AJP - Endo Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 290: E456-E462, 2006. First published October 4, 2005; doi:10.1152/ajpendo.00311.2005
0193-1849/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/E456    most recent
00311.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sims, N. A.
Right arrow Articles by Seibel, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sims, N. A.
Right arrow Articles by Seibel, M. J.

Perinatal testosterone surge is required for normal adult bone size but not for normal bone remodeling

Natalie A. Sims,1 Karen Brennan,2 Jenny Spaliviero,2 David J. Handelsman,2 and Markus J. Seibel2

1Department of Medicine at St. Vincent's Hospital, The University of Melbourne and St. Vincent's Institute, Fitzroy, Victoria; and 2ANZAC Research Institute, University of Sydney, Sydney, New South Wales, Australia

Submitted 11 July 2005 ; accepted in final form 3 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although testosterone (T) has striking effects on mature skeletal size and structure, it is not clear whether this depends exclusively on adult circulating levels of T or whether additional early-life factors also play a role. We have compared the androgen-deficient hypogonadal (hpg) mutant mouse with intact, orchidectomized, and T-treated non-hpg mice to determine relative contributions of adult and perinatal T to bone growth and development. At 3 wk of age, although trabecular and cortical bone structure was normal, bone turnover was significantly altered in hpg male mice; osteoid volume (OV/BV) and osteoblast surface (ObS/BS) were significantly lower and osteoclast surface (OcS/BS) significantly higher in hpg mice compared with age-matched non-hpg mice, pointing to a role for the perinatal T surge in determining bone turnover levels before sexual maturity. At 9 wk of age, the hpg bone phenotype mimicked closely that of age-matched non-hpg mice that had been orchidectomized at 3 wk of age, including low trabecular bone mass and high bone turnover. These bone phenotypes of hpg and orchidectomized non-hpg mice were all prevented by replacement doses of T or dihydrotestosterone (DHT), suggesting that these are determined by adult sex steroid hormones. In contrast, a short bone phenotype that could not be prevented by T or DHT treatment was observed in 9-wk-old hpg mice yet not in intact or castrated non-hpg mice. These data suggest a role for the perinatal T surge in determining adult bone length and confirms that adult circulating T determines adult bone density.

hypogonadism; skeleton; androgens

SKELETAL SIZE, SHAPE, AND INTERNAL STRUCTURE are determined by three major processes: embryonic development, growth before sexual maturity, and remodeling of the trabecular network throughout life. These three pathways depend on complex intercellular communication between the cells within bone, particularly osteoblasts (bone-forming cells), osteoclasts (bone-resorbing cells), and chondrocytes (cartilage cells). Androgens are known to influence proliferation and function of all three of these cell types and are required for both skeletal growth and maturation.

Androgens play an important role in trabecular remodeling. In most mammalian species, trabecular bone density is significantly higher in males (41). Low bone density is observed in men with androgen deficiency regardless of whether the hormone deficit is congenital, e.g., idiopathic hypogonadotropic hypogonadism (IHH) (5, 7, 8) or Klinefelter's syndrome (KS) (5), or acquired during adult life, e.g., castration (6, 38, 39). Although osteoporosis due to castration is associated with high bone turnover (6, 38, 39), this is not consistently reported in early-onset (and sometimes less complete) androgen deficiency (5, 7, 8, 12, 17). By contrast, in rodent models of complete congenital androgen deficiency, such as androgen insensitivity due to androgen receptor (AR) mutations or orchidectomy, low trabecular bone mass due to high bone turnover is observed consistently (15, 41).

Bone size and geometry are also influenced by sex steroid hormone levels, as reflected in sexual dimorphism in bone length and width in humans and rodents. Bone width and cortical bone growth are modified by defects in either estrogen or androgen actions (1, 15, 18, 22, 31, 35, 40). In contrast, bone length is unaltered in the absence of ARs (15, 18, 40) but is modified by complete blockade of estrogen action due to absence of either estrogen receptors (ER) or aromatase in humans (1, 35) and mice (22, 31). Thus, although both estradiol and testosterone (T) regulate appositional bone growth, it has been suggested that only estrogen, possibly acting via the growth hormone (GH)-IGF-I axis, is required for normal longitudinal bone growth (24, 41).

Although no sexual dimorphism is observed in bone size before sexual maturity, it is possible that differences in sex steroid secretion during development may determine adult bone size, shape, and internal structure. The adult male-specific secretion pattern of GH is determined by a peak of testicular androgen secretion during the perinatal period (14), a process known as hormonal imprinting, due to the perinatal androgen surge present in all male mammals. Because GH also regulates bone size and shape (29), perinatal T may therefore also determine the quality and quantity of mature bone. Although studies in gonadectomized rodents provide much information about the need for androgens in bone growth and turnover during and after sexual maturation, any role of the perinatal androgen surge is overlooked in such models.

The androgen-deficient (hpg) mouse is a naturally occuring mutant (2) with a large deletion in the gonadotropin-releasing hormone (GnRH) gene (19). This produces complete, permanent functional elimination of hypothalamic GnRH secretion so that male mice lack T secretion from birth onward, including both the perinatal T surge and sexual maturation. As a result, hpg mice show persistence of an immature reproductive system with undetectable blood LH and FSH, very low blood T, infantile testes, and immature androgen-dependent organs (33). The functional consequences of GnRH deficiency are fully rectified by transfer of the GnRH gene (20) and, to a lesser extent consistent with the later onset of functional GnRH replacement, by intracerebral transplantation of fetal GnRH neurons (16, 28) or GnRH-secreting tumor cells (27) or even frequent GnRH injections (3). The latter findings also confirm that, despite low level of expression of GnRH and/or its receptor in some extracranial sites, all physiological effects of GnRH are replicated by restoring GnRH action within the hypothalamo-pituitary unit. Although homozygous hpg mice have complete postnatal androgen deficiency (21), they retain normal androgen sensitivity with T replacement rectifying all androgen-dependent deficits in mature somatic organs and tissues (13, 26, 34, 36, 37). Hence, treatment of hpg mice with T from weaning to emulate sexual maturation represents a model for loss of the perinatal androgen surge without loss of T during sexual maturation.

We have utilized this mouse model to make a comparison between complete androgen deficiency either from birth (the hpg mouse) or after the perinatal period (orchidectomy) to determine the role of the perinatal androgen surge in determining adult bone size and trabecular structure. To do this, we examined the role of androgens in early skeletal growth by comparing immature 3-wk-old hpg and non-hpg male mice; the role of androgens in the determination in bone structure in early mature life by comparing 9-wk-old hpg and non-hpg male mice, and then determining whether exposure to the perinatal androgen surge makes a difference by comparing hpg mice with castrated non-hpg males. Finally, we determined whether androgen replacement using aromatizable T or its nonaromatizable metabolite dihydrotestosterone (DHT) could prevent the effects of congenital androgen deficiency and thereby identify which effects of T on the skeleton relate to loss of sexual maturation and which are due to lack of the perinatal testosterone surge.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The hpg mouse line is a naturally occuring mutant (2) extensively used as a model of complete postnatal androgen deficiency with preservation of androgen sensitivity (10, 32, 37). The hpg colony is bred from fertile heterozygotes originally from F1 hybrids of two inbred strains, C3H/HeH and 101/H, as previously described (32). Animals with at least one wild-type allele (non-hpg) were used as controls for all parts of the study, as previously described (32). There was no significant difference in any marker of bone size, structure, or turnover between heterozygous and homozygous non-hpg mice. Animals were maintained under standard conditions at the ANZAC Research Institute, and all experiments were approved by the Central Sydney Area Health Service Animal Welfare Committee within National Health and Medical Research Council of Australia (NHMRC) guidelines for animal experimentation.

Skeletal specimens were collected from 3- and 9-wk-old male mice. Samples were fixed in 10% buffered formalin and embedded in methylmethacrylate (29). Double fluorochrome labeling was performed as described previously (29). Sections (5 µm) were stained with toluidine blue or Xylenol orange for analysis of fluorochrome labels (11). Histomorphometry was carried out in the secondary spongiosa of the proximal tibia according to standard procedures using the Osteomeasure system (OsteoMetrics, Decatur, GA). Tibial cortical thickness, periosteal mineral appositional rates, and proximal tibial growth plate widths were measured as described previously (29). Femoral and tibial length and femoral middiaphysial diameter were determined from contact X-rays that were scanned and measured using NIH Image 1.62, as described previously (31).

Femoral cortical (Ct) and trabecular (Tb) bone mineral density (BMD), femoral circumference, and femoral cortical thickness were measured by perpheral quantitative computer tomography (Stratec X-CT Research SA+, version 5.5) using methods adapted from Schmidt et al. (25). Metaphysial scans of the distal femur were taken at a resolution of 70 µm; trabecular and cortical measurements (including circumference) were taken at a distance proximal to the distal growth plate of 5 and 25% of the length of the femur, respectively; Tb.BMD was determined as the inner 45% of the total area (peel mode 20). Interassay coefficients of variation (CVs) were <1%.

Serum osteocalcin levels were measured by a mouse-specific IRMA (Immutopics), which utilizes polyclonal goat anti-mouse antibodies against midregion, COOH-, and NH2-terminal osteocalcin. The intra-assay CV for this assay was 4.6%, and the interassay CV was 5.2%. Circulating levels of mouse tartrate-resistant acid phosphatase 5b (TRAP5b) were determined by ELISA on the basis of a polyclonal antibody raised against intact mouse osteoclast TRAP5b (Suomen Bioanalytiikka Oy, Oulu, Finland). This assay had an intra-assay CV of <6% and an interassay CV of <8%. All measurements were done in duplicate.

To compare the effects of congenital hypogonadism to gonadectomy after the perinatal period, 3-wk-old non-hpg mice were sham operated or orchidectomized. To determine the effects of T, non-hpg mice were orchidectomized at 3 wk of age (ORX), and both non-hpg and hpg mice were treated from 3 to 9 wk of age with T or DHT by subdermal implantation of 1-cm Silastic implants filled with crystalline steroids (Sigma) as described previously (9, 32). We have previously shown that these implants provide reproducible and predictable steady-state blood T or DHT levels for up to 8 wk (33). Mice were killed by cardiac puncture under anesthetic at 9 wk of age, when tissues were collected for analysis.

Statistically significant effects of genotype or treatment were determined by one- or two-way ANOVA followed by Fisher's post hoc test using the StatView software package. All data are presented as means ± SE. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of postnatal androgen deficiency on bone growth and skeletal maturation in immature mice. At 3 wk of age, no significant difference was detected in body weight (mean g ± SE: non-hpg 12.3 ± 0.5, hpg 11.5 ± 0.7), femoral circumference (Fem.Ci), cortical thickness (Ct.Th), cortical BMD (Ct.BMD), or femoral length (Fem.L) between non-hpg and hpg mice (Fig. 1, A–C); yet growth plate width was significantly reduced (Fig. 1D). Furthermore, there was no significant difference in trabecular bone structure or density, including trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), or trabecular BMD (Tb.BMD) between non-hpg and hpg mice at 3 wk of age (Fig. 2, A and B), confirming that androgens prior to sexual maturity do not determine bone size or trabecular structure at this age.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1. Femoral diameter (Fem.D), cortical width (Ct.Wi.), cortical BMD (Ct.BMD), and femoral length (Fem.L) were not significantly altered by the hpg mutation (filled bars) at 3 wk of age, yet growth plate width (G.Pl.Wi) was significantly reduced in the hpg mutant mice compared with non-hpg mice (ctrl). Values are means ± SE on 8–10 mice per group. Effect of mutation: *P < 0.05 vs. control of same age.

 

Figure 2
View larger version (46K):
[in this window]
[in a new window]
  		Fig. 2. Comparison of trabecular bone structure and turnover in 3-wk-old hpg and control (ctrl) mice. A: representative sections of proximal tibiae of control and hpg mutant mice at 3 wk of age stained with modified von Kossa stain (mineralized bone appears black); bar = 500 µm. B: trabecular bone volume (BV/TV), thickness (Tb.Th), and number (Tb.N) were not significantly different in hpg mice compared with non-hpg mice. C: bone turnover was significantly altered by hpg mutation. In hpg mice, osteoid volume (OV/BV) and osteoblast surface (ObS/BS) were significantly lower and osteoclast surface (OcS/BS) significantly higher in hpg mice compared with age-matched non-hpg mice. Values are means ± SE of 8–10 mice per group. Effect of mutation: *P < 0.05, **P < 0.01 vs. age-matched control.

 
Although there was no difference in trabecular structure between the non-hpg and hpg mice at 3 wk of age, there was a striking difference in trabecular bone remodeling levels between the two mouse strains. In the 3-wk-old hpg mice, osteoclast surface (Fig. 2C) was significantly greater than in non-hpg mice; yet this was not associated with a similar rise in bone formation, as usually occurs when bone resorption is increased. Rather, in the hpg mice, histomorphometric indicators of bone formation, including osteoblast surface (ObS/BS) and osteoid volume (OV/BV), were significantly lower than levels observed in non-hpg mice (Fig. 2C). A similar effect of the mutation on biochemical markers of bone turnover was observed, but this was not statistically significant (TRAP5b mean U/l: non-hpg 26.4 ± 1.5, hpg 30.5 ± 1.6, P = 0.075; osteocalcin mean ng/ml: non-hpg 888 ± 39, hpg 816 ± 59, P = 0.312). Thus in hpg mice there is disrupted coupling of osteoblast and osteoclast formation such that bone resorption is high and bone formation is low. At the 3-wk time point, this imbalance in bone turnover has not yet resulted in a significant change in trabecular structure (Fig. 2, A and B).

In untreated 9-wk-old animals, a significant difference in trabecular structure and density was observed between non-hpg and hpg mice (Fig. 3, A and B). In hpg mice, Tb.BMD (Fig. 3A) was significantly lower than in non-hpg controls. Not only was the normal growth-associated increase in Tb.Th between 3 and 9 wk inhibited, but there was also a significant reduction in Tb.N in the hpg mice, resulting overall in significantly lower BV/TV at 9 wk compared with age-matched non-hpg mice. This was also reflected in a significant reduction in Tb.BMD (Fig. 3B). At 9 wk of age, hpg mice also demonstrated significantly elevated bone turnover compared with non-hpg mice (Fig. 3C). Serum levels of TRAP5b, a marker of bone resorption, were also significantly elevated in 9-wk-old hpg mice (mean U/l: non-hpg 12.7 ± 1.2, hpg 19.7 ± 2.0, P = 0.007). In contrast, serum osteocalcin, a marker of bone formation, was significantly reduced compared with non-hpg mice (mean ng/ml: non-hpg 362 ± 35, hpg 203 ± 18, P = 0.0003). This phenotype is similar to the trabecular bone defect reported in ORX mice.


Figure 3
View larger version (92K):
[in this window]
[in a new window]
  		Fig. 3. Low bone mass and high turnover in 9-wk-old hpg mutant mice are similar to those in control mice orchidectomized at 3 wk (ORX), and testosterone (T) treatment is able to prevent this low bone mass. A: representative von Kossa-stained proximal tibial sections from 9-wk-old control and hpg mice. Control mice were sham operated (Sham) or orchidectomized (ORX) at 3 wk of age and treated with vehicle (ORX) or T (ORX + T) from 3 wk of age. Hpg mice were treated with vehicle (hpg), T (hpg + T), or dihydrotestosterone (hpg + DHT) from 3 wk of age; bar = 500 µm. The structure of untreated hpg and ORX control mice appeared very similar. T and DHT treatment in both control (ORX + T) and hpg mice (hpg + T, hpg + DHT) prevented the low bone mass. B: trabecular bone mineral density (Tb.BMD), trabecular bone volume (BV/TV), and trabecular number (Tb.N) were significantly lower in ORX and hpg mice compared with Sham control animals; T and DHT treatment in both hpg and control ORX mice prevented the low bone mass associated with ORX or hpg mutation. C: osteoblast surface (ObS/BS) and osteoclast surface (OcS/BS) were both high in ORX and hpg mice. This elevation and mineral appositional rate (MAR) were all suppressed by T and DHT treatment in both ORX and hpg mice. Values are means ± SE of 6–12 mice per group. Effect of sex steroid depletion: +P < 0.05; ++P < 0.01; +++P < 0.001 in ORX or hpg vs. Sham. Effect of T or DHT treatment: *P < 0.05; ***P < 0.001 vs. untreated ORX or hpg.

 
Comparison of postnatal with postweaning androgen-deficiency. To determine whether the phenotype of 9-wk-old hpg mice is exclusively due to the lack of T during sexual maturation, these mice were compared with age-matched ORX non-hpg mice.

In trabecular bone, the low Tb.BMD, BV/TV, and Tb.N observed in 9-wk-old hpg mice were not significantly different from the structural defects observed in age-matched ORX non-hpg mice (Fig. 3, A and B). The low trabecular bone volume and density induced by congenital androgen deficiency or orchidectomy at weaning was associated with a high level of both ObS/BS and OcS/BS (Fig. 3C) as well as osteoid volume (OV/BV), osteoid surface (OS/BS), osteoid thickness (OTh), osteoblast number (NOb/BPm), and osteoclast number (NOc/BPm) (data not shown). We could detect no significant differences in trabecular structure (Fig. 3, A and B), bone remodeling parameters (Fig. 3C), or bone appositional growth (Fig. 4) between 9-wk-old hpg mice and age-matched ORX non-hpg mice, indicating that androgen-related effects on bone width and trabecular structure are determined by physiological events after 3 wk of age.


Figure 4
View larger version (54K):
[in this window]
[in a new window]
  		Fig. 4. Effects of hpg mutation and sex steroid depletion or treatment on accrual of bone width and cortical thickness at 9 wk of age. Femoral circumference (Fem.Ci) and cortical thickness (Fem.Ct.Th), and tibial cortical thickness (Tib.Ct.Th) and tibial periosteal appositional rate (Ps.MAR) were significantly lower in hpg mice and ORX non-hpg mice compared with Sham non-hpg mice of the same age. T and DHT treatment of both hpg mice and ORX non-hpg mice prevented this reduction in bone width. Values are means ± SE of 6–12 mice per group. Effect of sex steroid depletion: {dagger}P < 0.05; ++P < 0.01; +++P < 0.001 in ORX or hpg vs. Sham. Effect of T or DHT treatment: *P < 0.05; **P < 0.01; ***P < 0.001 vs. untreated ORX or hpg.

 
In contrast, bone length and markers of longitudinal bone growth differed between ORX non-hpg mice and age-matched hpg mice, suggesting a role for the perinatal T surge in longitudinal bone growth. At 9 wk of age, both femoral and tibial lengths were significantly lower in the hpg mice than in age-matched ORX non-hpg mice (Fig. 5). This difference may relate to a lower level of chondrocyte proliferation, early in bone growth, suggested by reduced growth plate width at 3 wk of age in the hpg mice (Fig. 1C) but resolved by 9 wk of age (Fig. 5C).


Figure 5
View larger version (63K):
[in this window]
[in a new window]
  		Fig. 5. Effects of sex steroid depletion or treatment on bone length and longitudinal bone growth at 9 wk of age. A: representative contact X-rays of each treatment group; bar = 500 µm. B: tibial and femoral lengths were significantly lower in hpg mice compared with Sham and ORX non-hpg mice of the same age. T and DHT treatment did not prevent this reduction in bone length in hpg mice, resulting in an even further reduction in tibial length. C: tibial growth plate width (G.P.Wi), but not proliferating zone width (Prol.Z.Wi), was significantly elevated in hpg mice compared with non-hpg mice. T and DHT treatment caused a reduction in both G.P.Wi and Prol.Z.Wi in non-hpg and hpg mice. Values are means ± SE of 6–12 mice per group. Effect of sex steroid depletion: +P < 0.05; +++P < 0.001 in ORX or hpg vs. Sham. Effect of T or DHT treatment: *P < 0.05 vs. untreated ORX sor hpg.

 
Androgen replacement in young ORX and hpg mice. A second method of determining whether perinatal or adult T is the more major regulator of bone structure was to restore normal levels of T in hpg mice and age-matched ORX non-hpg mice as described previously (13, 26, 34, 36, 37). Both T and DHT treatments were used to separate out any difference between effects of a pure (nonaromatizable) androgen (DHT) from potential effects of T via its aromatized metabolite estradiol.

Treatment with replacement doses of either T or DHT from 3 to 9 wk of age prevented the reduction in Tb.BMD, BV/TV, and Tb.N associated with maturation in hpg mice and, at the same dose, prevented the bone loss observed in ORX non-hpg mice. The effects of T and DHT on trabecular bone in hpg mice were not significantly different (Fig. 3, A and B) and were both associated with an inhibition of bone turnover. In non-hpg mice, T inhibited the orchidectomy-induced increase in all markers of bone turnover (Fig. 3C and data not shown). In hpg mice, T and DHT treatment significantly reduced bone formation, indicated by ObS/BS, OV/BV, OS/BS, OTh, NOb/BPm, mineral appositional rate (MAR), mineralizing surface (MS/BS), and bone formation rate (BFR/BS) to levels equivalent of that of 9-wk-old intact non-hpg mice (Fig. 3C and data not shown). Similarly, with bone resorption, both T and DHT reduced NOc/BPm and OcS/BS until they were not significantly different from intact non-hpg mice at the same age (Fig. 3C and data not shown). No significant differences between the effects of T or DHT were detected on trabecular density, structure, or histomorphometric markers of bone turnover.

In cortical bone, both T and DHT treatment from the age of 3 wk completely prevented the low FemDi and CtTh (in both femora and tibia) associated with orchidectomy or postnatal androgen deficiency by maintaining periosteal bone growth (Ps.MAR) at a level similar to that observed in intact non-hpg mice (Fig. 4).

Supporting a role for the perinatal androgen surge in longitudinal bone growth, neither T nor DHT treatment was able to prevent the short bone phenotype observed in hpg mice. Instead, T worsened this phenotype, and a further impairment of femoral longitudinal growth was observed in both hpg and control mice (Fig. 5, A and B). This shortened length was associated with both a significantly narrower growth plate (Fig. 5B) and a significant reduction in the proliferating compartment (Fig. 5C), confirming the known anti-proliferative effect of both T and DHT on chondrocyte proliferation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The hpg mouse, with complete postnatal androgen deficiency but preserved androgen sensitivity, provides a unique model for studying the importance of sex steroids from the time of birth. This naturally occurring mutant allows the study of sex steroid response without the complications of surgical or chemical gonadectomy and, in contrast to knockouts (KO) of ARs and ERs, these mice respond normally to natural ligands.

The perinatal androgen surge, which transiently results in mature male levels of T, has been shown to be required for normal function of the mature mouse (32) and rat (23) prostate and for longitudinal bone growth (14). However, its role in determining trabecular bone mass and bone turnover has not yet been established. A key difference between wild-type and hpg mice from birth until 3 wk of age is the low (castrate equivalent) circulating T levels in hpg male mice (33), whereas wild-type mice demonstrate a surge in testicular T secretion in the perinatal period reaching mature male levels in the non-hpg mice (4, 9). The shortened adult bone length and disordered bone turnover at 3 wk of age in hpg mice appear to be a result of the lack of the perinatal androgen surge.

The reduced growth plate width at 3 wk of age is consistent with the shortened bones observed in 9-wk-old hpg mice compared with intact non-hpg mice and is consistent with a role for the perinatal T surge in determining GH secretion in male mice (14). The shortened bone phenotype of the hpg mice was not observed in the absence of a functional AR (15), indicating that perinatal T surge may affect longitudinal growth by aromatization of T, as suggested by reduced bone length in adult male ER{alpha}beta–/– and aromatase KO mice (22, 31) or by the effects of estrogen or perinatal T on the GH-IGF-I axis (14). The shortened bones, reduced chondrocyte proliferation, and reduced GH secretion in the hpg mice are also consistent with reduced bone size in GH receptor (GHR)-deficient mice (29).

Adult hpg mice also demonstrated high bone turnover and low bone mass, which did not appear to relate to the lack of perinatal T. Although consistent with the bone phenotype of the adult male AR KO and aromatase KO mice (15, 22), this aspect of the phenotype is quite different from that observed in the absence of GHR, which demonstrated normal trabecular bone structure and turnover (29). Thus, although perinatal T imprinting may regulate longitudinal growth via GH-IGF-I, this growth factor mechanism does not appear to be the mediator of either the altered bone turnover at 3 wk or the high turnover osteopenia observed at 9 wk in the hpg mice. Rather, the trabecular structure of hpg mice at 9 wk of age is remarkably similar to that of age-matched ORX non-hpg mice. Notably, this phenotype is consistent with the effects of both early- (5, 7, 8) and late-onset (6, 38, 39) androgen deficiency on bone mass in men. The only significant difference in the bone phenotype between the hpg mice and ORX mice is the reduced longitudinal growth in the former, which we attribute to a mild impairment in chondrocyte proliferation detected before orchidectomy (at 3 wk). The similarities between the hpg and ORX non-hpg mice indicate that the trabecular and cortical bone phenotypes of the hpg mutation relate to the effects of the mutation on postpubertal GnRH-dependent pituitary gonadotropin and testicular androgen secretion associated with sexual maturation rather than the absence of the perinatal T surge. Furthermore, the phenotype is distinct from the increased bone mass and low bone turnover observed in the male ER{alpha}beta–/– (31), suggesting that it is not the absence of aromatized T (i.e., estradiol) that has caused the osteopenia associated with the hpg mutation. These results confirm that T itself, through effects mediated via the AR, plays a crucial role in determining the rate of juvenile bone turnover, which itself determines adult cortical and trabecular structure.

T treatment is able to prevent orchidectomy-induced bone loss in the absence of estrogen receptor alpha (which is the only estrogen responsive receptor in male bone) (30), implying that aromatization is not required for the bone protective effects of T on the trabecular bone. Here, we also observed that the high bone turnover and low bone mass observed in adult hpg and ORX mice are prevented equally by either T or DHT treatment. This result and the mechanism by which T and DHT prevent the bone loss via effects involving the AR are consistent with effects observed in ORX rats (42) and the ER{alpha}–/– (30, 31) and AR KO (15) mice. For example, in the absence of ER{alpha}, which is the only ER required for estrogen action in male bone, the effects of T and DHT on orchidectomy-induced bone loss and the associated high level of bone remodeling are identical (30, 31), confirming that T does not require aromatization for its effect on trabecular bone.

The only effect of congenital androgen deficiency remaining uncorrected by T or DHT was the reduction in bone growth, which was exaggerated by this treatment protocol. This is consistent with our suggestion that the growth defect in hpg mice is largely due to the absence of the perinatal T surge.

In conclusion, this study demonstrates that testosterone, via both its aromatizable and nonaromatizable metabolites, is able to prevent trabecular bone loss and partially reduce the impaired periosteal bone growth observed in growing congenitally androgen-deficient (hpg) mice, confirming a central role for testosterone in the regulation of skeletal growth and maturation. We also demonstrate that perinatal androgen imprinting via an androgen receptor-dependent mechanism is a key determinant of mature bone length in males.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
N. A. Sims is supported by an NHMRC Career Development Award. This work was supported by a grant from the University of Melbourne and Program Grant 345401 to N. A. Sims.


   ACKNOWLEDGMENTS
 
We thank Ingrid Kriechbaum for excellent assistance in preparation and staining of histological sections, Emma Walker for assistance with peripheral quantitative computer tomography, James de Winter for performing the biochemical serum assays, and the University of Melbourne Department of Medicine, Royal Melbourne Hospital, for use of the Stratec-SA+.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Sims, Dept. of Medicine at St. Vincent's Hospital, 41 Victoria Pde, Fitzroy VIC 3065, Australia (e-mail: nsims{at}unimelb.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Carani C, Qin K, Simoni M, Faustini-Fustini M, Serpente S, Boyd J, Korach KS, and Simpson ER. Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med 337: 91–95, 1997.[Free Full Text]
  2. Cattanach BM, Iddon CA, Charlton HM, Chiappa SA, and Fink G. Gonadotrophin-releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 269: 338–340, 1977.[CrossRef][Medline]
  3. Charlton HM, Halpin DMG, Iddon C, Rosie R, Levy G, McDowell IFW, Megson A, Morris JF, Bramwell A, Speight A, Ward BJ, Broadhead J, Davey-Smith G, and Fink G. The effects of daily administration of single and multiple injections of gonadotropin-releasing hormone on pituitary and gonadal function in the hypogonadal (hpg) mouse. Endocrinology 113: 535–544, 1983.[ISI][Medline]
  4. Crawford BA, Singh J, Simpson JM, and Handelsman DJ. Androgen regulation of circulating insulin-like growth factor-I during puberty in male hypogonadal mice. J Endocrinol 139: 57–65, 1993.[Abstract]
  5. De Rosa M, Paesano L, Nuzzo V, Zarrilli S, Del Puente A, Oriente P, and Lupoli G. Bone mineral density and bone markers in hypogonadotropic and hypergonadotropic hypogonadal men after prolonged testosterone treatment. J Endocrinol Invest 24: 246–252, 2001.[ISI][Medline]
  6. Diamond TH, Higano CS, Smith MR, Guise TA, and Singer FR. Osteoporosis in men with prostate carcinoma receiving androgen-deprivation therapy: recommendations for diagnosis and therapies. Cancer 100: 892–899, 2004.[CrossRef][ISI][Medline]
  7. Finkelstein JS, Klibanski A, Neer RM, Greenspan SL, Rosenthal DI, and Crowley WF Jr. Osteoporosis in men with idiopathic hypogonadotropic hypogonadism. Ann Intern Med 106: 354–361, 1987.[ISI][Medline]
  8. Guo CY, Jones TH, and Eastell R. Treatment of isolated hypogonadotropic hypogonadism effect on bone mineral density and bone turnover. J Clin Endocrinol Metab 82: 658–665, 1997.[Abstract/Free Full Text]
  9. Handelsman DJ, Spaliviero JA, Simpson JM, Allan CM, and Singh J. Spermatogenesis without gonadotropins: maintenance has a lower testosterone threshold than initiation. Endocrinology 140: 3938–3946, 1999.[Abstract/Free Full Text]
  10. Haywood M, Tymchenko N, Spaliviero J, Koch A, Jimenez M, Gromoll J, Simoni M, Nordhoff V, Handelsman DJ, and Allan CM. An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice. Mol Endocrinol 16: 2582–2591, 2002.[Abstract/Free Full Text]
  11. Horn D, Rivas P, McCluskey B, Mundy GR, and Gutierrez G. A new staining technique for undecalcified bone sections that enhances visualization of fluorochromes (Abstract). J Bone Miner Res 17: S416, 2002.
  12. Horowitz M, Wishart JM, O'Loughlin PD, Morris HA, Need AG, and Nordin BE. Osteoporosis and Klinefelter's syndrome. Clin Endocrinol (Oxf) 36: 113–118, 1992.[Medline]
  13. Jacobson JD and Ansari MA. Immunomodulatory actions of gonadal steroids may be mediated by gonadotropin-releasing hormone. Endocrinology 145: 330–336, 2004.[Abstract/Free Full Text]
  14. Jansson JO, Ekberg S, Isaksson O, Mode A, and Gustafsson JA. Imprinting of growth hormone secretion, body growth, and hepatic steroid metabolism by neonatal testosterone. Endocrinology 117: 1881–1889, 1985.[Abstract]
  15. Kawano H, Sato T, Yamada T, Matsumoto T, Sekine K, Watanabe T, Nakamura T, Fukuda T, Yoshimura K, Yoshizawa T, Aihara K, Yamamoto Y, Nakamichi Y, Metzger D, Chambon P, Nakamura K, Kawaguchi H, and Kato S. Suppressive function of androgen receptor in bone resorption. Proc Natl Acad Sci USA 100: 9416–9421, 2003.[Abstract/Free Full Text]
  16. Krieger DT, Perlow MJ, Gibson MJ, Davies TF, Zimmerman EA, Ferin M, and Charlton HM. Brain grafts reverse hypogonadism of gonadotropin releasing hormone deficiency. Nature 29: 468–471, 1982.
  17. Luisetto G, Mastrogiacomo I, Bonanni G, Pozzan G, Botteon S, Tizian L, and Galuppo P. Bone mass and mineral metabolism in Klinefelter's syndrome. Osteoporos Int 5: 455–461, 1995.[CrossRef][ISI][Medline]
  18. Marcus R, Leary D, Schneider DL, Shane E, Favus M, and Quigley CA. The contribution of testosterone to skeletal development and maintenance: lessons from the androgen insensitivity syndrome. J Clin Endocrinol Metab 85: 1032–1037, 2000.[Abstract/Free Full Text]
  19. Mason AJ, Hayflick JS, Zoeller RT, Young WS III, Phillips HS, Nikolics K, and Seeburg PH. A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 234: 1366–1371, 1986.[Abstract/Free Full Text]
  20. Mason AJ, Pitts SL, Nikolics K, Szonyi E, Wilcox JN, Seeburg PH, and Stewart TA. The hypogonadal mouse: reproductive functions restored by gene therapy. Science 234: 1372–1378, 1986.[Abstract/Free Full Text]
  21. O'Shaughnessy PJ, Baker P, Sohnius U, Haavisto AM, Charlton HM, and Huhtaniemi I. Fetal development of Leydig cell activity in the mouse is independent of pituitary gonadotroph function. Endocrinology 139: 1141–1146, 1998.[Abstract/Free Full Text]
  22. Oz OK, Zerwekh JE, Fisher C, Graves K, Nanu L, Millsaps R, and Simpson ER. Bone has a sexually dimorphic response to aromatase deficiency. J Bone Miner Res 15: 507–514, 2000.[CrossRef][ISI][Medline]
  23. Rajfer J and Coffey DS. Effects of neonatal steroids on male sex tissues. Invest Urol 17: 3–8, 1979.[ISI][Medline]
  24. Riggs BL, Khosla S, and Melton LJ III. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev 23: 279–302, 2002.[Abstract/Free Full Text]
  25. Schmidt C, Priemel M, Kohler T, Weusten A, Muller R, Amling M, and Eckstein F. Precision and accuracy of peripheral quantitative computed tomography (pQCT) in the mouse skeleton compared with histology and microcomputed tomography (microCT). J Bone Miner Res 18: 1486–1496, 2003.[CrossRef][ISI][Medline]
  26. Sciote JJ, Horton MJ, Zyman Y, and Pascoe G. Differential effects of diminished oestrogen and androgen levels on development of skeletal muscle fibres in hypogonadal mice. In: Acta Physiol Scand 172: 179–187, 2001.[CrossRef][ISI][Medline]
  27. Silverman AJ, Roberts JL, Dong KW, Miller GM, and Gibson MJ. Intrahypothalamic injection of a cell line secreting gonadotropin-releasing hormone results in cellular differentiation and reversal of hypogonadism in mutant mice. Proc Natl Acad Sci USA 89: 10668–10672, 1992.[Abstract/Free Full Text]
  28. Silverman AJ, Silverman RC, and Gibson MJ. Repair of reproductive defects by neural transplantation. Biol Reprod 42: 29–38, 1990.[Abstract]
  29. Sims NA, Clement-Lacroix P, Da Ponte F, Bouali Y, Binart N, Moriggl R, Goffin V, Coschigano K, Gaillard-Kelly M, Kopchick J, Baron R, and Kelly PA. Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5. J Clin Invest 106: 1095–1103, 2000.[ISI][Medline]
  30. Sims NA, Clement-Lacroix P, Minet D, Fraslon-Vanhulle C, Gaillard-Kelly M, Resche-Rigon M, and Baron R. A functional androgen receptor is not sufficient to allow estradiol to protect bone after gonadectomy in estradiol receptor-deficient mice. J Clin Invest 111: 1319–1327, 2003.[CrossRef][ISI][Medline]
  31. Sims NA, Dupont S, Krust A, Clement-Lacroix P, Minet D, Resche-Rigon M, Gaillard-Kelly M, and Baron R. Deletion of estrogen receptors reveals a regulatory role for estrogen receptors-beta in bone remodeling in females but not in males. Bone 30: 18–25, 2002.[Medline]
  32. Singh J and Handelsman DJ. Imprinting by neonatal sex steroids on the structure and function of the mature mouse prostate. Biol Reprod 61: 200–208, 1999.[Abstract/Free Full Text]
  33. Singh J, O'Neill C, and Handelsman DJ. Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology 136: 5311–5321, 1995.[Abstract]
  34. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, and Korach KS. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 331: 1056–1061, 1994.[Abstract/Free Full Text]
  35. Smithson G, Beamer WG, Shultz KL, Christianson SW, Shultz LD, and Kincade PW. Increased B lymphopoiesis in genetically sex steroid-deficient hypogonadal (hpg) mice. J Exp Med 180: 717–720, 1994.[Abstract/Free Full Text]
  36. Spaliviero JA, Jimenez M, Allan CM, and Handelsman DJ. Luteinizing hormone receptor-mediated effects on initiation of spermatogenesis in gonadotropin-deficient (hpg) mice are replicated by testosterone. Biol Reprod 70: 32–38, 2004.[Abstract/Free Full Text]
  37. Stepan JJ, Lachman M, Zverina J, Pacovsky V, and Baylink DJ. Castrated men exhibit bone loss: effect of calcitonin treatment on biochemical indices of bone remodeling. J Clin Endocrinol Metab 69: 523–527, 1989.[Abstract]
  38. Stoch SA, Parker RA, Chen L, Bubley G, Ko YJ, Vincelette A, and Greenspan SL. Bone loss in men with prostate cancer treated with gonadotropin-releasing hormone agonists. J Clin Endocrinol Metab 86: 2787–2791, 2001.[Abstract/Free Full Text]
  39. Vanderschueren D, Van Herck E, Suiker AM, Visser WJ, Schot LP, Chung K, Lucas RS, Einhorn TA, and Bouillon R. Bone and mineral metabolism in the androgen-resistant (testicular feminized) male rat. J Bone Miner Res 8: 801–809, 1993.[ISI][Medline]
  40. Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, and Ohlsson C. Androgens and bone. Endocr Rev 25: 389–425, 2004.[Abstract/Free Full Text]
  41. Wakley GK, Schutte HD Jr, Hannon KS, and Turner RT. Androgen treatment prevents loss of cancellous bone in the orchidectomized rat. J Bone Miner Res 6: 325–330, 1991.[ISI][Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
E. Tucker, K. O'Donnell, M. Fuchsberger, A. A. Hilton, D. Metcalf, K. Greig, N. A. Sims, J. M. Quinn, W. S. Alexander, D. J. Hilton, et al.
A Novel Mutation in the Nfkb2 Gene Generates an NF-{kappa}B2 "Super Repressor"
J. Immunol., December 1, 2007; 179(11): 7514 - 7522.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/3/E456    most recent
00311.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sims, N. A.
Right arrow Articles by Seibel, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sims, N. A.
Right arrow Articles by Seibel, M. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.