, A. & STOHLMAN, F. Jr. (1968).
Fetal and neonatal erythropoiesis. Ann. N.Y. Acad. Sci. 149, 544-559.
MARTIN, C. E., CAKE, M. H., HARTMANN, P. E. & COOK, I. F. (1977). Relationship between
foetal corticosteroids, maternal progesterone and parturition in the rat. Acta endocrinol. 84
167-176.
MATOTH, Y. & ZAIZOV, R. (1971). Regulation of erythropoiesis in the fetal rat. Israel J.
Med. Sc. 7, 839-843.
Erythropoiesis regulation in foetal rat
293
A. (1980). Plasma erythropoietin levels in fetal and newborn rats: response to
hypoxia. Expl Hemat. 8, 5, 615-619.
MILKOVIC, K., PAUNONIC, J., KNIEWALD, Z. & MILKOVIC, S. (1973). Maintenance of the
plasma corticosterone concentration of adrenalectomized rat by the foetal adrenal glands.
Endo. 93, 115-118.
MILKOVIC, S., MILKOVIC, K. & PAUNOVIC, J. (1973). The initiation of foetal adrenocorticotrophic activity in the rat. Endo, 92, 380-384.
NAGEL, J. (1968). Le tissu hematopoietique dans le foie foetal de rat enfinde gestation. I.
Evolution normale au cours du dernier quart de la gestation. Archs Anat. micr. Morph.
Exp. 57, 99-105.
NAGEL, J. (1972). Le tissu hematopoietique dansle foie foetal derat enfinde gestation. IV.
Consequences de son evolution sur la population erythrocytaire circulante. Archs Anat.
micr. Morph. Exp. 61, 301-312.
NAGEL, J. & JACQUOT, R. (1968). Le tissu hematopoietique dans le foie foetal de rat enfinde
gestation. II. Influence de la laparotomie et de la surrenalectomie maternelle. Archs Anat.
micr. Morph. Exp. 57, 99-105.
NAGEL, J. & JACQUOT, R. (1969). Le tissu hematopoietique dans le foie foetal de rat en fin
de gestation. III. Influence de l'hypophysectomie foetale. Archs Anat. micr. Morph. Exper.
58, 97-104.
RIFKIND, R. A., CHUI, D. & EPLER, H. (1969). An ultrastructural study of early morphogenetic events during the establishment of fetal hepatic erythropoiesis. / . Cell. Biol. 40,
343-365.
SINGER, J. W., SAMUELS, A. I. & ADAMSON, J. W. (1976). Steroids and hematopoiesis. I. The
effect of steroids on in vitro erythroid colony growth: structure/activity relationships.
/. cellphysiol. 88, 127-134.
URABE, A., HAMILTON, J. & SHIGERU, S. (1979). Dexamethasone and erythroid colony
formation: contrasting effects in mouse and human bone marrow cells in culture. Brit.
J. Haematol. 43, 479-480.
ZALMAN, F., MALONEY, M. A. & PATT, H. M. (1979). Differential response of early erythropoietic and granulopoietic progenitors to dexamethasone and cortisone. /. exp. Med. 149,
67-72.
MEBERG,
(Received 15 July 1980, revised 5 March 1981)