INTRODUCTION

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IN ADDITION TO THEIR VIRILIZING effects in the reproductive tracts of males, androgens have anabolic effects in some skeletal muscles, but the mechanism of androgen control of muscle size is poorly understood (5, 23). In adult animals, castration produces atrophy of the rat levator ani and bulbocavernosus muscles, frog flexor carpi radialis and flexor carpi centralis muscles, and several guinea pig muscles, such as the sternomastoid, spinotrapezius, and latissimus dorsi muscles, whereas the readministration of androgenic hormones to castrated animals results in hypertrophy of these muscles (5, 11, 16, 20). Electrical stimulation of rat gastrocnemius muscle results in an increase in the number of androgen receptors (ARs), whereas the administration of an androgen antagonist suppresses the hypertrophy seen in exercised gastrocnemius muscle (7, 8). As recently demonstrated in men, the administration of supraphysiological amounts of androgenic steroids (600 mg/wk testosterone enanthate) for 10 wk resulted in an increase in skeletal muscle mass and strength (3). In addition to animal data (5, 11, 16, 20), it is likely that the anabolic effects of androgens are directed by the same receptor that mediates androgen action in the urogenital tract. Furthermore, Kreig et al. (12) have also reported that the number of ARs varies among skeletal muscles, which may explain the regional differences in the physiological response to androgen administration.

The purpose of this study, therefore, was to assess the effects of castration and androgen replacement on AR levels in the rat bulbocavernosus, levator ani, and plantaris muscles. These findings have been reported in abstract form (1).

	METHODS

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Animals and hormonal manipulations. Eighteen 16-wk-old male Holtzman/Sprague-Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). The animals were housed in the animal facilities of the University of Texas Southwestern Medical Center under a 12:12-h light-dark cycle with free access to standard laboratory chow and water. Animal care and use protocols were approved by the institutional review board of the University of Texas Southwestern Medical Center and were in accordance with the guidelines of the National Institutes of Health.

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Tissue extraction. Animals were killed with an overdose of ether. The prostate gland and three skeletal muscles (levator ani, bulbocavernosus, and plantaris) were immediately removed, weighed, and minced. Tissue fragments were suspended (0.025-0.1 g/0.9 ml) in buffer containing 110 mM SDS, 100 mM dithiothreitol, 80 mM Tris (pH 6.9), 0.002% bromphenol blue, and 10% glycerol and were homogenized on ice in an all-glass Dounce homogenizer (10 strokes of a loose pestle, followed by 20 strokes of a tight pestle). The homogenate was then sonicated (at setting 7; model W350, Branson) for 10 s, boiled for 5 min, and centrifuged at 200,000 g for 30 min. Aliquots of the supernatant were used immediately for Western blotting or were stored at 80°C and thawed only once.

Immunoblot assay. Extracts were boiled for 3 min immediately before loading on the gel. Aliquots (15-500 µg protein) of each sample were applied to 1.5-mm × 20-cm × 20-cm 8% polyacrylamide gels containing 3.5 mM SDS and 0.2% N,N'-methylene-bis-acrylamide (2).

Statistics. Descriptive statistics include means ± SE. A one-way ANOVA was used to determine significance. If an F ratio was found to be significant, all pairwise multiple comparisons were made via the Student-Newman-Keuls method. P < 0.05 was selected to indicate statistical significance.

	RESULTS

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Body weight. There were no significant differences in body weight among groups after treatment [P > 0.05; N: 489.0 ± 10.0 (SE) g; C: 456.7 ± 13.4 g; C+DHT: 486.0 ± 13.2 g].

Muscle weights. Castration for 2 wk caused a significant decrease in the weight of the levator ani and bulbocavernosus muscles (and in the weight of the ventral prostate) but had no effect on the weight of the plantaris muscle (Table 1). Similarly, treatment of the C+DHT group for 1 wk with DHT resulted in growth of the bulbocavernosus and levator ani muscles back to the normal range but had no effect on the weight of the plantaris muscle (Table 1).

AR levels. In the N male animals, AR levels were approximately threefold higher in the bulbocavernosus and levator ani muscles than in the plantaris muscle (Fig. 1 and Table 2). Castration caused a significant (P < 0.05) decrease in the AR level in the bulbocavernosus muscles, an average 51% decline (P = 0.09) in the levator ani muscle, and a nonsignificant (P > 0.05) decrease in the plantaris muscle. However, the administration of DHT to the C+DHT group caused a significant rise in AR levels (compared with those in the C group) in both the levator ani and bulbocavernosus muscles and a nonsignificant (P > 0.05) increase in the AR level in the plantaris muscle (Table 2).

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Western blot of the levator ani (A), bulbocavernosus (B), and plantaris muscles. Protein (104 µg) was loaded into each lane.

	DISCUSSION

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The response of skeletal muscles to androgen deprivation and/or administration is muscle and species specific. For example, the flexor carpi radialis muscle of the male frog Xenopus laevis atrophies in response to castration and returns to normal size within 7 days of testosterone administration (6), whereas these procedures have no discernible effect in the sartorius muscle of the same species. Similarly, in the guinea pig the temporalis muscle is highly sensitive to androgens (14). The temporalis muscle of the female guinea pig contains type IIa fibers predominantly, whereas in the male, testosterone causes hypertrophy of the muscle and an increase in type IIb fibers (9, 14). It is believed that the pectoral and shoulder girdle muscles are the most androgen-dependent muscles in humans (23).

In the rat, the bulbocavernosus and levator ani muscles, which play a major role in copulation and/or ejaculation (4), are known to be particularly sensitive to androgen levels (16). Atrophy of these muscles after castration is associated with a loss of contractile proteins that continues for ~45 days and is reversed by androgen administration (16, 22). In the present study, the weight of the bulbocavernosus and levator ani muscles dropped significantly within 2 wk of castration and was no different from the control weight within a few days of androgen treatment. In contrast, the plantaris muscle was unaffected by either castration or DHT treatment. A similar lack of androgen responsiveness has been observed in the extensor digitorum longus and soleus muscles of male mice and the flexor digitorum brevis muscle of rats (13, 21).

The findings in the present study confirm and extend the conclusions of Kreig et al. (12). One could postulate that androgen responsiveness of muscles in rats correlates with the AR level and imply that the presence of some critical amount of AR separates major androgen target tissues from tissues with minor or no responsiveness. It is striking that, in the two muscle groups known to respond dramatically to androgen in the rat (i.e., bulbocavernosus and levator ani), the levels of AR are only ~5% as high as in the ventral prostate, which was used for the control for this experiment. The cause for variation in AR levels in different muscles and the minimal AR level needed to exert a major effect are unclear.

The findings in the present study do provide some insight into the mechanisms by which AR levels in muscles are regulated. That is, the administration of DHT caused a significant increase in the AR level compared with the C rats. This finding is in contrast to the report by Rance and Max (15) that castration caused an increase and that testicular testosterone caused a decrease in the level of AR in the levator ani muscle. The differences in these two studies are almost certainly due to the fact that it is difficult to measure AR levels in the presence of ligand. The effects of androgens on AR vary among tissues. Namely, androgen administration causes downregulation in AR levels in the rat penis (19), whereas, in the rat adrenal and prostate, androgens appear to upregulate AR levels (2). The nature of the upregulation by androgens is not entirely clear and may be the passive consequence of the fact that its ligand-occupied receptor is more stable and has a slower half-life (10). Regardless of the mechanism, it is intriguing to speculate that the upregulation of AR levels via the administration of pharmacological amounts of androgens might convert some muscles that normally have a minor or no response to muscles with enhanced androgen responsiveness. The present study was too short in duration to determine whether such changes do occur.

The anabolic potencies of different androgens vary. Although DHT is one of the most potent, naturally occurring anabolic hormones that has been identified (11), the synthetic androgens 7-methyl-19-nortestosterone and 19-nortestosterone are even more potent anabolic steroids (18). Because athletes may selfadminister synthetic androgens for many months or years at even higher doses than used in this study (17), it is possible that upregulation of AR levels might be an important part in the anabolic effects of androgen abuse.

In summary, these data are unique in that they demonstrate changes in skeletal muscle mass that may be associated with the observed changes in AR levels. Future work should examine the effects of different androgens and lengths of administration on the response of the AR in various skeletal muscles.