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.

Tropism of serotonergic neurons towards glial targets in the rat ependyma.

During development, recognition mechanisms between neurons and their targets are necessary for the formation of the neuronal network. Neural connections are synaptic or non-junctional. Both types of communication can be found between neurons and glial elements in the periventricular walls. Serotonergic fibers form synaptic contacts on the specialized ependymocytes of the subcommissural organ, a structure which forms the roof of the third ventricle at its junction with the aqueduct. A network of non-junctional fibers containing both GABA and serotonin spread between the cilia of the classical ependymocytes in the ventricles. These anatomical, morphological and biochemical features suggest a tropism and specific recognition mechanisms between glial elements and serotonergic neurons. This hypothesis can be tested by the study of the innervation of the subcommissural organ and the classical ependyma by grafted embryonic neurons after a chemical destruction of the serotonergic endogenous innervation. Solid implants or cell suspensions prepared from embryonic metencephalon were transplanted to either the third ventricle or the periventricular gray matter in 5,7-dihydroxytryptamine denervated rats. Grafted serotonergic neurons were able to reinnervate the classical ependyma and the subcommissural organ. The fibers forming the supraependymal plexus were non-junctional and contained both serotonin and GABA while those innervating the subcommissural organ formed synaptic contacts and contained only serotonin. The signals capable of inducing the ependymal innervation were specific for serotonergic neurons since catecholaminergic neurons present in the grafts were unable to innervate either classical or specialized ependymocytes. These results demonstrate that glial cells are targets for serotonergic neurons and that the morphological and biochemical characteristics of the serotonergic innervation are closely related to the target cell phenotype.

Testosterone-induced masculinization of the rat levator ani muscle during puberty.

Previous study of the development of the levator ani muscle (LA) revealed that the sex difference in muscle fiber number is under the control of testosterone during the perinatal period. In this study, the development of the LA muscle was examined in both sexes to test the hypothesis that testosterone action determines a further sex difference at the onset of puberty. This period is of key importance because plasma testosterone concentration rises sharply between Postnatal Days 40 and 60 in the male, while in the female testosterone level stays very low. In the male, a transient increase in the satellite cell number occurs between Days 42 and 47, followed by a persistent myonuclei increase. Hence, when the pubertal process has been completed, the myonuclei number is increased by about 50%. Moreover, sexual dimorphism related to the muscle fiber width becomes more marked: before puberty (Day 30), the mean cross-sectional area of muscle fibers is 2-fold higher in males than in females, while between Days 30 and 78, a further hypertrophy is observed which leads to a mean cross-sectional area about 10-fold higher in the male. In the female, during the same period, there is neither satellite cell increase nor myonuclei increase and the fiber cross-sectional area remains constant. Treatment with testosterone before the onset of puberty induces a marked but transient satellite cell increase not only in male but also in female LA muscle as early as 3 days after the time of injection. This cell proliferation is followed by a subsequent increase in the myonuclei number. Results indicate that (1) the levator ani muscle in the male exhibits high sensitivity to testosterone during the pubertal process; during this period, sexual dimorphism related to fiber width and myonuclei number is emphasized; (2) the myonuclei increase is subsequent to the testosterone-induced satellite cell proliferation; and (3) the female LA muscle presents the same sensitivity to testosterone as the male. Hence, the female muscle does not acquire specific male characteristics, owing to the very low concentration of circulating androgens.

[Muscular reconstruction by myogenic cell graft]. / Reconstruction musculaire par greffe de cellules myogéniques.

We have developed an experimental model to test the ability of cultured satellite cells to form new muscle fibers when grafted in an irreversibly injured muscle. Association of X-irradiation to autotransplantation reduced extensor digitorum longus muscles of adult rats to a cystic structure formed by a peripheral rim of surviving muscle fibers surrounding a central space devoid of myofibers. Grafting autologous satellite cells, multiplied and labelled in vitro, into this central space gave rise to new myotubes which developed and matured to form fully differentiated myofibers. Mecanophysiological recordings confirmed the improvement of functional parameters particularly a 4 fold increase of twitch and tetanic tensions in grafted muscles. Furthermore, we examined the role of old basal lamina in architectural organisation of the reconstituted muscle by electron microscopy and immunofluorescence using antibodies to laminin, fibronectin, type IV collagen and heparan sulfate proteoglycan. We observed the persistence of immunoreactivity to all components; anti-laminin antibodies for example, evidenced a clear fascicular organization of ghost basal lamina which are progressively repopulated by the grafted cells. We finally investigated in vivo proliferation of the grafted cells by an autoradiographic study of H3-thymidine incorporation in the regenerated fibers and demonstrate that grafted cells proliferate for, at least, 1 week after cell grafting.

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.

Satellite cell proliferation and increase in the number of myonuclei induced by testosterone in the levator ani muscle of the adult female rat.

We injected adult female rats with 0.25 mg/100 g body weight of testosterone to test the sensitivity to androgens of the levator ani muscle (LAM). Testosterone induced marked hypertrophy characterized by fiber size enlargement, but did not increase the number of fibers. Morphological observations and quantitative data indicated that hypertrophy was accompanied by satellite cell proliferation between Days 1 and 3 after testosterone treatment, and by an increase in the number of myonuclei, which started between Days 2 and 3 after treatment. On Day 30, this increase reached 80% of the initial number of myonuclei. The new myonuclei seemed to result from satellite cell proliferation and from the fusion of some of them with preexistent muscle fibers. These results strongly suggest that testosterone-induced cell proliferation might have a role in developing the sexual dimorphism of the LAM. They also indicate the need to reconsider the currently accepted notion that sexual dimorphism is owing to female LAM involution.

The levator ani of the female rat: a suitable model for studying the effects of testosterone on the development of mammalian muscles.

The present results give evidence by using cytochemical markers of motor end-plates (ChE, AchR) and ultrastructural techniques that the Levator ani (LA) muscle is also present in adult females: it is composed of differentiated and innervated fibres. A significant difference, both on the number of fibres per muscle (n) and on their average cross-sectional area (acsa), was observed between sexes:-male: n = 5300 +/- 687, acsa: 522 +/- 68.6 microns 2;-female: n = 565 +/- 246.9, acsa: 68 +/- 8.6 microns 2. These results suggest that testosterone could control, at least partially, the number and the diameter of muscle fibres during development.