Wet weights, total bone alkaline phosphatase activity and specific alkaline phosphatase activity were determined on demarrowed femurs of normal, ‘hypophysectomized’ and pituitary-grafted chick embryos at selected intervals of incubation. In normal bones, all parameters noted above increased progressively through developmental time. ‘Hypophysectomy’ by means of surgical decapitation significantly retarded the normal increase of femur wet weight, total and specific alkaline phosphatase activity; in embryos bearing pituitary transplants, there was a return towards normal values. The possible role(s) of the pituitary in skeletal maturation and enzyme synthesis or activation is discussed.

Alkaline phosphatases have been studied histochemically and biochemically in many types of invertebrate and vertebrate embryos. In general, high enzyme activity is associated with early cell proliferation, followed by tissue-specific patterns of activity-for example, the activity of embryonic chick liver decreases (Moog, 1944), while that of vertebrae increases (Bose, 1960). Among tissues characterized by high alkaline phosphatase activity are those secreting an extracellular matrix. Numerous studies on developing or regenerating bone in vertebrates indicate that the enzyme is involved in the early ontogenetic processes of bone differentiation. Further, McWhinnie & Saunders (1966) reported that although all tissues of the chick limb bud have alkaline phosphatase, the bone enzyme exceeds that of other limb components and increases through development, paralleling the process of mineralization.

Since alkaline phosphatase is characteristically related to calcifying tissues, one might inquire whether hormones regulating skeletal development also influence its activity. For some years, it has been recognized that the pituitary plays a role in embryonic events-for example, carbohydrate metabolism (Konigsberg, 1954; Thommes & Aglinskas, 1966), water balance (Thommes & McCarter, 1966) and sexual differentiation (Woods & Weeks, 1969). It was Fugo (1940), however, who first noted that long bone lengths were less than normal in pituoprivic chick embryos and this was later confirmed by Betz (3rd toe lengths, 1968). More recently, Mehall (1970) and Thommes, Hajek & McWhinnie (1973) have shown that bone differentiation, growth and mineralization are modified in the absence of the pituitary. It would also appear that the pituitary has an influence on the enzyme, alkaline phosphatase, for in its absence, enzyme activity fails to accumulate in the duodenum (Hinni & Watterson, 1963 ; Bellware & Betz, 1970; Hart & Betz, 1972) and several other tissues (Manwell & Betz, 1966).

In view of the possible role of the pituitary in both bone development and enzyme activity regulation, the present investigation was undertaken to evaluate hypophyseal influence on long bone growth and alkaline phosphatase activity in the developing chick embryo.

White Leghorn chick embryos, incubated at 38 ±0·5 °C in a ‘Jamesway’ incubator were used. At 33–38 h of incubation, ‘hypophysectomy’ was accomplished by the partial decapitation method of Fugo (1940). Using steel needles, a transverse cut was made through the mesencephalon, and the severed region, including the prosencephalon, was discarded. Sham-operated controls consisted of embryos whose brain parts had been separated, but whose forebrain tissue was left to heal in situ. Eggs of control and ‘hypophysectomized’ embryos were sealed and returned to the incubator. At selected intervals from days 10·5 to 18·5 the embryos were killed; a few day-20·5 embryos were also obtained. Embryos were removed from the shell and separated from extra-embryonic membranes. Incomplete cases of ‘hypophysectomy’, as revealed by the presence of eyes or upper beak, were discarded.

In some cases, pituitary grafts from day-10·5 embryonic donors were made onto the chorioallantoic membranes of day-10·5 ‘hypophysectomized’ animals; the grafts used did not include brain tissue or hypothalamic elements. These embryos were killed at intervals and their femurs treated as those from the control and pituoprivic groups.

Femurs were immediately dissected and cleaned of adhering tissue. Bones were demarrowed by expressing marrow through transverse cuts made in the diaphysis, and the marrow cavity was flushed with cold Ringer’s solution under pressure. Bone samples were weighed, stored at —20 °C, and used for enzyme analysis within 2 weeks after isolation. Subsequent to thawing, femurs were homogenized in distilled water with a Kontes all-glass Duall homogenizer at 4 °C, and used for alkaline phosphatase (APase) determination. The final concentration of homogenates was 1 mg bone/ml. Enzyme activity was determined by a modification of the method of King & Armstrong (1934), as described by McWhinnie & Saunders (1966). Enzyme activity was calculated as total units of activity per femur g phenol liberated/30 min) and units of specific activity (μ g phenol liberated/10 μ g protein/30 min). The protein content of homogenates was determined by the method of Lowry, Rosebrough, Farr & Randall (1951), as modified by Oyama & Eagle (1956).

Statistical methods employed were Analysis of Variance and the Duncan New Multiple Range Test (Duncan, 1955).

The removal of the pituitary Anlage through surgical decapitation has marked effects on skeletal development. The increase of bone weight and enzyme activity is severely inhibited. Providing ‘hypophysectomized’ animals with a source of hypophyseal factors through pituitary transplants tends to return these parameters towards normal.

Femurs of normal embryos progressively increase in wet weight from days 10·5–20·5, with an elevation in mass particularly observable from days 17·5–20·5 (Fig. 1). The increase in femur growth during the last 10 days of incubation is 44-fold as wet weights increase from 3–122 mg. In contrast, femurs of pituo-privic embryos exhibit an atypical growth pattern. While weights increase normally during days 10·5–12·5, subsequently in developmental time, these values become progessively less than normal (Fig. 1). On days 13·5 of incubation, while no statistically significant difference is yet noted between femurs of these 2 groups, a trend towards retarded bone weight is already observable (23 % less than normal). Subsequently, wet weights of femurs from operated animals are significantly (P < 0·05) below controls. In contrast to the 44-fold elevation in normal femur mass through days 10·5–20·5, that of ‘hypophysectomized’ embryos is only 20-fold. By day 20·5, femoral weight of operated embryos (54 mg) is 56 % less than normal (122 mg).

Fig. 1.

Wet weights of femurs of normal, ‘hypophysectomized’, and ‘hypophysectomized’-pituitary grafted chick embryos. Vertical bars represent standard errors of means. ○—○, Intact;• – – • hypophysectomized; ◼––.◼, hypophysectomized + pituitary graft.

Fig. 1.

Wet weights of femurs of normal, ‘hypophysectomized’, and ‘hypophysectomized’-pituitary grafted chick embryos. Vertical bars represent standard errors of means. ○—○, Intact;• – – • hypophysectomized; ◼––.◼, hypophysectomized + pituitary graft.

When decapitated embryos bear pituitary grafts, there is a tendency for skeletal mass to increase (Fig. 1). While in 11·5-to 13·5-day embryos, there is no significant weight difference among femurs of normal, ‘hypophysectomized’, and pituitary-grafted animals, on days 14·5 and 15·5, there are significant 17·8 % and 25·6 % increases above the pituoprivic level for the graft-bearing embryos.On days 16 · 5 and 20 · 5 of incubation, the increases above the operated embryo level are 16 · 7 % and 42 · 7 %, respectively.

The lower than normal bone mass in ‘hypophysectomized’ animals is paralleled by effects on total alkaline phosphatase activity. In normal femurs, enzyme activity rises in total bone mass from 212 to 15450 units from days 10 · 5 – 20 · 5 (72-fold increase; Table 1). In contrast, enzyme activities of bones from pituoprivic embryos are lower (Fig. 2). This difference first appears on day 13·5 when total femoral activity in normal animals is 2330 units, with that of operated embryos being 1028 units (56 % less). This difference then becomes more pronounced, and by day 18 · 5, total enzyme activity (3095 units) is 72% below controls (10540 units). Thus, the overall increase in APase activity between days 10·5 and 18·5 for operated animals is only 14-fold.

Table 1.

The influence of ‘hypophysectomy’ on total alkaline phosphatase activity in the femur of the embryonic chick

The influence of ‘hypophysectomy’ on total alkaline phosphatase activity in the femur of the embryonic chick
The influence of ‘hypophysectomy’ on total alkaline phosphatase activity in the femur of the embryonic chick
Fig. 2.

Total alkaline phosphatase activity in femurs of normal, ‘hypophysectomized’, and ‘hypophysectomized’-pituitary grafted chick embryos. Vertical bars represent standard errors of means. ○—○, Intact; •– –•, hypophysectomized; ◼ – .– ◼, hypophysectomized + pituitary graft.

Fig. 2.

Total alkaline phosphatase activity in femurs of normal, ‘hypophysectomized’, and ‘hypophysectomized’-pituitary grafted chick embryos. Vertical bars represent standard errors of means. ○—○, Intact; •– –•, hypophysectomized; ◼ – .– ◼, hypophysectomized + pituitary graft.

Pituitary transplantation to operated embryos elevates the total enzyme content of femurs. On days 16·5 and 18·5, enzyme activities are 79·2% and 89·6% greater, respectively, than ‘hypophysectomized’ levels (P < 0·01). In spite of this increase, total bone enzyme activities are only partially returned to normal by pituitary grafts. They remain, at these two developmental times, 55 % below normal (Table 1).

To preclude the possibility that the low APase activity was not simply a consequence of decreased bone mass, enzyme activities as a function of protein concentrations were determined. Through days 10·5–20·5, the specific APase activity of bones from normal embryos increases 5-fold (Fig. 3). From 10·5 to 12·5 days of incubation, the specific APase activity of femurs from ‘hypophysectomized’ embryos remains indistinguishable from controls. However, subsequently, not only does activity lag behind that of controls, but actually decreases from the level on day 12·5 (Table 2). For example, on day 13·5 not only is APase specific activity 28·8 % below control values, but it is also 9·2 % below the value for bones of operated embryos on the preceding day. Later, APase specific activity is as low as 75·1 % below values for bones of normal day 20·5 animals, and 37·6 % (16·5-day embryos) below the highest activity obtained for ‘hypophysectomized’chicks (day 12·5).The latter part of embryonic development in these animals is thus characterized by a fluctuating plateau of APase specific activity which becomes progressively separated from the sustained increase shown by normal femurs.

Table 2.

The influence of ‘hypophysectomy’ on the specific activity of alkaline phosphatase in the femur on the embryonic chick

The influence of ‘hypophysectomy’ on the specific activity of alkaline phosphatase in the femur on the embryonic chick
The influence of ‘hypophysectomy’ on the specific activity of alkaline phosphatase in the femur on the embryonic chick
Fig. 3.

Specific alkaline phosphatase activity in femurs of normal, ‘hypophysectomized’, and ‘hypophysectomized’-pituitary grafted chick embryos. Vertical bars represent standard errors of means. ○—○, Intact; •– –•, hypophysectomized; ◼ – .– ◼, hypophysectomized + pituitary graft.

Fig. 3.

Specific alkaline phosphatase activity in femurs of normal, ‘hypophysectomized’, and ‘hypophysectomized’-pituitary grafted chick embryos. Vertical bars represent standard errors of means. ○—○, Intact; •– –•, hypophysectomized; ◼ – .– ◼, hypophysectomized + pituitary graft.

Partial recovery of the abnormally low APase specific activity in ‘hypophysectomized’ embryos is effected by hypophyseal transplants. On days 16·5 and 18·5 the pituitary-grafted embryos have bone enzyme activities 88·6 % and 61·3 %, respectively, above the values for pituoprivic embryos (Table 2). These activities, however, remain 32·0 % and 49·7 % below normal at comparable developmental times.

Growth patterns, cellular differentiations, matrix synthesis, and ossification in the developing skeleton may be regulated by the changing hormonal environment within the embryo. Data presented here indicate that the presence or absence of the pituitary gland influences the growth of long bones, total bone alkaline phosphatase content, and the specific activity of this enzyme in bone.

In normal embryos, femur growth is roughly linear through the latter half of incubation. Accompanying the increase in mass is an increase in total alkaline phosphatase activity. There is also a rise in the specific activity of the enzyme which indicates a synthesis, or activation, of the enzyme exceeding that fraction which is concomitant with mass increase.

The role of APase in bone formation has never been clarified. It has been long recognized, however, that osteoblastic tissues have high enzyme activity, and that its inhibition leads to bone defects. While early literature tended to link its activity to transfer of phosphorus from a carbohydrate donor to a calcium acceptor for bone salt precipitation (Robison, 1936), this concept has not withstood further investigation. More recently, the enzyme has been linked to elaboration of organic matrix; i.e. sites of matrix synthesis, collagen production, and ectopic calcification are also areas of high APase activity (Firschein & Urist, 1971). APase may also remove a pyrophosphate crystal poison inhibiting mineralization. Whatever the linkage between this enzyme and mineralization, it is clear that APase activity is an index of the ‘normalcy’ of bone development.

The removal of the pituitary from the embryo exerts profound effects on long bones. Studies by Mehall (1970) have shown that ‘hypophysectomy’ results in a failure of bone rudiments to increase in length, as well as histological changes in the developing skeleton. As reported by Thommes et al. (1973), total bone mineral, bone calcium, and matrix are all less than normal. The present information confirms and extends these observations. Bones of pituoprivic embryos, by the end of incubation, have less than half the total mass of normal bones. Total APase activity is also low (one-quarter that of normal femurs).

That this enzymological defect is not a consequence of low bone weight alone may be adduced by an examination of APase specific activity. After day 13-5 not only does activity lag behind that of normal bones, but also decreases from the level it had attained previously. These data suggest that though matrix elaboration itself has been curtailed by ‘hypophysectomy’ (Thommes et al. 1973), APase activity relative to bone protein is more strongly retarded. Thus, while the impaired matrix synthesis of bones from ‘hypophysectomized’ individuals rises, although at a reduced rate, the synthesis (or activation) of APase is even more impaired, resulting in a decreased enzyme/protein ratio. This drop in APase specific activity may be due to defects in matrix elaboration and ossification (Thommes et al. 1973). It is also possible that among an array of bone APase isoenzymes, the synthesis (or activation) of some are selectively inhibited, while others are not affected in the absence of the pituitary.

Dependence of bone development on the pituitary (or a normal hypothalamic-pituitary unit) is first apparent on days 13·5, since at this time the bones of ‘hypophysectomized’ embryos have abnormally low weights and enzyme activities. This developmental period may be a critical one in regard to hypophyseal function, for many other systems become pituitary-dependent from days 10·14. For example, in the absence of the pituitary, the following become abnormal at this time: (1) blood cholesterol levels (Thommes & Shulman, 1967), (2) adrenal ascorbic acid and cholesterol levels (Case, 1952), (3) levels of allantoic fluid corticosteroids (Woods, DeVries & Thommes, 1971), (4) feather development (Goeringer, 1959), (5) thyroid structure and function (Martindale, 1941; review by Thommes, 1958), (6) yolk-sac membrane glycogen (Thommes & Aglinskas, 1966), (7) liver glycogen (Konigsberg, 1954), and (8) water balance (Thommes & McCarter, 1966). Moreover, during this critical developmental period, trophic interrelationships between the pituitary and the thyroid, adrenals and gonads are seemingly established (Martindale, 1941; Woods & Weeks, 1969; Woods et al. 1971). Hence, whatever developmental events are regulated by the pituitary alone, or the pituitary in concert with another endocrine gland, may be expected to become abnormal at this time in its absence. While the basic mechanism(s) involved in establishment of such dependencies are unknown, one could consider the timing of (1) activation of hypothalamus, i.e. synthesis and secretion of release factors, (2) synthesis and release of hypophyseal hormones, (3) maturation of receptor sites for trophic hormones in interrelated endocrine glands, and (4) maturation of receptor sites for various hormones in various responding tissues.

That the pituitary gland is, at least in part, a regulator for bone development is shown by experiments wherein pituitary transplants to the chorioallantoic membrane of ‘hypophysectomized’ embryos were made. In each case a return towards normal levels was evident; bone mass, total bone APase content, and APase specific activity were elevated above levels attained by bones of pituoprivic embryos.

The inability of pituitary grafts to return bone development fully to normal in ‘hypophysectomized’ embryos is not an isolated observation; for example, whole body weight (Thommes & McCarter, 1966; Brasch & Betz, 1971) and duodenal APase activity (Hart & Betz, 1972) are also only partially restored to normal. The role of ‘higher brain centers’ (i.e. hypothalamus) in this regard is unclear. There is evidence that the release of TSH and ACTH by the embryonic chick pituitary is independent of the hypothalamus (see reviews: Szentágothai, Flerkó, Mess & Halász, 1968; Betz, 1971), but little is known concerning hypothalamic regulation of the release of other adenohypophyseal hormones in the chick embryo. However, histological and histochemical studies of hypophyseal transplants indicate that in the absence of ‘higher brain centers’, normal function is maintained (Szentágothai et al. 1968; Brasch & Betz, 1971). Based upon this evidence, it is presumptions to assume that normal hypothalamic-pituitary relationships are not necessary for bone development, since all or some adenohypophyseal hormones may be released in abnormal amounts in the absence of the hypothalamus. Moreover, the grafting procedure per se may so affect the transplant that abnormal hormone titers are produced. Therefore, it remains unknown whether the absence of a functional hypothalamic-adenohypophyseal relationship in either ‘surgically decapitated’ or pituitary transplanted embryos, results in the observed bone changes.

The pituitary hormones which may affect bone development are undefined. Possible hormones include thyrotrophin, ACTH and somatotrophin. Each of these influences bone development in higher vertebrate embryos, or bone metabolism in adults, either directly or through trophic action on other endocrine glands. For example, thyroid hormone stimulates bone differentiation, matrix elaboration and mineralization (Fell & Mellanby, 1955, 1956). The relation between defective bone development and adrenal function is more tenuous, for as shown by Buño & Goyena (1955) and Siegel, Smith & Gerstl (1957), glucocorticoids arrest bone growth and mineralization. In its probable absence in this study, similar results occurred.

The role of somatotrophin (STH) in the regulation of bone growth in the embryonic chick is equivocal. Several investigators have reported results which vary from positive to negative ‘growth effects’ on developing bone. In vivo or in vitro addition of somatotrophin has been indicated to increase bone length, thickness, wet and dry weights, mitotic index, total nitrogen, and sulfur uptake (Blumenthal, Hsieh & Wang, 1954; Hay, 1958; Sobel, 1958; Ito, Takamura & Endo, 1959, 1960). In contrast, Chen (cited by Fell, 1954, 1955; Hay, 1958) found that in vitro treatment of chick embryo bones with somatotrophin brought about no change in rudiment length, while Vogel (1965) could not restore normal growth rates in surgically decapitated (‘hypophysectomized’) embryos with in vivo injection of an undefined bovine growth ‘factor’.

The species specificity of growth hormones could explain these seemingly conflicting results (Geschwind, 1966; Tashjian & Levine, 1969) in the embryonic bird. Moreover, the presence of a growth hormone in either the adult or embryonic chicken is problematical.

The role of STH in regulation of growth in fetal mammals also remains equivocal. Jost (1954) found that the pituitary does not influence growth (total body weight) in fetal rabbits, and his data were supported by the work of Bearn (1971), in which pituoprivic fetal rabbits showed normal body growth and skeletal ossification. Moreover, although Heggestad & Wells (1965) reported that fetal rat growth during the last 3 days of gestation was STH-dependent, Bearn (1968) has suggested that size differences between normal and decapitated fetuses may be due to abnormalities in placental circulation caused by the operative procedure. Studies on the human fetus also support the concept that the pituitary plays little or no role in growth. For example, growth in anencéphalie individuals is not retarded (Talbot & Sobel, 1947; Seckel, 1960) and Lessof (1964; cited by Laron, Pertzelan & Frankel, 1971) has reported that pituitary dwarfs grow normally until 2-3 years of age. There is, however, some evidence that STH may be operative in the fetus; i.e. (1) in the 12-week human fetus, growth hormone is present in the pituitary (Makler, 1968) and released into circulation (Laron et al. 1966) and (2) genetically HGH-deficient siblings or pituitary dwarfs with biologically inactive HGH, have low birth weights and crown-rump lengths (Laron et al. 1971).

These mammalian data are as yet inconclusive, for they do not clearly indicate whether or not growth (body weight, crown-rump lengths, etc.) is abnormal in the absence of the pituitary and/or STH. In contrast, ‘hypophysectomy’ of the embryonic chick markedly affects body weight and bone development. Whether these phenomena are related to the cleidoic nature of the ‘enclosed’ chick embryo system versus the relative ‘openness’ of the mammalian fetal-maternal relationship, awaits further investigation.

This study confirms and extends observations (Thommes et al. 1973) that the pituitary gland (or a functioning hypothalamic-pituitary unit) is essential for normal bone development. Those parameters which show an hypophyseal dependence are bone growth, bone wet, dry and ash weights, total calcium content, total bone alkaline phosphatase content, and alkaline phosphatase specific activity. The nature of the pituitary hormone(s) exerting this regulation, and their interactions with developing osseous tissues at the cellular and molecular levels, are yet to be determined.

This investigation was supported by the Brown-Hazen Fund of the Research Corporation of America, and partially by Training Grant 5-T01-HD00293 from the National Institute of Child and Human Development.

Bearn
,
J. G.
(
1968
).
The thymus and the pituitary-adrenal axis in anencephaly. A correlation between experimental foetal endocrinology and human pathological observations
.
Br. J. exp. Path
.
49
,
136
144
.
Bearn
,
J. G.
(
1971
).
The role of the foetal pituitary in organo-genesis
.
In Hormones in Development
(ed.
M.
Hamburgh
&
E. J. W.
Barrington
),pp.
121
134
.
New York
:
Appleton-Century-Crofts
.
Bellware
,
F. T.
&
Betz
,
T. W.
(
1970
).
The dependence of duodenal differentiation in chick embryos on pars distalis hormones
.
J. Embryol, exp. Morph
.
24
,
335
355
.
Betz
,
T. W.
(
1968
).
The effects of embryonic pars distalis grafts and albumen on the growth of chick embryos
.
J. Embryol. exp. Morph
.
20
,
431
436
.
Betz
,
T. W.
(
1971
).
The pars distalis and avian development
.
In Hormones in Development
(ed.
M.
Hamburgh
&
E. J. W.
Barrington
), pp.
75
94
.
New York
:
Appleton-Century-Crofts
.
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
.
Bose
,
A.
(
1960
).
Localization of alkaline phosphatase in the development of the vertebral column in the chick
.
Experientia
16
,
144
146
.
Brasch
,
M.
&
Betz
,
T. W.
(
1971
).
The hormonal activities associated with the caudal regions of the cockerel pars distalis
.
Gen. comp. Endocr
.
16
,
241
256
.
Buño
,
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
.
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
.
Fell
,
H. B.
&
Mellanby
,
E.
(
1955
).
The biological action of thyroxin on embryonic bones grown in tissue culture
.
J. 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. Physiol., Lond
.
133
,
89
100
.
Firschein
,
H. E.
&
Urist
,
M. R.
(
1971
).
The induction of alkaline phosphatase by extra-skeletal implants of bone matrix
.
Calc. Tissue Res
.
7
,
108
113
.
Fugo
,
N. W.
(
1940
).
Effects of hypophysectomy in the chick embryo
.
J. exp. Zool
.
85
,
271
297
.
Geschwind
,
I.I.
(
1966
).
Species specificity of anterior pituitary hormones
.
In The Pituitary Gland. Vol. 2. Anterior Pituitary
(ed.
G. W.
Harris
&
B. T.
Donovan
), pp.
589
612
.
California
:
University of California Press
.
Goeringer
,
G. C.
(
1959
).
Modified development of the integument of hypophysectomized chick embryos. I: The epidermis. II: The feather germs
.
Ph.D. Dissertation, Northwestern University
.
Hart
,
D. E.
&
Betz
,
T. W.
(
1972
).
On the pars distalis hormonal activities involved in duodenal development in chick embryos
.
Devi Biol
.
27
,
84
99
.
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
.
Heggestad
,
C. B.
&
Wells
,
L. J.
(
1965
).
Experiments on the contribution of somatotrophin to prenatal growth in the rat
.
Acta Anat
.
60
,
348
361
.
Hinni
,
J. B.
&
Watterson
,
R. L.
(
1963
).
Modified development of the duodenum of chick embryos hypophysectomized by partial decapitation
.
J. Morph
.
113
,
381
426
.
Ito
,
Y.
,
Takamura
,
K.
&
Endo
,
H.
(
1959
).
The stimulative effect of pituitary growth hormone on 35S-sulfate incorporation in the chick embryo femur in tissue culture
.
Endocrinol, 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
.
Endocrinol, jap
.
7
,
327
335
.
Jost
,
A.
(
1954
).
Hormonal factors in the development of the foetus
.
Cold Spring Harb. Symp. quant. Biol
.
19
,
167
181
.
King
,
E. J.
&
Armstrong
,
A. R.
(
1934
).
A convenient method for determination of serum and bile phosphatase
.
Can. Med. Ass. J
.
31
,
376
381
.
Konigsberg
,
I. R.
(
1954
).
The effects of early pituitary removal by ‘decapitation’ on carbohydrate metabolism in the chick embryo
.
J. exp. Zool
.
125
,
161
191
.
Laron
,
Z.
,
Pertzelan
,
A.
&
Frankel
,
J.
(
1971
).
Growth and development in the syndromes of familial isolated absence of HGH or pituitary dwarfism with high serum concentration of an immunoreactive but biologically inactive HGH
.
In Hormones in Development
(ed.
M.
Hamburgh
&
E. J. W.
Barrington
), pp.
573
585
.
New York
:
Appleton-Century-Crofts
.
Laron
,
Z.
,
Pertzelan
,
A.
,
Mannheimer
,
S.
,
Goldman
,
J.
&
Guttman
,
S.
(
1966
).
Lack of placental transfer of human growth hormone
.
Acta endocr., Copenh
.
53
,
687
692
.
Lowry
,
O. H.
,
Rosebrough
,
N. J.
,
Farr
,
A. L.
&
Randall
,
R. J.
(
1951
).
Protein measurement with the Folin-phenol reagent
.
J. biol. Chem
.
193
,
265
275
.
Makler
,
M. T.
(
1968
).
Growth hormone in human development
.
Nature, Lond
.
217
,
1149
1150
.
Manwell
,
C.
&
Betz
,
T. W.
(
1966
).
The effect of embryonic partial decapitation on the developmental sequences of some proteins in the chicken
.
J. Embryol. exp. Morph
.
16
,
83
89
.
Martindale
,
F. M.
(
1941
).
Initiation and early development of thyrotropic function in the incubating chick
.
Anat. Rec
.
79
,
373
393
.
Mcwhinnie
,
D. J.
&
Saunders
,
J. W.
, Jr
. (
1966
).
Developmental patterns and specificities of alkaline phosphatase in the embryonic chick limb
.
Devi Biol
.
14
,
169
191
.
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
.
Moog
,
F.
(
1944
).
Localizations of alkaline and acid phosphatases in the early embryogenesis of the chick
.
Biol. Bull. mar. biol. Lab., Woods Hole
86
,
51
80
.
Oyama
,
V. I.
&
Eagle
,
H.
(
1956
).
Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau)
.
Proc. Soc. exp. Biol. Med
.
91
,
305
307
.
Robison
,
R.
(
1936
).
Chemistry and metabolism of compounds of phosphorus
.
A. Rev. Biochem
.
5
,
181
204
.
Seckel
,
H. P. G.
(
1960
).
Concepts relating the pituitary growth hormone to somatic growth of the normal child
.
Am. J. Dis. Child
.
99
,
349
379
.
Siegel
,
B. V.
,
Smith
,
M. J.
&
Gerstl
,
B.
(
1957
).
Effects of cortisone on the developing chick embryo
.
A.M.A. Archs Pathol
.
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
.
Szentágothai
,
J.
,
Flerkó
,
B.
,
Mess
,
B.
&
Halász
,
B.
(
1968
).
The development of the hypothalamus-TSH-thyroid relationship
.
In Hypothalamic Control of the Anterior Pituitary
, pp.
177
184
.
Budapest
:
Akadémiai Kaidó
.
Talbot
,
N. B.
&
Sobel
,
E. H.
(
1947
).
Endocrine and other factors determining the growth of children
.
Adv. Pediat
.
2
,
238
.
Tashjian
,
A. H.
&
Levine
,
L.
(
1969
).
Taxonomic specificity of growth hormones and thyrocalcitonins as measured immunologically
.
In Progress in Endocrinology. Proc. 3rd Int. Congr. Endocrin
. (ed.
C.
Gual
&
F. J. G.
Ebling
), pp.
440
452
.
Amsterdam
:
Excerpta Medica
.
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.
&
Aglinskas
,
A. S.
(
1966
).
Endocrine control of yolk sac membrane glycogen in the developing chick embryo. II. Effects of hypophysectomy
.
Gen. comp. Endocrinol
.
7
,
179
185
.
Thommes
,
R. C.
,
Hajek
,
A. S.
&
Mcwhinnie
,
D. J.
(
1973
).
The influence of ‘hypophysectomy’ by means of surgical decapitation on skeletal growth in the developing chick embryo
.
J. Embryol. exp. Morph
.
29
,
503
513
.
Thommes
,
R. C.
&
Mccarter
,
C. F.
(
1966
).
Adenohypophyseal control of water balance in the developing chick embryo
.
Am. Zool
.
6
,
517
.
Thommes
,
R. C.
&
Shulman
,
R. W.
(
1967
).
Endocrine control of lipid metabolism in the developing chick embryo. I. Blood cholesterol
.
Gen. comp. Endocrinol
.
8
,
54
60
.
Vogel
,
N. W.
(
1965
).
Growth in chick embryos hypophysectomized by ‘decapitation’
.
Proc. Penn. Acad. Sci
.
39
,
57
60
.
Woods
,
J. E.
,
Devries
,
G. W.
&
Thommes
,
R. C.
(
1971
).
Ontogenesis of the pituitaryadrenal axis in the chick embryo
.
Gen. comp. Endocrinol
.
17
,
407
415
.
Woods
,
J. E.
&
Weeks
,
R. L.
(
1969
).
Ontogenesis of the pituitary-gonadal axis in the chick embryo
.
Gen. comp. Endocrinol
.
13
,
242
254
.