/. Embryol. exp. Morph. Vol. 43, pp. 17-28, 1978
Printed in Great Britain © Company of Biologists Limited 1978
17
Re-evaluation of the
presence of multiple haemoglobins during
the ontogeny of the mouse
ByKOJI SHIMIZU 1 AND TOMOMASA WATANABE 1
From the Department of Morphology and the Department of Biochemistry,
Institute for Developmental Research, Aichi Prefectural Colony, Kasugai
SUMMARY
s
A
In mice, three alleles, Hbb , Hbb and Hbbp, have been found at the /?-chain locus of
haemoglobin. Analytical Polyacrylamide gel electrophoresis revealed the presence of almost
the same four haemoglobin bands (E-T, E-IT, E-III and X) in all of the lysates of peripheral
blood cells from 12-day-old foetuses of C57BL/6J (Hbb% A/He (Hbbd) and PON (Hbb"). All
were embryonic-type haemoglobins; the most anodal band (X) seemed to be a product of
polymerization of E-I. Electrophoretic patterns of the haemolysates from 12-day-old foetuses
were not affected by alkylation with iodoacetamide or maleic anhydride. Almost the same
electrophoretic patterns in subunit analysis, using acid starch and acid-urea Polyacrylamide
gels, were obtained in 12-day-old foetuses of C57BL/6J, A/He and PON. E-I comprised aand x-chains, E-Il, a- and y-chains, and E-Ill, a- and z-chains.
In adults, two haemoglobin bands, s and Y, were present in the haemolysate of C57BL/6J,
while three, dmaj, dmin and Y, existed in A/He, and three, pmaj, pmin and Y, in PON. All
were adult-type haemoglobins. Haemoglobin Y, which was not demonstrable after alkylation of the haemolysates, seemed to be a product of polymerization of major haemoglobins
(s, dmaj and pmaj). Each haemoglobin possessed common a-chains, while the /?-chains of
major haemoglobins were different from those of minor haemoglobins.
Developmental changes of the haemolysates from foetuses of C57BL/6J, A/He and PON
were similar to each other. Haemolysates of the liver cells of developing foetuses contained
only adult-type haemoglobins. Our techniques used here were not able to demonstrate the
presence of foetal-type haemoglobins.
INTRODUCTION
Electrophoretic studies of mouse haemolysates have revealed the existence of
several haemoglobin patterns, classified as 'single' or 'diffuse'. These patterns
are controlled by two alleles at the Ebb locus, Hbbs and Hbbà, which determine
the structure of the /?-chain of mouse haemoglobin. A third allele at the Hbb
locus, Hbb9, was first found by Morton (1962, 1966). This allele was also
demonstrated in Asian feral mice by Gilman (1974), and in two Japanese strains
of mice, PON and Mol-A, by Watanabe, Shimizu, Goto & Ogasawara (1976).
1
Authors' address: Institute for Developmental Research, Aichi Prefectural Colony,
Kasugi, Aichi 480-03, Japan.
18
K. SHIMIZU AND T. WATANABE
No other alleles have been reported (Hutton, 1969). As most vertebrates, mice
have both embryonic and adult haemoglobins during the course of development. The nomenclature of embryonic haemoglobins in mice is confusing
(Schalekamp, Harrison & Paul, 1975), but three have been demonstrated in
foetal haemolysates (Fantoni, Bank & Marks, 1967; Gilman & Smithies, 1968:
Melderis, Steinheidev & Ostertag, 1974). Recently, Schalekamp et al. (1975)
have reported foetal haemoglobins also in Porton white Swiss mice. The socalled 'single' adult haemoglobin has been split by Barker (1968) into two
components through analytical Polyacrylamide gel electrophoresis. In this paper,
we re-evaluate some features of multiple haemoglobins during the ontogeny of
C57BL/6J (Hbbs), A/He (Hbb*) and PON (Hbb*) mice, using Polyacrylamide
and starch gel electrophoresis, chromatography on DEAE-Sephadex column,
and immunology.
MATERIALS AND METHODS
Animals. Mice of three strains, C57BL/6J (C57), A/He (A), and PON, were
used. C57 and A were imported from the United States. PON is an inbred
strain derived from F x mice of C57 x Pink, which is a Japanese fancy mouse
stock maintained in Laboratory of Animal Genetics, Nagoya University.
Preparation of haemolysates. Adult mice were bled by decapitation. Foetuses
from 12 to 17 days of gestation (the day on which vaginal plugs were observed
being taken as day zero) were washed thoroughly with buffered saline (pH 7-2),
and bled from the umbilical vessels for peripheral blood cells. Livers of foetuses
were excised, washed thoroughly, and then disaggregated by pipetting. Citrated
ice-cold buffered saline (pH 7-2) was used for washing and cell collection as
follows. Blood cell suspensions were washed three times in at least a tenfold
volume of isotonic ice-cold buffered saline (pH 7-2). Following the final washing, the cells were resuspended in a double volume of ice-cold deionized water,
and left overnight at 4 °C to haemolyse. The stroma was removed by centrifugation at 10000 g for 60 min, and clear supernatant was used as fresh haemolysate. Cyanmethemoglobin was obtained by mixing 1 vol. of haemoglobin
solution (ca. 40 mg/ml) with 2 vol. of a solution containing 2 % K3Fe(CN6),
0-5 % KCN, and 0-1 % NaHC0 3 (Moss & Ingram, 1968).
Alkylation of mouse haemoglobin. Alkylation of mouse haemoglobin was
carried out as described by Petras & Martin (1969). Fresh haemolysate {ca.
40 mg/ml) was mixed with equal volumes of iodoacetamide (6-84 mg/ml) and
maleic anhydride (16-5 mg/ml, neutralized with NaOH).
Analytical Polyacrylamide gel electrophoresis. Analytical Polyacrylamide gel
electrophoresis in alkaline gels was carried out by the procedure of Barker
(1968), but without KCN in the reservoir buffer (Tris-glycine buffer, pH 9-3).
Elution of each haemoglobin from alkaline gels was performed (Bruns &
Ingram, 1973). Analytical Polyacrylamide gel electrophoresis in acid-urea gels
was carried out (Panyim & Chalkley, 1969).
Multiple haemoglobins
in mouse ontogeny
19
Starch gel electrophoresis. Starch gel electrophoresis in alkaline gels was
carried out by the method of Gilman & Smithies (1968). To separate each
haemoglobin component into subunits (polypeptide chains), starch gel electrophoresis in 1-4 M formate buffer (ju, = 0-05 in gels, JU, = 0-2 in bridges) at
pH 1-9 was performed (Muller, 1960).
DEAE-Sephadex column chromatography. Column chromatography was
carried out on diethylaminoethyl (DEAE)-Sephadex (Pharmacia A-50) column
(2-0x21 cm), using 0 0 8 M trihydroxymethylaminomethane (Tris)-HCl buffer
in a linear gradient from pH 8-6 to 7-4. About 100 mg haemoglobin from fresh
haemolysates or cyanmethemoglobin forms in starting buffer was placed on the
column, and elution was accomplished at 20 °C. The optical density (o.D.) of
the effluent fractions was measured at 280 nm, using LKB Uvicord III 2089
UV-absorptio-meter.
Immunology. Male rabbits were immunized by five subcutaneous injections
of mouse haemoglobin at weekly intervals (1 ml of about 50 mg/ml haemoglobin
solution in buffered sahne, pH 7-2) mixed with equal volume of Freund's complete adjuvant (Difco). Complement inhibition of antisera was performed at
56 °C for 30 min. The maximum dilution of antisera which gave positive
reactions in ring precipitin tests was about 64. Antiserum against the major
(minor) haemoglobin component of adult A mice was obtained by absorbing
antiserum to haemoglobin of adult A mice with the minor (major) haemoglobin
component of adult A mice. Antiserum against the major or minor haemoglobin
component of adult PON mice was obtained similarly. Ouchterlony tests were
carried out (Ouchterlony, 1958).
RESULTS
Ontogenic changes of haemoglobins. When subjected to analytical Polyacrylamide gel electrophoresis in alkaline gels, the lysates of peripheral blood
cells from 12-day-old foetuses of C57, A and PON all contained four embryonictype haemoglobin bands, E-I, E-II, E-III and X. Adult-type haemoglobins were
not demonstrable in the haemolysates of peripheral blood cells from foetal mice
until 12 days of gestation, unless the foetal liver was injured by careless preparation of the circulating blood cells. Foetal liver cells contained only adult-type
haemoglobins.
Electrophoresis revealed two adult-type haemoglobin bands in the haemolysate of adult C57, s (major component) and Y (minor component); three in
the haemolysate of adult A, dmaj (major component), dmin (minor component)
and Y (minor component); and three in the haemolysate of adult PON, pmaj
(major component), pmin (minor component) and Y (minor component).
Electrophoretically, s was slightly different from dmaj and pmaj, and obviously
different from dmin and pmin. Differences between dmaj and pmaj were not
demonstrable by this technique, but those between dmin and pmin were clearly
20
K. S H I M I Z U A N D T. W A T A N A B E
+
C57
PON
A
C57
C57
PON
A
C57
36(39)
dmin
dmaj
24 42 60(62)
v
_
20
30,
40
50
Fraction number
60
4
I
pmin
, pmaj
16(18)
59
43(44)
Fig. 1. Comparisons of cyanmethemoglobins and fresh haemolysates of adult mice
of three types on electrophoretograms in alkaline starch gels (A), and on chromatograms in alkaline DEAE-Sephadex columns (B). In (B), electrophoretograms of each
peak by chromatography are shown on the right. Each number indicates a fraction
Multiple haemoglobins in mouse ontogeny
21
B
é
C57BL/6J
E-III
E-II
E-I
X
•
»4 ,,
A/He
>dmin
umin
E-II
E-I
X
dmin
dmaj
1
te
dmin
•dmaj
•
13
dmin
^ dmaj
^jdmin
™ dmaj
14
13L
14L
D
PON
i
pmin
pmaj
Y
•
i pmin
dmin
dmaj
pmin
pmaj
Y
I pmaj
3
4
+
C57
PON
Fig. 2. Effects of alkylation on electrophoretograms of haemoglobins in developing
mouse fetuses. (A), Electrophoretograms of adult haemoglobins in mice of three
types. 1 = haemolysate, 2 = haemolysate after treatment with 2-mercaptoethanol,
3 = haemolysate after treatment with iodoacetamide, 4 = haemolysate after treatment with maleic anhydride. (B), Electrophoretograms of embryonic haemoglobins
from 12-day-old foetuses of A/He. (C), Electrophoretograms of haemoglobins from
peripheral blood cells (13 and 14) and liver cells (13L and 14L) of 13- and 14-day-old
foetuses of A/He. Each number indicates days of gestation. (D), Electrophoretograms of cyanmethemoglobins after treatment with maleic anhydride in adult mice
of three types.
detectable. Haemoglobin Y, detectable at the time when adult-type haemoglobins appear in the foetal circulation, always migrated slower anodally than
haemoglobin X. We were unable to demonstrate foetal-type haemoglobins
either in the haemolysates of peripheral blood cells or in the lysate of liver cells
of feotuses with Hbbs (C57), Hbbd (A) or Hbb* (PON).
Electrophoretic behaviours of E-III and pmin of PON were similar. Coelectrophoresis of equal amounts of the haemolysates of PON 12-day-old
foetuses and adults demonstrated that they were electrophoretically indistinguishable from each other. Later experiments in subunit analysis will show
that although they share one polypeptide chain in common, they differ in
another chain.
dmaj
pmaj
C57
pmin
pm
dmin
0
pmaj
dmaj
dmin
Gl
E™!
11
pmaj
1C57
PON
Fig. 3. Some immunological features of each haemoglobin of adult mice of three types by Ouchterlony tests.
C57
dmin
w
W
>
>
H
3
d
>
N
C/3
Multiple haemoglobins in mouse ontogeny
s
pmaj
dmaj
dmin
pmin
E-I
23
E-II E-III
(PON)
« *
-4H*>
E-III E-II
(C57)
E-I
E-I
E-II
(A)
dmaj
E-III
dmin
dmin
pmaj
dmaj
pmin
x
(C57)
j
(A)
Fig. 4. Subunit analysis of each haemoglobin revealed by electrophoresis at pH 1-9
in starch gels. (A), Electrophoretogram of each haemoglobin isolated by electrophoresis in alkaline starch gels. Embryonic haemoglobins were obtained from
12-day-old foetuses of PON. (B) and (C), Electrophoretograms of each haemoglobin
isolated by electrophoresis in alkaline Polyacrylamide gels. Embryonic haemoglobins
were obtained from 12-day-old foetuses of C57BL/6J (B) or A/He (C).
Cyanmethemoglobin form of mouse haemoglobin. Fig. 1A shows that differences between cyanmethemoglobin from the haemolysates of adult A and PON
mice were not always clear in alkaline starch gel electrophoretograms. Fresh
haemolysates from all three types of adult mice sometimes lacked haemoglobin
band Y after Polyacrylamide gel electrophoresis in alkaline buffer which did not
contain KCN, but cyanmethemoglobins from all three types of adult mice
always produced haemoglobin Y, whether or not KCN-containing buffer was
used. After chromatography on DEAE-Sephadex column with a pH gradient,
24
K. SHIMIZU AND T. WATANABE
s
dmaj
dmin
pmaj pmin
E-I
E-II
E'III
X
Fig. 5. Subunit analysis of each haemoglobin revealed by electrophoresis at pH 3-2
in Polyacrylamide gels with 6-25 M urea. Each haemoglobin was isolated by analytical Polyacrylamide gel electrophoresis at alkaline pH. Embryonic haemoglobins
were obtained from 12-day-old foetuses of A/He.
almost no effect of the conversion of adult C57 haemoglobin to cyanmethemoglobin was seen (Fig. IB). On the other hand, with cyanmethemoglobin from
adult A or PON mice, the third peak, which contained almost all the haemoglobin Y, was the highest while with fresh haemolysates, the second peak,
which included almost all the major haemoglobin, was the highest (Fig. 1B).
No effect of the conversion of haemoglobin into cyanmethemoglobin was
detected in the case of 12-day-old C57, A or PON foetuses.
Alkylation of mouse haemoglobin. Electrophoretic mobilities were not
changed when the haemolysates of adult C57, A and PON were treated with
iodoacetamide, but were increased towards the anode when the haemolysates of
adult A and PON were treated with maleic anhydride (Fig. 2 A). Even when
treated with 2-mercaptoethanol (0-1 % in final concentration), haemoglobin Y
was always found electrophoretically in the haemolysates of adult mice, but was
never demonstrable after alkylation with iodoacetamide or maleic anhydride in
electrophoretograms of the haemolysates using the reservoir buffer which did
not contain KCN (Fig. 2 A). However, when KCN-containing buffer was used
in electrophoresis, it was difficult to obtain such obvious results as shown in
Fig. 2 A, and sometimes haemoglobin Y was found. When cyanmethemoglobins
from adult A or PON mice were treated with maleic anhydride, electrophoretic
mobilities of each haemoglobin component were changed, but haemoglobin Y
was always demonstrable (Fig. 2D).
On the other hand, electrophoretic mobilities of the haemolysates from
12-day-old foetuses of C57, A and PON were not altered, and haemoglobin X
was always observed in electrophoretograms after alkylation with iodoacetamide
or maleic anhydride (Fig. 2B). Only adult-type haemoglobins, dmaj or pmaj,
were affected in their mobilities in electrophoretograms of the haemolysates
Multiple haemoglobins
in mouse ontogeny
25
from 13- or 14-day-old foetuses of A or PON, which possess both embryonicand adult-type haemoglobins in their circulation, after alkylation with maleic
anhydride (Fig. 2C).
Ouchterlony tests. One strong precipitation line, which had a tendency to split
into two, was observed between antiserum to adult C57 haemolysate (anti-C57)
and major haemoglobins (adult C57 haemolysate, dmaj or pmaj), but no line
was seen between anti-C57 and minor haemoglobins (dmin and pmin) (Fig. 3 A).
Only one strong line was found between antiserum to adult A haemolysate
(anti-A) and major haemoglobins, while two weak lines were seen between
anti-A and minor haemoglobins; one of the two seemed to be identical to the
line formed between anti-A and major haemoglobins (Fig. 3B). Similarly, in
reactions with anti-serum against adult PON haemolysate (anti-PON), one
strong reactant was detected in major haemoglobins, but two weak reactants
were present in minor haemoglobins (Fig. 3C). Only one precipitation line was
found between antiserum against dmaj (anti-dmaj) or antiserum against pmaj
(anti-pmaj) and major haemoglobins, while no line was present between antidmaj or anti-pmaj and minor haemoglobins (Fig. 3D and E). In reactions with
antiserum to dmin (anti-dmin) or antiserum to pmin (anti-pmin), only one precipitin reactant was detected in minor haemoglobins, and none in major
haemoglobins (Figs. 3 F and G). Unexpectedly, one precipitation line was also
found between anti-dmin or anti-pmin and adult C57 haemolysate, which does
not have a minor haemoglobin (Fig. 3 F and G).
Subunit analysis. Haemoglobins s, dmaj, dmin, pmaj and pmin possessed
common a-chains (Figs. 4A-C and 5); the ^-chains of dmaj and pmaj were
electrophoretically quite alike, although they differed slightly from the /?-chain
of s. The /^-chains of dmin and pmin resembled each other closely, while their
/?-chains differed from those of dmaj and pmaj. Subunit analysis by acid-urea
Polyacrylamide gel electrophoresis was not necessarily suitable for separation
of polypeptide chains of haemoglobin s (Fig. 5), though rather good separations
were obtained in dmaj, dmin, pmaj and pmin. The subunit composition of
haemoglobin Y was indistinguishable from that of s or dmaj or pmaj (Fig. 4C).
As to subunit compositions of embryonic haemoglobins, E-I, E-II and E-III
possessed common a-chains, but their /?-chains were different from each other
(Figs. 4A-C and 5). E-I comprised a- and x-chains, E-II, a- and y-chains, and
E-III, a- and z-chains. The same electrophoretic patterns were obtained from
the haemolysates of 12-day-old C57, A and PON foetuses (Fig. 4A-C). The
subunit composition of haemoglobin X was not distinguishable from that of
E-I (Fig. 4B).
DISCUSSION
Fantoni et al (1967), using CM-cellulose column chromatography with pH
gradient and with 8 M urea, demonstrated the presence of three embryonic-type
haemoglobins in the yolk-sac erythroid cells from 12-day-old C57 foetuses.
26
K. SHIMIZU AND T. WATANABE
Gilman & Smithies (1968) found three embryonic haemoglobins in the haemolysates from 12-day-old foetuses by two-dimensional starch gel electrophoresis.
Recently, Melderis et al. (1974) confirmed the presence of three embryonic
haemoglobins in the peripheral blood cells of 12-day-old BALB/c foetuses
(Hbb^, by the technique of CM-cellulose column chromatography with 8 M
urea. However, Barker (1968) reported the presence of four haemoglobins in the
haemolysates of 12-day-old C57 and C3H/HeAu (Hbbd) foetuses. She designated them haemoglobin bands, 1, 2, 4 and 6 from cathode to anode, which
seemed to correspond to bands E-III, E-II, E-I and X, respectively. Recently,
Schalekamp et al. (1975) have reported the presence of foetal-type haemoglobins
in the peripheral blood cells and liver cells of foetal Porton white Swiss mice
(Hbbd) by CM-cellulose column chromatography. They also described that
band 6 reported by Barker (1968) seemed to correspond to one of their foetal
haemoglobins. Barker (1968) mentioned that only band 6 of the four original
haemoglobins persisted and was maintained throughout adult life, and that
band 6 might be an artifact similar to haemoglobin A3 in the human. As we
shall discuss in subunit analysis of embryonic haemoglobins, haemoglobin X
may be a product of polymerization or aggregation of E-I. Haemoglobin Y also
seemed to be a product of polymerization or aggregation of adult major
haemoglobin. Barker (1968) probably termed both haemoglobins X and Y in
this paper as haemoglobin 6 in her paper. Furthermore, we think it possible
that foetal haemoglobins, F-I and F-II, reported by Schalekamp et al. (1975),
may correspond to haemoglobins X and Y described here.
Conversion of some haemoglobins into cyanmethemoglobin forms is known
to promote extra components (Riggs, Sullivan & Agee, 1964; Riggs, 1965;
Manwell, Baker & Betz, 1966; Schalekamp et al, 1975), although cyanmethemoglobin is considered to be more stable than other haemoglobin forms
in some animals (Hashimoto & Wilt, 1966; Moss & Ingram, 1968). We confirmed here that adult-type haemoglobins of mice in the forms of cyanmethemoglobin had a strong tendency to produce haemoglobin Y, although there is a
tendency to polymerize or aggregate in mouse haemoglobins of 'diffuse' type
(Hbb*) (Riggs, 1965; Bonaventura & Riggs, 1967; Watanabe et al. 1976).
Alkylated agents such as iodoacetamide, iodoacetate and maleic anhydride
prevent polymerization of mouse haemoglobins of 'diffuse' type (Riggs, 1965;
Bonaventura & Riggs, 1967; Petras & Martin, 1969; Hutton, 1969; Watanabe
et al. 1976). Both iodoacetate and maleic anhydride alter the electrophoretic
mobilities of haemoglobins of adult mice with Hbbd and Hbbp, because they
possess free sulphydryl groups (Riggs, 1965; Bonaventura & Riggs, 1967;
Petras & Martin, 1969; Hutton, 1969; Watanabe et al. 1976). After alkylation
of the haemolysates of adult mice, haemoglobin Y was never detectable in
electrophoretograms using the reservoir buffer which did not contain KCN. It
is probable that KCN may promote the production of haemoglobin Y in the
haemolysates of adult mice. Furthermore, haemoglobin Y was always found in
Multiple haemoglobins
in mouse ontogeny
27
electrophoretograms when cyanmethemoglobins from adult mice were treated
with alkylated agents and run. However, electrophoretic mobilities of embryonic
haemoglobins were not altered and haemoglobin X was always detectable in
electrophoretograms even after thorough alkylation. Electrophoretic mobilities
of adult haemoglobins only were altered after alkylation of the haemolysates
of A foetuses, which possess both embryonic and adult haemoglobins. There is
a possibility that embryonic haemoglobins of mice may not have free sulphhydryl groups.
We have succeeded in obtaining antisera against adult-type mouse haemoglobins, and against s, dmaj, dmin, pmaj andpmin. However, the immunological
technique used here was not powerful enough to clarify each haemoglobin fully.
As to subunit compositions of adult haemoglobins, our results were consistent with those described already by Gilman (1974). Haemoglobin Y may be a
product of polymerization or aggregation of a major haemoglobin, since its
subunit composition proved to be indistinguishable. As to subunit compositions
of embryonic haemoglobins, all previous authors have stated that E-I possesses
x- and y-chains, E-II, a- and y-chains, and E-III, a- and z-chains (Fantoni et al.
1967; Gilman & Smithies, 1968; Melderis et al. 1974). Our results have demonstrated, however, that E-I, E-II and E-III possess common a-chains, and their
/Mike chains are different from each other, so we have concluded that E-I
comprises a- and x-chains, E-II, a- and y-chains, E-III, a- and z-chains. As the
subunit composition of haemoglobin X was indistinguishable from that of E-I,
haemoglobin X may be a product of polymerization or aggregation of E-I.
The authors are indebted to Dr W. Ostertag of Max-Planck-Institut für Experimentelle
Medizin for his kind criticism.
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{Received 5 January 1977, revised 11 August 1977)