Full text of DOI:10.1359/jbmr.2001.16.5.823

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 5, 2001
© 2001 American Society for Bone and Mineral Research
The Hormonal Milieu in Early Stages of Bone Cell
Differentiation Modifies the Subsequent Sex-Specific
Responsiveness of the Developing Bone
to Gonadal Steroids
E. BERGER,1 I. BLEIBERG,2 Y. WEISMAN,3 B. LIFSCHITZ-MERCER,1 L. LEIDER-TREJO,1 A. HAREL,4,5
A.M. KAYE,6 and D. SOMJEN4
ABSTRACT
We have established previously that rat bone tissue, as well as rat and human-derived bone cells in culture,
show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of
creatine kinase (CK) and DNA synthesis. This response could be modified by manipulation of the endocrine
environment during early stages in rat development. To further examine the influence of changing hormonal
steroid milieu and vitamin D status on the action of gonadal steroids in developing bone tissue, we used two
models of ectopic bone formation: demineralized tooth matrix (DTM) implanted under the skin, and femoral
bone marrow (BM) transplanted under the kidney capsule of a syngeneic recipient mouse. The response to
gonadal steroids in ossicles developed from implanted DTM depended on the recipient’s gender; injection of
estradiol 17␤ (E2; 5 ␮g) into young female mice 21 days after DTM implantation increased, 24 h later, CK
activity in the newly formed ossicles by ϳ60%, whereas injection of dihydrotestosterone (DHT; 50 ␮g) had no
effect on CK activity. In contrast, in male mice, DHT but not E2 increased CK activity in the ossicles by ϳ50%.
This sex-specific response was abolished in gonadectomized mice resulting in a similar response of the ossicles
to both E2 and DHT. When DTM was implanted into vitamin D–deficient female mice, there was a lower basal
CK activity and a significantly diminished response to E2 in the newly formed bone tissues. When BM, which
contains mesenchymal and stromal cells and committed osteoprogenitor cells, was transplanted into 6-week-
old intact or gonadectomized female or male mice, the response of the newly formed bone ossicles, 21 days
after transplantation, to E2 or to DHT was according to the gender of the donor. Bone formed from BM
obtained from female mice responded to E2 only and those formed from male BM responded to DHT only.
Ossicles developed from BM obtained from gonadectomized mice showed lack of response to either gonadal
steroid. Furthermore, only ϳ25% of the BM transplants obtained from castrated (CAST) male donors
developed into ossicles. Ossicles formed from BM obtained from vitamin D–deficient female donors showed
lack of response to gonadal steroids. These findings suggest that the manipulation of the hormonal milieu in
early stages of the differentiation sequence of bone cells modifies the subsequent selective responsiveness of the
developing bone tissue to gonadal steroids. (J Bone Miner Res 2001;16:823–831)
Key words: bone marrow, bone, cartilage, implants, estrogen, androgens, creatine kinase, vitamin D
1Institute of Pathology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.
2Department of Cell Biology and Histology, Tel-Aviv University, Tel-Aviv, Israel.
3Bone Disease Unit, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.
4Institute of Endocrinology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel.
5Deceased.
6Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel.
823

824
BERGER ET AL.
INTRODUCTION
Preparation and implantation of DTM
DTM particles were prepared by washing mice teeth with
distilled water and then dehydrating by ethanol and ether.
The particles were then pulverized in a mortar in the pres-
ence of liquid nitrogen and sieved to select particles of
about 75–300 ␮. The particles were washed with 0.5 M HCl
and dehydrated. All steps were carried out at 4°C.(27)
Mice at the age of 6 weeks were anesthetized and two
samples of 2 mg of DTM each were implanted under the
E HAVE established previously that rat bone in vivo
(1,2)
Wand cultured rat-embryo calvaria cells,
human
osteoblast-like cells,(3) and human untransformed bone
cells(4) show sex-specific response to gonadal steroids; es-
tradiol 17␤ (E2) stimulated creatine kinase (CK) activity
and DNA synthesis only in female bone cells, whereas
testosterone or dihydrotestosterone (DHT) stimulated CK
activity only in male bone cells. This sex-specific response skin (subcutaneously) over the pectoralis muscle.
of bone cells to gonadal steroids can be modified by ma-
nipulation of the endocrine environment in early develop-
Transplantation of femoral BM
ment as we showed in young rats after gonadectomy,(5) in
prenatally or neonatally androgenized female rats,(6) and in
Mice were killed by ether and BM was prepared by
androgen-receptor–deficient (Tfm) male rats.(6) It also was
pushing the femoral marrow out with a needle and keeping
it in the cold. The BM was obtained from either 6-week-old
intact female mice or 6-week-old intact male mice, ovari-
ectomized (OVX) female mice, castrated (CAST) male
mice, and vitamin D–deficient mice.
Two samples of femoral BM were transplanted under the
renal capsules (one sample per kidney) of a syngeneic recipient
mouse, separating it physically from other possible migrating
cells, either intact female mice or intact male mice, OVX mice
or CAST mice, and vitamin D–deficient mice.
shown that in vitamin D–deficient rats, E2 or DHT failed to
stimulate DNA synthesis in diaphyseal (Di) bone and the
CK response was markedly lower.(7–9) To further examine
the role of gender and/or the hormonal milieu on the action
of gonadal steroids in developing bone tissues, we used two
experimental models of ectopic bone formation in mice: (1)
implantation of demineralized bone or tooth matrix (DTM)
particles under the skin(10,11) (DTM particles contain colla-
gens and bone morphogenic proteins (BMPs), which induce
bone formation by pluripotent mesenchymal cells of the
recipient)(12-18); (2) transplantation of mouse femoral bone
marrow (BM) under the renal capsule of syngeneic recipi-
ent, resulting in the formation of osteogenic tissue origi-
nated from stromal and/or osteoprogenitor cells from the
donor mouse.(19–23) These experimental approaches enabled
us to dissociate between the influence of the animals’ gen-
der from that of the hormonal milieu on the response of the
newly formed ossicles to gonadal steroids.
Morphology
Three weeks after implantation, mice were killed and
implants were collected, fixed in 10% neutral buffered for-
malin, decalcified, dehydrated, and paraffin embedded. Sec-
tions (3 ␮m thick) were stained for hematoxylin and eosin.
Pictures were taken at ϫ100 enlargement.
In the present study we tested the hormonal regulation of
the development of the two ectopic bone tissues, because it
is well established that hormones such as gonadal steroids
and vitamin D(7,24,25) regulate bone formation. We exam-
ined the effects of these hormones on the specific activity of
the brain isozyme of CK in the two ectopic tissues. This
enzyme has been used as a general genomic response
marker for gonadal steroids. This enzyme is involved in
cellular energy buffering and is related closely to changes in
cell replication rate in various cell types.(24–26)
Hormonal treatment
Three weeks (or other times if indicated) after implanta-
tion of DTM or transplantation of BM, female mice, male
mice, OVX female mice, or CAST male mice were injected
intraperitoneally (ip) with 5 ␮g of E2 or 50 ␮g of DHT.
Twenty-four hours later the mice were killed; the implants
or transplants as well as bone, cartilage, or BM from the
host mice were collected for enzyme measurements.
To examine the effect of vitamin D deficiency and vita-
min D treatment on the response of the newly formed
ossicles to the gonadal steroids, vehicle (0.01% ethanol), 3
ng/g body weight (BW) 1,25-dihydroxyvitamin D3
[1,25(OH)2D3], or 30 ng/g BW 24,25-dihydroxyvitamin D3
[24,25(OH)2D3] were injected daily for 3 weeks into vita-
min D–depleted donors before obtaining the BM for trans-
plantation followed, in some experiments, by gonadal ste-
roids injections as described previously.
MATERIALS AND METHODS
Animals
C57Bl/6J mice were obtained from the colony at Tel-
Aviv University, Sackler School of Medicine, and used at
the age of 6 weeks. Intact male or female (immature) or
gonadectomized mice (2 weeks postsurgery starting at the
age of 6 weeks) as well as vitamin D–deficient mice were
obtained as described previously for rats, namely, at the age
of 4 weeks, the mice were switched to grow in the dark on
a vitamin D–deficient diet supplemented with Ca (0.75%)
and phosphate (0.55%).(7)
Enzyme extraction and assay
At the end of the hormonal treatment, bones formed from
the implants, the mice bone, and cartilage were collected
and homogenized in a cold homogenizing buffer using an
Ultra Torax (Janke and Kunkel, Staufer Brisgov, Germany)
and the CK was obtained by centrifugation at 14,000g as

BONE IMPLANTS AND GONADAL STEROIDS
825
FIG. 2. Age-dependent stimulation by gonadal steroids of CK-
specific activity in different organs in female mice. Female mice at the
ages of 9, 12, 15, and 19 weeks were injected with 5 ␮g of E2 or 50 ␮g
of DHT and killed 24 h later. Mouse organs Ep cartilage, Di bone, and
BM were collected and homogenized and enzyme was extracted and
assayed as described in the Materials and Methods section. Results are
expressed as experimental/control Ϯ SEM for n ϭ 5–10. *p Ͻ 0.05;
**p Ͻ 0.01. The basal levels (in ␮mol/minute per milligrams protein)
of CK at 9 weeks were Ep ϭ 7.9 Ϯ 1.3, Di ϭ 11 Ϯ 1.5, and BM ϭ
0.94 Ϯ 0.1; at 12 weeks were Ep ϭ 5.6 Ϯ 1.7, Di ϭ 8.6 Ϯ 0.6, BM ϭ
0.62 Ϯ 0.13; at 15 weeks were Ep ϭ 2.3 Ϯ 0.4, Di ϭ 8.6 Ϯ 1.5, BM ϭ
1.38 Ϯ 0.28; at 19 weeks were Ep ϭ 2.0 Ϯ 0. 5, Di ϭ 6.3 Ϯ 1.5, and
BM ϭ 0. 78 Ϯ 0.19.
FIG. 1. The effect of E2 and DHT on CK-specific activity in imma-
ture (6 weeks old) and gonadectomized male and female mouse organs.
Animals were injected with 5 ␮g of E2 or 50 ␮g of DHT and killed 24 h
later. Mice organs: Ep cartilage, Di bone, and BM were collected and
homogenized, and enzyme was extracted and assayed as described in
the Materials and Methods section. Results are expressed as experi-
mental/control Ϯ SEM for n ϭ 5–10. *p Ͻ 0.05; **p Ͻ 0.01. The
following were the basal levels (in ␮mol/minute per milligram pro-
teins) of CK in immature female mice: Ep ϭ 5.9 Ϯ 1.0, Di ϭ 12.6 Ϯ
1.1, and BM ϭ 0.67 Ϯ 0.13; in immature males: Ep ϭ 5Ϯ0.6, Di ϭ
16.4 Ϯ 1.25, and BM ϭ 0.5 Ϯ 0.08; in gonadectomized females: Ep ϭ
2.6 Ϯ 0.45, Di ϭ 9.3 Ϯ 0.45, and BM ϭ 1.7 Ϯ 0.07; in gonadecto-
mized males: Ep ϭ 5.1 Ϯ 0.7, Di ϭ 11.9 Ϯ 0.8, and BM ϭ 2.4 Ϯ 0.5.
described previously.(1,6) Enzyme activity was measured
using the CK kit (Sigma-Aldrich, Rehovot, Israel) in a
computerized automatic spectrophotometer (Kontron AG,
Milan, Italy). A unit of enzyme activity was defined as the
amount yielding 1 ␮mol of adenosine triphosphate (ATP)/
minute at 30°C and the specific activity was defined as units
per milligram of protein.(1)
three different experiments with at least five implants or
organs in each.
Reagents
All reagents were of analytical grades and were obtained
from Sigma Co., Ltd.
Protein was assayed by the Coomassie brilliant blue dye
binding assay(28) using bovine serum albumin (BSA) as the
standard.
RESULTS
The effect of gonadal steroids on CK activity in mouse
bone and cartilage
Statistical analysis
The significance of differences between experimental and
Mouse bone responded to E2 or DHT sex specifically as
control means was evaluated by Student’s t-test for at least previously shown in rats.(1) E2 stimulated CK-specific ac-

826
BERGER ET AL.
FIG. 3. Morphology of ossicles formed by either (A) DTM origi-
nated from females and transplanted in females or (B) BM obtained
from females and implanted into females. The details are given in the
Materials and Methods section.
FIG. 4. Time-dependent stimulation by gonadal steroids of CK-
specific activity in ossicles originated from DTM implantation and
from BM transplantation. BM and DTM were implanted in intact
female mice for different time periods. At the different times mice were
injected with 5 ␮g of E2 or 50 ␮g of DHT and killed 24 h later. The
ossicles (BM and DTM) were collected homogenized and enzyme was
extracted and assayed as described in the Materials and Methods
section. Results are expressed as experimental/control Ϯ SEM for n ϭ
5–10. *p Ͻ 0.05; **p Ͻ 0.01. The basal levels (in ␮mol/minute per
milligram protein) were BM ϭ 0.74 Ϯ 0.06 and DTM ϭ 4.1 Ϯ 0.3.
tivity only in female Di bone by 40–50%, whereas DHT
stimulated CK-specific activity only in male Di bone by
80–120% (Fig. 1). In contrast to rat epiphyseal (Ep) carti-
lage, mouse Ep cartilage also responded in a sex-specific
pattern (Fig. 1), which means Ep cartilage from female mice
responded to E2 while Ep cartilage from male mice to DHT
only. BM from female mice responded to both gonadal
steroids, whereas from male mice there was no significant
response to E2. OVX female mice or CAST male mice
responded to both gonadal steroids that is, loosing the sex
specificity of the response in either Ep or Di, or male BM
(Fig. 1). When female mice were used at different ages, the
basal activity of CK in Ep cartilage, Di bone, and BM
declined with age (Fig. 2), but their sex-specific response
remained similar while in BM the response declined with
age (Fig. 2).
Gonadal steroids stimulation of CK activity in ossicles
induced by DTM implants
When DTM from female mice was implanted in female
mice, their constitutive CK-specific activity was changed
with time after implantation. They responded only to E2 and
its magnitude was changed with time after implantation
(Fig. 4). There was a significant response 21 days after
The features of the ossicles formed by the implanted
DTM or BM
Ossicles formed by implanted DTM show new bone implantation, and, therefore, this time point was chosen for
tissues rich in osteocytes surrounded by a layer of osteo- analysis of their hormonal responsiveness. The response to
blasts (Fig. 3A, thin arrow). We also can see hematopoietic gonadal steroids in ossicles developed from implanted DTM
tissue between bone trabeculae. The DTM particle not cov- depended on the recipient gender; injection of E2 into im-
ered by osteoblasts is seen in the upper left corner of Fig. 3 mature female mice 21 days after DTM implantation in-
(thick arrow). Ossicles formed by implantation of BM under creased, 24 h later, CK-specific activity in the newly formed
the renal capsule show bone tissue rich in osteocytes and ossicles by 60% (Fig. 5), whereas DHT had no effect on CK
covered by osteoblasts (Fig. 3B). New hematopoietic tissue activity. In contrast, in male mice, DHT but not E2 increased
is observed in the ossicle.
CK activity in the newly formed ossicles by 50% (Fig. 5).

BONE IMPLANTS AND GONADAL STEROIDS
827
FIG. 6. Gonadal steroid stimulation of CK-specific activity in os-
sicles formed from DTM implanted into intact or vitamin D–depleted
female mice. DTM from intact mice was implanted into intact or
vitamin D–depleted female mice for 3 weeks, and mice were injected
with 5 ␮g of E2 or 50 ␮g of DHT and killed 24 h later. The ossicles
(DTM) were collected and homogenized and enzyme was extracted and
assayed as described in the Materials and Methods section. Results are
expressed as experimental/control Ϯ SEM for n ϭ 5–10. *p Ͻ 0.05;
**p Ͻ 0.01. The following were the basal levels (in ␮mol/minute per
milligrams protein): into intact female ϭ 4.15 Ϯ 0.52 and into vitamin
D–depleted female ϭ 2.23 Ϯ 0.17.
FIG. 5. Gonadal steroid stimulation of CK-specific activity in os-
sicles formed from DTM implanted into either intact male or female
mice or gonadectomized male and female mice. DTM was implanted
into intact male or female mice or gonadectomized male and female
mice for 3 weeks and mice were injected with 5 ␮g of E2 or 50 ␮g of
DHT and killed 24 h later. The ossicles (DTM) were collected and
homogenized and enzyme was extracted and assayed as described in
the Materials and Methods section. Results are expressed as experi-
mental/control Ϯ SEM for n ϭ 5–10. *p Ͻ 0.05; **p Ͻ 0.01. The basal
levels (in ␮mol/minute per milligrams protein) were DTM into intact
male ϭ 4.53 Ϯ 0.77, DTM into intact female ϭ 4.15 Ϯ 0.52, DTM into
gonadectomized male ϭ 6.47 Ϯ 1.29, and into DTM gonadectomized
female ϭ 5.32 Ϯ 0.8.
male mice, the response of the newly formed ossicles to E2
or DHT, 3 weeks after transplantation, was determined by
the gender of the donor; ossicles formed from BM obtained
from female mice responded to E2 by a 30–100% increase
in CK activity (Fig. 7), whereas in those formed from
transplanted male BM, the enzymatic activity was induced
This sex-specific response of ossicles induced by DTM was only by DHT by 40–70% (Fig. 7). When BM from intact
abolished in gonadectomized male and female mice, result- female mice was transplanted into OVX mice, the ossicles
ing in a similar response (70–90%) of the ossicles to both formed had high CK-specific activity and responded to E2
E2 and DHT (Fig. 5). When DTM was implanted into but not to DHT by a 300% increase in CK activity. When
vitamin D–depleted female mice, there was a significant BM was transplanted into CAST mice, the CK activity was
lower CK basal activity compared with intact DTM im- low but still responded only to E2 by 40% (Fig. 8). When
planted into intact mice and a significantly diminished sex- BM from intact male mice was transplanted into OVX mice,
specific response to E2 in the newly formed ossicles (20% the basal CK activity in the ossicles was low but was
compared with 60% stimulation; Fig. 6).
stimulated significantly by DHT and not by E2 (Fig. 8). BM
obtained from OVX or CAST mice and transplanted either
into intact male or intact female mice resulted in decreased
basal CK activity with no response to either gonadal ste-
roids (data not shown). Furthermore, only ϳ25% of BM
Gonadal steroids stimulation of CK activity in ossicles
formed by BM transplantation
When BM from mice was transplanted under the kidney transplanted obtained from CAST male donors developed
capsule of 6-week-old intact or gonadectomizes female or into bone ossicles.

828
BERGER ET AL.
FIG. 8. Gonadal steroid stimulation of CK-specific activity in os-
sicles formed from BM implanted into gonadectomized animals. BM
from male or female mice was implanted into gonadectomized male
(CAST) or female (OVX) mice for 3 weeks and mice were injected
with 5 ␮g of E2 or 50 ␮g of DHT and killed 24 h later. The ossicles
(BM) were collected and homogenized and enzyme was extracted and
assayed as described in the Materials and Methods section. Results are
expressed as experimental/control Ϯ SEM for n ϭ 5–10. *p Ͻ 0.05;
**p Ͻ 0.01. The basal levels (in ␮mol/minute per milligram protein)
were from intact female into OVX female ϭ 0.38 Ϯ 0.08, from intact
male into OVX female ϭ 0.15 Ϯ 0.06, from intact female into CAST
male ϭ 0.061 Ϯ 0.013, and from intact male into CAST male ϭ
0.068 Ϯ 0.020.
FIG. 7. Gonadal steroid stimulation of CK-specific activity in os-
sicles formed from BM transplanted into different mice. BM from male
or female mice was implanted into intact male or female mice for 3
weeks and mice were injected with 5 ␮g of E2 or 50 ␮g of DHT and
killed 24 h later. The ossicles (BM) were collected and homogenized
and enzyme was extracted and assayed as described in the Materials
and Methods section. Results are expressed as experimental/control Ϯ
SEM for n ϭ 5–10. *p Ͻ 0.05; **p Ͻ 0.01. The basal levels (in
␮mol/minute per milligrams protein) were from female into intact
female ϭ 0.74 Ϯ 0.06, from male into intact female ϭ 0.22 Ϯ 0.03,
from female into intact male ϭ 0.45 Ϯ 0.11, and from male into intact
male ϭ 0.44 Ϯ 0.09.
BM obtained from intact female mice and transplanted
into vitamin D–depleted female mice developed into bone
tissue, which had low basal CK activity with no response to
gonadal steroids (Fig. 9). Ossicles formed from BM ob-
tained from vitamin D–deficient female donors transplanted
into either intact female mice or into D-depleted female
mice showed both low basal CK activity and lack of re-
sponse to gonadal steroids (Fig. 9). BM from vitamin
vitamin D treatment, up to a 2-fold increase in CK activity
(Fig. 10).
DISCUSSION
The investigation of the effects of gonadal steroids on
D–depleted female mice that had been replenished by daily bone cells was stimulated in the last decade by the finding
injections of either 1,25(OH)2D3 or 24,25(OH)2D3 and then of low concentrations of receptors for estrogen(29) and an-
transplanted into intact female mice developed into bone drogens(30) in bone cells and demonstration by several lab-
tissues with higher basal CK activity compared with BM oratories of direct effects of these hormones on the stimu-
with no vitamin D treatment, and the newly formed ossicles lation of bone cell proliferation and function. We have used
showed a higher response to E2 compared with BM with no markers of cell proliferation ([3H]thymidine incorporation

BONE IMPLANTS AND GONADAL STEROIDS
829
cultures obtained from female fetuses androgenized in
utero.(6) However, in vitro treatment of female rat-embryo
calvaria cells in culture with either testosterone or E2 did not
change their response to E2.(6) These observations raised the
question whether exposure to a certain hormonal steroid
environment in early critical stages of bone cell lineage
differentiation determines the subsequent responsiveness of
rat bone cells to gonadal steroids. The results presented in
this article show that in mice, as in rat bone, there was a
sex-specific response of bone to gonadal steroids. However,
in contrast to rat Ep cartilage,(2) which responds to both E2
and DHT, mouse cartilage responded to gonadal steroids
sex specifically (Fig. 1).(2) This may reflect a different
pattern of differentiation, growth, and/or receptor develop-
ment in the skeletal tissues of rats and mice. BM shows a
peculiar behavior; BM originated from females responds to
both hormones, whereas BM from males responds only to
androgens and not to estrogens. It might be because of
different modulation of the receptors to these hormones,
which has to be analyzed further. Gonadectomy of mice
abolished the sex-specific response of bone and cartilage in
both sexes and male BM to gonadal steroids. It also ac-
quired a response to the less dominant steroid, probably by
counteracting the effects of each sex steroid on the action by
the other, by an unknown mechanism that might involve
modulation of receptors or their coactivators and/or core-
pressors (Fig. 1) similar to what was described for the rats.
The results of this study, in which we used experimental
models of ectopic bone formation, indicate that the hor-
monal milieu of the donors’ BM mesenchymal stem cells or
osteoprogenitor cells determines the development and the
subsequent response. In contrast, ossicles originated from
DTM implants responded sex specifically to E2 or DHT,
depending on the gender of the host, with no dependence on
the gender of the DTM itself. Gonadectomy, resulted in loss
of sex specificity of the response of the ossicles and they
responded to both hormones, similar to rat skeletal tissues(5)
(Fig. 5(8)). The results obtained by these models suggest that
the critical time point for the appearance of active receptors
to gonadal steroids in the skeletal tissues is in the early
stages of bone development (i.e., on stem cells differentia-
tion into osteoprogenitor cells). If the hormonal levels in the
milieu are normal at this stage, even if implanted into a
gonadectomized animal, there will be a normal sex-specific
response. On the other hand, BM taken from gonadecto-
FIG. 9. Gonadal steroid stimulation of CK-specific activity in os-
sicles formed from BM obtained from intact or vitamin D–depleted
female mice implanted into intact or vitamin D–depleted female mice.
BM from intact female or vitamin D–depleted female mice was im-
planted into intact or vitamin D–depleted female mice for 3 weeks and
mice were injected with 5 ␮g of E2 or 50 ␮g of DHT and killed 24 h
later. The ossicles (BM) were collected and homogenized and enzyme
was extracted and assayed as described in the Materials and Methods
section. Results are expressed as experimental/control Ϯ SEM for n ϭ
5–10. *p Ͻ 0.05; **p Ͻ 0.01. The basal levels (in ␮mol/minute per
milligram protein) were from intact into D-depleted ϭ 0.120 Ϯ 0.006,
from D-depleted into D-depleted ϭ 0.25 Ϯ 0.04, from D-depleted into
intact ϭ 0.218 Ϯ 0.080, and from D-depleted into D-depleted ϭ
0.249 Ϯ 0.037.
into DNA) and energy metabolism associated with cell mized mice, implanted into intact male or female mice, will
division (the specific activity of CK(26)) to show that pre- lose the sex specificity of the response to gonadal steroids,
pubertal rat bone in vivo, as well as cultured rat-embryo which now respond to both. When gonadectomized mice
calvaria cells,(1,2) human osteoblast-like cells,(3) and human were replenished before the experiments, the results were
untransformed bone cells,(4) show a sex-specific response to similar to those obtained using intact mice (data not shown).
gonadal steroids. Estrogens are active only in female-
Previously, we have shown that there was a mutual in-
derived cells and androgens are active only in male cells. teraction between vitamin D analogs and gonadal steroids in
This sex-specific response of bone cells to gonadal steroids affecting the development and responsiveness of the skele-
can be modified and broadened by manipulation of the tal system.(7–9) Vitamin D depletion resulted in a decreased
hormonal milieu as we showed in prepubertal rats after response of the CK activity and DNA synthesis of rat bones
gonadectomy,(5) in prenatally or neonatally androgenized to gonadal steroids, but the sex specificity of the reduced
female rats,(6) and in androgen-receptor deficient (Tfm) response was retained.(7) Replenishment with 1,25(OH)2D3
male rats.(6) The broadening of sex hormone specificity of but not 24,25(OH)2D3 restored the gonadal steroid respon-
rat bone responsiveness by in vivo changes in the hormonal siveness. In addition, both in vivo(7) and in vitro,(8) pretreat-
milieu can be maintained in vitro, as shown in calvaria cell ment with vitamin D analogs increased the responsiveness

830
BERGER ET AL.
FIG. 10. Gonadal steroid stimulation of CK-specific activity in ossicles formed from BM obtained from intact or vitamin D–depleted female
mice or vitamin D–depleted after replenishment female mice and transplanted into intact female mice. BM from females either intact, from vitamin
D–depleted, vitamin D–depleted and injected for 3 weeks with 1,25 (3 ng/g), or vitamin D–depleted and injected for 3 weeks with 24,25 (30 ng/g)
was implanted into intact female mice and then the hosts were injected with 5 ␮g of E2 or 50 ␮g of DHT and killed 24 h later. The ossicles (BM)
were collected and homogenized and enzyme was extracted and assayed as described in the Materials and Methods section. Results are expressed
as experimental/control Ϯ SEM for n ϭ 5–10. *p Ͻ 0.05; **p Ͻ 0.01. The basal levels (in ␮mol/minute per milligram protein) were from intact ϭ
0.74 Ϯ 0.06, from vitamin D–depleted ϭ 0.184 Ϯ 0.029, from vitamin D–depleted ϩ 1,25 ϭ 0.397 Ϯ 0.032, from vitamin D–depleted ϩ 24,25 ϭ
0.342 Ϯ 0.027.
and the sensitivity of bone cells to gonadal steroids because the control of differentiation or selective proliferation of
of an increased number of the nuclear receptors for E2.(8,9) particular cell types, probably mesenchymal stem cells or
Reciprocally, estrogen increases the concentration of osteoprogenitor cells, which preferentially respond sex spe-
1,25(OH)2D3 receptors in rat uterus.(31) Therefore, it is cifically to a gonadal steroid, or (2) the regulation by a
possible that in responsive tissues each of these hormones steroid hormone of the genes for the synthesis of receptors
augment and/or modulate the receptors for the other hor- for the same or the other hormones.(9)
mones. Thus, the relationship between vitamin D and go-
In summary, this study shows that gonadal steroids stim-
nadal steroids also was studied in the ectopic bone forma- ulate sex specifically CK specific-activity in bone and in
tion systems presented in this article. Normal development cartilage of mice as well as in two ectopic bones formed as
of ossicles induced by DTM required normal levels of the result of DTM implantation or BM transplantation in
vitamin D in the host, because DTM transplanted into these mice. The response of ossicles formed from DTM
D-depleted mice had lower basal CK activity and a much depended on the hormonal milieu of the host mice, whereas
reduced sex-specific response to gonadal steroids (Fig. 6). the response of ossicles formed from BM transplantation
Moreover, BM obtained from intact mice, when trans- was dependent on the hormonal milieu of the donor mice. In
planted into D-depleted mice responded to gonadal steroids all systems, the response to gonadal steroids depends on
as when transplanted into intact mice, but BM obtained normal levels of both gonadal steroids and vitamin D in the
from D-depleted mice, when transplanted into intact mice, early stages of skeletal development.
had a lower basal enzyme activity and a significantly re-
duced sex-specific response to gonadal steroids (Fig. 9).
Replenishment of mice donors of the BM with 1,25(OH)2D3
restored the normal CK basal activity and response to go-
ACKNOWLEDGMENTS
nadal steroids in the ossicles (Fig. 10). The ossicles formed
had even significantly enhanced responsiveness to E2. These
observations suggest that the vitamin D status of the hor-
monal milieu, in which mesenchymal stem cells and osteo-
progenitor cells differentiate, had a significant effect on the
subsequent responsiveness to gonadal steroids of the newly
formed bone. At present, it is not clear which mechanism
determines the sex-specific responses of bone to gonadal
steroids. Possible mechanisms involved may include : (1)
This article is in partial fulfillment of the Ph.D. thesis for
The Sackler Faculty of Medicine, Tel-Aviv University, Tel-
Aviv, Israel for E.B.
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Address reprint requests to:
Dalia Somjen, Ph.D.
Institute of Endocrinology
Tel-Aviv Sourasky Medical Center
Tel-Aviv 64239, Israel
17. Kubler N, Reuther J, Kirchner T, Pfaff M, Muller-Hermelink Received in original form March 13, 2000; in revised form No-
HK, Albert R, Sebald W 1994 IgG monoclonal antibodies that vember 30, 2000; accepted December 27, 2000.