Satellite cells of the rat soleus muscle in the process of compensatory hypertrophy combined with denervation

Compensatory hypertrophy was induced in the rat soleus muscle by sectioning the tendon of the ipsilateral gastrocnemius and plantaris muscle. Seven days after tenotomy of synergistic muscles, when soleus hypertrophy attains about 40%, the number of satellite cells (expressed as percentage of all muscle nuclei found in the same cross-sections) as revealed by electron microscopy, was increased from 5.8+/-0.06% in the normal soleus muscle to 16.6+/-1.26%. After four days' denervation of the soleus muscle the percentage of satellite cells was increased to 7.2+/-0.62%. In experiments where hypertrophy of the soleus muscle was combined with denervation three days after tenotomy of synergists, and examined after another four days (during which time it loses, as has previously been shown, over 40% of its predenervation weight), the number of satellite cells was greatly increased to 29.9+/-3.42%. This increase is apparently due to two independent processes which take place during the first postoperative period: a) mitotic division of satellite cells during the early stages of compensatory hypertrophy and b) pinching off of muscle nuclei from rapidly atrophying muscle fibres due to subsequent denervation. Activation of satellite cells was mainly manifested by expansion of smooth and especially of rough endoplasmic reticulum, a rich Golgi complex, high pinocytotic activity, increased number of ribosomes and by nuclear changes. Concomitantly with the increased number of satellite cells, proliferation of fibroblasts, macrophages and mast cells could be observed.     

去神经后小鼠骨胳肌胞纳的增加和卫星细胞增殖

用生物化学和体外培养法研究了小鼠骨胳肌的胞纳增加和卫星细胞增殖的关系。结果表明：（１）去神经４ｄ或６ｄ的肌肉可引起胞纳的增和卫星细胞的增殖;（２）放线菌素Ｄ抑制正常肌肉的卫星细胞激活和胞纳作用;（３）在去神经的肌肉中,放线菌素Ｄ抑制了卫星细胞增殖的同时还抑制了胞纳的增加,但不能去神经肌肉的萎缩。上述结果：肌肉的卫星细胞增殖和胞纳增加可能发生于去神经后某些因素的出现,或者胞纳的增加即是卫星细胞增殖的     

Prenatal and postnatal development of the small intestine in the insectivore Suncus murinus

The development of the small intestine in the insectivore Suncus murinus was noted during the period from 21 days' gestation to 20 days after birth. At 21 days of gestation, the proximal small intestine exhibited the beginning of villus formation, whereas the distal small intestine preserved the stratified epithelium. Stratified epithelium in the distal small intestine changed into a single layer by 24 days' gestation. At 26 days' gestation, each epithelial cell was immature; but by 28 days mature-looking epithelial cells were found. The shape of the villi changed from cuboid to columnar during the same period. The connective-tissue cores of the villi began to develop at 7 days after birth in the proximal small intestine and at 15 days after birth in the distal small intestine. Crypts appeared at 15 days after birth. Endocytosis of epithelial cells took place at 28 days of gestation. In the proximal small intestine, supranuclear vesicle clusters were observed first at birth; they began to decrease both in number and size at 10 days' gestation and then disappeared completely by 20 days after birth. In the distal small intestine, large supranuclear vacuoles were observed first at 28 days of gestation. Although these vacuoles invariably were found up to 15 days after birth, they also disappeared completely by 20 days. Epithelial cells showed a structure similar to those of the adult after weaning.     

TSH-stimulated increases in calcium uptake and calmodulin levels in thyroid cells

This is the first report to show that polypeptide hormone increases cellular calmodulin contents. In cultured porcine thyroid cells, 6 days' exposure to TSH (above 0.02 mU/ml) increased cellular calmodulin contents. Six days' exposure to TSH also increased calcium uptake in thyroid cells. This TSH-stimulated increase in calcium uptake was partly due to the increase in cellular calmodulin contents.     

Estradiol-induced adenohypophyseal growth reaction and beta-adrenergic receptors.

The effect of administration of estradiol benzoate on beta-adrenergic receptors of rat adenohypophyseal cells was studied. Twenty days' administration of estradiol benzoate was followed by an increase of adenohypophyseal weight and a decrease in specific binding of 3H-dihydroalprenolol (3H-DHA). In contrast to thyroid hormone treatment which induced an increase in 3H-DHA binding, thyroid hormone treatment decreased both the growth reaction and the reaction of beta-adrenergic receptors after estradiol. Although the relationship between the adenohypophyseal receptors and the growth reaction is unclear, changes in beta-adrenergic receptors after hormonal therapy can be one of pathophysiological conditions that may influence this reaction.     

Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation

 The objectives of these studies were to determine if (1) hypertrophy-stimulated myogenic regulatory factor (MRF) mRNA increases occur in the absense of proliferating satellite cells, and (2) acute hypertrophy occurs without satellite cell proliferation. Adult and aged quails were exposed to 0 or 2500 Rads gamma irradiation, and then wing muscles were stretch-overloaded for 3 or 7 days. MRF mRNA levels in stretch-overloaded and contralateral anterior latissimus dorsi (ALD) muscles were determined after 3 days; hypertrophy was determined after 7 days. The elimination of proliferating cells in irradiated muscles was verified histologically by bromodeoxyuridine incorporation. Relative levels of MRF4, MyoD, and myogenin mRNA were elevated 100%–400% in stretch-overloaded ALD muscles from irradiated adult quails indicating that satellite cell proliferation was not a prerequisite for MRF mRNA increases. Myogenin was the only MRF that exhibited mRNA increases that were lowered by irradiation. This suggests that satellite cells contribute only to myogenin mRNA increases in non-irradiated adult muscles following 3 days of stretch-overload. Stretch-overloaded ALD muscles from aged quails had a relative increase in myogenin mRNA of ∼150%. The myogenin increase was the same in non-irradiated and irradiated aged animals and also the same as that in stretch-overloaded muscles from irradiated adult quails. Together, these data indicate that attenuated increases in MRF expression in muscles from aged animals are attributable to lower satellite cell MRF expression. ALD muscle masses and protein contents in adult irradiated quails approximately doubled after 7 days of stretch-overload demonstrating hypertrophy despite the elimination of satellite cell proliferation. Received: 5 June 1998 / Accepted: 19 November 1998     

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.     

Skeletal muscle denervation increases satellite cell susceptibility to apoptosis

Peripheral motor nerve trauma severely compromises skeletal muscle contractile function. Satellite cells respond to denervation by dividing multiple times, ultimately fusing with other satellite cells or myocytes to form new muscle fibers. After chronic denervation, satellite cell numbers decline dramatically, impairing the ability to regenerate and repair myofibers. This satellite cell depletion may contribute to the mechanical deficit observed in denervated or reinnervated muscle. Apoptosis, an evolutionarily conserved form of cell suicide, is a potential mechanism for satellite cell depletion in denervated skeletal muscle. This work tested the hypothesis that skeletal muscle denervation increases satellite cell susceptibility to apoptotic cell death. Adult rats underwent sciatic nerve transection to denervate the distal hindlimb musculature; rats of similar age without the operation served as controls. Two, 6, 10, or 20 weeks after denervation (n = 6 each group), the gastrocnemius and soleus were excised, enzymatically digested, and plated for satellite cell culture. After reaching 95 percent confluence, satellite cells were treated for 24 hours with tumor necrosis factor-alpha (20 ng/ml) and actinomycin D (250 ng/ml), known pro-apoptotic agents. Immunostaining for activated caspases, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and hematoxylin and eosin staining were performed to identify apoptotic satellite cells. Percentages of apoptotic cells were quantified histomorphometrically. In addition, the presence or absence of bcl-2 and bax was determined by Western blot analysis of control, 6 weeks of denervation, and 10 weeks of denervation specimens. At 6 and 10 weeks after nerve transection, TUNEL and caspase activity were increased more than two-fold in satellite cells isolated from denervated muscle compared with those isolated from control muscle (p < 0.05). In all experimental groups, retention of adherence to the collagen-coated substrate was strongly associated with satellite cell survival. Western blot analysis revealed that adherent satellite cells from all groups expressed both bcl-2 and bax. These data support the authors' hypothesis that skeletal muscle denervation increases satellite cell susceptibility to apoptotic cell death. Apoptosis may play a causative role in the depletion of satellite cells in long-term denervated skeletal muscle.     

Trachea enhances neurite regeneration from adult rat nodose ganglia in vitro

Trachea is intensely innervated with vagal afferent nerve fibers, and may play an important role in vagus nerve regeneration after axonal injury caused by trauma and surgical operation. We investigated the effects of tracheal tissue on neuronal cell survival and neurite regeneration in adult rat nodose ganglia (NG) in vitro. Co-culture with trachea significantly increased the average number of neurites regenerated from transected nerve terminals of NG explants, from 73.7 to 154.2 after 3 days, from 68 to 186.7 after 5 days, and from 31 to 101.5 after 7 days in culture. Dissociated NG neurons could continue to survive and extend neurites only in the co-existence with satellite cells in collagen gel. Co-cultured trachea improved the ratios of survival and neurite-bearing cells of NG neurons, from 56.7% and 11.1% to 72.3% and 37.6% after 4 days, and from 41.1% and 20.3% to 56.4% and 47.2% after 7 days in culture, respectively. These results imply that tracheal tissue secretes a factor, which could enhance neuronal cell survival and neurite regeneration in NG in the presence of satellite cells in vitro.     

Concomitant increases in myonuclear and satellite cell content in female trapezius muscle following strength training

A skeletal muscle fibre maintains its cytoplasmic volume by means of hundreds of myonuclei distributed along its entire length. Therefore it is hypothesised that changes in fibre size would involve modifications in myonuclear number. In this study, we have examined whether 10 weeks of strength training can induce changes in the number of myonuclei and satellite cells in female trapezius muscles. Biopsies were taken pre- and posttraining from the upper part of the descending trapezius muscle of nine subjects. Muscle samples were analysed for fibre area and myonuclear and satellite cell number using immunohistochemistry. There was a 36% increase in the cross-sectional area of muscle fibres. The hypertrophy of muscle fibres was accompanied by an approximately 70% increase in myonuclear number and a 46% increase in the number of satellite cells. Myonuclei number was positively correlated to satellite cell number indicating that a muscle with an increased concentration of myonuclei will contain a correspondingly higher number of satellite cells. The acquisition of additional myonuclei appears to be required to support the enlargement of multinucleated muscle cells following 10 weeks of strength training. Increased satellite cell content suggests that mitotic divisions of satellite cells produced daughter cells that became satellite cells. Accepted: 30 November 1999     

Loss And Survival of Spiral Ganglion Neurons in the Guinea Pig After Intracochlear Perfusion with Aminoglycosides

Loss of cochlear hair cells results in a loss of ganglion cells and further neurodegenerative changes throughout the auditory pathway. Understanding more about the early stages of ganglion cell loss in vivo may lead to ways of ameliorating or preventing the loss of these neurons. To examine these stages, the effects of intracochlear perfusion with aminoglycoside antibiotics on the organ of Corti and spiral ganglion cells were evaluated in young adult guinea pigs at survival periods ranging from 1 hour to 12 weeks, using immunocytochemical and ultrastructural techniques. At 1 hour survival a base-to-apex gradient of damage was indicated in the cochlea by the appearance of severely damaged hair cells and injured ganglion cells in the basal coil while in the apical coil, hair cells were damaged but intact and ganglion cells appeared normal. By 4 hours the appearance of severely disrupted hair cells and damaged ganglion cells had extended throughout the cochlea. The ultrastructural appearance of many injured ganglion cells demonstrated features characteristic of cell death including condensed cytoplasm, non-marginal clumping of nuclear chromatin, and wrinkled nuclear membrane. Despite the loss of many ganglion cells, a population of these cells remained at 12 weeks survival. These contained large amounts of rough endoplasmic reticulum, were unmyelinated apart from the central process and were surrounded by satellite cells. These features are typical of ganglion cells during development, before the onset of hearing. Immunolabelling of cochlear whole mounts after hair cell destruction with protein gene product 9.5 (PGP 9.5) revealed the presence of neural elements in the organ of Corti at up to 12 weeks survival. These may associated with the remaining ganglion cells. In these surviving ganglion cells, the intense labelling with PGP 9.5 together with the increase in rough endoplasmic reticulum, indicates the presence of active protein synthesis which may be connected with their survival.     

Satellite cells say NO to radiation

Skeletal muscles are commonly exposed to radiation for diagnostic procedures and the treatment of cancers and heterotopic bone formation. Few studies have considered the impact of clinical doses of radiation on the ability of satellite cells (myogenic stem cells) to proliferate, differentiate and contribute to recovering/maintaining muscle mass. The primary objective of this study was to determine whether the proliferation of irradiated satellite cells could be rescued by manipulating NO levels via pharmacological approaches and mechanical stretch (which is known to increase NO levels). We used both SNP (NO donor) and PTIO (NO scavenger) to manipulate NO levels in satellite cells. We observed that SNP was highly effective in rescuing the proliferation of irradiated satellite cells, especially at doses less than 5 Gy. The potential importance of NO was further illustrated by the effects of PTIO, which completely inhibited the rescue effect of SNP. Mechanical cyclic stretch was found to produce significant increases in NO levels of irradiated satellite cells, and this was associated with a robust increase in satellite cell proliferation. The effects of both radiation and NO on two key myogenic regulatory factors (MyoD and myogenin) were also explored. Irradiation of satellite cells produced a significant increase in both MyoD and myogenin, effects that were mitigated by manipulating NO levels via SNP. Given the central role of myogenic regulatory factors in the proliferation and differentiation of satellite cells, the findings of the current study underscore the need to more fully understand the relationship between radiation, NO and the functionality of satellite cells.     

Mitotic activity of rat muscle satellite cells in response to serum stimulation: relation with cellular metabolism

Cellular and molecular adaptations of satellite cells isolated from rat hindlimb muscles (n = 10) were investigated in response to serum stimulation. Flow cytometry analysis of the amounts of DNA and RNA indicated that 97.7 +/- 0.7% of satellite cells were in G0 at the end of the isolation procedure, whereas 93.2 +/- 2.0% of cells were cycling after serum exposure. The length of cell division was 34.0 +/- 2.8 h. Myoblast proliferation was asynchronous, suggesting the existence of heterogeneous cell populations in skeletal muscle. Myoblast proliferation was also accompanied by a significant increase in c-met expression, and major adaptations of energetic and proteolytic metabolisms, including an increase in the relative contribution of glycolytic metabolism for energy production, an increase in proteasome and matrix metalloproteinases 2 and 9 activities, and a decrease in plasminogen activator activities. Our data suggest that, along with molecular adaptations leading to cell cycle activation itself, adaptations in energetic and proteolytic metabolisms are crucial events involved in satellite cell activation and myoblast proliferation.     

Effect of fasting and refeeding on in vitro muscle cell proliferation in rainbow trout (Oncorhynchus mykiss)

The effects of short-term fasting and refeeding were studied on satellite cells extracted from white epaxial muscle of juvenile rainbow trout (1-3 g body weight). In vitro changes in the proliferation of satellite cells were analyzed using bromodeoxyuridine (BrdU) incorporation over a 24-h period. Proliferation in fed control fish was characterized by an initial basal proliferation rate of 5-10% BrdU-labeled nuclei x day(-1), followed by an exponential increase at a rate of +18-20% x day(-1), up to a maximum of 60-70% BrdU-labeled nuclei x day(-1). Characteristics of satellite cells extracted from starved fish, namely extraction yield, morphology, and proliferation, were different from those of fed fish. Fasting (8-10 days) completely suppressed initial proliferation of satellite cells in vitro over a period of 4 days. After this delay, proliferation resumed and changes in proliferation rates over time were similar to those of the control group. In fish fed for 4 days after an 8-day fast, the initial proliferation rate and the changes in proliferation rates over time were completely restored. These findings demonstrate that satellite cells express different behavior depending on feeding status, which could be due to the presence of different satellite cell populations.     

Regulation of myogenic stem cell behaviour by vessel cells: The "ménage à trois" of satellite cells,periendothelial cells and endothelial cells

In skeletal muscle, satellite cells, that are responsible of muscle repair, are localized close to capillaries. Although angiogenesis is known for a long time to be crucial for muscle repair and satellite cell survival, cellular interplays between vessel cells and satellite/myogenic cells have been poorly explored. We analyzed the interrelationships between myogenic cells, endothelial cells, and periendothelial cells that includes smooth muscle cells and endomysial fibroblasts. We found that endothelial cells strongly stimulate myogenic cell growth and, inversely, myogenic cells increase angiogenesis. VEGF plays a essential role in this bidirectional interaction. On the contrary, periendothelial cells promote the return to quiescence of a subset of muscle precursor cells to quiescence that ensures self-renewal of adult muscle stem cells. We have shown that Angiopoietin-1/Tie-2 signalling controls the entry into quiescence. We propose that during muscle regeneration, i.e. while vessels are not stabilized, endothelial cells and myogenic cells interact with each other to promote both myogenesis and angiogenesis, that have been shown to be concomitant processes in several models. On the other hand, once homeostasis of muscle is reached, the proximity of satellite cells and periendothelial cells allows the responsiveness of satellite cells, that bear Tie-2 receptor, to the secretion of Angiopoietin-1 by periendothelial cells, that, in the same time, stabilize vessels by promoting quiescence of endothelial cells.     

Activation of satellite cells induced by chronic neostigmine administration in the rat

Muscle fibrillation has been suggested as a possible trigger for activation of satellite cells, a well known phenomenon associated with denervation. In order to test such a hypothesis fibrillation has been induced in normally innervated muscles by chronic administration of neostigmine and the response of satellite cells has been observed with a scanning electron microscope. The results show that satellite cells protrude from the profile of the muscle fiber, become partially separated from the latter, and align in rows. Elongated structures and presumable new muscle fibers are observed after 14 days of treatment. It is concluded that the overactivity of muscle fibers which is induced during fibrillation causes activation and differentiation of satellite cells. This result is consistent with that of a previous experiment showing that satellite cells are activated during acute increase in workload.     

Activation of Satellite Cells Induced by Chronic Neostigmine Administration in the Rat

Muscle fibrillation has been suggested as a possible trigger for activation of satellite cells, a well known phenomenon associated with denervation. In order to test such a hypothesis fibrillation has been induced in normally innervated muscles by chronic administration of neostigmine and the response of satellite cells has been observed with a scanning electron microscope. The results show that satellite cells protrude from the profile of the muscle fiber, become partially separated from the latter, and align in rows. Elongated structures and presumable new muscle fibers are observed after 14 days of treatment. It is concluded that the overactivity of muscle fibers which is induced during fibrillation causes activation and differentiation of satellite cells. This result is consistent with that of a previous experiment showing that satellite cells are activated during acute increase in work-load.     

Satellite cells of normal and dystrophic muscle

The structure, the ultrastructure and the number of myonuclei and satellite cells in Duchenne's muscular dystrophy and in control muscles were compared in order to determine the possible changes in the satellite cells population. The bioptical fragments were obtained from 16 healthy (control) and from 16 dystrophic male children from 12 to 96 months of age. The biopsies were embedded in paraffin and in Durcupan and the sections were stained with ematossilin-eosin, P.A.S. for the light microscope observation and with uranil-acetate and lead-citrate for the electron microscope study. Moreover the semithin sections were stained according to the method of Ontell (1974) that is specific for the satellite cells identification. The morphological aspects of the dystrophic muscles are the same previously reported by other authors. The quantitative analysis of the myonuclei and satellite cells in control and dystrophic muscles was carried out on five random sections of each biopsy. The whole number of nuclei (myonuclei and satellite cell nuclei) and the number of the satellite cells nuclei were evaluated and the mean values in controls and dystrophic muscles were compared with the t Student test. The obtained results show that: 1) in the control muscles the satellite cells number is nearly the same in all ages considered; 2) in the dystrophic muscles the satellite cells number is in a statistically significant way greater than in control muscles and show a moderate trend to increase with aging; 3) in the dystrophic muscles the whole number of nuclei (myonuclei and satellite cells) is greater than in control in a statistically significant way and this increase is due to the number of satellite cells.     

Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells

The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context.     

Satellite cells of striated muscle after compression injury so slight as not to cause degeneration of the muscle fibres

Summary Electron microscopy of musculus bulbi rectus superior in the rat reveales increase in the number of. muscle satellite cells during the first 10–12 hours following compression injury so slight as not to cause degeneration of the muscle fibre. These satellite cells are situated close to the muscle nuclei and, in contrast to those in the intact rectus superior, they have a pale nucleus with a conspicuous nucleolus, sparse, pale cytoplasm containing a few small mitochondria, a mainly agranular endoplasmic reticulum and numbers of pinocytotic vesicles along their cell membrane. Later on, between 24 and 48 hours, the cytoplasm increases greatly in amount and extendes in long processess over a considerable length of the muscle fibre. An increase in the number of mitochondria and free ribosomes, the appearance of polysomes and great numbers of Golgi complexes and a decrease in the number of pinocytotic vesicles is noted. On the 4th day, some of the satellite cells have still further extended their cytoplasm beneath the muscle basement membrane, while others are apposed to the muscle fibre only with a minor part of their membrane. On the 9th day, only satellite cells comparable in number and structure with those in intact muscles are found.The possibility is discussed that the increase and subsequent decrease in the number of these satellite cells as well as the changes in their morphology at different periods after the injury reflect morphogenesis of satellite cells originating from the nuclei of the muscle fibre by mitotic division even after a trauma, too slight to cause any visible muscle injury. The observations are interpreted as giving new support to the hypothesis that muscle satellite cells may be of importance in posttraumatic muscle regeneration.