7α-Methyl-19-Nortestosterone (MENT) vs. Testosterone Implants for Hypogonadal Osteoporosis: a Preclinical Study in the Aged Male Orchidectomized Rat Model

Abstract

Purpose

Overt male hypogonadism induces not only osteoporosis but also unfavorable changes in body composition, which can be prevented by testosterone (T) replacement. In this preclinical study, the potential of synthetic androgen 7α-methyl-19-nortestosterone (MENT) as alternative treatment for male hypogonadism was evaluated in comparison with T.

Methods

11-month-old male rats were orchidectomized (orch) and left untreated for 2-months. Subsequently, the effects of 4-months MENT (12 µg/day) and T (72µg/day) treatment on bone, muscle and fat were analyzed by microcomputed tomography, dual-energy X-ray absorptiometry, dynamic bone histomorphometry and muscle fiber typing.

Results

At the onset of treatment orch rats were clearly hypogonadal. This was evidenced by significant reductions of androgen-sensitive organ weight, lean mass, cortical thickness and trabecular bone volume compared with sham-operated aged-matched controls (sham). MENT and T restored weight of androgen-sensitive organs to a similar extent, with a superior anabolic action of MENT on levator ani muscle. Both androgens not only fully rescued hypogonadal loss of lean mass, but also restored muscle fiber type composition and trabecular bone volume. Cortical bone loss was similarly prevented by MENT and T, but without full recovery to sham. Both androgens stimulated periosteal bone formation, but with a stronger effect of T. In contrast, MENT more strongly suppressed endocortical bone formation and bone turnover rate and reduced fat mass and serum leptin to a greater extent than T.

Conclusion

MENT and T are both effective replacement therapies to stimulate bone and muscle in hypogonadal rats, with stronger lipolytic action of MENT.

INTRODUCTION

The major clinical complication of male androgen deficiency or hypogonadism is osteoporosis [1, 2]. Similar to bone loss in postmenopausal women or female animal models of osteoporosis [3, 4], severe bone loss also occurs in men suffering from testicular dysfunction [5]. Osteoporosis in hypogonadal men is characterized by increased biochemical markers of bone turnover resulting in sustained loss of bone mass as well as increased fracture risk at several sites including spine and hip [6]. Moreover, hypogonadal men also lose muscle mass and gain fat mass [7, 8]. These changes in body composition may further increase their frailty and fracture risk. Similar to hypogonadal men, sex steroid deficiency – as induced by orchidectomy – in aged male rats increases bone turnover and induces subsequent trabecular and cortical bone loss [9, 10]. In these hypogonadal rats, muscle mass – assessed by lean body mass – is lowered as well, with a concomitant gain of fat mass [11]. Furthermore, similar to hypogonadal men [12, 13], conventional replacement with testosterone (T) prevents bone loss as well and body composition changes in these rodents [11]. As a result, the aged orchidectomized rat model has become a well-validated model for preclinical evaluation of different androgens in comparison with testosterone on bone and body composition.

The synthetic androgen 7α-methyl-19-nortestosterone (MENT) is a potential alternative for androgen replacement in male hypogonadism. MENT is a more potent androgen than T, with lower dose shown to be sufficient for restoration of the androgen sensitive organs in rats [14]. Moreover, MENT suppresses the gonadotrophins and, therefore, has the potential to be used in male contraception as well as male hypogonadism [15]. In contrast with T, however, MENT cannot be 5α-reduced due to steric hindrance of an additional methyl group [16]. Such resistance to 5α-reduction of MENT may avoid overstimulation of the prostate. MENT indeed prevents bone and muscle loss, when administered immediately following castration, at relatively low doses without overstimulation of prostate [17]. In analogy with most studies that evaluated the effect of T in orchidectomized male rats, MENT has only been evaluated immediately after orchidectomy and not at a later stage of hypogonadism. Therefore, it is unknown to what extent MENT as well as other androgens are able to reverse changes in bone as well as body composition in hypogonadal rats. Therefore we performed the current study, which addresses hormone replacement after the animals have been hypogonadal for 2 months. Moreover, the effect of MENT in hypogonadal rats has not yet been compared with equivalent doses of the conventional T replacement. Likewise, MENT effects on bone have only been evaluated in just one randomized clinical trial. In this short small single-center trial, however, MENT failed to maintain lumbar spine bone density in hypogonadal men, despite restoration of sexual function [18]. These observations indicate the need for further preclinical evaluation of the effects of this compound on non-reproductive tissues. In this context, the aim of our current study was to evaluate the ability of MENT to reverse changes of bone and body composition in rats, who have been hypogonadal for 2 months, in comparison with T replacement.

MATERIALS AND METHODS

Animals

Male 11-months-old aging Wistar rats were obtained from Bio Services (Uden, The Netherlands) and housed under standard conditions: a 12-h light/dark cycle, in an air-conditioned room, standard diet (1% calcium, 0.76% phosphate) and water ad libitum.

Experimental design

Fifty-six rats were randomly divided into seven groups (8 rats/group). A baseline group was immediately killed at the start of the experiment (Base – 11m). The other rats were either sham-operated (sham, 2 groups) or orchidectomized (orch, 4 groups) using pentobarbitone sodium anesthesia (Nembutal, 50 mg/kg body weight, CEVA Santé Animale, Brussels, Belgium) and buprenorfine hydrochloride as peroperative analgesic (Temgesic, 0.05 mg/kg body weight, Schering-Plough, Brussels, Belgium), and left untreated for a period of two months. Subsequently, one group of sham and orch rats was sacrificed at 13 months of age to assess the effects of castration during 2 months. At that time, treatment was initiated in the other groups for a period of 4 months. Orchidectomized rats received a pump with 7α-methyl-19-nortestosterone (MENT, 12 µg/day) or testosterone (T, 72 µg/day; Serva, Heidelberg, Germany), whereas sham-operated rats received no treatment. Doses of androgens were compared according to the traditional Hershberger assay [19]. The doses of MENT and T were selected on the basis of their normal physiological effect on the wet weight of the seminal vesicles (SV), ventral prostate (VP) and levator ani (LA) in a preliminary experiment in castrated aged male rats. Weighing of these reproductive organs was also used to evaluate biological andrological potency of both androgens at the end of the study since considerable variations in the serum concentrations of testosterone are not unusual in normal rats [20]. MENT and T were administered through subcutaneously implanted Alzet mini-osmotic pumps (model 20004, Charles River Laboratories, Brussels, Belgium). Pumps were replaced every 4 weeks. Sham-operated rats received an empty plastic tube instead of a pump following similar anesthesia and surgical procedures. MENT was kindly provided by the Population Council (New York, USA).

Body weight and food intake were determined weekly during the experimental period. At sacrifice, rats were anesthetized with Nembutal and sacrificed by exsanguination via the abdominal aortic artery. Serum was collected and stored at −20°C until assay. All rats were injected intraperitoneally with the fluorochrome calcein at a 10-day interval and were killed 1 day after the second injection. The left and right femora were dissected for microCT (µCT) measurement and dynamic histomorphometry. Also the wet weights of the SV, LA muscle and VP were determined at death. In addition, both the soleus muscle and tibialis anterior were dissected and used for muscle fiber typing. All experimental procedures were conducted after obtaining formal approval from the ethical committee of the Catholic University Leuven.

Dynamic bone histomorphometry

One femur was cleaned from surrounding tissue, immersed in Burckhardt’s fixative (24 h, 4 °C), kept in 100% ethanol and embedded in methylmethacrylate. Cross-sections of the undecalcified femur perpendicularly to the long axis were prepared at 200 µm thickness in the mid-diaphyseal region using the contact-point precision band saw (Exakt, Norderstedt, Germany). Then, sections were ground to a final thickness of 25 µm using a grinding system (Exakt). Sections were left unstained and subjected to dynamic histomorphometry. Three sections in the middiaphyseal region were measured by fluorescence microscopy, and the bone formation rate per bone perimeter (BFR/B.Pm., µm²/µm/day) was assessed at both the endocortical and periosteal bone surfaces. The BFR was obtained by the product of mineral apposition rate (MAR, µm/day) and mineralizing perimeter per bone perimeter (Min.Pm./B.Pm., %). The mineralizing perimeter was calculated as followed: Min.Pm. = [dL + (sL/2)]/B.Pm., where dL represents the length of the double labels and sL is the length of single labels along the entire endocortical or periosteal bone surfaces. The MAR (µm/day) was calculated as the mean width of double labels, divided by interlabel time (10 days). All measurements were performed with a Kontron Image Analyzing computer (KS400 3.00, Kontron Bildanalyze, Munich, Germany) and a Zeiss microscope with drawing attachment. Specific software was developed in collaboration with the manufacturer. Histomorphometric parameters are reported according to the recommended American Society for Bone and Mineral Research nomenclature [21].

Microcomputed tomography (µCT)

µCT analysis was done ex vivo on the femur by using a Skyscan 1172 scanner (Skyscan, Kontich, Belgium). Each specimen was imaged using an X-ray tube voltage of 70 kV and current of 140 µA with a 0.5-mm aluminium filter. The scanning angular rotation was 180° and an angular increment of 0.8°, frame averaging of 2 and a voxel size of 10 µm were used. The image slices were reconstructed using the cone-beam reconstruction software (NRecon, v.1.4.4.0, Skyscan, Kontich, Belgium). Reconstructed datasets were segmented into binary images by using simple global thresholding methods. Trabecular bone of the distal femur was selected for analysis by drawing contours with the “CT-analyser” software (Skyscan, Kontich, Belgium), commencing at a distance of 2 mm from the growth plate and extending a further longitudinal distance of 3 mm in the proximal direction. The number of slices was 300, each with the same thickness as the voxel size, 10 µm. Cortical bone was analyzed starting at a distance of 11 mm from the growth plate and extending a further longitudinal distance of 1 mm in the proximal direction (100 slices, 10 µm pixel size). Micromorphological information was obtained from image stacks, allowing 3D parameters of bone microstructure to be calculated, including trabecular bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb. Sep), total cross-sectional area (T. CSA), cortical area (Ct.Ar), medulary area (Med. Ar) and cortical thickness (Ct. Th).

Whole-body dual-energy X-ray absorptiometry (DEXA)

Before sacrifice, rats were anesthetized and lean body mass and fat mass were measured in vivo with a QDR 4500A fan beam X-ray bone densitometer (Hologic, Inc., Waltham, MA, USA) using a specific software program (version v8.19a) from the manufacturer for rats in the body weight range of 200–750g.

Assays

Serum osteocalcin and C-termininal telopeptides of type I collagen (CTX-I) were measured by an in-house radioimmunoassay (RIA) (CV intra-assay: <5.9%; CV inter-assay: <5.2%) [22] and by a RatLaps ELISA kit (Immunodiagnostic Systems Nordic A/S, Herlev, Denmark) (CV intra-assay: <10%; CV inter-assay: <15%), respectively. After acid-ethanol extraction, serum IGF-I concentrations were analyzed by an in-house RIA (CV intra-assay: <7.6%; CV inter-assay: <10.8%) [23] in the presence of an excess of IGF-II (25ng/tube). LH and leptin were determined in serum by the Rat LH ELISA Kit (Biovendor Gmbh, Heidelberg, Germany) (CV intra-assay: <5%; CV inter-assay: <5%) and the Rat Leptin ELISA Kit (Crystal Chem Inc, Downers Grove, IL, USA) (CV intra-assay: ≤10%; CV inter-assay: ≤10%), respectively.

Serum levels of T and MENT were measured by specific extraction type radioimmunoassay as described earlier [24, 25]. Briefly, serum samples (0.1 – 0.2 ml) were extracted with ethyl ether and the extracts transferred to new test tubes and dried under a stream of nitrogen gas. The residues were dissolved in assay buffer. A 7 – 8 point standard curve ranging from 1 to 500 picogram/tube was prepared. Antiserum diluted with assay buffer was added so as to obtain 40 – 50 % binding of the tracer in the absence of cold T or MENT. The 125-I – iodohistamine conjugated MENT and 3-H Testosterone tracers diluted with assay buffer were used in the assays. The tubes were incubated at 4 C overnight and bound and free tracer were separated by using dextran – charcoal method. Intra and interassay variations were less than 10%.

Fiber typing

Transverse sections (4 µm) were cut from the mid-belly area of soleus muscles with a Leica CM1850 cryostat (Leica, Nussloch, Germany) at −20 °C and mounted on glass slides, each slide containing duplicate serial sections. After a 10-min fixation in 4% paraformaldehyde in PBS cryosections were treated with 10 mM NH₄Cl for 30 min after which slides were pre-hybridized in 1% BSA in PBS for 30 min. Thereafter, sections were incubated for 1 h primary monoclonal antibodies against human myosin heavy chain I (A4.840 supernatant, Developmental Studies Hybridoma Bank, Iowa, USA) and IIa (N2.261 supernatant, Developmental Studies Hybridoma Bank), followed by adding (1 h) the appropriate conjugated antibodies (type I: FITC anti-mouse IgM Southern Biotechnology Associates, Birmingham, AL, USA; type IIa: Alexa Fluor-350 anti-mouse IgG1 Molecular Probes, Leiden, The Netherlands). Cover slips were mounted with Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA, USA). Slides were examined using a Nikon E1000 fluorescence microscope (Nikon, Boerhavedorp, Germany) equipped with a digital camera. Epifluorescence signal was recorded utilizing a FITC and a DAPI filter for type I and IIa muscle fibers respectively. Three to five digital images were taken from each section and the fibers were identified as type I fibers (green staining), type IIa fibers (blue staining) and type IIb/x fibers (no staining). Fiber type distribution as percentage number and average fiber size per type were calculated for each of the fiber categories. However, due to the small number of type IIb/x fibers in the soleus muscle, results for type IIb/x fibers are not included in this paper.

Statistical analysis

Statistical analysis of the data was performed using NCSS software (NCSS, Kaysville, UT, USA). Differences between treatment groups were assessed following one-way analysis of variance followed by Fisher’s least significant difference multiple comparison test. Data are represented as means ± SE, and P < 0.05 was accepted as significant.

RESULTS

Rats were hypogonadal at onset of treatment

Androgen deficiency as induced by orchidectomy has a significant impact on androgen sensitive organs as well as bone and body composition. At the onset of treatment, the weight of the seminal vesicles (SV), ventral prostate (VP) and levator ani (LA) were all drastically reduced in Orch versus Sham rats (Table 1). Moreover, lean body mass was significantly decreased (−8%) together with a non-significant increase of fat mass (+17%) (Table 1). Cortical thickness was also reduced 2 months following castration (−5%) as a result of an enlargement of the medullary cavity (+22%) (Table 2). Finally, severe loss of trabecular bone volume (−58%) and number (−60%) completed the picture of androgen deficiency in these 13-month-old Orch rats.

Table 1.

Weight of androgen-sensitive tissues, body composition and food intake.

	Baseline			Experimental Groups
	Base – 11m	Sham – 13m	Orch – 13m	Sham – 17m	Orch – 17m	Orch + MENT	Orch + T
Seminal vesicles (g)	1.87 ± 0.08	1.84 ± 0.10	0.18 ± 0.01a	1.72 ± 0.16	0.15 ± 0.01b	2.07 ± 0.09b,c	1.81 ± 0.10c
Ventral prostate (g)	0.79 ± 0.05	0.74 ± 0.05	0.08 ± 0.01a	0.72 ± 0.06	0.06 ± 0.01b	0.82 ± 0.04c	0.81 ± 0.05c
Levator ani (g)	1.10 ± 0.08	1.29 ± 0.04	0.63 ± 0.02a	1.24 ± 0.04	0.46 ± 0.02b	1.79±0.07b,c,d	1.50 ± 0.05b,c
Body weight (g)	589 ± 13	652 ± 8	629 ± 10	669 ± 20	621 ± 23	607 ± 9b	646 ± 9
Fat mass (g)	79 ± 7	114 ± 8	133 ± 11	109 ± 13	149 ± 19b	72 ± 4b,c	106 ± 9c
Fat percentage (%)	13.3 ± 1.0	17.5 ± 1.3	21.2 ± 1.6	16.1 ± 1.7	23.5 ± 2.2b	11.9 ± 0.8c	16.4 ± 1.4c
Lean mass (g)	502 ± 12	519 ± 14	478 ± 12a	542 ± 16	455 ± 10b	517 ± 12c	520 ± 11c
Lean percentage (%)	85.4 ± 2.0	79.6 ± 1.4	75.9 ± 1.6a	81.2 ± 1.8	73.6 ± 2.2b	85.0 ± 0.8c	80.6 ± 1.4c
Food intake (g/day)	NA	NA	NA	31.0 ± 0.5	28.1 ± 0.4b	29.5 ± 0.4b,c,d	33.3 ± 0.5b,c

Data are reported as mean ± SE (n = 8 rats/group). One baseline group was sacrificed at the start of the experiment (Base – 11m). The other baseline groups were orchidectomized (Orch) or sham-operated (Sham) at 11 months of age, left untreated and sacrificed at 13 months (13m) of age. The experimental groups were also orchidectomized or sham-operated at 11 months of age, left untreated for 2 months, and subsequently received 4-months treatment with either vehicle (12µg/day), MENT (12µg/day) or with T (72µg/day) before sacrifice at 17 months (17m).

^ap < 0.05 vs. Sham – 13m;

^bp < 0.05 vs. Sham – 17m;

^cp < 0.05 vs. Orch – 17m;

^dp < 0.05 vs. Orch + T

Table 2.

Cortical and trabecular bone microstructure of the femur.

	Baseline			Experimental Groups
	Base – 11m	Sham – 13m	Orch – 13m	Sham – 17m	Orch – 17m	Orch + MENT	Orch + T
T. CSA (mm2)	13.9 ± 0.5	14.9 ± 0.3	16.0 ± 0.6	16.7 ± 0.5	15.2 ± 0.4b	15.8 ± 0.4	16.4 ± 0.6
Ct. Ar. (mm2)	8.9 ± 0.3	9.8 ± 0.3	9.8 ± 0.3	11.2 ± 0.3	8.6 ± 0.2b	10.1 ± 0.2b,c	10.1 ± 0.3b,c
Med. Ar. (mm)	5.03 ± 0.33	5.16 ± 0.17	6.28 ± 0.43a	5.52 ± 0.49	6.57 ± 0.31b	5.63 ± 0.29c	6.33 ± 0.62
Ct. Th. (µm)	744 ± 16	787 ± 13	745 ± 9a	828 ± 20	680 ± 11b	775 ± 12b,c	754 ± 24b,c
Trab. BV/TV (%)	14.4 ± 1.2	15.7 ± 2.2	6.6 ± 1.1a	11.1 ± 1.2	4.5 ± 0.8b	9.8 ± 0.7c	11.2 ± 0.9c
Trab. N. (1/mm)	1.81 ± 0.17	1.95 ± 0.31	0.78 ± 0.12a	1.24 ± 0.15	0.46 ± 0.08b	1.05 ± 0.10c	1.21 ± 0.11c
Trab. Th. (µm)	79.7 ± 1.6	82.5 ± 2.3	84.1 ± 2.6	90.9 ± 2.7	98.4 ± 3.0b	93.9 ± 2.3	94.2 ± 3.3
Trab. Sep. (µm)	365 ± 22	340 ± 34	526 ± 40a	423 ± 33a	644 ± 37b,c	443 ± 25b,c	437 ± 21b,c

Data are reported as mean ± SE (n = 8 rats/group). One baseline group was sacrificed at the start of the experiment (Base – 11m). The other baseline groups were orchidectomized (Orch) or sham-operated (Sham) at 11 months of age, left untreated and sacrificed at 13 months (13m) of age. The experimental groups were also orchidectomized or sham-operated at 11 months of age, left untreated for 2 months, and subsequently received 4-months treatment with either vehicle (12µg/day), MENT (12µg/day) or with T (72µg/day) before sacrifice at 17 months (17m).

^ap < 0.05 vs. Sham – 13m;

^bp < 0.05 vs. Sham – 17m;

^cp < 0.05 vs. Orch – 17m

MENT and T action on androgen sensitive organs in hypogonadal rats

Both androgens restored the wet weight of the SV, VP and LA. MENT even increased the weight of the SV and LA above Sham level (Table 1). Overall, MENT and T action were similar except for a superior effect of MENT on LA compared with T-treated rats (Table 1). MENT also suppressed LH more than T (Table 3). The T levels were lower in Orch groups with or without androgen replacement (Fig. 1). T concentrations remained significantly lower than sham and also in orch rats receiving T. However the concentration of T at the end of T treatment (0.37 ng/ml) was comparable with the concentration of MENT at the end of MENT treatment (0.27 ng/ml).

Table 3.

Serum parameters.

	Baseline			Experimental Groups
	Base – 11m	Sham – 13m	Orch – 13m	Sham – 17m	Orch – 17m	Orch + MENT	Orch + Testo
Osteocalcin (ng/ml)	72.8 ± 3.0	75.3 ± 3.5	93.5 ± 3.9a	71.8 ± 4.8	71.1 ± 4.5b	34.2 ± 2.5 b,c,d	57.1 ± 4.7b,c
CTX-I (ng/ml)	25.5 ± 1.6	24.7 ± 1.7	26.7 ± 2.8	25.8 ± 3.7	19.9 ± 1.4	15.5 ± 1.0b,d	28.8 ± 4.6c
LH (ng/ml)	1.20 ± 0.24	1.34 ± 0.23	2.45 ± 0.15a	0.48 ± 0.05	2.44 ± 0.21b	0.25 ± 0.18c,d	1.22 ± 0.16b,c
Leptin (ng/ml)	4.21 ± 0.73	6.74 ± 1.59	8.83 ± 1.05	7.51 ± 0.79	8.89 ± 1.52b	4.41 ± 0.49b,c,d	7.80 ± 0.53
IGF-I (ng/ml)	1073 ± 35	1107 ± 33	964 ± 36a	921 ± 27	756 ± 22b	823 ± 24b,d	978 ± 42c

Data are reported as mean ± SE (n = 8 rats/group). One baseline group was sacrificed at the start of the experiment (Base – 11m). The other baseline groups were orchidectomized (Orch) or sham-operated (Sham) at 11 months of age, left untreated and sacrificed at 13 months (13m) of age. The experimental groups were also orchidectomized or sham-operated at 11 months of age, left untreated for 2 months, and subsequently received 4-months treatment with either vehicle (12µg/day), MENT (12µg/day) or with T (72µg/day) before sacrifice at 17 months (17m).

^ap < 0.05 vs. Sham – 13m;

^bp < 0.05 vs. Sham – 17m;

^cp < 0.05 vs. Orch – 17m;

^dp < 0.05 vs. Orch + T

Figure 1.

Testosterone concentrations at the end of experiments in sham-operated (Sham) and orchidectomized rats treated with vehicle (Orch) (12µl/day) or testosterone (Orch + Testo) (72µg/day). MENT concentration in orchidectomized rats treated with MENT (Orch + MENT) (12µg/day). ^a p < 0.05 vs. Sham, ^b p < 0.05

MENT and T effects on food intake, body weight and fat mass in hypogonadal rats

Orchidectomy lowered body weight and food intake, but increased fat mass at 17 months of age (Table 1). Only T, not MENT rescued body weight and food consumption (Table 1). The changes in food intake were paralleled with similar changes in serum IGF-I (Table 3). Although both androgens normalized the orchidectomy-induced gain of fat mass, only MENT decreased fat tissue below Sham level with a concomitant significant reduction of serum leptin levels (Table 1, 3).

MENT and T effects on muscle in hypogonadal rats

Both MENT and T fully prevented the Orch-induced decrease of lean body mass (Table 1). In parallel, androgen deficiency as induced by orchidectomy lowered the proportional number of type I fibers in the soleus muscle but not in the tibialis anterior muscle and was associated with a concomitant increase in type IIa fibers in this muscle (Fig. 2), while the mean cross-sectional area of the soleus and the tibialis anterior muscle was not affected (data not shown). The proportion of type I and IIa muscle fibers in the soleus muscle was equally restored to Sham level following MENT and T treatment (Fig. 2). The number of type IIb/x fibers in soleus was negligible and too small to allow a valid quantitative analysis.

Figure 2.

Percentage of (A) Type I fibers and (B) Type IIa fibers in the soleus muscle of sham-operated (Sham) and orchidectomized rats treated with vehicle (Orch) (12µg/day), MENT (Orch + MENT) (12µg/day) or testosterone (Orch + T) (72µg/day). ^a p < 0.05 vs. Sham.

MENT and T effects on bone in hypogonadal rats

Both androgens completely restored the orchidectomy-induced deterioration of the trabecular bone microstructure. In fact, parameters of trabecular bone microstructure (trabecular bone volume, number and thickness) in MENT- and T-treated animals were not different from Sham rats at 17 months of age (Table 2, Fig. 4).

Figure 4.

(A) Representative 3D models of trabecular microstructure, (B) trabecular bone volume, (C) trabecular bone number and (D) trabecular thickness of sham-operated (Sham) and orchidectomized rats treated with vehicle (Orch) (12µl/day), MENT (Orch + MENT) (12µg/day) or testosterone (Orch + Testo) (72µg/day). ^a p < 0.05 vs. Sham.

As expected androgen deficiency in Orch rats further reduced cortical thickness at 17 months of age (−18% vs. Sham) (Table 2). This resulted from a significant increase of the medullary area (+19%) and a reduction of the cortical bone area (−23%) (Table 2). Both MENT and T equally prevented these cortical changes, however, without full recovery to Sham level (Table 2). However, differences between MENT and T with respect to their cortical bone effects were observed. Only MENT fully prevented the enlargement of the medullary area (Table 2), and clearly suppressed endocortical bone formation (Fig. 3), as well as biochemical markers of bone turnover such as serum CTX-I and osteocalcin (Table 3). T on the other hand increased periosteal bone formation more than MENT, together with a normalization of endocortical bone formation (Fig. 3). In the end the effects of both androgens on total cross-sectional area, cortical bone area and thickness were comparable (Table 2).

Figure 3.

Dynamic histomorphometry. (A) Representative cross-sections of the rat femur; the lower panels represent magnification of white rectangle; (B) Periosteal and (C) endocortical bone formation rate of sham-operated (Sham) and orchidectomized rats treated with vehicle (Orch) (12µg/day), MENT (Orch + MENT) (12µg/day) or testosterone (Orch + Testo) (72µg/day). ^a p < 0.05 vs. Sham, ^b p < 0.05 vs. Orch, ^c p < 0.05 vs. Orch + T.

DISCUSSION

This study is the first to provide evidence on the capacity of MENT to reverse osteoporosis in a preclinical animal model of male hypogonadism. The effects of MENT were also compared with T replacement, the conventional treatment for male hypogonadism. MENT is approximately 10-fold more potent than testosterone at the androgen receptor [26]. In line with previous studies [14, 17], MENT proved also a more potent androgen than T in vivo, with the weight of the ventral prostate maintained with a much lower dose of MENT (12 versus 72 µg/day for T) in aged male orchidectomized rats. In contrast with MENT, however, T action is amplified into the more potent androgen DHT by 5α-reductase in the prostate [27]. Nevertheless, the greater potency of MENT is also observed in other androgen-sensitive organs such as the SV and even more in the LA muscle. In this context and in line with previous findings [17], MENT fully restored the integrity of the reproductive organs as well as levator ani in hypogonadal animals without overstimulation of the prostate or amplification by 5α-reductase in the reproductive tract. In accordance with earlier reports [15, 28], MENT also suppressed LH more strongly than T. Such strong suppression of LH by MENT may be of clinical interest in its potential use as a male contraceptive. The effect on LH also reflects its greater androgenic potency since androgen action on LH in rats is mainly androgen receptor dependent. In fact, only DHT but not 17β-estradiol has been shown to lower LH levels in this animal model [29].

The most important question addressed in this preclinical study in hypogonadal rats was whether enhanced androgenic potency of MENT – in comparison with T – would be translated into favorable effects on bone and body composition. The aged orchidectomized rat model is a well-characterized preclinical model for overt male hypogonadism as induced by chemical or surgical castration in the elderly [5, 30]. Similar to overt hypogonadism in men, castration in aged male rats results in loss of cortical and trabecular bone mass as well as lean body mass and gain of fat [31]. In addition, androgens – when administered immediately after orchidectomy – prevent these unfavorable changes in this model [17]. However, what is unknown is if, and to what extent, androgens are also able to restore hypogonadal osteoporosis as well as changes in body composition at a later stage of hypogonadism, the focus of the current preclinical study. Interestingly, in our hypogonadal rat model, MENT reduced fat mass as well as corresponding serum leptin levels not only to a greater extent than T but also below age-matched sham-operated rats. These findings confirm similar dose-dependent reductions in fat mass and leptin levels following administration of MENT immediately after castration in rats [17], and are consistent with a comparable effect of MENT on fat mass in hypogonadal men [18]. MENT administration also appears to translate in a stronger lipolytic effect in hygonadal rats in comparison with T replacement. Another feature of hypogonadism is muscle wasting [7, 8]. In this context, MENT is likely more anabolic than T, as suggested by the fact that it stimulates the levator ani more than T. In line with its well documented effects on lean body mass when administered immediately after orchidectomy [17], MENT – similar to T [32] – is also able to restore lean body mass in overt hypogonadal rats. However, in contrast with its action on the levator ani, the effect of MENT on lean body mass is not superior to T. As suggested by other preclinical studies [33, 34], androgen action on levator ani may not be representative of changes in skeletal muscle tissue. Moreover, mice with muscle-selective disruption of androgen receptor neither have reductions in skeletal muscle weight nor changes in muscle fiber size [33]. Hence, relevant muscle-specific androgen actions may not impact on muscle size or weight, but on other functional aspects such as muscle fiber type composition [35]. In fact, both MENT and T equally prevent the orchidectomy-induced shift of type I slow oxidative towards type IIa fast oxidative muscle fibers in the soleus muscle. Such a shift of fiber type composition in slow twitch muscles is in line with some but not all earlier observations [36–38], and may also reflect relevant changes in muscle performance.

Similar to hypogonadal osteoporosis in men, the aged rat model used in this study is also characterized by rapid loss of cortical and trabecular bone mass. MENT has been shown to prevent such bone loss when administered immediately after orchidectomy [17]. Yet, it is not known to what extent MENT is able to restore bone deficits at a later stage, and if such effect would be superior to T. In this study, both androgens restored trabecular bone mass and prevented further loss of cortical bone. The overall effects of MENT and T on cortical and trabecular bone mass were again not significantly different. Interestingly, both androgens also stimulated periosteal bone formation, although T had a more pronounced effect than MENT. To our knowledge, such an activation of periosteal bone formation by androgens has been demonstrated mainly in younger growing animals [39–41]. The reduction of periosteal bone formation as well as the stimulatory action of T – as demonstrated in this preclinical study and one earlier study [31] – may be of clinical interest, although similar changes are difficult to assess in humans. While the importance of periosteal apposition in establishing structural strength during growth and its maintaining during aging has been well established, the periosteal surface has often been neglected in osteoporosis research [42], mainly because of difficulties in assessing therapeutic effects of drugs or hormones on this envelope. In contrast with the actions on the periosteal surface, both androgens also suppressed endosteal bone formation and net resorption, although MENT did so more strongly than T. It would seem, therefore, that both androgens have differential actions at the different envelopes in hypogonadal animals, which may be reflected by different responses in bone markers (such as a decrease of CTX-I which was observed in MENT but not T-treated rats).

The small but significant differences in endpoints between MENT and T observed in this study are probably not only related to differences in biological androgenic potency. Since both MENT and T are aromatizable androgens [27], some of the favorable androgen actions may be mediated indirectly, by estrogens following aromatization. The superior effect of estrogens compared to non-aromatisable androgens on male cortical bone has been previously established in this model [29]. In accordance with the well established role of estrogen and estrogen receptor-α on male fat mass [43], the lipolytic effect of estradiol has also been shown to be superior to DHT in aged hypogonadal rats [29]. Therefore, the capacity of MENT to be aromatized into estrogens may be higher than T and this may have contributed to the small but significant differences between both androgens. Although the relevance of aromatization of MENT was not assessed in this study, observations consistent with estrogen mediated action of MENT have been made in mice with complete disruption of androgen receptor, with small but significant increases in trabecular bone mass following administration of MENT (unpublished observations). Thusfar only one clinical study has evaluated whether MENT has bone sparing effects in hypogonadal men [18].

In contrast with the findings of this and one other preclinical study, MENT failed however to fully maintain lumbar bone density in these hypogonadal men. However the number of hypogonadal men included in this study was small and the duration of the study very short. Also the effects of MENT were not compared with testosterone replacement.

To the best of our knowledge our study is the first one to show curative actions of both MENT and T on relevant muscle and bone endpoints in a well established model for hypogonadal osteoporosis. However our study has some limitations. There is some discrepancy between the effect of MENT and T on respective serum concentrations en reproductive organs. However reproductive hormones were measured at the end of the experiment, at the very end of pumping duration and therefore are lower than expected. Also the effect of MENT and T on bone formation was only evaluated at periosteal and endosteal surfaces but not on cancellous surfaces.

In conclusion, in this preclinical animal model of male hypogonadism, the more potent androgen MENT was equally effective as T in reversing unfavorable changes in bone and muscle tissue, even when administered in a later stage. Its lipolytic effect was found to be superior to T, with similar effects on the reproductive organs.