/. Embryol. exp. Morph. Vol. 29, 3, pp. 503-513, 1973
503
Printed in Great Britain
The influence of 'hypophysectomy'
by means of surgical decapitation on skeletal
growth in the developing chick embryo
By ROBERT C. THOMMES 1 , ANTHONY S. HAJEK
AND D. J. McWHINNIE
From the De Paul University, Chicago
SUMMARY
Wet, dry, and ash weights and calcium content were determined on demarrowed tibiae
of normal and 'hypophysectomized' chick embryos from days 105 to 19-5 of incubation.
All parameters noted above increased progressively through developmental time in normal
embryos. VHypophysectomy' significantly affected bone wet and dry weights, and total
mineral and calcium content. The data indicate that mineralization and matrix synthesis
are retarded in the absence of the pituitary.
INTRODUCTION
In the developing chick embryo, 'hypophysectomy' by surgical decapitation
has been shown to retard growth (Fugo, 1940; Case, 1952; Vogel, 1957, 1965;
Thommes & McCarter, 1966; Betz, 1967, 1968). This retardation may be as
great as 53-55% during the latter half of development (Yogel, 1957, 1965;
Betz, 1967; Thommes & McCarter, 1966). Partial restoration of growth in
'hypophysectomized' embryos has been achieved by transplanting pituitaries
from donor chick embryos onto the chorioallantoic membrane of pituoprivic
hosts of the same age (Thommes & McCarter, 1966; Betz, 1967, 1968). Moreover, Enemar (1967) has demonstrated a growth-promoting influence of chick
embryo pituitaries transplanted into amphibian tadpoles.
Although little specific data on pituitary regulation of skeletal development
exists, it might be anticipated that the generalized effects on growth noted
above include changes in bone growth. Fugo (1940), for example, noted that
limb bones of 'hypophysectomized' embryos appeared shorter than normal
although they maintained their normal proportions. Betz (1968), also, observed
that the 3rd toe length of 'hypophysectomized' animals was 23-26% below
normal by day 20 of incubation, while those of operated embryos bearing
pituitary transplants were only 10-13 % below that of intact controls. More
1
Author's address: Department of Biological Sciences, De Paul University, 1036 W.
Belden Avenue, Chicago, Illinois 60614, U.S.A.
33
E M B 29
504
R. C. THOMMES AND OTHERS
recently, Mehall (1970) reported that 'hypophysectomy' retarded the normal
increase in long bone length and width, and in size of articular-epiphyseal caps,
the zone of cartilage hypertrophy and the epiphyseal plate, and caused periosteal
disorganization. Hypophyseal transplants reduced these skeletal defects in
pituoprivic animals.
The present investigation was undertaken to evaluate the effects of ' hypophysectomy' by surgical decapitation on several other gross parameters of
skeletal growth. These studies include an analysis of matrix, total mineral, and
calcium content in the long bones of normal and 'hypophysectomized' chick
embryos.
MATERIALS AND METHODS
Embryos were obtained from White Leghorn eggs incubated at 38 ± 0-5 °C
in a ' Jamesway' incubator. The age of the embryos at the time of 'hypophysectomy' and sacrifice represents the actual time the eggs remained in the
incubator.
Experimental embryos were 'hypophysectomized' at 33-38 h of incubation
by the partial decapitation method of Fugo (1940). A transverse cut was made
through the mesencephalic portion of the brain and the severed portion removed.
Eyes, upper beak and all other derivatives of the prosencephalon, including
both rudiments of the hypophysis (Rathke's pouch and infundibulum), were
eliminated. Controls consisted of intact embryos.
Embryos were killed at 24 h intervals from 12-5 to 19-5 days of incubation.
Incompletely 'hypophysectomized' animals, characterized by the partial or
complete presence of prosencephalic derivatives, were discarded. Tibiae were
isolated from hind limbs and cleaned of adhering tissue. They were demarrowed
by expressing marrow mechanically through a transverse cut, followed by
flushing the marrow cavity with Ringer's solution under pressure. All bones were
stored in isotonic Ringer's solution at - 2 0 ° C until used, to prevent tissue
deterioration and mineral loss.
After determination of wet weight, bones were dried at 55 °C in a forceddraft oven until constant weight was attained (24 h), and ashed at 500 °C for
4-6 h, or until the gray-ash stage was reached. The residue was moistened with
dilute HNO 3 , dried, and heated at 500 °C until the ash was white (1-2 h). The
mineral residue was dissolved in 1-0 ml of 0-08 M-HCI, and diluted with distilled
water. Total calcium was determined at 422-8 nm on a Beckman DU spectrophotometer with flame attachment.
Statistical methods employed were Analysis of Variance (with log transformations) and the Duncan New Multiple Range Test (Duncan, 1955).
505
Pituitary and chick embryo bone growth
ON
0-6
Total calcium
0-4
11-25
Ash wcisiht
7-50
2-75
Dry weisiht
150
7-5
50
40
Wet weiulit
30
20
10
12-5
13-5
J4o
15-5
16-5
17-5
]S-5
19-5
Days of incubation
Fig. I. Comparison of total calcium, ash, dry and wet weights of demarrowed
bones of control and hypophysectomized chick embryos.
, Control; - - , hypophysectomized.
RESULTS
Bone development in normal chick embryos is characterized by progressive
mineralization and matrix elaboration. From days 12-5 to 19-5, wet, dry and
ash weights and total calcium increase with an elevation noted from days
16-5-19-5 (Table 1, Fig. 1). That rates of mineralization and matrix synthesis
remain similar during development is shown by the stability of the ratios, ash/dry
weight and calcium to ash and dry weights (Table 2, Fig. 2).
VHypophysectomy' markedly influenced bone development. Abnormally low
tibial wet, dry and ash weights, as well as total calcium content, were a con33-2
506
R. C. THOMMES AND OTHERS
Table 1. Mean weights and total calcium content of demarrowed tibiae of
'hypophysectomizeaf' and control chick embryos
Age at
time of
o Q (* f i fi f*r*
Parameter
Total calcium (mg)
Ash weight (mg)
Dry weight (mg)
Wet weight (mg)
sacrince
(days)
12-5
13-5
14-5
15-5
16-5
17-5
18-5
19-5
12-5
13-5
14-5
15-5
16-5
17-5
18-5
19-5
12-5
13-5
14-5
15-5
16-5
.17-5
18-5
19-5
12-5
13-5
14-5
15-5
16 5
17-5
18-5
19-5
Control
r
Mean±s.E.M.
(10)* 0040 ± 0003
(10) 0086 ± 0006
03) 0160 ± 0 0 1 5
(8) 0-237 ± 0020
(20) 0-273 ± 0 0 1 7
(12) 0-427 ± 0004
(11) 0-529 ± 0 0 1 4
(10) 0-657 ± 0 0 6 3
' Hypophysectomized'
r
Mean±s.E.M.
(9)=* 0045±0005
(ID 0080 ±0007
(13)
(14)
(8)
(13)
(8)
(4)
(9)
(9)
0-4 ± 0 1
1-7 ±0-1
(M)
2-4 ± 0 1
(13)
(8)
(19)
3-1 ±0-3
3-6±0-2
6-7 ±0-4
9-3 ±0-5
11-8 ± 1 1
0-9 ± 0 1
3 0 ±0-2
5O±O-3
6-1 ±0-6
7-1 ±0-3
12-7 ±0-6
19 6 ± 1 0
22-2 ±2-2
2 0 ±0-3
7-9 ±0-7
12-7 ±1-1
12-5 ±1-9
15-6 ±1-1
28-6 ±1-9
42-1 ±3-5
46-5 ±3-7
(14)
OD
(ID
(9)
(8)
(10)
(12)
(9)
(19)
(13)
(9)
(10)
(9)
(9)
(11)
(8)
(19)
(12)
(10)
(8)
(9)
OD
(9)
(14)
(8)
(4)
(10)
(12)
(12)
(13)
(9)
(14)
(8)
(4)
(8)
(12)
(13)
(13)
(8)
(13)
(8)
(4)
0111 f ±0008
0-140f±0-010
0-257 ±0010
0-359$ ±0-020
0-327f± 0-054
0-513f± 0-063
0-3 ±01
0-9 ± 0 1
l-2J±01
l-7f±0-2
2-4J±0-3
5-1 f± 0-5
5-2f±0-4
8-0f±l-1
1-2 ±0-1
2-3 ±0-2
2-8f±0-2
3-6f±0-4
5-4J±0-6
10-3t±10
8-5f±0-7
14-1 f± 1-7
3-5f±O-5
6-4J±0-9
7-9f±0-7
10-9±0-8
144 + 0-8
19-7f±l-9
17-2f±l-5
29-4f±4-5
* Number of samples.
t Statistically significant at P < 001.
t Statistically significant at P < 005.
sistent consequence of this operation. By day 18-5, growth retardation was 56 %
when the dry mass of tibiae from operated embryos was compared with that
of controls. Ash weights were significantly depressed by day 16-5. While all
weight values of bones from 'hypophysectomized' animals slightly increased on
day 17-5, on days 18-5 and 19-5, the experimental values were again much lower
(P < 0-01) than those of intact embryos (Table 1, Fig. 1). Total calcium content
Pituitary and chick embryo bone growth
507
18
•S 12
u
.op
4
60 r
.2P 40
20
12 5
13-5
14-5
15-5
16-5
17-5
18-5
19-5
Days of incubation
Fig. 2. Comparison of the percentages of calcium to ash and dry weight and the
percentages of ash to dry weights of demarrowed bones of control and hypophysectomized chick embryos.
, Control; - - , hypophysectomized.
was also very low; the reduction was statistically significant by day 14-5, and
was 39-2% below controls by 18-5 days. These data indicate that lack of
pituitary influence during the latter half of development causes a general retardation in bone growth.
In addition to the abnormally slow bone growth in the absence of the hypophysis, an influence on specific skeletal compartments was noted. In 12-5- to
14-5-day tibiae, the amount of calcium relative to total bone mineral (ash) was
elevated (P < 0-01) above that of intact embryos. However, by day 15-5 the
calcium/ash ratio approached control levels, and paralleled the normal pattern
through the remainder of the incubation periods studied (Table 2, Fig. 2). These
data indicate that during early ossification in pituoprivic embryos, there may
be an abnormal balance of mineral ions available for bone crystal formation.
508
R. C. THOMMES AND OTHERS
Table 2. Mean values of calcium per dry and ash weights and ash per dry weights of
demarrowed tibiae of' hypophysectomized' and control chick embryos
(Control
Age at time
' Hypophysectomized'
A
Parameter
(days)
Samples
Mean±s.E.M.
Samples
Mean±s.E.M.
% Ca/ash
12-5
13-5
14-5
15 5
16-5
17-5
18-5
19-5
12-5
13-5
14-5
15-5
(9)
(10)
(13)
(8)
(21)
(11)
(11)
(10)
(9)
(JO)
(U)
(6)
(ID
(13)
(14)
(9)
(14)
(9)
(4)
(8)
(10)
(13)
(14)
16-7* ±2-2
8-9* ±1-5
9-2* ±0-5
91 ±0-6
8-4 ±0-4
71 ±0-3
6-2 ±0-5
6-4 ±0-7
4-3* + 0-5
16-5
17-5
18-5
19-5
(21)
(13)
(11)
(10)
12-5
13-5
14-5
15-5
16-5
17-5
18-5
19-5
(7)
(10)
(12)
(8)
(21)
(.10)
(U)
(10)
ll-2±0-8
5-5 ±0-4
6-9 ±0-5
7-6 ±0-5
7-4 ±0-4
6-5 + 0-2
5-7 ± 0 1
61 ±0-3
51 ±0-5
2-9 ±0-1
3-5 ±0-1
3-7 + 0-1
3-8 ± 0 1
3-5±01
2-9 ±0-1
30 ± 0 1
42-3 ±5-3
54-3 ±2-9
49-8 ±1-1
48-4 ± 1 - 5
51-5 ±0-1
52-4 ±1-7
50-8 ±0-9
49-5 ±1-8
% Ca/dry
% ash/dry
(8)
(10)
(12)
(8)
(4)
(7)
(ID
(13)
(14)
(10)
(14)
(9)
(4)
3-6f±0-5
3-8±01
3-8 + 01
3-9 ±0-4
3-3±O-l
3-8*±O-l
3-7f±0-2
23-7* ±2-3
39-4* ±1-8
42-4f±l-5
42-6f±l-7
44-0f±20
49-6 ±0-8
62-6* ± 1 0
57-1 f ± 0-8
* Statistically significant at P < 001.
t Statistically significant at P < 005.
The calcium/dry weight ratio in bones of 'hypophysectomized' animals was
slightly but consistently greater than that of controls from days 13-5 to 16-5,
and was significantly higher the last few days of development. In contrast, the
ash/dry weight ratio was lower than normal (P < 0-05) in bones of experimental
animals from days 12-5 to 16-5. By day 18-5, however, this relationship had
reversed, and the ratio became significantly greater (P < 0-05) than that of
normal embryos (Table 2, Fig. 2).
DISCUSSION
Long-bone growth in the embryonic chick is accompanied by progressive
elevations in bone mass, as demonstrated by wet, dry, ash and calcium determinations. 'Hypophysectomy' results in abnormal bone development; during
the last half of incubation the long bones of pituoprivic embryos were significantly lower in wet, dry, ash, and calcium values than controls.
Pituitary and chick embryo bone growth
509
Since dry weight includes both mineral and matrix phases of bone, the lower
dry weights of tibiae from ' hypophysectomized' embryos might indicate that
either mineralization and/or matrix synthesis have been impeded. Total mineral
and calcium content are less than normal, indicating that bone crystal deposition
is retarded in the operated embryos; matrix synthesis is also inhibited. While
normal tibiae increased 35 % and 43 % in calcium and dry weight respectively
(a ratio less than 1) from days 17-5-19-5, those of experimental animals only
increased 30 % and 27% (a ratio greater than 1). 'Hypophysectomy' thus results
in a failure of bone mass to increase normally as a consequence of reduced
mineralization and matrix elaboration.
An analysis of ratios between bone compartments reveals that they are differentially affected by 'hypophysectomy'. From days 12-5 to 14-5, bone calcium
is greater than normal, even though total mineral is reduced. This change in the
calcium/mineral ratio may be due to either an influx of calcium to the mineral
compartment, or depletion of other mineral components in bone crystal. This
unbalanced mineral content returns to normal the last week of development.
When the calcium/dry weight ratio is examined it must be recognized that
dry weight includes both mineral and matrix. If, as noted above, calcium increases in operated embryos on days 12-5-14-5, an elevation in the calcium/dry
weight ratio might be anticipated. This was observed; the lack of statistical
significance may be due to masking of small changes in calcium by the larger
amount of matrix present. Subsequently, as matrix synthesis becomes more
limited, the calcium/dry weight ratio is elevated. Similar interpretations may be
drawn from an analysis of the ash/dry weight ratio.
Whether the nature of the pituitary influence on bone development is via the
gland itself, or through trophic hormones, is not established. However, events
related to several pituitary-endocrine axes correspond in time to the bone
changes observed in surgically decapitated embryos. 'Hypophysectomy' modifies
the following adrenal parameters between days 10 and 14: (1) ultrastructural
differentiation and A5-3/?-hydroxysteroid dehydrogenase levels (Straznicky,
Hajos & Bonus, 1966); (2) adrenal ascorbic acid content (Case, 1952); (3) cortical
cord development and free cholesterol levels (Adjovi, 1970); (4) allantoic fluid
levels of corticosteroids (Woods, DeVries & Thommes, 1971). Moreover, stimulation of ACTH release by use of corticosteroid synthesis inhibitors leads to an
initial hypertrophy of the pituitary (Stoll, Faucounau & Maraud, 1964) and
decreased conversion of cholesterol to adrenal steroids (Adjovi & Eidelman,
1969) on day 12. Whether or not establishment of the pituitary-adrenal axis
during days 10-14 relates to bone changes following 'hypophysectomy' is
problematical, since glucocorticoids may arrest skeletal growth and ossification
(Karnofsky, Ridgway & Patterson, 1951; Buno & Goyena, 1955; Siegel, Smith
& Gerstl, 1957; Sobel & Freund, 1958; Reynolds, 1966; Badran & Provenza,
1969); in their probable absence in this study, similar results occurred.
The day 10-14 period is also critical in thyroid development. At this time,
510
R. C. THOMMES AND OTHERS
thyroxin is present in relatively large amounts and iodide uptake sharply increases (Trunnell & Brayer, 1953; Trunnell & Wade, 1955); Vhypophysectomy'
eliminates the latter (Mess & Straznicky, 1970). Moreover, the pituitarythyroid axis may be established at this time, i.e. pituitary grafts to the chorioallantoic membrane now stimulate the thyroid in ' hypophysectomized' embryos
(Studitskii, 1946), and conversely, thyroid grafts now respond to pituitary TSH
(Martindale, 1941). Additionally, the thyroids of' hypophysectomizedn embryos
are arrested in growth at this time (Fugo, 1940), and retain the vascular pattern
characteristic of younger embryos (Thommes, 1958). Electron-microscopic data
also indicate that thyroidal fine-structure becomes pituitary-dependent about
day 11 (Hajos, Straznicky & Mess, 1964).
Thyroid hormones are known to influence skeletal development. Fell &
Mellanby (1955, 1956), Fell, Galton & Pitt-Rivers (1958) and Lawson (1961 a, b)
have shown that lack of, or presence of, thyroid hormones depresses or stimulates, respectively, growth, differentiation, ossification and matrix formation in
chick embryo bones. Moreover, Lengemann (1962) has demonstrated that incorporation of radiocalcium in embryonic chick tibiae is inhibited by thyroxin.
Therefore growth, matrix synthesis, mineralization, and mineral balance may be
regulated by thyroid hormones in vivo. Such an analysis allows interpretation of
the present data, since the pituitary-thyroid axis appears to be established during
that time when 'hypophysectomy' first influences bone development.
The possibility that pituitary somatotrophin (STH) may regulate bone growth
in the embryonic chick cannot be excluded. As reported by Ito, Takamura &
Endo (1959, 1960), the addition of mammalian STH to bone organ cultures
increases bone length and sulfur uptake into matrix. STH also increases wet
and dry weights and total nitrogen of embryonic bone in vitro (Hay, 1958),
elevates the mitotic index and bone thickness (Blumenthal, Hsieh & Wang,
1954), and alleviates cortisone-induced skeletal teratologies in vivo (Sobel, 1958).
In contrast, other studies indicate that growth hormone may have little influence
on avian bone development. For example, Chen (cited by Fell, 1954, 1955 and
Hay, 1958) could detect no change in bone length with STH treatment in vitro,
and Vogel (1965) could not restore depressed growth rates of 'hypophysectomized' embryos to normal with an undefined bovine growth 'factor'. Additionally, it is not certain that the avian pituitary possesses a growth hormone comparable to that found in mammals, although Enemar (1967) observed a 'growth
hormone effect' in tadpoles receiving transplants of the caudal region of the
embryonic chick pars distalis. The role of STH in avian embryonic growth and
bone elaboration is thus unclear: (1) since there is uncertainty as to the presence
or absence of STH in birds and (2) since a lack of response in the chick embryo
to non-avian STH's may be a result of the species-specific nature of growth
hormones. It is also unclear whether the striking effects of 'hypophysectomy'
in the chick embryo are a result of the cleidoic nature of the avian egg, for
in the relatively 'open' maternal-fetal relationship of mammals, 'hypophy-
Pituitary and chick embryo bone growth
511
sectomy' appears to have little effect on growth (see McWhinnie & Thommes,
1973).
The role of the hypothalamus in adenohypophyseal regulation of embryonic
systems remains problematical. Some data on the chick embryo indicate that
in the absence of the hypothalamus, adenohypophyseal function is maintained;
for example, pituitaries transplanted to 'hypophysectomized' embryos (no
hypothalamus present) produce sufficient TSH to cause an abrupt rise in thyroidal
iodide-trapping (Mess & Straznicky, 1964); ACTH secretion may also be independent of the hypothalamus (Betz, 1967; Woods et al. 1971). However,
these data are as yet inconclusive, and it is unknown whether secretion of different adenohypophyseal hormones may be independent of, or partially or
totally dependent upon, hypothalamic release factors. Hence, the possibility
that normal bone development in the chick embryo may require a functional
hypothalamic-adenohypophyseal relationship cannot be excluded.
In conclusion, this investigation indicates that the pituitary gland (or hypothalamus-pituitary complex) is essential for skeletal growth and development
in the chick embryo. In its absence, bone matrix synthesis is severely retarded
and a lag in bone crystal deposition occurs. Whether the nature of this pituitary
influence is direct or through its trophic hormones remains to be elucidated.
This investigation was supported in part by Training Grant 5-TO1-HD00293 from the
National Institute of Child Health and Human Development, and the Brown-Hazen Fund
of the Research Corporation of America.
REFERENCES
Y. (1970). Morphogenese et activite de la glande cortico-surrenale de Pembryon de
Poulet normal et decapite. Archs Anat. microsc. Morph. exp. 59, 185-200.
ADJOVI, Y. & EIDELMAN, S. (1969). Action d'un inhibiteur de la sterodio-genese, l'elipten,sur
la cortico-surrenale de Pembryon de poulet (Leghorn blanc). C. r. Seanc. Soc. Bio/. 163,
2588-2593.
BADRAN, A. & PROVENZA, D. V. (1969). Studies of the growth-inhibitory action of cortisone
on chick embryos. Teratology 2, 221-233.
BETZ, T. W. (1967). The effects of embryonic pars distalis grafts on the development of
hypophysectomized chick embryos. Gen. comp. Endocr. 9, 172-186.
BETZ, T. W. (1968). The effects of embryonic pars distalis grafts and albumen on the growth
of chick embryos. /. Embryol. exp. Morph. 20, 431-436.
BLUMENTHAL, H. T., HSIEH, K.-M., & WANG, T.-Y. (1954). The effect of hypophyseal growth
hormone on the tibia of the developing chick embryo. Am. J. Path. 30, 771-785.
BUNO, W. & GOYENA, H. (1955). Effect of cortisone upon growth in vitro of femur of the chick
embryo. Proc. Soc. exp. Biol. Med. 89, 622-625.
CASE, J. F. (1952). Adrenal cortical-anterior pituitary relationships during embryonic life.
Ann. N.Y. Acad. Sci. 55, 147-158.
DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, 1—42.
ENEMAR, A. (1967). Ontogeny of the hypophyseal growth-promoting activity in the chick.
/. Endocr. 37, 9-15.
FELL, H. B. (1954). The effect of hormones and vitamin A on organ cultures. Ann. N.Y.
Acad. Sci. 58, 1183-1187.
FELL, H. B. (1955). The effect of hormones on differentiated tissues in culture. In The
Hypophyseal Growth Hormone, Nature and Actions (ed. R. W. Smith Jr., O. H. Gaebler
& C. N. H. Long), pp. 138-148. New York: McGraw-Hill.
ADJOVI,
512
R. C. THOMMES AND OTHERS
H. B., GALTON, V. A. & PITT-RIVERS, R. (1958). The metabolism of some thyroid
hormones by limb-bone rudiments cultivated in vitro. J. Physiol., Lond. 144, 250 256.
FELL, H. B. & MELLANBY, E. (1955). The biological action of thyroxin on embryonic bones
grown in tissue culture. /. Physiol., Lond. 127, 427-447.
FELL, H. B. & MELLANBY, E. (1956). The effect of L-triiodothyronine on the growth and
development of embryonic chick limb-bones in tissue culture. J. Physio/., Lond. 133, 89100.
FUGO, N. W. (1940). Effects of hypophysectomy in the chick embryo. J. exp. Zool. 85, 271 297.
FELL,
HAJOS, F., STRAZNICKY, K. & MESS, B. (1964). The effect of TSH on the ultrastructure of
embryonic chick thyroid. Acta biol. Acad. Sci. hung. 15, 237-249.
HAY, M. F. (1958). The effect of growth hormone and insulin on limb-bone rudiments of the
embryonic chick cultivated in vitro. J. Physiol., Lond. 144, 490-504.
ITO, Y., TAKAMURA, K. & ENDO, H. (1959). The stimulative effect of pituitary growth
hormone on a5S-sulfate incorporation in the chick embryo femur in tissue culture. Endocr.
jap. 6, 68-69.
ITO, Y., TAKAMURA, K. & ENDO, H. (1960). The effect of growth hormone on the incorporation of labeled sulfate into chick embryo femur in tissue culture. Endocr. jap. 7, 327-335.
KARNOFSKY, D. A., RIDGWAY, L. P. & PATTERSON, P. A. (1951). Growth-inhibiting effect of
cortisone acetate on the chick embryo. Endocrinology 48, 596-616.
LAWSON, K. (1961O). The differential growth response of embryonic chick limb-bone rudiments to triiodothyronine in vitro. I. Stage of development and organ size. J. Embryol.
exp. Morph. 9, 42-51.
LAWSON, K. (19616). The differential growth response of embryonic chick limb-bone rudiments to triiodothyronine in vitro. II. Growth rate. /. Embryol. exp. Morph. 9, 534-555.
LENGEMANN, F. W. (1962). Effects of thyroxin upon strontium and calcium metabolism of
embryonic bone grown in vitro. Endocrinology 70, 774-780.
MARTINDALE, R. M. (1941). Initation and early development of thyrotropic function in the
incubating chick. Anat. Rec. 79, 373-393.
1
MCWHINNIE, D. J. & THOMMES, R. C. (1973). The influence of 'hypophysectomy on bone
growth and alkaline phosphatase activity in the chick embryo. J. Embryol. exp. Morph. 29,
515-527.
MEHALL, A. G. (1970). Capacity of anterior pituitary grafts to correct modified growth and
development of selected long bones of hypophysectomized chick embryos. Ph.D. Dissertation,
University of Illinois.
MESS, B. & STRAZNICKY, K. (1964). Functional differentiation of the thyroid gland investigated
by ml-autoradiography in decapitated chick embryos bearing hypophyseal homografts.
Acta biol. Acad. Sci. hung. 15, 77-85.
MESS, B. & STRAZNICKY, K. (1970). Differentiation and function of the hypophyseal-target
organ system in chicken embryos. Stud. Biol. Hung. 9, 21—45.
REYNOLDS, J. J. (1966). The effect of hydrocortisone on the growth of chick bone rudiments in
chemically defined medium. Expl Cell Res. 41, 174-189.
SIEGEL, B. V., SMITH, M. J. & GERSTL, B. (1957). Effects of cortisone on the developing chick
embryo. A.M.A. Archs Path. 63, 562-570.
SOBEL, H. (1958). Antagonistic effects of cortisone and growth-hormone on the developing
chick embryo. Proc. Soc. exp. Biol. Med. 97, 495-498.
SOBEL, H. & FREUND, O. (1958). The action of cortisone on the embryonic cartilage and
muscle in vitro. Experientia 14, 421-422.
STOLL, R., FAUCOUNAU, N. & MARAUD, R. (1964). Nouvelles donnees sur la fonction sur-
renalienne de l'embryon de Poulet, etudiee a l'aide de la metopirone (Su 4.885). C. r.
Seanc. Soc. Biol. 158, 1866-1888.
STRAZNICKY, K., HAJOS, F. &BOHUS, B. (1966). Relationship between the ultrastructure and
cortical activity of the embryonic adrenal gland in the chicken. Acta biol. Acad. Sci. hung.
16, 261-274.
STUDITSKII, A. N. (1946). Endocrine correlation in the embryonal development of the
vertebrates. Nature, Lond. 157, 427-430.
Pituitary and chick embryo bone growth
513
THOMMES, R. C. (1958). Vasculogenesis in selected endocrine glands of normal and hypophysectomized chick embryos. I. The thyroid. Growth 22, 243-264.
THOMMES, R. C. & MCCARTER, C. F. (1966). Adenohypophyseal control of water balance in
the developing chick embryo. Am. Zool. 6, 517.
TRUNNELL, J. B. & BRAVER, F. T. (1953). Factors governing the development of the chick
embryo thyroid. I. Determination of the time at which I.131 collection begins. J. clin.
Endocr. Metab. 13, 88-94.
TRUNNELL, J. B. & WADE, P. (1955). Factors governing the development of the chick embryo
thyroid. II. Chronology of the synthesis of iodinated compounds studied by chromatographic analysis. / . clin. Endocrinol. Metab. 15, 107-117.
VOGEL, N. W. (1957). Free tissue cholesterol and growth in chick embryos hypophysectomized
by 'decapitation'. Anat. Rec. 127, 382.
VOGEL, N. W. (1965). Growth in chick embryos hypophysectomized by 'decapitation'. Proc.
Perm. Acad. Sci. 39, 57-60.
WOODS, J. E., DEVRIES, G. W. & THOMMES, R. C. (1971). Ontogenesis of the pituitary-adrenal
axis in the chick embryo. Gen. comp. Endocr. 17, 407-415.
(Received 18 July 1972, revised 15 November 1972)