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Histochemical study of aberrant parathyroid glands associated with the thymus of the mouse.

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Histochemical Study of Aberrant Parathyroid Glands
Associated with the Thymus of the Mouse'
CHRISTIANNA SMITH AND CAROL P. CLIFFORD
Department of Zoology, Mount Holyohe College, South Hadley, Mass.
on dry ice before storing in the deep freeze
(-18°C). In these studies, the thymuses
were removed with the surrounding connective tissue and neighboring vessels so
that the surfaces were not injured.
When the study was initiated, one thymus had not only aberrant parathyroid
glands but an accessory duct associated
with it which was strongly reactive with
the technique for nonspecifh esterase
(figs. 2, 3 ) . Therefore the procedure
adopted was ( 1 ) to cut serial frozen sections in the cryostat at -18"C, 10 I. in
thickness (the sections were cut horizontally from ventral to dorsal) and (2) to
mount every other section on a slide to be
treated with the technique for the demonstration of sites of leucine aminopeptidase
MATERIAL AND METHODS
activity and alternate sections on another
The mice included 45 fetuses (14, 15, slide, for nonspecific esterase. The para16, 18 and 19 days old) and 33 young and thyroid cells were also reactive with the
old mice (1, 4, 7, 10, 14 days, four-five latter technique so that they were readily
weeks of age and retired breeders, 8-14 identifiable. For the demonstration of leumonths old). Five thymuses of retired cine aminopeptidase, the method of Nachbreeders were from mice of the C57B1/6 las, Crawford and Seligman ('57) was
strain, Roscoe B. Jackson Memorial Labo- used; for nonspecific esterase, the techratory. All other mice (males and fe- nique of Pearse ('53, as modified by Padymales) were from five inbred lines of kula ('55).
Rests in 68 animals were weighed by
Mount Holyoke College stock (courtesy of
Doctor K. Stein); the majority of these the paper weight method.
(48) were from one line, number 25 (table
OBSERVATIONS
1). The ages of the fetal mice were deWith the method for demonstrating sites
termined either by their lengths (Gruneberg, '43) or from the date of the appear- of leucine aminopeptidase activity, the
ance of the vaginal plug. According to cells of most rests were characterized by
Snell ('41), the largest percentage of mice the presence of dark blue granules; some,
are born on the twentieth day (strains among the fetuses, were rose colored. At all
C57B1 and dba). The old,est fetuses ex- fetal ages investigated, the cells of the
amined in this study were taken from
1 Supported by research grant C-lSSS(C9) .from the
National Cancer Institute, National Institutes O f
mothers who had started their nest build- Health,
Public Health Service.
ing.
ZThis exception is included but cannot be proved
be real as the thymus was not cut through comThe mice were killed by cervical dis- to
pletely to its dorsal surface and the parathyroid
location; the thymuses were excised or the glands were just appearing.
preliminary report of this study was published
fetuses dissected immediately and frozen as SA
an abstract, Anat. Rec., 139: 321, 1961.
Attention has been redirected recently
to the presence of accessory parathyroid
glands associated with the thymus by Van
Dyke's work on the rat ('59). During some
histochemical studies of the thymus of the
mouse using the technique for the demonstration of sites of leucine aminopeptidase
activity, we noted that parathyroid rests,
deeply stained by the presence of dark blue
granules with this method, were made
distinctly visible. According to Pearse and
Tremblay ('58) this positive reaction indicates functioning of the parathyroid cells.
Seventy-eight mice of varying ages were
investigated; except one fetus: all possessed accessory parathyroid glands in the
region of the thymus3
229
1
1 day
4 days
7 days
10 days
14 days
4-5 weeks
4-5 weeks
4-5 weeks
retired breeders
breeders retired
14 days
14 days
15 days
16 days
18 days
18 days
19 days
19 days
Age
3
3
3
5
4
3
3
3
3
3
6
6
6
5
~3)'
7
6
6
Number
of
animals
5.0
3.0
4.0
2.0
3.3
5.3
1.7
4.7
5.3
5.8
2
1.7
2.1
2.6
5.5
3.0
5.2
5.3
Left
lobe
5.7
7.3
2.7
4.7
3.3
6.2
4.7
4.3
3.7
5.4
1.8
1.8
2.1
3.0
3.0
2.4
3.1
3.0
Right
lobe
21.2
9.0
9.0
10.7
10.3
6.7
6.7
6.6
11.5
6.4
3.8
3.5
4.2
5.6
8.5
5.4
8.3
8.3
~~~d
Average number of
rests per thymus
One 18 day old fetus had no rests (see footnote 2).
C57B1/6
Postnatal
25
25
25
25
25
25
66
44,16
25
Fetal
25
55
25
25
25
55
25
55
Line
TABLE 1
3-10
2-9
2-5
1-6
3-4
2-9
0-9
1-8
1-7
2-12
1-3
1-5.
0-5
1-5
2-6
1-5
1-8
1-7
sing1e
lobes
numbers
15.7
8.7
15.7
6.3
11.7
28.5
10.7
34.0
42.0
56.6
4
9.3
6.3
5.6
14.0
17.0
13.3
20.8
Left
lobe
38.6
13.7
23.7
8.7
14.0
16.7
35.8
16.3
18.0
53.0
4.2
9.5
8.1
4.8
6.0
10.3
8.0
17.7
:%7
29.4
32.4
24.4
20.3
28.4
64.3
27.0
52.0
95.0
95.2
8.2
18.8
14.4
10.4
20.0
27.3
21.3
38.5
Total
Average number of
sections per thymus
1-10
1-14
2-6
2-6
2-10
1-29
1-12
2-22
1-30
140
1-6
1-14
1-7
1-8
4-17
1-18
1-6
1-21
Range:
number
of
sections
per lobe
27.5
31.3
36.5
30.5
44.7
55.9
42.6
57.7
105.5
85.0
21.3
53.8
33.4
18.6
23.5
50.5
25.6
46.2
3
3
3
3
3
2
3
2
3
5
6
6
3
5
2
7
6
3
0.058
0.070
0.121
0.110
0.163
0.340
0.082
0.303
0.464
2.960
0.052
0.094
0.052
0.033
0.043
0.138
0.073
0.127
Average
Weight
extent
single
Nuomfber Grams
rests
in micra animals average
Parathyroid rests associated with the thymus of the mouse. Inbred lines other than 25 are italicized
231
PARATHYROID RESTS WITH MOUSE THYMUS
parathyroid glands themselves were highly
reactive. With the technique for nonspecific esterase activity, fetal parathyroid
cells were less deeply stained than adult
cells.
The data include (1) the position of
the rests in relation to the thymus, (2) the
number of rests associated with the left
and right lobes, (3) the number of sections involved, (4) the extent of the rests
in micra, and (5) the weights of the rests.
Excepting item one, the averages of the
data for each group are given in table 1.
Some parathyroid rests were well developed glands, nodules or elongated cords;
others were isolated small clusters of cells,
often only in single sections. Such minute
rests were present in young and old mice.
Those on the surface of the thymus were
extracapsular (figs. 4, 6, 9, 11) and were
often located in a triangular (fig. 4) or
slightly hollow depression (figs. 4, 6). Some
which appeared imbedded in the thymus
were at the base of an interlobular septum
(fig. 5). A rest within the thymus with no
connection at any level with the surface or
a septum was a rarity (fig. 7). More accessory glands were located dorsally than
ventrally. Rests were seen in the following
positions with descending frequency: interlobar or medial (usually associated with
one lobe or the other) (fig, 4), anterior,
posterior or lateral. In any one thymus the
individual rests tended to be arranged in
groups; for example, in some thymuses,
most were interlobar.
Although after birth, the number of
aberrant parathyroid glands associated
with the left and right lobes varied somewhat, the total number of sections involved was not significantly different. The
number of rests per lobe ranged from 012; the number per whole thymus, 2-21.
These single lobes with no rests were in a
15 day old fetus and a young adult mouse.
There appeared to be some correlation of
the amount of aberrant glands with sex but
the differences were not statistically significant; in inbred line 25, the averages for
mice from one day old through four-five
weeks old were higher for the females (12
mice) than the males (seven mice) in regard to numbers per thymus ( 11.4 7.1 0,
7.4 2.7 d), extent in micra (39.4 -C
17.8 ?,27.0 +- 12.2 d) and weight in grams
(0.139 2 0.070, 0.123 2 0.11 3 ) . Variations were also present between inbred
lines (table 1). Statistically, the differences between the weights of the parathyroid rests of the 14 and 19 day old
fetuses of lines 25 and 55 were significant.
This was also true for rests of line 66 when
compared with those o€ 25, 44 and 16,
young adult mice. Between the retired
breeders, line 25 and C57B1/6, the difference in weights was not significant (figs.
9, 10). The distribution of the number
of the aberrant glands according to lobes
and to the whole thymus is given in table
2. Up to 11 aberrant glands were present
with the thymuses before birth. After
birth, 12 rests were with two thymuses
of four-five week old mice and one with
a thymus of a retired breeder; 15, with
a one-day old and two four-five week old
ones; 21, with one thymus of a retired
breeder, C57B1/6 strain.
Both weight and average extent of the
accessory glands increased with age after
birth (line 25) but before birth, unexplained fluctuations were present (table 1,
fig. 1).
An accessory duct was present in only
one five week old mouse (figs. 2, 3).
*
*
DISCUSSION
In his study of the accessory parathyroid glands in the rat, Van Dyke ('59)
TABLE 2
Distribution of rests by lobes
Number
of rests
Fetal
Postnatal
Fetal
Postnatal
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
1 27 13 15 14 12 4 1 1
1 4 9 14 9 8 8 5 2 4 1
1
0
9
3
2
2
Distribution of rests by thymuses
7 2 9 4 5 2 1
7 5 3 3 5 1 3
3
1
CHRISTIANNA SMITH AND CAROL P. CLIFFORD
I
I
7
10
-
Fig. 1 Weights of the parathyroid rests associated with the thymus of the mouse, inbred
line 25 (paper weight method).
emphasized that these were postnatal in
development and that they increased in
size and possible numbers with age and
after such experimental procedures as
feeding sodium phosphate, after parathyroidectomy or thyro-parathyroidectomy. He
concluded that certain foci within the thymus of the rat might “display phenomena
of deZayed differentiation resulting in accessory parathyroid tissue” (p. 195).
Among 73 normal rats (newborn through
19 months) 62% possessed aberrant
glands. Van Dyke also made the statement
that “Since all such parathyroid tissue may
not reside within the thymus, it is possible that accessory parathyroid glands
may be present even to a greater extent
in adult and old rats, normally, than our
studies have indicated. Some tissue might
well have been unavoidably neglected in
the mediastinum during excision” (p. 192).
The present study of parathyroid rests
associated with the thymus of the mouse
was based on data from 78 normal animals
and included rests in close proximity to
the surfaces of the thymus (figs. 4, 6, 8,
9, 11, 12), as well as interlobular (figs.
5, 9, 10). The use of histochemical techniques for the demonstration of sites of
leucine aminopeptidase and nonspecific
esterase activity made it possible to detect
rests composed of a few cells and those
displaying weak activity as in some fetuses.
The rests varied in number in any one
mouse from a few (two, fetal and post-
natal) to many (21, one retired breeder).
The average numbers of accessory glands
for the four fetal groups (14, 15, 16, 18
days) were somewhat less than those for
the postnatal ones (table 1). However, there
was no increase in the average number of
rests after the first day of extrauterine life
that could be correlated with age (line 25).
It is interesting to note that the average
number of rests for the 19 day old fetuses
was 8.3 per thymus and for all the postnatal mice, 8.8. The difference between
these two averages was not significant.
From these limited data it would seem
that the number of parathyroids associated
with the thymus of the mouse was probably determined by the time the animal
was born. On the other hand, it must be
recognized that in individual mice (table
2 ) including one one-day old one, a larger
number of rests than were found in the
fetal mice (12, 15, 21) accompanied 21%
of the thymuses of the young and adult
mice. An increase in volume as indicated
by the number of sections involved and
by the weights (paper weight method) in
inbred line 25 occurred with increasing
age. Although there were no obvious strain
differences in regard to the numbers of
rests associated with the thymus, variations in size were present. The questions
arise as to whether the amount of accessory parathyroid cells in the mouse is correlated with that of the parathyroid glands
233
PARATHYROID RESTS WITH MOUSE THYMUS
and whether the total quantity varies with
the strain.
The simplicity of the structure of the
thymus of the mouse (little lobulation)
made it possible to place accurately the
position of the rests in relation to the
thymus. They were most numerous dorsally and medially (interlobar) but could
be found anteriorly, posteriorly or laterally.
That the accessory glands were often
grouped would suggest that they were distributed according to the growth shiftings
of the individual pharyngeal regions.
Crisan (’35) described cords of parathyroid cells on the dorsal and medial surfaces
of the thymic lobes of a 13-14 day old
fetus of a white mouse and at the cranial
ends of the thymuses of 16-17 and 17-18
day old fetuses. Crisan concluded that
these were rests of parathyroid cords,
thinned and broken, when the parathyroid
gland separated from the thymus. Our
observations on the 14-19 day old fetuses
confirm those of Crisan. The presence,
therefore, of aberrant parathyroid glands
in the mouse in the region of the thymus
correlates with their embryonic developmpnt
.11--*1.
SUMMARY
1. Inbred lines of mice were inVeStigated for the presence of accessory parathyroid glands associated with the thymus.
2- Such parathyroid rests were consistently present in fetal, young and old mice.
3. These accessory glands increased in
size from one day of age through 12-14
months. Unexplained fluctuations were
present before birth.
4. The average number of rests for the
19-day old fetuses and for all young and
adult mice was not significantly different.
5. Twenty-one per cent of the postnatal
mice had larger numbers of rests per thymus than were present in any fetal mouse.
6. The data suggest that the size of the
parathyroid rests varies with the inbred
line or strain.
LITERATURE CITED
Crisan, C. 1935 Die Entwicklung des thyreoparathyreo- thymischen Systems der weissen
Maus. Z. Anat. Entwickl., Gesch., 104: 327-358.
Gruneberg, H. 1943 The development of some
external features in mouse embryos. J. Hered.,
34: 89-92.
Nachlas, M. M., D. T. Crawford and A. M. Seligman 1957 The histochemical demonstration
of leucine aminopeptidase. J. Histochem. and
Cytochem., 5: 264-278.
Padykula, H. 1955 Personal communication.
Pearse, A. G. E. 1953 Histochemistry. Little,
Brown and Co., Boston.
Pearse, A. G. E., and G. Tremblay 1958 Leucine aminopeptidase in rat parathyroid and
its relation to varathmoid hormone Droduction.
Nature, 181: i532-1533.
Snell, G. D. 1941 Biology of the laboratory
mouse. The Blakiston CO., Philadelphia. Chap.
2 , 55-88.
va&~~~’=d
J
h
;:$:
&l’Tzt’
of accessory parathyroid glands in the
~~~~~~~~~~
Anat. Rec., 134: 185-204.
rat.
PLATE 1
EXPLANATION O F FIGURES
Photomicrographs of sections of thymus treated with the technique for
the demonstration of sites of leucine aminopeptidase activity (except
where noted) but otherwise unstained. All sections cut at 10 p . In some
sections diffusion from the parathyroid rests into the thymus has occurred.
Parathyroid rests and accessory duct epithelium within an anterior,
interlobular septum of a thymus of a five-week old mouse, inbred
line 44. Thymus (T) at left and tip of a thymic lobe at right; parathyroid rest (P) extends through 10 sections; accessory duct (AD)
is less reactive with this technique than the parathyroid cells. X 500.
Section adjacent to (2) treated with the technique for the demonstration of sites of nonspecific esterase activity. Thymus (T) at left
contains more reactive epithelial and less reactive mesenchymal
reticular cells. The accessory duct epithelium (AD) is more reactive
than the parathyroid cells (P). X 500.
Parathyroid rests (P) in the interlobar septum ( s ) of a thymus (T)
of a C57B1/6 mouse, retired breeder about 12 months old. Parathyroid rests in depressions on the surface of the thymus but not under
the capsule (c, capsular cell). Larger rest extends through four sections; smaller, six sections. x 60.
Parathyroid rest (P) at the base of an interlobular septum (is) of a
thymus (T) of a five-week-old mouse, inbred line 25. Rest extends
through four sections. x 195.
Parathyroid rest (P) in a shallow depression on the anterior surface
of a thymus (T) of a mouse, inbred line 25, retired breeder, 12Yz
months old. Rest extends through eight sections; capsule (c). x 195.
Parathyroid rest (P), completely within a thymus (T) of a five-weekold mouse, inbred line 25 (same as fig. 5 ) . Rest extends through
nine sections. Previous sections of this rest are through a solid cord
of cells. x 195.
234
PARATHYROID RESTS WITH MOUSE THYMUS
Christianna Smith and Carol P. Clifford
PLATE 1
235
PLATE 2
EXPLANATION O F FIGURES
8
Parathyroid rest ( P ) on the surface of the thymus and associated
with an interlobular septum (is) of a thymus ( T ) of a C57B1/6
mouse, retired breeder, about 12 months old. Rest extends through 32
sections. x 60.
9
Parathyroid rest ( P ) , the same as figure 8; interlobular septum ( i s ) ,
capsule ( c ) . X 195.
10
Parathyroid rest ( P ) in a n anterior, medial interlobular septum of
a thymus (T) of a mouse, inbred line 25, retired breeder, 12Y2
months old. Rest extends through 13 sections. For comparison with
figure 9 to illustrate strain difference i n size. x 195.
11 Parathyroid rest ( P ) on the posterior surface of a thymus ( T ) of a
19-day-old fetus, inbred line 25. Rest extends through four sections;
capsule ( c ) . x 195.
12 Parathyroid rest ( P ) on the anterior surface of a thymus ( T ) of a n
18-day-old fetus, inbred line 55. Rest extends through six sections.
For comparison with figure 11 to illustrate strain difference i n size.
X 195.
236
PARATHYROID RESTS WITH MOUSE THYMUS
Christianna Smith and Carol P. Clifford
PLATE 2
23 7
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