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.     

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.     

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.     

Exogenous testosterone reverses age-related atrophy in a spinal neuromuscular system

Aging is associated with a variety of pathologies, including motor dysfunctions and reductions in sexual behavior. In male rats, declines in sexual behavior during the aging process may be caused in part by the loss of the lumbar spinal cord motoneurons that innervate the penile musculature. Alternatively, declining sexual behavior may be caused by the precipitous reductions in circulating testosterone that occur during aging. In this paper, we report two experiments examining these issues. In Experiment 1, we counted motoneurons in the lumbar motor nuclei and measured several androgen-sensitive morphological properties of the penile muscles and their innervating motoneurons at several time points during the aging process. Motoneuron number in the lumbar nuclei did not change over time, even with very advanced age. In contrast, the penile muscles and their innervating motoneurons underwent profound atrophy, with muscle weight and motoneuron dendritic length declining to less than 50% of young adult levels. In Experiment 2, we treated aged animals with exogenous testosterone, and then examined their penile neuromuscular systems for morphological changes. Testosterone treatment, both acute and chronic, completely reversed age-related declines in the weight of the penile muscles and in the soma size and dendritic length of their innervating motoneurons. Together, these data suggest that reductions in male sexual behavior during the aging process are caused primarily by declines in testosterone levels rather than motoneuron loss. Furthermore, they raise the possibility that testosterone treatment could play an important role in maintaining neuronal connectivity in the aging body.     

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.     

Novel instrumented probe for measuring 3D pressure distribution along the vaginal canal

We developed an intravaginal instrumented probe (covered with a 10 × 10 matrix of capacitive sensors) for assessing the three-dimensional (3D) spatiotemporal pressure profile of the vaginal canal. The pressure profile was compared to the pelvic floor (PF) digital assessment, and the reliability of the instrument and repeatability of the protocol was tested. We also tested its ability to characterize and differentiate two tasks: PF maximum contraction and Valsalva maneuver (maximum intra-abdominal effort with downward movement of the PF). Peak pressures were calculated for the total matrix, for three major sub-regions, and for 5 planes and 10 rings throughout the vaginal canal. Intraclass correlation coefficients indicated excellent inter- and intra-rater reliability and intra-trial repeatability for the total and medial areas, with moderate reliability for the cranial and caudal areas. There was a moderate correlation between peak pressure and PF digital palpation [Spearman’s coefficient r = 0.55 (p < 0.001)]. Spatiotemporal profiles were completely different between tasks (2-way ANOVAs for repeated measures) with notably higher pressures (above 30 kPa) for the maximum contraction task compared to Valsalva (below 15 kPa). At maximum contraction, higher pressures occurred in the mid-antero-posterior zone, with earlier peak pressure onsets and more variable along the vaginal depth (from rings 3 to 10-caudal). During Valsalva, the highest pressures were observed in rings 4–6, with peak pressure onsets more synchronized between rings. With this protocol and novel instrument, we obtained a high-resolution and highly reliable innovative 3D pressure distribution map of the PF capable of distinguishing vaginal sub-regions, planes, rings and tasks.     

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.     

Group chorusing as an intragroup signal in the greater ani,a communally breeding bird

Group living often requires maintaining dynamic and varied relationships with fellow group members, while simultaneously monitoring and interacting with external competitors. Group members in many social species vocalize together to produce duets or choruses—coordinated, often conspicuous vocal displays—that may play a role in these interactions. Compared with male–female duets, however, relatively little research exists on the function and adaptive significance of group choruses, which involve three or more individuals. Here we investigate chorusing behavior in the greater ani (Crotophaga major), a communally breeding cuckoo that nests in stable social groups of four to eight unrelated individuals. Groups may remain together for several years on the same nesting territory, and groups occasionally destroy each other's clutches in conflicts over high-quality territories. We asked whether the raucous, highly stereotyped choruses performed by ani groups are primarily used for intra- or intergroup communication, and whether they contain information about the identity of the social group and the number of birds vocalizing. Behavioral observations and acoustic recordings from three breeding seasons revealed that choruses typically occurred during social interactions within the group (78% of choruses) or in response to a predator or extra-group individual (17%) and only rarely in intergroup interactions (4%). Consistent with this finding, choruses did not reliably reflect the number of birds vocalizing, and we found only limited evidence for group-specific acoustic signatures (driven by a single group whose choruses were highly distinct). These results suggest that group choruses play an important role in intra-group signaling, potentially in contexts such as group formation, reinforcement of social bonds within the group, and/or collective decision-making, and they motivate new research questions about the role of collective signaling in social evolution.     

Breeding status affects motoneuron number and muscle size in naked mole‐rats: Recruitment of perineal motoneurons?

Naked mole‐rats live in large colonies and exhibit a strict reproductive hierarchy. Each colony has one breeding female and one to three breeding males; all other individuals are nonreproductive subordinates. Subordinates show a remarkable lack of sex differences in behavior and anatomy, but can become reproductive if removed from the colony. We recently reported that the striated perineal muscles and their innervating motoneurons, which are sexually dimorphic in all other mammals examined to date, are not dimorphic in subordinate naked mole‐rats. Here we asked whether sexual differentiation of this neuromuscular system occurs when a subordinate becomes a breeder. The size and number of cells within Onuf's nucleus (homologue of the rat spinal nucleus of the bulbocavernosus) as well as perineal muscle volume were examined in subordinate and breeding naked mole‐rats of both sexes. Sex differences in perineal motoneurons were not observed, regardless of social status. To our surprise, however, counts of motoneurons in Onuf's nucleus were increased ～30% in breeders of both sexes. This was accompanied by a reciprocal decrease in cells in Onuf's nucleus that were characterized by small soma size, and lacked a clear nucleus or nucleolus. Although not exhibiting typical motoneuron morphology, some of these small cells were positive for the motoneuron marker, SMI‐32. The neuronal changes correlate with increased perineal muscle volumes in breeders. We propose that small, relatively undifferentiated cells are recruited to the pool of large Onuf's nucleus motoneurons when subordinate naked mole‐rats become breeders. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006