/. Embryol. exp. Morph. 97, 87-94 (1986)
87
Printed in Great Britain © The Company of Biologists Limited, 1986
The role of ultimobranchial bodies in the modulation of
the response of chick embryos to 1,25-dihydroxycholecalciferol
ROBERTO NARBAITZ AND JAFFAR SOLEIMANI RAD
Department of Anatomy, Faculty of Health Sciences, University of Ottawa,
451 Smyth Road, Ottawa, Canada
SUMMARY
Ultimobranchial bodies (UBBs) were dissected from 17-day-old chick embryos and grafted
onto the chorioallantoic membrane of 8-day-old embryos. The embryos with UBB grafts as well
as sham-grafted controls were injected on the 10th day of incubation with 100 ng 1,25(OH)2D3
dissolved in ethyl alcohol or with an equal volume of ethyl alcohol alone; embryos were
sacrificed on the 13th day. Grafted UBBs showed ultrastructural characteristics typical of
actively secreting glands. A histological study of the tibiae from all embryos showed that while
the grafted embryos responded to the injection of 1,25(OH)2D3 with a peripheral rim of
undermineralized bone trabeculae, sham-grafted embryos never did so. These results confirm
the original hypothesis that the presence of differentiated UBBs is a precondition for the
production of undermineralized bone (osteoid) by 1,25(OH)2D3.
In a second series of experiments, similarly treated embryos were sacrificed on the 10th, 11th,
12th and 13th day; the levels of calcium and inorganic phosphate were determined in their
blood. The injection of 1,25(OH)2D3 produced in all embryos hypercalcaemia and hypophosphataemia. However, the hypophosphataemic response was more prolonged in the embryos
with UBB grafts than in sham-grafted ones. These results suggest that the grafted UBBs
prolonged the hypophosphataemic response, probably by secreting calcitonin and thus reducing
the rate of bone resorption. It is also probable that the prolonged hypophosphataemia produced
or contributed to the undermineralization of the peripheral (subperiosteal) trabeculae.
INTRODUCTION
lar,25-dihydroxycholecalciferol (1,25(OH)2D3) is probably the most active metabolite of vitamin D 3 ; the mechanisms by which its synthesis is regulated and the
mechanisms by which it acts on target tissues are so similar to those observed in
steroid hormones in general that it has been suggested that it should be considered
a steroid hormone (DeLuca, 1978).
The injection of large doses of 1,25(OH)2D3 to chick embryos produces hypercalcaemia and it has been suggested that it does so by increasing the resorption of
shell mineral through the chorioallantoic membrane (Narbaitz & Tolnai, 1978).
In support of this suggestion, it has been shown that, after the injection of
Key words: ultimobranchial bodies, calcitonin, vitamin D, chick embryo, calcium, phosphate,
1,25-dihydroxycholecalciferol.
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R. NARBAITZ AND J. SOLEIMANI R A D
1,25(OH)2D3, carbonic anhydrase activity in the chorioallantoic membrane increases following a time course similar to the one followed by the increase in blood
calcium observed after the injection of the same dose of 1,25(OH)2D3 (Narbaitz,
Kacew & Sitwell, 1981). Carbonic anhydrase appears to be required for the
solubilization and/or the absorption of shell mineral (Tuan, 1983) and has been
localized histochemically in the 'villus-cavity' cells of the chorionic epithelium
(Rieder, Gay & Schraer, 1980) which are precisely the same cells that have been
shown to be targets for 1,25(OH)2D3 (Narbaitz, Stumpf & Sar, 1981).
In addition to hypercalcaemia, the injection of 1,25(OH)2D3 to the chick
embryo produces hypophosphataemia and interrupts the mineralization of newly
formed bone trabeculae at the periphery of the diaphysis of long bones (Narbaitz
& Tolnai, 1978). The mechanisms responsible for these changes are still unclear.
Narbaitz & Fragiskos (1984) supplied significant experimental evidence leading
to the conclusion that bone undermineralization induced by 1,25(OH)2D3 is
secondary to the concomitant humoral alterations. However, this conclusion was
contradicted by the results of one of their experiments: while they succeeded in
inducing hypercalcaemia and hypophosphataemia by injecting 1,25(OH)2D3 as
early as the 9th day of incubation they failed to obtain bone lesions when injecting
the hormone before the 11th day (Narbaitz & Fragiskos, 1984). Based on the fact
that ultimobranchial bodies (UBBs) are believed to begin their differentiation
precisely on the 11th day of incubation (Stoeckel & Porte, 1969) the above
discrepancy may be explained by suggesting that the presence of functional UBBs
might be a precondition for the production of undermineralized bone (osteoid) by
1,25(OH)2D3.
The present experiments have been conducted in an attempt to find experimental support for this hypothesis.
MATERIALS AND METHODS
Fertile eggs from White Leghorn hens were used in all our experiments. They were incubated
in a forced-draught incubator at 37-5 °C and 60% R.H. UBBs from 17-day-old embryos were
used as grafts: they were dissected under sterile conditions and kept in Hanks' solution until
grafted. 8-day-old embryos were used as hosts; a small window was drilled in the shell, the shell
membranes were opened and three UBBs were deposited on the chorioallantoic membrane
close to large blood vessels. In sham-operated embryos the procedure was similar, but only a
small amount of Hanks' solution was deposited on the chorioallantoic membrane. Both grafted
and sham-grafted embryos were injected either with 100 ng 1,25(OH)2D3 in 0-05 ml absolute
ethyl alcohol or with a similar volume of ethyl alcohol alone (1,25(QH)2D3 was kindly supplied
by Dr M. Uskokovic, Hoffmann La Roche Laboratories, Nutley, NJ). All injections were made
into the yolk sac through the same window that was used for grafting. The dose of 1,25(OH)2D3
used in our experiments was selected according to previous experience (Narbaitz & Fragiskos,
1984) that indicated that this was the minimal dose capable of inducing bone defects in 11-dayold embryos. Embryos were sacrificed on the 13th day of incubation. The chorioallantoic grafts
were dissected and fixed in half-strength Kamovsky's fixative (Karnovsky, 1965); UBBs from
17-day-old control embryos were similarly fixed and used as controls. All tissues were postfixed
in osmium tetroxide, dehydrated in ethyl alcohol and embedded in Araldite. 1 jum sections were
stained with toluidine blue for light microscopy and thin sections with uranyl acetate and lead
citrate according to Reynolds (1963) for electron microscopy. One tibia from each embryo was
fixed and embedded as indicated above; 2/zm cross sections through the mid-diaphysis were
UBBs and response of chick embryos to 1,25-(OH)2D3
89
stained with alizarin red (Narbaitz & Fragiskos, 1984) and observed by light microscopy in order
to determine the degree of mineralization of bone trabeculae.
In a second series of experiments more than 200 embryos were divided into four groups which
received the following treatments: (a) grafted and injected with 1,25(OH)2D3; (b) grafted and
injected with ethyl alcohol; (c) sham-grafted and injected with 1,25(OH)2D3; and (d) shamgrafted and injected with ethyl alcohol. As in the first series of experiments all grafts were made
on the 8th day and all injections on the 10th day. Eggs were opened on the 11th, 12th or 13th day
and embryos bled from a branch of the allantoic artery. Sera were separated by centrifugation;
colorimetric tests were used for both the determination of total calcium (Calcium colorimetric
assay kit, D.K. 1100-20, Fisher Scientific Limited) and of inorganic phosphate (Catalized
Phosphorus kit, MC6500, American Monitor Corporation, Indianapolis, IN). Statistical
significance of the differences between different means was established using ANOVA followed
by Tuckey's HSD test (Spence, Cotton, Underwood & Duncan, 1983).
RESULTS
In each of the grafted embryos at least one of the grafted UBBs survived and
was well vascularized. The histological and ultrastructural characteristics of the
grafted UBBs did not differ, when examined 5 days after implantation, from those
of the UBBs from control 17-day-old embryos. In both cases the glands were
formed by cords of cells rich in mitochondria and containing large numbers of
secretory granules; granules were rounded or oval-shaped, were very electron
dense and measured between 100 and 250 nm (Fig. 1). In both the grafts and the
control UBBs some of the cells contained granules with morphological characteristics similar to those described above but of larger size (250-400 nm) (Fig. 2).
The number of granules per cell as well as the proportion of cells with large and
small granules varied from zone to zone in any one gland and from gland to gland
in each experimental group and no quantification was attempted. Granules with
different degrees of electron density were sometimes found (Fig. 2) and were
interpreted as representing stages in their maturation process. In all cases the
connective tissue separating the glandular cords contained cells with large granules
which, following previous literature (Belanger, 1971), were identified as pseudoeosinophils.
Seven out of eleven embryos holding successful grafts and receiving a dose of
1,25(OH)2D3 on the 10th day had tibiae with a peripheral ring of undermineralized
trabeculae (Fig. 4; compare with Fig. 3). Undermineralization was never observed
in any one of the more than 30 sham-grafted embryos injected with 1,25(OH)2D3
at the same age.
The concentrations of Ca 2+ and Pj in the serum from 10-day-old embryos that
had received UBB grafts did not differ significantly from those of control embryos
of the same age. These values were averaged and are included in Table 1 as
preinjection means for the 10th day.
Table 1 shows that the injection of 1,25(OH)2D3 produced a significant hypercalcaemia 24 h after the injection; the concentration of calcium returned to the
initial values on the second day. Similar response to 1,25(OH)2D3 was observed in
the embryos previously grafted with UBBs. The concentration of P, in the serum
diminished significantly 24 h after the injection of 1,25(OH)2D3 and returned to
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R. NARBAITZ AND J. SOLEIMANI RAD
UBBs and response of chick embryos to 1,25-(OH)2D3
91
normal on the 2nd day; however, in embryos that had been previously grafted with
UBBs the hypophosphataemia was more prolonged and phosphate values
returned to normal only on the 3rd day after the injection.
DISCUSSION
Chorioallantoic grafts have been extensively used by experimental embryologists (Hamburger, 1947). The grafts are known to become vascularized very
rapidly and, in the case of endocrine glands, to function and affect correspondingly
the host embryo (Betz, 1967; Woods & Weeks, 1969). In our present experiments
it was found that UBBs from 17-day-old embryos grafted into 8-day embryos
survived well and that after 4 days in the host they still presented all the
ultrastructural characteristics of actively secreting glands. Thus, their cells
contained numerous secretory granules of size and morphological characteristics
similar to those that in adult (Stoeckel & Porte, 1967; Belanger, 1971) and
embryonic (Stoeckel & Porte, 1969) glands have been identified as calcitonin
granules. Moreover, the presence in the cells of rough endoplasmic reticulum,
abundant mitochondria and granules in different stages of maturation strongly
suggests that the grafted glands were capable of producing the hormone. In order
to find out to what extent calcitonin was being actually secreted into the blood
stream of the host we would have had to determine the levels of calcitonin in the
host's blood; we did not attempt to do this because the radioimmunoassay
techniques presently available are not sensitive enough to detect circulating levels
of calcitonin in the chick embryo before the 17th day of incubation (Baimbridge &
Taylor, 1980).
Despite the above reservation, our results demonstrate without doubt that the
grafted UBBs were capable of influencing their hosts. Thus, many of the embryos
with successful grafts which were injected with 1,25(OH)2D3 on the 10th day
responded by interrupting the mineralization of their bone trabeculae while shamgrafted embryos injected at the same age and with the same dose never did so.
This finding strongly supports our original hypothesis in the sense that the
presence of functional UBBs is a precondition for the production of bone
undermineralization by 1,25(OH)2D3 (see Introduction).
The presence of the UBB grafts not only facilitated the production of undermineralized bone but also modified somewhat the humoral response to 1,25(OH)2D3:
the ensuing hypophosphataemia lasted for 2 days in the embryos with grafts
instead of 1 day as it did in the case of sham-grafted embryos. The mechanism(s)
by which the grafted UBBs prolonged the hypophosphataemic response is (are)
not clear. However, it is known that in long bones the bone trabeculae at the inner
(endosteal) surface of the diaphysis are continuously being resorbed by osteoclasts
Fig. 1. Electron micrograph showing part of a cord of cells of a UBB from a control
17-day-old embryo. Note the large number of secretory granules, e, pseudo-eosinophil
cell in the connective tissue.
Fig. 2. Electron micrograph showing cells from UBB graft 5 days after implantation. It
shows a group of cells containing large secretory granules.
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R. N A R B A I T Z AND J. S O L E I M A N I R A D
Fig. 3. Cross section of a tibia from a 13-day-old embryo that was sham-grafted on the
8th day and injected with 1,25(OH)2D3 on the 10th day. Most trabeculae are well
mineralized as indicated by their deep staining by alizarin, m, marrow cavity. Plasticembedded, undecalcified. Alizarin stain.
Fig. 4. Cross section of a tibia from a 13-day-old embryo that had received UBB grafts
on the 8th day and was injected with 1,25(OH)2D3 on the 10th day. Note that most
trabeculae at the periphery of the bone (p) are undermineralized as indicated by their
lack of alizarin staining, m, marrow cavity. Plastic-embedded, undecalcified. Alizarin
stain.
Table 1. Calcium and inorganic phosphate concentrations in the sera of grafted and
sham-grafted embryos at various intervals after the administration ofl,25(OH)2D3
Time after
injection
(h)
0
24
48
72
Total calcium (mg dl )
Inorganic phosphate (mg dl )
Graft+ 1,25
Sham+ 1,25
Graft+ 1,25
Sham+ 1,25
a
a
a
6-19±0-30a
5-31±0-42b
5-87 ± 0-71a
6-19±0-28a
9-58 ± 0-32
12-28 ± 0-74b
10-30 ±0-52a
9-82 ± 0-25a
9-68 ± 0-40
12-13 ±0-98b
10-10 ±0-41 a
9-75 ± 0-29a
6-27 ± 0-37
5-15±0-43b
5-19±0-41b
5-97±0-44a
Figures are means of at least 10 samples ± S.D.
Grafts or sham-grafts were made on the 8th day and injection of 1,25(OH)2D3 was on the 10th
day.
Means within a column with a different superscript are significantly different, F<0-01;
ANOVA followed by Tuckey HSD test.
UBBs and response of chick embryos to 1,25-(OH)2D3
93
while new trabeculae are being continuously laid down at the outer (subperiosteal)
surface (Fell, 1925; Romanoff, 1960; Volpi et al. 1981). During this remodelling
process the phosphate from the resorbed trabeculae contributes to the pool from
which the new trabeculae obtain their mineral. Since calcitonin is known to reduce
osteoclastic bone resorption (Talmage, Cooper & Toverud, 1983) it can be
suggested that in our experiments the secretion of this hormone by the grafted
UBBs might have reduced resorption of the endosteal trabeculae and thus
contributed to prolonging the hypophosphataemic response.
Calcitonin, parathyroid hormone and 1,25(OH)2D3 are known to affect the
process of bone mineralization but there is no convincing evidence that they do
this through direct action on bone cells (see review by Stern, 1980); rather, they
appear to affect mineralization by altering the concentration of calcium and/or
phosphate in the plasma (Stern, 1980). For this reason it is probable that in our
experiments it was the prolonged hypophosphataemia that produced or contributed to the undermineralization of the bone trabeculae formed after the injection
of 1,25(OH)2D3.
This project was supported by a grant from the Medical Research Council of Canada to the
senior author. The skilful technical assistance of Mr Vijay K. Kapal is gratefully acknowledged.
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(Accepted 3 June 1986)