SAN FERNANDO VALLEY STATE COLLEGE
Erythropoiesis in the Neottatal Mouse
i•
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Janet Ruth Beecher Baas
January, 1970
The thesis of Janet Ruth Be
}her Ba�s)
pr:oved:
Committee Chairman
San Fernando Valley State College
January, 1970
TABLE OF CONTENTS
Abstract
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Introduction.
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Materials and Methods.
Figure 1.
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Figure 5
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Figure 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . 16
Table I
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Table II
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Results
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Discussion
Literature Cited
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17
ABSTRACT
ERYTHROPOIESIS IN THE NEONATAL MOUSE
by
Janet Ruth Beecher Baas
Master of Science
January, 1970
An electronic cell sizing and counting system was employed for the
first time to study developmental changes in the peripheral blood pic­
ture of neonatal mice.
Among the parameters studied were erythrocyte
volume and number, hemoglobin level, mean corpuscular hemoglobin, and
mean corpuscular hemoglobin concentration.
The system used determined
several of these blood values simultaneously and permitted much more
rapid and accurate measurements than have previously been obtained using
standard hemocytometric techniques.
The erythrocytes of the neonatal period were macrocytic and hypo­
chromic.
The mean volume and mean corpuscular hemoglobin decreased
'after birth, while the erythrocyte count, hemoglobin content and mean
corpuscular hemoglobin concentration rose.
Adult values for each of
these parameters were reached by the 41st day after birth.
The eryth­
rocyte population gradually became more homogeneous as the largest cells
were eliminated with time,
When supplemental iron was administered to female mice during or
just before pregnancy, the erythrocyte distributions of the offspring
were the same as in normal newborn mice, but the erythrocyte number was
higher.
On the basis of evidence reported in this study together with
that obtained by other investigators, a model for the production of
hypochromic macrocytes in neonatal mice is described.
It is suggested
that the immediate cause of the macrocytosis is the skipping of cell
divisions during erythrocyte production.
v
INTRODUCTION
During the first two months following birth, extensive changes
occur in the blood picture of the mouse.
Blood values which have pre-
viously been examined during this period include circulating erythrocyte and leukocyte number, erythrocyte volume, hematocrit (ratio of
packed erythrocyte volume to total blood volume ) , reticulocyte percentage and hemoglobin level.
Results of the early investigations of mouse blood v1ere review·ed
by Scarborough in 1931.
Although a limited number of studies were com-
pleted at that time, normal values for erythrocyte, leukocyte and reticulocyte numbers were established for adult mice.
It was also noted
that the total number of circulating erythrocytes increased from birth
to adulthood.
A study of the increase in blood volume during the first three
II
weeks following birth vlas made by Gruneberg (1941).
He found that
blood volume per unit body weight decreased immediately after birth,
but then gradually rose until about the third week.
As body weight
continued to increase after the third week, there was a slow decline in
blood volume per unit '\veight.
It was also found that the hemoglobin
concentration of the blood declined after birth, but thereafter, gradually rose to the adult level.
A comprehensive study of the blood picture of young adult mice was
made in 1951 by Russell et al.
Their study included 18 of the inbred
strains of mice maintained at Roscoe B. Jackson Laboratory, Bar Harbor,
Maine.*
The study established the normal values for the formed elements
*Jackson Laboratory produces many strains of mice which are \videly used
in biological research.
1
of the blood in each strain, and differences between strains were shown
to result from genetic differences among the inbred strains used.
Russell and Fondal (1951) made erythrocyte volume and number determinations for normal and anemic mice of the C57BL/ 6 inbred strain obtained from J ackson Laboratory.
The number of cells \o7aS determined by
hemocytometry and average cell volumes were calculated by dividing the
:hematocrit value by the erythrocyte count. Blood samples were examined
on the 16th day of gestation and the 1st, 14th and 28th days after·
birth.
They observed an increase in erythrocyte number and a decrease
in average erythrocyte volume during this period.
Gyllensten and Swanbeck (1959) reported changes in the hemoglobin
content and erythrocyte number from birth through the 30th day in various oxygen concentrations.
These mice were also of the C57BL/6 strain
.from the Jackson Laboratory.
Scribner et al. (1968) studied day-to-day changes in the blood
--
picture of CF-1 mice (Carworth Farms, New York) through the 14th day
follmving birth.
Average cell volume \vas calculated by dividing the
hematocrit value by the erythrocyte number and hemoglobin concentration
determined with a spectrophotometer using the cyanomethemoglobin method.
In 1963, Russell reviewed the techniques used in the study of
.mouse blood.
In the past, the method used most often for determining
erythrocyte number involved the dilution of a known volume of blood, a
·portion of which is then enumerated using a Neubauer counting chamber
(hemocytometer).
Once this value is obtained, average cell volume may
be determined from the hematocrit.
More recent studies of blood employ
electronic counting and sizing, which simultaneously evaluates both
erythrocyte number and volume.
Electronic methods have not previously
3
been used in studies of neonatal mice.
In the operation of electronic
counters, relatively non-conducting particles (erythrocytes), suspended
in a conducting solution are dra\vn through the small orifice of a glass
probe by a pump.
Current is supplied to the orifice by a set of elec­
trodes and the passage of cells causes a transient voltage drop by in­
creasing the orifice resistance (Mattern et al. , 1957; Brecher et al. ,
1962).
Since individual cells passing through the orifice replace an
equivalent volume of suspending medium, the electrical pulses produced
are proportional to the cell volume.
The number of pulses are simul­
taneously recorded on a scaler and displayed as a distribution of spikes
on an oscilloscope screen.
The height of each spike is directly related
to the size of the cell responsible for producing the spike.
By means
of a threshold control, only those pulses greater than a selected mag­
nitude are amplified and counted.
This system permits much more rapid
and accurate cell measurements than standard hernocytometric methods and
requires far less initial blood sample (Mattern et al. , 1957; Russell,
1963).
In the studies to be reported here, a modified electronic count­
ing and sizing system was employed to follow changes in the peripheral
blood picture of C57BL/6J mice during the first 6 \<Teeks following
birth.
A pulse height analyzer was connected to the electronic count­
ing and sizing system which permits the recording of a very precise cell
volume distribution.
This instrument sorts pulses from �he electronic
counter according to their size and keeps a running tally in its memory
of the number of pulses of each size.
The contents of the analyzer
memory are displayed on an oscilloscope screen at the end of a single
cell sample measurement.
The oscillograph shows the number of cells vs.
cell volume for the entire volume distribution.
It is not possible to
4
obtain the entire volume distribution immediately with the ordinary
commercially available sizing system, the Coulter counter, as only
small sections o f the distribution can be obtained with a single meas­
urement.
Beca��e the pulse height analyzer forms a complete distribu­
tion with only one measurement much smaller cell samples are needed
than with the ordinary electronic counter.
The results of several
measurements may be stored in different parts of the analyzer memory
and compared directly on the oscilloscope display.
In addition, a
digital prin-t-out attachment prints the exact number of cells for each
volume at the end of each measurement.
Lucarelli et al. (1964) reported that abnormally large erythrocytes
(macrocytes) which are deficient in hemoglobin (i.e. , hypochromic) are
released into circulation during the neonatal period in rats.
et al. (1959) made similar observations in neonatal mice.
Jacobson
Macrocytosis
in adults is usually a symptom of an anemia and has also been observed
under certain conditions of induced erythropoietic stress, such as
severe bleeding or phenylhydrazine (an intravascular hemolysant) treat­
ment.
Macrocytosis is the result of skipped cell divisions during the
development of erythrocytes in the bone marrow· (Stahlman et al. 1964).
Jacobson et al.
(1959) also found that the naturally occurring anemia
of newborns could be alleviated by treating pregnant mothers \vith iron
during pregnancy.
How:ever, no attempt was made to examine the effects
of iron treatment on erythrocyte volume.
In the studies to be reported,
cell volumes and nmubers were examined in neonatal mice from mothers
which had received supplementary iron.
5
MATERIALS AND HETHODS
Animals
All mice used in these studies were of the C57BL/6J inbred strain,
obtained from the Roscoe B. Jackson Laboratory, Bar Harbor, Maine.
The
animals were bred periodically and the offspring \vere also employed in
the studies.
Mice 0-24 hours old were termed 1 day old, after 24-48
hours, two days old, and so on.
Cell Sizing and Counting
'
The following instruments \vere used tor cell counting and size
distribution measurements:
a Coulter transducer \vith a 90!1 long
x
90!1
diameter jeweled orifice; a RIDL preamplifier (model 31-25), amplifier
(model 30-30) and pulse shaper (model 52-58); a Packard multichannel
pulse height analyzer (model 15); and a Honr<?e digital print-out
· (model 11C 10-40) (Zucker and Gassen, 1969) (Fig. 1).
The multichannel
analyzer system \vas calibrated with 3. 711 polystyrene spheres and human
and mouse erythrocytes.
The components of the electronic counting and sizing system are
·shown diagrammatically in Fig. 2.
The Coulter transducer is supplied
with a regulated constant current source.
Thus, as a cell passes
through the orifice there is an increase in resistance and because
current is constant the voltage changes.
The changing voltage output
goes to the preamplifier, which is needed because of the weak pulses
generated by the small cells.
The linear amplifier further amplifies
the signal so that it enters the range of the multichannel analyzer.
The pulse then goes both to the discriminator and, v1ith a delay, to the
gate \vhich allmvs pulses to pass to the multichannel analyzer.
discriminator determines l·lhich P!-tlses are to be counted.
The
A pulse which
6
r,ig. 1.
Photograph of electronic counting and sizing instruments used
in this study.
7
Fig. 2.
Diagrrumnatic representation of the electronic cell s�z�ng and
counting system. The Coulter transducer is supptied with a
constant current source. As a cell passes through the orifice
the voltage changes. The changing voltage output goes to the
pre-amplifier and then to the linear amplifier. The signal
then goes both to the gate and discriminator. The pulses are
then counted by the multichannel analyzer and displayed either
on the counting or overflo·w scaler, The amplitude of the
pulses is recorded in the memory of the analyzer. The entire
memory of the analyzer may be read out by the digital print­
out.
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is greater than a pre-set lower threshold level will cause the mono­
stable to allow the gate to open and the pulse to pass through for a
pre-set amount of time.
Pulses below the lmoJer threshold are not
counted, those above the pre-set upper threshold are counted but only
as overflm., counts on the overflm., scaler.
In the multi-channel
analyzer each pulse a�mplitude is first converted into an equivalent
time interval.
a
This time interval determines the number of pulses from
periodic oscillator.
This number is counted by the address scaler.
These periodic pulses are a quantified measure of the amplitude of the
input signal.
The final position of the address scaler identifies the
channel in which the pulse will be stored in the magnetic core memory.
The contents of the entire memory can be viewed on the built-in oscil­
loscope (Fig. 3), photographed, and read out on the digital print-out.
The total number of counts is displayed on the counting scaler.
Blood examined with the counting system was diluted in Hank's
Balanced Salt Solution (HBSS) which had been filtered through a 0. 22�
Millipore filter.
The filtered HBSS was tested on the counter before
use and only used for experimentation if fewer than 50 counts occurred
in 0. 5 ml.
In order to reduce the effect of small day-to-day changes
in the counting system, human erythrocytes from the same individual
\'Jere examined with the counter before each analysis.
When necessary,
the system was adjusted so the mode of the hmnan erythrocyte size dis­
tribution remained constant.
Blood for all experiments was drawn with a 0. 003 ml micropipette
rinsed in heparin, from the cut tail of each mouse.
The blood to be
examined with the counter was diluted in HBSS to 10, 000-20, 000 cells
per 0. 5 ml.
This di.lution minimized the effect of coincidence loss
Fig. 3.
Photograph of multi-channel analyzer oscilloscope screen
showing a typical 14 day size distribution of mouse erythrocytes.
10
(i. e., the ability to resolve two or more cells as individuals as they
pass through the orifice).
A size distribution for 100, 000 cells was
made on each diluted blood sample.
In addition, a total cell count for
0. 5 ml of the sample was made to permit the determination of the total
number of cells in the original, undiluted sample.
The mean cell volumes for each of the erythrocyte size distributions were calculated from the digital tapes \vith an Olivetti-Underwood
Programma 101 desk calculator.
A special program was developed for the
calculator to analyze the type of data received from the digital print­
out (Zucker and Gassen, 1969).
Hemoglobin Determinations
The oxyhemoglobin concentration of the blood was determined colorimetrically with a Bausch and Lomb Spectronic 20 colorimeter (Schalm,
1961).
Blood was diluted in 0.04% NH 0H and optical density read at a
4
"mvelength of 578 nm.
sample.
The 0. 04% NH 0H reagent uas used as a blank
4
}lean corpuscular hemoglobin (NCR), the amount of hemoglobin
per cell, and mean corpuscular hemoglobin concentration (HCHC), the
amount of hemogl obin per �3, were calculated from cell sizing and
counting measurements and hemoglobin determinations.
Imferon Studies
In order to examine the effects of iron supplementation on the
peripheral blood picture of mice, mothers received injections of irondextran (Imferon, Lakeside Laboratories).
Tv1o procedures w·ere employed.
Initially, mated female mice were injected intran1uscularly with the
iron-dextran solution at the 13th-14th day of gestation.
Dosage ranged
from one injection of 0.1 cc (5 mg) to two injections of 0.2 cc (20 mg).
In later experiments, however, one injection of 0. 15 cc (7.5 mg iron)
11
was given two days before mating.
Over 90% of the iron is utilized in
the first two weeks after injection, and only a small amount of the
iron is excreted in the following months (Golberg, 1958).
RESULTS
Changes in mean cell volume during the fi rst 17 days following
birth are shown in Fig. 4.
The mean erythrocyte volume of newborn mice
was about twice that of adult mice.
This value decreased almost lin­
early from 98!13 at birth to the adult value of 45.5fl3 by about the 17th
day (Table I).
values.
Table I also gives erythrocyte count and hemoglobin
Fig. 5 shows erythrocyte size distributions obtained f rom the
electronic counting system during this period.
Not only is there a
reduction in average cell size, but the entire population becomes much
more uniform with time.
Concurrent with the change in erythrocyte size is a change in
erythrocyte number.
The number of circulating blood cells increased
steadily from 3.73 x 106fmm3 at birth to the adult value of 9.66 x 106
/mm3 by about the 41st day (Fig. 6).
The amount of hemoglobin measured at birth was 8. 6 gm/100 cc blood,
but during the following tw·o weeks lower levels were recorded (Fig. 7).
Thereafter, the hemoglobin value gradually rose to the adult level of
13.3 gm/100 cc blood by about the 39th day.
shown in Table II.
MCH and MCHC values are
NCR declined from 2. 30 x 10-11gm/cell at birth to
the adult value of 1.37 x 10-llgm/cell by the 17th day.
MCHC rose con­
currently from the birth value of 2.40 x w-13gmf!l 3 to the adult value
of 2.98 x w-13gm/ !l3 by the 17th day.
Thus, erythrocyte volume, MCH,
and NCHC reach adult values simultaneously by about the 17th day, but
12
Fig. b,..
Changes in mean volume of erythrocytes of mice during the
first 17 days follm.;ring birth. (Each point represents the
average for five mice).
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TABLE I
Erythrocyte volume, erythrocyte number and hemoglobin concentration of
newborn mice.
Values given are derived from Figures 4, 6, and 7.
Age
{days)
Volume
(IJ.3)
Number
(x 106/mm3)
Hemoglobin
( gm /100 cc
blood)
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
41
Adult
98 ± 9. 27
87 ± 8. 13
84 ± 9. 60
76 ± 7. 85
65 ± 7. 05
59 ± 6. 41
50 ± 5. 75
49 ± 6. 02
46 ± 5. 38
3. 73
4. 21
4. 28
4. 29
4. 68
4. 86
5. 41
6. 08
6. 25
6. 76
7. 42
7. 64
7. 81
8. 16
8. 23
8. 55
8. 73
8. 81
9. 22
9. 58
9. 68
9. 66
8. 6
7. 6
7. 5
8. 2
7. 7
8. 0
7. 7
8. 1
8. 7
9.'�
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45.5
4. 20
9.0
8. 9
10. 5
9. 9
11. 0
12. 4
13. 0
12. 7
13. 4·
13. 1
13. 3
±one standard deviation
No further determinations were made. After this time mean volume
stays the same, while the remaining abnormally large cells are
eliminated.
14
Fig. 5.
Volume distributions of mouse erythrocytes from the electronic
counting and sizing system. The distributions become narrm·1er
and less skewed to right while decreasing in mean volume.
Thus, the largest cells are being eliminated and the popula­
tion is becoming more homogeneous with time.
15
Fig. 6.
Changes in erythrocyte count of mice during the first 41 days
following birth. Each point represents the average for five
mice).
BL'eTMJNIC eRYTHROCYTE VOI.UM£ /JISTR/81/T/ON.S
MY 12
ADIILT
v
1.11 1'1.1 119 J06 2JO zu
VOL VME {M.I)
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Fig. 7.
Changes in hemoglobin concentration of mice during the first
39 days following birth.
(Each point represents the average
for five mice).
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TABLE II
Mean corpuscular hemoglobin and mean corpuscular hemoglobin concentra­
tion
Age
(days)
1
3
5
7
9
11
13
15
17
19
21
23
25
27
29
31
33
35
37
39
Adult
MCH
(x 10-llgm/cell)
2. 30
1. 81
1. 69
1. 90
1. 65
1. 60
1. 41
1. 33
1. 38
1. 38
1. 21
1. 16
1. 34
1. 21
1. 33
1. 45
1. 48
1. 44
1. 45
1. 37
1. 37
MCHC
(x 1Q-13 flJ.3)
2. 46
2. 08
2. 01
2. 50
2. 53
2. 71
2. 81
2. 71
3. 00
3. 00
2. 63
2. 52
2. 91
2. 63
2.89
3. 15
3. 20
3. 13
3. 15
2. 98
2. 98
18
adult values for erythrocyte nTh�ber and hemoglobin concentration are
not reached for another three weeks.
Only three of nine mice injected with Imferon during pregnancy
survived the injections.
Because it is often difficult to tell if a
mouse is pregnant at the 13th or 14th day of gestation, an additional
number of non-pregnant mice were injected; all survived.
were injected tv10 days before mating, no deaths occurred.
When the mice
It may be
that pregnancy somehmv- makes toleration of the injection difficult.
Erythrocyte size distributions and erythrocyte counts for newborn mice
from Imferon-injected mothers v1ere compared to normal newborns on the
day of birth.
No differences in size distributions were noted.
!low­
ever, the erythrocyte count of 4.40 x 106/mm3 (average for 7 animals)
was higher than the normal 1st day count of 3.73 x 106 cell /mm3•
DISCUSSION
Erythrocyte volume distributions from birth through achievement of
adult cell volume have not previously been obtained for C57BL/ 6J mice.
Although no direct comparison can be made, the general pattern of de­
creasing average erythrocyte size from birth through the first few weeks
of life, described by other investigators, was confirmed in the present
studies (Russell and Fondal, 1951; Scribner et al. , 1968).
The rate of
decrease in mean cell volume varies from strain to strain, and stable
adult values may not be reached in some strains until the 8th or lOth
week (Russell and Bernstein, 1966).
The modified method of electronic cell sizing employed in this
study permits detailed examination of the entire distribution of cell
volumes.
The progressive changes in cell volumes can be seen very
19
clearly.
In earlier studies, mean erythrocyte volume was calculated by
dividing the hematocrit v alue by the number of erythrocytes.
not possible
to
It was
determine the distribution of cell volumes about the
It can be seen in Fig. 5 that the volume distributions from day
mean.
1 through day 17 become narrower and less skewed to the right while
simultaneously decreasing in average volume.
The narrowing of the dis­
tributions indicated a gradual decrease in the heterogeneity of the
cells.
This is also clear from an examination of the changes in stand­
ard deviations of mean cell volumes given in Table I.
The fact that
the distributions become less skewed to the right suggests the pro­
gressive elimination of cells of the largest volumes.
Thus, while the
modal erythrocyte volume of the 17 day mouse is the same as the adult,
the volume distribution is slightly more skewed to the right than the
adult distribution and therefore still contains a greater number of
large cells than the adult.
The number of circulating erythrocytes increased steadily from
birth.
The rate of increase in the present study corresponds closely
with that reported earlier for C57BL/6 mice by Russell and Fondal (1951)
.and Gyllensten and Swanbeck (1959).
The absolute values reported in
this study are slightly higher than those of Gyllensten and Swanbeck
(1959) and slightly lower than those of Russell and Fondal (1951).
Differences in reported blood values during the neonatal period can re­
sult from variations in the time of examination (i.e., early or late on
the first day, etc.) since the blood picture changes very rapidly
during this period.
reported values.
Variations in method can also cause differences in
Previous investigators used hemocytometry for cell
20
number determinations while the more accurate electronic cell counting
method was employed in the present study.
The hemoglobin values reported in this study are similar to those
reported by Gyllensten and Swanbeck (1959).
The immediate decline in
hemoglobin concentration after birth followed by a gradual increase to
adult values is a general pattern in mammals (Holman, 1956).
Wintrobe
(1967) suggested that in human infants the primary reason for the drop
in hemoglobin concentration immediately after birth may be the altered
oxygen saturation of the blood.
The low MCHC value for mice at birth found in these studies is
consistent with findings of other investigators (Jacobson !:,! al. , 1959;
Scribner et al. , 1968).
Thus, the neonatal period in mice is character­
ized by an anemia in which there is marked hypochromia and macrocytosis.
The macrocytic hypochromia disappears at the 17th day with the attain­
ment of adult MCH, MCHC and erythrocyte volume�
The anemic condition
persists, hmvever, until adult cell numbers and hemoglobin levels are
reached by the 41st day after birth.
Erythrocytes are derived from a precursor called a stem cell.
The
origin of the stem cell in hemopoietic organs is unknown (Matioli,
1969).
The yolk sac stem cell is thought to be the common precursor of
all stem cells by some while others believe that stem cells evolve in­
dependently from endogenous precursors in each hemopoietic organ.
The
liver is the main site of erythropoiesis in the mouse from the 13th day
of gestation until birth, after which the bone marrow becomes the pri­
mary site.
Late in fetal life, the liver stem cells which previously
had been stem cells for a line differentiating into erythrocytes can be
seen di fferentiati.ng into hepatic cells (Lucarelli et al. , 1968).
The
21
regulatory mechanism is unknown,
The stem cell undergoes a series of
divisions in which cell volume is approximately halved with each mito­
sis, since little or no growth occurs between each division (Borsook,
1964).
The last two stages of development are called polychromatic and
orthochromatic erythroblasts.
The nucleus is lost at the orthochromatic
stage and the resulting reticulocyte is released into the blood stream
where it matures to
an
erythrocyte.
The differentiation of erythrocytes in the bone marrow is regulated
by the hormone erythropoietin, produced by the kidney,
Erythropoietin
may also initiate and govern the rate of hemoglobin synthesis (Stohlman,
� al., 1964).
Under conditions of sever� erythropoietic stress, eryth­
ropoietin production increases and hemoglobin synthesis is accelerated.
When a critical cytoplasmic hemoglobin concentration (CHC) is reached,
a feedback mechanism shuts off further nucleic acid synthesis and cell
division.
Therefore, the number of cell divisions is determined by the
rate of hemoglobin synthesis.
When hemoglobin synthesis is accelerated,
the critical CHC is reached more rapidly, one or more divisions are
skipped and macrocytes are released into the peripheral blood.
If the
rate of hemoglobin synthesis is decreased, as in iron deficiency, addi­
tional divisions and microcytes (abnormally small erythrocytes) result.
It has been shown that neonatal rats and mice both pass through a
period of intense erythropoiesis (reticulocytosis 90%) in the face of
mild anemia (Lucarelli et al., 1964; Scribner et al. , 1968),
Since the
natural anemia of newborn nuce is alleviated by iron injections to the
mother during pregnancy, it is thought to be the result of insufficient
i ron available to both mother and fetus during gestation (Jacobson
et al., 1959).
In the light of the model for production of macrocytes,
22
it seemed possible that iron supplements during pregnancy might result
in erythrocyte sta.ge skipping in offspring of iron t reated mothers
since the young are iron deficient and under intense erythropoietic
stress.
�1us,
in this study the blood of offspring from iron treated
mothers was comp ared to normal blood. on the first day of birth.
:No
difference in erythrocyte volume t.o.ras found, although the erythrocyte
number of newborns f rom iron treated mothers \vas greater thah in nor­
mals.
Since the erythrocytes of newborn mice are twice adult size at
birth,
it may be that they have already skipped a stage during their
normal production.
If this is true,
the iron supplements would not be
expected to cause additional stage skipping
ever,
•
.
It should be noted,
that in the case of iron deficiency anemia,
microcytosis is predicted by the model.
how­
as in the ne'\<rborns,
This incongruity might be
explained if regulation of fetal and neonatal erythropoiesis differs
f rom the adult.
Feldman (1969),
There is evidence that this is so.
Eleiberg and
using the in vivo cloning method for hemopoietic cells,
showed that fetal mouse liver cells are able to form clones under con­
ditions of inhibition of erythropoietin production,
c lones of bone marrow cells is prevented.
while formation of
Nephrectomy in adult rats
results in virtually complete cessation of erythrocyte p roduction,
but
the same operation has little effect on neonatal erythropoiesis
·(Lucarelli et al.,
1964).
Nephrectomy produces a progressively greater
suppression of e rythrocyte product.ion. as the animal matur.es and the
adult response is reached by the JOth day.
Hypertransfusion of the
adult rodent severely suppresses erythropoiesis but only slightly sup­
presses it in the newborn (Stohlm9.n,
1967).
It thus seems clear that
erythropoiesis is not controlled by renally produced erythropoietin as
in the adult, particularly since erythropoiesis starts in the embryo before the kidney is established as a functional unit (Jacobson et al. ,
1959),
Although there is some evidence that, in adults, erythropoietin
may be produced extrarenally, the amount of erythropoietin from these
sources appears inadequate to support the intense erythropoiesis of the
neonatal period (Lucarelli et al, , 1964).
Since it appears that the regulation of erythropoiesis in adults
and ne\vborns differs, it has been suggested that the CHC for terminIf the
ating cell division is also different (Lucarelli� al., 1964).
CHC in neonatal mice is lmver than in adult, then the appearance of
macrocytosis instead of microcytosis would be explained,
Examination
of the volume distributions in this study show the life span of the
largest erythrocytes is shortened.
This is consistent with the finding
that macrocytes produced in response to severe phenylhydrazine anemia
in adults have a shortened life span (Stohlman, 1961).
�fuile the iron supplement had no effect on cell volume, increased
erythrocyte numbers at birth were observed.
It seems likely that
erythrocyte production is at a near maximum rate at birth and that
additional iron enables an increased number of cells to be produced.
On the basis of evidence reported in this study and that obtained
by other investigators, a possible model for the production of hypochromic macrocytes in neonatal mice has been described in this paper.
It has been proposed that neonatal erythropoiesis is regulated by a
different, though unknoWl1, mechanism than adult erythropoiesis,
Thus,
macrocytes might normally be produced in the newborn under conditions
which \vould produce microcytes in the adult.
It is therefore suggested
that the immediate cause of macrocytosis in neonatal mice is the
24
skipping of cell divisions during erythrocyte production.
25
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