/. Embryo!, e.xp. Morph. Vol. 29, 3, pp. 515-527, 1973
Printed in Great Britain
515
The influence of 4 hypophysectomy'
on bone growth and alkaline phosphatase activity
in the chick embryo
By D. J. McWHINNIE 1 AND ROBERT C. THOMMES
From the De Paul University, Chicago
SUMMARY
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.
INTRODUCTION
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
1
Author's address: Department of Biological Sciences, De Paul University, 1036 West
Belden Avenue, Chicago, Illinois 60614, U.S.A.
516
D. J. MCWHINNIE AND R. C. THOMMES
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.
MATERIALS AND METHODS
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 chorioallantoicmembranes 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 - 2 0 °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
Pituitary effects on bone alkaline phosphatase
517
120
100
W)
40
20
10-5
11-5
I
12 5
I
13-5
I
14-5
15-5
16-5
17-5
18-5
I
19-5
I
20-5
Days of incubation
Fig. I. Wet weights of femurs of normal, 'hypophysectomized', and 'hypophysectomized'-pituitary grafted chick embryos. Vertical bars represent standard
errors of means. O—O, Intact; # - - • , hypophysectomized; •
• , hypophysectomized + pituitary graft.
of activity per femur (jug phenol liberated/30 min) and units of specific activity
(fig phenol liberated/10 fig 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).
RESULTS
The removal of the pituitary Anlage through surgical decapitation has marked
effects on skeletal development. The increase of bone weight and enzyme activity
518
D. J. MCWHINNIE AND R. C. THOMMES
Table 1. The influence of'' hypophysectomy' on total alkaline phosphatase activity
in the femur of the embryonic chick
Alkaline phosphatase activity (/tg phenol liberated/femur/30 min ± S.E.M.)
Days of
incubation
10-5
11-5
12-5
13-5
14-5
15-5
16-5
17-5
18-5
19 5
20-5
r
(13) *
03)
(14)
(14)
(13)
(JO)
(14)
(19)
(14)
(13)
(14)
A
Pituitary-grafted
' Hypophysectomized'
Control
,
,
212 ±16
551 ±74
905 ± 54
2330+184
3890 ±379
4658 ±161
6803 ±267
8274 ±288
10540 ±665
13652 ±1087
15451 + 1028
,
A
(16)
—
(14)
(6)
(18)
—
(14)
—
(8)
—
—
224 ±10
—
840 ±37
1028 ±66f
1215 ± 1541
—
2095 ±1691
—
3095 ± 341 f
—
—
^
^
r
^
.
—
—
—
.—
—
—
(12) 3755 + 205}
—
(6) 5870 ± 320%
—
—
—
—
* Number of samples.
t Difference between control and 'hypophysectomized' embryos; significant at P < 0 0 1 .
% Difference between 'hypophysectomized' and pituitary-transplanted embryos; significant at P < 0 0 1 .
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 pituoprivic 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).
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.
519
Pituitary effects on bone alkaline phosphatase
16000 i -
o
CO
12000
\ libci
rj
14000
alkal inc p Iiospl
i activity I
5
10000
SOOO
6000
4000
2000
10-5
11-5
12-5
13-5 14-5 15-5 16-5
Days of incubation
17-5
18-5 1 9 5
20-5
Fig. 2. Total alkaline phosphatase activity in femurs of normal, ' hypophysectomized', and 'hypophysectomized'-pituitary grafted chick embryos. Vertical bars
represent standard errors of means. O—O, Intact; # — # , hypophysectomized;
•
• , hypophysectomized + pituitary graft.
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
34
E M B 29
520
D. J. MCWHINNIE AND R. C. THOMMES
Table 2. The influence of' hypophysectomy' on the specific activity of alkaline
phosphatase in the femur on the embryonic chick
Alkaline phosphatase activity (/tg phenol liberated/10 /*g femur
protein/30 min ± S.E.M •)
A
Days of
Incubation
10-5
115
12-5
13-5
14-5
.15-5
16-5
17-5
18-5
19-5
20-5
Control
*
(22)*
(22)
(27)
(10)
(15)
(4)
(15)
(17)
(15)
01)
(15)
4-92 ± 0 1 5
7-42 ±0-23
8-77 + 0 1 9
11-53 ±0-45
12-67 ±0-38
1411 ± 0 1 4
15-65 ±0-41
18-23 ±0-79
21-66 ±0-72
23-45 + 0-74
25-99 ±0-85
'.Hypophysectomized
*—,
> t
(21)
(14)
(10)
(8)
(21)
(26)
(9)
(13)
(8)
(4)
(4)
4-85 ±0-30
7-36 ±0-83
905 ±0-62
8-22±0-40f
6-47±0-47f
6-61±0-44f
5-65 ± 0-71 f
6-75±0-90f
6-75±0-60f
7-33+ 102+
6-49±0-82t
Pituitary-grafted
,
v.
^
—
.—.
—
—
—
—
—
—.
—
__
(12) 10-65+ 0-30J
—
—
(6) 10-90±H2t
—
—
—
—
* Number of samples.
t Difference between control and 'hypophysectomized' embryos; significant at P < 0 0 1 .
% Difference between 'hypophysectomized' and pituitary-transplanted embryos; significant at P < 0 0 1 .
controls (10540 units). Thus, the overall increase in APase activity between
days 10-5 and 18-5 for operated animals is only 14-fold.
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
Pituitary effects on bone alkaline phosphatase
521
25 r-
20
o
15
10-5
11-5 12 5
13-5 14-5 15-5 165
Days of incubation
17-5
18-5
195 20-5
Fig. 3. Specific alkaline phosphatase activity in femurs of normal, 'hypophysectomized', and 'hypophysectomized'-pituitary grafted chick embryos. Vertical bars
represent standard errors of means. O—O, Intact; • — # , hypophysectomized;
• — • , hypophysectomized + pituitary graft.
APase specific activity which becomes progressively separated from the sustained
increase shown by normal femurs.
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.
34-2
522
D. J. MCWHINNIE AND R. C. THOMMES
DISCUSSION
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
Pituitary effects on bone alkaline phosphatase
523
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 hypothalamicpituitary 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 at. 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: Szentagothai,
Flerko, Mess & Halasz, 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
524
D. J. MCWHINNIE AND R. C. THOMMES
function is maintained (Szentagothai et ah 1968; Brasch & Betz, 1971). Based
upon this evidence, it is presumptious to assume that normal hypothalamicpituitary 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 hypothalamicadenohypophyseal 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 Buno & 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 Beam
(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,
Beam (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
Pituitary effects on bone alkaline phosphatase
525
the pituitary plays little or no role in growth. For example, growth in anencephalic 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.
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(Received 31 July 1972, revised 15 November 1972)