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Keywords:

  • pubococcygeus muscle;
  • dihydrotestosterone;
  • cross-sectional area;
  • estradiol benzoate;
  • testosterone propionate

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Effects of gonadal hormones on dimorphic striated muscles such as the bulbocavernosus/levator ani complex related to male penile erection have been widely studied. However, the action of these hormones on pelvic nondimorphic muscles is not known. In the present study, the sensitivity of the male rat pubococcygeus muscle (Pcm) to gonadal hormones was studied measuring the cross-sectional area (CSA) of its fibers. For this, two experiments were done: in the first, the effect of castration, and in the second the effect of gonadal hormone administration was analyzed. We found that castration after 6 weeks significantly reduced the average CSA of the fibers of this muscle and that castration after 2 or 6 weeks reduced the percentage of fibers with higher CSAs, but only castration after 6 weeks increased the percentage of fibers with the lowest CSA. In comparison with castrated animals implanted with an empty Silastic capsule, Silastic implants of testosterone propionate or dihydrotestosterone significantly increased the average CSA of Pcm fibers, and the treatment with testosterone propionate, estradiol benzoate, or dihydrotestosterone decreased the percentage of fibers with low CSAs and increased the percentage with larger CSAs. Our results could be considered for therapy in patients with damage of the Pcm, and suffering urinary incontinence or ejaculatory dysfunctions. Anat Rec, 291:586–592, 2008. © 2008 Wiley-Liss, Inc.

Effects of gonadal hormones on dimorphic striated muscles related to male reproductive physiology have been studied widely, particularly the bulbocavernosus/ levator ani complex implicated in mechanisms of penile erection. It has been shown that androgens affect muscle weight (Tucek et al.,1976; Gutmann and Carlson,1978; Souccar et al.,1982; Foster and Sengelaub,2004; Axell et al.,2006), size of muscle fibers (Venable,1966; Tucek et al.,1976; Gutmann and Carlson,1978; Rand and Breedlove,1992; Jordan et al.,1995; Axell et al.,2006), neuromuscular junction size (Bleisch and Harrelson,1989; Balice-Gordon et al.,1990), and acetylcholine receptor number and cholinesterase activity (Tucek et al.,1976; Bleisch et al.,1982; Bleisch and Harrelson,1989) of this muscle complex. These morphological parameters have also been correlated with greater contractile speed of the muscle fibers (Gutmann and Carlson,1978; Souccar et al.,1982).

In the present study, we explored the effect of gonadal hormones on a nondimorphic muscle, the pubococcygeus (Pcm). In humans, this muscle is considered part of the levator ani complex (Shafik,1975; Jundt et al.,2005), is important in supporting organs and tissues of the pelvic area, and has been related with the prolapse of pelvic viscera and incontinence (Ayoub,1979; Fritsch et al.,2004). Although in rats the Pcm is not considered part of the levator ani complex (Poortmans and Wyndaele,1998; Yuan et al.,2003), it performs similar functions as in humans. It originates from the inner face of the pelvic bone at the level of the acetabulum and inserts in the third and fourth caudal vertebrae (Manzo et al.,1997), its fibers are attached in part to the ventrolateral portion of the external urethral muscular complex (Pacheco et al.,2003), and it is innervated by means of the somatomotor branch of the pelvic nerve (s-mbPn; Manzo et al.,1997). In male rats, Pcm plays an important role in the micturition process, regulating bladder tension during voiding and triggering a spinal reflex mechanism promoting continence (Manzo et al.,1997). The Pcm is also important for copulatory performance. Although its denervation by transection of the s-mbPn does not alter copulatory behavior (Lucio et al.,1994), it modifies the seminal plug formed from ejaculate and results in less efficient induction of pregnancy (Manzo et al.,2000).

Our group has previously found that male rat Pcm motoneurons retrogradely labeled with horseradish peroxidase–wheat germ agglutinin (HRP-WGA) show a decrement in morphometric measures after 6 weeks of castration. In castrated animals, 4 weeks of treatment with testosterone propionate (TP) or estradiol benzoate (EB) was able to recover the morphometric alterations, but not dihydrotestosterone (DHT; Manzo et al.,1999b). These findings indicate that centrally the Pcm neuromuscular system exhibits hormone sensitivity. However, possible effects of these hormones on the muscle itself are still not known. In the present study, we explored in a first experiment the effect of 2 and 6 weeks of castration on the cross-sectional area (CSA) of Pcm fibers in male rats, and in a second experiment, we investigated the effect of TP, EB, and DHT treatments on CSA of the Pcm fibers of castrated animals.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Throughout the study, animals were treated and maintained according to the Policy on the Use of Animals in Neuroscience Research (The Society for Neuroscience), the Policy on Human Care and Use of Laboratory Animals (National Institutes of Health), and the guidelines of the Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, for the treatment of animals in research.

Animals and Groups

Forty-two adult male Wistar rats, approximately 90 days old were used. They were housed on a 12:12 hr light/dark reverse cycle with ad libitum access to food (Harlan-México) and water.

For experiment 1, a group of intact males (INT; n = 6), a group of males castrated for 2 weeks (CAS2; n = 6), and a group of males castrated for 6 weeks (CAS6; n = 6) were formed. Castration was performed under intraperitoneal anesthesia with sodium pentobarbital (45 mg/kg body weight; Anestesal, Smith Kline, México). By means of an abdominal midline longitudinal incision, the vas deferens ducts and the corresponding arteries and veins were ligated and sectioned, the testis were removed, and the animals were sutured and placed in a warm chamber (25–28°C) until they recovered from anesthesia. Intact and castrated animals were killed by an overdose of sodium pentobarbital to continue with the histological procedure.

For experiment 2, four groups of animals that had been castrated for 2 weeks were implanted in the dorsum with a subcutaneous Silastic capsule (1 cm long, 3.06 mm outer diameter, 1.58 mm inner diameter; Dow Corning Corporation, Midland, MI). It is known that endogenous androgen levels have dropped notably 2 weeks after castration (Rivier et al.,1989), making it possible to explore the action of exogenous hormones while minimizing the possible combined effect of endogenous and exogenous hormones. One group was implanted with an empty Silastic capsule (CAS2+EmC; n = 6); in a second group, the Silastic capsule was filled with TP (CAS2+TP; n = 6; 31 mg); in a third group with EB (CAS2+EB; n = 6; 11.3 mg); and in a fourth group with DHT (CAS2+DHT; n = 6; 4.7 mg). The hormones (Sigma-Aldrich Química, México D.F) and doses were chosen in accordance with previous reports showing effects on Pcm motoneurons, perigenital skin, and pelvic nerves (Manzo et al.,1999b,2003). After 4 weeks of Silastic capsule implantation, animals were weighed then killed by an overdose of sodium pentobarbital to continue with the histological procedure. Note that, by this time, all animals had been castrated for 6 weeks.

Pcm Extraction and Histological Procedure

Both Pcms were carefully extracted after partial removal of the pubic bone. Immediately after extraction, each Pcm was placed on a piece of Manila paper and dried at room temperature. Muscles were then immersed in Helly's fixer for 24 hr, washed with distilled water for 2 hr, and dehydrated in alcohols. A piece of muscle approximately 4 mm in length was obtained from a region close to the origin (Fig. 1A), imbedded in paraffin, and transversally cut on a microtome (7 μm). Serial sections were stained with hematoxylin and eosin (Fig. 1B).

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Figure 1. Macro- and microphotographs of the pubococcygeus (Pcm) from an intact male rat. A: The triangular shape of the muscle and the approximate level (solid line) from which the histological sections were obtained. B: Hematoxylin-eosin stained section of Pcm showing transversal fiber morphology: peripheral cellular nuclei (double left arrowheads), a blood vessel (v), and a muscle spindle (asterisk).

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Neither individual muscle weight nor number of fibers was considered in this study as the procedure for removing the Pcm at its long origin at the pelvic bone could not be standardized. Thus, reliable comparisons using these parameters were not possible.

Morphometry

From each block of muscle, right and left, one section was selected. A measuring grid 12 compartments in length by 8 compartments in width was placed in the center of the section image on the computer screen always at the same magnification, and the CSA of a fiber in every fifth compartment was measured (25 fibers per muscle, that is, 50 fibers per animal) using a light microscope (Olympus BH-2) and software for morphology (SigmaScanPro for Windows, version 4.0; Aspire Software International, Leesburg, VA). For each group, we calculated the mean CSA of Pcm fibers as well as the percent frequency distribution of fibers categorized according to CSA bins, each bin with a CSA range of 500 μm2 (Gutmann and Carlson,1978; Nnodim,1999; Dedkov et al.,2001).

Statistical Analysis

Body weight and average CSA values were compared among groups using one-way analyses of variance (ANOVA) and Dunnet tests for post hoc contrasts (GB-STAT for Windows, version 5.0; Dynamic Microsystems, Silver Springs, MD). A Student's t-test was used to compare the values for the CAS6 and CAS2+EmC groups. A two-way ANOVA was used to compare the percentage of the CSA according to fiber size bin and experimental manipulation. Newman-Keuls tests for post hoc contrasts were done. Differences were considered significant when P < 0.05. Descriptive statistics are expressed as mean ± standard error of the mean (SEM).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Pcm Characteristics

Macroscopic examination showed that the Pcm is fleshy along its complete length and that it is wider at its origin than at its insertion, giving it a triangular shape (Fig. 1A). During microscopic screening of the cross-sections peripheral cellular nuclei, blood vessels and occasionally muscle spindles were observed (Fig. 1B). Smooth musculature was never seen. The muscle fibers presented either a polyhedral or an oval transversal morphology with peripheral nuclei.

Experiment 1

Body weights on the day of euthanasia were 378 ± 18 g for the INT, 299 ± 10 g for the CAS2, and 357 ± 17 g for the CAS6 group. A one-way ANOVA reported the differences among groups to be significant [F(2,15) = 6.84, P < 0.007], and post hoc tests showed that the body weight values for the CAS2 animals were significantly lower than those for the INT group.

The average value of the CSA of Pcm fibers for the CAS2 group (castrated for 2 weeks) was similar to that for the INT animals, but was significantly lower in the CAS6 group (castrated for 6 weeks) [F(2,15) = 4.39; P < 0.03] (Fig. 2). Furthermore, when the CSAs of the Pcm fibers were grouped in 500-μm2 bins, and the percentage of fibers for each bin and treatment was obtained and compared with a two-way ANOVA [F(12,105) = 4.84; P < 0.0001] (Fig. 3), we found that (1) the INT group had the greatest CSA range (500–3,999 μm2) with a significantly bigger percentage of fibers with 1,000–1,499 μm2 than with a higher CSA range (2,500–3,999 μm2); (2) the CAS2 group had the second highest range, reaching a maximum of 2,999 μm2, with a significantly higher percentage of fibers in the range 1,500–1,999 μm2 in comparison with the other ranges of CSA; (3) the CAS6 group had a range from only 500–1,999 μm2, with a significantly lower percentage of fibers in the range 1,500–1,999 μm2 in comparison with the other ranges of CSA; and (4) in comparison with the INT group, the CAS6 group had a significantly larger percentage of fibers in the range of 500–999 μm2, and the CAS2 group showed a significantly larger percentage of fibers in the range of 1,500–1,999 μm2.

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Figure 2. Mean cross-sectional area (CSA) values of pubococcygeus (Pcm) fibers in intact animals (INT), and in animals 2 weeks after castration (CAS2) or 6 weeks after castration (CAS6). Comparisons were done using a one-way analysis of variance and Dunnet post hoc tests. *P < 0.05. Means ± SEM are shown.

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Figure 3. Percent of pubococcygeus (Pcm) fibers distributed in bins according to their cross-sectional area (CSA; μm2) in intact animals (INT) and in animals castrated for 2 (CAS2) or for 6 weeks (CAS6). Comparisons were done with a two-way analysis of variance followed by Newman-Keuls tests. Different lower cases represent significant differences (P < 0.05) within each treatment, whereas significant differences between the two castrated groups and INT are represented as (*) P < 0.05 and (**) P < 0.01. Means ± SEM are shown.

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Experiment 2

Body weights on the day of euthanasia were 386 ± 19 g for the CAS2+EmC (empty capsule) group, 389 ± 14 g for the CAS2+TP, 274 ± 4 g for the CAS2+EB, and 402 ± 11 g for the CAS2+DHT groups. A one-way ANOVA reported the differences among groups to be significant [F(3,20) = 20.30, P < 0.0001], and post hoc tests showed that the body weight values for the CAS2+EB animals were significantly lower than for the CAS2+EmC group.

The average value of the CSA of Pcm fibers for the CAS2+EmC group was 1,096 ± 42 μm2. This value was similar to that for the CAS6 group (1,101 ± 80 μm2) from experiment 1 (Student's t-test: t = 0.054; df = 8.51; P = 0.96), and was significantly lower than for the castrated animals treated with TP or DHT for 4 weeks [F(3,20) = 17.99; P < 0.001], but not than for the castrated animals treated with EB (Fig. 4).

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Figure 4. Mean cross-sectional area (CSA) of pubococcygeus (Pcm) fibers in animals castrated for 2 weeks and either implanted with an empty Silastic capsule for 4 weeks (CAS2+EmC), with testosterone propionate for 4 weeks (CAS2+TP), with estradiol benzoate for 4weeks (CAS2+EB), or with dihydrotestosterone for 4 weeks (CAS2+DHT). Note that all animals had been castrated for 6 weeks. Comparisons were done using a one-way analysis of variance and Dunnet post hoc tests. **P < 0.01 compared with CAS2+EmC. Means ± SEM are shown.

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When the CSAs were grouped in ranges, we found significant differences among groups in the percentage of fibers for each bin [F(18,140) = 9.69; P < 0.0001], (Fig. 5); (1) the range of CSAs for the CAS2+EmC (control group) was 500–1,999 μm2, with a significantly higher percentage of fibers in the bins of 500–999 and 1,000–1,499 μm2; (2) castrated animals treated with TP showed a greater variability in fiber CSA (500–2,999 μm2), with a significantly higher percentage of fibers in the bins of 1,000–1,499 and 1,500–1,999 μm2; (3) the fibers of animals treated with EB were in the CSA range of 500–2,499 μm2, with a significantly higher percentage of fibers in the bins of 1,000–1,499 and 1,500–1,999 μm2; (4) treatment with DHT showed a CSA distribution of 1,000–2,999 μm2, with a significantly higher percentage of fibers in the bins 1,500–1,999 and 2,000–2,499 μm2; (5) compared with the CAS2+EmC group, TP and EB treatments promoted a significantly lower percentage of fibers in the range of 500–999 μm2, DHT treatment resulted in a significantly lower percentage of fibers in the range of 1,000–1,499 μm2, and TP, EB, and DHT treatments resulted in a larger percentage of fibers in the range of 1,500–1,999 μm2, although only significantly so for TP and DHT.

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Figure 5. Percent of pubococcygeus (Pcm) fibers distributed in bins according to their cross-sectional area (CSA; μm2) in animals castrated for 2 weeks and either implanted with an empty Silastic capsule for 4 weeks (CAS2+EmC), treated with testosterone propionate (CAS2+TP), treated with estradiol benzoate (CAS2+EB), or treated with dihydrotestosterone (CAS2+DHT) for 4 weeks. Note that all animals had been castrated for 6 weeks. Comparisons were done with a two-way analysis of variance followed by Newman-Keuls tests. Different lower cases represent significant differences (P < 0.05) within each treatment, whereas significant differences between the various groups compared with CAS2+EmC are represented as (*) P < 0.05 and (**) P < 0.01, respectively. Means ± SEM are shown.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

In pelvic muscles that participate in penile erection such as the levator ani, castration reduces muscle mass and the CSA of its fibers. Likewise, treatment with testosterone reaching at supraphysiological plasmatic levels increases both measures consistent with the large force exerted by this muscle (Tucek et al.,1976; Gutmann and Carlson, 1979; Godinho et al.,1987; Monks et al.,2004; Axell et al.,2006). Now we have found that Pcm in males is also sensitive to castration and testosterone replacement. The CSA of Pcm fibers was reduced by castration after 6 weeks (CAS6 and CAS2+EmC groups) and treatment with TP or DHT for 4 weeks prevented this effect. Also we found that the effects of castration occurred gradually, because the reduction in CSA was more prominent in animals after 6 than after 2 weeks. This finding is in agreement with studies of the bulbocavernosus muscle in which castration also gradually reduces muscle weight (Araki et al.,1991). It is unlikely that castration affected the number of Pcm fibers, as counting fibers in other muscles such as the levator ani (Tucek et al.,1976) has shown neither castration nor androgen treatment to affect this parameter, even 1 year after castration.

In the second experiment, we found that, in castrated animals, the mean CSA was significantly increased only by TP and DHT and not by EB. However, an effect of EB on the CSA of Pcm fibers was revealed when we analyzed the percentage of fibers according to CSA bins. Using this analysis, we found that all three gonadal hormones administered were effective in ameliorating the effects of 6 weeks of castration (CAS2+EmC group), increasing the percentage of fibers with higher CSAs and reducing the percentage of fibers with low CSAs. With this type of analysis, we also observed that DHT induced a greater hypertrophy of Pcm than TP or EB, increasing the percentage of fibers with the highest CSA. This effect could be explained by the greater efficacy of DHT (Krieg et al.,1976) or by the low dose of TP since a dose six times higher has been reported to induce strong hypertrophy of the levator ani muscle (Gutmann and Carlson,1978). Another possibility is the differential solubility of the two forms of the hormones used (salt or pure), although both should have had sufficient time to reach the muscle.

The doses and the forms of the hormones used by us here have been shown to restore noncontact erection (Manzo et al.,1999a), the number of urine marks and sniffing by males in response to female presence (Manzo et al.,2002), the threshold stimulus needed to trigger a response in the proximal and distal branches of the scrotal and dorsal penile nerves (Manzo et al.,2003), the size of the cutaneous sensory field related to the scrotal nerve (Manzo et al.,2003), and to modify Pcm motoneuron activity (Manzo et al.,1999b). Now we can add that these hormones can also avoid the reduction of the CSA of Pcm fibers after 6 weeks of castration. According to some studies, the CSA of muscle fibers is generally correlated with their activity, which is promoted by the activity of the respective motoneuron (Witzmann et al.,1982; Deschenes et al.,1997; Nnodim,1999,2001; Brown et al.,2001; Dedkov et al.,2001; Gomes et al.,2004; Dow et al.,2004,2006). Thus, doses of gonadal hormones that promote an increment in the size of the soma of the levator ani motoneurons are also effective in increasing the CSA of the fibers of this muscle (Jordan et al.,1995).

Although EB did not increased significantly the mean CSA it was able to reduce the percentage of fibers in the lowest CSA bin (500–999 μm2) and to increase those in the ranges 1,500–1,999 and 2,000–2,499 μm2 as also did TP and DHT. The results thus show that EB had a modest but visible effect on the CSA of Pcm fibers. In previous studies, a lack of effect of estrogens on the CSA of bulbocavernosus/levator ani complex fibers has been reported (Jordan et al.,1995; Fargo et al.,2003), although they are able to recover the electromyographic properties of this muscle in castrated male rats (Fargo et al.,2003; Foster and Sengelaub,2004).

The CSA of muscle fibers can change because of a change in the number of myofibrils composing them, a process directly related to protein synthesis in the muscle (Jackman and Kandarian,2004). Thus, it is possible that gonadal hormones could be affecting this process. In this regard, it is known that, whereas castration reduces the weight (Antonio et al.,1999), fiber size (Nnodim,1999; Monks et al.,2004), and synthesis of contractile proteins (Ferry et al.,1999) of the bulbocavernosus/levator ani muscle complex, the administration of androgens reverses or prevents the castration effects (Balice-Gordon et al.,1990; Venable,1966; Antonio et al.,1999; Monks et al.,2004), recovering the intracellular physiology (Gori et al.,1969) to promote muscle protein synthesis and the incorporation of satellite cells into the muscle fibers (Nnodim,2001) through the activation of receptors found in muscle fibers, fibroblasts close to neuromuscular junctions (Monks et al.,2004) and in muscle satellite cells (Sinha-Hikim et al.,2003,2004). In addition, estrogen receptors are also present in the bulbocavernosus/levator ani muscle complex (Dubé et al.,1976; Dionne et al.,1979), and participate in the metabolism of this muscle (Max and Knudsen,1980; Knudsen and Max,1980). In other muscles such as the soleus, vastus, or plantaris, estrogens reduce the atrophy resulting from immobility (Sugiura et al.,2006), protect from the oxidative stress following exercise (Tiidus and Bombardier,1999) and increase the number of satellite cells incorporated into fibers when the muscles are exercised (Tiidus et al.,2005). Similarly to these studies, our results suggest that estrogens have the potential to repair muscle damage.

In summary, although we did not count the number of nuclei of the muscle fibers, our results show that gonadal hormones act directly on the Pcm to preserve or increase the CSA of fibers, possibly by promoting the incorporation of satellite cells, and/or by modifying the electrical properties of the motoneuron and muscle fibers. In this regard, we need to conduct further studies to determine whether the Pcm contains androgen and estrogen receptors, and if the incorporation of satellite cells within muscle fibers is modified by gonadal hormones. Finally, the present results could be useful in developing noninvasive (pharmacological) therapy in men with urinary incontinence following prostatectomy.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

We thank Carolina Rojas for her excellent technical and bibliographical assistance. This work was supported by a fellowship from CONACyT México to M.A.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED