Although the pelvic striated musculature of the female rat undoubtedly plays an important role on copulatory, urodynamic, and parturitional processes, there is little experimental data regarding its morphological characteristics. It is known that the pubococcygeus muscle (Pcm) in the female rat is the largest striated muscle of the pelvic floor. It originates at the inner face of the pelvic bone at the level of the acetabulum and inserts in the third and fourth caudal vertebrae (Brink and Pfaff, 1980). The Pcm is surrounded by connective tissue close to its caudal insertion on the peritoneal surface of the vaginal wall (Martínez-Gómez et al., 1992). Pcm motoneurons are located in segments L6-S1 (Cuevas et al., 2006), and their axons travel from the spinal cord in the somato-motor branch of the pelvic nerve (Pacheco et al., 1989). Contraction of the muscle, elicited by electrical stimulation of the somato-motor branch of the pelvic nerve, increases intravaginal pressure. The reflexive activation of Pcm motoneurons by mechanical stimulation of the clitoral sheath, perineum, or lower vagina is inhibited by stimulation of the cervix (Martínez-Gómez et al., 1992). In addition, Pcm denervation alters the urodynamic parameters in rats (Kamo et al., 2004), and the motoneurons are sensitive to gonadal hormones (Cuevas et al., 2006), however, it is not known if the Pcm fibers in female rats are also sensitive to these hormonal effects.
The effects of gonadal hormones on striated muscles have been widely studied in male rats, those involved in reproduction and micturition such as bulbocavernosus (Breedlove and Arnold, 1981) and Pcm (Manzo et al., 1997). It has been shown that androgens affect muscle weight (Axell et al., 2006), the size of muscle fibers (Alvarado et al., 2008), neuromuscular junction size, acetylcholine receptor number, and cholinesterase activity of these muscles (Bleisch and Harrelson, 1989). These morphological changes are correlated with an increase in the speed of the contraction of the muscle fibers (Souccar et al., 1982). In a previous study, we analyzed the effect of gonadal hormones on the cross-sectional area of Pcm in male rats (Alvarado et al., 2008) and we found that: (1) 6 weeks castration, there was a significant increased the percentage of fibers with smaller cross-sectional area, and (2) subsequent administration of testosterone propionate (TP) or dihydrotestosterone (DHT) decreased the percentage of fibers with smaller cross-sectional areas and increased the percentage of those with larger cross-sectional areas, suggesting that Pcm fibers in males are sensitive to gonadal hormones. In this study, we explored the effect of gonadal hormones in the Pcm fibers of female rats.
MATERIALS AND METHODS
Throughout the study, animals were treated and maintained according to 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.
Animals and Groups
Fifty-four adult female Wistar rats, ∼90 days old (initial body weight, 250–260 g), which had undergone three previous regular estral cycles, were used. They were housed on a 14:10 hr light/dark cycle with ad libitum access to food (7001 Teklad 4%, Harlan-México) and water. They were divided for two experiments.
For Experiment 1, groups of intact females in proestrous phase (INT; n = 6), and females ovariectomized 2 (Ovx-2; n = 6), or 6 (Ovx-6; n = 6) weeks previously were used. Bilateral ovariectomies were performed under intraperitoneal anesthesia with sodium pentobarbital (30 mg/kg body weight; Anestesal, Smith Kline, México). For histological examination, intact and ovariectomized animals of approximately the same age were weighed and euthanized by an overdose of anesthesia.
For Experiment 2, six groups females ovariectomized 2 weeks previously were implanted with a subcutaneous Silastic capsule (20 mm long, 3.18 mm outer diameter, 1.98 mm inner diameter, sealed with 5 mm wooden plugs; Dow Corning Corporation, Midland, MI) into the dorsum. The contents of the capsules for the different groups were as follows: Group 1 received an empty capsule (EmC; n = 6); Group 2, progesterone (P4; n = 6; 75.6 mg); Group 3, testosterone propionate (TP; n = 6; 31 mg); Group 4, dihydrotestosterone (DHT; n = 6; 4.7 mg); Group 5, estradiol benzoate (EB; n = 6; 11.3 mg); and Group 6, 17β-estradiol (E2; n = 6; 9 mg). The hormones were obtained from Sigma–Aldrich Química, México. Hormone replacement was performed through Silastic capsule, in which construction was chosen according to the method described by Stratton et al. (1973), Lapolt et al. (1986), Wilkinson et al. (1985), and Legan et al. (1975) to achieve capsule release rates similar to those studies. Four weeks after the Silastic capsule implantation, animals were weighed and euthanized (approximately at the same age) by an overdose of anesthesia to obtain histological specimens for analysis.
Pcm Extraction and Histological Procedure
As evidenced in other studies (Brink and Pfaff, 1980; Poortmans and Wyndaele, 1998), dissection and extraction of the Pcm is complicated, however, after partial removal of the pelvic bone, both Pcms were carefully extracted, without a reliable complete separation of the muscle mass from the inner face of the pelvic bone. Both muscles were placed on a piece of Manila paper immediately before fixation to prevent muscle shirking. Muscles were immersed in Bouin–Duboscq fixer for 24 hr and dehydrated in alcohols. A piece of muscle ∼4 mm in length from a region close to the origin was obtained, imbedded in paraffin, and cut transversally on a microtome (section thickness 7 μm). Serial sections were stained with hematoxylin and eosin. This procedure was similar to that used before (Alvarado et al., 2008). As we focused our study in the cross-sectional area values of a sample of Pcm fibers, neither density (weight/volume) nor total fiber number was considered.
One section was selected from each block of muscle on the right and left sides. Five images per section were chosen and analyzed using a grid with 12 × 8 compartments (100 × 100 μm each compartment), which was placed in the center of each image. The image was displayed on the computer screen at 10× magnification. Five compartments per image, in which a complete cross-sectional area of a fiber could be observed, were selected for measurement. Twenty-five fibers per muscle (50 fibers per animal) were analyzed using a light microscope (Olympus BH-2) and software for morphology (Sigma ScanPro for Windows, version 4.0; Aspire Software International, Leesburg, VA). The average cross-sectional area average of Pcm fibers was calculated for each animal within each group and a grand mean was determined for comparisons among groups. The percentage of fibers grouped in bins (500 μm2 per bin) was also analyzed.
In Experiment 1, body weight and cross-sectional area average values were compared among groups using one-way analysis of variance (ANOVA) and Tukey–Kramer post hoc tests. A two-way ANOVA was used to compare the percentage of the cross-sectional area according to fiber size bin and experimental manipulation.
In Experiment 2, body weights and cross-sectional area average values of INT, Ovx-2, EmC, P4, TP, DHT, EB, and E2 were compared using a one-way ANOVA and Tukey–Kramer post hoc tests. A two-way ANOVA was used to compare the percentage of the cross-sectional area according to fiber size bin and experimental manipulation. Differences were considered significant when P < 0.001. Descriptive statistics are expressed as mean ± standard error of the mean (S.E.M.).
The muscle is wider at its origin than at its insertion, giving it a triangular shape. It is surrounded by connective tissue that is attached to the lateral surface of the vagina. During microscopic screening of the cross-sections, blood vessels, muscle spindles, but not smooth musculature was observed. The muscle fibers presented either oval or polyhedral morphology with peripheral nuclei.
Body weights on euthanasia day were similar for INT and Ovx-2 groups, but it was significantly higher in the Ovx-6 group [F(2, 15) = 41.4, P < 0.001] (Fig. 1).
In comparison to INT, the cross-sectional area average of Pcm fibers for the Ovx-2 and Ovx-6 groups was significantly higher and similar [F(2, 15) = 69.3, P < 0.001] (Fig. 1). Furthermore, when the cross-sectional area of the Pcm fibers was grouped in 500 μm2 bins, and the percentage of fibers for each bin and treatment was obtained, we found that: (1) the INT group had a distribution of cross-sectional area values that reached a maximum of 2,000–2,499 μm2, with a bigger percentage of fibers in both 500–999 and 1,000–1,499 μm2 bins; (2) the Ovx-2 group had the widest range of cross-sectional area values, with a bigger percentage of fibers in the bins of 1,000–1,499 and 1,500–1,999 μm2; (3) the Ovx-6 group had a cross-sectional area reaching a maximum of 2,000–2,499 μm2 with a higher percentage of fibers in the bin of 1,500–1,999 μm2, followed by the 1,000–1,499 μm2 bin; and (4) in comparison with the INT group, Ovx-2 and Ovx-6 groups had a lower percentage in the 500–999 μm2 bin, and a significantly higher percentage of fibers in the 1,500–1,999 μm2 bin [F(5, 90) = 93.2, P < 0.001] (Fig. 2).
Body weights on the euthanasia day for INT, Ovx-2, and EB groups were similar, but significantly different when compared to EmC, P4, TP, DHT, and E2 groups, where E2 group had the lowest weight [F(7, 40) = 45.8, P < 0.001] (Fig. 3).
In comparison to Ovx-2 or EmC groups, treatment with TP produced the most significant increase on cross-sectional area values, followed by DHT, and treatment with P4 or EB did not produce significant differences. However, treatment with E2 significantly reduced the cross-sectional area values of the already increased by ovariectomy, reaching values as those observed in the INT group [F(7, 40) = 61.9, P < 0.001] (Fig. 3). When the cross-sectional area of the Pcm fibers was grouped in 500 μm2 bins, and the percentage of fibers for each bin and treatment was obtained, we found that: (1) the range of cross-sectional areas for the EmC group was from 500 to 2,499 μm2 with a higher percentage of fibers in the 1,500–1,999 μm2 bin; (2) animals treated with TP showed a fiber cross-sectional area distribution from 1,000 to 3,999 μm2, there were no fibers in the 500–999 μm2 bin, and a higher percentage of fibers in 1,500–1,999, 2,000–2,499, and 2,500–2,999 μm2 bins; (3) treatment with DHT showed a cross-sectional area distribution from 500 to 2,499 μm2, with a higher percentage of fibers in the 1,500–1,999 μm2 bin; (4) treatment with P4 showed a cross-sectional area distribution from 500 to 2,999 μm2, with a higher percentage of fibers in the 1,000–1,499 and 1,500–1,999 μm2 bins; (5) animals treated with EB showed a cross-sectional area distribution from 500 to 2,999 μm2, with a higher percentage of fibers in 1,000–1,499 μm2 bin; and (6) animals treated with E2 showed a cross-sectional area distribution from 500 to 2,499 μm2, with a higher percentage in the1,000–1,499 μm2 bin. Hence, when compared to the EmC group: (i) TP treatment presented an absence of fibers within the 500–999 μm2 bin, a significant reduction in the percentage of fibers for the 1,000–1,499 and 1,500–1,999 μm2 bins, a significant increase in the percentage of fibers for the 2,000–2,499 μm2 bin, and presence of fibers distributed from 2,500 to 3,999 μm2; (ii) DHT treatment significantly increased the 2,000–2,499 μm2 bin; (iii) P4 treatment did not produce significant changes; (iv) EB treatment significantly increased the percentage of fibers in 1,000–1,499 μm2 bin, and reduced the percentage of fibers in 1,500–1,999 μm2 bin; and (v) E2 treatment significantly increased the percentage of fibers for the 500–999 and 1,000–1,499 μm2 bins and reduced the percentage of fibers for the 1,500–1,999 μm2 bin [F(30, 210) = 23.0, P < 0.001] (Fig. 4).
From this study, it can be inferred that Pcm fibers of female rats are sensitive to gonadal hormones. Two weeks after ovariectomy, there was an increase in the average cross-sectional area of the muscle fibers, together with a reduction in the percentage of fibers within lower value bins (500–999 μm2) and an increase in the percentage of fibers with higher value bins (1,500–1,999 μm2). Furthermore, this pattern can be modified by hormone replacement treatment.
The ovaries are the principal source of progesterone, testosterone, and estradiol in female mammals (Arlt, 2006), and their removal reduces the plasma levels of these hormones (Lu and Judd, 1982; Feng et al., 2004; Sitnick et al., 2006; Moran et al., 2007), in addition to inducing atrophy of the reproductive tissue (Fisher et al., 1998). Moreover, it has been reported that the lack of gonadal hormones after ovariectomy increases body weight, and the cross-sectional area of the fibers within plantaris, gastrocnemius, soleus, and extensor digitorum longus muscles (Suzuki and Yamamuro, 1985; Fisher et al., 1998; McClung et al., 2006; Moran et al., 2006, 2007; Sitnick et al., 2006), though not in peroneus longus and genioglossus (Joubert and Tobin, 1989; Fisher et al., 1998; Liu et al., 2009). According to the present results, the Pcm should be added to the list of skeletal muscles, in which ovariectomy increases the cross-sectional area of fibers.
However, our results reveal no correlation between the body weight change after ovariectomy and the cross-sectional area increment of Pcm fibers. Two weeks after ovariectomy, body weight remained similar to that of intact animals, whereas fiber cross-sectional area significantly increased. Body weight increased significantly only 6 weeks postovariectomy, whereas the muscle fiber cross-sectional area values did not change after the first 2 weeks. Furthermore, though P4 treatment increased body weight, it did not affect muscle fiber cross-sectional area.
It is known that after ovariectomy plasma testosterone becomes predominant over estradiol (Handa et al., 1986), and this could be related to the increase of the cross-sectional area in female Pcm fibers observed in this study. Androgens induce a strong hypertrophy of the male levator ani muscle (Venable, 1966; Gutmann and Carlson, 1978). Furthermore, androgen treatment increases the fiber size by promoting an increase in the number of myofibrils, a process that has been directly related to protein synthesis in some muscles (Jackman and Kandarian, 2004). Androgens also promote the incorporation of satellite cells into muscle fibers (Nnodim, 2001) through activation of receptors within these cells (Sinha-Hikim et al., 2004) and in the fibroblasts close to neuromuscular junctions (Monks et al., 2004). As androgen receptor expression is enhanced in the synaptic myonuclei, it has been claimed that receptors within the muscle fibers may regulate the expression of skeletal muscle-specific genes related to controlling the cross-sectional area size of muscle fibers.
Castration in male rats obviously reduces plasma testosterone levels (Handa et al., 1986) and also the cross-sectional area of Pcm fibers (Alvarado et al., 2008). After castration, treatment with DHT promotes a stronger increment on the cross-sectional area of Pcm fibers than TP (Alvarado et al., 2008). In the ovariectomized animals, in which the cross-sectional area of the Pcm fibers had already increased, TP rather than DHT treatment resulted in the greater increment. A greater affinity for the androgen receptors and better solubility of the free form of DHT were given as the explanation for the differential androgenic effects in male rat (Alvarado et al., 2008). Thus, if the Pcm fibers of female rats have androgen receptors, one would hypothesize that their affinity would be greater for TP rather than for DHT. It is known that in the female rat, DHT is converted to 5α-androstane3β,17β-diol, which has a low binding affinity for androgen receptors and a preferential binding to estrogen receptors (Simard and Labrie, 1987). These observations indicate the presence of a mechanism through which estrogen receptors could modulate the cross-sectional area growing produced by androgens.
In relation to estrogen effects in Pcm fibers of female rat, we found that 4 weeks of EB treatment, in animals with 2 weeks of previous ovariectomy (which fiber cross-sectional area values were already increased), did not show any additional significant increment or reduction in the mean cross-sectional area values. In contrast, E2 treatment showed a significant reduction in the mean cross-sectional area values, which was correlated with the increase in the percentage of fibers with smaller cross-sectional areas. These findings are in agreement with previous studies, which have shown that estradiol reduces fiber cross-sectional area in the extensor digitorum longus and soleous muscles (Suzuki and Yamamuro, 1985), suggesting an inhibitory action on muscle fiber growth and function (Moran et al., 2007). Estradiol treatment not only is associated with lower rates of skeletal muscle protein synthesis (Toth et al., 2001) but also promotes the formation of lysosomes in muscle cells, which promote the breakdown of the cellular constituents such as proteins (Sloane, 1980). It has been also suggested that estradiol indirectly affects skeletal muscle through the down-regulation of the insulin-like growth factor I (IGF-I), which participates in skeletal muscle fiber growth (Kalu et al., 1994; Fisher et al., 2000). After ovariectomy estradiol plasma levels are reduced, whereas IGF-I levels are increased (Borski et al., 1996). This could therefore represent another mechanism by which the cross-sectional area of Pcm fibers is increased after ovariectomy.
On the other hand, studies of motoneurons in a sexually dimorphic motor nucleus in the lumbar cord supplying the bulbocavernosus and levator ani muscles exhibit a hormone-sensitive plasticity in a number of features such as soma size (Breedlove and Arnold, 1981), dendritic length (Kurz et al., 1986), synapse number and size (Matsumoto et al., 1988), synaptic efficiency (Tanaka and Arnold, 1993), input resistance (Vyskocil and Gutmann, 1977), neuromuscular junction dimensions (Balice-Gordon et al., 1990), and neuromuscular activity (Foster and Sengelaub, 2004). This suggests the possibility of an indirect route by which gonadal hormones can modulate muscle mass or fiber size. We have previously observed that Pcm motoneurons in both male (Manzo et al., 1999) and female rats (Cuevas et al., 2006) change their morphology and their antidromic labeling after castration, and that estradiol treatment reversed these effects. The different effects found between E2 and EB can be explained through two possibilities: (1) a different release rate from the Silastic capsule; or (2) a greater affinity of E2 to estrogen receptors within skeletal muscle of the rat (Dionne et al., 1979), because EB has a 6- to 10-fold less binding affinity compared to E2 (Matthews et al., 2000).
According to McKenna and Nadelhaft (1986) in male and female rats, as in the human, the pelvic floor is composed of muscles having origin on the inner surface of the bony pelvis and inserting onto the caudal coccygeal vertebrae. As the striated muscles of the pelvic floor in humans form a very important functional complex, which contributes to continence, micturition, defecation, orgasm, and the support of the pelvic organs (Graber and Kline-Graber, 1979; Poortmans and Wyndaele, 1998), they are collectively referred to as the levator ani muscle; however, this name has been used in the rat to refer to a completely different muscle, and therefore, they should be referred individually as pubococcygeus, iliococcygeus, and coccygeus muscles.
Finally, considering the differences that exist on the cross-sectional area of Pcm fibers between intact male (Alvarado et al., 2008) and female rats, plus the differential androgenic and estrogenic treatment effects on this muscle and its physiological participation in different reproductive processes of both sexes (Martínez-Gómez et al., 1992; Manzo et al., 1997), we can conclude that the Pcm of the rat is sexually dimorphic. This is important to consider during hormonal treatments in human diseases, where this pelvic muscle is implicated.
The authors would like to thank Dr. Leanne Fraser and Carolina Escobar for their technical assistance.