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/. Embryol. exp. Morph. Vol. 22, 1, pp. 99-105, August 1969
99
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The effect of thyroid function on hemoglobin
differentiation in the chick
By ROBERT W. ATHERTON
Department of Physiology, University of California, Berkeley
The switching off of the synthesis of embryonic hemoglobin (EHb) and the
turning on of adult hemoglobin (AHb) synthesis has served as one of several
major examples of biochemical differentiation. However, the control mechanisms
underlying this transformation remained elusive, except in the case of Rana
catesbeiana (Moss & Ingram, 1965, 1968 a, b) where the classical thyroxineinduced metamorphosis effected a precocious shift from EHb to AHb. In the
chick embryo such a dramatic metamorphosis does not occur, but a change
from EHb to AHb can be detected electrophoretically on the sixth day of
incubation (Hashimoto & Wilt, 1966; Manwell, Baker & Betz, 1966).
Normal thyroid development can be seen on the second day in the chick
embryo, appearing as a medial outgrowth from the pharyngeal floor. By the
sixth day, when AHb is first recognized electrophoretically, the thyroid concentrates 131I 500 times more than does muscle (Olonoski, Lengeman & Comar,
1960). At 7 days, but not before, TSH has been detected in vitro (Tixier-Vidal,
1956). On the eighth day, yolk-sac absorption can be inhibited by injection of
100 /ic of131!, and 50 JLLC of 131I will delay hatching and significantly reduce body
and thyroid uptake of isotope (Maraz, 1966). By 8 days, anterior pituitary
tissue placed on the chorioallantoic membrane causes an accelerated thyroid
morphogenesis (Studitskii, 1938), indicating the presence of biochemical interrelationships. Thus, in the chick, there is evidence that the thyroid-pituitary axis
is anatomically developed and biochemically active during and shortly following
the switch from EHb to AHb. Experimentally it has been shown that the chick
embryo, hypophysectomized at 33-36 h of development, exhibited a normal
switch from embryonic to adult hemoglobin (Manwell & Betz, 1966). It would
appear that the lack of embryonic TSH does not affect the differentiation of
hemoglobin. However, evidence is available that egg yolk has a thyroid stimulating effect that increases the weight, function and histological structure of the
gland (Tsuji, 1922).
In the present study, electrophoretic patterns of hemoglobin and erythrocyte
morphology were investigated in the chick rendered hypo- or hyper-thyroid by
1
Author's address: Department of Physiology-Anatomy, University of California,
Berkeley, California 94720, U.S.A.
7-2
100
R. W. ATHERTON
the administration of thyroxine, 131I, thiourea, and tapazole (l-methyl-2mercaptoimidazole). Because of the close functional relationship between thyroid activity and growth hormone, the effect of growth hormone on hemoglobin
differentiation was also investigated.
MATERIALS AND METHODS
Incubation was started within 3 days after receipt of eggs of the species,
G. gallus, from Kimber Farms in Niles, California. Montgomery Ward incubators were maintained at 37-7 °C and 80 % relative humidity. Thyroxine and
growth hormone were injected into the air sac according to the method of
Romijn, Fung & Lokhorst (1952). This method was chosen for several reasons.
First, by 8 days in development, this route will allow both thyroxine and thiourea
to reach the embryo and affect embryonic respiration (Romijn et al. 1952). By
this time, vascularization is sufficiently established to allow diffusion and
absorption to facilitate transport. That compounds may reach the embryo
before vascularization is complete is shown by the finding that propylene
glycol (0-05 ml), injected from day zero to day 7 of incubation, results in a high
incidence of mortality. Up to the fourth day when vascularization is incomplete,
mortality increases rapidly (> 90%) and falls by day 7 (~ 30%) (Gebhardt,
1968). Thyroxine and thiourea, when injected into the air sac prior to incubation,
have significant effects on the thyroid of newly hatched chicks (McCartney &
Shaffner, 1949). In the present research, pilot studies indicated that doses of
thyroxine (30 /^g/005 ml of saline) increased mortality (40 %) over control
figures. The minimal dose at which thyroxine was effective was determined to be
2-5/*g/0-05ml.
To induce hypothyroidism, 131I, thiourea, and tapazole were injected by the
same route as the two hormones; the doses injected are indicated in Table 3.
Single injections were administered at 24, 48, 72, and 96 h. incubation and
multiple injections were given from day 3 through day 8. A minimum of twelve
embryos was used in each control and experimental group.
Blood smears were stained with Wright's Stain and also phosphate buffer
(pH = 6-4). Five hundred cells from each embryo were catalogued according to
the number of erythroblasts, mitotic figures, and the distribution (percentage) of
polychromatic erythrocyte cell types (Lucas & Jamroz, 1961). Electrophoresis of
the cyanomethemoglobin derivative was carried out on starch gel (Manwell,
1963).
The role of thyroid hormones in differentiation of other systems was determined indirectly by administering 50 /.ic of 131I at 9 days and measuring body
and brain weights at 19 days, since total body growth and brain development
have been considered useful parameters of thyroid function in the developing
rat (Geel & Timiras, 1967).
Chick hemoglobin differentiation
101
RESULTS
Body and brain weights were reduced by early injections of 131I (Table 1),
indicating that a state of hypothyroidism was effectively induced. The electrophoretic diagram of the hemoglobin bands presented in Table 2 was adapted from
Table 1. Effects of early injections o/ 1 3 1 / on total body and brain
weight at 19 days of incubation
Dry brain weight (mg)*
Wet body weight (g)
Control
Experimental
0086±0005
211 ±2-4
0072±0006f
J7-7± l-7f
* Dried at 105 °C, 4 days.
t / test: P = 0-05
Table 2. Diagrammatic distribution of chick (embryo and adult)
hemoglobins on starch-gel electrophoresis *
Hashimoto & Wilt (J966), PH
Embryo
=
Manwell, Baker & Betz (1966), pH = 8-5
8-6
Embryo
Adult
Adult
+
A
X
Minor adult
X X X X X
X X X X X X
X X X X X
X X X X X X
Ex
X X X X X X
E2
X X X X X X
E3
4'
A2f
A2s
x x x x x x
Major anodal
x x x x x x
Major cathodal
X X X X X
xxxxx
Major adult
A3
x x x x x x
Minor
Width of bands not indicative of proportions present
the literature. For comparative purposes, these results are incorporated in Fig. 1,
together with the results of the present experiments. The various hormonal
treatments—thyroxine (1, 2, 3 and 4 days), growth hormone (3 and 4 days), and
thyroxine plus growth hormone (3 and 4 days)—did not alter the electrophoretic
patterns, indicating that apparently endocrine influences have not induced a
precocious shift to AHb. Further, single injections of 131I and thiourea did not
prolong the presence of EHb beyond 5 days of development. Because it is conceivable that the number and type of cells might change before any differences in
electrophoretic patterns could be detected, it was decided to employ the more
102
R. W. ATHERTON
sensitive method of establishing the mitotic index of dividing blood cells, the
number of erythroblasts, and the differential counts of the polychromatic
erythrocyte series (Table 3). Endocrine treatment did not alter significantly (by
the t test) the mitotic index; some increase of mitotic activity was noted
after growth hormone and thyroxine, but not after the combined treatment.
When the mean number of erythroblasts decreased the mitotic index increased—
an inverse ratio that is not expected. Because of the slight changes observed in
Minor adult
E,
E,
Major adult
E3
1 cm
T4
1
T4
2
T4
3
A
C
5D
5D
5D
5
GH
T
GH
Fig. 1. Electrophoretic patterns of adult chickens, hormone-injected embryos, and
controls. A = adult. C5 = 5 day control.
T —T
4
* = T4 injected on day 1-3; sampled day 5.
Gri, T,
CJH.
= T4 or growth hormone injected on day 3; sampled day 5.
mitotic indices and the number of erythroblasts at 5 days, a more detailed
differential count of primary, middle and late polychromatic erythrocytes was
done at 9 days, when the erythrocytes are more distinguisahble. It was confirmed
that there is a distinct distribution (percentage) of the cell types as previously
reported (Lucas & Jamroz, 1961), but no significant effect of stimulation or
inhibition of erythropoiesis by thyroid agents was observed (Table 4).
In another series of experiments, multiple injections of thyroxine and tapazole
were administered (daily doses of 0-09/*g/005 ml) covering the entire critical
Chick hemoglobin differentiation
103
period of hemoglobin differentiation, that is, between 3 and 8 days. As in the
case of single injections, the multiple injections did not modify the date of
appearance of AHb differentiation.
Table 3. Hormone and anti-thyroid treatment in chick
embryos and embryonic blood response
Treatment
Saline
Growth hormone
Thyroxine
Thyroxine and
growth hormone
Thiourea
Dose*
(/</0-05 ml)
Time
sampled
5
5
5
5
1000
2-5
2-5 + 1000
3000
2000
50/<ct
days
days
days
days
5 days
5 days
Daily, 5-19 days
Hb
type
EHb
EHb
EHb
EHb
Mitotic Mean no. of
index erythroblasts
7-2
24-5
19-8
5-2
191
10-2
12-6
21-3
EHb
EHb
AHb
* Given at 24 h of incubation.
f Given at 9 days of incubation
Table 4. Distribution of blood cell types in the 9-day-old embryo
{expressed as percentage)
Polychromatic cell types*
Treatments
Primary
Middle
Late
Control
Thyroxinej
Tapazole
17-6±2-6
14-5 ±1-0
14-0 ±1-8
19-3±2-2
17-8 ± 2 0
200 ±1-5
62-8±2-5
65-9 ± 1 0
65-5 ±2-0
* Each cell type was significantly different (t test; P = 005) from one another, but a given
cell type within different treatment groups was not.
t Dose: 009/<g/005 ml in saline.
DISCUSSION
Contrary to the data reported for R. catesbeiana, thyroxine treatment did
not alter the electrophoretic pattern of hemoglobin differentiation in G. gallus.
Various morphological parameters of blood differentiation similarly showed no
changes consequent to thyroxine and growth-hormone treatment. These findings
indicate that erythropoietic tissue is not competent to react to these hormones
during thyroid morphogenesis in this species, although the literature and the
present research suggest that the thyroid is beginning to function at the time of
hemoglobin differentiation. It is suggested that some other mechanism, probably
not directly involving the thyroid or other endocrines, is responsible for the signal
to alter this particular protein synthesis. It would be of interest to study embryonic blood cells, in vitro without yolk, in order to elucidate further the mechanism
by which hemoglobin differentiation occurs.
104
R. W. ATHERTON
SUMMARY
Function of the thyroid gland in both the tadpole and the chick has been
reported early in development. In the tadpole, early thyroid treatment has been
shown to induce precocious differentiation of hemoglobin. The present study
was conducted to ascertain the role of the thyroid in the same transformation
(from embryonic (EHb) to adult (AHb) hemoglobin) in the chick. At various
periods prior and during the time of normal synthesis of AHb (6 days), thyroxine, 131I, thiourea, or tapazole were injected into the air sac. Several erythropoietic parameters were measured at 5, 6, 7, 8, and 9 days to determine the
thyroid effect. Because of the known relation between growth hormone and
thyroxine during development, growth hormone was administered both separately and in conjunction with thyroxine, but was found to have no effect on
erythropoietic differentiation. The results demonstrate that despite the presence
of thyroid function in the chick in early embryonic stages, hemoglobin differentiation, the number of erythroblasts, mitotic figures, and differential counts do not
depend upon thyroid function in this species as in R. catesbeiana. It is suggested
that the mechanism(s) that trigger hemoglobin differentiation in the chick are
probably non-endocrine in origin.
RESUME
Veffet de la fonction thyroidienne sur la differentiation de
l'hemoglobine chez le Poulet
La fonction de la glande thyroide a ete etudiee sur les jeunes stades de
developpement du Tetard et du Poulet. Chez le Tetard, un traitement precoce de
la thyroide a entraine une differenciation precoce de l'hemoglobine. Le present
travail a ete effectue pour determiner le role de la thyroide sur la meme transformation (depuis l'hemoglobine embryonnaire (EHb) jusqu'a l'hemoglobine
adulte (AHb)) chez le Poulet. A differents moments, avant et pendant la peroide
de synthese normale de l'AHb (6 jours), on a injecte, dans la poche d'air, de la
thyroxine, 131I, thiouree ou du tapazole. Differents parametres erythropoietiques
ont ete mesures apres 5, 6, 7, 8 et 9 jours pour determiner l'effet de la thyroide.
Etant donne la relation connue qui existe entre l'hormone de croissance et la
thyroxine pendant le developpement, l'hormone de croissance a ete administree
separement et simultanement avec la thyroxine, mais n'a jamais eu d'effects sur la
differenciation erythropoietique. Les resultats montrent que malgre la presence
de fonction thyroidienne dans l'embryon a de jeunes stades embryonnaires, la
differenciation de l'hemoglobine, le nombre des erythroblastes, les figures
mitotiques et les comptages differentiels ne dependent pas de la fonction thyroidienne ni chez le poulet ni chez Rana catesbeiana. II est suggere que le (ou les)
mecanismes qui font demarrer la differenciation de l'hemoglobine chez le
poulet, sont d'origine non-endocriniennes.
Chick hemoglobin differentiation
105
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{Manuscript received 30 September 1968, revised 6 December 1968)