A M . ZOOLOGIST, 7:843-855 (1967).
Parathyroid Glands of Amphibians. I.
Parathyroid Structure and Function in the Amphibian, with Emphasis on
Regulation of Mineral Ions in Body Fluids
John R. Cortelyou and Dolores J. McWhinnie
Department of Biological Sciences, De Paul University, Chicago, Illinois 60614
SYNOPSIS. Parathyroid glands of amphibians develop from the pharyngeal pouches
and are located adjacent to the external jugular vein and hypoglossal nerve. They
are composed of whorls of compact epithelial cells covered by a vascular capsule of
connective tissue. Possibly two cell types are present: (a) peripheral clear cells, which
are implicated in elaboration of hormone and contain secretory granules, and (b) dense
reserve cells. During winter, the parathyroids undergo degeneration, characterized by
morphological details which vary with the species; this is followed by regeneration in
the spring. Parathyroidectomy may cause hyperexcitability, but rarely does tetanic
death occur. Parathyroidectomized amphibians, like mammals, become hypocalcemic
and hyperphosphatemic. In contrast to higher vertebrates, however, parathyroprivic
anurans show an elevated urine phosphorus, which is presently interpreted as a reflection of the increased plasma load due to absence of a hormonally-induced, soft-tissue
accumulation of phosphate. Further, parathyroidectomized frogs are hypercalciuric,
possibly because of the lack of hormone-mediated mesonephric reabsorption. In both
mammals and amphibians, treatment with exogenous parathormone induces hypercalcemia, hypercalciuria, and hypophosphatemia. The absence of change in the urine
phosphorus content of hormone-treated frogs, associated with its decline in plasma,
may be a consequence of metabolic variations based upon the heterothermic nature
of the Amphibia.
STRUCTURE OF THE GLAND
The parathyroid glands of amphibians
were discovered by Leydig in 1852, and
Maurer (1888) described their development
from the ventral surfaces of the pharyngeal
pouches. The glands develop during larval
stages in anurans, but at the time of metamorphosis in urodeles (Baldwin, 1918);
they are not found in neotenous axolotls or
Necturus. In general, two pairs are present
in the Anura (except Bombina bombina
and Alytes obstetricus; von Brehm, 1963a),
whereas a single pair is found in urodeles
(e.g., Salamandra atra, S. maculosa, Triton
alpestris, Scholz, 1933; Triturus viridescens, Cortelyou, unpublished observations).
Scholz (1933), however, found 4 glands in
several species (Triton cristatus, T. taeniatus).
Anuran parathyroids are located in the
pre-cardial region, lateral and posterior to
the thyroid, dorso-lateral to the hypoglossal
nerve, and adjacent to the external jugular
This work was supported by research grants Nos.
G-13403 and GB-1485 from the National Science
Foundation.
vein; the vascular Kiemenreste lies medial
to the parathyroids (Waggener, 1929; Cortelyou, et ah, 1960). In urodeles, they are
found in a similar location (Scholz, 1933).
Accessory parathyroid tissue has been identified in some species of anurans (von
Brehm, 1963a).
The parathyroid glands of these lower
vertebrates are composed of compact whorls
of epithelial cells. Surrounding the parenchyma is a capsule of connective tissue containing fibroblasts, collagenous fibers,
nerves, and capillaries. Immediately under
the capsule is a basement membrane attached to the epithelial cells. Since the
gland is generally avascular (Schlumberger
and Burk, 1953; Isono, 1960; Lange and
von Brehm, 1965), except in Rana catesbeiana (Waggener, 1929), R. temporaria,
Xenopus laevis (von Brehm, 1963a), and
R. clamitans (Rogers, 1965), the pathway
for egress of hormone to the surrounding
capillary bed is unknown. However, in
some toads (Hara, et ah, 1959; Isono, 1960;
Boschwitz, 1961) and frogs (Lange and von
Brehm, 1965; Rogers, 1965) intercellular
canals are present, which are often filled
843
844
JOHN R. CORTELYOU AND DOLORES J. MCWHINNIE
with a dense material, possibly representing
the secretory product (Yamada, 1963).
The cells of the parathyroids are packed
closely, with an average of 10 mju, intercellular distance, and are interdigitated in R.
temporaria and Bufo vulgaris (Lange and
von Brehm, 1965). The parathyroid cells
of R. esculenta have smoother surfaces, and
occasionally the membranes are discontinuous; this has been suggested as an indication of communication between cells and
the external environment (Montsko, et al.,
1963a). Attachment zones with tonofibrils
have been observed in parathyroid cells of
R. clamitans (Rogers, 1965).
Although only a single cell type has been
definitively identified in amphibian parathyroids (Shapiro, 1933; Cortelyou, et al.,
1960; Boschwitz, 1961; Montsko, et al.,
1963a), there is some evidence that a functional difference exists in R. clamitans between dark and light parathyroid cells
(Rogers, 1965). The dark cells, forming the
main mass of the gland, are rich in organelles and secretory granules; light cells,
found near blood vessels of the subcapsular
plexus and parenchyma, have few mitochondria, Golgi bodies, and secretory granules, and these have been interpreted as
inactive by Rogers (1965).
A somewhat different localization of active and inactive cells was described for
toads and R. temporaria by Isono (1960)
and Lange and von Brehm (1965). Peripheral cells, designated as clear cells, are larger
and have a lighter nucleus than the elongated, central, dense cells. These cell types
also vary in fine structure and cytochemical
characteristics. Dense cells contain few mitochondria and poorly developed organelles. In contrast, the clear cells have welldeveloped organelles, including numerous
mitochondria, lipid droplets, vesicular and
lamellar Golgi, membrane-bound multivesicular compound bodies, and endoplasmic reticulum composed of rough vesicles,
tubules or flattened sacs, and free clusters
of ribosomes (Montsko, et al., 1963a; Lange
and von Brehm, 1965). Moreover, the clear
cells contain a higher proportion of oxidative enzymes, basophilia, lipids, and glycogen than is evident in dense cells (Isono,
el al., 1959; von Brchin, 1963«). As a consequence of these differences in ultrastructure and chemical components, it was inferred that the clear cells are responsible
for elaboration of hormone, while the dense
cells are interpreted to be resting or reserve
cells (von Brehm, 1963a).
Further evidence for the involvement of
clear cells in synthesis of parathormone was
adduced by the presence of membranebound secretory granules, 0.1 to 1.5 /*. in
diameter. These have been observed in B.
vulgaris (Hara, et al, 1959; Isono, 1960;
Lange and von Brehm, 1965), R. esculenta
(Montsko, et al., 19636), R. temporaria
(Lange and von Brehm, 1965), and R. clamitans (Rogers, 1965). The origin of the
granules, which contain dense paniculate
material, is unknown, and no clear association exists between them and the endoplasmic reticulum or Golgi apparatus.
However, it has been suggested by Lange
and von Brehm (1965) that the dense material occasionally seen in the Golgi substance of parathyroid cells in R. temporaria
represents prosecretory granules. No granules have been seen in the Golgi apparatus
of R. esculenta, but Montsko, et al. (19636)
described the budding of vesicles from the
Golgi material, which became filled with
paniculate material and are then indistinguishable from typical secretory granules.
The secretory granules may be identical to
the DDD-diazo-blue B granules in the clear
cells of the parathyroids of B. vulgaris
(Yamada, 1963). These granules have also
been observed on the outer surface of the
cell membrane, and within the intracellular channels, indicating that they may be
the secretory product of the gland. Since,
however, the DDD-diazo-blue B reaction
(2,2'-dihydroxy-6,6'-dinaphthyl disulfide)
identifies disulfide and sulfhydryl groups,
neither of which are numerous in parathormone, Yamada (1963) has suggested that
the hormone is associated with a sulfhydrylrich carrier protein.
Recent electron microscopic studies on
the parathyroid glands of R. pipiens have
been done in our laboratories. Low magnifications (Fig. 1) show large nuclei containing 1 to 2 nucleoli. Mitochondria are
PARATHYROID GLANDS OF AMPHIBIANS
845
present, as are large and small vesicles
which are variously filled with homogene-
ous material. Occasonally, however, fine
dense particulates have been observed with-
FIG. 1. Survey electron micrograph of a portion of
the parathyroid gland of liana pipiens. Nuclear
profiles (X) are oval with indentations, nucleoli
(n), and dispersed chromatin. Numerous mitochondria (M) and vesicles (V) are present. Cell mem-
branes
bound
contain
ments.
(C) are extensively folded. MembranemultivesicuJar compound bodies (MVC)
particulates, vesicles, and membrane fragX 7,500.
846
JOHN R. CORTELYOU AND DOLORES J. MCWHINNIE
in these vesicles.
Additionally, large membrane-bound bodies are seen, which contain small vesicles,
remnants of membranes, and particulates
(Figs. 1, 2); these appear to be indistinguishable from the multivesicular com-
FIG. 2. Low power view of frog parathyroid cells.
Many mitochondria (M), nuclei (N), vesicles (V),
and large multivesicular compound bodies (MVC)
are scattered through the cytoplasm. Note the
widening between cells to torm expanded intercellular channels (IC) in which slender cytoplasmic
projections (P) are often seen. X 14,000.
PARATHYROID GLANDS OF AMPHIBIANS
847
pound bodies described by Lange and von
Brehm (1965) in the parathyroid cells of
R. temporaria. Early stages of the multi-
vesicular bodies are represented by membrane-bound structures partially filled with
small vesicles and membrane fragments
FIG. 3. Several frog parathyroid cells showing
prominent mitochondria (M). Note the vesicles (V)
of various sizes which appear to be filled with
homogeneous material. Several multivesicular compound bodies (MVC) are present which are only
partially filled with small, clear vesicles, X H.Q00.
JOHN R. CORTELYOU AND DOLORES J. MCWHINNIE
(Fig. 3) - The function of these bodies remains unknown, but it has been suggested
(Lange and von Brehm, 1965) that they are
related to the involution of mitochondria.
FIG. 4. High power electron micrograph of a secretory granule (S) in a parathyroid cell oC liana pipiens. The granule is membrane-bound and rilled
with fine, dense particulates. Also shown are mito-
chondria (M) with cristae, clusters of free ribosomes (R), intercellular channels (IC) the nucleus
(X), and cell membrane (C). X 56,000.
PARATHYROID GLANDS OF AMPHIBIANS
849
The scant endoplasmic rcticiilum in the 1965). The number and density of secretory
parathyroid cells of R. pipiens is largely granules in the parathyroid cells of it. escuvesicular. Numerous ribosomes, many free lenta which have been stimulated by inin clusters (Fig. 4), and mitochondria with jection of EDTA also increased, accomclearly defined cristae (Figs. 3, 4) are scat- panied by a rise in the number of ribotered throughout the cytoplasm. Irregular somes (Montsko, et al., 1963ft). These
intercellular channels (Figs. 2, 4), which events have been interpreted differently:
are similar to those observed in B. viridis Montsko, et al. (19636) presented the view
(Boschwitz, 1961), B. vulgaris (Hara, et al., that the parathyroids of R. esculenta have
1959; Isono, 1960), R. damitans (Rogers, a single cell type, and that hyperfunction is
1965) and R. temporaria (Lange and von attained through the elaboration of organBrehm, 1965) may provide a means of trans- elles. In contrast, von Brehm (1964) and
port for the secretory product to the sub- Lange and von Brehm (1965) have stated
capsular capillary network. The Golgi sub- that the parathyroids of R. temporaria,
• stance of the parathyroid cells of R. pipiens stimulated to hyperfunction by injection of
is similar to that of R. temporaria (Lange phosphate salt or warm-acclimation, beand von Brehm, 1965) and R. damitans come so by conversion of resting dense cells
(Rogers, 1965), consisting of curved stacks to active clear cells.
of flattened cisternae with numerous vesicles at their expanded distal portions.
SEASONAL CHANCES IN STRUCTURE OF GLANDS
Secretory granules of similar appearance
For many years, it has been reported that
to those described in parathyroid gland
cells of R. esculenta (Montsko, et al., parathyroid glands of anuran amphibians
19636), R. damitans (Rogers, 1965), and undergo cyclical histological changes in reR. temporaria (Lange and von Brehm, sponse to seasonal fluctuations. Similar
1965) are present in R. pipiens (Fig. 4). studies have not been done with urodeles,
The granules are membrane-bound, and and although Scholz (1933) occasionally obdensely filled with fine, paniculate grains, served a lumen in the parathyroids of Salawhich are packed to yield a crystalline ap- mandra atra and 5. maculosa, he attributed
pearance. In contrast to the smaller secre- this to captivity rather than to seasonal
tory granules, the membrane of large gran- change. The parathyroids of many frogs
ules is closely applied to the participate and toads degenerate in winter (Romeis,
1926; Waggener, 1929; Boschwitz, 1965).
contents.
In
general, the cells show nuclear chromatoExperimental manipulation of blood calcium levels, which presumably control the lysis and pyenosis, vacuolation, and loss of
functional state of the parathyroids, induces structural integrity. However, certain
changes in cellular structure which strength- species-differences exist with respect to the
en the view that the clear cells are involved morphological details and timing of the
in synthesis of hormone. Injecting calcium seasonal changes. In R. pipiens (Cortelyou,
salts into B. vulgaris or R. temporaria (von et al., 1960) the entire gland becomes reticuBrehm, 1964) increased the number of dense larized except for a layer of apparently
cells, and decreased that of the clear cells; viable cells under the capsule; these cellular
those remaining became vacuolated. De- changes begin in late fall and continue
pressing the parathyroids of R. esculenta by through winter. Formation of a central
injecting calcium salts (Montsko, et al., lumen has been reported only in R. cates19636) decreased the number of secretory beiana (Waggener, 1929) and B. viridis
granules and other organelles. In contrast, (Boschwitz, 1965).
stimulation of the parathyroids of R. temAlthough cytolysis appears to be initiated
poraria by injecting phosphate salts in- in the center of the gland in B. viridis
creased the ratio of clear/dense cells, and (Boschwitz, 1965), R. catesbeiana (Wagincreased the number of secretory granules gener, 1929) and R. pipiens (Cortelyou, et
in the clear cells (Lange and von Brehm, al., 1960), observations reported by Romeis
850
JOHN R. CORTELYOU AND DOLORES J. MCWHINNIE
(1926) and von Brehm (1963&, 1964) indicate that vacuolation is predominantly in
peripheral cells of the parathyroids of R.
temporaria. Unlike Romeis (1926), however, von Brehm (1964) did not observe
extensive reticularization. Similar peripheral changes were reported in the glands of
B. vulgaris, in contrast to the observation
of Hara and Yamada (1963) that in February only the central cells of the gland in
this species were vacuolated. It is possible
that these differences in the location of
cellular changes are due to variations in
time sequences at which the animals were
studied, differences between species, and the
latitudinal position at which the investigations were done.
The vacuoles are lipid-free in parathyroids of R. catesbeiana (Waggener, 1929)
and R. temporaria (Romeis, 1926), but in
those of B. vulgaris, Hara and Yamada
(1963) identified neutral and acidic lipids.
They suggested that the vacuoles are not
indicative of degeneration, but represent a
source of phospholipids for the reconstruction of mitochondria and Golgi substance,
and thus a reaction preparatory to increasing the metabolic rate on emergence from
hibernation. Until further study, however,
the possibility remains that the vacuolar
contents are end-products of organelle degeneration.
The association between the clear cells
and activity of the gland has been elucidated by studies of their fluctuations with
season or temperature. Parathyroids of
normal winter, or cold-acclimated R. temporaria and B. vulgaris have predominantly
dense cells and few, vacuolated clear cells,
while those from summer or warm-acclimated animals, have many clear cells, and
few dense cells (Isono, 1960; Lange and von
Brehm, 1965). Further, the DDD-diazo-blue
B granules of the clear cells are numerous
in summer and absent in winter (Yamada,
1963). Isono, et al., (1959) have suggested
that the spring decline in glycogen within
the clear cells indicates that it is being
utilized as an energy source during reactivation, and during the active summer months.
A simultaneous decrease in lipids in the
clear cells has been reported (von Brehm,
1963b). The greater activity of the glands
in summer is also indicated by the increased
capillary diameters (Isono, 1960).
Regardless of the conflicting details of
seasonal variations in amphibian parathyroid glands, it is clear that during winter,
morphological and cytochemical changes
occur. Vacuolation has been variously interpreted as degeneration (Romeis, 1926;
Waggener, 1929; Cortelyou, et al, 1960),
activation (Isono, 1960; Hara and Yamada,
1963; Boschwitz, 1965), or production of
hormone exceeding its need (Lange and
von Brehm, 1965). If it is true, however,
that the clear cells are responsible for elaboration of hormone, and that they decrease
in number and become vacuolated, (a) during winter, (b) with cold-acclimation, or
(c) when activity is depressed by injection
of calcium, it is more probable that the
vacuolation and reticularization of the
gland seen in many winter toads and frogs
represents cellular degeneration and a decrease in hormonal synthesis and/or secretion.
Regeneration of the gland from central
growth areas (Romeis, 1926), from discrete
subcapsular growth centers (Waggener,
1929), from a layer of viable cells under the
capsule (Cortelyou, et al., 1960; Boschwitz,
1965), or by conversion of dense to clear
cells (Lange and von Brehm, 1965) is completed in spring.
GLANDULAR FUNCTION—PARATHYROIDECTOMY
Early studies of amphibian parathyroids
implicated the glands in such activities as
control of pigmentation, retardation of
growth (Thompson and Huxley, 1934),
accessories of thyroid function, detoxification of metabolic by-products (Uhlenhuth,
1918, 1921), and depression of heart activity
(Takacs, 1927). However, experimental investigations, either with ablation of gland
or addition of hormone, have clearly indicated that, as in mammals, the parathyroids
of amphibians regulate mineral metabolism.
Parathyroidectomy of most amphibians
does not cause tetany and death. Parathyroprivic 5. atra and S. maculosa showed no
PARATHYROID GLANDS OF AMPHIBIANS
tetany through one year (Scholz, 1935), and
no overt hyperexcitability has been observed in R. temporaria (Romeis, 1926), R.
pipiens (Cortelyou, et al., 1960), or Triturns
viridescens (Cortelyou, unpublished). In
contrast, Boschwitz (1961) reported that
parathyroidectomized B. viridis were hyperexcitable, and tetany occurred in 2 to 8
days.
Waggener (1930) found severe tetany in
parathyroidectomized R. catesbeiana, and
death followed in about 66 hours. There is
recent evidence, however, which does not
support these observations. Yoshida and
Talmage (1962) parathyroidectomized R.
catesbeiana, but made no mention of hyperexcitability although the frogs were observed for 2 days. Further, Houston and
Husbands (1965) stated that no hyperexcitability, tetany, or death occurred in
parathyroprivic bullfrogs through 7 days
after ablation of glands. In light of these
data, the early results of Waggener (1930)
on this species must be held open to
question.
Although no spontaneous nervous activity has been observed in parathyroidectomized tree or grass frogs, muscle-nerve
preparations have shown that some increase
in excitability occurs. Kuffler (1945) reported that parathyroprivic Hyla aurea
show a lowered threshold of synapses and
motor endplates. Isolated gastrocnemius
muscle-sciatic nerve preparations have been
studied by Cortelyou, et al. (1960) using
parathyroidectomized R. pipiens. Myographs were made of responses to stimuli
of graded frequencies at 2 to 5 days, and
2 to 12 weeks after ablation of glands.
Approximately 50% of the frogs studied
during the first week showed increased
excitability; the motor responses of treppe,
incomplete, and complete tetanus occurred
at lower stimulating frequencies than were
required for normal or sham-operated animals. Hyperexcitability was further evidenced by a protracted increase in relaxation time. However, by 2 weeks after parathyroidectomy, the responses generally returned to normal.
These subtle changes in neuro-muscular
responses are reflections of blood calcium
851
fluctuations induced by parathyroid ablation. Although Zhenevskaya (1948) found
no change in blood calcium in parathyroidectomized R. ridibunda, and only a
slight decrease in R. temporaria parathyroidectomized in spring, hypocalcemia occurs in both bullfrogs and grass frogs. Waggener (1930) found the normal whole blood
calcium of R. catesbeiana to be 11.9 mg%.
After glands were removed, and prior to
death, calcium values had decreased to
7.8 mg%. In a recent study on the same species, where no tetany or death occurred
after parathyroidectomy, Houston and Husbands (1965) determined plasma calcium
levels at daily intervals. At 24 hours, an
18% decrease was seen, and by the 6th day,
plasma calcium had fallen 43% from the
normal level.
Similarly, grass frogs become hypocalcemic after removal of glands. Cortelyou
(1958, 19626) and Cortelyou and co-workers
(I960) studied calcium changes in whole
blood and plasma of parathyroidectomized
R. pipiens. During the first 4 days, whole
blood calcium decreased 68% below the
control level of 10.3 mg%. This gradual
decline stands in contrast to the rapid fall
in blood calcium of mammals, which
reaches its lowest limit at 24 to 48 hr. The
low titer obtained on the fourth day with
R. pipiens was maintained through the
first several weeks, followed by a steady rise
to just sub-normal levels by the twentyninth week. Some parathyroidectomized
mammals also show a tendency to return to
normal blood calcium levels, but the increase is more rapid.
Similar results were obtained when plasma calcium changes were studied, although
the decline was less marked and slower
than with total blood calcium. During the
first day after parathyroidectomy, plasma
calcium was not significantly different from
the control level of 6.6 mg%. However, by
48 hr, it had dropped to 5.7 mg%, and by
the seventh day it was 38% below normal.
Hence, whether whole blood or plasma is
studied, a decrease in calcium occurs during
the first to second week after parathyroidectomy. This hypocalcemia is interpreted as
a decline in the ability to maintain normal
852
JOHN R. CORTEI.YOU AND DOLORES J. MCWHINNIE
calcium levels in the absence of parathormone-induced mobilization from bone.
In contrast to mammals, the hypocalcemia in parathyroprivic R. pipiens is not reflected by a well-defined hypocalciuria.
Rather, wide variations in urine calcium
levels occur, which represent both hypoand hypercalciuria, with a predominance
of the latter (Cortelyou, et ah, 1960; Cortelyou, 1962b). Immediately following removal of glands, hypercalciuria occurs, but
this is followed by a marked decrease in
urine calcium at 24 hr. Subsequently,
through 8 days, hypercalciuria again appears, but declines below normal from days
8 to 18. By three weeks after ablation of
glands, urine calcium approximates the
control level.
These results may indicate that the absence of parathormone impairs the ability
of the mesonephros to reabsorb calcium,
although this remains unresolved. It is
postulated that the fluctuations in urine
calcium represent attempts to maintain
mineral equilibrium. As plasma calcium
falls through the first 8 days, urine is hypercalciuric; these changes may be caused by
the expected decrease in mobilization from
bone coupled with a lack of calcium reabsorption. When, however, plasma calcium reaches its lowest limits (second to
third week), a homeostatic mechanism may
be established which elevates the renal
threshold to calcium, resulting in the observed drop in calcium excretion, and initiating the gradual increase in blood calcium
levels. Although a well-defined hypocalciuria has been established for parathyroprivic mammals, it is of interest to note
that Buchanan and co-workers (1959) also
found a transient hypercalciuria in parathyroidectomized mice, although urine calcium did return to normal within 14 hr.
Similar, but delayed responses, are evident
in the frog.
Following parathyroidectomy in mammals, hyperphosphatemia occurs; it has
long been held that since parathormone
may prevent tubular reabsorption of phosphate, in its absence increased reabsorption
causes a heightened blood phosphate and a
decrease in urine phosphate. Parathyroidec-
toniized amphibians show quite similar
changes in blood phosphorus. Houston and
Husbands (1965) reported that by the sixth
day after ablation of glands in R. catesbeiana, plasma phosphate had increased 100%.
Cortelyou (1960a, 19626) has shown with
parathyroidectomized R. pipiens that, by 2
hr, plasma phosphorus has increased 100%
from the normal value of 3.62 to 7.41 mg%.
This hyperphosphatemia persists 2 weeks,
with a gradual decline toward control levels
by day 17.
Unlike mammals, however, hypophosphaturia has not been observed in parathyroprivic amphibians. In the only studies done
on urine phosphorus, Cortelyou (1960a,
19626) reported a significant hyperphosphaturia in parathyroidectomized R. pipiens
within 2 hr after removal of glands; this
persisted 10 days, being characterized, like
the hypercalciuria, by wide fluctuations.
After day 10, the fluctuations subsided, and
urine phosphorus decreased to normal. The
duration of the hyperphosphaturia probably precludes trauma as its cause. Although Beutner and Munson (1960)
ascribed a transient increase in urine phosphate in parathyroidectomized rats to
trauma, it decreased in several minutes,
whereas the increase in frogs is maintained
significantly longer.
It is unlikely that the elevated plasma
phosphate is dependent upon decreased
blood calcium levels, since a marked hyperphosphatemia occurs in 2 hr after ablation
of glands, while plasma calcium remains
normal for 24 hr. Conversely, if the hypocalcemia were dependent upon the increased blood phosphorus, it is improbable
that over 24 hr would elapse before it appeared. Moreover, the hyperphosphatemia
may not be caused by uninhibited phosphate reabsorption in the absence of parathormone since hyperphosphaturia occurs
simultaneously and persists many days. It
may also be noted that after urine phosphorus declines to normal, i.e., about the
tenth day, plasma phosphorus remains high
for several more days. One might inquire
as to the source of this phosphate in the
absence of hormone-influenced mobilization
from bone. On the basis of data from hor-
PARATHYROID GLANDS OF AMPHIBIANS
mone-treated animals, to be discussed in the
subsequent paper, it is tentatively suggested
that the hormone-induced accumulation of
phosphate in mitochondria of soft tissues
is eliminated in parathyroidectomized frogs;
the consequence of this is a slow release of
phosphate to the vascular bed and a sustained, low-level hyperphosphatemia. In
turn, the hyperphosphaturia may reflect
the increased plasma phosphate load.
GLANDULAR FUNCTION—ADDITION OF
PARATHYROID EXTRACT
Further information on regulation of
mineral ions in amphibians by parathyroid
hormone can be gained from studies of
animals treated with parathyroid extract
(PTE). If hormone induces mobilization of
calcium from amphibian bone, this should
be reflected by increases in the levels of
calcium in blood and urine, as found in
mammals. Although Irving and Solms
(1955) reported no change in the blood calcium of Xenopus laevis, it is likely that the
PTE dose they used (3 units and less) was
below the quantity required to reach threshold. In contrast, Waggener (1930) found
that PTE-treated R. catesbeiana were markedly hypercalcemic with substantially
higher doses.
Several studies on the effect of varying
doses of PTE on plasma and urine calcium
levels in R. pipiens have been reported by
Cortelyou (1960b, 1962«, 1967). Frogs were
injected with 5 units of PTE on alternate
days through 14 days, and calcium levels
were determined daily. A brief hypercalcemia was observed at 24 hr, but plasma calcium remained normal during the remainder of the experimental period. Urine calcium, however, was significantly elevated
from the control level by 24 hr, and the hypercalciuria persisted 14 days. A transient
hypercalcemia was also obtained if a single
injection of 5 units was given; at 4 hr, plasma calcium had increased significantly, but
earlier or later, it remained normal. Hypercalciuria also appeared at 4 hr, and was
maintained through 24 hr, but had declined
to normal by 48 hr.
Ten and 30 units of PTE induced a
853
marked and persistent hypercalcemia for 48
hr after treatment, and this was accompanied by increases in calcium excretion.
The magnitude of the hypercalciuria was
greatest with 30 units, and it persisted for
48 hr, while with 10 units, calcium excretion returned to normal by the second day.
These data indicate that 5 units of PTE
may be the threshold for observation of
gross changes in blood calcium, since it
produced only a weak hypercalcemia. In
contrast, urine calcium responses showed a
dose-dependency in both the duration and
magnitude of the hypercalciuria. The elevated urine calcium subsequent to treatment with higher doses of PTE may be due
to the increased load caused by the simultaneous hypercalcemia. However, hypercalciuria also occurred with 5 units of
PTE, without an elevated blood calcium.
It is conceivable that individual tissue sensitivities to PTE exist, and that as the "key"
organ of response, bone may be induced to
mobilize calcium with the low dose of 5
units; the liberated calcium may then be
lost to urine to maintain blood homeostasis.
When the threshold of mesonephric sensitivity is reached with higher doses of PTE,
some induced reabsorption may occur
which simultaneously increases the plasma
load and sustains the hypercalciuria. This
concept also allows a rationale for the elevated urine calcium following parathyroidectomy, since hormone is not present to
promote reabsorption.
Although PTE induces hypercalcemia
and hypercalciuria in both mammals and
amphibians, the regulation of phosphorus
levels in body fluids appears to differ in
higher and lower vertebrates. Subsequent
to PTE-treatment of mammals, hypophosphatemia and hyperphosphaturia result; it
has been held that these changes are caused
by hormonal inhibition of tubular reabsorption. The typical mammalian pattern may
be somewhat modified in amphibians.
Schrire (1941), for example, reported that
Xenopus laevis responds to PTE by an increase in blood phosphorus. No studies of
urine phosphate were made, however, and
no explanation for the. hyperphosphatemia
was offered.
854
JOHN R. CORTELYOU AND DOLORES J. MCWHINNIE
rus changes in totally parathyroidectomized Rana
More extensive investigations on phospipiens. Anat. Rec. 137:346.
phorus in PTE-treated R. pipiens have been
Cortelyou, J. R. 1960b. The effects of commercially
made by Cortelyou, et al. (1967). In the
prepared parathormone on calcium and phosinitial studies, 5 units of PTE were injected
phorus levels in unoperated Rana pipiens. Anat.
on alternate days for 14 days, and blood
Rec. 138:341.
and urine phosphorus levels were deter- Cortelyou, J. R. 1962a. Changes in plasma and
urine calcium and phosphorus of Rana pipiens
mined daily. Plasma phosphorus declined
subsequent to different dose levels of Haroidin.
from the control level of 3.94 mg% to yield
Am. Zoologist 2:400.
a significant hypophosphatemia through Cortelyou, J. R. 1962fc. Phosphorus changes in
the 14-day period. In contrast, no change
totally parathyroidectomized Rana pipiens. Endocrinology 70:618-621.
occurred in urine phosphorus. In a second
series of studies, 5, 10, or 30 units of PTE Cortelyou, J. R. 1967. The effect of commercially
prepared parathyroid extract on plasma and urine
were administered, and phosphorus was
calcium levels in Rana pipiens. Gen. Comp. Endetermined at intervals for 2 days. With
docrinol. (In press.)
all PTE doses, plasma phosphorus de- Cortelyou, ]. R., A. Hibner-Owerko, and J. Mulroy.
1960. Blood and urine calcium changes in totally
creased, the hypophosphatemia appearing
parathyroidectomized Rana pipiens. Endocrinolat 2 hr and persisting 48 hr. Instead of the
ogy 66:441 -450.
expected hyperphosphaturia, however, no Cortelyou,
J. R., P. Quipse, and D. J. McWhinnie.
significant increase in urine phosphorus
1967. Parathyroid extract effects on phosphorus
occurred after PTE-treatment. These data
metabolism in Rana pipiens. Gen. Comp. Endocrinol. 9:76-92.
stand in contrast to those obtained with
mammals, and leave an unbalanced ac- Hara, J., H. Isono, and A. Fujii. 1959. Electron
microscopic observations of the parathyroid gland
count of phosphorus distribution; for if
of Bufo vulgaris japonicus. Acta Sch. Med. Gifu
PTE-induced dissolution of bone mineral
7:1548-1556.
does occur, and the liberated phosphorus Hara, }., and K. Yamada. 1963. Chemocytological
studies on a sudanophil substance in parathyroid
is detected neither in blood nor urine, what
cells of the toad (Bufo vulgaris japonicus). Anat.
is its ultimate fate? Recent evidence, to be
Rec. 145:377-383.
discussed in the next paper, clearly indiHouston, A. H., and L. E. Husbands. 1965. Some
cates that the site of phosphorus distribuobservations upon parathyroprivic Rana calesbeition is not only hormonally influenced, but
ana. Rev. Canad. Biol. 24:163-168.
is strongly modified by the aquatic and Irving, J. T., and C. M. Solms. 1955. The influence
of parathyroid hormone upon bone formation in
heterothermic nature of the amphibian,
Xenopus laevis. S. African J. Med. Sci. 20:32.
and the effects of temperature upon this
Isono, H. 1960. Histological study of the parathyorganism.
roid gland in the toad (Bufo vulgaris japonicus).
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