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The origin of osteoclasts in estrogen-stimulated bone resorption of the pubic symphysis of the mouse.

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The Origin of Osteoclasts in Estrogen-stimulated
Bone Resorption of the Pubic Symphysis
of the Mouse'
JAMES A. CORWINZ AND JAMES R. MOREHEAD
Department of Anatomy, Tufts University School of Medicine,
Boston, Massachusetts 021 11
ABSTRACT
Four-week-old female mice were injected with estradiol, and the
following day received intraperitoneally one p C / g m body weight of thymidinemethyL3H. At various intervals following the injection of thymidinezH, pairs of
mice were sacrificed, the pubic symphyses excised and prepared for radioautographs of transverse sections. Examination of the radioautographs revealed that
the highest percentage of labeled cells was to be found in the osteoprogenitor
cell population at all time intervals examined. The number of osteoclasts and
the percentage of labeled osteoclasts rose progressively. The percentage of labeled
osteoblasts rose and then fell, but was at all times much less than that of the
osteoprogenitor cell population.
Since the osteoprogenitor cell population was the only one in which the percentage of labeled cells was high enough to account for the rise in the percentage
of labeled osteoclasts, the conclusion was drawn that the labeled osteoclasts arose
from the labeled osteoprogenitor cells.
In support of the above conclusion, and in keeping with the theory that the
osteoprogenitor cell undergoes "differential" mitosis, one daughter cell going on
to become an osteoclast or an osteoblast, and one daughter cell remaining in the
osteoprogenitor cell population, the number of silver grains over the labeled osteoclast nuclei was observed to be roughly one-half that over the labeled osteoprogenitor cell nuclei.
The changes noted in the pubic symphysis during pregnancy or following the administration of estrogen have been investigated extensively since Hisaw's ('25, '26,
'29) demonstration that the ovary, and specifically the corpus luteum, was involved
in symphyseal relaxation. Burrows ('35)
demonstrated that estrogen alone was sufficient to produce symphyseal relaxation,
i t was later shown by Steinetz et al. ('65)
that the presence of growth hormone was
essential to bring about changes in the
pubic symphysis. Talmage ('47) showed
that estrogen priming was required for relaxin to have an effect on the symphysis.
Hall ('47) noted that osteoclasts increased
in number in association with the resorption of the pubic bones following the administration of estrogen. Further studies
have been directed toward the estrogenrelaxin relationship, and the ligamentous
changes that occur after hormone adminisANAT. REC., 171: 509-516.
tration. Only occasional mention has been
made of the osteoclast population, notably
by Crelin ('69), who concluded that osteocytes, released during resorption and dedifferentiated to osteoprogenitor cells, are
the source of osteoclasts. No work has been
done to determine whether osteoprogenitor
cells are the primitive precursor for osteoclastogenesis in the pubic symphysis, as
has been done in the long bones.
In contrast, a substantial amount of research has been directed toward determining the mode of action of parathyroid hormone (PTH) and the cell of origin of
osteoclasts in normal and in PTH-stimulated bone. The currently accepted theory
Received .Tune 24. '70. Acceuted Mav 17. '71.
1 Supported in pa& by USPHS pan; GM01451.
zThe contents of this paper reflect the personal
views of the author, and are not to be construed as a
statement of official Air Force policy. The work re.ported herein represents a portion of a Thesis submitted by the first author in partial fulfillment of the
requirements for the degree of Master of Science.
509
510
JAMES A. CORWIN AND JAMES R. MOREHEAD
of osteoclastogenesis is based on the work
of Young (’62). According to this theory,
both the osteoclast and the osteoblast originate from a mesenchymal precursor, the
“osteoprogenitor cell.” Young (‘62) considered this cell as identical to the “reticulum
cell” and “mesenchymal cell” described by
others. Scott (’67), using electron microscopic radioautography, essentially confirmed Young’s findings. In addition, she
was able to subdivide the osteoprogenitor
pool into two populations, identifiable as
pre-osteoblasts and pre-osteoclasts on the
basis of fine structure. Using radioautographic techniques, Talmage et al. (’65)
studied PTH-induced resorption in the long
bones of rats. They concluded that the precursor cell of PTH-induced osteoclasts was
identical to Young’s osteoprogenitor cell.
Talmage (’67) strengthened this argument
using tritiated cytidine radioautography.
These studies indicated that PTH stimulates proliferation of the osteoprogenitor
cell line exclusively.
The action of estrogen on the pubic symphysis is unique, apparently, in that elsewhere in the bodies of rats, mice, and birds,
estrogen stimulated proliferation of bone
and not osteoclastic resorption (Selye, ’32;
Urist et al., ’50; Gardner, ’40). Therefore,
it was thought to be of interest to determine
whether the osteoclasts that are seen in the
pubic bones following stimulation by estrogen were derived from the same cell as
those in the long bones following the administration of parathyroid hormone.
%
two
I,animals were sacrificed, using a
chloroform atmosphere, at each of the following time periods: 2, 4, 8, 12, 24,30, 36,
48 hours. Two untreated animals were saciificed on day 28 to serve as controls.
The pubic symphyses and rami were excised by cutting across the midpoints of the
obturator foramina. Tissues were fixed for
24-36 hours in 10% neutral buffered forrnalin, decalcified for six hours in RDO,’
washed, dehydrated, embedded and sectioned at 5 p. NTB-3 emulsion was applied
to the mounted sections according to the
technique of Kopriwa and Leblond (’62).
Following an exposure time of 14 days at
4 “C, the emulsion was developed with
Kodak D-170 (Dolmi developer), and the
sections were stained through the emulsion
with hematoxylin and alcoholic eosin.
The metaphyseal region adjacent to the
symphysis was examined under oil immersion. In order to ascertain the relative numtier of each cell type in the population, a
total of 1,000 cells was counted in each
symphysis. Osteoprogenitor, osteoblast, and
clsteoclast cells were categorized using
Young’s classification of cell types. The
percentage of labeled cells of each type
was determined by examining 1,000 osteoprogenitor cells, 1,000 osteoblasts, and 200
osteoclasts. To determine whether or not
osteoprogenitor cells divide prior to forming osteoblasts, grain counts were made on
cells of these types which appeared to be
maximally labeled (see DISCUSSION).
MATERIALS AND METHODS
Ligamentous proliferation, proliferation
or swelling of fibrocartilage caps, resorption of the dorsal walls of the pubic bones,
and a decrease in the number of metaphyseal trabeculae were noted. These observations were in agreement with the findings
of other workers (Steinetz et al., ’65; Crelin
and Levin, ’55; Haines, ’57; Hall, ’50).
The examination of cell populations revealed a preliminary fall in the number of
osteoprogenitor cells to a level that was
maintained throughout later time periods.
There was a progressive rise in the number
Four-week-old female mice of the Ajax
strain, weighing 10-15 gm at the start of
the experiment, were used in this investigation. The animals were kept in stainlesssteel cages with Purina Laboratory Chow
and tap water available ad libitum. The environment was maintained at 21-24”C,
40-60% humidity, with natural lighting.
At 28 days of age, each of 16 experimental animals was injected with 0.025
ml (0.005 mg) of estradiol 17-beta cyclopentyl propionate in oil. Injections were
made subcutaneously in the interscapular
region. On day 29, each of the 16 animals
was injected intraperitoneally with thymidine-rneth~l-~H,
one pc/gm body weight.
Following the administration of thymidine-
RESULTS
3 The experiments were conducted according to the
“Rules Regarding Animal Care” as established by the
American Medical Association.
4 Obtained from DuPage Kinetic Laboratories, Downers Grove, Illinois.
J Obtained from Eastman Kodak Company, Roches
ter, New York.
511
ESTROGEN-STIMULATED OSTEOCLASTOGENESIS
TABLE 1
Differential cell count
Hours post- 2
thymidine-3H
Osteoprogenitor
Ave.
1
Osteoblast
Ave.
Osteoclast
Ave.
2
633
621
627
326
364
345
41
15
28
4
541
530
536
384
391
387
75
79
77
8
513
526
520
407
379
393
80
95
87
12
498
453
475
421
481
451
81
66
74
24
521
500
510
402
411
407
77
89
83
30
581
513
548
298
314
306
121
173
147
36
546
510
528
352
421
386
102
68
85
48
504
Control
556
526
105
391
541
422
453
437
22
21
21
1 The figures given represent the number of cells of each type that was seen in a count of 1,000
cells per animal.
%Add24 hours for the time post-estrogen.
of osteoclasts. The number of osteoblasts
peaked and then decreased (table 1). The
concurrent opposing changes in cell populations suggested the osteoprogenitor cells
were converted to osteoblasts and osteoclasts.
In order to shed further light on this, the
percentage of each cell type that was
labeled following the injection of thymidine-'H was determined. The percentage of
labeled osteoprogenitor cells (fig. 1) rose
from 16% at two hours to about 25%
thereafter. At two hours, the precentage of
labeled osteoblasts was only about 2% ,
and there were no labeled osteoclasts. At
later time periods, the percentage of labeled
osteoclasts, however, rose progressively to
a plateau of about 12.5% at 30 and 36
hours (table 2).
The population of osteoprogenitor cells
showed a fairly constant mitotic rate which
suggested that these cells were dividing
and maintaining a steady population in the
face of constant loss, apparently to the
osteoclast and osteoblast cell lines. If such
were the case, labeled osteoblasts and
labeled osteoclasts should have had only
one-half as much label as the undivided
osteoprogenitor cell. Grain counts over
maximally labeled cells of the three types,
which were presumed to be the undivided
cells of the population, confirmed this. No
more than one nucleus was ever labeled in
an osteoclast.
DISCUSSION
In this study, the preliminary shift in the
number of cells of each type was thought
to reflect the differentiation (modulation)
of osteoprogenitor cells into osteoblasts and
osteoclasts in response to estrogen (table
1). Inasmuch as there was not a progressive fall in the number of osteoprogenitor
cells after four hours, the initial drop apparently reflected the modulation of osteoprogenitor daughter cells that had been
formed prior to the administration of thymidine-'H. After these daughter cells had
modulated into osteoblasts and/or osteoclasts, the osteoprogenitor cells would have
had to undergo mitosis in order to provide
new precursors for the specialized bone
cells, and in order to maintain the basal
population of osteoprogenitor cells. In keeping with this concept, the total number of
osteoprogenitor cells, as well as the percentage of them that were labeled, remained fairly constant after four hours.
512
JAMES A. CORWIN AND JAMES R. MOREHEAD
TABLE 2
Percentage of labeled cells
Hours postthymidine-3H
2
4
Osteoprogenitor 1
labeled % 3
Osteoblast f
labeled % 3
Osteoclast 2
labeled % s
0
0
18
11
1.4
13.4
26
22
2.4
2
0
1.0
183
138
16
116
153
0
0
8
310
280
29.5
64
62
6.3
16
15
7.75
12
230
277
25.3
54
78
6.6
18
16
8.85
24
211
182
19.6
55
34
4.45
24
20
11.0
30
284
24 7
26.5
24
20
2.2
24
28
13.0
36
245
207
22.6
65
16
4.0
29
22
12.5
48 4
Based on examination of
Based on examination of
3 Percentages are based on
4 Unusable for this part of
1
1,000 cells.
200 cells.
the average of the two figures.
the experiment.
This constant percentage of labeled cells
within a relatively steady population was
thought to indicate the maintenance of a
stable population, some of which were differentiating into specialized cells. The
number of osetoclasts rose progressively,
reflecting the estrogen-stimulated osteoclastogenesis.
In order to determine the cell type of
origin for the osteoclast, one needed to
know which cells divided in response to
estrogen. Therefore, the percentage of
labeled cells of each type was determined
for each time period. The most important
observation to be made from the tabulated
data is that at all times the percentage of
labeled osteoprogenitor cells was much
greater than the percentage of either
labeled osteoblasts or labeled osteoclasts
(table 2). The small number of labeled osteoblasts, and the lack of labeled osteoclasts
at two hours suggest that the osteoprogenitor cell is the only cell, or the principal cell,
to synthesize DNA following estrogen
stimulation.
At later time periods, the increase in the
percentage of labeled osteoblasts and osteoclasts could not have been due to the incorporation of label by these cells, inasmuch
as th~midine-~H
is cleared from the system
within 15-45 minutes, even following intraperitoneal injection. Thus, these labeled
cells must have arisen from a previously
labeled cell. The only cell that was labeled
in sufficient quantity to account for the
magnitude of the increase in the percentage of labeled osetoblasts or of labeled osteoclasts was the osteoprogenitor cell.
Assuming that the osteoprogenitor cell
is the precursor of the osteoclast, and that
the osteoprogenitor cell must divide, undergoing "differential" mitosis (Ris, '55), with
one daughter cell maintaining the basal
population, and one daughter cell becoming an osteoclast, labeled osteoclast nuclei
should have only one-half as much label
as the most heavily labeled osteoprogenitor
cell (which presumably would not have
divided). In order to confirm this hypothesis, the grain counts were made only on
maximally labeled cells, so as to examine
only those cells that had not divided, even
if they could have done so. The data indicated that the osteoclast was indeed formed
from the daughter cells of osteoprogenitor
cells, since osteoclast nuclei were typically
overlain by one-half as many silver grains
as were osteoprogenitor cell nuclei. In light
of the fact that only one labeled osteoclast
nucleus was seen per cell, and in keeping
ESTROGEN-STIMULATED OSTEOCLASTOGENESIS
with the above hypothesis that the osteoprogenitor cells must divide, osteoclasts
are thought to arise from the fusion of both
labeled and unlabeled osteoprogenitor cells
(fig. 3). That they do not derive from the
fusion of osteoblasts is supported by the
small number of labeled osteoblasts relative
to the large number of labeled osteoclasts
that are seen at later time periods.
Considering the diametrically opposed
roles of the osteoblast and osteoclast, the
fusion of osteoblasts per se to form an osteoclast would be untenable unless the osteoblast were to undergo a change in its
functional capacity. Current theory considers such a modulation of cellular metabolism to be a fact of bone physiology.
According to this theory, as demonstrated
by the experiments of Heller, McLean, and
Bloom ('50), Young ('62), Talmage et al.
('65), Talmage ('67), Crelin and Koch
('67), and Crelin ('69), all cells of the
osteogenic and chondrogenic series are interrelated, and can transform from one
specialized cell into another as the hormonal environment shifts. The common
link among these cells is a primitive cell
that Young ('62) has termed the "osteoprogenitor" cell. The in vitro studies of Crelin
and Koch ('67), that demonstrated the
transformation of chondrocytes into osteoprogenitor cells from which derived the osteoid series of cells, led these authors to
suggest that Young's progenitor cell could
more appropriately be called an osteochondroprogenitor cell.
Implicit in the experiments that have
been cited regarding the source of osteoclasts has been the concept that the osteoprogenitor cell (of whatever source) has
the potential, following estrogen stimulation, to become an osteoclast. The experiment that is the subject of this paper was
designed specifically to evaluate this concept. Hence, the effect of estrogen on the
osteoprogenitor cell was studied, with the
full realization that other events were occurring simultaneously.
The observations that have been made
are in accord with those of Young ('62),
which were made on normal bone, and
which were supported by Talmage et al.
('65) and by Talmage ('67) in studies of
parathyroid hormone stimulated bone.
Their studies demonstrated that osteoclasts
513
originate from a mesenchymal precursor
cell that Young ('62) termed an osteoprogenitor cell in order to indicate its commitment as an osteogenic cell. The work reported herein has indicated that osteoclasts
arise from the osteoprogenitor cell in the
pubic symphysis of the mouse following
estrogen stimulation similarly as they do
following parathyroid hormone stimulation.
LITERATURE CITED
Burrows, H. 1935 Separation of the pubic
bones following the administration of oestrogens to male mice. J. Physiol., 85: 159-160.
Crelin, E. S. 1969 The development of the
bony pelvis and its changes during pregnancy
and parturition. Trans. N.Y. Acad. Sci., 31:
1049-1058.
Crelin, E. S., and W. E. Koch 1967 A n autoradiographic study of chondrocyte transformation into chondroclasts and osteocytes during
bone formation in vitro. Anat. Rec., 158: 473484.
Crelin, E. S.,and J. Levin 1955 The prepuberal
symphysis and uterus in the mouse: Their response to estrogen and relaxin. Endocrinology,
57: 730-747.
Gardner, W. U. 1940 Modifications of bones of
animals receiving sex hormones. Anat. Rec.,
76: 22 (Abstract).
Haines, A. L. 1957 The effect of estrogen on
cartilage and bone in castrate CsH mice. Yale
J. Biol. Med., 30: 121-136.
Hall, K. 1947 The effects of pregnancy and
relaxin o n the histology of the pubic symphysis in the mouse. J. Endocr., 5: 174-182.
1950 The effect of oestrone and progesterone on the histological structure of the
symphysis of the castrated female mouse. J.
Endocr., 7 : 54-63.
Heller, M., F. C. McLean and W. Bloom 1950
Cellular transformations in mammalian bones
induced by parathyroid extract. Am. J. Anat.,
87: 315448.
Hisaw, F. L. 1925 The influence of the ovary
on the resorption of the pubic bones of the
pocket-gopher, Geomys bursarius ( Shaw). J.
Exp. Zool., 42: 411441.
1926 Experimental relaxation of the
pubic ligament of the guinea pig. Proc. SOC.
Exp. Biol. and Med., 23: 661-663.
-1929 The corpus luteum hormone. I.
Experimental relaxation of the pelvic ligaments
of the guinea pig. Physiol. Zool., 2: 59-79.
Kopriwa, B. M., and C. P. Leblond 1962 Improvements in the coating technique for radioautography. J. Histochem. Cytochem., 10: 269284.
Ris, H. 1955 Cell Division. In: Analysis of
Development. B. H. Willier, P. A. Weiss and
V. Hamburger, eds. W. B. Saunders Co., Philadelphia, pp. 91-125.
Scott, B. L. 1967 T h ~ m i d i n e - ~electron
H
microscopic autoradiography of osteogenic cells in
fetal rats. J. Cell. Biol.. 35: 115-126.
514
JAMES A. CORWIN AND JAMES R. MOREHEAD
Selye, H. 1932 Action of parathyroid hormone
in the epiphyseal junction of the young rat.
Arch. Path., 14: 60-65.
Steinetz, B. G.,J. P. Manning, M. Butler and V.
Beach 1965 Relationship of growth hormone,
steroids, and relaxin in the transformation of
pubic joint cartilage to ligament in hypophysectomized mice. Endocrinology, 76: 876-882.
Talmage, R. V. 1947 The role of estrogen in
the estrogen-relaxin relationship in the relaxation of the symphysis pubis of the guinea pig.
Anat. Rec., 99: 571 (Abstract).
1967 A study of the effect of parathyroid hormone on bone remodeling and on calcium homeostasis. Clin. Orthop., 54: 163-173.
Talmage, R. V., S. B. Doty, C. W. Cooper, C. Yates
and J. Neueschwander 1965 Cytological and
biochemical changes resulting from fluctuations
i n endogenous parathyroid hormone levels. In:
The Parathyroid Glands, P. J. Gaillard, R. V.
Talmage and A. M. Budy, eds. W. B. Saunders
Co., Philadelphia, pp. 107-124.
Urist, M. R., A. M. Budy and F. C. McLeon 1950
Endosteal bone formation in estrogen-treated
mice. J. Bone and Joint Surg., 32A: 143-162.
Young, R. W. 1962 Cellular proliferation and
specialization during endochondral osteogenesis i n young rats. J. Cell Biol., 14: 357-370.
PLATE 1
EXPLANATION O F FIGURES
1
Radioautograph. In the center of the field is a trabeculum of bone
along which are a number of osteoprogenitor cells and osteoblasts. A
labeled osteoprogenitor cell is seen (arrow). Immediately to its left is
a n unlabeled cell of the same type. X 1,250.
2 Radioautograph. In the center of the field, within a large lacuna, is a
trinucleate osteoclast. This photomicrograph has been focused so a s to
reveal cytoplasmic detail. Note the vacuolated cytoplasm, and of special interest, apparent fusion with a spindle-shaped cell. x 2,000.
3 The same field as in figure 2, but focused o n the silver grains. Note
that the silver mains are over only one nucleus of the osteoclast.
x 2,000.
ESTROGEN-STIMULATED OSTEOCLASTOGENESIS
PLATE 1
James A. Corwin and James R. Morehead
515
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