close

Вход

Забыли?

вход по аккаунту

?

Effects of multiple doses of thyroxine on chick embryos.

код для вставкиСкачать
Effects of Multiple Doses of Thyroxine on
Chick Embryos’”
A. ELIZABETH ADAMS
Department of Zoology, Mount Holyoke College,
South Hadley, Massachusetts
Aspects of the reactions of the chick embryo to exogenous thyroid hormone in attempts to create a hyperthyroid state have
been studied by a number of investigators.
(For references see Adams, ’58a; Honjo
and Kuth, ’59). At autopsy the growth of
the embryo had been increased, decreased
or unaffected and length of incubation
period shortened or normal. The thyroid
glands had generally been decreased in
size, though one report (Romijn, Fung and
Lokhorst, ’52) recorded thyroid weight
larger than in controls. It is not surprising
that results varied since different dosages
had been used, and time, number and locus
of injections, time of autopsy, and breeds
differed.
The present series of experiments were
undertaken in an effort to create a marked
and consistent pattern of hyperthyroidism
in chick embryos by giving multiple injections of thyroxine amounting to a larger
total dose than previously used.
MATERIALS AND METHODS
Four series of chick embryos furnished
the data of the present experiments. In
three series the breed was White Plymouth
Rock. Eggs for series 1 were purchased
from W. H. Atkins, South Amherst, Massachusetts; those of series 2 and 3 from the
Poultry Department, University of Massachusetts, because the former source was
not available. The 4th series (White Leghorn) was also secured from the University of Massachusetts and used because
White Plymouth Rock eggs were not obtainable. The eggs were incubated at 38°C
in controlled humidity, turned twice daily,
and candled prior to each injection.
A 0.2 ml dose of a solution of Squibb’s
crystalline thyroxine (TH) of the desired
strength was injected into the yolk sac by
the method of Adams and Bull (’49). In
series 1 and 3, a dose of 0.25, 2.5, 5.0, and
10.0 vg was injected on days 0, 7, 10 and
14 or days 4, 7, 10, and 14, respectively,
amounting to a total of 17.75 clg per egg.
In series 2, injections were begun on day
10 with a dose of 2.5 ug, followed by 5.0 vg
on day 12, 7.5 ug on day 14, and 10.0 vg
on day 16, a total of 25.0 ug per egg. In
series 4, a 3.0 vg dose was given daily for
7 ox 8 days, beginning on day 10, amounting to a total of 21.0 or 24.0 vg per egg.
For controls, eggs were either injected with
0.2 ml of sterile alkalinized distilled water
( C ) or untreated (N). No difference was
observed between treated and untreated
controls so their data were combined (CN).
Many embryos died before the 18th day
of incubation on which the majority were
autopsied. A few were killed on days 16,
17, 19 and 20. No TH-exposed embryos
hatched. At autopsy body weights and
lengths of left third toes were taken. At
dissection, the thyroids and certain other
organs were removed, weighed and preserved for histological study. The thyroids
were fixed in Bouin, sectioned at 5 p, and
stained in hematoxylin and eosin, or Mallory’s triple connective tissue stain, or
periodic acid-Schiff (PAS) alone or with
methylene blue. The heights of thyroid
epithelia, diameters of thyroid follicles and
their lumina were measured with an ocular
micrometer at a magnification of 1000.
These data were taken on follicles in the
central region of sections chosen from the
center of the gland. Each measured section was at least 5 sections from the next
Supported by U.S.P.H.S. Grant A-3080 ( C l ) .
The author acknowledges with appreciation the
technical assistance of Margaret A. Seeck.
Some of the data of this paper appear in the
abstracts of the American Society of Zoologists
in the Anat. Rec., 237: 334, (1960), under the
title “Reaction of thyroids of chick embryos to
large doses of thyroxine.”
63
64
A. ELIZABETH ADAMS
0
u
0
@?
0
3
0
$1
c9
3
3
3
N
v!
0
+I
2
3
b
8tv
m
1
*
$1
rl
tv
P)
2+I
(4
0
m
9
2
,-
tl
rl
m
m
m
rl
rl
P$1
"!
tl
2
c?
3
rl
0
II)
v!
0
$1
o!
H
3
n
0.1
4
r:
0
+I
r!
w
n
m
W
v
0
tv
r!
r!
rl
+I
Q?
3
$1
v!
2
0
n
n
3
W
H
0,
v!
+I
LD
xmr:
r-
3
m
3
2
-2
v)
3
mg
CN
CN
(9) 0.69f0.083 ( 3 ) 2.8320.32
(1) 1.3S2
18
18
19
20
3
17.75
24.00
3
17.75
(1) 4.70
( 2 ) 5.13
(6) 3.3620.13
P
s
2.09
5.21
17.09
18.97
4.67r0.84 12.86i1.00
3.66i0.76 17.34i0.07
3.63
3.68
4.17
4.39
3.72kO.U4 5.5320.25
3.96k0.29 4.3620.24
3.23e0.51 17.2050.82 4.23-CO.50 4.1420.13
3.17t0.114 5.09k0.08
P
14.16
18.60
25.93
24.82
P
10.30
18.28
16.16
5.71i0.324 14.7320.77
3.53iO.2g4 17.67k0.28
3.42-C0.194 16.0420.42
4.17k0.23 12.9142.96
3.77iO.3O4 9.39a0.88
P
CN
Lumen diameter
TH
6.30
_____
13.29~k0.54~
25.17-CO.90
11.82%0.714 27.06k0.45
12.65iO.7g4 25.24-CO.49
11.03i0.034 23.17k2.08
12.6620.16 18.72C2.23
P
CN
Follicle diameter
TH
1
In one embryo tissue removed as right thyroid was thymus, so total thyroid weight was considered twice left thyroid.
2 0 n e thyroid was lost, so weight of other one was doubled.
3 At sectioning, two of the thyroids had non-thyroid tissue attached. Sections were projected, paper weights determined and proportionate correction
made for thyroid tissue weight. In one embryo, only one thyroid was removed at dissection, so its weight was doubled.
Differences between experimental and control data are significant.
(1) 0.63
(4) 0.7520.14
(3) 0.98k0.15 (8)4.5020.26
18
2
25.00
CN
Epith. height
TH
4.84k0.68 13.46-CO.32 4.07-CO.13 4.43k0.18
(2) 1.00-t-0.172 (2) 3.8920.65 6.5121.65 20.1950.55
1
17.75
4
TH4
Relative
m g / l O O gm body wt.
Thyroid weight
(4) 0.5820.031 ( 2 ) 1.6020.08
mg
TH4
Absolute
17
16
autopsy
21.00
4
17.75
3
P9
Series
dog&
Average data with probable errors, aTranged according to day of autopsy, for thyroids of chick embryos exposed to thyroxine (TH) and of their controls
(CN).Figures in parentheses indicate numbers of embryos
TABLE 2
66
A. ELIZABETH ADAMS
one measured. For epithelial height and
one follicular diameter, two cells opposite
each other along an axis through the
center of a follicle and the follicular width
along the axis were measured. The second
follicular diameter was taken along the
axis perpendicular to the first passing
through the center of the follicle. Diameters of lumina were calculated by subtracting the sum of the two cell heights
from the follicular diameter in the first
axis measured.
OBSERVATIONS
Mortality following the injection of thyroxine was high and always exceeded that
in controls. In the 4 series, 24 embryos
from the 97 TH-injected eggs were alive
at examination on days 16 through 20. Of
the 42 controls (treated and untreated),
303 embryos were living when the eggs
were opened on these days and one embryo
hatched on the 21st day.
On autopsy days in the 4 series, average
body weight and average length of left
third toe in TH-exposed embryos sometimes were greater, sometimes less than
those of controls (table 1 ) . When data on
body weights and left third toes warranted
statistical analysis, differences between experimental and control data were significant only in toe length in series 3, day 16,
and series 4, day 17. As two other criteria
of development, the degree of retraction
and average weight of the yolk sac were
recorded at killing. The process of retraction ordinarily begins on the 19th day of
incubation, rarely on the 18th. Of the embryos in eggs opened on days 16 through
20 (both living and dead embryos were
examined), 17 of 33 TH-exposed embryos
showed earlier and more advanced retraction than did 19 of 27 controls. In fact, in
14 of the 30 experimental embryos examined on days 16, 17 and 18, the process
was well under way, whereas only in 6 of
the 23 controls had it begun and this was
on day 18. As for the average yolk sac
weights, the differences between those of
experimental and control embryos were not
significant (table 1) .
In the case of the thyroids, however,
average absolute and relative (mg thyroid/
100 gm body weight) weights of TH-exposed embryos were significantly less than
those of corresponding controls, no matter
on which day the experiment began or
embryos were killed, or the total of the
dose given (table 2 ) . Histologically the
thyroids of experimental embroys were
“cellular,” cords were usually present,
follicles were small and colloid was
scanty while those of controls were “follicular,” follicles were large and wellsupplied with colloid (figs. 1-6). Measurements of follicles were usually in line with
the above observations. Average epithelial
cell height was significantly less in series
4, day 17, and series 3, day 18, in experimental thyroids compared with controls
but not on other autopsy days. Average
follicular and lumen diameters (the latter
a n indication of colloid content) were with
two exceptions significantly less in experimental embryos compared with controls.
In Mallory-stained sections, the colloid of
control thyroids was yellow or orange
(stored colloid) and blue (colloid recently
secreted or being resorbed) whereas in THexposed embryos, the small amount of thyroid colloid was typically blue. Vacuolation was common in the colloid bordering
the apical ends of thyroid epithelium in
controls, especially in the large follicles
found at the periphery of the gland. In
periodic acid-Schiff (PAS) alone or with
methylene blue, granules of PAS-positive
material similar in staining reaction to the
colloid were more usual and more numerous in the cytoplasm of the thyroid epithelium of control embryos than in that of experimental ones. These granules retained
their color after treatment with saliva, an
indication that they were not glycogen.
Thus, signs of activity were many in control thyroids in contrast to their paucity in
experimental ones.
DISCUSSION
The attempt to create a marked hyperthyroidism in chick embryos by administering via several injections a large total dose
of thyroxine into incubating eggs resulted
in (1 ) a high embryonic mortality; (2) no
consistent modification of growth of the
embryos; and ( 3 ) a marked reduction in
size and differentiation of the thyroid
glands compared with controls.
Six are not included in tables 1 and 2 because
there were no matching experimental embryos.
CHICK EMBRYOS EXPOSED TO THYROXINE
It is probable that the large amount of
injected thyroxine (17.75 to 25.0 pg) was
mainly responsible for the embryonic
deaths although the multiple injections (4,
7, or 8) may also have proved a hazard.
In previous investigations high mortality
has usually been attributed to the size of
the dose of thyroxine and/or the time of
injection, but there is considerable discrepancy in the data reported (Greenwood
and Chaudhuri, '28; Hanan, '28a, b; Guelin-Schedrina, '33, '34; Booker and Sturkie,
'49; McCartney and Shaffner, '49; Beyer,
'52; Romijn et al., '52; Vidal, '53a; Adams,
'58a). For example, Beyer claimed that a
preincubation dose of thyroxine must not
be larger than 0.025 ug, though McCartney
and Shaffner had secured hatched chicks
after doses of 6.0 and 12.0 ug given prior
to incubation. Similarly Hanan stated that
doses larger than 0.025 ug given on day 8
were toxic, but Romijn et al. reported that
0.1 ug injected on that day did not prevent
hatching. Vidal recorded a mortality of
35.0% and Adams one of 100.0% following 5.0 Vg given on the 7th day. The doses
mentioned and others not cited, however,
typically fell below those of the present
experiments. Time of injection cannot be
correlated satisfactorily with present data.
Guelin-Schedrina claimed that embryos
three days old tolerated doses fatal to older
embryos and Booker and Sturkie observed
that 1.0 Erg of thyroxine was toxic when
injected after day 6 but tolerated prior to
that day. Since in the present experiments,
injections were started prior to incubation
or on day 4 or day 10 and mortality percentages in the 4 series were very similar,
the time of injection seems a n unlikely
factor in the mortality.
Since the effects on growth of the chick
embryo in previous investigations using
relatively small doses of thyroid hormone
had ranged from a reduced body weight
(Willier, '24; Guelin-Schedrina, '33, '34;
Vidal, '53a; Adams, '58a) to a n increased
(Beyer, '52; Adams, '58a; Honjo and Kuth,
'59) or an unaffected one (Hanan, '28b;
Guelin-Schedrina, '34; Romijn et al., '52;
Adams, '58a), it was thought that a large
total dose of thyroxine spread within the
incubation period might produce consistent results. It proved otherwise. In no
series where numbers warranted analysis
67
were the differences between average body
weights of TH-exposed and control embryos
significant. A nearly similar situation also
occurred in measurements of left third toe,
although there was a slightly significant
increase on day 16, series 3, and a decrease
on day 17, series 4, in the experimental
embryos. It is a question why the large
dose elicited such indecisive effects on
growth. Since small doses speeded metabolism (Hanan, '28a, b; Beyer, '52; Romijn
et al., '52), it was thought that the large
dose might exaggerate this effect, causing
a rapid utilization of the food supply in
the yolk sac. This should result in smaller
yolk sac weights and either an enhanced
or reduced embryonic growth depending
on the rate of the metabolism (Adams,
'58a). The data on yolk sac weights gave
no clue to yolk use. Sometimes those of
TH-treated embryos were larger, sometimes smaller than those of controls, but
the differences were not significant. The
only suggestion that data on the yolk sac
offered as to a possible speeded develop
ment of the embryo was seen in the earlier
and more advanced degree of this organ's
retraction in a number of TH-exposed embryos compared with the process in their
controls. This was in line with a similar
result reported by Vidal ('53a) and the
shortened incubation period observed by
this investigator as well as by GuelinSchedrina ('34) and Beyer ('52) following
small doses of thyroxine.
The decisive result obtained after the
large dose of thyroxine was the retardation
of the growth and differentiation of the
thyroid glands of the chick embryos exposed to the hormone. Their average absolute and relative (mg thyroid/100 gm body
weight) thyroid weights, epithelial heights
(with one exception), sizes and colloid content of follicles, and secretory activity were
markedly and usually significantly reduced
compared with those of their controls killed
the same day. The weight decreases were
more pronounced than those following a
single smaller dose of hormone (Booker
and Sturkie, '49; McCartney and Shaffner,
'49; Vidal, '53b; Adams, '58a, table 4).
Data are lacking for a comparison of epithelial height and follicular size in the reports of previous investigators except for
the author's study in 1958. In this work,
68
A. ELIZABETH ADAMS
measurements of thyroid cell height and
follicular diameters were made on days 16
and 18 through 21 in embryos from eggs
g of thyinjected on day 14 with 10.0 M
roxine (Adams, '58a, table 5). The average follicular diameters were smaller than
those of controls, but less so than in the
present experiments. The average thyroid
cell heights, however, were consistently
higher than those of their controls, whereas in the present experiments they were
consistently lower. Factors probably involved in causing this difference are : ( 1)
one injection of 10.0 pg in contrast to 4, 7
or 8 injections totaling 17.75 to 25.0 vg in
the present experiments, and ( 2 ) injection
on day 14, when secretory activity had
been in progress for at least 4 days allowing for considerable colloid secretion and
storage. In the present experiments the
first injections were given before secretion
started (days 0 or 4) or when it was just
beginning (day 10) (Bradway, '29; Sun,
'32; Hopkins, '35; Fugo, '40; Martindale,
'41; Venzke, '49) and therefore an almost
complete blockage of thyroid growth and
differentiation occurred.
One further point should be mentioned.
There appeared to be no significant difference in the response of the thyroids to the
injected thyroxine when administration of
the hormone was started on or prior to the
10th day of incubation. This was evident
when the data for day 18 in series 1, 2, 3,
were compared (table 2). Thus it was
probably of no advantage to begin treatment before day 10 when according to the
evidence the thyroid begins to function
(see references p. 68).
It is probable that the marked retardation of growth and differentiation of the
thyroids of chick embryos after thyroxine
injections was the result of an inhibition
of the manufacture and release of the thyrotrophic hormone of the pars anterior via
a "feed-back" mechanism. The exogenous
thyroxine would block the pituitary's activity, thus resulting in inactivity of the embryo's own thyroid. Some evidence for this
interpretation was seen in the tendency of
the thyroid-blockage to lessen and thyroid
activity to be restored when injections of
thyroxine ceased. This was apparent in a
comparison of the thyroids on day 16 (last
injection, day 14, fig. 1) with those of day
18 (last injection, day 14, figs. 3 and 5).
This phenomenon was also observed by
Guelin-Schedrina ('34), Vidal ('53b) and
Adams ('58a,b). Whether the hypothalamus is also involved in the mechanism
is an additional question. The present
method of hypophysectomy of the chick
embryo by "decapitation" has produced
data interpreted largely from the standpoint of endocrine relationships (hypophysis-thyroid; hypophysis-adrenal; hypophysis-gonad : Fugo, '40; Martindale, '4 1;
Case, '52; HAmori, Mess and Szekely, '59;
Watterson, Brown and Bartha, '59), but
the hypothalamus is also removed in this
operation.
SUMMARY
Four series of eggs (White Plymouth
Rock and White Leghorn) received totals
of 17.75, 21.0, 24.0 and 25.0 vg of thyroxine via multiple injections into the yolk
sacs. The injections were given on days
0, 7, 10, 14 of incubation (series l ) , days
10, 12, 14, 16 (series 2), days 4, 7, 10, 14
(series 3) and on days 10 through 16 or
17 (series 4). Controls received injections
of sterile alkalinized distilled water or
were untreated. At autopsies on days 16
through 20, body weight, yolk sac weight
and length of left third toe were determined and degree of yolk sac retraction
was recorded. At dissection thyroids and
certain other organs were removed,
weighed and preserved for histological
study.
Body and yolk sac weights and toe
lengths of the TH-exposed embryos varied
from those of controls but not significantly.
Yolk sac retraction often started earlier
and advanced more rapidly than in controls, suggesting a speeding of embryonic
development following thyroxine administration. The thyroid glands of the TH-exposed embryos were consistently retarded
in growth and differentiation as indicated
by significantly smaller weights and, with
rare exceptions, lower epithelial cell
heights, smaller follicular size and less
secretory activity. The thyroid reaction is
interpreted as due to an inhibition of formation and release of thyrotrophic hormone of the pars anterior by the exogenous
thyroxine.
CHICK EMBRYOS EXPOSED TO THYROXINE
LITERATURE CITED
Adams, A. E. 1958a Effects of thyroxine on
chick embryos. Anat. Rec., 131: 445-464.
1958b The thyroid glands of thyroxinetreated chick embryos. Ibid., 131: 526.
Adams, A. E., and A. L. Bull 1949 The effects
of antithyroid drugs on chick embryos. Ibid.,
104: 421-444.
Beyer, R. E. 1952 The effect of thyroxin upon
the general metabolism of the intact chick embryo. Endocrinology, 50: 497-503.
Booker, E. E., and P. D. Sturkie 1949 The
effect of thyroxine and iodinated casein on the
development of the chick thyroid. Poultry Sci.,
28: 147-148.
Bradway, W. 1929 The morphogenesis of the
thyroid follicles of the chick. Anat. Rec., 42:
157-167.
Case, J. F. 1952 Adrenal cortical anterior pituitary relationships during embryonic life. Ann.
N. Y. Acad. Sci., 55: 147-158.
Fugo, N. W. 1940 Effects of hypophysectomy
in the chick embryo. J. Exp. Zool., 85: 271297.
Greenwood, A. W., and A. C. Chaudhuri 1928
An experimental study on the effect of thyroxin
upon sexual differentiation in the fowl. Brit.
J. Exp. Biol., 5: 378-384.
Guelin-Schedrina, A. 1933 Effets produits par
l’injection intravasculaire de thyroxine chez
l’embryon de Poulet. C. R. SOC.Biol., 113:
717-719.
1934 Effets de la thyroxine sur l’embryon de Poulet. Ann. Physiol. Physicochim.
Biol., 10: 453461.
Hamori, J., B. Mess and G. Szekely 1959 Onset
of thyroidal I accumulation in normal and decapitated chick embryos. Acta Biol. Acad. Sci.,
Hung., 10: 207-214.
Hanan, E. B. 1928a The effect of certain endocrine substances upon the prenatal development of the chick embryo. Anat. Rec., 38: 14.
69
1928b Effect of thyroxin on growth rate
and carbon dioxide production of chick embryo.
Proc. SOC.Exp. Biol. Med., 25: 422425.
Honjo, S., and T. Kuth 1959 The effects of
thyroxine and thiouracil upon the time and
order of appearance of ossification centers of
chick embryos. Jap. J. Vet. Sci., 21: 9-18.
Hopkins, M. L. 1935 Development of the thyroid gland in the chick embryo. J. Morph., 58:
585-6 13.
Martindale, F. M. 1941 Initiation and early
development of thyrotropic function in the incubating chick. Anat. Rec., 79: 373-393.
McCartney, M. G., and C. S. Shaffner 1949
Chick thyroid size and incubation period as in5uenced by thyroxine, thiouracil and thyroprotein. Poultry Sci., 28: 223-228.
Romijn, C., K. F. Fung and W. Lokhorst 1952
Thyroxine, thiouracil and embryonic respiration in White Leghorns. Ibid., 31: 684-691.
Sun, T. P. 1932 Histo-physiogenesis of the
glands of internal secretion-thyroid, adrenal,
parathyroid, and thymus-of
the chicken embryo. Physiol. Zool., 5: 384-396.
Venzke. W. C. 1949 Momhommesis of the thvroid ’glands of chicken embryos. Am. J. Vet.
Res., 10: 272-281.
Vidal, A. 1953a Influence de la thyroxine sur
le dCveloppement de l’embryon de Poulet. Ann.
Endocr., 14: 437443.
1953b Influence de la thyroxine sur la
diffbrenciation de la thyroyde embryonnaire du
Poulet. Ibid., 14: 444-449.
Watterson, R. L., D. A. Brown and A. Bartha
1959 Reduced endocrine activity of the adrenal cortex of hypophysectomized chick embryos as indicated by modified development of
the duodenum. Anat. Rec., 133: 347-348.
Willier, B. H. 1924 The endocrine glands an&
the development of the chick. I. The effects
of thyroid grafts. Am. J. Anat., 33: 67-103.
PLATE 1
EXPLANATION O F FIGURES
Sections of thyroids of chick embryos of series 3 exposed to 4 injections of thyroxine (TH)
in doses of 0.25, 2.5, 5.0 and 10.0 pg (total 17.75 pg) on days 4, 7, 10 and 14, respectively,
and of their controls ( c ) injected with sterile distilled water. Glands were fixed in Bouin’s
fluid, cut at 5 p and stained in periodic acid Schiff and methylene blue. Photographed with
yellow Wratten filter G . X 400 k.
t583, 16-day-old TH-exposed embryo. Thyroid weight 0.64 mg; cell height 4.41 p ; follicular diameter 12.71 p; lumen diameter 2.85 p . Note cords of cells, small follicles,
minute amounts of colloid i n follicles. Final injection was given on day 14. Compare
figure 2.
c592, 16-day-old control embryo. Thyroid weight, 1.68 mg; cell height 4.33 p; follicular
diameter 20.34 f i ; lumen diameter 11.46 p. Note typical “follicular” pattern and variation in follicular size and colloid content. Compare figure 1.
t579, 18-day-old TH-exposed embryo. Thyroid weight 0.49 mg; cell height 2.95 p ; follicular diameter 10.99 p ; lumen diameter 5.27 p. Note prominent cords, small follicles and
limited amount of colloid, but more than in t583 (fig. 1). The final injection was given
on day 14. Compare figure 4.
c567, 18-day-old control embryo. Thyroid weight 3.09 mg; cell height 5.27 p; follicular
diameter 23.02 p; lumen diameter 13.34 p. Note typical “follicular” pattern, varying
sizes of follicles and amounts of colloid. Gland appears well-developed and active. Compare figure 3.
t553, 18-day-old TH-exposed embryo. Thyroid weight, 1.08 mg; cell height 4.77 p ; follicular diameter 17.14 p ; lumen diameter 7.30 p. Note variations in follicular size and
amount of colloid. There is more colloid than at day 16 (fig. 1). Final injection was
given on day 14. Cord formation not very evident. Compare figure 6.
c593, 18-day-old control embryo. Thyroid weight 2.46 mg; cell height 5.37 p ; follicular
diameter 26.55 p; lumen diameter 16.40 p. Note “follicular” pattern. Follicles are mostly
medium or large with good supply of colloid. (Colloid has fallen out of some follicles.)
Compare figure 5.
70
CHICK EMBRYOS EXPOSED TO THYROXINE
A. Elizabeth Adams
PLATE 1
71
Документ
Категория
Без категории
Просмотров
2
Размер файла
675 Кб
Теги
effect, dose, embryo, chick, thyroxine, multiple
1/--страниц
Пожаловаться на содержимое документа