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Sex steroids are critical for skeletal development and maturation during puberty as well as for skeletal maintenance during adult life. However, the exact time during puberty when sex steroids have the highest impact as well as the ability of bone to recover from transient sex steroid deficiency is unclear. Surgical castration is a common technique to study sex steroid effects in rodents, but it is irreversible, invasive, and associated with metabolic and behavioral alterations. Here, we used a low dose (LD) or a high dose (HD) of gonadotropin-releasing hormone antagonist to either temporarily or persistently suppress sex steroid action in male mice, respectively. The LD group, a model for delayed puberty, did not show changes in linear growth or body composition, but displayed reduced trabecular bone volume during puberty, which fully caught up at adult age. In contrast, the HD group, representing complete pubertal suppression, showed a phenotype reminiscent of that observed in surgically castrated rodents. Indeed, HD animals exhibited severely impaired cortical and trabecular bone acquisition, decreased body weight and lean mass, and increased fat mass. In conclusion, we developed a rodent model of chemical castration that can be used as an alternative to surgical castration. Moreover, the transient nature of the intervention enables to study the effects of delayed puberty and reversibility of sex steroid deficiency. NEW & NOTEWORTHY We developed a rodent model of chemical castration, which can be used as an alternative to surgical castration. Moreover, the transient nature of the intervention enables to study the effects of delayed puberty and reversibility of sex steroid deficiency.

Mechanical stress determines bone mass and structure. It is not known whether mechanical loading affects expression of bone regulatory genes in a combined deficiency of estrogen and vitamin D. We studied the effect of mechanical loading on the messenger RNA (mRNA) expression of bone regulatory genes during vitamin D and/or estrogen deficiency. We performed a single bout in vivo axial loading with 14 N peak load, 2 Hz frequency and 360 cycles in right ulnae of nineteen weeks old female control Wistar rats with or without ovariectomy (OVX), vitamin D deficiency and the combination of OVX and vitamin D deficiency (N = 10/group). Total bone RNA was isolated 6 hours after loading, and mRNA expression was detected of Mepe, Fgf23, Dmp1, Phex, Sost, Col1a1, Cyp27b1, Vdr, and Esr1. Serum levels of 25(OH)D, 1,25(OH) 2 D and estradiol were also measured at this time point. The effect of loading, vitamin D and estrogen deficiency and their interaction on bone gene expression was tested using a mixed effect model analysis. Mechanical loading significantly increased the mRNA expression of Mepe, and Sost, whereas it decreased the mRNA expression of Fgf23 and Esr1. Mechanical loading showed a significant interaction with vitamin D deficiency with regard to mRNA expression of Vdr and Esr1. Mechanical loading affected gene expression of Mepe, Fgf23, Sost, and Esr1 independently of vitamin D or estrogen, indicating that mechanical loading may affect bone turnover even during vitamin D deficiency and after menopause., (© 2020 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals LLC on behalf of Orthopaedic Research Society.)

Objective: Long-term androgen deprivation therapy (ADT) negatively influences bone. The short-term effects on bone and mineral homeostasis are less known. Therefore, we aimed to investigate the early effects of ADT on calcium/phosphate homeostasis and bone turnover., Design: Prospective cohort study., Methods: Eugonadal adult, male sex offenders, who were referred for ADT to the endocrine outpatient clinic, received cyproterone acetate. Changes in blood markers of calcium/phosphate homeostasis and bone turnover between baseline and first follow-up visit were studied., Results: Of 26 screened patients, 17 were included. The median age was 44 (range 20-75) years. The median time interval between baseline and first follow-up was 13 (6-27) weeks. Compared to baseline, an 81% decrease was observed for median total testosterone (to 3.4 nmol/L (0.4-12.2); P < 0.0001) and free testosterone (to 0.06 nmol/L (0.01-0.18); P < 0.0001). Median total estradiol decreased by 71% (to 17.6 pmol/L (4.7-35.6); P < 0.0001). Increased serum calcium (P < 0.0001) and phosphate (P = 0.0016) was observed, paralleled by decreased PTH (P = 0.0156) and 1,25-dihydroxyvitamin D3 (P = 0.0134). The stable calcium isotope ratio (δ44/42Ca) decreased (P = 0.0458), indicating net calcium loss from bone. Bone-specific alkaline phosphatase and osteocalcin decreased (P < 0.0001 and P = 0.0056, respectively), periostin tended to decrease (P = 0.0500), whereas sclerostin increased (P < 0.0001), indicating suppressed bone formation. Serum bone resorption markers (TRAP, CTX) were unaltered., Conclusions: In adult men, calcium release from the skeleton occurs early following sex steroid deprivation, reflecting early bone resorption. The increase of sclerostin and reduction of bone formation markers, without changes in resorption markers, suggests a dominant negative effect on bone formation in the acute phase.

The selective estrogen receptor modulator tamoxifen exerts estrogen agonistic or antagonistic actions on several tissues, including bone. The off-target effects of tamoxifen are one of the most widely recognized pitfalls of tamoxifen-inducible Cre recombinases (CreERs), potentially confounding the phenotypic findings. Still, the validation of tamoxifen induction schemes that minimize the side effects of the drug has not been addressed. Here, we compared the side effects on the skeleton and other androgen-responsive targets of a shortened tamoxifen regimen (2 doses of 190 mg/kg body weight by oral gavage) to a standard protocol (4 doses) and determined their efficiency in inducing CreER-mediated gene deletion. In addition, both a vehicle- and a 10-dose group, which served as a positive control for tamoxifen side effects, were also included. For this purpose, we generated male mice with a floxed androgen receptor (AR) and a neuron-specifically expressed CreER. Treatment with two doses of tamoxifen was the only regimen that did not diminish androgenic bioactivity, as assessed by both seminal vesicles and levator ani/bulbocavernosus muscle weights and serum testosterone concentrations. Similarly, trabecular and cortical femoral bone structure were dramatically altered by both the standard and high-dose protocols but not by the shortened version. Serum osteocalcin and bone-gene expression analyses confirmed the absence of effects on bone by 2 doses of tamoxifen. This protocol decreased AR mRNA levels efficiently and specifically in the nervous system. Thus, we optimized a protocol for tamoxifen-induced CreER gene deletion in mice without off-target effects on bone and male reproductive organs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: frailty is associated with an increased risk of fragility fractures. Less is known, however, about the association between frailty and bone health., Methods: men aged 40-79 years were recruited from population registers in eight European centres for participation in the European Male Aging Study. Subjects completed a comprehensive assessment which included quantitative ultrasound (QUS) scan of the heel (Hologic-SAHARA) and in two centres, dual-energy bone densitometry (dual-energy x-ray absorptiometry, DXA). Frailty was defined based on an adaptation of Fried's phenotype criteria and a frailty index (FI) was constructed. The association between frailty and the QUS and DXA parameters was determined using linear regression, with adjustments for age, body mass index and centre., Results: in total, 3,231 subjects contributed data to the analysis. Using the Fried categorisation of frailty, pre-frail and frail men had significantly lower speed of sound (SOS), broadband ultrasound attenuation (BUA) and quantitative ultrasound index (QUI) compared to robust men (P< 0.05). Similar results were seen using the FI after categorisation into 'high', 'medium' and 'low' levels of frailty. Using the Fried categorisation, frail men had lower femoral neck bone mineral density (BMD) compared to robust men (P < 0.05), but not lower lumbar spine BMD. Using the FI categorisation, a 'high' level of frailty (FI > 0.35) was associated with lower lumbar spine BMD (P < 0.05) when compared to those with low (FI < 0.2), but not lower femoral neck BMD. When analysed as a continuous variable, higher FI was linked with lower SOS, BUA and QUI (P < 0.05)., Conclusions: optimisation of bone health as well as prevention of falls should be considered as strategies to reduce fractures in frail older people., (© The Author 2016. Published by Oxford University Press on behalf of the British Geriatrics Society.)

Estrogens and androgens influence the growth and maintenance of the mammalian skeleton and are responsible for its sexual dimorphism. Estrogen deficiency at menopause or loss of both estrogens and androgens in elderly men contribute to the development of osteoporosis, one of the most common and impactful metabolic diseases of old age. In the last 20 years, basic and clinical research advances, genetic insights from humans and rodents, and newer imaging technologies have changed considerably the landscape of our understanding of bone biology as well as the relationship between sex steroids and the physiology and pathophysiology of bone metabolism. Together with the appreciation of the side effects of estrogen-related therapies on breast cancer and cardiovascular diseases, these advances have also drastically altered the treatment of osteoporosis. In this article, we provide a comprehensive review of the molecular and cellular mechanisms of action of estrogens and androgens on bone, their influences on skeletal homeostasis during growth and adulthood, the pathogenetic mechanisms of the adverse effects of their deficiency on the female and male skeleton, as well as the role of natural and synthetic estrogenic or androgenic compounds in the pharmacotherapy of osteoporosis. We highlight latest advances on the crosstalk between hormonal and mechanical signals, the relevance of the antioxidant properties of estrogens and androgens, the difference of their cellular targets in different bone envelopes, the role of estrogen deficiency in male osteoporosis, and the contribution of estrogen or androgen deficiency to the monomorphic effects of aging on skeletal involution., (Copyright © 2017 the American Physiological Society.)

Semaphorin molecules regulate cell adhesion and motility in a wide variety of cell types and are therefore involved in numerous processes including axon guidance, angiogenesis, cardiogenesis, tumor growth, and immune response. Increasing evidence points to a role of transmembrane, membrane-associated and soluble semaphorins during bone development as well as in the control of normal bone homeostasis. Within bone, semaphorins are implicated in the communication between different cell types by relaying signals in an autocrine or paracrine way. Semaphorins are not only involved in bone resorption but also in bone formation. Therefore, targeting semaphorin-induced signaling in bone may constitute an interesting new therapeutic strategy in osteoporosis. However, all the pioneering research on semaphorins is performed in mice and it remains to be established to what extent semaphorin signaling pathways are conserved between mice and men. In addition, knowledge of semaphorin signaling in bone mostly arises from loss/gain of function studies of one single semaphorin and/or receptor. However, different semaphorin molecules are co-expressed in bone and their signaling pathways are likely to interact in a complex and coherent way that needs proper understanding before targeting semaphorin signaling can be therapeutically exploited., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Bone is a biomechanical tissue shaped by forces from muscles and gravitation. Simultaneous bone and muscle decay and dysfunction (osteosarcopenia or sarco-osteoporosis) is seen in ageing, numerous clinical situations including after stroke or paralysis, in neuromuscular dystrophies, glucocorticoid excess, or in association with vitamin D, growth hormone/insulin like growth factor or sex steroid deficiency, as well as in spaceflight. Physical exercise may be beneficial in these situations, but further work is still needed to translate acceptable and effective biomechanical interventions like vibration therapy from animal models to humans. Novel antiresorptive and anabolic therapies are emerging for osteoporosis as well as drugs for sarcopenia, cancer cachexia or muscle wasting disorders, including antibodies against myostatin or activin receptor type IIA and IIB (e.g. bimagrumab). Ideally, increasing muscle mass would increase muscle strength and restore bone loss from disuse. However, the classical view that muscle is unidirectionally dominant over bone via mechanical loading is overly simplistic. Indeed, recent studies indicate a role for neuronal regulation of not only muscle but also bone metabolism, bone signaling pathways like receptor activator of nuclear factor kappa-B ligand (RANKL) implicated in muscle biology, myokines affecting bone and possible bone-to-muscle communication. Moreover, pharmacological strategies inducing isolated myocyte hypertrophy may not translate into increased muscle power because tendons, connective tissue, neurons and energy metabolism need to adapt as well. We aim here to critically review key musculoskeletal molecular pathways involved in mechanoregulation and their effect on the bone-muscle unit as a whole, as well as preclinical and emerging clinical evidence regarding the effects of sarcopenia therapies on osteoporosis and vice versa., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Androgens are well known to enhance exercise-induced muscle hypertrophy; however, whether androgens also influence bone's adaptive response to mechanical loading remains unclear. We studied the adaptive osteogenic response to unilateral in vivo mechanical loading of tibia in adult male mice in both a long- and a short-term experimental set-up. Mice were divided into four groups: sham operated, orchidectomized (ORX), T (ORX+T), or nonaromatizable dihydrotestosterone (ORX+DHT) replacement. Significant interactions between androgen status and osteogenic response to mechanical loading were observed. Cortical thickness increased by T (0.14 vs 0.11 mm sham, P<.05) and DHT (0.17 vs 0.11 mm sham, P<.05). However, T partially (+36%) and DHT completely (+10%) failed to exhibit the loading-related increase observed in sham (+107%) and ORX (+131%, all P<.05) mice. ORX decreased periosteal bone formation, which was restored to sham levels by T and DHT. However, both androgens completely suppressed the loading-related increase in periosteal bone formation. Short-term loading decreased the number of sclerostin-positive osteocytes in sham, whereas in control fibulas, ORX decreased and T increased the number of sclerostin-positive osteocytes. Loading no longer down-regulated sclerostin in the ORX or T groups. In conclusion, both T and DHT suppress the osteogenic response to mechanical loading.

Sex steroids are chief regulators of gender differences in the skeleton, and male gender is one of the strongest protective factors against osteoporotic fractures. This advantage in bone strength relies mainly on greater cortical bone expansion during pubertal peak bone mass acquisition and superior skeletal maintenance during aging. During both these phases, estrogens acting via estrogen receptor-α in osteoblast lineage cells are crucial for male cortical and trabecular bone, as evident from conditional genetic mouse models, epidemiological studies, rare genetic conditions, genome-wide meta-analyses, and recent interventional trials. Genetic mouse models have also demonstrated a direct role for androgens independent of aromatization on trabecular bone via the androgen receptor in osteoblasts and osteocytes, although the target cell for their key effects on periosteal bone formation remains elusive. Low serum estradiol predicts incident fractures, but the highest risk occurs in men with additionally low T and high SHBG. Still, the possible clinical utility of serum sex steroids for fracture prediction is unknown. It is likely that sex steroid actions on male bone metabolism rely also on extraskeletal mechanisms and cross talk with other signaling pathways. We propose that estrogens influence fracture risk in aging men via direct effects on bone, whereas androgens exert an additional antifracture effect mainly via extraskeletal parameters such as muscle mass and propensity to fall. Given the demographic trends of increased longevity and consequent rise of osteoporosis, an increased understanding of how sex steroids influence male bone health remains a high research priority.

Bone is an endocrine tissue expressing androgen and estrogen receptors as well as steroid metabolizing enzymes. The bioactivity of circulating sex steroids is modulated by sex hormone-binding globulin and local conversion in bone tissue, for example, from testosterone (T) to estradiol (E2) by aromatase, or to dihydrotestosterone by 5α-reductase enzymes. Our understanding of the structural basis for gender differences in bone strength has advanced considerably over recent years due to increasing use of (high resolution) peripheral computed tomography. These microarchitectural insights form the basis to understand sex steroid influences on male peak bone mass and turnover in cortical vs trabecular bone. Recent studies using Cre/LoxP technology have further refi ned our mechanistic insights from global knockout mice into the direct contributions of sex steroids and their respective nuclear receptors in osteoblasts, osteoclasts, osteocytes, and other cells to male osteoporosis. At the same time, these studies have reinforced the notion that androgen and estrogen defi ciency have both direct and pleiotropic effects via interaction with, for example, insulin-like growth factor 1, inflammation, oxidative stress, central nervous system control of bone metabolism, adaptation to mechanical loading, etc., This review will summarize recent advances on these issues in the fi eld of sex steroid actions in male bone homeostasis.

Purpose of Review: This review provides an update on the associations of testosterone, estrogens, sex hormone binding globulin, GH-IGF-I, osteocalcin and mechanical loading with relevance to skeletal health., Recent Findings: The simple concept of a dual model of action of androgens, i.e. either directly via the androgen receptor or indirectly by estrogens, is proving more complicated because of novel interactions of these hormones and their receptors with other hormonal as well as mechanical signals., Summary: Testosterone - in contrast with estrogen - is not uniformly associated with fracture risk in men. However, androgen receptor mediated action is clearly important for trabecular bone maintenance in male mice whereas both estrogens and androgens regulate cortical bone growth. The osteoblast and osteocyte appear to be involved in such androgen receptor mediated action on bone in male mice. Studies in mice also showed an unexpected interaction between osteocalcin and testosterone production in males and, vice versa, between ovarian production of follicle-stimulating hormone with testosterone and potentially bone formation.

We sought to determine the influence of single-nucleotide polymorphisms (SNPs) in RANKL, RANK, and OPG on volumetric bone mineral density (vBMD) and bone geometry at the radius in men. Pairwise tag SNPs (r (2) ≥ 0.8) for RANKL (n = 8), RANK (n = 44), and OPG (n = 22) and five SNPs near RANKL and OPG strongly associated with areal BMD in genomewide association studies were previously genotyped in men aged 40-79 years in the European Male Ageing Study (EMAS). Here, these SNPs were analyzed in a subsample of men (n = 589) who had peripheral quantitative computed tomography (pQCT) performed at the distal (4%) and mid-shaft (50%) radius. Estimated parameters were total and trabecular vBMD (mg/mm(3)) and cross-sectional area (mm(2)) at the 4% site and cortical vBMD (mg/mm(3)); total, cortical, and medullary area (mm(2)); cortical thickness (mm); and stress strain index (SSI) (mm(3)) at the 50% site. We identified 12 OPG SNPs associated with vBMD and/or geometric parameters, including rs10505348 associated with total vBMD (β [95% CI] = 9.35 [2.12-16.58], P = 0.011), cortical vBMD (β [95% CI] = 5.62 [2.10-9.14], P = 0.002), cortical thickness (β [95% CI] = 0.08 [0.03-0.13], P = 0.002), and medullary area (β [95% CI] = -2.90 [-4.94 to -0.86], P = 0.005) and rs2073618 associated with cortical vBMD (β [95% CI] = -4.30 [-7.78 to -0.82], P = 0.015) and cortical thickness (β [95% CI] = -0.08 [-0.13 to -0.03], P = 0.001). Three RANK SNPs were associated with vBMD, including rs12956925 associated with trabecular vBMD (β [95% CI] = -7.58 [-14.01 to -1.15], P = 0.021). There were five RANK SNPs associated with geometric parameters, including rs8083511 associated with distal radius cross-sectional area (β [95% CI] = 8.90 [0.92-16.88], P = 0.029). No significant association was observed between RANKL SNPs and pQCT parameters. Our findings suggest that genetic variation in OPG and RANK influences radius vBMD and geometry in men.

The aim of this study was to determine the influence of insulin-like growth factor binding protein (IGFBP)-1, IGFBP-3, and IGF-I on calcaneal ultrasound parameters in middle-aged and elderly European men. Men aged 40-79 years were recruited from population registers for participation in the European Male Ageing Study (EMAS). Subjects were invited by letter to complete a postal questionnaire and to attend for an interviewer-assisted questionnaire, quantitative ultrasound (QUS) of the calcaneus, and a fasting blood sample from which serum levels of IGFBP-1, IGFBP-3, IGF-I, estradiol (E(2)), and SHBG were assayed. The questionnaires included the Physical Activity Scale for the Elderly (PASE) and questions about smoking and alcohol consumption. Estimated bone mineral density (eBMD) was derived as a function of the QUS parameters speed of sound and broadband ultrasound attenuation. Height and weight were measured in all subjects. 3057 men, mean age 59.7 years (standard deviation 11.0) were included in the analysis. After adjusting for age, center, and BMI, higher levels of IGFBP-1 were associated with lower eBMD. Higher levels of both IGFBP-3 and IGF-I were associated with higher eBMD. After further adjustment for PASE score, current smoking, alcohol consumption, free E(2), and SHBG, IGFBP-3 and IGF-I, though not IGFBP-1, remained significantly associated with eBMD. IGFBP-1 was associated with bone health, though the effect could be explained by other factors. IGFBP-3 and IGF-I were independent determinants of bone health in middle-aged and elderly European men.

Introduction: In this study, we aimed to investigate the association between single nucleotide polymorphisms (SNPs) within two genes involved in the NF-κB cascade (GPR177 and MAP3K14) and bone mineral density (BMD) assessed at different skeletal sites, radial geometric parameters and bone turnover., Methods: Ten GPR177 SNPs previously associated with BMD with genome-wide significance and twelve tag SNPs (r(2)≥0.8) within MAP3K14 (±10 kb) were genotyped in 2359 men aged 40-79 years recruited from 8 centres for participation in the European Male Aging Study (EMAS). Measurement of bone turnover markers (PINP and CTX-I) in the serum and quantitative ultrasound (QUS) at the calcaneus were performed in all centres. Dual energy X-ray absorptiometry (DXA), at the lumbar spine and hip, and peripheral quantitative computed tomography (pQCT), at the distal and midshaft radius, were performed in a subsample (2 centres). Linear regression was used to test for association between the SNPs and bone measures under an additive genetic model adjusting for study centre., Results: We validated the associations between SNPs in GPR177 and BMD(a) previously reported and also observed evidence of pleiotrophic effects on density and geometry. Rs2772300 in GPR177 was associated with increased total hip and LS BMD(a), increased total and cortical vBMD at the radius and increased cortical area, thickness and stress strain index. We also found evidence of association with BMD(a), vBMD, geometric parameters and CTX-I for SNPs in MAP3K14. None of the GPR177 and MAP3K14 SNPs were associated with calcaneal estimated BMD measured by QUS., Conclusion: Our findings suggest that SNPs in GPR177 and MAP3K14 involved in the NF-κB signalling pathway influence bone mineral density, geometry and turnover in a population-based cohort of middle aged and elderly men. This adds to the understanding of the role of genetic variation in this pathway in determining bone health.

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 hormone-insulin-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 (+40%). Concomitantly, in 5 week old male versus female mice, periosteal and endocortical bone formation was higher (+70%) and lower (-47%), 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., (Copyright 2010 American Society for Bone and Mineral Research.)

Traditionally, the stronger male skeleton was considered to result from higher androgen levels in men compared to women. However, the regulation of male bone growth by sex steroids appears more complex than originally anticipated. Based on clinical observations and studies in animal models, not only androgens and androgen receptor (AR), but also estrogens and estrogen receptor-alpha (not ERbeta) are required for optimal bone mineral acquisition during male growth. In addition, both sex steroids are involved in the maintenance of male skeletal health. In fact, bone loss and fracture risk have been associated with estrogen exposure in elderly men. Overall, a compelling body of evidence suggests that both androgens and estrogens are crucial for male skeletal growth and maintenance., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

In female mice, estrogen receptor-alpha (ERalpha) mediates the anabolic response of bone to mechanical loading. Whether ERalpha plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR-ERalpha knockout (KO) mice and corresponding male and female single KO and wild-type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERalpha inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR-ERalpha KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow-induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERalpha activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling., (2010 American Society for Bone and Mineral Research)

Introduction: Sex steroids have distinct effects on bone growth and maintenance in men and women, mediated through their respective steroid receptors. Though most evidence is derived from animal studies, several concepts have been confirmed in humans by detection of specific mutations. In this report we describe changes in bone size and volumetric bone density in a complete androgen insensitive subject (CAIS) due to a mutation in the androgen receptor during 5 years of estrogen treatment., Materials and Methods: We present a case report of a 31 year old XY female with CAIS with a longitudinal follow-up for 5 years of areal and volumetric bone parameters. Areal and volumetric bone parameters were determined using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Sex steroids, LH, FSH and IGF-I were determined by immunoassay., Results: Complete androgen insensitivity syndrome was genetically confirmed by detection of the mutation Asp767Tyr in the androgen receptor gene. Bone size at presentation was found to be intermediate between male and female reference values. Low areal and volumetric bone density (both trabecular and cortical) was observed at baseline and improved gradually with estrogen treatment (+2% to 6.5%). Upon estrogen treatment, endosteal contraction (-1%) was demonstrated, with increasing cortical thickness (+3%), cortical area (+5%) and unchanged periosteal circumference., Conclusions: During adult life, estrogens mediate endosteal bone apposition and volumetric bone density, without marked influence on periosteal bone apposition. The finding of a bone size intermediate between male and female supports testosterone as an essential mediator for periosteal bone expansion, but not as the sole stimulus for bone expansion during growth.

Disruption of the androgen receptor (AR) in male mice reduces cortical bone expansion and muscle mass during puberty and results in high bone turnover-related cancellous osteopenia. We hypothesized that voluntary wheel running during growth is able to rescue the effects of AR disruption on bone. To this end, 5-week-old AR knockout (ARKO) mice were randomized to a running group (cage with running wheel) and a sedentary group (cage without wheel) and followed-up until 16 weeks of age. Voluntary wheel running in ARKO mice did not influence body weight, muscle mass or periosteal bone expansion. Interestingly, voluntary running significantly reduced bone turnover in ARKO mice and prevented cancellous bone loss due to a preservation of trabecular number. Thus, voluntary running in ARKO mice was able to reduce cancellous bone resorption, suggesting that sustained exercise may potentially compensate the effects of androgen disruption on cancellous bone.

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.

Purpose of Review: The present review will focus on the most important recent findings with respect to skeletal androgen action. Many studies have indicated that part of the androgen action may be related to the conversion of androgens into estrogens. Therefore, some of the most recent findings of skeletal estrogen action relevant for male skeletal physiology will also be discussed., Recent Findings: Androgens and estrogens stimulate bone formation and inhibit bone resorption. Sex steroids may interact with different receptors, target cells and other bone anabolic pathways. Androgen receptor and estrogen receptor signalling appear to be important for male bone formation during growth. Sex steroid signalling may involve genomic and nongenomic pathways, interaction with mechanical loading, the growth hormone/insulin-like growth factor-I axis and/or other bone anabolic pathways. Estrogen receptor alpha in osteoclasts appears to regulate bone resorption in women but not men, whereas androgen receptor signalling in osteoblasts may only partly regulate bone resorption in males., Summary: The latest developments indicate that androgens and estrogens are important for male bone metabolism and homeostasis and therefore selective estrogen receptor alpha and androgen receptor signalling remain interesting drug targets for the stimulation of bone formation and male skeletal integrity.

Unlabelled: 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.

This study reports the preclinical evaluation of the bone and muscle protective potential of the synthetic androgen 7alpha-methyl-19-nortestosterone (MENTtrade mark), as assessed in the aged orchidectomized rat model. Aged (13-month-old) orchidectomized Wistar rats were treated with different doses of MENT (4, 12 or 36 microg/day) subcutaneously for 16 weeks via mini-osmotic pumps. Analysis of the effects of androgen deficiency versus MENT replacement was performed using quantitative computed tomography (pQCT), dual energy X-ray absorptiometry (DEXA) and biochemical markers of bone turnover. At the end of the study period, prostate weight in orchidectomized rats treated with low- (4 microg/day) or mid-dose (12 mug/day) MENT remained significantly lower compared to the sham-operated animals (-47% and -25%, respectively). High-dose MENT (36 microg/day), on the other hand, induced prostate hypertrophy (+21% versus sham). Low-, mid- and high-dose MENT were found to be effective in suppressing the acceleration of bone remodeling following orchidectomy, as assessed by osteocalcin and deoxypyridinoline. In addition, low-, mid- and high-dose were able to prevent the orchidectomy-induced bone loss, as evaluated by DEXA at the femur and total-body and by pQCT at the femur. Compared to sham-operated animals, the low- and mid-dose MENT groups showed no decline in lean body mass and no muscle atrophy (as measured by m. quadriceps weight) at 16 weeks, whereas high-dose MENT was associated with a significant decline in lean body mass (-8.5% versus sham) and quadriceps weight (-10.6%). We conclude that, in the aged orchidectomized rat model, low- and mid-doses of the synthetic androgen MENT have bone and muscle protective effects and do not induce prostate hypertrophy. The bone protective action of high-dose MENT, however, occurs at the expense of muscle wasting and prostate hypertrophy. Our findings support the need for human studies to explore the potential of MENT as an option for androgen replacement in aging men.

Unlabelled: Based on data searches and life-table analyses, we determined the long-term (remaining lifetime) and short-term (10- and 5-year) absolute risks of hip fracture after sustaining a Colles' or spine fracture and searched for potential gender-related differences. In aging men, Colles' fractures carry a higher absolute risk for hip fracture than spinal fractures in contrast to women. These findings support the concept that forearm fracture is an early and sensitive marker of male skeletal fragility., Introduction: Colles' fracture occurrence has been largely ignored in public health approaches to identify target populations at risk for hip fracture. The aim of this study was to estimate the long-term and short-term absolute risks of hip fracture after sustaining a Colles' or spine fracture and to search for potential gender-related differences in the relationship between fracture history and future fracture risk., Materials and Methods: To determine the long-term (remaining lifetime) and short-term (10- and 5-year) absolute risks of hip fracture, we applied life-table methods using U.S. age- and sex-specific hip fracture incidence rates, U.S. age-specific mortality rates for white women and men, pooled hazard ratios for mortality after Colles' and spine fracture, and pooled relative risks for hip fracture after Colles' and spine fracture, estimated from cohort studies by standard meta-analytic methods., Results: Our results indicate that the estimated remaining lifetime risks are dependent on age in both genders. In women, remaining lifetime risks increase until the age of 80 years, when they start to decline because of the competing probabilities of fracture and death. The same pattern is found in men until the age of 85 years, the increment in lifetime risk being even more pronounced. As expected, the risk of sustaining a hip fracture was found to be higher in postmenopausal women with a previous spine fracture compared with those with a history of Colles' fracture. In men, on the other hand, the prospective association between fracture history and subsequent hip fracture risk seemed to be strongest for Colles' fracture. At the age of 50, for example, the remaining lifetime risk was 13% in women with a previous Colles' fracture compared with 15% in the context of a previous spine fracture and 9% among women of the general population. In men at the age of 50 years, the corresponding risk estimates were 8%, 6%, and 3%, respectively. Similar trends were observed when calculating 5- and 10-year risks., Conclusions: In aging men, Colles' fractures carry a higher absolute risk for hip fracture than spinal fractures in contrast to women. These findings support the concept that forearm fracture is an early and sensitive marker of male skeletal fragility. The gender-related differences reported in this analysis should be taken into account when designing screening and treatment strategies for prevention of hip fracture in men.

Unlabelled: The role of androgen receptor-mediated androgen action on bone was investigated in testicular feminized male (Tfm) mice. Cortical bone was found to be unresponsive to testosterone (T) in orchidectomized Tfm mice, whereas cortical thickness as well as trabecular BMD and structure were fully maintained by T in the corresponding Tabby control mice. These data show an essential role for androgen receptor-mediated androgen action in periosteal bone formation., Introduction: Androgens can affect the male skeleton both directly-through activation of the androgen receptor (AR)-and indirectly-through stimulation of estrogen receptors after aromatization. We assessed the importance of AR-mediated androgen action on bone in a mouse model of androgen resistance., Materials and Methods: Eight-week-old androgen-resistant testicular feminized male (Tfm) and Tabby control mice were orchidectomized (ORX) and treated for 4 weeks with a slow-release testosterone (T) pellet (delivering 167 microg/day) or a placebo pellet. A comprehensive analysis of the skeletal effects of androgen deficiency and replacement was performed using histomorphometry, QCT, and biochemical assessment of bone turnover., Results: As expected, T increased trabecular BMD, volume, number, and width in ORX Tabby mice. In ORX Tfm mice, however, T had less effect on trabecular BMD and no effect on trabecular bone structure. T action on trabecular bone was associated with opposite changes in bone turnover: trabecular and endocortical bone turnover and serum levels of osteocalcin were all reduced by T in ORX Tabby mice, but not in ORX Tfm mice. T also increased cortical thickness (+16%), area, and density in ORX Tabby mice, but not in Tfm mice, resulting in greater bone strength in the Tabby control strain. The positive effects of T on cortical bone reflected a stimulatory effect on periosteal bone formation (+137%), which was again absent in Tfm mice., Conclusions: These data show that, in male mice, AR-mediated T action is essential for periosteal bone formation and contributes to trabecular bone maintenance.

Loss of estrogens or androgens increases the rate of bone remodeling by removing restraining effects on osteoblastogenesis and osteoclastogenesis, and also causes a focal imbalance between resorption and formation by prolonging the lifespan of osteoclasts and shortening the lifespan of osteoblasts. Conversely, androgens, as well as estrogens, maintain cancellous bone mass and integrity, regardless of age or sex. Although androgens, via the androgen receptor (AR), and estrogens, via the estrogen receptors (ERs), can exert these effects, their relative contribution remains uncertain. Recent studies suggest that androgen action on cancellous bone depends on (local) aromatization of androgens into estrogens. However, at least in rodents, androgen action on cancellous bone can be directly mediated via AR activation, even in the absence of ERs. Androgens also increase cortical bone size via stimulation of both longitudinal and radial growth. First, androgens, like estrogens, have a biphasic effect on endochondral bone formation: at the start of puberty, sex steroids stimulate endochondral bone formation, whereas they induce epiphyseal closure at the end of puberty. Androgen action on the growth plate is, however, clearly mediated via aromatization in estrogens and interaction with ERalpha. Androgens increase radial growth, whereas estrogens decrease periosteal bone formation. This effect of androgens may be important because bone strength in males seems to be determined by relatively higher periosteal bone formation and, therefore, greater bone dimensions, relative to muscle mass at older age. Experiments in mice again suggest that both the AR and ERalpha pathways are involved in androgen action on radial bone growth. ERbeta may mediate growth-limiting effects of estrogens in the female but does not seem to be involved in the regulation of bone size in males. In conclusion, androgens may protect men against osteoporosis via maintenance of cancellous bone mass and expansion of cortical bone. Such androgen action on bone is mediated by the AR and ERalpha.

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 5alpha-dihydrotestosterone, 17beta-estradiol, or vehicle. Both ERalpha and AR but not ERbeta activation preserved the amount of trabecular bone. ERalpha 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 17beta-estradiol could not be mediated by the AR, and the effects of 5alpha-dihydrotestosterone were increased rather than decreased in ER-inactivated mice. ERalpha, but not AR or ERbeta, activation resulted in preserved thickness, volumetric density, and mechanical strength of the cortical bone. ERalpha 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 ERalpha activation but not by AR activation, suggesting that insulin-like growth factor I might mediate these effects of ERalpha activation. Thus, the in vivo bone-sparing effect of ERalpha 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.