J. Embryol. exp. Morph. Vol. 20, 3, pp. 431-6, November 1968
Printed in Great Britain
431
The effects of embryonic pars distalis grafts and
albumen on the growth of chick embryos
By T. W. BETZ1
Department of Zoology, University of Illinois, Urbana
and Department of Biology, Carleton University, Ottawa
Normal chick embryos swallow and utilize the albumen in the egg between
the 11th and 15th day of incubation (Witschi, 1949). They use approximately
two-thirds of the yolk by the end of incubation (Romanoff & Romanoff, 1949).
Thus the embryo has access to 4 g of protein in the albumen and 3 g of protein
in the yolk (Romanoff & Romanoff, 1949). Embryos which have been hypophysectomized by partial decapitation, which is the only practical way of
hypophysectomizing chick embryos (Betz, 1965 a, see also the reviews of Benoit
(1962) and of Hinni & Watterson (1963), do not develop an upper beak and do
not swallow much of the albumen since most of it remains in the egg (Witschi,
1959; Betz, 19656).
The rate of growth of hypophyseoprivic embryos between the 14th and 18th
day of incubation is reduced to 53 % of the normal rate (Vogel, 1965). At 18-5
(Thommes, 1967) and 20 days of incubation (Fugo, 1940; Case, 1952; Betz,
1967) these embryos are only one-half as heavy as normal embryos. These
results contradict those of Wolff & Stoll (1937) and Stoll (1939). Witschi (1959)
said, in comparing the body weight of normal and hypophysectomized embryos,
' . . .it is evident that obstruction of alimentary intake must dwarf the fetus from
the 1 lth day on. On the other hand, if the albumen is swallowed the chick must
gain weight even if the albumen were not assimilated. Therefore, the question
of somatotrophic hormone (STH) deficiency remains undecided'. Thommes
(1967) reported that pars distalis grafts restored the body water content of
hypophysectomized chick embryos to normal by 18-5 days of incubation if the
data were expressed as the ratio of grams of water in the body to grams of dry
body weight; however, both the wet and dry body weights were subnormal.
Betz (1967) reported that most of the characteristic defects of hypophysectomized chick embryos are prevented by pars distalis grafts. Even though the growth
of these embryos is improved it is still subnormal. Thus, the limited growth of
these embryos may be due either to reduced albumen ingestion or failure of the
pars distalis grafts to produce normal amounts of STH and other hormones.
This paper reports the results of experiments which distinguish between the
effects of hormones and of albumen ingestion on the growth of chick embryos.
1
Author's address: Department of Biology, Carleton University, Ottawa 1, Ontario.
432
T. W. BETZ
MATERIAL AND METHODS
The experimental design is summarized in Table 1. White Leghorn eggs from
the University of Illinois poultry farm were used in this study. The embryos in
groups one, four and six were treated as described by Betz (1967). The rudiment
of the upper beak in group two was resected at stage 12 (Hamburger & Hamilton,
1951) of incubation. The albumen in group three was removed with a pipette
through a window in the shell on the fourth day of incubation. The embryos in
groups five and seven were hypophysectomized at stage 12 by resecting the head
at the posterior end of the mesencephalon. This results in a pituitaryless embryo
with a non-patent esophagus. The amount of albumen ingested by groups one,
Table 1. Experimental groups for the effects of graft hormones
and albumen on the growth of chick embryos
Group
number
1
2
3
4
5
6
7
Treatment
Normal
Upper beakless
Albumenless
Hypophysectomized, one graft,
and a patent esophagus
Hypophysectomized, one graft,
and a non-patent esophagus
Hypophysectomized with a
patent esophagus
Hypophysectomized with a nonpatent esophagus
Amount of
Amount of albugraft hormones men swallowed
Normal
Normal
Normal
Some
All
Some
None
Some
Some
None
None
Some
None
None
two, four and six was estimated by marking the albumen with two drops of
sterile carmine suspension (0-1 g/ml of Ringer's solution) which were added to
each egg on the fourth day of incubation through a window in the shell. The
eggs were placed in egg cartons and rotated five times. This, it was found, was
enough to insure an even distribution of the dye in the albumen.
At the end of 20 days of incubation the embryos were killed and the amount
of dyed meconium in the ventriculus, the amount of albumen remaining in the
egg and the amount of depot fat in the embryos were scored by visual estimation.
Then the embryos were fixed according to the method of Betz (1967). The length
of the third toe was measured twice to the nearest 0-1 mm according to the
criteria of Hamburger & Hamilton (1951). The duplicate measurements were
within 1 % of each other. The fixed bodies of the normal and hypophysectomized embryos (in which parts of the head are missing) were made comparable
by severing the necks between the fifth and sixth cervical vertebrae. Then the
headless bodies were weighed. The amount of albumen ingested by the different
groups was variable. Therefore the gut was removed and the bodies were
Number of specimens.
7
6
5
z
Albumen less (12)
Hypophysectomized with one
+ +
graft and patent esophagus (12)
Hypophysectomized with one
—
graft and non-patent esophagus (12)
Hypophysectomized with patent
+
esophagus (12)
Hypophysectomized with
—
non-patent esophagus (11)
3
4
3rd toe
0/
Normal
/o
22-2
100
(20-5-23-5);2
201
90
(15-2-22-9)
—
—
201
90
(15-5-230)
19-4
87
(160-220)
171
77
(160-18-5)
165
74
(150-190)
The range within the group.
+ +
Upper beakless (10)
2
+++++
albumen
ingested
Normal (12)1
Treatment
1
Group
number
19-7
(151-260)
13-6
(10-3-17-7)
—
14-2
(11-9-16 5)
10-9
(7-6-13-8)
8-8
(7-0-10-6)
90
(7-1-10-1)
Headless
Normal Headless, Normal
0/
gutless
/o
/o
100
100
16-2
(12-2-21.0)
10-7
66
69
(81-13-6)
—
—
75
65
10-6
72
(6-9-13-7)
9-3
57
55
(6-8-11-6)
7-3
45
44
(5-5-9-6)
7-3
45
45
(5-1-9-2)
A
Body weights
3-9
(2-9-5-0)
2-4
(1-8-3-1)
—
2-6
(1-9-3-2)
2-2
(1-6-2-9)
1-7
(1-3-2-2)
1-7
(1-4-2-3)
Dried
°5
44
44
^
3
^
*
^
S
^
o
56
62
66
62
ioo
Normal
o/
Table 2. The mean length (mm) of the 3rd toe, weight (g) of the modified bodies and amount of albumen ingested
by the embryos in the normal and experimental groups
434
T. W. BETZ
weighed in order to see if the gut contents significantly affected the differences
between the groups. The headless, gutless bodies were dried to a constant weight
at 60 °C. All weights were determined twice to the nearest 0-1 g. The duplicate
weights did not vary more than 1 %. The mean values of the third toe lengths
and the body weights of the groups were calculated and in order to compare the
groups the mean values of the experimental groups were expressed as a percentage of the normal mean value.
The albumenless embryos in group three were Hyline strain 934F. This
group was compared to normal Hyline embryos. Therefore, the mean body
weight of group three expressed as a percentage of the normal value is comparable to those of the other groups. Only fresh and dry body weights were
determined.
RESULTS
The embryos in group three were small but otherwise normal in their development. These embryos even retracted their yolk sacs and reduced their vitelline
and chorioallantoic circulations as did the embryos in groups one, two, four
and five. This was not accomplished by the embryos in groups six and seven.
None of the embryos were achondroplastic, nor were there any apparent
differences in the relative amounts of depot fat or any dimorphic somatic
differences. The data concerning the amount of albumen ingested, the length of
the third toe and the body weights are summarized in Table 2.
The amount of albumen swallowed by groups two and four was the same,
two-fifths of the normal amount, but it is twice the amount swallowed by group
six. The lengths of the third toe of the embryos in groups two, four and five were
reduced to a similar extent, but were still longer than those of groups six and
seven. The differences in the length of the third toe indicate that the skeletal
growth of groups two, four and five was greater than that in groups six and
seven. The body weights of groups two and four were not different from each
other although they were abnormal. Group five was actually lighter than two
and four but they were all significantly heavier than groups six and seven. On
the basis of the percentage of normal value group three was slightly heavier than
groups two and four.
For all the groups, the contents of the gut and the water content of the body
did not appear to make an appreciable contribution to the differences among
them.
DISCUSSION
Apparently the growth of the embryos in groups two, three, four and five was
retarded because of dietary restriction. For some unknown reason these
embryos were unable to use the protein in the yolk to attain normal growth.
The additional dwarfing of group five, even though the amount of skeletal
growth was the same as groups two and four, was apparently due to the absence
Growth of chick embryos
435
of albumen. However, the embryos of group three which also did not have any
albumen were slightly heavier than those of groups two and four. Perhaps the
Hyline embryos normally use more of the protein in the yolk for growth than
do the White Leghorn embryos.
The pars distalis grafts in group four apparently produced normal amounts
of hormones because these embryos grew the same amount as the embryos in
group two which had intact partes distales.
The embryos in groups six and seven were significantly smaller than those in
the other groups. The small amount of albumen swallowed by the hypophysectomized embryos in group six had no effect on their growth.
Simpson, Asling & Evans (1950) and Nalbandov (1963) argue that the growth
of vertebrates depends upon growth hormone and that other hormones and
nutrition are only permissive in the process. These data do not show the
extent to which the thyroid and adrenocortical hormones may be involved in
the growth of chick embryos.
The data confirm and extend the work of Betz (1967) and they support the
conclusion of Hadorn (1961), Herrmann, Clark & Landauer (1963) and
Allenspach (1966) that the lethality of the syndrome of the crooked neck dwarf
mutant in chick embryos is not due to chronic starvation caused by esophageal
atresia as proposed by Pun (1954). However, these results would explain the
reduced body size which is a characteristic of these mutant embryos.
SUMMARY
The growth which occurs in hypophysectomized chick embryos is independent
of pars distales hormones and it is not affected by albumen ingestion. However,
in embryos with intact partes distales and in hypophysectomized embryos with
pars distalis grafts the additional growth which occurs depends on hormones.
If the dietary intake of these embryos is variously restricted, the amount of
growth is proportionately limited.
RESUME
Les effets de la grejfe de pars distalis embryonnaire et de Valbumen,
sur la croissance d'embryons de poulet
La croissance d'embryons de poulet hypophysectomises est independante des
hormones de la adenohypophyse et n'est pas affectee par l'ingestion d'albumen.
Neammoins, chez les embryons a adenohypophyse intacte et chez es embryons
hypophysectomises porteurs de adenohypophyse greffee, la croissance additionnelle observee est sous la dependance d'hormones. Si l'absorption alimentaire
de ces embryons est reduite de maniere variable, le degre de croissance est proportion nellement limite.
I wish to acknowledge the expert technical assistance of Mr James Bell and particularly of
Miss Blanche Vachon. I am especially indebted to Professor A. E. Wilhelmi for a critical
review of the manuscript.
436
T. W. BETZ
This research was supported in part by a grant to Dr Ray L. Watterson from the Graduate
School of the University of Illinois and by a grant from the National Research Council of
Canada and the Ontario Department of University Affairs.
REFERENCES
ALLENSPACH, A. L. (1966). The reopening process of the esophagus in the normal chick
and the crooked neck dwarf mutant. J. Embryol. exp. Morph. 15, 67-76.
BENOIT, JEAN A. A. (1962). Le role de l'hypophyse dans le developpement des embryons de
vertebres amniotes a sang chaud. Ann. Biol. 1, 297-335.
BETZ, T. W. (1965 a). Correction of defects induced by 'hypophysectomy' in chicken embryos
by grafts of embryonic anterior pituitary glands. Doctoral Dissertation. University of
Illinois.
BETZ, T. W. (19656). Unpublished data.
BETZ, T. W. (1967). The effects of embryonic pars distalis grafts on the development of hypophysectomized chick embryos. Gen. Comp. Endocrinol. 9, 172-86.
CASE, J. F. (1952). Adrenal cortical-anterior pituitary relationships during embryonic life.
Ann. N.Y. Acad. Sci. 55, 147-58.
FUGO, N. W. (1940). Effects of hypophysectomy in the chick embryo. /. exp. Zool. 85, 271-97.
HADORN, E. (1961). Developmental Genetics and Lethal Factors. London: Methuen and Co.,
Ltd.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. /. Morph. 88, 49-92.
HERRMANN, H., CLARK, E. M. & LANDAUER, W. (1963). Muscle development in the crooked
neck dwarf mutant and in the acetylpyridine-treated chick embryo. Acta Embryol. Morph.
Exptl. 6, 169-74.
HINNI, J. B. & WATTERSON, R. L. (1963). Modified development of the duodenum of chick
embryos hypophysectomized by partial decapitation. /. Morph. 113, 381-426.
NALBANDOV, A. V. (1963). Symposium on growth: endocrine causes of growth and growth
stasis. /. An. Sci. 22, 558-60.
PUN, C. F. (1954). The crooked neck dwarf lethal syndrome in the domestic fowl. /. exp.
Zool. 126, 101-34.
ROMANOFF, A. L. & ROMANOFF, A. J. (1949). The Avian Egg. New York: Wiley.
SIMPSON, M. E., ASLING, C. W. & EVANS, H. M. (1950). Some endocrine influences on skeletal
growth and differentiation. Yale J. Biol. Med. 23, 1.
STOLL, R. (1939). L'agenesie de l'hypophyse et de la thyroide est sans influence sur le developpement de l'embryos de poulet. C.R. Soc. Biol. 130, 926-8.
THOMMES, R. C. (1967). Personal communication.
VOGEL, N. W. (1965). Growth in chick embryos hypophysectomized by 'decapitation'.
Proc. Penn. Acad. Sci. 39, 57-60.
WITSCHI, E. (1949). Utilization of the egg albumen by the avian fetus. Contrib. Ornithol. Biol.
Wissensch. Winter, 111-22. Heidelberg.
WITSCHI, E. (1959). See comments in discussion of A. Raynaud, In Comparative Endocrinology, A. Gorbman ed., p. 476. New York: Wiley and Sons.
WOLFF, E. & STOLL, R. (1937). Le role de l'hypophyse dans le developpement embryonnaire
du poulet d'apres l'etude des cyclocephales experimentaux. C.R. Soc. Biol. 126, 1215-17.
{Manuscript received 25 March 1968)