The relative importance of AR and ER activation has been studied in pubertal male AR knockout and WT mice after orchidectomy and androgen replacement therapy, either with or without an aromatase inhibitor. AR activation dominates normal trabecular bone development and cortical bone modeling in male mice. Moreover, optimal periosteal bone expansion is only observed in the presence of both AR and ER activation.Introduction: Androgen receptor (AR)-mediated androgen action has traditionally been considered a key determinant of male skeletal growth. Increasing evidence, however, suggests that estrogens are also essential for 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 AR knockout (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 an aromatase inhibitor (anastrazole) were administered to orchidectomized ARKO and WT mice. Trabecular and 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 ARKO mice. Administration of an aromatase inhibitor did not affect T action on trabecular bone. AR inactivation and orchidectomy had similar negative effects on cortical thickness (−13% and −8%, respectively) and periosteal bone formation (−50% and −26%, respectively). In orchidectomized WT mice, both DHT and T were found to stimulate periosteal bone formation and, as a result, to increase cortical thickness. In contrast, the periosteum of ARKO mice remained unresponsive to either DHT or T. Interestingly, administration of an aromatase 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 significant decrease 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 bone and periosteal bone growth in male mice. Moreover, optimal stimulation of periosteal growth is only obtained in the presence of both AR and ER activation.

Osteoporosis and muscle frailty are important health problems in elderly men and may be partly related to biological androgen activity. This androgen action can be mediated directly through stimulation of the androgen receptor (AR) or indirectly through stimulation of estrogen receptor-alpha (ERalpha) following aromatization of androgens into estrogens. To assess the differential action of AR and ERalpha pathways on bone and body composition, AR-ERalpha double-knockout mice were generated and characterized. AR disruption decreased trabecular bone mass, whereas ERalpha disruption had no additional effect on the AR-dependent trabecular bone loss. In contrast, combined AR and ERalpha inactivation additionally reduced cortical bone and muscle mass compared with either AR or ERalpha disruption alone. ERalpha inactivation--in the presence or absence of AR--increased fat mass. We demonstrate that AR activation is solely responsible for the development and maintenance of male trabecular bone mass. Both AR and ERalpha activation, however, are needed to optimize the acquisition of cortical bone and muscle mass. ERalpha activation alone is sufficient for the regulation of fat mass. Our findings clearly define the relative importance of AR and ERalpha signaling on trabecular and cortical bone mass as well as body composition in male mice.

Although it is well established that males acquire more bone mass than females, the underlying mechanism and timing of this sex difference remain controversial. The aim of this study was to assess the relative contribution of sex steroid versus growth hormoneinsulin-like growth factor 1 (GH-IGF-1) action to pubertal bone mass acquisition longitudinally in pubertal mice. Radial bone expansion peaked during early puberty (3 to 5 weeks of age) in male and female mice, with significantly more expansion in males than in females (þ40%). Concomitantly, in 5 week old male versus female mice, periosteal and endocortical bone formation was higher (þ70%) and lower (À47%), respectively, along with higher serum IGF-1 levels during early puberty in male mice. In female mice, ovariectomy increased radial bone expansion during early puberty as well as the endocortical perimeter. In male mice, orchidectomy reduced radial bone expansion only during late puberty (5 to 8 weeks of age), whereas combined androgen and estrogen deficiency modestly decreased radial bone expansion during early puberty, accompanied by lower IGF-1 levels. GHRKO mice with very low IGF-1 levels, on the other hand, showed limited radial bone expansion and no skeletal dimorphism. From these data we conclude that skeletal sexual dimorphism is established during early puberty and depends primarily on GH-IGF-1 action. In males, androgens and estrogens have stimulatory effects on bone size during late and early puberty, respectively. In females, estrogens limit bone size during early puberty. These longitudinal findings in mice provide strong evidence that skeletal dimorphism is determined by independent and time-specific effects of sex steroids and IGF-1. ß

Androgens may regulate the male skeleton either directly by stimulation of the androgen receptor (AR) or indirectly by aromatization of androgens into estrogens and, thereafter, by stimulation of the estrogen receptors (ERs). To directly compare the effect of ER activation on bone in vivo with the effect of AR activation, 9-month-old orchidectomized wild-type and ER-inactivated mice were treated with the nonaromatizable androgen 5␣-dihydrotestosterone, 17␤-estradiol, or vehicle. Both ER␣ and AR but not ER␤ activation preserved the amount of trabecular bone. ER␣ activation resulted both in a preserved thickness and number of trabeculae. In contrast, AR activation exclusively preserved the number of trabeculae, whereas the thickness of the trabeculae was unaffected. Furthermore, the effects of 17␤-estradiol could not be mediated by the AR, and the effects of 5␣-dihydrotestosterone were increased rather than decreased in ER-inactivated mice. ER␣, but not AR or ER␤, activation resulted in preserved thickness, volumetric density, and mechanical strength of the cortical bone. ER␣ activation increased serum levels of insulin-like growth factor I, which were positively correlated with all the cortical and trabecular bone parameters that were specifically preserved by ER␣ activation but not by AR activation, suggesting that insulin-like growth factor I might mediate these effects of ER␣ activation. Thus, the in vivo bone-sparing effect of ER␣ activation is distinct from the bone-sparing effect of AR activation in adult male mice. Because these two pathways are clearly distinct from each other, one may speculate that a combined treatment of selective ER modulators and selective AR modulators might be beneficial in the treatment of osteoporosis. S ex steroids are important not only for the maintenance of the female skeleton, but also for the male skeleton. The relative contribution of androgens versus estrogens in the regulation of the male skeleton is unclear. Testosterone replacement therapy increases bone mineral density (BMD) in hypogonadal men (1), but several clinical studies indicate that BMD is correlated more to serum levels of estradiol than to serum levels of testosterone in males (2-4). A previous clinical study, which directly compared estrogen versus testosterone effects on bone, showed that estrogens play the dominant role in the regulation of bone resorption markers, whereas both estrogens and testosterone contribute to the maintenance of markers for bone formation (5).The effects of testosterone can be exerted either directly by means of the androgen receptor (AR) or indirectly by aromatization to estrogens and further by estrogen receptor (ER)␣ and͞or ER␤. All three sex steroid receptors are expressed both in growth-plate cartilage and in bone (6-11). Functional studies using sex steroid receptor-inactivated animal models have demonstrated that ER␣ but not ER␤ is important for the regulation of appendicular longitudinal skeletal growth in male mice (12-14), and a recent report indicates that AR-inactivate...