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Transient and permanent effects of androgen during synapse elimination in the levator ani muscle of the rat.

Young male rats were castrated at 7 days of age, and treated with testosterone propionate daily from 7 to 34 days of age. At 13 months of age, motor axons and terminals innervating the levator ani (LA) muscle were stained with tetranitroblue tetrazolium (TNBT). The number of separate axons innervating individual muscle fibers was counted, and muscle fiber diameter was measured. Previous studies have shown that this androgen treatment increases muscle fiber diameter and delays synapse elimination, measured as (1) a greater percentage of muscle fibers innervated by multiple axons and (2) larger motor units. The present results indicate that the androgenic effect on synapse elimination is permanent, in that high levels of multiple innervation persisted for 12 months after the end of androgen treatment. In contrast, the effect on muscle fiber diameter was not maintained for this period. This dissociation of androgenic effects on the pattern of innervation from androgenic effects on muscle fiber diameter offers further evidence that the androgenic maintenance of multiple innervation is not dependent on muscle fiber size. In addition, circulating testosterone levels were measured at 50 and 60 days of age in animals similarly treated with androgen or oil from 7 to 34 days of age. By 60 days of age, testosterone levels in hormone-treated animals had dropped below detectability, comparable to levels in oil-treated controls. This provides additional evidence that androgen treatment during juvenile development can have permanent effects on the adult pattern of innervation in the LA muscle.

Aging↗

Androgen locally regulates rat bulbocavernosus and levator ani size.

Adult male rats were gonadectomized, and small Silastic capsules filled with hormone were sutured to each bulbocavernosus and levator ani muscle complex (BC/LA). In the first experiment, one capsule contained testosterone (T), while the capsule on the contralateral muscles contained the antiandrogen hydroxyflutamide (hFl). The intent of this treatment was to provide a focus of androgenic stimulation to the muscles on one side. After 30 days, animals were sacrificed, and the BC/LA muscle pairs were removed, weighed, and compared. BC/LAs receiving T treatment were heavier than those receiving hFl treatment (p less than 0.0001), with an average weight difference of 12%. Muscle fibers from T-treated BCs were significantly larger in diameter than those from contralateral, hFl-treated BCs. These results indicate that androgen exerts its anabolic effect by acting locally upon a cell population within or near the BC/LA. When hFl and blank capsules were implanted in castrated males, the hFl-treated muscles were significantly heavier (by 9%), demonstrating an anabolic effect of hFl in the absence of androgen, and refuting the idea that hFl may have caused local toxic effects in the first experiment. Gonadectomized animals given T versus blank capsules had T-treated muscles that were 8% heavier than the blank-treated side. Muscle weights were also compared in animals receiving bilateral denervation of the BC/LA at the time of T and hFl capsule implantation and gonadectomy; local testosterone treatment failed to affect BC/LA weights in these denervated muscles.

Animals↗

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.

Animals↗

Nitrergic and VIPergic neurons in the choroid and ciliary ganglion of the duck Anis carina.

Immunohistochemistry for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP), and NADPH diaphorase histochemistry, were applied to investigate neurons in the choroid and the ciliary ganglion of the muscovy duck Anis carina. Up to 1000 neurons in the choroid stained for NADPH diaphorase and showed virtually complete colocalization for nNOS immunoreactivity. Almost all of them co-stained for VIP, while about 90% of VIP immunoreactive cell bodies showed colocalization for nNOS. Two-thirds of the neurons were located, mostly singly, at nodes of a wide-meshed nerve plexus in the suprachoroid and were only rarely grouped in ganglia of up to 3 neurons. Numerous varicose nNOS/NADPH-diaphorase-positive nerve fibers were seen around large arterial blood vessels. These fibers derived mainly from paravascular cell bodies that represented about one-third of all choroidal neurons and also displayed costaining for nitrergic markers and VIP. Colocalization of nNOS/NADPH-d and VIP could be demonstrated in most of the perivascular fibers, while slightly more VIP-positive axons in the suprachoroid plexus did not costain for nNOS/NADPH-d. Small-caliber blood vessels and those localized in the choriocapillaris were not endowed with VIP/nNOS/NADPH-diaphorase-positive fibers. A few reactive neuronal cell bodies were also found in ciliary nerves, while most ciliary axons were unstained. In the ciliary ganglion a small subpopulation of neurons showed VIP/nNOS/NADPH-diaphorase colocalization. There were also nNOS/NADPH-d-positive cap-like terminals on ciliary ganglion cells. The presence of VIP/nNOS/NADPH-diaphorase positive neurons and nerve fibers in both the choroid and ciliary ganglion, and in the choroidal perivascular plexus, indicates peripheral nitrergic and VIPergic control of blood flow in the choroid of the duck.

Animals↗

Effect of castration and testosterone administration on the neuromuscular junction in the levator ani muscle of the rat.

The ultrastructure of the neuromuscular junction (n.m.j.) of the androgen-sensitive levator ani muscle was studied in normal adult male rats, in 8-month-old rats castrated at the age of one month and in castrated rats treated with testosterone propionate (TP). Castration does not result in significant changes of the n.m.j. The density of synaptic vesicles and the postsynaptic junctional folds remain practically normal inspite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in pre- and postsynaptic structures. There is slow progressive depletion of synaptic vesicles, appearance of cisternae and coated vesicles in axon terminals, and coalescence of coated vesicles with the plasma membrane. Coated vesicles are also found inside Schwann cells and among junctional folds. Dense core vesicles appear both in the axon terminals and in the postsynaptic area. Collateral sprouting of terminal axons with the formation of new immature junctions is observed. After 35 days of TP administration depletion of synaptic vesicles continues. Glycogen beta-particles, mostly freely dispersed, occasionally seen in axon terminals 7 days after TP administration, subsequently increase in number. In the endplate zone of the muscle fibre increased protein synthesis is indicated by a rapid increase in ribosomes and irregularly located myofilaments and myofibrils. The appearance of n.m.j. after testosterone administration resembles that described after nerve stimulation; the degree of change is however less pronounced.

Animals↗

Testosterone control of endplate and non-endplate acetylcholinesterase in the rat levator ani muscle.

The time course of effects of castration (5-60 days) and testosterone treatment (15-60 days) of adult male rats were examined on the endplate (+EP) and non-endplate (-EP) acetylcholinesterase (AChE) of the androgen-dependent levator ani (LA) muscle. The thiocholine method was used to determine the enzyme activity. Castration caused LA muscle atrophy within 5 days but reduced the -EP and +EP AChE after 10 and 20 days, respectively. Following 30 days castration -EP and +EP AChE reached respectively 41% and 35% of control activity. Testosterone retrieval restored the control values of both muscle weight and total AChE after 15 and 60 days, respectively. Recovery of the +EP AChE preceded that of -EP AChE by 30 days. The results showed that in the rat LA muscle, +EP and -EP AChE depend on a continuous testosterone regulation that predominates at +EP region spreading thereafter to -EP region. Those data suggest a hormone regulation of AChE exerted indirectly through the synthesis and release of neurotrophic substances.

Acetylcholinesterase↗

Electrophysiological and contractile properties of the levator ani muscle after castration and testosterone administration.

Electrical and contractile properties of the levator ani muscle were studied in normal rats, in castrated rats and in castrated rats treated with testosterone. 2. No significant changes in the frequency of miniature end-plate potentials were found 6 months after castration. The frequency increased already 6 h after testosterone treatment; an increase of about 100% was observed after 7 days of testosterone treatment. 3. Castration led to a 2-fold increase of the input resistance of the muscle fibres. After 7 days of testosterone treatment the input resistance was only slightly higher than normal. 4. The weight of the muscle was decreased to 18% of the control value after 6 months castration. It increased to 46% after 7 days of testosterone treatment. 5. The muscles of castrated animals revealed a prolongation of contraction time and marked changes in maximal rate of tension development and half relaxation time. Partial recovery of these parameters was found after 7 days of testosterone treatment. 6. Long-term castration did not induce any denervation-like changes of action potential parameters, and no tetrodotoxin resistance was found in spite of marked muscle atrophy.

Anal Canal↗

MRI evaluation of the levator ani muscle: anatomic correlations and practical applications.

A comparative study of serial anatomic sections in the transverse, frontal and sagittal planes with corresponding MRI sections of the pelvis allowed the authors to define the most suitable sectional planes and MRI modes for a morphologic study of the levator ani muscle. This study shows the value of MRI examination in the assessment of anorectal malformations.

Adolescent↗

Biofeedback is effective treatment for levator ani syndrome.

PURPOSE: The effects of biofeedback (BF) on pain relief and anorectal physiology in patients with levator ani syndrome (LAS) were prospectively studied. METHOD: Sixteen consecutive patients (9 men, 7 women; mean age, 50.1 (range, 39-66) years) with LAS were treated with BF from July 1993 to October 1995. Mean duration of pain was 32.5 (standard error of the mean, 6.7) months. All underwent a full course of BF using a manometric balloon technique. Mean follow-up was 12.8 (standard error of the mean, 2.6) months. Pain score and anorectal physiology tests were administered prospectively by an independent observer before and after BF. RESULTS: After BF, the pain score was significantly improved (before BF: median, 8 (range, 6-10); after BF: median, 2 (range, 1-4); P < 0.02). Analgesic requirements were also significantly reduced (all 16 patients needed nonsteroidal anti-inflammatory drugs (NSAID) before BF; only two patients needed NSAID after BF; P < 0.03). There were no significant changes to the anorectal physiology parameters after BF. To date, there have been no side effects or regressions. CONCLUSION: Although BF had a negligible effect on anorectal physiologic measurements in LAS, it was effective in pain relief, with no side effects.

Anal Canal↗

Perineal excision of rectal prolapse with posterior levator ani repair in elderly high-risk patients.

Perineal excision of rectal prolapse with simultaneous posterior levator ani repair was used to treat 41 elderly patients with rectal procidentia. The majority of the patients had significant associated risk factors. This procedure was performed with minimal morbidity and no mortality. A significant improvement in anal continence was seen in 78 percent of patients. The recurrence rate of rectal prolapse was 4.8 percent.

Aged↗

The role of the levator ani muscle in evacuation, sexual performance and pelvic floor disorders.

This paper reviews the role of the levator ani muscle (LAM) in evacuation, sexual performance and pelvic floor disorders. The LAM fixes the vesical neck, anorectal junction and vaginal fornices to the side wall of the pelvis by means of the suspensory sling and hiatal ligament. On contraction it shares in the mechanism of evacuation (urination, defecation). During the sexual act vaginal distension by the erect penis evokes the vaginolevator and vaginopuborectalis reflexes, with a resulting LAM contraction. The LAM also contracts upon stimulation of the clitoris or cervix uteri, an action mediated through clitoromotor and cervicomotor reflexes. LAM contraction leads to upper vagina ballooning, which acts as receptacle for semen collection, to uterine elevation and straightening and to elongation and narrowing of the vagina. These actions enhance the sexual response and prepare the uterus and vagina for the reproductive process. During ejaculation LAM contraction facilitates semen ejection. Levator subluxation and sagging leads to levator dysfunction syndrome, which may present as pudendal canal syndrome.

Female↗

The histomorphologic structure of the levator ani muscle and its functional significance.

The levator ani muscle (LAM), unlike other striated muscles has resting myoelectric activity, the cause of which is not precisely known. In a recent study this activity was suggested to be related to the presence of smooth muscle bundles in the LAM. The present study investigated this point in 25 cadaveric specimens (10 neonates, 15 adults). Histologic examination of the LAM was performed in 12 specimens, three to six slices being taken from the lateral to the medial side of each muscle, processed and stained with H&E and Masson's trichrome stain. The remaining 13 specimens were studied by direct dissection and photographed. Microscopic examination of specimens from adult cadavers showed that the lateral part of the LAM consisted of purely skeletal muscle bundles, mostly of the small-caliber type and few of the large or intermediate type. As we proceeded medially, smooth muscle bundles started to appear. Examination of slices medial to the midportion of the LAM showed that the muscle began to be separated into two layers: a deep (pelvic) one formed of smooth fibers and a superficial (perineal) one of skeletal fibers, separated by a neurovascular plane. Microscopic examination of specimens from neonates showed that the whole LAM consisted of skeletal fibers; no smooth fibers or separation into two layers were identified. By direct dissection of the adult specimens the medial part of the LAM consisted of two layers, superficial and deep, which were separated by fascia containing vessels and nerves. The muscle was thicker at its medial part and tapered laterally, with loss of demarcation, into two layers. Specimens from neonates showed no differentiation of the LAM, into two layers. It is suggested that the deep layer of the LAM, which was formed of smooth fibers, acts involuntarily, supporting the pelvic viscera by its tone and responding to variations in intra-abdominal pressure by adaptation of this tone. The superficial layer of the LAM, made of skeletal fibers, appears to represent the functional mobile part that acts voluntarily during urination or defecation. The presence of the smooth fibers in the adult LAM and not in neonates seems to be adaptational, caused by the action of intra.abdominal pressure and visceral weight. The skeletal fibers pass into various histologic stages before they transform to smooth fibers.

Adult↗

MR imaging of levator ani muscle recovery following vaginal delivery.

Our aim was to quantify the changes that occur in the levator ani muscles (LA) after vaginal delivery using magnetic resonance imaging. Fourteen women underwent MRI 1 day postpartum. Six of them were also scanned 1, 2, 6 weeks and 6 months after delivery. LA signal intensities and thickness, in areas of the urogenital and the levator hiatus were assessed in the transverse plane. Perineal body position was measured in the sagittal plane. One day postpartum a higher T2-signal intensity of the LA compared to the obturator internus muscle was found in all women and a lower T1-signal intensity in 8 of 12 women. By 6 months these differences were present in only 1 woman in the left LA. An elevation in perineal body position of 13.4 +/- 7.3 mm (P < 0.05), as well as a decrease in the area of the urogenital hiatus by 27% (P<0.05) and of the levator hiatus by 22% (P < 0.05) by 2 weeks postpartum suggest a return of normal LA geometry. LA thickness showed interindividual variations, and a complete loss of LA tissue was found in 1 woman. Changes in LA signal intensity, topography and thickness during the puerperium can be documented using MR imaging.

Adult↗

Testosterone-induced development of the rat levator ani muscle.

The perinatal development of the levator ani (LA) muscle in male and female rats was investigated by measuring the total number of muscle units (MU) (i.e., mononucleate cells, clustered or independent myotubes, and muscle fibers) in transverse semithin sections of the entire muscle and the MU cross-sectional area in 22-day-old fetuses (F22), 1-day-old (D1 = day of birth), 3-day-old (D3), and 6-day-old (D6) newborns. Male muscle contained 350 +/- 64 MU on F22, twice that of the female. The number of MU increased markedly in males from F22, but changed little in females; the number of MU in males was 760% that of females on D6. The MU cross-sectional area was greater in males on F22 (120.8 micron(s)2 +/- 7.5) and D1 (155.2 micron(s)2 +/- 64.8) than in females (F22: 89.2 micron(s) +/- 14.2, D1: 64.1 micron(s)2 +/- 19.7) and dropped to about 30 X micron(s)2 in both sexes on D6. Female rats given a single injection of testosterone propionate (TP) before D7 showed a significant increase in the number of fibers, but no increase in cross-sectional area. TP given after D7 had no effect on the fiber number, but increased the average cross-sectional area. The increase in fiber number induced by postnatal TP treatment was a permanent effect, still quantifiable in 15-month-old females. We conclude that the sexual dimorphism of the rat LA muscle is principally due to a dramatic increase in the MU number in male muscles during the perinatal period, rather than to involution of the fibers in female muscles as it is widely accepted. This increase seems to be, at least partly, under the control of testosterone.

Animals↗

Effect of denervation on the androgen-induced expression of actin and CPK mRNAs in the levator ani muscle of the rat.

In the adult male rat, the castration-induced atrophy of the levator ani (LA) muscle was found to be associated with a decrease in the relative levels of both actin and creatine phosphokinase (CPK) mRNAs. The typical recovery of these two sequences following 5 days of testosterone propionate (TP) replacement therapy was not impaired by the bilateral denervation of the LA. This indicated that TP was the sole trophic factor regulating the plasticity of these two mRNAs and challenged the hypothesis that androgen action might be neuronally mediated. The observation that denervation led to a severe repression of both actin and CPK messages only in the absence of TP replacement therapy suggested that the nerve impulse could play an accessory role in the control of their expression.

Abdomen↗

Sexual activity influences organ weight and androgen metabolism of rat prostate, bulbocavernosus/levator ani muscle and kidney.

Previously we have shown that rats living under heterosexual conditions (HE-rats) have significantly higher weights of androgen target organs like prostate and bulbocavernosus/levator ani muscle (BCLA) than rats living under homosexual conditions (HO-rats). Knowing that androgen metabolism is an important regulator of androgenic action, we have measured in vitro by thin-layer chromatography the testosterone 5 alpha-reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase (3 alpha (beta)-HSDH) activity in prostate and BCLA of both groups. Furthermore, we looked for weight differences of the kidney from HE- and HO-rats. The main results are: (1) The mean apparent Michaelis constant (Km) of 5 alpha-reductase in prostate was identical in both groups, being 0.22 and 0.24 microM for HE- and HO-rats, respectively. (2) The mean 5 alpha-reductase activity was significantly (P less than 0.001; n = 18) lower in prostate of HE- (11.1 +/- 0.5 (SEM) pmol 5 alpha-reduced metabolites X mg protein-1 X h-1 1) than HO-rats (13.9 +/- 0.4). (3) The mean apparent Km of 3 alpha (beta)-HSDH was identical in HE- and HO-rats, being 3.7 and 4.3 microM, respectively. (4) The mean 3 alpha (beta)-HSDH activity was significantly (P less than 0.001; n = 20) lower in prostate of HE- (1.58 +/- 0.05 (SEM) nmol 3 alpha (beta)-reduced metabolites X mg protein-1 X h-1) than HO-rats (1.85 +/- 0.05). (5) The mean 3 alpha (beta)-HSDH activity was significantly (P less than 0.001; n = 24) lower in BCLA of HE- (284 +/- 9.6 (SEM) pmol 3 alpha (beta)-reduced metabolites X mg protein-1 X h-1 than HO-rats (422 +/- 18.7). (6) Besides prostate and BCLA, also the absolute as well as relative weights of the kidney were significantly higher in HE- than HO-rats. (7) It will be discussed that despite various significant differences in androgen metabolism, other factors might be responsible for the organ weight differences of prostate, BCLA and kidney between HE- and HO-rats.

3-Hydroxysteroid Dehydrogenases↗