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Department of Morphological Sciences (G.M.), The B. Rappapport Faculty of Medicine, Technion, Haifa 31096, Israel; Molecular Endocrine Laboratory Soroka Medical Center (Y.S., M.P.), Ben-Gurion University of the Negev, Beer-Sheva 84101, Israel; and Felsenstein Medical Research Center (M.P.), Institute for Endocrinology and Diabetes, Schneider Children’s Medical Center, Beilinson Campus, Petach Tikva 49202, Israel
Address all correspondence and requests for reprints to: Prof. Moshe Phillip, Director, Institute for Endocrinology and Diabetes, Schneider Children’s Medical Center of Israel, 14 Kaplan Street, Petach-Tikva, 49202, Israel.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility that androgens increase the serum IGF-I levels by a direct stimulatory effect on the liver has been previously ruled out by our group (11). Another possibility is that sex steroids stimulate the growth centers of the bones, either directly or through local production of IGF-I or other growth factors. Despite the well-known importance of local growth factors in regulating skeletal growth (12), there is almost no information on their possible role in mediating the effects of gonadal steroids.
In the present study, we used an in vitro organ culture of mandibular condyle to study the direct effect of sex hormones on bone growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[3H]-thymidine incorporation
Cultures were incubated for the last 18 h of the culture
period in the presence of 5µCi/ml of
methyl-[3H]-thymidine (5.0 Ci/mmol, Amersham International plc, Little Chalfont, Buckinghamshire, UK).
TCA-insoluble material was precipitated with cold 5% TCA (containing 1
mM thymidine). Explants were then washed in acetone and
ether, air dried and dissolved in Soluene 350 (Packard, Downers Grove,
IL). Radioactivity was counted in scintillation fluid containing 0.2%
glacial acetic acid. Our preliminary trials showed that
10-6 M is the optimal dose of both
testosterone and estradiol for thymidine incorporation into DNA assay
(data not shown).
Morphology and morphometric studies
Paraffin sections (6 µm) were deparaffinized in xylene,
hydrated in graduated ethanols, and stained in hematoxylin/eosin.
Stained sections served for morphometric studies. Histomorphometric
determinations of the total length of the cartilaginous zone were
performed with an Olympus Corp. Cue-2 image analysis
system using appropriate morphometry software (Olympus Corp., Lake Success, NY). The system consists of a Zeiss
Universal R photomicroscope (x10 objective) fitted with a Panasonic
WV-CD50 camera and a Sony 14" color monitor connected to an
IBM-compatible PC. Each point represents the average of measurements
done on four slides from each of three different experiments.
Significance is analyzed according to the two-tailed Student’s
t test.
Immunohistochemistry
Deparaffinized paraffin sections were exposed for 2 h at
room temperature to specific antibodies: sheep anti-IGF-I polyclonal
antibody (catalog no. AB1011, Chemicon, no cross-reaction with IGF-II),
rabbit anti- IGF-I receptor (anti-IGF-I-R)(anti 
-subunit) (catalog
no. SC-712, Santa Cruz Biotechnology, Inc., Santa Cruz,
CA). This was followed by incubation with an appropriate biotinylated
second antibody, with streptavidine-peroxidase conjugate and AEC as a
substrate (Histostain-SP kit, Zymed Laboratories, Inc.,
San Francisco, CA). Counterstaining was done with hematoxylin.
Nonspecific binding was blocked with 10% nonimmuned serum (rabbit or
goat). As negative controls slides were incubated with the relative
nonimmune serum, i.e. mouse serum for non clonal Abs and
Rabbit serum for polyclonal antibodies.
In situ hybridization
Paraffin sections (6 µm) were loaded on precleaned
poly-L-lysine coated slides, deparaffinized with xylene,
hydrated with graduated ethanols, and treated with 3%
H2O2 in methanol to neutralize endogenous
peroxidase. Sections were then treated for 15 min with 12.5 µg/ml
proteinase K, rinsed with 2 mg/ml glycine, and acetylated in 0.5%
acetic anhydride in 0.1 M Tris, pH 8.0. Sections were then
postfixed with 4% paraformaldehyde/PBS and prehybridized for 10' in
2 x SSC followed by 1 h in hybridization buffer: 50%
formamide, 0.5 mg/ml salmon sperm DNA, 4 x SSC, 1 x
Denhardt’s solution. Hybridization was done overnight (18 h) at 42 C
in maximal humidity with 5 ng/µl digoxygenin (Dig)-labeled
probe (see below). At the end of the incubation period, slides were
rinsed in SSC at increasing stringency conditions and then with 0.1
M Tris 0.15 M NaCl, pH 7.5. Hybrids were
detected using anti-(Dig) antibodies conjugated with peroxidase
(Boehringer Mannheim, Mannheim, Germany) and AEC as a
substrate and counterstained with hematoxylin. As negative controls we
used sections from the highly expected positive tissue reacted with the
same concentration of Dig-labeled pSPT18-Neo antisense RNA transcribed
with T7 from PvuII linearized pSPT18-Neo DNA (supplemented
with the kit).
Dig-labeled antisense RNA probes for in situ hybridization
We used probes for mouse IGF-IR cloned in pBluescript
SK+ amp+, for mouse IGF-I cloned in pGEM3
amp+ (386 bp). After linearization, antisense RNA was
transcribed using (Sp6/T7) Dig-RNA labeling kit (Boehringer Mannheim), following the company’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that sex steroids had
a direct stimulatory effect on DNA synthesis in male and female-derived
condyles. The effect of the steroid hormones on the proliferative
activity in the mandibular condyles were basically sex specific,
although male-derived condyles were less responsive to estradiol (A)
than female-derived condyles were to testosterone (B).
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shows that compared with
the control (A), the testosterone-treated male condyle (B) was much
bigger, and it’s cellular population was composed of a large
chondroprogenitor zone followed by a normal developing gradient of
chondrocytic cells. The estradiol-treated condyles (C) were similar to
the controls.
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) the
testosterone-treated condyles were more than twice the size of
untreated controls, and the estradiol-treated condyles were 25%
smaller than the controls.
Effects of gonadal steroids dosage and duration of treatment on
condyle development
Figure 3, shows the effect of
incubation of the mandibular condyles for 3 days in the presence of
10-5, 10-6, and 10-7
M testosterone. A maximal stimulatory effect on condylar
growth was seen at a concentration of 10-6 M
(A), whereas incubation in the presence of 10-5
M caused severe changes in the condyle: most of the
cartilaginous tissue was replaced by bone, leaving only a thin
chondroprogenitor zone adjacent to ossifying tissue (B). A dose of
10-7 M testosterone (C) apparently induced
only proliferative activity, as indicated by the crowded
chondroprogenitor zone, but no parallel increase in the mature
chondrocytic populations. It is noteworthy that the male condyles were
less responsive to 10-6 M estradiol, but
10-5 M estradiol also enhanced the
ossification process.
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shows the effect of 7-days
treatment with testosterone on the morphologic characteristics of the
mandibular condyles. In comparison to the 7-day control culture (A),
which still showed a large cartilaginous zone, 7-days testosterone
treatment (B) was associated with replacement of the cartilaginous
component by bone, very much resembling the appearance of the condyle
treated with 10-5 M testosterone (Fig. 3B).
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) were almost
negligible, and estradiol only slightly increased IGF-I levels in the
progenitor cells (Fig. 5B), whereas in the testosterone-treated condyle
(Fig. 5C) a positive reaction was seen in both chondroprogenitor and
chondrocytic cell populations. The stimulation of IGF-I-R by
testosterone was even greater (Fig. 6).
Neither the untreated control (Fig. 6A), nor the estradiol-treated
condyles (Fig. 6B) revealed any positive reaction, whereas the
testosterone-treated condyles (Fig. 6C) contained an appreciable amount
of IGF-I-R mainly in the young chondrocytes (yc).
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and 8)
indicated that IGF-I mRNA was almost absent in both the untreated
control (A) and estradiol-treated condyles (B), whereas 3 days of
testosterone treatment resulted in significantly increased IGF-I mRNA
levels (C). This increase was mainly seen in the upper portion of the
condyle, namely, in the chondroprogenitor zone, and young chondroblast
cells. Testosterone also increased the expression of IGF-I-R compared
with estradiol and to the nontreated control (Fig. 8, A–C.). However,
unlike the effect of testosterone on the distribution of IGF-I mRNA,
induction of IGF-I-R mRNA appeared in the more mature cellular
population, i.e. in the chondrocytic cell layer (C).
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).
The anti-IGF-I treated condyles (Fig. 9B), showed a marked reduction in
the cartilaginous zone of the condyle as compared with the untreated
control (Fig. 9A) accompanied with ossifying tissue "climbing" up
to the surface of the condyle, leaving only an "island" of
cartilage surrounded by bone. Immunoblocking of IGF-I in the
testosterone-treated condyle resulted mainly in the disappearance of
the young chondroblasts and chondrocytes with apparent overcrowding of
the chondroprogenitor zone (Fig. 9C). Morphometric studies of the
cartilaginous portion of the mandibular condyle however, did not
demonstrate a significant decrease in the size. In contrast,
immunoblocking of IGF-I in the control group caused a significant
reduction (30%) in the cartilaginous part.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, testosterone and estradiol were found to have a
direct, sex-specific stimulatory activity on male- and female-derived
chondroprogenitor cell proliferation. This observation is supported by
a previous animal model study showing testosterone stimulation of
metacarpal weight and length, in association with an increment in
thymidine incorporation in the proliferative zone of the growth plate
(18), and other studies showing sex-specific effects of steroids
hormones on skeletal growth (19). The stimulatory effect of
testosterone in the presence of a scant dihydrotestosterone effect on
[3H] thymidine incorporation in our experiments (Fig. 1),
could be explained by a different biological effect of testosterone and
dihydrotestosterone on chondroprogenitor cell proliferation. Both
androgens act through binding and activation of the androgen receptor,
but it has been suggested that the receptor may interact with different
genes when bound to dihydrotestosterone vs. testosterone,
perhaps under the direction of tissue specific cofactors (20).
Growth plate activity is characterized by the continuous production of new cells balanced by an absorptive process at the metaphyseal interface (21, 22); the chondrocytes grow along vertical columns exhibiting three main phases of differentiation (reserve, proliferative, and hypertrophic cells) (23). In vivo studies of hypophysectomized prepubertal lambs have shown that testosterone can stimulate growth in the absence of GH (24) and that direct administration of testosterone increases unilateral rat tibial epiphyseal growth plate width (25). In the present study, we confirmed a direct effect of testosterone and showed that it is mediated, at least partially, by local stimulation of IGF-I and IGF-I-R as demonstrated by the increment in the relevant mRNA and protein levels. Under optimal conditions (10-6 M testosterone for 3 days) mandibular condyles, a model for the in vitro studies of endochondral ossification, were shown to double in size compared with controls. Furthermore they were composed of all the cellular layers in proper proportions, morphologically resembling "young" mandibular condyles. Thus, testosterone exhibits a general stimulatory effect, resulting in coupling of the proliferation and differentiation processes essential for normal skeletal growth. However, testosterone’s effects on these two activities seem to be differentially regulated: immunoblocking of IGF-I in the testosterone-treated condyles resulted in an uncoupling of the proliferation and differentiation activities. The cell population of chondroblasts and young chondrocytes was markedly reduced, and the chondroprogenitor zone seemed to be overcrowded, indicating proper proliferating activity. Our previous studies using authoradiograpy of tritiated thymidine labeled condyles have shown that the only proliferating cell population in the condyle is the chondroprogenitor cell layer (26). This finding may indicate that testosterone-induced cell differentiation is IGF-I-mediated, but testosterone-induced cell proliferation is sustained regardless of the local IGF-I blocking. We speculate that IGF-I is mainly secreted by the younger cell population of the EGP and exerts its effect mainly on the more mature cell layers.
These results are further supported by our finding of overtreatment
with testosterone, which only slightly affected proliferation but led
to a shift in the differentiation pathway from chondrogenesis to
osteogenesis, resulting in complete ossification of the whole condyle.
Lowering the dose of testosterone to 10-7 M
did not affect its stimulation of cellular proliferation, though the
latter was not followed by a parallel increase in differentiation,
hence culminating in an overcrowded chondroprogenitor zone (see Figs. 3 and 4).
The IGF-I-mediated effect of testosterone on the differentiation of the mandibular condyle is similar to that described for GH-induced IGF-I activity in the EGP. Hormonal activity (i.e. GH) induces local production of IGF-I, which acts in an autocrine/paracrine fashion to stimulate growth (27).
We have previously reported that daily injections of testosterone for 5 days had no effect on IGF-I mRNA abundance in the EGP of hypophysectomized rats (11). However, in this work, RNA was extracted from the entire area and studied by solution hybridization, a procedure that could lead to a dilution effect on mRNA derived from other cells in the organ removed. The technique used in the present study, however, allows identification of local changes within the skeletal growth centers. Two previous reports had failed to demonstrate IGF-I mRNA in the EGP chondrocytes of rat and mice using in situ hybridization (28, 29), although Lazowski (30) did show IGF-I mRNA in the proliferating and hypertrophic chondrocytes, as did Nilsson (31) before, using the same technique. Our present study demonstrates a low but detectable level of IGF-I mRNA in the condyles of the untreated mice, which significantly increased following 3 days incubation with testosterone. Therefore, the differences among the earlier reports may be attributable to differences in the technique used and animal age and pubertal status.
Using in situ hybridization and immunohistochemistry
techniques, we were also able to show that testosterone increased the
levels of both IGF-I and IGF-I-R mRNA and protein. Therefore,
testosterone seems to have a dual effect on local IGF-I activity in the
skeletal growth center by stimulating both IGF-I production and
cellular sensitivity to IGF-I. A different cellular distribution of the
protein and mRNA of IGF-I-R was observed (Figs. 6 and 8). IGF-I-R
protein is most prominent in the young chondrocytes, whereas IGF-I-R
mRNA is more abundant in the chondrocytes. This difference could be due
to different posttranslational modification of the IGF-I-R gene.
Similar observations have been reported in other skeletal
development-related genes. One example could be the collagen type I
gene. It has been shown that in mature chondrocytes collagen type I
mRNA is abundant, despite the fact that these cells produce and secrete
only type II collagen (32).
Interestingly, a differential dosage effect of both estrogen and
testosterone is well documented in humans. Ross et al. (33)
observed a 2-fold increase in the growth rate of the ulna following
treatment with ethinyl-estradiol 0.1 mg/kg·day. Higher dosages of 0.4
and 0.8 mg/kg·day were not effective, indicating a biphasic estrogen
effect on growth. A study in tall girls showed that treatment with high
doses of estrogen causes acceleration in bone maturation, with a
decrease in ultimate height (34, 35, 36, 37, 38). Pubertal growth has also been
studied in hypopituitary boys receiving therapy with testosterone
enanthate at dosages ranging between 100 and 250 mg/month (39, 40, 41, 42, 43). The
total pubertal height gain was 2-fold higher with the lower dose
compared with the others. It is possible that estrogens and androgens
play different roles in the regulation of growth in the EGP. Aromatase
is expressed widely in human bone tissue (44) in addition to other
tissues. It is likely that some of the effect testosterone has on the
EGP is mediated through its conversion to estrogen especially when high
doses of testosterone are used. Until recently, only one
"classical" form of the estrogen receptor (ER) was known to exist.
Since the discovery of the new ER-ß form (45), the original form was
defined as ER-. By using the in situ hybridization
technique, Kusec et al. have recently demonstrated that
ER- could be identified in both human and rabbit growth plate
chondrocytes (46). Indeed, bone maturation and EGP closure are
interrupted in diseases where estrogen is deficient or not effective
even in the presence of testosterone, as was recently described by
Bilezikian et al. in a patient with aromatase deficiency
(47) and in a patient with estrogen receptor deficiency by Smith
et al. (48). We assume, therefore, that some of the effects
observed in the testosterone treated mandibular condyle in our study,
were due to local aromatization of testosterone. Studies using
different nonaromatizing androgens are needed to better understand this
mechanism of interaction.
In addition, the bioactivity of the IGFs in bone tissue is modulated by several insulin-like growth-factor binding-proteins (IGFBPs), mainly IGFBP-3, -4, -5 (49). It is therefore possible that some regulatory effect of the sex steroids on the epiphyseal growth plate may be also mediated via regulation of the local production of the IGBPs.
In conclusion, the present study shows that testosterone, but not estradiol, has a direct stimulatory effect on the growth of the male-derived mandibular condyle. Although the mandibular condyle was used as a model of endochondral ossification, it should be stressed out that it might not necessarily represent other growth centers such as those of the longitudinal bones. The stimulatory effects of testosterone (especially its effect on differentiation) were partially inhibited by immunoblocking of IGF-I. Overtreatment with testosterone (regarding both dosage and duration) causes enhanced calcification at the expense of chondrogenic activity, resulting in reduced overall condylar growth. These results imply that to achieve optimal stimulatory effects on skeletal growth, testosterone treatment must be administrated under tightly controlled conditions.
Further studies are needed to better understand the interactions among the different hormones and growth factors, and their receptors and binding proteins, which are involved in the bone growth and maturation.
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Acknowledgments |
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Received June 2, 1998.
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References |
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in human and rabbit skeletal
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