close

Вход

Забыли?

вход по аккаунту

?

Thyroxine and the development of the tibia in the embryonic chick.

код для вставкиСкачать
Thyroxine a n d the Development of the Tibia
in the Embryonic Chick
B . K. HALL
Department o f Biology, L i f e Sciences Centre, Dalhousie University,
Halifax, Nova Scotia, Canada
ABSTRACT
Embryonic chicks were treated with exogenous L-thyroxine or
with thiourea at eight days of incubation and the subsequent development of
the tibia studied.
The weight of the tibia was 62% lower than that from normal embryos, and
the length of the tibia 24% below normal in the embryos treated with thiourea.
This reduced rate of growth was shown to be due to a reduction in the rate of
maturation of chondroblasts into chondrocytes, reduced chondrocyte hypertrophy
and defective deposition of acid mucopolysaccharide into the cartilage matrix.
Osteogenesis pet se was unaffected. It was concluded that thyroxine plays a role
in the control of chondrocyte maturation and in cartilage matrix production during normal development.
The epiphyses of the tibiae from the embryos treated with thiourea were extensively eroded, invaded by marrow and more fragile than those from untreated
embryos, indicating that thyroxine is essential for the maintenance of the integrity of the articular cartilage. An abnormal core of bone developed within the
proximal epiphysis of these embryos.
Exogenous thyroxine at concentrations as low as 100 pg/embryo also reduced
the growth of the tibia below that seen in untreated embryos. Evidently the cells
of the skeleton are sensitive to both lowered and increased levels of circulating
thyroxine.
The role played by the hormone thyroxine in the differentiation and growth
of embryonic long bones is poorly understood. The present communication deals
with the effect of thyroxine on the development of the embryonic chick tibia from
the eighth to the eighteenth day of incubation. Embryos were either made hyperthyroid by the application of exogenous
thyroxine or were made hypothyroid by the
in vivo injection of thiourea, an anti-thyroid agent.
Thiourea was first used as an antithyrogenic agent in the embryonic chick
by Grassowicz ('46). In 1949 Adams and
Bull published an account of the inhibition
of general body growth and of the length
of the tibia (tibiotarsus) which followed
single or multiple injections of 2 mg
thiourea or of 0.5 to lmg thiouracil commencing on the eighth day of incubation.
They found that multiple injections (at 8,
14 and 18 days) were no more effective
than were single injections; that overall
ANAT. REC., 176: 49-64.
body growth was inhibited by up to 18%;
and that the length of the tibia was shorter
than normal. No other aspeots of skeletal
growth were studied by them. Subsequently, Adams and Buss ('52) provided
further information on this reduction in
growth in hypothyroid chick embryos and
also studied changes in thyroid weight,
histology and mitotic activity. Romanoff
and Laufer ('56) treated 11 day embryos
with from 2 to 10 mg of thiourea and
studied the subsequent development of the
liver, thyroid and adrenal glands in some
detail. No further detailed information on
the response of long bones of chick embryos in the in vivo application of antithyroid agents such as thiourea is available.
The respnse of whole long bones of
the embryonic chick to exogenous uhymxine in vitro has been studied by Fell
and Mellanby ('55) who found an initial
enhancement of 'cartilage maturation folReceived Oct. 5, '72. Accepted Jan.22, '73.
49
50
B. K. HALL
lowed by retardation of bone growth, and
by Lawson ('61) who confirmed growth
retardation in both the tibia and the radius.
Melcher ('71 ) has shown that hypertrophic
chondrocytes of Meckel's cartilage maintained in vitro may be induced to commence synthesis of DNA in response to
thyroxine, Evidently thyroxine enhances
both proliferation and maturation of
chondrocytes.
The response of chondrocytes from embryonic chicks isolated in vitro and exposed to thyroxine #has been studied by
Pawelek ('69) who found an enhancement
of the differentiation of non cartilage-making clones in chondrocytes and a concomitant stimulation of the synthesis of chondroitin sulphate by the differentiating cells
(see also Dorfman and Schiller, '58). Thus
exogenous thyroxine exerts a direct effect
on the differentiation and maturation of
cartilage in vitro. Thyroxine also enhances
the formation of ectopic cartilage and
bone (Somogyi and Kovass, '69).
The aim of the present study was to
render chick embryos hypothyroid by treatment with thiourea, or hyperthyroid by
treatment with thyroxine, and to examine
the differentiation, growth, histology and
gross histochemistry of the tibia in an endeavour to provide information additional
to that currently available on the in vivo
effects of thyroxine on skeletogenesis and
to correlate these data with that obtained
in the above in vitro studies.
MATERIALS AND METHODS
Eggs were incubated in a forced-draft
Leahy incubator at 37 k: 0.5"C and 54 &
2% relative humidity.
At eight days of incubation 153 eggs
were injected with thiourea (Fisher Scientific) and 152 eggs with L-thyroxine (sodium salt, British Drug Houses, lot no.
0.476590). Doses used/embryo were:
thiourea 1 pg to 5 mg (see table l ) , and
rhyroxine 100 pg to 10 mg (see table 3 ) .
The chemicals were dissolved in sterile
saline (0.9% NaCl supplemented with 1%
ETOH for thyroxine) and injected in a
final volume of 0.5 em3 through a pinhole
in the shell and shell membrane and onto
the chorio-allantoic membrane. All injectiom were carried out with a 2.5 em3 presterilized Tuberculin syringe. The thio-
urea-treated embryos were examined at
two-day intervals from 10 to 18 days, the
thyroxine-treated embryos at 14 and 18
days of incubation. Three hundred and
forty eight contml uninjected embryos
were also examined.
All embryos were examined for survival.
The surviving elmbryos were removed from
their shells, cleared of adherent yolk and
extraembryonic membranes, blotted on
filter paper and weighed to the nearest
0.01 g. The tibiae were dissected out,
cleaned of adherent connective tissue and
muscle, weighed to the nearest 0.01 mg
and their lengths measured to the nearest
0.01 mm.
Tibiae from embryos at each age examined were treated by the Alizarin Red S
method of Evans ('48) to visualize cenlters
of ossification, to assess gross morphology
and to determine the proportion of bone to
cartilage in treated versus untreated embryos (by measurement of the linear dimensions of the osseous shaft and of the
unossified cartilage model and epiphyses).
At least three tibiae per dose per age
were fixed in 80% ethanol, embedded in
52°C m.p. paraffin, serially sectioned and
used for histological and histochemical
analysis. The Alcian Blue 8GX-Chlorantine
Fast Red 5B (ABCR) method of Lison
('54) was used as the routine histological
stain. Alcian blue stains the non-sulphated
acid mucopolysaccharides of the cartilage
matrix bright blue whilst chlorantine red
stains bone matrix bright red. Light green
in Mason's trichrome procedure (Pantin,
'60) was used to visualize the distribution
of collagen and to supplement the ABCR
method for general hisitology.
Further information on the distribution
of acid mucopolysaccharides, mucoproteins, glycoproteins and glycogen was obtained from ethanol-stable metachromasia
after 0.01% toluidine blue (Ham and
Harris, '50) and from the periodic acidSahifF (PAS) procedure (Barka and Anderson, '65).
Sites of calcification were visualized with
alizarin red S and sites of alkaline phosphatase by the Gomori method (Barka and
Anderson, '65). As the final steps in the
Gomori methods also visualize calcium,
sections were decalcified with 5% EDTA
and the dkaline phosphatase reactivated
51
THYROXINE AND THE CHICK TIBIA
with 1% sodium veronal (Schajowicz and
Cabrini, '56) before the Gomori method
was applied.
RESULTS
Thiourea-treated embryos
Body weight
At ten days of incubation, i.e. two days
post-injection with thiourea, the embryos
treated with 1 or 5 mg were significantly
smaller ( 1 6 % , P > 0.02) than untreated
embryos. Lower doses of thiourea retarded
body weight by an average of 8% but were
not statistically different from controls.
The initial effect of thiourea on embryonic
growth was thus restricted to doses above
1 mg/embryo (table 1, fig. 15).
By 12 days of incubation all doses
(0.001 to 5 mg) significantly retarded
body weight below control values by an
average of 21% (table 1). Analysis of
variance indicated no significant difference between doses, ind&ating that all
doses were equally effective in slowing embryonic growth.
By 14 days of incubation all doses of
thiourea except the lowest used (0.001
mg) retarded body weight below controls
by an average of 20% (P > 0.02 or lower,
table 1). At 16 and 18 days of incubation
the effect of thiourea on body weight could
be divided into two dose groups: 1 to 5 mg
and 0.1 to 0.001 mg. All doses produced
significantly lower body weights than normal but the higher doses produced significantly greater reductions below normal
(35% ) than did the lower doses ( 1 8 % , fig.
15). From the fourteenth day of incubation
onwards a dose of thiourea as low as 0.01
mg was sufficient to retard body weight
by some 1 8 % . The subnormal body weight
of 35% of control values seen after doses
of thiourea above 1 mg may have been due
to pharmacological effelcts of thiourea
rather than to its effect on thyroxine levels
(see DISCUSSION).
Growth of the tibia
At ten days of incubation and over the
range of doses used,, the tibiae were significantly lighter (by 44% P > 0.001) and
TABLE I
Survival rate, number of survivors per age and body weight of survivors for
embryos treated with thiourea at eight days of incubation
Dose
Embryonic age (days)
10
mg
5
2.5
1.0
0.5
0.1
0.05
0.01
0.001
Control
5
2.5
1.0
0.5
0.1
0.05
0.01
0.001
Control 1
69 (5)
78 (5)
79 ( 5 )
84 (4)
87 (6)
100 (20)
12
Survival ( %
48 (9)
40 (4)
71 (9)
75 (9)
77 (10)
77 (7)
75 (4)
96 (24)
P > 0.01.
UP > 0.02.
7 P > 0.05.
16
18
)
28 (7)
46 (4)
40 (4)
65 (5)
60 (3)
59 (3)
67 (5)
96 (24)
Body weight ( 9 ) 2
3.23f0.10 3 6.08f0.153
3.23f 0.183
1.72?~0.13~
3.45C00.063
6.50f0.495
3.22k 0.07 6.81 C 0.43
1.8220.11 3.36f0.053 6.80f0.256
6.732 0.39
1.81k0.10 3.38k0.13 3 6.56f0.35
2.01k0.08 3.2920.20 7.23-CO.12
2.04&0.04 4.19f0.05 8.22f0.12
1.69i0.06
No. of survivors examined in parentheses.
Mean + Standard error.
P > 0.EOOl.
4P > 0.001.
1
2
3
14
30 (4j
92 (12)
9.4720.57
12.37? 0.15
10.952 0.817
13.20k 0.31
12.38% 0.80
16.01 % 0.34
16.28 2 0.44
16.34f 0.95
17.26* 0.71
20.35i0.24
12.30k0.175
12.18k0.834
14.4020.17
52
B. K. HALL
shorter (by 20%, P > 0,001) than were
those from control embryos. It may be recalled that at this same age body weight
was lighter by 16%, indicating that the
growth of the tibia was much more affected
by thiourea than was the rest of the body
(see fig. 15). The doses of thiourea which
did not reduce body weight at ten days (i.e.,
below. 1 mg thiourea) did significantly reduce the weight of the tibia and as a consequence the relative tibia weight (tibia
weight (mg)/g body weight-tibia weight)
was very much lower than normal (table
2). A similar situation was seen at 12 days
of incubation, where the weight and length
of the tibia were below control vdves by
45 and 24% respectively and where the
relative tibia weight W;LS substantially below normal. By 14 days of incubation the
lowest dose of thiourea used (0.001 mg)
produced a smaller tibia weight (39% )
than did the higher doses, and this same
trend was intensified at 16 and 18 days
(cf. the similar effect on growth of the rest
of the body). At 18 days of incubation, the
weight of the tibia was 62% below normal
after treatment with from 1 to 5 mg
thiourea and 45% below normal after
treatment with lower doses. Length of the
tibia displayed the same pattern (figs. 1-3,
15). Even though its size was reduced the
form of growth curve for the tibia from the
treated embryos paralleled that from control embryos. Thus despite the greater depression in the growth of the tibia compared with that of the remainder of the
body the proportionality of tibia weight to
body weight was maintained. Over the 12
to 18 day period the reduction in tibia
weight exceeded that of the remainder of
the body by a factor of 2.1 : 1. The general
trend in the relationship between weight
and length of the tibia was that at the
lower doses (0.001 to 0.1 mg) weight was
depressed to a greater extent than was
length and that at the higher doses length
was more depressed.
The effect of thiourea on the rate of
ossification of the tibia was assessed by
examining the “alizarin preparations” and
calculating the percentage of the cartilaginous model replaced by bone. Aside from
an early initiation of ossification at ten
days in embryos treated with 0.1 to 5 mg
thiourea the relative amounts of bone in
the tibiae from the treated embryos was
essentially normal. Absolute amounts of
bone were, of course, less than normal.
Histogenesis and histochemisty
of the tibia
Changes in the morphology of the tibiae
from the treated embryos were assessed
using the “alizarin preparations” and the
histological sections and are summarized
in figure 16.
At ten days of incubation the proximal
epiphyses of a majority of the tibiae were
bent, the bending originating at the junction of the articular and proliferating
chondrocyte zones (see fig. 4 for zones), A
cenltral area of the same zone of the distal
epiphysis was eroded and the resting cavity
infiltrated by elements of the marrow. In
some specimens the cartilaginous shaft
had fractured. Such features were not seen
in untreated tibiae. Osteogenesis appeared
normal.
The reduction in the growth rate of the
tibia described above was due to a reduction in the number of chondrocytes maturing and undergoing hypertrophy. At ten
days of incubation the zone of hypertrophic chondrocytes made up 56% of the
total length of the tibia in control embryos
but only 46% in the tibiae from the
treated embryos. Evidently treatment with
thiourea resulted in a slowing of hypertrophy of the chondrocytes. The proliferating zone was little affected indicating that
the rate of production of chondroblasts was
normal.
By 12 days of incubation bone had
begun to form within the cavity in the
distal epiphysis, the cartilaginous shaft had
undergone more erosion than was normal
and the articular surface of the proximal
epiphysis had broken down.
The acid mucopolysaccharides of the
cartilage matrix were visualized after
alcian blue or toluidine blue. Up to 12 days
of incubation their distribution appeared
normal, although after toluidine blue the
matrix was more fibrillar than usual. From
12 days of incubation onwards the distribution of matrix acid mucopolysaccharides
was distinctly abnormal. Alcian blue staining was very patchy and the matrix did not
stain uniformly with toluidine blue.
Glycogen appeared as the chondrocytes
53
THYROXINE AND THE CHICK TIBIA
TABLE 2
Tibia weight ( m g ) , length ( m m ) and weight/g body weight for control embryos
and for embryos treated with thiourea at eight days o f incubation.
Values are means -C S.E.M.
Age (days)
Dose ?.
10
6.602 0.61
2.5
1.0
0.5
0.1
0.05
0.01
0.001
Control
5
2.5
1.0
0.5
0.1
0.05
0.01
0.001
Control
5
2.5
1.o
0.5
0.1
0.05
0.01
0.001
Control
1
2
5.75 2 0.37
6.00 2 0.40
12
14
Tibia weight ( m g ) *
10.9720.84
25.602 1.40
12.32k 1.17
12.55f0.40
27.75 f1.20
12.662 0.41
26.8222.00
13.1020.54
28.4022.14
26.4021.74
2 8 . O O k 1.63
13.14C 0.55
13.90k 1.14
35.0022.85
22.96 2 0.65
46.61 2 0.83
16
44.30 2 3.92
77.30 -C 1.78
51.20k 8.21
80.40 rt 2.79
70.25
* 5.42
70.00f 1.62
69.50'1.77
99.65* 1.51
109.7824.47
110.70k 5.34
112.302 5.44
125.00C5.12
207.9823.87
16.17k 0.75
20.27 k 0.23
17.172 1.20
20.85%0.31
18.80f0.33
22.00 C _ 0.60
22.10 k 0.53
22.00 -+- 0.49
22.36 f0.44
26.05 f0.07
~
5.752 0.65
6.10k0.38
10.7120.17
6.74 rt 0.23
6.30 2 0.33
6.54 2 0.15
6.722 0.32
6.9720.18
8.36 k 0.15
Tibia length ( m m ) I
9.4220.18
13.20-+-0.10
9.75 f0.30
10.01 & 0.23
12.80 % 0.62
10.30k0.11
12.40k0.55
9.0020.12
12.80~0.21
12.37-+0.22
9.66f 0.23
12.60 k 0.23
9.72f 0.33
14.10%0.15
12.69 2 0.06
16.69 f0.10
18.70f 0.66
18.90 2 0.35
21.74f0.09
2 Tibia ( m g / g body weight - tibia wt. ( m s > >
7.86
6.83
8.49
9.44
7.70
7.34
8.62
6.72
9.44
7.94
7.93
7.87
8.43
6.63
11.48
7.90
7.84
8.61
6.39
11.51
6.10
8.53
9.78
11.54
11.09
11.44
10.52
14.03
All si nificantly different from control: P
No. 08ernbryos: see table 1.
18
12.66
12.33
13.90
13.79
13.94
14.70
20.87
> 0.001.
underwent hypertrophy as is the normal some it lay alongside the epiphysis, consituation. The distribution of muco- and nected to it by the perichondrium and surglycoproteins as visualized by the PAS re- rounding mesenohyme. In others the proaction was not uniform and paralleled the liferating zone of the distal epiphysis had
toluidine blue staining. Calcification and broken down (fig. 8); chondrocytes had
alkaline phosphatase distribution was become freed from the intercellular matrix
where extensive vacuolization was apparnormal.
The severity of the deviations from ent (fig. 5) and free red blood cells had
normal increased with increasing em- invaded the zone of several points (fig. 6).
bryonic age. Bone was prominent within Long blood vessels running from the base
the proximal epiphysis by 14 days and in of the articular cartilage almost to the end
some tibiae from 16 day embryos the of the hypertrophic zone were present
proximal epiphysis had become detached (fig, 7).
The distribution of the acid mucopolyfrom the diaphysis. Erosion of both proximal and distal epiphyses was very exten- saccharides stained by alcian blue was
sive (fig. 4). In some specimens rhe articu- most abnormal, especially in the articular
lar zone, especially of the distal epiphysis, and proliferating zones (fig. 8). Some
had become completely detached from the clumps of chondrocytes had normal
remainder of the cartilage (fig. 7) and in amounts of alcian blue - staining mate-
54
B. K. HALL
rial surrounding them, others had none,
giving the cartilage a mottled appearance
(figs. 9, 10). The articular zone stained
intensely with toluidine blue. PAS and
Masson’s trichrome (fig. 11) - more intensely than did the remainder of the cartilage. The cells of the proliferating zone
were widely separated from one another,
the matrix consisting of little other than
sparsely distributed fibers (fig. 8).
By 18 days of incubation the shaft of
bone in the center of the distal epiphysis
was extensive in all specimens treated with
0.1 or more mg of thiourea (figs. 12, 13).
It ran from the base of the articular zone
proximally into the marrow cavity where
it was intimately connected with the
diaphyseal bone. This core of bone was
heavily calcified and contained a well developed marrow cavity, partially filled with
fibrous material connecting the bone to the
epiphyseal cartilage (fig. 14). These fibres
stained orthochromatically after toluidine
blue, violet after PAS and green after Masson’s trichrome.
The histogenesis of the tibiae in embryos treated with thiourea was then characterized by: increased resoprtion of the
cartilage model and of the epiphyses,
fragile epiphysed articular zones, extensive erosion of the articular surfaces, abnormal deposition of acid mucoplysaccharides into the cartilage matrix and the
deposition of a central core of bone within
the proximal epiplhysis (fig. 16).
Thyroxine-treated embryos
L-thyroxine at or a b v e 10 ag/eight-day
embryo was lethal in 100% of cases
(table 3). The L. Dsowas 100 ng/embryo.
At doses below 10 ng/embryo survival was
comparable to that of control embryos,
Only at one dose (10 ng/embryo) was
body weight significantly lower than that
of control embryos at 14 days of incubation (table 3). Both the weight and the
length of the tibia were significantly below
control values for all doses at both 14 and
18 days of incubation (P > 0.001, table 3).
Variance analysis on these data indicated
no significant difference between doses.
The average reduction of 33% in weight
and 14% in length was very similar to
that obtained after 1 &g thiourea. Thus
thyroxine significantly reduced growth of
the tibia without affecting growth of the
remainder of the body.
The cytodflerentiation of the cells of the
tibia h m thyroxine-treated embryos appeared normal. Histochemically those embryos treated with doses of thyroxine below
100 ng appeared normal. In those embryos
given a dose of 1 &gand examined at 14
days of incubation the distribution of sulphated acid mucopolysaccharides (as
visualized by tduidine blue) and of mucoand glycoproteins (as visualized by PAS)
appeared normal, but the non-sulphated
acid mucopolysaccharides were very unevenly distributed, especially in the articular zone. The matrix surrounding many of
TABLE 3
Body weight; weight, length and relative weight of the tibia from control embryos and
from embryos treated with thyroxine at eight days of incubation and examined at
14 or 18 days of incubation. Values are means 2 standard error
Dose
1 Pg
100 ng
10 n g
1 ng
100 Pg
Control
I PLg
Control
1
2
Tibia
N
Body weight
Weight 2
Lengths
9
mg
mm
mg/gl
718
7/8
24/25
14 days of incubation
27.42 1.92
7.50f0.28
7.81 % 0.25
33.0& 3.09
6.92f0.522
29.721.71
32.722.00
7.9040.14
7.34 f 0.33
31.32 2.01
8.2240.12
46.62 0.83
14.0C 0.27
14.4 & 0.56
13.9&0.21
14.7& 0.24
14.5 C 0.40
16.7+- 0.10
7.93
8.53
8.66
8.35
8.60
11.47
2/10
12/13
18 days of incubation
13.7140.29*
120.02 7.07
20.3540.24
207.92 3.87
22.6& 0.28
26.0 2 0.07
17.82
20.86
7/18
4/8
7/a
Based on wet weight of
two tibiae ( m g ) / g body weight
Significantly different from control ( P < 0.001).
- tibia
wt (mg).
THYROXINE AND THE CHICK TIBIA
the chondrocptes was unstained after
alcian blue, indicating minimal deposition
of acid mucopolysaccharide. By 18 days of
incubation the sulphated acid mucopolysaccharides were also unevenly distributed,
especially in the proliferative zone. Thus
1 pg of exogenous thyroxine resulted in impaired deposition of acid mucopolysaccharide into the cartilage matrix. With
lower doses the gross histochemistry of the
tibia (collagen distribution, calcification,
alkaline phosphatase, glycogen distribution) was normal.
DISCUSSION
It was evident that when thyroxine synthesis was inhibited in the embryonic chick
by the in vivo application of thiourea, the
growth of the tibia was retarded to a much
greater extent than was the growth of other
organs (by a factor of 2 : 1). Many tissues
of the embryo evidently depend on thyroxine for their normal development. This
is no doubt because of the s t i m u l a t q
effect which thyroxine has on general
metabolism (MacLean and Urist, '68;
Romanoff, '60; Pawelek, '69) ; on oxidative
metabolism, glycolysis and mucopolysaccharide synthesis (Hoch, '62; Dziewiatkowski, '64; Dorfman and Schiller, '58); and
on collagen synthesis (Kivirikko et al., '67;
Blumenkrantz and Prockop, '70).
The growth of the skeleton may be more
affected than other organs in hypothyroid
embryos: ( a ) because skeletal cells are
especially dependent on thyroxine or have
a lower threshold to thyroxine or (b) because the skeleton contains large amounts
of acid mucopolysaccharide and collagen,
both of which are especially sensitive to
circulating levels of thyroxine. The fact
that long bones or their constituent cells
respond to thyroxine when isolated in vitro
(see Introduction) indicates that the hormone can act directly on the skeleton. The
direct action of thyroxine on skeletal cells
in vivo has been clearly demonstrated by
Dratman and Kuhlenbeck ('69). They injected C'"-labelled thyroxine into newborn
rats and found intense labelling of fibroblasts and of osteoblasts six minutes postinjection. Subsequently cartilage matrix
was labelled, especially near blood vessels
and areas of cartilage erosion. Dziewiatkowski ('64) has shown that SS5uptake is
55
diminished after thiouracil injection, indicating that the sulphated acid mucopolysaccharides of the cartilage matrix are very
sensitive to lowered thyroxine levels, and
Vaughan ('70) indicated ehat protein synthesis of skeletal cells was stimulated by
thyroxine, probably at the level of RNA
synthesis.
From studies on normal and hyperthyroid rats and mice i t has been shown that
thyroxine stimulates the proliferation and
maturation of osteoblasts and chondroblasts and so increases the rate of osteogenesis and chondmgenesis and that the
increased cartilage proliferation is followed
by enhanced cartilage resorption, further
increasing endochondral ossification (Levai
et al., '69; Becks et al., '46; Silberberg and
Silberberg, '40. ) By contrast in hypothyroid
rats and in cretinism epiphyseal cartilage
persists and the formation of epiphyseal
centers of ossification is delayed (Levai
et al., '69; Hamburger and Lynn, '64;
Vaughan, '70).
Thyroxine controls the mobilization of
Ca by osteogenic cells and so controls the
rate of remodelling of bone (Frost, '64;
MacLean and Urist, '68; Vaughan, '70;
Urist et al., '63).
The present results extend the above
findings to the embryonic chick. The cartilaginous model of the tibia was the most
affected. Initially (at 10 to 12 days of incubation, i.e., 2 to 4 days post treatment
with thiourea) maturation of the chondrocytes was reduced. Subsequently (to 18
days of incubation) formation of the cartilage matrix was inhibited. The fact that,
in the hypothyroid embryo, the integrity of
the articular cartilage was lost; that erosion
of the cartilage was extensive; that there
was excessive vascularization of the epiphyses; and that the distribution of matrix
acid mucopolysaccharides was abnormal
indicated that thyroxine normally maintains cartilage integrity, controls the rate
of erosion and resorption of the cartilage
model and plays a role in synthesis or deposition of acid mucopolysaccharide-protein complex into the m a t i x of the long
bones of the embryonic chick.
The formation of bone in the epiphysis
of thiourea-treated embryos, preceded as
it was by premature invasion of the epiphysis by blood vessels and the formation of
56
B. K. HALL
marrow in the erosion cavity, was reminiscent of the replacement of marrow by bone
in the hypothyroid dwarf (Vaughan, ' 7 0 ) ,
of osteopetrosis resulting from stimulating
of the parafollicular cells (thyrocdcitonin)
in the mouse (Marks and Walker, '69)
and of the formation of medullary bone in
adult laying hens and in the metatarsus as
it repairs a fracture (Sturkie, '65). Evidently the normal integrity of the marrow
and marrow cavity of the tibia is also dependent on normal levels of circulating
thyroxine.
The degree to which the tibial growth
was below normal, could, at 16 and 18 days
of incubation, be divided into two groups
and related to the concentration of
thiourea administered. After injection of 1
or 5 mg thiourea the weight of the tibia
was much lower than normal than after
lower concentrations of thiourea (fig. 15).
A possible interpretation is that the higher
doses of thiourea had a pharmacological
effect in addition to the inhibition of
thyroxine synthesis. Evidently eight days
were required (treatment at day 8, fast effect at day 16) before this pharmacological
effect was manifested, for no such dichotomy of response was observed in 14 day
tibia examined. A more precise indication
of this possible pharmacological effect of
high doses of thiourea could be obtained
by co-injection of thiourea and thyroxine to
determine whether tibial development
could be restored to normal. However, such
an approach may not be practical as there
is evidence that thiourea not only suppresses synthesis of thyroxine but also inactivates any circulating thyroxine present
in the embryo (Sturkie, '65). We are currently assaying circulating thyroxine levels
in the embryonic chick, both with and
without added thiourea and thyroxine, to
determine the feasibility of thyroxine replacement therapy. The problem is compounded by our finding (table 3 ) that
exogenous thyroxine itself, at very low concentrations (100 pg) inlhibits skeletal development, although not to the same extent as does thiourea. As rhe body weight
in the thyroxine-treated embryos was
normal, the inhibition of skeletogenesis
may have reflected negative feedback to
the thyroid and a consequent lowering of
the total amount of circulating thyroxine,
even though additional hormone has been
administered. In any event our results indicate that skeleton of the embryonic chick
is sensitive to very small levels of exogenous thyroxine and that the hormone
balance of the embryo must be very precise if skeletogenesis is to be normal.
ACKNOWLEDGMENTS
This research as been supported by the
National Research Council of Canada
(grant A5056). The technical assistance of
Miss Joan Calder and Miss Josephine
Smith is gratefully acknowledged.
LITERATURE CITED
Adams, A. E., and A. L. Bull 1949 The effects
of anti-thyroid drugs on chick embryos. Anat.
Rec., 104: 421443.
Adams, A. E., and J. M. Buss 1952 The effect
of a single injection of an anti-thyroid drug
on hyperplasia in the thyroid of the chick
embryo. Endocrinol., 50: 234-253.
Barka, T., and P. J. Anderson 1965 Histochemistry, Theory, Practice and Bibliography.
Harper and Row, New York.
Becks, H., M. Simpson, H. Evans, R. Ray, C . Li
and W. C. Asling 1946 Response to pituitary
growth hormone and thyroxine of the tibias
of hypophysectomized rats after long post-operative intervals. Anat. Rec., 94: 631-656.
Blumenkrantz, N., and D. J. Prockop 1970
Variations in the glycosylation of the collagen
synthesized by chick embryo cartilage: effects
of development and several hormones. Biochim.
Biophys. Acta, 208: 461466.
Dorfman, A., and S. Schiller 1958 Effects of
hormones on the metabolism of acid mucopolysaccharides of connective tissue. Recent Prog.
Horm. Res., 14: 427453.
Dratman, M. B., and H. Kuhlenbeck 1969 Interaction of thyroxine with developing skeletal
tissues of the newborn rat. Anat. Rec., 163: 180.
Dziewiatkowski, D.D. 1964 Effect of hormones
on the turnover of polysaccharides in connective tissue. Biophys. J., 4: 215-238.
Evans, H. S. 1948 Clearing and staining small
vertebrates in toto for demonstration of ossification. Turtox News., 26: 4247.
Fell, H. B., and E. Mellanby 1955 The biological action of thyroxine on embryonic bones
grown in tissue culture. J. Physiol., 127: 427447.
Frost, H. M. 1964 Dynamics of bone remodeling. In: Bone Biodynamics. H. M. Frost, ed.
Little Brown and Co., Boston, pp. 335-352.
Grassowicz, N. 1946 Influence of thiourea on
development of the chick embryo. Proc. Soc.
Exp. Biol. Med., 63: 151-152.
Ham, A. W., and W. R. Harris 1950 Histological techniques for the study of bone and some
notes on the staining of cartilage. In: Handbook of Microscopical Techniques. R. McC.
Jones, ed. Hoeber, New York, pp. 269-284.
THYROXINE AND THE CHICK TIBIA
Hamburger, M., and E. Lynn 1964 The influence of temperature on skeletal maturation of
hypothyroid rats. Anat. Rec., 150: 163-172.
Hoch, F. L. 1962 Biochemical actions of thyroid hormones. Physiol. Rev., 42: 605-673.
Kivirikko, K. I., 0. Laitnen, J. Aer and J. Halme
1967 Metabolism of collagen in experimental
hyperthyroidism and hypothyroidism in the rat.
Endocrinol., 80: 1051-1061.
Lawson, K. 1961 The differential growth response of embryonic chick limbbone rudiments
to triiodothyroxine in vitro. 1. Stage of development and organ size. J. Embryol. exp. Morph.,
9: 42-51.
Levai, G., F. Moricz., P. Szerze, G. Petranyi Jr. and
J. Laczko 1969 The effect of thyrotropic hormone treatment on the epiphyseal cartilage of
the white rat. Acta Morphol. Acad. Sci. Hung.,
17: 7-15.
Lison, L. 1954 Alcian Blue 8G with chlorantine
fast red 5B: a technic for selective staining of
mucopolysaccharides. Stain Technol., 29: 131138.
McLean, F. C., and M. R. Urist 1968 Bone
Fundamentals of the Physiology of Skeletal
Tissues. Univexsity of Chicago Press, Chicago.
Marks, S. C. Jr., and D. G. Walker 1969 The
role of the parafollicular cell of the thyroid
gland in the pathogenesis of congenital osteopetrosis in mice. Am. J. Anat., 126: 299-314.
Melcher, A. H. 1971 In vitro effect of oxygen,
hydrocortisone, and triiodothyronine on cells
of Meckel's cartilage. Israel. J. Med. Sci., 7:
374-3 76.
Pantin, C. F. A. 1960 Notes on Microscopical
57
Techniques for Zoologists. Cambridge University Press, Cambridge.
Pawelek, J. M. 1969 Effects of thyroxine and
low oxygen tension on chondrogenic expression
in cell culture. Devel. Biol., 19: 52-72.
Romanoff, A. L. 1960 The Avian Embryo.
Structural and Functional Development. The
MacMillan Company, New York.
Romanoff, A. L., and H. Laufer 1956 The
effect of injected thiourea on the development
of some organs of the chick embryo. Endocrinol., 59: 611-619.
Schajowicz, F., and R. L. Cabrini 1956 Chelating agents as histological and histochemical
decalciiiers. Stain Technol., 31: 129-134.
Silberberg, M.,and R. Silberberg 1940 Changes
in the skeletal tissues of mice following the
administration of thyroxin. Growth., 4: 305314.
Somogyi, A., and K. Kovacs 1969 Der EinfluBeiniger Hormone auf die heteroplastische
Knorpel-und Knochenbildung im Herzmuskel
der Ratte. W. Roux' Archiv., 163: 248-258.
Sturkie, P. D. 1965 Avian Physiology. Cornell
University Press, Ithaca.
Urist, M. R., N. S. MacDonald, M. J. Moss and
W. A. Skoog 1963 Rarefying disease of the
skeleton: observations dealing with aged and
dead bone in patients with osteoporosis. In:
Mechanisms of Hard Tissue Destruction. R. F.
Sognnaes, ed., Amer. Assoc. Adv. Sci., Washington D. C., pp. 385-446.
Vaughan, J. M. 1970 The Physiology of Bone.
The Clarendon Press, Oxford.
PLATE 1
EXPLANATION OF FIGURES
1-3
The tibiae from 12 day (fig. l), 14 day (fig. 2 ) , and 16 day (fig. 3)
thiourea-treated embryos. From left to right the tibiae represent
embryos treated with 5, 1.0, 0.1, 0.01 or 0.001 mg thiourea. In figure
1 a control tibia is also shown (extreme right). Alizarin Red S. X 1.
4
The proximal epiphysis of the tibia from an 18 day embryo treated
with 5 mg thiourea a t eight days. Note the extensive erosion of the
articular cartilage (arrow, and see figs. 5, 6). a, articular zone;
p. proliferative zone; h, hypertrophic zone. Alcian Blue-Chlorantine
Fast Red. x 16.
5
The articular cartilage of figure 4 a t higher magnification ( X 217).
Note the mottled appearance of the intercellular matrix, with some
unstained areas indicating abnormal deposition of acid mucopolysaccharide into the matrix.
6 T h e area indicated by the arrow in figure 4 at higher magnification
( x 217). Note that the cartilage matrix a t lower left is exposed to
the invading red blood cells (arrows).
58
THYROXINE AND THE CHICK TIBIA
B. K. Hall
PLATE 1
59
PLATE 2
EXPLANATION OF FIGURES
7
The distal epiphysis of the tibia from a n 18 day embryo treated with
1 mg thiourea. The articular cartilage has broken off leaving the proliferative zone exposed (arrow). Note the extensive vascular invasion
of the epiphysis. Toluidine Blue, X 15.6.
8
The junction of the proliferative zone (left) and the articular zone
of the tibia of an embryo treated as that in figure 7. The proliferative
zone has been disrupted whilst the articular zone remains normaI.
Masson’s trichrome. x 87.
9
Hypertrophic chondrocytes from the tibia of a n untreated 18 day
embryo. Note the pronounced cellular hypertrophy and the uniform
staining of the matrix (cf. fig. 10). Alcian Blue-Chlorantine Fast
Red. x 217.
10 Hypertrophic chondrocytes from the tibia of an 18 day embryo
treated with 5 mg thiourea. Note the reduced cellular hypertrophy
and uneven distribution of acid mucopolysaccharide within the matrix
(cf. fig. 9). Alcian Blue-Chlorantine Fast Red. x 217.
11
The articular zone from the tibia of a n 18 day embryo treated with
0.01 mg thiourea. The articular surface is eroded (cf. fig. 4 ) and
the staining of the articular cartilage more intense than elsewhere.
Toluidine Blue. x 16.
12 The distal epiphysis from the same tibia as in figure 4. Note the
central core of bone (see fig. 13). The arrow indicates the site of
figure 14. Alcian Blue-Chlorantine Fast Red. x 16.
13 A higher magnification of the bone from figure 12. Note trabeculae of
bone lined with osteoblasts. x 110.
14 The fibrous material within the central bone and its connection to
the cartilage (see fig. 12 for orientation). Toluidine Blue. x 110.
60
THYROXINE AND THE CHICK TIBIA
B. K. Hall
PLATE
2
61
PLATE 3
EXPLANATION OF FIGURE
15 Body weight ( O - - - - O ) , tibia weight (.---a),
and tibia length
(A,-.-.-A) of embryos treated with thiourea of eight days of incubation and examined at 10, 12, 14, 16 and 18 days of incubation.
Values plotted as percentage of control values.
62
PLATE 3
THYROXINE AND THE CHICK TIBIA
B. K. Hall
100
90
-
80
70
i'.
0
60
\0-0-0
I
10
4
O/..
*I
001
*001
-
0 90
Q:
- 80
-
k
2 70
- 60
0
c,
-
50
\
0
- 80
9Q
s
-
70
- 60
- 40
50
I
I
I
I
10
I
.I
I
I
=01 -001 10
THIOUREA
I
I
I
I
I
*I
-01
-001
(my)
63
64
B. K. HALL
Fig. 16 A diagrammatic representation of the tibia from a normal embryo (left) and
fmm a thiourea-treated embryo (right). Bone, black; cartilage, hatched; marrow, unshaded.
Note: the erosion of the articular cartilage, invasion of the epiphysis by marrow and blood
vessels, and core of bone within the proximal epiphysis of the treated embryo.
Документ
Категория
Без категории
Просмотров
0
Размер файла
1 121 Кб
Теги
development, embryonic, tibial, chick, thyroxine
1/--страниц
Пожаловаться на содержимое документа