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.     

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.     

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

Summary 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 in spite of marked atrophy of the muscle. TP administration for 7 days results in marked changes in preand 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 -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.The authors wish to acknowledge the skillful technical assistance of Mrs. L. Vedralová     

Effect of foreign innervation on the androgen-sensitive levator ani muscle of the rat

Summary Cross-union of the tibial with the pudendal nerve innervating the androgen-sensitive levator ani (LA) muscle of male rats, results in reversal of the histochemical muscle fibre pattern concerning myofibrillar ATPase, succinate dehydrogenase and phosphorylase enzyme activities. The homogeneous muscle fibre pattern of the LA muscle is changed to a mosaic pattern of muscles normally innervated by the tibial nerve. The success of the hetero-reinnervation is shown by practically full recovery of muscle weight and of isometric twitch-contraction properties of the LA muscle. Castration of 2-months duration, i. e. lack of the male sex hormone, leads to marked atrophy but no change in histochemical muscle fibre pattern. Hetero-reinnervation of the LA muscle results in change of histochemical enzyme pattern even if the cross-union of nerves is performed after long periods of castration leading to very marked decrease of muscle fibre size. However, testosterone application alone after castration increases markedly muscle fibre size but does not lead to reversal of muscle fibre pattern. The myotropic hormonal influence on the target (LA) muscle is therefore primarily of myogenic origin and specificity of hormonal action is maintained even with a foreign nerve innervating the muscle. The experiments, thus, provide evidence for the differentiation of specific neural influences affecting muscle fibre pattern and hormonal influences in respect to the myotropic action of the sex hormone on the androgen-sensitive LA muscle.     

Cytomorphometry of skeletal muscle: The influence of age and testosterone on the rat M. levator ani

Summary The levator ani muscle of the rat was examined by correlated light and electron microscopic morphometry. Corrections were made for shrinkage, compression, and differences in stretching. Age, castration, and subsequent testosterone treatment do not affect the fiber number, the filament lattice, and the size of the filaments and myonuclei. The fibers in intact growing males increase in width and length. The number of myonuclei rises, although relatively slower than the amount of contractile material.Castration, performed at six weeks, partially suppresses fiber growth. The increase of mean fiber width is more strongly inhibited than that of fiber length. Myonuclear multiplication is almost completely arrested in castrates, and the amount of contractile material per myonucleus is lower than in intact males of equal age.Testosterone, administered at about two months following orchidectomy, highly accelerates the transversal fiber growth, but fiber length is not significantly influenced. Between the fourth and seventh day of treatment a marked increase in myonuclear number occurs.Analysis of the frequency distribution of the individual fiber widths, which is logarithmic-normal in intact males, revealed that the hormonal influence on the net result of protein anabolism and catabolism markedly differs in the various fibers of a single muscle.With the technical assistance of Tineke J. Hoogenboezem.     

The influence of age and testosterone on the ribosomal population in the m. levator ani and a thigh muscle of the rat

Summary The influence of age, castration, and subsequent testosterone treatment on the population of (poly)ribosomes in rat skeletal muscle fibers was studied, using a procedure which clearly differentiates ribonucleoprotein particles from glycogen granules. The m. levator ani, known to be highly reactive to testosterone, was compared with a thigh muscle.The effect of increasing age is about the same in both muscles: the concentration of intermyofibrillar ribosomes decreases, in contrast to the ribosomal abundance in the paranuclear cones of sarcoplasm, which remains approximately constant. Castration and testosterone treatment do not affect the ribosomal concentration in the thigh muscle, but in the m. levator ani the following effects were observed. Castration, performed at six weeks, elicits a marked decrease of the paranuclear ribosomes, but the intermyofibrillar concentration does not noticeably differ from the intact controls. Testosterone, administered at three months following orchidectomy, causes a rapid rise in the paranuclear population. The concentration of the intermyofibrillar ribosomes shows a transient increase. Very early, the concentration of glycogen granules is augmented also, both in between and within the myofibrils. These observations are related to quantitative changes of the contractile system reported previously. It is emphasized that the effects discussed highly depend on the age of orchidectomy.With the technical assistance of Tineke J. Hoogenboezem.     

Passive biomechanical properties of human cadaveric levator ani muscle at low strains

The objective of this study was to measure and model the passive biomechanics of cadaveric levator ani muscle in the fiber direction at low strains with a moderately slow deformation rate. Nine levator ani samples, extracted from female cadavers aged 64 to 96 years, underwent preconditioning and uniaxial biomechanical analysis on a tensile testing apparatus after the original width, thickness, and length were measured. The load extension data and measured dimensions were used to calculate stress–strain curves for each sample. The resulting stress–strain curves up to 10% strain were fit to four different constitutive models to determine which model was most appropriate for the data. A power-law model with two parameters was found to fit the data most accurately. Constitutive parameters did not correlate significantly with age in this study; this may be because all of the cadavers were postmenopausal.     

Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.     

A novel pathway for MuSK to induce key genes in neuromuscular synapse formation

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.     

Anisotropic effects of the levator ani muscle during childbirth

Pelvic floor dysfunction and pelvic organ prolapse have been associated with damage to the levator ani (LA) muscle, but the exact mechanisms linking them remain unknown. It has been postulated that factors such as vaginal birth and ageing may contribute to long-term, irreversible LA muscle damage. To investigate the biomechanical significance of the LA muscle during childbirth, researchers and clinicians have used finite element models to simulate the second stage of labour. One of the challenges is to represent the anisotropic mechanical response of the LA muscle. In this study, we investigated the effects of anisotropy by varying the relative stiffness between the fibre and the matrix components, whilst maintaining the same overall stress–strain response in the fibre direction. A foetal skull was passed through two pelvic floor models, which incorporated the LA muscle with different anisotropy ratios. Results showed a substantial decrease in the magnitude of the force required for delivery as the fibre anisotropy was increased. The anisotropy ratio markedly affected the mechanical response of the LA muscle during a simulated vaginal delivery. It is apparent that we need to obtain experimental data on muscle mechanics in order to better approximate the LA muscle mechanical properties for quantitative analysis. These models may advance our understanding of the injury mechanisms of pelvic floor during childbirth.     

Pre‐ and postsynaptic mechanisms in Hebbian activity‐dependent synapse modification

We have used a three compartment tissue culture system that involved two separate populations of cholinergic neurons in the side compartments that converged on a common target population of myotubes in the center compartment. Activation of the axons from one population of neurons produced selective down‐regulation of the synaptic inputs from the other neuronal population (when the two inputs innervated the same myotubes). The decrease in heterosynaptic inputs was mediated by protein kinase C (PKC). An activity‐dependent action of protein kinase A (PKA) was associated with the stimulated input and this served to selectively stabilize this input. These changes associated with PKA and PKC activation were mediated by alterations in the number of acetylcholine receptors at the neuromuscular junction. These results suggest that neuromuscular electrical activity produces postsynaptic activation of both PKA and PKC, with the latter producing generalized synapse weakening and the former a selective synapse stabilization. Treatment of the neuronal cell body and axon to increase PKC activity by putting phorbal ester (PMA) in the side chamber did not affect synaptic transmission (with or without stimulation). By contrast, PKA blockade in the side compartment did produce an activity‐dependent decrease in synaptic efficacy, which was due to a decrease in quantal release of neurotransmitter. Thus, when the synapse is activated, it appears that presynaptic PKA action is necessary to maintain transmitter output. Published 2002 Wiley Periodicals, Inc. J Neurobiol 52: 241–250, 2002     

Synapse elimination occurs late in the hormone-sensitive levator ani muscle of the rat

Using tetranitroblue tetrazolium (TNBT) to stain neuromuscular synapses, we compared the development of the adult pattern of innervation in two fast-twitch muscles in the rat: the androgen-sensitive levator ani (LA) and the extensor digitorum longus (EDL), which is not thought to be androgen sensitive. We found that about 18% of adult LA muscle fibers, but only about 2% of adult EDL fibers, are multiply innervated. Moreover, synapse elimination occurs substantially later in the LA compared with the EDL. At 2 weeks after birth, the EDL is already predominantly singly innervated, whereas the LA is still predominantly multiply innervated. The apparent delay in the normal time course of synapse elimination in the LA corresponds to a similar delay in other aspects of neuromuscular development (the time course of appearance of axonal retraction bulbs, the growth of fibers, and the development of adult motor terminal morphology). Finally, motor terminals change during synapse elimination from morphologies resembling growth cones to the adult form of neuromuscular synapses. Because the period of synapse elimination is significantly different for muscles that differ in their androgen sensitivity, hormonal sensitivity may represent an important property of motoneurons or muscle fibers influencing the normal time course of neuromuscular synapse elimination in rats. Thus, androgen might regulate the normal ontogenetic process of synapse elimination.     

An activity-dependent determinant of synapse elimination in the mammalian brain

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Phosphorylation of p70s6 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.     

Protein phenotype and gene expression in the rat perineal levator ani muscle

The rat perineal levator ani (LA) and bulbocavernosus (BC) muscles are homogeneously type 2B fibers as determined by Ca, Mg-ATPase activity. The LA and extensor digitorum longus (EDL) muscles contain similar quantities of creatine kinase and several glycolytic enzymes despite significant differences in fiber composition. The LA muscles synthesizes and accumulates only the fast isoforms of protein C, myosin heavy chain and myosin light chains.     

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.     

Importance of target innervation in recovery from axotomy-induced loss of androgen receptor in rat perineal motoneurons

In adult male rats, axotomy of the spinal nucleus of the bulbocavernosus (SNB) motoneurons transiently down-regulates androgen receptor (AR) immunoreactivity. The present study investigates the importance of target reinnervation in the recovery of AR expression in axotomized SNB motoneurons after short (up to 5 days) and long (1 to 6 weeks) periods of recovery. In the long-term recovery experiment, animals were divided into two groups. In one, the two stumps of the cut pudendal nerve, which carries the axons of the SNB motoneurons, were sutured together immediately after axotomy. In the second group, the proximal stump was ligated immediately after axotomy to prevent target reinnervation. Axotomy of the SNB motoneurons caused a significant down-regulation in AR immunoreactivity within 3 days. At 6 weeks, AR immunoreactivity was still depressed in ligated animals but had recovered to control levels in resutured animals. The recovery in the resutured group was coincident with the first signs of reinnervation of the target perineal muscles, although reinnervation seemed to lag behind AR immunoreactivity. SNB soma size was significantly reduced 2 weeks after axotomy and returned to control levels after 6 weeks of recovery only in the resutured animals. These findings suggest that the target perineal muscles play a role in the regulation of AR expression and androgen sensitivity in the SNB motoneurons, perhaps mediated by muscle-derived trophic factors. © 1995 John Wiley & Sons, Inc.     

Overexpression of the CT GalNAc transferase in skeletal muscle alters myofiber growth, neuromuscular structure, and laminin expression.

Carbohydrates have been shown to mediate or modulate a number of important events in the development of the nervous system; however, there is little evidence that they participate directly in the development of synapses. One carbohydrate structure that is likely to be important in synaptic development of the neuromuscular junction is the CT carbohydrate antigen [GalNAcbeta1,4[NeuAcalpha2,3]Galbeta1(-3GalNAc or -4GlcNAc)]. The synaptic localization of the CT antigen is due to the presence of the terminal beta1,4 GalNAc linkage, and such linkages are localized to the neuromuscular junction in many species. Here we show that an enzyme that can create the synaptic CT structure, the CT GalNAc transferase, is also confined to the neuromuscular junction in mice. Using transgenic mice, we show that overexpression of the CT GalNAc transferase in extrasynaptic regions in skeletal myofibers caused as much as a 60% reduction in the diameter of adult myofibers and an order of magnitude increase in satellite cells. Neuromuscular junctions of transgenic mice had severely reduced numbers of secondary folds, Schwann cell processes were present in the synaptic cleft, and secondary folds were often misaligned with active zones. In addition, multiple presynaptic specializations occurred on individual myofibers. In addition, some normally synaptic proteins, including laminin alpha4, laminin alpha5, utrophin, and NCAM, were expressed along extrasynaptic regions of myofibers. One of the muscle proteins that displayed increased glycosylation with the CT antigen in the transgenic mice was alpha-dystroglycan. These experiments provide the first in vivo evidence that a synaptic carbohydrate antigen has important roles in the development of the neuromuscular synapse and suggest that the CT antigen is involved in controlling the expression of synaptic molecules.