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Keywords:

  • estrogen receptors;
  • androgens;
  • bone;
  • transgenic;
  • males

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Androgens may regulate the male skeleton either directly via activation of the androgen receptor (AR) or indirectly via aromatization of androgens into estrogen and, thereafter, via activation of estrogen receptors (ERs). There are two known estrogen receptors, ER-α and ER-β. The aim of this study was to investigate the relative roles of ER-α, ER-β, and AR in the maintenance of trabecular bone in male mice. Seven-month-old male mice, lacking ER-α (ERKO), ER-β (BERKO), or both receptors (DERKO), were orchidectomized (orx) and treated for 3 weeks with 0.7 μg/mouse per day of 17β-estradiol or vehicle. No reduction in trabecular bone mineral density (BMD) was seen in ERKO, BERKO, or DERKO mice before orx, showing that neither ER-α nor ER-β is required for the maintenance of a normal trabecular BMD in male mice. After orx, there was a pronounced decrease in trabecular BMD, similar for all groups, resulting in equal levels of trabecular BMD in all genotypes. This reduction was reversed completely in wild-type (WT) and BERKO mice treated with estrogen, and no significant effect of estrogen was found in ERKO or DERKO mice. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR and by an estrogen-induced activation of ER-α. These results indicate that AR and ER-α are redundant in the maintenance of the trabecular bone in male mice. In contrast, ER-β is of no importance for the regulation of trabecular bone in male mice.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

MORBIDITY FROM osteoporosis in the aging population is substantial in both men and women. However, few studies have been designed for the elucidation of the mechanism(s) behind male osteoporosis. It is obvious that androgens are important both for the acquisition of bone during skeletal growth and for the maintenance of trabecular bone in adults. The effects of testosterone on the skeleton can be exerted either directly via the androgen receptor (AR) or indirectly via aromatization to estrogen and further via estrogen receptors (ERs). Orchidectomy (orx) results in bone loss, which is prevented by treatment with androgens.(1,2) However, estrogen treatment also prevents orx-induced bone loss.(1,2) Humans, as well as rodents, with impaired aromatase activity, suffer from decreased bone mineral density (BMD),(3–8) which can be prevented by treatment with estrogen.(3,5,9,10) Furthermore, several studies have presented stronger correlations between BMD and estrogen than with testosterone.(11–13) These experimental and clinical data support the importance of estrogen in the maintenance of normal BMD in males. There are two known ERs, denoted α (ER-α) and β (ER-β). Previously, we have shown that ER-α, but not ER-β, mediates important effects of estrogen in the regulation of skeletal growth in male mice.(14) However, the relative importance of ER-α and ER-β in the regulation of adult bone metabolism in male mice is unknown. The lack of ER-α in male mice results in elevated serum levels of estrogen and testosterone, probably because of disturbed feedback regulations.(15) Therefore, to avoid confounding effects of elevated sex steroids, the ER-inactivated and wild-type (WT) mice used in this study were orx. The mice were substituted with 17β-estradiol or vehicle and the ER specificity in the regulation of the adult bone metabolism in males was investigated.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Animals

Male double heterozygous (ER-α+/−β+/−) mice were mated with female double heterozygous (ER-α+/−β+/−) mice, resulting in WT, ER-α−/−ER-β+/+ (ERKO), ER-α+/+ER-β−/− (BERKO) and ER-α−/−ER-β−/− (DERKO) offspring with a mixed C57BL/6J/129 background.(16,17) Genotyping of tail DNA was performed at 3 weeks of age as previously described.(14) Animals had free access to fresh water and food pellets (B & K Universal AB, Sollentuna, Sweden) consisting of cereal products (76.9% barley, wheat feed, wheat, and maize germ), vegetable proteins (14.0% hipro soya), and vegetable oil (0.8% soya oil). All mice were orx at the age of 7 months. The mice were left to recover for 10 days after orx. After recovery, mice were injected subcutaneously (sc) with 0.7 μg/mouse per day of 17β-estradiol benzoate (Sigma, St. Louis, MO, USA) for 5 days/week during 3 weeks. Control mice received injections of vehicle oil (olive oil; Apoteksbolaget, Göteborg, Sweden). Treatment with 0.7 μg/mouse per day of 17β-estradiol benzoate resulted in 17β-estradiol levels of ∼220 pmol/liter, which could be regarded as physiologically relevant concentrations of 17β-estradiol.

Dual X-ray absorptiometry

Measurement of bone mineral content (BMC) and areal BMD (aBMD) of total body in vivo and femur in vitro was performed with the Norland Medical Systems pDEXA Sabre (Norland Medical Systems, Fort Atkinson, WI, USA) and the Sabre Research software (v3.6; Norland Medical Systems, Fort Atkinson, WI, USA) as previously described.(14,18)

Peripheral quantitative computerized tomography

Computerized tomography (CT) was performed with the Stratec pQCT XCT Research M (v5.4B; Norland Medical Systems) operating at a resolution of 70 μm as previously described.(18) Cortical parameters were determined with a middiaphyseal peripheral quantitative CT (pQCT) scan of the tibias. Trabecular BMD was determined with a metaphyseal pQCT scan of the proximal tibias and defined as the inner 45% of the total area.

Histomorphometry

The left tibia was fixed in 10% phosphate-buffered formalin, embedded in methacrylate resin, sectioned, and stained by Goldner's trichrome method. Analysis of trabecular bone was restricted to an area 0.25-2 mm in a diaphyseal direction from the growth plate, maintaining separation between the analysis area and the cortical wall. Histomorphometric parameters measured were trabecular bone volume (BV/TV; %), trabecular thickness (Tb.Th; μm), trabecular separation (Tb.Sp; μm), and trabecular number (Tb.N; mm−1).(19)

Mechanical testing

After pQCT measurements, the humerus was applied to mechanical testing using Mechanical Tester 8841 (Instron, Canton, MA, USA). Three-point bending force was measured by placing the bone horizontally with the anterior surface upward and applying pressing force vertically to the midshaft of the bone. Each bone was compressed with a constant speed of 2 mm/minute until failure. Breaking force (maximal load) was defined as bending load at failure. Maximal stress (sigma) and elastic or Young's modulus (E) were calculated as previously described.(20)

Radioimmunoassay

Serum insulin-like growth factor (IGF) I levels were measured by double antibody IGF binding protein-blocked radioimmunoassay (RIA).(21) Serum osteocalcin levels were measured using a monoclonal antibody raised against human osteocalcin (Rat-MID osteocalcin ELISA; Osteometer Biotech A/S, Herlev, Denmark). The sensitivity of the osteocalcin assay was 21.1 ng/ml and intra- and interassay coefficients of variation (CVs) were <10%. Levels of c-telopeptide were measured in serum by ELISA, which measures degradation products of type I collagen, generated by osteoclastic bone resorption.(22) The sensitivity of the ELISA was <0.1 ng/ml. The average intra- and interassay CVs were <12%. 17β-estradiol was measured using an RIA detecting estradiol (DiaSorin, Saluggia, Italy) with sensitivity below 5 pg/ml at 95% confidence limit.

Tartrate-resistant acid phosphatase 5b activity

Tartrate-resistant acid phosphatase (TRAP) 5b, purified from human osteoclasts as described,(23) was used as antigen to develop a polyclonal antiserum in rabbits.(24) The antiserum was incubated on anti-rabbit immunoglobulin G (IgG)-coated microtiter plates (EG & G Wallac, Turku, Finland) for 1 h. Diluted mouse serum samples were incubated in the wells for 1 h, and bound enzyme activity was detected using 8 mmol/liter of 4-nitrophenylphosphate (4-NPP) as substrate in 0.1 M of sodium acetate buffer, pH 6.1, for 2 h at 37°C. The enzyme reactions were terminated by adding 25 μl of 0.32 M of NaOH, and A405 was measured using Victor2 equipment (EG & G Wallac).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

aBMD and BMC as determined by dual-energy X-ray absorptiometry

In vivo analysis of the total body aBMD in 7-month-old intact male mice revealed a slight decrease in ERKO (−2.7%) and DERKO (−3.6%) but not in BERKO compared with WT mice (p < 0.05; one-way analysis of variance [ANOVA], followed by Student-Newman-Keul's multiple range test; Fig. 1). After orx, the total body aBMD decreased to a similar extent in all four genotypes. Treatment with 17β-estradiol increased total body aBMD in WT and BERKO as compared with vehicle treatment, whereas no estrogenic effect was found in ERKO or DERKO mice (Fig. 1). After death the left femur was excised and analyzed by dual-energy X-ray absorptiometry (DXA) in vitro. Treatment with 17β-estradiol increased the femoral aBMD in WT and BERKO, whereas no effect was seen in ERKO or DERKO mice (Fig. 2A). The estrogen-induced increase in aBMD in WT and BERKO mice resulted in a similar increase in femur BMC (Fig. 2B). Thus, the effect of 17β-estradiol on femoral BMC in orx mice is ER-α-mediated. In contrast, no effect of treatment with 17β-estradiol was seen on the femur area (Fig. 2C).

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Figure FIG. 1.. Total body aBMD measured using DXA. Seven-month-old male mice were orx and treated with vehicle (V) or 17β-estradiol (E) for 3 weeks (intact animals [n = 14], orx animals [n = 4-6]). Values are given as means ± SEM;++p < 0.01 versus intact mice and **p < 0.01 versus vehicle, Student's t-test.

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Figure FIG. 2.. Femur (A) aBMD (B) BMC, and (C) area measured using DXA. Seven-month-old male mice were orx and treated with vehicle (V) or 17β-estradiol (E) for 3 weeks (n = 4-6). Values are given as means ± SEM; *p < 0.05 and **p < 0.01 versus vehicle, Student's t-test.

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The results obtained from DXA measurements are a combination of effects on trabecular bone and cortical bone parameters. To be able to distinguish between effects of 17β-estradiol on trabecular and cortical bone, the mice were analyzed by pQCT.

Trabecular BMD as determined by pQCT

The trabecular volumetric BMD (tvBMD) was measured in the metaphyseal region of the proximal tibias using pQCT. Measurements of tvBMD before orx revealed a slight increase in ERKO and DERKO (p < 0.01; one-way ANOVA, followed by Student-Newman-Keul's multiple range test), whereas no difference was seen in BERKO compared with WT mice (Figs. 3A and 3B). After orx, the tvBMD decreased dramatically to the same level in all genotypes. Treatment with 17β-estradiol prevented this decrease in WT and BERKO mice, whereas no effect of 17β-estradiol was seen in ERKO or DERKO mice (Figs. 3A and 3B).

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Figure FIG. 3.. (A) tvBMD of tibias measured using pQCT. (B) Representative pQCT scans. Seven-month-old intact (I) male mice were orx and treated with either vehicle (V) or 17β-estradiol (E) for 3 weeks. Intact animals (n = 14) and orx animals (n = 4-6). Values are given as means ± SEM,++p < 0.01 versus intact mice, *p < 0.05, and **p < 0.01 versus vehicle, Student's t-test. H, high bone mineral density; L, low bone mineral density.

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Histomorphometry

Histomorphometry of the metaphyseal part of the proximal tibias was performed to confirm the effects on trabecular bone as detected by pQCT. Trabecular bone volume as a ratio to total bone volume (BV/TV) was well correlated to tvBMD as measured using pQCT (R = 0.76; p = 3.1 × 10−8). Estrogen treatment increased the BV/TV in WT. Statistical analysis in which ER-α+/+ (WT and BERKO) and ER-α−/− (ERKO and DERKO) mice were analyzed independently indicated that 17β-estradiol treatment increased BV/TV in ER-α+/+ animals but not in ER-α−/− animals (p < 0.01; two-way ANOVA, followed by Student-Newman-Keul's multiple range test; Table 1), confirming our results of the effect of 17β-estradiol on tvBMD as measured using pQCT. Tb.Sp was decreased and Tb.N was increased after estrogen treatment in WT and BERKO mice, whereas no effect was seen in ERKO or DERKO mice (Table 1).

Table Table 1.. Histomorphometric Parameters
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Cortical bone parameters (pQCT)

Cortical bone parameters (BMC, area, and thickness) were measured by a middiaphyseal pQCT scan of the tibias. These cortical bone parameters were decreased in 7-month-old intact ERKO and DERKO but not in BERKO mice compared with WT mice (data not shown). Cortical BMC was increased after estrogen treatment in WT and BERKO but not in ERKO or DERKO mice (Table 2). Similar tendencies were seen regarding both cortical thickness and cross-sectional area (Table 2). Statistical analysis in which ER-α+/+ (WT and BERKO) and ER-α−/− (ERKO and DERKO) mice were analyzed independently showed that 17β-estradiol treatment increased all these cortical bone parameters in orx ER-α+/+ (BMC, 24 ± 4% and p < 0.01; thickness, 16 ± 3% and p < 0.01; cross-sectional area, 19 ± 3% over vehicle and p < 0.01; two-way ANOVA, followed by Student-Newman-Keul's multiple range test), whereas no effect was seen in ER-α−/− mice.

Table Table 2.. Cortical Bone Parameters
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Mechanical strength

The mechanical strength was measured using three-point bending of the humerus. Maximal load, a measurement of the strength of the bone, was increased after 17β-estradiol treatment in orx ER-α+/+ (WT and BERKO) mice (28 ± 8% over vehicle and p < 0.01 two-way ANOVA, followed by Student-Newman-Keul's multiple range test). No effect of 17β-estradiol treatment was seen in ER-α−/− (ERKO and DERKO) mice, showing an ER-α-mediated effect. The qualitative bone parameter maximal stress and elastic modulus were not affected by 17β-estradiol treatment in any genotype (data not shown).

Serum levels of biochemical bone parameters and IGF-I

Serum parameters were measured at death after 3 weeks of treatment with estrogen or vehicle to orx mice. No major effects of 17β-estradiol treatment were seen on the serum levels of osteocalcin or c-telopeptide (Table 3). The activity of TRAP 5b, an osteoclast-specific enzyme, was increased after 17β-estradiol treatment in WT and BERKO mice, whereas no effect was detected in ERKO or DERKO mice as compared with vehicle treatment (Table 3). The activity of TRAP 5b was correlated to tvBMD, as determined using pQCT (R = 0.76; p = 4.4 × 10−8) as well as BV/TV, determined by histomorphometry (R = 0.75; p = 1.1 × 10−7). Serum levels of IGF-I were not significantly affected by estrogen treatment in any of the genotypes (Table 3).

Table Table 3.. Serum Parameters
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Estrogen is of importance for the regulation of skeletal growth and maturation in female mice and an increasing amount of data indicates that estrogen is of importance in male mice as well. Previously, we have shown that the skeletal growth of the long bones in male mice is dependent on estrogen, because combined loss of both ER-α and ER-β results in impaired longitudinal bone growth. This phenotype also was seen in male ERKO, but not in male BERKO mice, showing an ER-α-mediated effect.(14) Male aromatase deficient mice (ArKO) have shorter femurs than their normal littermates (NLMs), further supporting the importance of estrogen in the regulation of longitudinal bone growth in male mice.(6) Interestingly, androgens acting directly on the AR were not able to substitute for the loss of ER-α in the stimulation of longitudinal bone growth in male mice.(14) Thus, ER-α and AR are not redundant regarding the regulation of longitudinal bone growth in male mice.

Our previous study, analyzing ER-inactivated young adult male mice with intact gonads, did not detect any major effect on the amount of trabecular bone. However, these ER-inactivated mice have increased levels of endogenous estrogen and testosterone, which might have confounded the results on the amount of trabecular bone.(15,25) Therefore, to avoid confounding effects of elevated sex steroids, the ER-inactivated and WT mice used in this study were orx. Androgens may regulate the amount of trabecular bone either directly via activation of AR or indirectly via aromatization into estrogens and further via activation of ERs (Fig. 4A). A protective role of estrogen on trabecular bone is supported by the fact that aromatase deficiency in male mice(6,9) as well as aromatase inhibition in male rats(7,8) results in a slight decrease in the amount of trabecular bone. Treatment with estrogen prevents this bone loss.(9,10) This study confirms some previous studies by showing that orx-induced trabecular bone loss is prevented by estrogen treatment.(1,2) However, for the first time, we have elucidated the ER specificity for this bone-sparing effect of estrogen in male mice. 17β-Estradiol treatment of orx male mice prevents trabecular bone loss in WT and BERKO but not in ERKO or DERKO mice. This finding clearly shows that the protective effect of estrogen on trabecular bone in adult male mice is mediated via ER-α, and ER-β is of no importance.

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Figure FIG. 4.. Effects of androgens on trabecular bone in male mice. (A) Three different possible pathways for androgens in the regulation of trabecular bone in male mice are indicated. (B) Our present data together with some previous findings show that the trabecular bone is preserved both by an activation of ER-α and by an activation of the AR.(1,25) Thus, we propose that the AR and ER-α are redundant in the regulation of trabecular bone in male mice. In contrast, ER-β is of no importance for the regulation of trabecular bone in male mice.

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The orx of adult male rats causes trabecular bone loss that can be prevented both by testosterone and by nonaromatizable androgen dihydrotestosterone (DHT).(1) These data show a protective role of androgens on the trabecular bone via activation of AR (Fig. 4B). In this study, the physiological role of AR in the regulation of trabecular bone was analyzed indirectly. The fact that the trabecular BMD is preserved in ER-α as well as in ER-α/β double-inactivated male mice with intact gonads indicates that a testicular factor, presumably testosterone acting via the AR, is able to maintain a normal trabecular BMD. This notion is supported by a recent study in which testosterone prevented orx-induced bone loss in ERKO mice.(25) Furthermore, the orx-induced trabecular bone loss in this study is similar in WT and DERKO mice. Thus, a testicular factor is able to maintain the trabecular bone in male mice devoid of all known ERs. Because a similar magnitude of orx-induced trabecular bone loss is seen in mice having functional ERs and in mice devoid of all known ERs, this testicular factor is most likely acting via the AR. Previous studies indicate that the serum levels of testosterone in orx rats are 5-10% of that in intact rats.(7) Therefore, one cannot exclude that a low background concentration of adrenal-derived androgens may have reduced the effect of removal of testicular androgens. Our present data together with previous findings(1,25) show that trabecular bone is preserved both by an activation of ER-α and by an activation of the AR (Fig. 4B). Neither male mice with ER-α inactivation nor rats with a nonfunctional AR suffer from any major trabecular bone loss.(26) Thus, we propose that the AR and ER-α are “redundant” in the regulation of trabecular bone in male rodents (Fig. 4B). In contrast, as discussed previously, no redundancy between ER-α and AR was seen for the regulation of longitudinal bone growth in male mice.

A recent in vitro study indicated that both androgens and estrogens are able to exert nongenomic effects via activation of either the AR or ERs.(27) We could not detect any effect of estrogen on the trabecular bone in orx male DERKO mice, suggesting that an in vivo effect of estrogen via the AR is unlikely. However, our study does not rule out the possibility that androgens might be able to exert effects via ERs. Treatment of orx AR-deficient mice with nonaromatizable DHT could test this possibility.

Young adult ERKO and DERKO but not BERKO mice with intact gonads have decreased cortical BMC compared with WT mice, showing that the estrogenic regulation of cortical BMC is mediated via ER-α.(14) Treatment of adult orx mice with estrogen resulted, in this study, in a small but significant increase in the cortical BMC in WT and BERKO male, whereas no effect of estrogen treatment was found in ERKO or DERKO mice. In addition, these data show that the effects of estrogen on adult cortical BMC are mediated via ER-α and not ER-β. The effects on cortical BMC were reflected by similar changes in mechanical strength as measured by three-point bending. Estrogen increased the maximal load in ER-α+/+ but not in ER-α−/− mice. However, there was no effect on qualitative bone parameters, including elastic modulus and maximal stress, indicating that the quality of the cortical bone is not affected by estrogen treatment.

Analysis of total body areal BMD using DXA revealed a small decrease in intact ERKO and DERKO mice compared with WT mice. The image produced by the DXA is two-dimensional and does not recognize changes in the third dimension. Therefore, the reduction in areal BMD seen in male ERKO and DERKO mice with intact gonads probably is caused by decreased size of the animals and does not reflect a true decrease in vBMD.

Serum levels of osteocalcin, a marker of bone formation, and c-telopeptide, a marker of bone resorption, were measured at the end of the study after 3 weeks of treatment with estrogen. No major effect of estrogen was detected for these markers in any genotype. On the contrary, the recently described new bone resorption marker, serum TRAP 5b activity,(28,29) was increased after estrogen treatment in WT and BERKO mice whereas the activity was unaffected by estrogen treatment in ERKO and DERKO mice. It is well known that the acute effect of estrogen in gonadectomized rodents is to decrease TRAP 5b.(30) In contrast, in this study we have found that TRAP 5b activity is increased after a prolonged estrogen treatment. Interestingly, the increased TRAP 5b levels were correlated with the amount of trabecular bone. Similarly, recently, we reported that long-term orx results in a decreased TRAP 5b activity, associated with a decrease in trabecular BMD.(30) Thus, one might speculate that TRAP5b reflects the amount of trabecular bone after a prolonged estrogen treatment. In summary, the trabecular bone is preserved both by a testicular factor, presumably testosterone acting via AR, and by an estrogen-induced activation of ER-α. These results indicate that AR and ER-α are redundant in the maintenance of the trabecular bone in male mice (Fig. 4B). In contrast, ER-β is of no importance for the regulation of trabecular bone in male mice.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We thank Anette Hansevi and Maud Pettersson for valuable technical assistance. We also thank SWEGENE Center for Bio-Imaging (CBI), Gothenburg University, for technical support regarding image analysis. This study was supported by the Swedish Medical Research Council, the Swedish Foundation for Strategic Research, the Lundberg Foundation, the Torsten and Ragnar Söderbergs Foundation, the Emil and Vera Cornell Foundation, Petrus and Augusta Hedlunds Foundation, Novo Nordisk Foundation, the Swedish Association Against Rheumatic Disease, the Swedish Cancer Fund, the National Institutes of Health (NIH) grant support AR31062, and Karo Bio AB.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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