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.     

Functional properties of muscle autografts substituted for the rat levator ani muscle

The present report deals with the functional properties (contraction parameters and neuromuscular transmission) of muscle grafts and transposed muscles substituted for the levator ani muscle in the rat. The experiments were divided into four main groups. Group I - the levator ani [LA] was excised and replaced in its own bed. Group II - the extensor digitorum longus, a fast muscle (with or without predenervation), and Group III - the soleus, a slow twitch muscle, were substituted for the LA. In group IV, the gracilis anterior muscle was either freely grafted in place of the LA or transposed a) with intact innervation, b) with its vascular supply intact or c) with preserved neuro-vascular supply. The optimum results of twitch and tetanic tension, and the amplitude of stimulation EMG responses was found in the case of LA resutured into its own bed and in the case transposition of the gracilis anterior muscle had been performed with its neuro-vascular supply intact in place of the LA. On the basis of these functional findings and morphological and anatomical observations (Grim et al. 1982), a surgical procedure is suggested for patients with anal incontinence (Grim et al. 1981, Dittertová-Vlasáková et al. 1982).     

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.     

Cytosolic androgen receptor in regenerating rat levator ani muscle.

The development of the cytosolic androgen receptor was studied after degeneration and regeneration of the rat levator ani muscle after a crush lesion. Muscle regeneration appears to recapitulate myogenesis in many respects. It therefore provides a model tissue in sufficiently in large quantity for investigating the ontogenesis of the androgen receptor. The receptor in the cytosol of the normal levator ani muscle has binding characteristics similar to those of the cytosolic receptor in other androgen-sensitive tissues. By day 3 after a crush lesion of the levator ani muscle, androgen binding decreased to 25% of control values. This decrease was followed by a 4-5 fold increase in hormone binding, which attained control values by day 7 after crush. Androgen binding remained stable at the control value up to day 60 after crushing. These results were correlated with the morphological development of the regenerating muscle after crushing. It is concluded that there is little, if any, androgen receptor present in the early myoblastic stages of regeneration; rather, synthesis of the receptor may occur after the fusion of myoblasts and during the differentiation of myotubes into cross-striated muscle fibres.     

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.     

Androgen receptor expression in satellite cells of the neonatal levator ani of the rat

Androgens are thought to mediate sexual differentiation of spinal nucleus of the bulbocavernosus (SNB) motoneurons via actions on androgen receptors (ARs) within their target muscles bulbocavernosus and levator ani (LA). However, the cells within these muscles which mediate masculinization of the SNB remain undefined. Until recently, myocytes were thought to be the most likely candidate cell type. However, genetic tests of AR function in myocytes have failed to support a sufficient role for these cells in producing masculine SNB morphology, suggesting the involvement of other cell types. To identify other candidate cell types in the LA, we evaluated whether satellite cells or fibroblasts express AR. Fluorescent immunohistochemistry and confocal microscopy were used to evaluate whether satellite cells and fibroblasts express AR in neonatal male and female rats in the LA and an adjacent sexually monomorphic control muscle (CM). We found that a small proportion of satellite cells in the LA express AR and that this proportion is significantly greater in the LA compared to the CM. No sex differences were found between the proportions of satellite cells expressing AR in either muscle. Less colocalization of satellite cells and AR was seen in postnatal day 3 muscle than in postnatal day 1 muscle. In contrast, only negligible amounts of fibroblasts labeled with S100A4 express AR in either the LA or the CM. Together, findings support satellite cells, but not fibroblasts, as a candidate cell type involved in the sexual differentiation of the SNB neuromuscular system. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 448–454, 2013.     

Depressed translational activity in the androgen sensitive levator ani muscle of the rat

The androgen-dependent levator ani (LA) muscle of the rat provides a suitable model to explore the molecular mechanism of steroid hormone action in target tissues. The objective of the present series of experiments was to study the effect of gonadectomy (GDX) and androgen replacement therapy on the in vitro protein synthetic capacity of the LA muscle. The incorporation of labeled methionine into the contractile protein fraction of the LA muscle maintained in organ culture decreases in a time-dependent manner following GDX. Translation of total polyadenylated mRNA in the rabbit reticulocyte translation system revealed that the decrease in protein synthetic capacity was not associated with differences in the template activity of the mRNA derived from GDX tissue. However, when polyribosomes were used to direct the same in vitro synthesis system, a significant time-dependent loss of translational activity was observed following GDX. The polyribosomes of the LA muscle of control and GDX rats were shown to contain equivalent amount of rRNA and mRNA of comparable translation efficiency. Collectively the results of these experiments indicate that the decrease in protein synthetic capacity of the LA muscle in androgen deficient rats is due, in part, to a repression of the translation process associated to the functional integrity of polyribosomes.     

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.     

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.     

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.     

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.     

Histochemical fiber types characteristics in the normal and the persistent levator ani muscle of the rat

Summary Morphological features of the persistent levator ani (LA) muscle of the female rat normally undergoing involution but maintained by application of testosterone propionate to newborn animals were compared with that of the normal LA muscle of untreated male rat. The two muscles differ in number and size of muscle fibers.Using histochemical methods for myofibrillar adenosine triphosphatase (ATPase), mitochondrial -glycerophosphate dehydrogenase (-GPDH) and succinic dehydrogenase (SDH) the distribution of these enzymes in individual muscle fiber types was studied.ATPase and -GPDH activities show a homogenously positive reaction in the muscle fibers of the male rat, whereas a small portion of fibers with low activity is found in the persistent muscle of female rat.The most pronounced difference concerns SDH activity, i.e. two kinds of fibre types are barely discernible with prolonged incubation in LA muscle of male rat, but three basic fibre types (white, intermediate and red) are clearly distinguished in LA muscle of the female rat.The results are discussed in relation to neural and hormonal influences on histochemical features in cross-striated muscle.This work was supported by a grant from the Muscular Dystrophy Association of America to the Institute of General Pathology, University of Padua, Italy.