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Number and size of rat thyroid C cellsNo effect of pinealectomy.

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Number and Size of Rat Thyroid C Cells: No Effect
of Pinealectomy
P.J. McMILLAN, U. HEIDBUCHEL, AND L. VOLLRATH
Department of Anatomy, Johannes Gutenberg-University, Mainz, Federal Republic of
Germany (U.H., L. V) and Department ofdnatomy, Loma Linda University, Loma Linda,
CA 92350 (PJ.M.)
ABSTRACT
A method for the estimation of the size and total number of calcitonin-containing cells (C cells) in the rat thyroid gland has been devised. The total
area, the number of C cells per unit area, and the areal fraction of C cells were
determined for the C cell region using ste serial sections. From these data it was
estimated that from 0.3 x lo6 to 1.0 x 102C cells were evenly divided between the
two thyroid lobes. Approximately 150 pm3 of cytoplasm were associated with each
of these cells. In comparison with sham-operated rats, pinealectomy had little effect
on the number of C cells. In a n experiment terminated in the summer, there was a
statistically insignificant decrease 6 weeks postsurgery; no effect was seen at 12
weeks. On the other hand, a slight increase in the number of C cells was seen in
January, 12 weeks postsurgery. The volume of cytoplasm per cell was not altered by
pinealectomy.
As C cells are restricted to what can be called the C
cell region of the thyroid gland (McMillan et al., 1974;
Wolfe et al., 1974), assessment of total C cell number
and average C cell volume requires a technique which
takes into consideration both the extent of the C cell
region and the density of the C cells within this region.
This paper describes a technique that permits estimation of quantitative data hitherto not available, i.e., C
cell number and size in the different thyroid lobes of the
rat. This method was used to test the influence of pinealectomy on C cells.
The need for this study derives from the fact that apart
from the pineal’s role in the reproductive system of
photoperiodic mammals (Reiter, 1982; Hoffmann et al.,
1981) its precise influence on other endocrine systems
remains to be established (cf. Vollrath, 1981). One of the
systems reported to be influenced by the pineal gland is
the parafollicular or C cells of the thyroid, which by way
of their calcitonin secretion play a n important role in
calcium homeostasis. Previous studies dealing with the
effects of the pineal gland on the C cells have yielded
somewhat contradictory results (see Discussion). Moreover, one aspect in particular has not been adequately
studied, i.e., how C cell number is affected by pinealectomy. This kind of information, however, is essential for
correctly interpreting both qualitative and quantitative
findings resulting from the experimental manipulation
of the pineal. In a n electron microscopic study, KrstiC
(1969) observed more C cells two months after pinealectomy, but the number of C cells had not actually been
counted. Two other papers claimed that pinealectomy
increases C cell numbers (Miline et al., 1968; Csaba and
Barath, 1974), but nonspecific staining reactions were
used and objective quantification was lacking.
The methods described in this study overcome these
shortcomings in two ways: 1) highly sensitive irnmunohistological staining reaction is used to demonstrate C
cells, and 2) C cell number and size are quantitated
0 1985 ALAN R. LISS, INC.
morphometrically. Pinealectomy was found to have no
effect on C cell size and a n inconsistent effect on C cell
number.
MATERIALS AND METHODS
Male rats of the Wistar (Han) strain (200-225 g) were
used in this study. They were maintained, four to a cage,
at a temperature of 22 & 2°C under LD 12:12 (lights on
7 A.M. to 7 P.M.), with free access to water and food
(Altromin rat pellets).
Two experiments were performed. In experiment 1
(May-July, 1980)40 rats were randomly assigned to four
groups. One group of 10 animals was pinealectomized
and another was sham-operated (Nembutal anesthesia)
6 and 12 weeks prior to sacrifice. In the sham-operated
animals, the dura mater anterior and to the right of the
confluence of sinuses was incised, but the pineal gland
was left undisturbed. At the time of sacrifice the presence or absence of the pineal gland was established by
inspection.
In experiment I1 (November-February, 1981) 12 rats
were pinealectomized and 12 were sham-operated as
above, 12 weeks prior to sacrifice. At the end of the
experiments the rats were anesthetized and the trachea
with attached thyroid and parathyroid were fixed in
Bouin’s fixative, in the first experiment by immersion
and in the second experiment by perfusion. The tissue
was embedded in butylmethacrylate-paraffin(Engen and
Wheeler, 1978), serially sectioned at 5 pm and every
20th section immunohistologically stained for calcitonin
and counterstained with hematoxylin. The immunohistological procedure followed that of Sternberger (19741,
using 10%normal goat serum to block nonspecific stain-
Received October 12, 1984; accepted December 13,1984.
Address reprint requests to Dr. Paul J. McMillan.
168
P.J. McMILLAN, U. HEIDBUCHEL, AND L. VOLLRATH
ing, anticalcitonin sera (which were gifts from L.J. Deftos and B.A. Roos), a peroxidase-antiperoxidase complex
obtained from Litton Bionetics Inc., and diaminobenzidine as the chromogenic agent. The calcitonin antiserum was used a t dilutions of 1:400 and 1:3200
containing 1% normal goat serum. Controls consisted of
normal rabbit serum and anticalcitonin serum adsorbed
with synthetic human calcitonin (300 pg/ml) (which was
a gift from Ciba to Dr. F. Fischer). In experiment 11, the
pituitary gland, adrenal glands and testes were also
taken and weighed.
Quantitation
In addition to C cell number and volume (see below),
data were obtained for the volumes of the thyroid, the
portion of the thyroid that contains C cells and the
parathyroids by circumscribing the respective organ and
region profiles on every sample section by means of a
Kontron MOP digitizer. Volumes were calculated by
multiplying the total area traced on all sections by the
interval between sections (0.1 mm).
To minimize the time required to obtain a n estimate
of C cell number, a sampling method was devised. It was
assumed that a reasonable but maximal (not corrected
for section thickness) estimate could be obtained if all of
the C cell nuclei in all of the sample sections (25-35)
were counted and the result multiplied by 20 (the number of sections between sample sections). An estimate of
the number of nuclei in the sample sections is given by
the C cell density (number of C cells per unit area) times
the total sample area. This times 20 would give the
desired count.
It was found that a n adequate estimate of the C cell
density could be obtained from three sections equally
spaced through the C cell region. The adequacy of this
sample was tested by determining the cell density for
every sample section for each of three randomly selected
rats. From the 20-30 density estimates for each thyroid
lobe, the mean density and standard deviation (SD) were
calculated. Then density estimates were obtained, based
on three randomly selected sections. Altogether 90 sets
of three (15 sets per lobe) were evaluated. Of these estimates, 96%were less than one SD away from the mean
and 63% less than one half the SD away from the mean.
Therefore, three sections evenly spaced through the C
cell region were accepted as a n adequate sample.
In the second experiment, estimation of the volume
occupied by calcitonin-containing cytoplasm per cell involved a two-step process. The areal fraction of stained
cytoplasm in the C cell region was first determined.
Then that estimate of the C cell volume fraction was
multiplied by the volume of the C cell region as determined previously and divided by the total number of C
cells to give the volume per cell. An image-array processor (Gould-DeAnza 5000) was used to estimate the areal
fraction (number of pixels in the C cell intensity “window”/total number of pixels). It was measured by systematically scanning the entire C cell area of three
representative sections. A correction for dirt and nonspecific staining was obtained by measuring the areal fraction in the intensity window of thyroid regions that did
not contain C cells. This correction averaged 5% of the
corresponding C cell fraction.
Statistical Analysis
Significance of group differences was determined using single classification analysis of variance after logarithmic transformation of the data. For correlation
studies, the nonparametric Spearman’s coefficient of
rank correlation was used.
RESULTS
With the immunological method used, C cells appear
as dark, polymorphic structures with dark blue nuclei
typically lying between follicular cells and the basement
membrane (Fig. la). The specificity of the reaction is
demonstrated by the fact that cells do not stain when
either normal rabbit serum or anticalcitonin serum adsorbed with calcitonin is used in place of the specific
antiserum (Fig. lb). The serum has been previously
checked for cross-reactivities and specificity (Deftos,
1971; Roos and Deftos, 1976). C cells occur singly or
adjacent to other C cells. Clusters consisting of more
than two to four C cells are rare. C cell profiles range
from being polygonal to cells with two small cylindrical
processes. The length of the processes is about twice the
width of the C cell body. The volume of calcitonin-stained
cytoplasm per C cell in the 12-week winter experiment
was the same for both control (146_+38pm3) and experimental (148532 pm3) rats.
C cells were found to be concentrated in the middle
and caudal portions of the thyroid lobes. They were
generally absent from the periphery of each lobe (Fig. 2)
and were never found in the rostal pole or isthmus. No
statistically significant differences exist between the
number of C cells in the right and left thyroid lobes. The
total number of C cells per thyroid averaged 0.6 x lo6
(range, 0.3-1.5 x lo6) or 63,000 per mm3 of thyroid
tissue. By comparing the volume of the thyroid in Figure 3 with the volume of the C cell region in Figure 4, it
can be seen that on the average only 20-25 percent of
the thyroid volume contains C cells. In agreement with
a previous study (McMillan et al., 19741, no immunoreactive cells were found in the parathyroid glands.
Figures 3-5 depict thyroid volume, volume of C cell
region, and C cells. per gland in sham-operated and pinealectomized rats. Due to some incomplete series of
sections, the number of animals given in each group
differs from that in Materials and Methods. One animal
in the sham series had some C cells anomalously located
in the thymus (McMillan et al., 1982). The total volume
of the C cell region and the number of C cells in the
thyroid gland of this animal were at the mean of those
of the other rats in this group in spite of the fact that
these were greatly reduced on the affected side. Data for
this animal have been included in the data for volume
of the C cell region but excluded from the final data on
C cell numbers and volume. Since the measured parameters for this anomalous rat were at the mean of the
other rats, inclusion or exclusion only alters the N used
for statistical analysis. To be conservative it was excluded from the final data. Variable results were obtained in comparing the number of C cells in control and
experimental animals (Fig. 5). Six weeks postsurgery in
the summer experiment, the number of C cells was
insignificantly reduced (F-ratio = 4.0, P = 0.06) and at
12 weeks no difference was seen (F-ratio = 1.4, P = .26).
169
THYROID C CELLS AND PINEALECTOMY
Fig. 1. Thyroid. Immunoperoxidase stain for calcitonin (a); adjacent section reacted with primary antiserum which was absorbed with
calcitonin 6).
Both are counterstained with hematoxylin. Magnification ~ 3 0 0 .
*I
E
€12
Ijl( 'I1
s
S Pr
Px
6wks
l2wks
l2wks
Fig. 3. Thyroid volume in summer experiment (6 and 12 weeks,
left) and winter experiment (12 weeks, right).
Parathyroid volume, pituitary weight, adrenal weight
and testes weight were not significantly (P > 0.05) affected by pinealectomy.
DISCUSSION
The data obtained in this study on rat C cells will be
of value in future experimental and comparative studies. Separate assessment of C cell numbers in the right
and left thyroid lobes revealed that there are no statistically significant differences. Hence calcitonin meaFig. 2. Cross-section of rat thyroid at the level of the parathyroid surements carried out in one thyroid lobe can be
immunohistcchemically stained for calcitonin and counterstained correlated with C cell number in the other, and hemithylightly with hematoxylin. The C cell region is outlined. Magnification
roidectomy will remove roughly half of the C cell popux32.
lation, the total population being on the order of 700,000
cells per gland. One exception (due to anomalous develIn the winter experiment, however, a marginally signif- opment) to the even distribution of C cells has been
icant increase (F-ratio = 4.7, P = 0.04) was observed 12 observed (McMillan et al., 1982). The number of C cells
weeks postsurgery.
per mm3 of thyroid tissue is 63,000 in the rat but 30,600
No correlation was found to exist between any of the in 8-week-old mice (Wechbanjong et al., 1979). For huC cell parameters and thyroid and parathyroid volumes. mans no such data are available, but judging from vis-
170
P.J. McMILLAN, U.HEIDBUCHEL, AND L. VOLLRATH
ual inspection (McMillan et al., 1974) C cells are much
scarcer than in rats and mice. The estimation of the
volume of C cell cytoplasm (150 pm3) provides for the
first time a baseline for comparison in studies of the
control of calcitonin storage. This estimate must be considered as a n upper limit, because of the thickness of
the sections used. The estimation of C cell volume was
only performed on perfusion-fixed tissue. This avoids the
probability that the superficial and deep cells would
shrink differentially in immersion fixation.
The pinealectomy experiments described in this study
were carried out since, according to the literature, the
pineal gland is capable of influencing two important
components of the calcium-regulating system of the body,
the parathyroid glands and the C cells.
With respect to the parathyroid glands, it had been
found that pinealectomy is followed by hypertrophy and
hyperplasia of parathyroid cells, whereas administration of pineal substances leads to a n atrophy (KrstiC,
1966, 1967, 1968; Miline and KrstiC, 1966), pointing to
a n inhibiting effect of the pineal gland on the parathyroids. By contrast, Kiss et al. (1969)observed a n atrophy
of the parathyroid and a decrease of 3H-methionine incorporation which suggests that the pineal may stimulate the parathyroid gland. Feedback of parathormone
on the pineal gland is suggested by the observation that
in guinea pigs the majority of pinealocytes can be influenced by microiontophoretically applied parathormone
(Semm et al., 1981).
Dramatic effects of the pineal gland can be demonstrated in parathyroidectomized rats. While parathyroidectomy alone does not lead to severe functional
changes, animals that are additionally pinealectomized
undergo convulsive attacks and most of them die within
14 hours (Reiter and Morgan, 1972; Reiter et al., 1972,
1973a and b, 1975; Pomerantz and Reiter 1973).
As the effect of pinealectomy o n parathyroid volume
had not been investigated in the previous studies, this
aspect could be conveniently examined in the present
study. According to our results, there is no statistically
significant difference in parathyroid volume either 6 or
12 weeks after pinealectomy. This suggests that if hypertrophy and hyperplasia of the intrinsic cells do occur
after pinealectomy (see above) they must be very slight.
Studies dealing with the interactions of the pineal
gland and the calcitonin-producing cells of the thyroid
point to mutual inhibition. Pinealectomy has been re-
ported to increase 1)C cell numbers (Miline et al., 1968;
KrstiC, 1968; Csaba and Barath, 19741, 2) the nucleocytoplasmic ratio in favour of the cytoplasm, and 3) the
number of electron-dense secretory granules (Csaba and
Barath 19751, although it should be noted that Soriano
(1973) found a decreased secretory activity of the C cells
after pinealectomy.
Pineal inhibition by calcitonin reduced the activity of
a number of histochemically demonstrable enzymes in
the pineal gland (KrstiC and Tarsoly, 1972). Moreover,
in the guinea pig, the majority of pinealocytes responsive to microiontophoretically applied calcitonin were
inhibited as assessed electrophysiologically (Semm et
al., 1981).
In view of these findings, it was somewhat surprising
to see that none of the C cell parameters investigated in
the present study showed consistent differences after
pinealectomy when compared to sham-operated controls. Despite these relatively negative data, it should
be noted that in the two sets of experiments opposing
tendencies were present: a decrease in C cell numbers
in the summer and a n increase in the winter. In addition, it should be taken into account that as C cell
numbers are highly variable between animals, a very
large number of experimental animals would be required to demonstrate positively a n effect of pinealectomy, especially if seasonal differences exist.
Nevertheless, under the conditions of these experiments, we believe that the pineal exerts little influence
on the number or size of C cells. We are all the more
confident with this conclusion, as the data were obtained
by applying a combination of a highly specific immunohistological technique and a morphometric method specifically designed for coping with the uneven distribution of C cells within the thyroid. That the C cell number
can be expected to change at all has been repeatedly
demonstrated. C cells show a n increase during development (Wechbanjong et al., 1979; Alumets et al., 1980),
with age (Peng et al., 19761, and when chronically stimulated with vitamin D and calcium (Young and Capan
1969). Whether C cells can be experimentally decreased
in number remains to be seen.
The problem of seasonal differences in C cells in nonhibernating species has received little attention in contrast to hibernators (cf. Nunez et al., 1967; Pearse and
Welsch, 1968; Nunez and Gershon, 1972). According to
Petko (1978), the area occupied by C cells in the rat
"E
E
6wks
l2wks
l2wks
Fig. 4. Volume of C cell region in summer experiment (6 and 12
weeks, left) and winter experiment (12 weeks, right).
6hs
l2wk1
12wkr
Fig. 5. Number of C cells in summer experiment (6 and 12 weeks,
left) and winter experiment (12 weeks, right).
THYROID C CELLS AND PINEALECTOMY
thyroid is large in autumn and small in spring; unfortunately, no experiments were carried out in the summer and winter. In the present study, statistically
significant differences that could be related to season
were not found for any of the parameters investigated.
It should be noted, however, that in our study, which
was not aimed at seasonal differences, the experiments
were carried out in seasons different from those of Petko’s study.
ACKNOWLEDGMENTS
The financial support of the Deutsche Forschungsgemeinschaft is gratefully acknowledged. Fr. Sabina Soda’s expert technical assistance is very appreciated, a s
is the secretarial help of Ms. Cherry Wendtland and Ms.
Terry McMillan.
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