Relative Impact of Androgen and Estrogen Receptor Activation in theEffects of Androgens on Trabecular and Cortical Bone in Growing

Male Mice: A Study in the Androgen Receptor KnockoutMouse Model

Katrien Venken, Karel De Gendt, Steven Boonen, Jill Ophoff, Roger Bouillon, Johannes V Swinnen, Guido Verhoeven,and Dirk Vanderschueren

ABSTRACT: The relative importance of AR and ER activation has been studied in pubertal male ARknockout and WT mice after orchidectomy and androgen replacement therapy, either with or without anaromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone mod-eling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both ARand ER activation.

Introduction: Androgen receptor (AR)–mediated androgen action has traditionally been considered a keydeterminant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essentialfor normal male bone growth. Therefore, the relative importance of AR-mediated and estrogen receptor(ER)–mediated androgen action after aromatization remains to be clarified.Materials and Methods: Trabecular and cortical bone was studied in intact or orchidectomized pubertal ARknockout (ARKO) and male wildtype (WT) mice, with or without replacement therapy (3–8 weeks of age).Nonaromatizable (dihydrotestosterone [DHT]) and aromatizable (testosterone [T]) androgens and T plus anaromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecularand cortical bone modeling were evaluated by static and dynamic histomorphometry, respectively.Results: AR inactivation or orchidectomy induced a similar degree of trabecular bone loss (−68% and −71%,respectively). Both DHT and T prevented orchidectomy-induced bone loss in WT mice but not in ARKOmice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivationand orchidectomy had similar negative effects on cortical thickness (−13% and −8%, respectively) and peri-osteal bone formation (−50% and −26%, respectively). In orchidectomized WT mice, both DHT and T werefound to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, theperiosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of anaromatase inhibitor partly reduced T action on periosteal bone formation in orchidectomized WT mice (−34%versus orchidectomized WT mice on T), but not in ARKO mice. This effect was associated with a significantdecrease in serum IGF-I (−21% versus orchidectomized WT mice on T).Conclusions: These findings suggest a major role for AR activation in normal development of trabecular boneand periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtainedin the presence of both AR and ER activation.J Bone Miner Res 2006;21:576–585. Published online on January 3, 2006; doi: 10.1359/JBMR.060103

Key words: androgen receptor knock-out mouse, puberty, orchidectomy, androgen receptor, estrogen re-ceptor, aromatization, periosteal bone growth, trabecular bone

INTRODUCTION

AS A RESULT of more pronounced periosteal bone for-mation, pubertal bone growth in men is associated

with more radial bone expansion than in women.(1) Thisadditional periosteal growth in males ultimately defines the

cross-sectional area of the bone and thereby confers greaterbone strength.(2) Androgen-mediated activation of the an-drogen receptor (AR) is considered a key determinant ofthis sex-specific pattern of periosteal growth.(3) In line withthis concept, androgen-resistant rodents show reducedbone size because of a reduction in cortical thickness andperiosteal perimeter.(4–6) A reduction in cortical bone areais also observed in the context of sex steroid deficiency, asThe authors state that they have no conflicts of interest.

Laboratory for Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 21, Number 4, 2006Published online on January 3, 2006; doi: 10.1359/JBMR.060103© 2006 American Society for Bone and Mineral Research

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induced by orchidectomy, in growing male rats and mice.(7)

Moreover, trabecular bone is markedly affected by sex ste-roid deficiency or androgen resistance.(5,6)

Androgens can stimulate the skeleton not only throughdirect activation of the AR, but also indirectly, after aro-matization into estrogens and subsequent activation of oneor both estrogen receptors (ER�/�). In fact, increasing evi-dence suggests that estrogens may be critically involved inmale skeletal growth. Men with natural mutations in theestrogen receptor � (ER�)(8) or the aromatase gene(9–13)

show low BMD and failure to establish peak bone mass.Likewise, growing male rats treated with an aromatase in-hibitor or male mice with inactivation of either the ER� orthe aromatase enzyme have reduced bone size, as reflectedby a decline in cortical area and periosteal perimeter.(14–16)

Transgenic mouse models offer the opportunity to studythe relative importance of sex steroid receptors during skel-etal growth and to determine the effects of orchidectomyand sex steroid replacement on trabecular and cortical bonecompartments. In orchidectomized androgen-resistantmice, trabecular and cortical bone are not responsive todihydrotestosterone (DHT) treatment.(6) Some(6) but notall(5) available data suggest only partial responsiveness totestosterone (T), supporting a role for aromatization of an-drogens into estrogens followed by ER activation. Intactmale ER� knockout mice (ER�KO), on the other hand,show a cortical bone deficit but an increase in trabecularbone volume as a result of enhanced T levels acting throughthe AR.(17) In this animal model, orchidectomy-inducedloss in trabecular and cortical BMD and cortical thickness isprevented by treatment with T,(17,18) consistent with a criti-cal role for androgen-mediated AR activation. Therefore, itremains uncertain to what extent androgen action on maletrabecular and cortical bone modeling depends on activa-tion of the AR and/or aromatization of androgens into es-trogens. To further address this question, we studied tra-becular and cortical bone phenotype and the response toorchidectomy and replacement therapy in growing ARknockout (ARKO) mice and corresponding wildtype (WT)mice. To define the importance of AR activation on tra-becular and cortical bone in growing male mice, we studiedthe effects of AR inactivation in comparison with orchidec-tomy. Additionally, to differentiate between direct (AR-mediated) and indirect (ER-mediated) effects, aromatiz-able and nonaromatizable androgens, as well as anaromatase inhibitor, were administered to orchidectomizedARKO and WT mice.

MATERIALS AND METHODS

AnimalsARKO mice were generated using the Cre/loxP technol-

ogy, as previously described.(19) Their genetic backgroundwas C57Bl6/N. Genotyping was performed using PCR am-plification.(19) Mice lived in conventional conditions: 12-hlight/dark cycle, standard diet (1% calcium, 0.76% phos-phate), and water ad libitum.

Experimental designAt the start of puberty (23 days of age), male WT and

ARKO littermates were randomly divided in groups. Mice

were either sham-operated (sham) or orchidectomized(orx) using sodium pentobarbital anesthesia. Orx mice weretreated during an experimental period of 5 weeks with ve-hicle (V), DHT, T, or T plus aromatase inhibitor (anastra-zole [Arimidex], 10 mg/kg/day). DHT (Fluka) and T(Serva) were administered using subcutaneous silastic im-plants (Silclear Tubing, Degania Silicone, Jordan Valley,Israel) in the cervical region. Vehicle animals receivedempty implants. Anastrazole is a potent, nonsteroidal,highly selective aromatase inhibitor with no intrinsic hor-monal activity.(20) The substance was obtained from AstraZeneca after formal approval of the protocol and was ad-ministered orally. Eight animals were included in eachgroup. Body weight was measured weekly. All mice wereinjected intraperitoneally with the fluorochrome calcein ata 5-day interval and were killed 1 day after the secondinjection. Before the first calcein injection, mice were put inmetabolic cages to collect urine for measurement of colla-gen cross-links. At death, serum was collected, stored at–20°C, and used for osteocalcin and IGF-I measurement.Femur and tibias were dissected and used to perform his-tomorphometric analysis. Efficacy of orx and DHT and Treplacement in WT mice was verified by measurement ofseminal vesicles wet weight immediately after death. Semi-nal vesicle weight expressed per gram of body weight was6.2 ± 0.3 mg/g in the DHT-treated group and 8.3 ± 0.2 mg/gin the T-treated group compared with 5.7 ± 0.2 mg/g insham-operated mice and 0.10 ± 0.04 in orx mice (p < 0.001).The greater androgenic potency of T may be explained bythe constant and continuous release from the implantsthroughout the experimental period. The physiological sig-nificance of anastrazole was assessed in 8-week-old femalemice that were given anastrazole (10 mg/kg/day, orally) for2 weeks. The weight of the uterus per gram of body weightin Arimidex-treated females was 2.5 ± 0.1 versus 6.0 ± 0.2mg/g in age-matched control females (p < 0.001).

The ethical committee of the Katholieke UniversiteitLeuven approved all experimental procedures.

Bone histomorphometry

One femur and tibia were cleaned from surrounding tis-sue, immersed in Burckhardt’s fixative (24 h, 4°C), kept in100% ethanol, and embedded in methylmethacrylate.

Longitudinal sections of the undecalcified tibia were cutat 4 �m thickness using a rotation microtome (RM 2155Autocut; Leica, Heidelberg, Germany) with a tungsten car-bide blade (Leica, Nussloch, Germany). Sections werestained by a modified Goldner technique and subjected tostatic histomorphometry. Measurements were performed inthe secondary spongiosa of at least three Goldner-stainedsections, as previously described.(5,18) In each section, threeconsecutive fields were measured along the vertical axis ofthe central metaphysis, starting at a regular distance of thegrowth plate. Trabecular width and trabecular numberwere calculated according to the parallel plate model de-veloped by Parfitt et al.(21)

Cross-sections of the undecalcified femur perpendicu-larly to the long axis were prepared at 200 �m thickness inthe mid-diaphyseal region using the contact-point precision

ANDROGEN AND ESTROGEN RECEPTOR ACTIVATION IN MALE BONE GROWTH 577

band saw (Exakt, Norderstedt, Germany). Sections wereground to a final thickness of 25 �m using a grinding system(Exakt). Sections were left unstained and subjected to dy-namic histomorphometry. Three sections in the mid-diaphyseal region were measured by fluorescence micros-copy, and the bone formation rate (BFR/B.Pm., �m2/�m/day) was assessed at both the endocortical and periostealbone surfaces. The BFR was obtained by the product ofmineral apposition rate (MAR, �m/day) and mineralizingperimeter per bone perimeter (Min.Pm./B.Pm., %).The mineralizing perimeter was calculated as follows:Min.Pm. � [dL + (sL/2)]/B.Pm., where dL represents thelength of the double labels and sL is the length of singlelabels along the entire endocortical or periosteal bone sur-faces. The MAR (�m/day) was calculated as the meanwidth of double labels, divided by interlabel time (5 days).The mineralizing perimeter is a measure for osteoblastnumber and MAR for the osteoblast activity. Also, thecross-sectional area (CSA), cortical area, cortical thickness,and endocortical and periosteal perimeters were measuredon cortical cross-sections. All measurements were per-formed with a Kontron Image Analyzing computer (KS4003.00; Kontron Bildanalyze, Munich, Germany) and a Zeissmicroscope with a drawing attachment. Specific softwarewas developed in collaboration with the manufacturer. His-tomorphometric parameters are reported according to therecommended American Society for Bone and Mineral Re-search nomenclature.(21)

Bone densitometry

Trabecular and cortical volumetric BMD was assessed exvivo by pQCT using the Stratec XCT Research M+ densi-tometer (Norland Medical Systems, Fort Atkinson, WI,USA). Slices of 0.2 mm thickness were scanned using avoxel size of 0.070 mm. One scan was taken 2 mm from thedistal end of the femur, using contmode 1, peelmode 20,and a density threshold of 280 mg/cm3. The trabecular boneregion was defined by setting an inner threshold corre-sponding to 30% of the total CSA. These metaphysealscans were performed to measure trabecular volumetricdensity. A second scan was taken 7 mm from the distal endof the femur (an area containing only cortical bone) usingseparation mode 1 and a density threshold of 710 mg/cm3.These mid-diaphyseal scans were performed to determinecortical volumetric density, cortical thickness, and endocor-tical and periosteal perimeters.

Whole body DXA

Body composition was analyzed in vivo by DXA (PIXI-mus densitometer; Lunar Corp., Madison, WI, USA) using

FIG. 1. (A) Trabecular BMD (Trab. BMD, mg/cm3), (B) tra-becular bone volume (bone area per total area, B.Ar./T.Ar., %),(C) trabecular number (mm−1), and (D) trabecular width (�m) insham-operated male wildtype (WT SHAM) and ARKO mice(ARKO SHAM) and orchidectomized WT mice (WT ORX).Mice were sham-operated (SHAM) or orchidectomized (ORX) at3 weeks of age and killed at 8 weeks of age. *p < 0.05 vs. WTSHAM (n � 6–8 mice/group). Pictures represent trabecular bone(blue-colored) in each group.

VENKEN ET AL.578

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ultra-high resolution (0.18 × 0.18 pixels, resolution of 1.6line pairs/mm) and software version 1.45. DXA was per-formed at the end of the experimental period.

Assays

Serum osteocalcin was measured by an in-house radio-immunoassay (RIA).(22) After acid-ethanol extraction, se-rum IGF-I concentrations were measured by an in-houseRIA(23) in the presence of an excess of IGF-II (25 ng/tube).Urinary collagen cross-links (deoxypyridinoline [DPD])were measured by high-performance liquid chromatogra-phy (HPLC) with fluorescence detection after acid hydro-lysis.(24) The concentration of DPD was corrected for cre-atinine excretion, which was measured colorimetrically.

Statistical analysis

Statistical analysis of data was performed using NCSSsoftware (Kaysville, UT, USA). One-way ANOVA, fol-lowed by Fisher’s least significant difference multiple com-parison test, and t-tests were performed to assess signifi-cance of difference between groups of the same genotypeand between respective WT and ARKO groups. Data arerepresented as mean ± SE. p < 0.05 was accepted as signifi-cant.

RESULTS

Effects of AR inactivation versus orchidectomy ontrabecular bone

AR inactivation resulted in a pronounced trabecularbone phenotype. Sham ARKO mice showed a significantreduction in trabecular BMD (−61%, p < 0.001; Fig. 1A)and trabecular bone volume (B.Ar./T.Ar., %) (−68%, p <0.05; Fig. 1B).The latter was the result of a significant de-crease in trabecular number (−63%, p < 0.05; Fig. 1C) butnot in trabecular width (Fig. 1D). Orchidectomy (orx) orandrogen deficiency in WT mice induced a trabecular bonephenotype similar to the phenotype observed with AR in-activation; trabecular BMD (−69%, p < 0.001), trabecularbone volume (−71%, p < 0.05), and number (−58%, p <0.05) were all significantly decreased after orx (Figs. 1A–1C). The reduction in trabecular bone volume was associ-ated with an increase in urinary DPD in sham ARKO mice(63 ± 8 nM/mM creatinine) and orx WT mice (52 ± 3 nM/mM) compared with sham WT mice (28 ± 3 nM/mM; p <0.001). Longitudinal growth, as assessed by femoral length,was not changed by AR inactivation (15.2 ± 0.2 mm in shamARKO mice) or orx (15.0 ± 0.1 in orx WT mice) comparedwith sham WT mice (15.2 ± 0.2 mm).

Effects of AR inactivation versus orx oncortical bone

Periosteal growth of the midfemoral shaft was assessedby dynamic histomorphometric analysis. Not only AR in-activation but also orx significantly decreased cross-sectional area (−13% and −9%, respectively, p < 0.05; Fig.2A), cortical area (−18% and −12%, respectively, p < 0.05;Fig. 2B), cortical thickness (−13% and −8%, respectively,p < 0.05; Fig. 2C), and periosteal perimeter (−6% and −5%,

FIG. 2. (A) Cross-sectional area (CSA, mm2), (B) cortical(cort.) area (mm2), (C) cortical thickness (�m), and (D) periostealperimeter (Pm., mm) in sham-operated male wildtype (WTSHAM) and ARKO mice (ARKO SHAM) and orchidectomizedWT mice (WT ORX). Mice were sham-operated (SHAM) or or-chidectomized (ORX) at 3 weeks of age and killed at 8 weeks ofage. *p < 0.05 vs. WT SHAM (n � 6–8 mice/group).

ANDROGEN AND ESTROGEN RECEPTOR ACTIVATION IN MALE BONE GROWTH 579

respectively, p < 0.05; Fig. 2D). This resulted from a signifi-cant decrease in periosteal bone formation rate (Ps.BFR/B.Pm.) in ARKO mice and orx WT mice (−50% and −26%,respectively, p < 0.05; Fig. 3A), which was associated with areduced periosteal osteoblast number in both sham ARKOmice and orx WT mice compared with sham WT mice, asindicated by a significantly reduced periosteal mineralizingperimeter (Ps.Min.Pm./B.Pm.) (−50% and −39%, respec-tively, p < 0.01; Fig. 3B). The periosteal MAR, a measurefor osteoblast activity, was not affected by either AR inac-tivation or orx (data not shown).

Sham ARKO mice and orx WT mice also gained signifi-cantly less body weight during puberty (from 3 to 8 weeksof age) compared with sham WT mice (p < 0.001; Fig. 4). Inaddition, lean body mass, but not fat mass, was significantlydecreased in sham ARKO and orx WT mice at the end ofpuberty (−12% and –15%, respectively, p < 0.001; Table 1).

Effects of DHT, T, and aromatase inhibitor ontrabecular and cortical bone modeling

Both DHT and T prevented the orx-induced loss of tra-becular BMD in WT mice (p < 0.001 versus orx +V WT;Fig. 5). Administration of the aromatase inhibitor (AI) didnot affect T action on trabecular BMD in WT mice. High

FIG. 3. (A) Periosteal bone formation rate per bone perimeter(Ps.BFR/B.Pm., �m2/�m/day) and (B) periosteal mineralizing pe-rimeter per bone perimeter (Ps.Min.Pm./B.Pm., %) in sham-operated male wildtype (WT SHAM) and ARKO mice (ARKOSHAM) and orchidectomized WT mice (WT ORX). Mice weresham-operated (SHAM) or orchidectomized (ORX) at 3 weeks ofage and killed at 8 weeks of age. *p < 0.05 vs. WT SHAM (n �6–8 mice/group). Pictures represent cortical cross-sections withcalcein labels (green) at the endocortical (Ec) and periosteal (Ps)bone surface of each group.

FIG. 4. Body weight during the experimental period. Mice weresham-operated (SHAM) or orchidectomized (ORX) at 3 weeks ofage and killed at 8 weeks of age. *p < 0.05, **p � 0.001, ***p <0.001 vs. ARKO sham and WT orx (n � 6–8 mice/group).

FIG. 5. Trabecular BMD in sham-operated (SHAM) and orchi-dectomized (ORX) WT and ARKO mice. ORX mice weretreated with either vehicle (V), dihydrotestosterone (DHT), tes-tosterone (T), or T + aromatase inhibitor (AI). Mice were sham-operated or orchidectomized at 3 weeks of age and treated for 5weeks. ap < 0.05 vs. SHAM, bp < 0.05 vs. ORX + V, cp < 0.05 vs.ORX + DHT.

TABLE 1. BODY COMPOSITION IN SHAM-OPERATED WT AND

ARKO MICE AND ORCHIDECTOMIZED WT MICE

WT SHAM ARKO SHAM WT ORX

Fat mass (g) 3.4 ± 0.2 3.3 ± 0.2 3.6 ± 0.1Lean body mass (g) 20.9 ± 0.7 18.4 ± 0.3* 17.7 ± 0.4*Body weight (g) 26.8 ± 0.9 23.1 ± 0.6* 22.4 ± 0.6*

Values are mean ± SE (n � 6–8 mice/group). Mice were sham-operated(SHAM) or orchidectomized (ORX) at 3 weeks of age and killed at 8weeks of age.

* p < 0.001 vs. WT SHAM.

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Fig 3 live 4/C

serum osteocalcin and urinary DPD levels, reflecting theorx-induced high bone turnover state, were suppressed af-ter treatment with DHT, T, and T + AI in WT mice (Table2). In contrast to the effects observed in WT mice, the lowtrabecular BMD in ARKO mice was not affected by non-aromatizable (DHT) and aromatizable (T) androgens orT + AI. The high rate of bone turnover in ARKO waspresent in each condition (Table 2).

Both DHT and T significantly increased the periostealmineralizing perimeter (Min.Pm) in orx WT mice (+101%and +141% versus orx + V WT, respectively, p < 0.001; Fig.6A), but not the periosteal MAR (p � 0.56; Fig. 6B). As aresult, the periosteal BFR was significantly enhanced byboth DHT and T in orx WT mice (+100% and +163%versus orx + V WT, respectively, p < 0.001; Fig. 6C), alongwith a significant increase in cortical area (+18% and +38%versus orx + V WT, respectively, p < 0.001) and corticalthickness (+17% and +27% versus orx + V WT, respec-tively, p < 0.001; Table 3). Also, the periosteal perimeter(+10% versus orx + V WT, respectively, p < 0.001) andCSA (+22% versus orx + V WT, respectively, p < 0.001)were significantly increased by T (Table 3). Administrationof T + AI partially reduced the effects of T on the periostealMin.Pm. (−25% versus orx + T WT, p < 0.001) and perios-teal BFR in orx WT mice (−34% versus orx + T WT, p <0.001; Figs. 6A and 6C), resulting in a reduced cortical area(−17% versus orx + T WT, p < 0.001), thinner cortex (−7%vs. orx + T WT, p < 0.001), and smaller periosteal perimeter(−9% versus orx + T WT, p < 0.001; Table 3). This effect ofT + AI on cortical bone of orx WT mice was associated witha significant decline in serum IGF-I levels (−21% versusorx + T WT, p � 0.01; Table 4). In contrast to the effectsobserved in WT mice, the periosteal bone surface ofARKO mice remained unresponsive to any treatment(Figs. 6A–6C; Table 3), and no significant changes in serumIGF-I could be detected (Table 4).

DISCUSSION

In this study in growing male mice, androgen unrespon-siveness, in the context of AR inactivation and sex steroiddeficiency induced by orx, induced a similar degree of tra-becular bone loss. Both conditions resulted in a significantreduction of trabecular bone volume with fewer and wide-

spread but normal-sized trabeculae. Aromatizable and non-aromatizable androgens prevented orx-induced trabecularbone loss to a similar degree in WT mice but not in ARKOmice. Moreover, inhibition of aromatase activity did notalter T action on trabecular bone. Taken together, thesefindings support the concept that AR activation is essentialfor normal development of trabecular bone in male mice.

It is generally accepted that both AR and ER� activa-tion, independently and to a similar degree, affect trabec-ular bone mass in male mice. This so-called “dual mode ofaction” of androgens on trabecular bone has been derivedfrom studies in male ER�KO and ARKO mice.(6,25) InER�KO mice, the increase in trabecular bone volume isnormalized to WT levels after administration of an antian-drogen, indicating that their enhanced trabecular bonemass is the result of the higher T levels acting directlythrough the AR.(17) In ER�KO mice, orx reduces trabec-ular bone mass to the same level as in WT controls, and thisorx-induced trabecular bone loss is fully prevented byT,(17,18) again showing the important role of AR-mediatedandrogen action for normal trabecular bone mass in theseanimals. In orx WT mice, on the other hand, 17�-estradiol(E2) also increases trabecular bone volume and even seemsto have an osteoanabolic effect.(18,25) However, the dosesused in these experiments are pharmacologic and not rep-resentative of the significantly lower physiological levels inmale mice (∼5 pg/ml). Further evidence for a possible rolefor aromatization of androgens into estrogens followed byER activation in male mice comes from data obtained byKawano et al.,(6) who showed that T partly prevents orx-induced bone loss in ARKO mice. However, this studyevaluated areal femoral BMD only and did not allow todistinguish between trabecular and cortical bone; in addi-tion, no aromatase inhibitor was used, and the experimentwas not designed to determine the role of aromatization inT action on trabecular bone. Finally, in androgen-resistanttesticular feminized male (Tfm) mice, administration ofpharmacological doses of T did not increase serum E2, tra-becular BMD, or trabecular bone volume.(5) This finding isconsistent with the very limited expression of the aromataseenzyme in rodents; the major sites of expression are thegonads and the brain and thus peripheral aromatization isvery poor.(26) With respect to trabecular bone, one mighttherefore hypothesize that the intrinsic response to andro-

TABLE 2. BONE TURNOVER MARKERS IN WT AND ARKO MICE

SHAM ORX + V ORX + DHT ORX + T ORX + T + AI

Serum OCWT 146 ± 22 177 ± 19 94 ± 10*† 111 ± 9† 129 ± 17†

ARKO 122 ± 13 182 ± 25 178 ± 15‡ 233 ± 18‡ 207 ± 25‡

Urinary DPDWT 28 ± 3 52 ± 3* 26 ± 3† 25 ± 3† 26 ± 3†

ARKO 63 ± 8‡ 54 ± 8 55 ± 7‡ 63 ± 4‡ 50 ± 6‡

Serum osteocalcin (OC, ng/ml) and urinary deoxypyridinoline (nM DPD/mM creatinine) are expressed as mean ± SE (n � 6–8 mice/group). Mice weresham-operated (SHAM) or orchidectomized (ORX) at 3 weeks of age and treated for 5 weeks with either vehicle (V), dihydrotestosterone (DHT),testosterone (T), or T + aromatase inhibitor (AI).

* p < 0.05 vs. SHAM.† p < 0.05 vs. ORX + V.‡ p < 0.05 vs. respective WT group.

ANDROGEN AND ESTROGEN RECEPTOR ACTIVATION IN MALE BONE GROWTH 581

gens and estrogens is regulated differently in rodents andhumans. Therefore, although both AR and ER� activationmay be able to increase trabecular bone mass, our studyshows that androgen acting directly through the AR is themajor determinant of normal trabecular bone development,at least in physiological conditions in male mice. Aromati-zation and subsequent ER activation, on the other hand,seem to play a less important role in trabecular bone de-

velopment (Fig. 7). Alternatively, one may also hypothesizethat androgens affect trabecular bone indirectly. In this re-spect, it has been shown that androgens are able to upregu-late the expression of the IGF-I receptor and, as a conse-quence, are able to enhance IGF-I action.(27,28) ARinactivation would therefore be associated with a decreasedresponsiveness to IGF-I. The concept that local actions ofIGF-I may be important for normal trabecular bone devel-opment is supported by the finding that loss of IGF-I re-ceptor signaling or IGF-I overexpression specifically in os-teoblasts is associated with marked alterations in trabecularbone volume of young growing mice.(29,30)

In this study, we found androgen resistance and sex ste-roid deficiency to have similar negative effects on corticalarea and thickness, as a result of a reduced bone formationat the periosteum. In WT controls, but not in ARKO mice,T and DHT stimulated periosteal bone formation with anincrease in cortical area. In ARKO mice, periosteal boneremained unresponsive to either aromatizable or nonaro-matizable androgens, again supporting the concept that ARactivation also plays a major role in cortical bone modelingin male mice (Fig. 7).

Overall, androgens are indeed considered a key determi-nant of male cortical bone growth.(7) This assumption isbased on observations in orx male rodents showing a de-crease in periosteal circumference.(31) Additionally, obser-vations of a female-like bone size in rodents and humanswith inactive ARs have provided further support for a keyrole of AR activation in male radial bone expansion.(4–6,32)

Interestingly, androgen resistance or deficiency also resultin reduced body weight and lean body mass (a surrogatemarker for muscle mass). Because muscle mass is an im-portant determinant of mechanical loading,(33) which, inturn, stimulates bone formation, one might speculate thatsome of the effects of androgens and AR activation areindirect through stimulation of muscle mass and subsequentincreased mechanical loading.

However, aromatization of androgens into estrogens andsubsequent ER activation may be important as well in theprocess of male cortical bone modeling. Our finding of ablunting of the effect of T on periosteal bone growth byanastrazole, an AI, supports this possibility. Moreover, thiseffect was only observed in WT mice and was paralleled bya significant decline in serum IGF-I levels. In previous stud-ies in male rats, administration of an AI was also associatedwith lower serum IGF-I levels and reduced CSA.(14,34) Asimilar decrease in serum IGF-I levels and cortical thick-ness has been observed in male ER�KO mice.(15) Likewise,administration of anastrozole to late pubertal boys de-creased serum E2 as well as serum IGF-I(35) and, morerecently, treatment with E2 was found to enhance periostealbone expansion in an aromatase-deficient adolescentboy.(11) All these studies lend support to the hypothesis thatestrogen-related changes in serum IGF-I may act to medi-ate estrogen action on the periosteal bone surface. It hasbeen well established that growth hormone (GH) andIGF-I are major determinants of postnatal growth.(36,37)

Mice with a disrupted GH receptor have dramatically de-creased serum IGF-I levels and severely retarded skeletalgrowth rates.(38,39) In GHRKO mice, we previously showed

FIG. 6. (A) Periosteal mineralizing perimeter per bone perim-eter (Ps.Min.Pm./B.Pm, %), (B) periosteal mineral appositionrate (Ps.MAR, �m/day), and (C) periosteal bone formation rateper bone perimeter (Ps.BFR/B.Pm., �m2/�m/day) in sham-operated (SHAM) or orchidectomized (ORX) WT and ARKOmice. ORX mice were treated with either vehicle (V), dihydrotes-tosterone (DHT), testosterone (T), or T + aromatase inhibitor(AI). Mice were sham-operated or orchidectomized at 3 weeks ofage and treated for 5 weeks. ap < 0.05 vs. SHAM, bp < 0.05 vs.ORX + V, cp < 0.05 vs. ORX + DHT, dp < 0.05 vs. ORX + T.

VENKEN ET AL.582

that the periosteal bone surface is extremely sensitive tochanges in circulating IGF-I levels, with periosteal bonegrowth being fully rescued after upregulation of serumIGF-I in GHRKO mice treated with E2, but not when ex-posed to T or DHT.(40) These findings suggest that theeffect of aromatization of androgens into estrogens andsubsequent ER activation is indirectly mediated throughalterations of the GH/IGF-I axis (Fig. 7). An intriguingobservation in this study was the lack of effect of the aro-matase inhibitor on cortical bone modeling in ARKO mice.This observation remains unexplained. One might hypoth-esize that the severe cortical phenotype in ARKO mice isnot further affected by inhibition of aromatase activity.Also, no changes in serum IGF-I were observed in ARKOmice that could have affected cortical bone growth.

In conclusion, this study in ARKO mice supports theconcept that AR activation is a major determinant of tra-becular bone development in physiological conditions,whereas aromatization and subsequent ER activation seemless important. Stimulation of bone formation at the peri-osteal bone surface and increases in bone size in male micepredominantly depend on activation of the AR as well.However, to obtain a maximal stimulation of periostealbone growth in male mice, both AR and ER activation arerequired.

FIG. 7. Summary. Androgen-mediated activation of the androgenreceptor (AR) is the major determinant of trabecular BMD, bonevolume, and number in growing male mice. Increases in corticalarea, periosteal perimeter (Pm), and periosteal bone formation rate(BFR) predominantly depend on AR activation as well. Aromati-zation of testosterone (T) to 17�-estradiol (E2) followed by estrogenreceptor � (ER�) activation also plays a role and is associated withchanges in serum IGF-I. Moreover, both AR and ER activation isneeded to become an optimal stimulation of periosteal growth.

TABLE 3. PARAMETERS OF CORTICAL BONE WIDTH IN WT AND ARKO MICE

SHAM ORX + V ORX + DHT ORX + T ORX + T + AI

CSA (mm2)WT 1.85 ± 0.04 1.69 ± 0.05* 1.80 ± 0.04 2.06 ± 0.07*†‡ 1.70 ± 0.03*§

ARKO 1.62 ± 0.03e 1.71 ± 0.04 1.84 ± 0.05 1.80 ± 0.04¶ 1.75 ± 0.03Cort.Ar. (mm2)

WT 0.74 ± 0.02 0.65 ± 0.02* 0.77 ± 0.03† 0.90 ± 0.02*†‡ 0.75 ± 0.02†§

ARKO 0.61 ± 0.02¶ 0.68 ± 0.02 0.69 ± 0.02¶ 0.70 ± 0.01¶ 0.67 ± 0.01¶

Cort.Thc. (�m)WT 183 ± 5 168 ± 2* 196 ± 5*† 214 ± 3*†‡ 200 ± 4*†§

ARKO 160 ± 3¶ 174 ± 3 172 ± 2¶ 176 ± 2¶ 171 ± 2¶

Ps.Pm. (�m)WT 5436 ± 73 5172 ± 82* 5334 ± 66 5678 ± 99*†‡ 5168 ± 54*§

ARKO 5085 ± 58¶ 5192 ± 62 5366 ± 91 5307 ± 55¶ 5215 ± 56

Cross-sectional area (CSA), cortical area (Cort.Ar.), cortical thickness (Cort.Thc.), and periosteal perimeter (Ps.Pm) are expressed as mean ± SE (n �

6–8 mice/group). Mice were sham-operated (SHAM) or orchidectomized (ORX) at 3 weeks of age and treated for 5 weeks with either vehicle (V),dihydrotestosterone (DHT), testosterone (T), or T + aromatase inhibitor (AI).

* p < 0.05 vs. SHAM.† p < 0.05 vs. ORX + V.‡ p < 0.05 vs. ORX + DHT.§ p < 0.05 vs. ORX + T.¶ p < 0.05 vs. respective WT group.

TABLE 4. SERUM IGF-I IN WT AND ARKO MICE

SHAM ORX + V ORX + DHT ORX + T ORX + T + AI

Serum IGF-IWT 325 ± 13 336 ± 21 338 ± 19 323 ± 20 256 ± 13*†‡§

ARKO 361 ± 14 363 ± 20 351 ± 16 346 ± 22 308 ± 6¶

Serum IGF-I (ng/ml) is expressed as mean ± SE (n � 6–8 mice/group). Mice were sham-operated (SHAM) or orchidectomized (ORX) at 3 weeks ofage and treated for 5 weeks with either vehicle (V), dihydrotestosterone (DHT), testosterone (T), or T + aromatase inhibitor (AI).

* p < 0.05 vs. SHAM.† p < 0.05 vs. ORX + V.‡ p < 0.05 vs. ORX + DHT.§ p < 0.05 vs. ORX + T.¶ p < 0.05 vs. respective WT group.

ANDROGEN AND ESTROGEN RECEPTOR ACTIVATION IN MALE BONE GROWTH 583

ACKNOWLEDGMENTS

This study was supported by Katolieke Universiteit Leu-ven Grant OT/01/39 and Fund for Scientific Research-Flanders, Belgium Grants G.0417.03, G.0458.05, andG.0171.03. DV and SB are Senior Clinical Investigators ofthe Fund for Scientific Research-Flanders, Belgium. SB is aholder of the Leuven University Chair in Metabolic BoneDiseases, supported by Roche and GSK.

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Address reprint requests to:Dirk Vanderschueren, MD, PhD

Laboratory for Experimental Medicine and EndocrinologyO&N 1, Campus Gasthuisberg

Herestraat 49, Bus 9023000 Leuven, Belgium

E-mail: dirk.vanderschueren@uz.kuleuven.ac.be

Received in original form November 25, 2005; revised form De-cember 19, 2005; accepted January 3, 2006.

ANDROGEN AND ESTROGEN RECEPTOR ACTIVATION IN MALE BONE GROWTH 585