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Endocrinology Vol. 140, No. 4 1901-1910
Copyright © 1999 by The Endocrine Society

ARTICLES

Testosterone Stimulates Insulin-Like Growth Factor-I and Insulin-Like Growth Factor-I-Receptor Gene Expression in the Mandibular Condyle—A Model of Endochondral Ossification

Gila Maor, Yael Segev and Moshe Phillip

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Puberty is associated with an increase in the plasma concentration of sex steroids, GH, and insulin-like growth factor-I (IGF-I). Gonadal steroid hormones are important for the normal pubertal growth spurt and skeletal growth. The mechanism by which gonadal steroids induce skeletal growth is still not fully understood. To better understand the direct effect sex steroids have on bone growth, we studied an isolated organ culture system of the mandibular condyle, derived from 3.5–5.5-week-old male and female mice. We found that testosterone 10-6 M, but not estradiol, stimulated thymidine incorporation into the DNA of male-derived condyle. Three days of testosterone treatment doubled the condyle size and increased the chondroprogenitor zone, while maintaining the normal gradient of the developing chondrocytes. Immunohistochemistry and in situ hybridization techniques showed that testosterone stimulated IGF-I and IGF-I-R and their messenger RNAs (mRNAs) mainly in the mature chondrocyte layer. Immunoneutralization of IGF-I in the testosterone-treated condyle caused the disappearance of the chondroblast and young chondrocyte layers, though the progenitor cell layer remained almost unaffected. Overtreatment with testosterone (dose or duration) accelerated condylar ossification. In the presence of testosterone 10-5 M (high dose), calcification "climbs" up to the chondroprogenitor zone, and most of the condylar chondrocytes are replaced by bone tissue. Similar changes occurred after 7 days of testosterone treatment (long duration) with 10-6 M. In conclusion, testosterone stimulates growth and local production of IGF-I and IGF-I-R in chondrocyte cell layers of an isolated organ culture of mice mandibular condyle. Part of the effect testosterone has on condylar growth is mediated by IGF-I.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PUBERTAL growth spurt response is determined by the combined effects of increased plasma concentrations of sex steroids, GH, and insulin-like growth factor-I (IGF-I) together with other endocrine, paracrine, and autocrine factors, on the epiphyseal growth plate (EGP) (1, 2, 3, 4, 5). The precise mechanisms by which the gonadal steroids induce skeletal growth remain unclear, mainly because it is impossible to isolate their effect from that of the other hormones and growth factors. The concomitant increase in the levels of gonadal steroids and GH during the pubertal growth spurt supports the notion that GH may mediate this process (6, 7). Craft and Underwood (8) suggested that GH plays a role in the androgenic stimulation of IGF-I, and Keenan et al. (9) found that androgens indirectly stimulate GH secretion via their aromatization to estrogen. Other authors claim that androgens directly stimulate skeletal growth independent of the GH-IGF-I axis. The finding of a pubertal growth spurt even in children with GH deficiency and precocious puberty (10) supports this notion.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Organ culture
Mandibular condyles of male and female ICR mice aged 3.5–5.5 weeks were grown in the BJGb culture medium (Fitton Jakckson Modification, Beth Haemek, Israel; catalog no. 01–010-1) as previously described (13, 14). In this specific in vitro system, the organ mandibular condyle was grown on a nitrocellulose membrane placed on a stainless mesh so that feeding and metabolism were performed via diffusion through the membrane. The medium was supplemented with 2% FCS, 300 µg/ml ascorbic acid, 20 mg/liter phenol red and antibiotics. The 2% FCS contained 17 pM testosterone and less than 1 pM estradiol. The tissues were incubated for 3–7 days at 37 C in 5% CO2/95% air and maximal humidity. Cultures were treated either with testosterone, dihydro-testosterone (DHT), estradiol, or vehicle (0.1% ethanol). For immunoblocking, IGF-I cultures were incubated for 3 days in the presence of anti-IGFI antibody diluted 1/500 (catalog no. AB101, Chemicon Co. USA) with or without additional steroids. At the end of the incubation period, the explants were thoroughly washed with cold Hanks’ buffer and processed according to the experimental aim.

[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 {alpha}-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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of sex steroids on thymidine incorporation by mandibular condyles
Figure 1Go 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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  		Figure 1. Effects of gonadal steroids on thymidine incorporation into DNA. Mandibular condyles derived from male (A) or female (B) mice were incubated in the presence of 10-6 M of either testosterone (T), estradiol (E), or dihydrotestosterone (DHT). Five microcuries of 3[H]-thymidine were added for the last 18 h of incubation. TCA-insoluble radioactivity was countered. Each point is an average of four different experiments each done in triplicate. The significance was calculated using two-tailed Student’s t test. Degree of significance are marked as follows: P value < 0.02: *, against control; + against testosterone; # against estradiol; P value < 0.005: **, against control, ++ against testosterone; ##, against estradiol; P value < 0.0005: ***, against control; +++, against testosterone; ###, against estradiol; P value nonsignificant: NS, against control; ’NS’, against estradiol. Results that are not marked are highly significant.

 
Effects of testosterone and estradiol on mandibular condyle morphology
Figure 2Go 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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  		Figure 2. Histological structure of steroid-treated condyles. Hematoxillin/eosin-stained paraffin sections of 3.5-week-old male-derived mandibular condyles were cultured for 3 days in the presence of the vehicle alone as a control (A), 10-6 M of T (B) or 10-6 M of E (C). Both control and estradiol-treated condyles are composed from only chondroprogenitor cells (pr), mature chondrocytes (mc), and hypertrophic cells (hyp), which are bordered by the primary spongiosa (psg) of the adjacent bone. In the T-treated condyle, the progenitor cells are accompanied by chondroblasts (cb) and young chondrocytes (yc), which endows the condyle with a younger/larger appearance, as presented in the morphometric analysis (D). Each point is an average of at least 12 measurements of the total cartilaginous zone length that were measured on sections from three different cultures. These results reveal a marked increase in the overall size of the T-treated condyle (D). ** P < 0.005.

 
According to the morphometric studies (Fig. 2DGo) 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 3Go, 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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  		Figure 3. Dose response effect of testosterone treatment on the histological appearance of the 3.5-week-old mandibular condylar. Condyles were incubated for 3 days in the presence of 10-6 M (A), and high 10-5 M (B) or low 10-7 M (C) concentrations of testosterone. An optimal effect appears in the presence of 10-6 M of T. 10-5 M causes induced ossification so the entire condyle is occupied by bone, and 10-7 M only increases proliferation (magnification, x190).

 
Figure 4Go 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. 3BGo).



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  		Figure 4. Long-term treatment experiment. Condyles (males, 3.5 weeks old) were incubated for 7 days in the presence of 0.1% ethanol as a control (A) or 10-6 M testosterone (B). Note the enhanced calcification induced by a long-term treatment with T (B-bone) (magnification, x190).

 
Effects of sex steroids on IGF-I and IGF-I-R levels in the condyle
Immunohistochemistry results showed that the levels of IGF-I in the control (Fig. 5AGo) were almost negligible, and estradiol only slightly increased IGF-I levels in the progenitor cells (Fig. 5BGo), whereas in the testosterone-treated condyle (Fig. 5CGo) a positive reaction was seen in both chondroprogenitor and chondrocytic cell populations. The stimulation of IGF-I-R by testosterone was even greater (Fig. 6Go). Neither the untreated control (Fig. 6AGo), nor the estradiol-treated condyles (Fig. 6BGo) revealed any positive reaction, whereas the testosterone-treated condyles (Fig. 6CGo) contained an appreciable amount of IGF-I-R mainly in the young chondrocytes (yc).



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  		Figure 5. Immunohistochemistry of IGF-I levels in the T and E treated condyles. Condyles (males, 3.5 weeks old) were incubated in the presence of 10-6 M of E (B), 10-6 M of T (C) or neither (A). 6 µm sections were reacted with anti IGF-I antibodies. Detection was done using biotinylated 2nd Ab, avidin-POD conjugate and AEC assay (see Materials and Methods for details). Positive reaction appears as red staining (see arrows). T increases IGF1 levels within the progenitor (pr) and chondrocytic cells-in both young and mature chondrocytes (yc, mc) (magnification, x190).

 


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  		Figure 6. Immunohistochemistry of IGF-I receptors (IGF-I-R) levels in the T- and E-treated condyles (males, 3.5 weeks old). Condyles were incubated in the presence of 10-6 M of E (B), 10-6 M of T (C), or neither (A). Six-micrometer sections were reacted with anti IGF-I-R ({alpha} subunit) antibodies. Detection was done as described above T increases the IGF-I-R levels mainly in the young chondrocytes population (yc) (magnification, x190).

 
Effects of sex steroids on IGF-I and IGR-I-receptor mRNA levels
To assess whether the increased levels of IGF-I and IGF-I-R, as determined by immunohistochemistry, are associated with increased expression of the respective mRNAs, in situ hybridization was performed. Results of in situ hybridization (Figs. 7Go and 8Go) 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. 8Go, 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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  		Figure 7. In situ hybridization of IGF-I mRNA in the T- and E-treated condyles. Deparaffinized sections from treated and control (males, 3.5 weeks old) condyles were obtained as described in Fig. 5Go (for immunohistochemistry). Sections were hybridized for 18 h at 42 C with 5 ng/µl digoxygenin-labeled antisense IGF-I RNA. Detection was done with antidigoxygenin-POD antibody and AEC as a substrate. Estradiol slightly increased. IGF-I mRNA expression (B) over the control (A).In the T-treated condyles (C), the expression of IGF-I mRNA is markedly increased in both the chondroprogenitor cells (cp) and the young chondrocytes (yc) (magnification, x240). For negative control see Fig. 8DGo.

 


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  		Figure 8. In situ hybridization of IGF-I receptor mRNA in the T- and E-treated condyles (males, 3.5 weeks old). This assay was performed as described above for the expression of IGF-I mRNA. T-treatment (C) increased the expression of IGF-I receptors mRNA over both control and E-treated condyles (A and B, respectively) mainly in the chondrocytes populations (ch). For negative control, T-treated section was incubated with Dig-labeled pSPT18-Neo antisense RNA under equal hybridization conditions (D) (magnification, x240).

 
Effect of immunoneutralization of IGF-I on testosterone-treated condyles
We have previously shown that, in the presence of GH, anti-IGF-I antibodies were able to block the induction of IGF-I. Incubation of the condyle for 3 days in the presence of anti-IGF-I antibody (diluted 1/500) resulted in marked morphological changes in the control and testosterone-treated condyles (Fig. 9Go). The anti-IGF-I treated condyles (Fig. 9BGo), showed a marked reduction in the cartilaginous zone of the condyle as compared with the untreated control (Fig. 9AGo) 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. 9CGo). 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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  		Figure 9. Immunoneutralization of IGF-I. Mandibular condyles were incubated for 3 days in the presence of anti IGF1 Abs (diluted 1/500) and either 0.1% of EtOH as a control (B), or 10-6 M of T (C). Anti-IGF-I significantly shrunk the cartilaginous zone of the control (B) condyle in comparison with the nontreated control (A- incubated with nonimmune sheep serum). T-treated condyle is more conserved (C), and the reduction in its total length (by anti IGF-I Ab) is not significant, as apparent from the morphometric studies presented in D (magnification, x190). ** P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The epiphyseal growth plate and the cartilaginous tissue are probably the target tissue for many growth factors including IGF-I, IGF-II, transforming growth factor ß, and basic fibroblast growth factor (15, 16, 17). Several lines of evidence indicate that IGF-I plays a major role in the regulation of skeletal growth and development through endocrine, autocrine and paracrine mechanisms.

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. 1Go), 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. 3Go and 4Go).

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. 6Go and 8Go). 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-{alpha}. By using the in situ hybridization technique, Kusec et al. have recently demonstrated that ER-{alpha} 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.


   Acknowledgments
 
We appreciate the technical assistance of Mrs. Irena Reiter and Mrs. Matilda Branman.

Received June 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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