Rat Levator Ani Muscle Research Articles

Histotopographic Organization of Levator Ani Muscle in Laboratory Rats

Morphological organization of the levator ani muscle still remains unclear due to methodological problems in the study of the pelvic floor muscles. Therefore, the study of the histological structure of muscle tissue m. levator ani in animals is of not only theoretical but also practical importance for the study and modeling of pelvic floor dysfunction conditions in animal models. The aim is to study the enzymohistochemical and ultramicroscopic organization of the levator ani muscle in white laboratory rats. Material and methods. The study was performed on 10 laboratory Wistar rats (5 females and 5 males) aged 12–14 months. The metabolic profile of muscle fibers was determined using the Nachlass succinate dehydrogenase test. Imaging and linear measurements were carried out on a Leica UC 7 microscope (Germany), using its software. Ultrathin sections were counterstained with uranyl acetate and lead citrate, viewed nd photographed using a Hitachi HT 7700 Exalens (Japan) electron microscope. Results. All types of muscle fibers are present in levator ani muscle: slow fibers and rapid muscle fibers of typs IIA and IIB. It has been established that the total cross-sectional area of white type fibers is 3 times greater than the total area of other fibers. Significant sex differences were found in relative square parameters of glycolytic fibers in m. levator ani (p=0,009). At the ultrastructural level, it was found that in some muscle fibers the mitochondrial apparatus is well developed, in others, mitochondria are few in number, have smaller sizes, are located singly between myofibrils, without forming clusters. In such fibers, there are large accumulations of glycogen between the myofibrils. Conclusion. In the laboratory rat, the levator ani muscle is heterogeneous both in metabolic activity of muscle fibers and in ultrastructural organization. This feature of the muscle involves not only static work in creating intra-abdominal pressure and retaining the pelvic organs, but also isotonic contraction, acting as a synergist to ensure the movement of the tail.

283 - ABNORMAL PROPRIOCEPTION AND CONDUCTION FUNCTION OF LEVATOR ANI MUSCLE IN RATS WITH STRESS URINARY INCONTINENCE

283 - ABNORMAL PROPRIOCEPTION AND CONDUCTION FUNCTION OF LEVATOR ANI MUSCLE IN RATS WITH STRESS URINARY INCONTINENCE

Morphological Description of Levator Ani Muscle in Rats

The aim of the research: the study of structural morphologic features of levator ani muscle in white laboratory rats. Materials and methods. Five male and five female laboratory rats 300–420 g of weight were observed as an object of investigation. The experimental animals were kept in standard conditions in vivarium with the free access to water and food. The methods of preparation, histochemistry, optical and electronic microscopy were used during the investigation. Results . It was stated that in male rats, levator ani muscle is represented by a single complex without a clear differentiation into compartments. In females, the iliocaudal and pubocaudal portions of levator ani muscle are clearly differentiated. The iliocaudal muscle is initially attached to the internal part of iliac bone, pubocaudal muscle is initially attached to the posterior part of pubic bone below pubic symphysis. The other ends of the muscles are attached through their tendons to the anterior surface of caudal vertebrae bodies Ca III –Ca VI . Meanwhile, iliocaudal muscle is attached more proximally to Ca III –Ca VI , and pubocaudal muscle is attached more distally to Ca V –Ca VI . The transverse sections of levator ani muscle in males and its pubocaudal portion in females are represented by polygonal muscle fibers, and the iliocaudal portion of levator ani muscle in females is represented by orbicular muscle fibers. Ultrastructural manifestations of sexual dimorphism among muscle fibers are not observed, they have got typical structure representative for transversal striated skeletal muscular tissue. On histochemical investigation the presence of I and II type of muscle fibers was determined, but glycolytic fibers (of the II type) prevailed. Conclusion . The obtained data indicate the presence of sexual dimorphism in levator ani muscle structure in white laboratory rats. This muscle is supposed to contribute more to provide tail movements than to strengthen small pelvis wall and protect organs.

Nonsteroidal Selective Androgen Receptor Modulators Enhance Female Sexual Motivation

Women experience a decline in estrogen and androgen levels after natural or surgically induced menopause, effects that are associated with a loss of sexual desire and bone mineral density. Studies in our laboratories have shown the beneficial effects of selective androgen receptor modulators (SARMs) in the treatment of osteoporosis and muscle wasting in animal models. A series of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-cyano-3-trifluoromethyl-phenyl)-propionamide analogs was synthesized to evaluate the effects of B-ring substitutions on in vitro and in vivo pharmacologic activity, especially female sexual motivation. The androgen receptor (AR) relative binding affinities ranged from 0.1 to 26.5% (relative to dihydrotestosterone) and demonstrated a range of agonist activity at 100 nM. In vivo pharmacologic activity was first assessed by using male rats. Structural modifications to the B-ring significantly affected the selectivity of the SARMs, demonstrating that single-atom substitutions can dramatically and unexpectedly influence activity in androgenic (i.e., prostate) and anabolic (i.e., muscle) tissues. (S)-N-(4-cyano-3-trifluoromethyl-phenyl)-3-(3-fluoro,4-chlorophenoxy)-2-hydroxy-2-methyl-propanamide (S-23) displayed full agonist activity in androgenic and anabolic tissues; however, the remaining SARMs were more prostate-sparing, selectively maintaining the size of the levator ani muscle in castrated rats. The partner-preference paradigm was used to evaluate the effects of SARMs on female sexual motivation. With the exception of two four-halo substituted analogs, the SARMs increased sexual motivation in ovariectomized rats, with potency and efficacy comparable with testosterone propionate. These results indicate that the AR is important in regulating female libido given the nonaromatizable nature of SARMs and it could be a superior alternative to steroidal testosterone preparations in the treatment of hypoactive sexual desire disorder.

Myoblast transplantation to defecation muscles in a rat model: a possible treatment strategy for fecal incontinence after the repair of imperforate anus

Infants with higher anorectal anomalies often develop fecal incontinence after surgical reconstruction mainly due to the incomplete development of defecation muscles. We investigated the possibility of defecation muscle regeneration by myoblast transplantation to improve fecal continence. Myoblasts from F344 female rats at ages of 1 day, 1, 2, 3, 4, 8, and 12 weeks were prepared by a preplating method. In vivo muscle differentiation of myoblasts was evaluated using immunofluorescence after transplantation of GFP-positive myoblasts into nude mice, the damaged thigh muscles, and the levator ani muscle of GFP-negative rats. The ratios of myoblasts obtained from 1 day, 1, 2, 3, 4, 8, and 12-week-old rats were 35, 71, 65, 61, 52, 44, and 23%, respectively. Myotube formation by transplanted myoblasts was observed in the back of nude mice. Myoblasts transplanted into damaged thigh muscles were integrated into recipient muscles with myofiber formation. Transferred myoblasts formed myotubes surrounding the levator ani muscle, although myofiber formation was not observed. Myoblasts were most efficiently obtained from juvenile rats. Myoblast transplantation may provide a novel treatment strategy for improving fecal continence after repair of anorectal anomalies in infants.

Quantitative analysis of motor neurons of the levator ani muscle in fetal rats with spina bifida occulta

Background With the combination of microsurgery and microinjection techniques, we investigated the development of motor neurons in the spinal cord of fetal rats with spina bifida occulta by injecting the retrograde trace FG into the levator ani muscle. Methods The fetal rats were divided into 3 groups. On the day 9 of gestation, 6 mature Wistar rats (weighing 250-300 g) in the control group (group 1) were subcutaneously injected with 0.5 mL of normal saline at their hind limbs at 9:00 am and 4:00 pm. At these 2 time points, 15 rats in the treatment group (group 2 and group 3) were subcutaneously injected with 20% sodium valproate solution (400 mg/kg of body weight) at their hind limbs, too. On the day 20 of gestation, pregnant rats were anesthetized with 10% chloral hydrate (300 mg/kg of body weight) intraperitoneally, and then fetal microsurgery and microinjection were performed to expose the levator ani muscle, whereas 5% FG was administered with microinjector. Twenty-four hours later, transcardial perfusion of 4% paraformaldehyde in phosphate-buffered saline (PBS) was given to the operated fetus. After the spine sample was stained with Alcian blue GX, the image of stained spine was measured using a computer system for the distance of the 2 cartilaginous ends of the vertebra arch. Then, the lumbosacral spinal cord was cryopreserved in 20% sucrose in PBS for a later serial transverse cryosection after 24 hours. The FG-labeled motor neurons were visualized with a wide-band ultraviolet-fluorescent filter, and the number of the FG-labeled motor neurons was recorded. Nine fetal rats survived in group 1. Eighteen fetal rats survived in the treatment group, including 7 (with no malformation) of 18 fetuses in group 2 and 11 fetuses with spina bifida occulta in group 3. Results The FG-labeled motor neurons in the ventral horn of normal spinal cord clustered at the dorsolateral and dorsomedial corner of the ventral horn. The FG-labeled motor neurons in the ventral horn of deformed spinal cord were less than that of normal spinal cord, and the motor neurons were scattered around the space between the dorsomedial and dorsolateral corners. The number of FG-labeled motor neurons was 244 ± 41 in group 3, 426 ± 36 in group 1, and 397 ± 20 in group 2. The data were stastistically significant if P < .05. Conclusion The motor neurons that innervate the levator ani muscle in fetal rats with spina bifida occulta are fewer than the normal fetal rats, and they are arranged in abnormal distribution.

Defective development of sensory neurons innervating the levator ani muscle in fetal rats with anorectal malformation

Defects of the pelvic nerve innervation of levator ani muscle are associated with poor postoperative anorectal function in patients with anorectal malformation (ARM). We have previously shown deficient development of motoneurons innervating the levator ani muscle in rats with ARM. In this study we investigate whether there is a deficiency in the development of sensory neurons that innervate the levator ani muscle in rats with ARM. ARM was induced by ethylenethiourea (ETU) in fetal rats. Retrograde tracer fluorogold (FG) was injected into the levator ani muscle. Serial transverse sections encompassing the entire length of the lumbosacral spinal cord were examined. The number of FG-labeled sensory neurons was scored and compared between fetuses with ARM and normal fetuses. The number of FG-labeled sensory neurons innervating the levator ani muscle in normal control fetuses, ETU-fed fetuses with no malformation, low type of imperforate anus, high type of imperforate anus, and high type of imperforate anus combined with neural tube defects were determined to be (mean +/- SEM) 11,804 +/- 2362, 10,429 +/- 1708, 2886 +/- 705, 1026 +/- 425, and 964 +/- 445, respectively. FG-labeled sensory neurons in fetuses with imperforate anus with or without neural tube defects were significantly fewer than in control and ETU-fed fetuses without malformation (p < 0.05). Defective sensory neurons innervating the levator ani muscle is a primary anomaly that coexists with the alimentary tract anomaly in ARM during fetal development. Nerve innervation deficiency of the pelvic muscles contributes to the poor postoperative anorectal functions in ARM patients.

Sexual dimorphism and androgen regulation of satellite cell population in differentiating rat levator ani muscle

The bulbocavernosus (BC) and levator ani (LA) muscles of rats show remarkable androgen-dependent sexual dimorphism. These muscles are additionally of interest because they are thought to indirectly mediate sexual differentiation of innervating spinal motoneurons. This sexual differentiation of the BC/LA is thought to be due to an increase in muscle units in the male rat during the first week after birth. We examined the cellular basis of this differentiation by studying satellite cells in the LA of postnatal day 2.5 rats, when sexual dimorphism is already prominent. Two experiments were performed in which LA satellite cells were measured: (1) wild-type (WT) males were compared with females and to Tfm androgen receptor mutant males, which are androgen insensitive despite producing masculine amounts of testosterone, and (2) females treated prenatally and/or postnatally with testosterone proprionate were compared with females receiving vehicle injections. Our results indicate that WT males have a larger LA and a greater number of satellite cells in the LA muscle than females or Tfm males. However, satellite cell density was similar for all three groups. Prenatal testosterone treatment masculinized LA size and resulted in a corresponding increase in satellite cell populations, while postnatal TP treatment resulted in a tendency for increased satellite cell density without a significant increase in LA size. Taken together, these studies indicate that satellite cells in the neonatal LA muscle are sexually dimorphic, and that this dimorphism likely results from perinatal actions of androgens on androgen receptors.

Muscle-Bound? A Tissue-Selective Nonsteroidal Androgen Receptor Modulator

Muscle-Bound? A Tissue-Selective Nonsteroidal Androgen Receptor Modulator

Distribution of sensory nerves in the levator ani muscle of young and aged male Wistar rats

Distribution of sensory nerves in the levator ani muscle of young and aged male Wistar rats Robert M. Santer, Andrew P. Carson-Stevens: School of Biosciences, Cardiff University, Cardiff CF10 3US, United Kingdom We have mapped the distribution of free nerve endings and muscle spindles by immunohistochemistry in the levator ani (LA) muscle of young and aged rats in order to investigate force transmission within this muscle. We have previously measured a near 40% decrease in tetanic and specific force generation in the rat LA in old age. The entire LA was removed bilaterally from terminally anaesthetised male rats (n = 6 per age group) of 3 and 24-27 month male Wistar rats, fixed in 4% paraformaldehyde and cryosectioned longitudinally or transversely at 12μm. Sections were incubated with primary polyclonal antisera against calretinin and neurofilament 200 followed by appropriate FITC-conjugated secondary antisera. The distribution of muscle spindles and free nerve endings was subsequently mapped in both the longitudinal and transverse planes. Muscle spindles were localised close to the LA’s myotendinous attachment to the penile bulb and also in the region of the midline raphé where the right and left LAs unite. Spindles were located deep within the LA whereas free nerve endings were distributed more widely in the muscle. In the aged animals the pattern of spindle distribution was unchanged but increased endomysial collagen(I) and decreased muscle fibre size did not permit quantitative assessment of nerve and muscle spindle numbers to be made. However, in spite of the expected signs of age-associated changes in LA muscle fibres, similar changes in the sensory innervation were not noted. Support of the Wellcome Trust is gratefully acknowledged.

Testosterone modulates Ca v2.2 calcium channels’ functional expression at rat levator ani neuromuscular junction

Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca 2+ channels (Ca v2.1), mediate evoked transmitter release. Here we report that N-type Ca 2+ channel (Ca v2.2) blocker ω-Conotoxin GVIA, as well as Ca v2.1 blocker ω-Agatoxin IVA, significantly reduced quantal content of transmitter release by ∼ 80% and ∼70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to ω-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca v2.2, as mediators of transmitter release in these synapses. Strikingly, the expression and localization of α 1B subunits, which form the pore of the Ca v2.2 channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca v2.2 and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.

Reinnervation of the rat levator ani muscle after neonatal denervation

After axonal injury on postnatal day 14 (P14), but not P21, motoneurons in the spinal nucleus of the bulbocavernosus (SNB) do not display their normal response to circulating testosterone levels. This could result from a permanent disruption of communication between motoneurons and their testosterone-sensitive target muscles. We assessed the extent of reinnervation of one of these target muscles, the levator ani (LA) muscle, 5 months after the pudendal nerve was cut either on P14 or P21. The number of motoneurons innervating the LA in control and nerve cut animals was determined using retrograde labeling procedures. Functional recovery of the LA muscle was determined via the testing of its in situ contractile properties. Compared to control muscles, reinnervated LA muscles were smaller, had fewer muscle fibers, generated a lower maximum tetanic tension, and were more fatigable. In spite of the fact that fewer motoneurons reinnervated the LA muscle after nerve cut on P14 than on P21, there were no differences in the weight or contractile properties of the LA muscle between these two groups. These data suggest that motoneurons that survived injury on P14 innervated more muscle fibers than normal and exhibited a similar ability to functionally reinnervate the target muscle as those motoneurons that survived injury on P21.

Phosphorylation of p70 s6 kinase is implicated in androgen-induced levator ani muscle anabolism in castrated rats

Androgens are known to increase muscle mass, strength and muscle protein synthesis. However, the molecular mechanisms by which androgens regulate skeletal muscle development remain poorly understood. The ribosomal protein kinase p70 s6k is a regulator of ribosome biogenesis and plays an important role in the regulation of growth-related protein synthesis. The phosphorylation of p70 s6k has been implicated in load-induced skeletal muscle hypertrophy. In the current study, we determined the effect of DHT on the phosphorylation of p70 s6k in the androgen-sensitive levator ani muscle of castrated rats. DHT induced a rapid increase in the phosphorylation of p70 s6k, which was detectable within 6 h after a single injection. Interestingly, DHT-induced phosphorylation of p70 s6k occurred only in androgen-sensitive muscles, but not prostate and seminal vesicle. Co-administration of flutamide, an AR antagonist, inhibited DHT-induced p70 s6k phosphorylation. While serum IGF-I levels were not changed by DHT treatment, IGF-I gene expression levels increased and the mRNA levels of IGFBP3 and IGFBP5 were suppressed in the LA muscle after DHT replacement in castrated rats. These results suggest that the phosphorylation of p70 s6k, likely via the IGF-I pathway, may play an important role in androgen-induced skeletal muscle hypertrophy.

Testosterone mediates satellite cell activation in denervated rat levator ani muscle

Denervation stimulates quiescent satellite cells in skeletal muscle to reenter the cell cycle. In the androgen-sensitive rat levator ani muscle (LA), this mitotic response to loss of neural input fails to occur in castrated animals. To elucidate the role of androgens in denervation-induced satellite cell proliferation, the denervated LA of castrated rats (Group A) was compared with that of animals infixed with testosterone implants after castration (Group B). Mean myofiber cross-sectional areas (Group A: 362.95 microm(2) +/- 27.74; Group B: 403.13 microm(2) +/- 53.87) and linear nuclear densities (Group A: 74.07 mm(-1) +/- 17.58; Group B: 104.13 mm(-1) +/- 4.06) were similar (P > 0.05) in both groups. The androgen-deprived myofibers of Group A, however, had a significantly lower nuclear content (271.0 +/- 74.91 vs. 1,285.80 +/- 81.74 in Group B; P < 0.05) on account of their considerably shorter mean length (3.44 mm +/- 0.29 vs. 12.31 mm +/- 0.92 in Group B; P < 0.05). The proportional representation of satellite cells in hormone-replaced, denervated muscle was more than twice that in the untreated group (Group B: 5.15 +/- 0.83% vs. Group A: 2.28 +/- 0.23%; P < 0.05). In absolute terms, the satellite cell number in Group B was approximately an order of magnitude greater than in Group A (408.4 x 10(3) vs. 38.08 x 10(3)). The results confirm the absence of testosterone as the factor responsible for the inability of satellite cells in the LA of castrated rats to respond mitotically to the withdrawal of neural input after denervation.

EVIDENCE OF APOPTOSIS IN THE CASTRATION-INDUCED ATROPHY OF THE RAT LEVATOR ANI MUSCLE

Apoptosis or programmed cell death has been previously reported in androgen-responsive tissue such as the prostate. We tested the hypothesis that apoptosis may also represent a component of the castration-induced atrophy of the sexually dimorphic levator ani (LA) muscle of the male rat. Gonadectomy (GDX) induced a severe decrease in the LA muscle wet weight accompanied by important modifications in cytoarchitecture including an increase in myofibrillar interspace and condensed mitochondria. These alterations were almost completely reversed after 7 days of testosterone propionate replacement therapy (GDX + TP). In GDX rats, internucleosomal DNA fragmentation was confirmed by both agarose gel electrophoresis and electron microscopy. DNA fragmentation was no longer detected in GDX + TP rats. In GDX rats, overexpression of the trpm-2/clusterin gene, used as an early marker of apoptosis, further confirmed that an apoptotic response, typical of androgen-responsive tissues, is taking place following androgen withdrawal.

Ciliary neurotrophic factor increases muscle fiber number in the developing levator ani muscle of female rats

In addition to its well characterized neurotrophic properties, ciliary neurotrophic factor (CNTF) also has myotrophic effects in several experimental paradigms. We have previously observed that the volume of the levator ani (LA) muscle is increased in female rats treated with CNTF during the perinatal period. In order to determine the cellular basis for the effect of CNTF on LA muscle volume, female rat pups were given daily perineal injections of CNTF or a control solution from postnatal day 1 through 6. Mean cross-sectional area of LA muscle fibers and LA fiber number were assessed on postnatal day 7. CNTF treatment increased LA muscle fiber number more than 300% while having no effect on LA fiber size. We conclude that CNTF prevents muscle fiber degeneration and/or increases myogenesis in the developing LA muscle.

Satellite cell numbers in senile rat levator ani muscle

Ageing in skeletal muscle results in motor frailty and a reduced capacity for self repair after injury. The contractile characteristics of muscle are determined principally by the myosin heavy chain (MHC) composition of its myofibers. During the restorative process, satellite cells play a central role. The present study compares the levator ani muscle of very old (32 months) and young (4 months) male WI/HicksCar rats in terms of structural integrity, MHC and satellite cell content. Myofiber typing was carried out by indirect immunohistochemistry using a panel of anti-MHC antibodies. Single myofibers for nuclear enumeration were isolated by an enzymatic technique while fiber cross-sectional areas and satellite cell frequencies were determined by computerized planimetry and electron microscopy. In both groups of rats, the myofiber population was homogeneously MHC type IIb-reactive. Cross-sectional data reflected a marked degree of atrophy in the muscle of the senile rats (710.05±63.6 μm 2, compared with 1519.98±79.0 μm 2 in young). The myofiber population was reduced by only about 6.7% with ageing and the representation of satellite cells, as a fraction of total sublaminal nuclei, was relatively stable (1.15 versus 1.91% in young; P>0.05). The results indicate that ageing had a considerable atrophic effect on the levator ani muscle but induced neither MHC isoform transition nor massive depletion of the satellite cell pool. They suggest that the well-documented impairment of the restorative capacity of senile muscle could be due more to alterations in the nature of microenvironmental cues than to quantitative aspects of its cellular capacity to respond.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7–P14) received 200 μg of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs. J. Comp. Neurol. 379:88-98, 1997. © 1997 Wiley-Liss, Inc.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7-P14) received 200 micrograms of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity at first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs.

Testosterone Treatment Results in Quiescent Satellite Cells Being Activated and Recruited into Cell Cycle in Rat Levator Ani Muscle

At puberty, the male levator ani (LA) muscle exhibits muscle fiber hypertrophy. This fiber enlargement can be provoked in the female LA muscle by testosterone treatment. In both cases the hypertrophic process is accompanied by an increase in the number of satellite cells and myonuclei. The present ultrastructural autoradiographic study was undertaken in order to investigate (1) whether satellite cells when stimulated by testosterone can undergo DNA synthesis by incorporating [ 3H]thymidine ([ 3H]Tdr); (2) whether the whole satellite cell population is committed in the cell cycle; and (3) whether the new myonuclei originate from fusion of the satellite cells with the preexistant myofibers. Thirty-day-old female rats treated with a single testosterone injection received a single injection of [ 3H]Tdr at 24, 28, 32, to 34 hr after testosterone treatment. LA muscles were removed 2 hr after [ 3H]Tdr injection. This first series of experiments allowed us to determine that onset of DNA synthesis in satellite cells occurs within the 34th and the 36th hour after testosterone treatment. To obtain a more precise timing, 30-day-old female rats treated with a single testosterone injection received multiple injections every 30 min either from Hour 32 to 33.5 or from Hour 34 to 35.5. LA muscles were removed 30 min after the last [ 3H]Tdr injection. This showed that the onset of satellite cell replication occurred between the 32nd and the 34th hour after testosterone treatment. However, only 30% of the satellite cell population was affected by this proliferative process regardless of the experimental protocol used. To confirm that the increase in myonuclei number results from incorporation of satellite cells into mature myofibers, 30-day-old female rats treated with a single injection of testosterone received a single injection of [ 3H]Tdr on the 60th hour after testosterone treatment. LA muscles were removed at 63, 84, to 108 hr after [ 3H]Tdr injection. We conclude that testosterone induces satellite cell proliferation at around the 33rd hour and that the increased number of myonuclei reported in our previous study is due to fusion of labeled satellite cells with myofibers.