中国林蛙胸腺类肌细胞的细胞学研究

目的通过对中国林蛙胸腺的组织细胞结构研究,探讨胸腺类肌细胞的结构特点及年周变化规律。方法选用崂山产中国林蛙(Rana chensinensis),雌雄兼有,逐月取材,应用H-E、铅苏木精、Grimelius嗜银和PAS等染色方法进行染色,超微结构用日立透射电子显微镜进行观察。结果类肌细胞是中国林蛙胸腺髓质中恒定出现的一种特征性细胞。细胞质包含许多类似于骨骼肌内的典型结构——肌纤维,根据结构特点可将细胞分为未成熟、成熟以及退化3种类型。肌细胞的出现伴有明显的年周变化,2、3月份开始逐渐增多,到9月份肌细胞的大小与数量达到最大,不同月份所含细胞类型也大有不同。结论类肌细胞是胸腺实质组织中的一种正常细胞,具有不同发育阶段的3种形态结构类型,细胞数量和类型具有年周变化特点,推测与胸腺的正常功能有密切关系。     

Comparison of the sarcoplasmic and myofibrillar proteins of twitch and tonic fibres of frog muscle (Rana esculenta)

Sarcoplasmic and myofibrillar proteins of a frog mixed muscle (distal cruralis bundle) were investigated and compared to their fast twitch muscle homologues. Histochemical reactions revealed two populations of fibres in this muscle, differing from fast twitch fibres by the intensity of their myofibrillar ATPase reaction and by their mitochondrial NADH dehydrogenase activity. The distribution of parvalbumins and LDH isoenzymes in the whole muscle showed some features of tonic muscle type. Myosin light chains pattern of cruralis bundle fibres was characterized by the lower proportion of the LC3 subunit. These results confirmed the heterogeneity of this frog muscle and the presence of tonic or intermediate fibres with their typical sarcoplasmic and myofibrillar proteinic composition.     

The properties of the extraocular muscles of the frog. III. Morphological, mechanical and pharmacological properties of the isolated retractor bulbi muscle.

Some morphological, physiological, and pharmacological properties of the retractor bulbi muscle of the frog were tested. The enzyme-histochemical investigation shows that the retractor bulbi muscle contains twitch muscle fibres only. Two types of twitch muscle fibres, which are especially different in their diameter and in the content of mitochondria, build the muscle in an irregular arrangement; tonic muscle fibres were not observed. On the average, the isolated retractor bulbi muscle has at room temperature a contraction time of 26 ms, a half-relaxation time of 28 ms, a fusion frequency of 75 stimuli/s, and a twitch-tetanus ratio of 0.28. The fatigability of this muscle is higher than in oculorotatory eye muscles but lower than in skeletal muscles of the frog. An increase of the extracellular K+-concentration elicits in retractor bulbi muscles a quickly transient contracture; the mechanical threshold of the muscle fibres is found in a range between 20 and 25 mM K+ in Ringer solution. Similar short-lasting contractures, which are probably caused by twitch fibres, rich in mitochondria, are also evoked by application of depolarizing drugs like acetylcholine. The properties of the retractor bulbi muscle are compared with those of the sartorius muscle of the frog, which likewise contains twitch muscle fibres only.     

Visualization of satellite cells in living muscle fibres of the frog

Satellite cells were visualized in living muscle fibres of the frog. Single fibres or bundles consisting of a few fibres were isolated after treatment with collagenase, and viewed under the light microscope. Subsequent electron microscopy of identified cells confirmed that they were satellite muscle cells. Under the light microscope, satellite cells appear as fusiform cells, tapering into long fine processes usually orientated parallel to the muscle fibre axis. Horseradish peroxidase injected into the muscle fibre was not transferred to the satellite cells.     

The subcommissural organ of the frog Rana perezi is innervated by nerve fibres containing GABA

The innervation of the frog subcommissural organ was studied by light-microscopic and ultrastructural immunocytochemistry using antisera against serotonin, noradrenaline, dopamine, gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, different GABA receptor subunits and bovine Reissner's fibre material (AFRU). In the proximity of the organ, serotonin- and noradrenaline-containing fibres were rare whereas dopamine-immunoreactive fibres were more numerous. Many GABA- and glutamic acid decarboxylase-containing nerve fibres were found at the basal portion of the ependymal cells of the subcommissural organ. Under the electron microscope, these GABA-immunolabelled nerve endings appeared to establish axoglandular synapses with secretory ependymal cells of the subcommissural organ. In addition, the secretory ependymal cells expressed high amounts of the beta2-subunit of the GABA(A) receptor. Since GABA-immunoreactive neurons were present in the frog pineal organ proper and apparently contributed axons to the pineal tract, we suggest that at least part of the GABAergic fibres innervating the frog subcommissural organ could originate from the pineal organ.     

Ultrastructural transformation of fast chicken muscle fibres induced by nerve cross-union

Summary The fast posterior latissimus dorsi (PLD) muscle of newly hatched chickens was transposed and cross-innervated by the slow-type nerve originally innervating the anterior latissimus dorsi (ALD) muscle. The innervation and the ultrastructure of the cross-innervated posterior latissimus dorsi (PLD-X) muscle was investigated from one week up to 18 months after the operation and compared with that of the control fast (PLD-C) and control slow (ALD-C) muscles. All nerve terminals in the PLD-X muscle were of the slow type. Yet the degree of ultrastructural transformation differed from fibre to fibre. Only about 30% of PLD-X fibres had transformed ultrastructure closely resembling the control slow fibres. In this group of maximally altered fibres, the myofibrils had large diameters, wide Z lines and indistinct M lines as the control slow fibres. The amount of mitochondria was increased to levels found in control slow fibres. The mean percentage of triads was also comparable to that of control slow fibres, being approximately by two thirds lower than in control fast fibres.The differences in the degree of ultrastructural transformation are presumably due to different plasticity of muscle cells at the time of cross-innervation. In the transposed PLD-X muscles large areas undergo degeneration and regeneration. It is suggested that an almost complete changeover of the fibre type is only brought about after cross-innervation of newly differentiating muscle cells, whereas partial alteration occurs after reinnervation of young myofibres.The skillful technical assistance of Dr. Z. Liková, Mrs. M. Sobotková, Ing. M. Doubek and Mr. H. Kunz is gratefully acknowledged.     

Ultrastructure of frog muscle fiber thick filaments at rest and during potassium contracture]

Isolated slow and intermediate frog muscle fibres were fixed in the rest state and under potassium contracture (50-100 mM KC1). The longitudinal and cross sections of two types of fibres were investigated. It was shown that at the rest the thick filaments of different fibres had similar length (1.6-1.65 mum), diameter (160-165 A) and the amount of subunits (12-13). Under potassium contracture the length of the thick filaments of both fibre types was shortened by 25-30% of the rest-length, the diameter of the slow fibres increased to 180-185 A, the diameter of the intermediate fibres to 200-220 A. The amount of subunits increased to 14-15 in slow fibres and to 17-18 in intermediate fibres. We believe that the ultrastructural changes observed in the thick filaments are a result of molecular transformation in these filaments, which seems to be important for maintaining the contracture.     

Immunocytochemical localization of taurine in different muscle cell types of the dog and rat

The presence and distribution of the amino acid taurine in different muscle cell types of the dog and rat was examined by immunocytochemical methods. The light microscope study revealed that smooth muscle cells were similarly immunoreactive for taurine, whereas skeletal muscle fibres showed wide differences in taurine immunoreactivity among individual cells. Some skeletal fibres were strongly immunoreactive whereas others did not display immunolabelling. Mononucleated satellite cells, found adjacent to skeletal fibres in a quiescent stage, were also immunostained. Other myoid cells, such as testicular peritubular cells showed a cytoplasmic and a nuclear pool of taurine. By means of electron microscope immunolabelling, the subcellular localization of taurine was studied in vascular and visceral smooth muscle cells. Taurine was present in most subcellular compartments and frequently appeared randomly distributed. Taurine was localized on myofilaments, dense bodies, mitochondria, the plasma membrane and the cell nucleus. Moreover, the labelling density within individual smooth muscle cells was variable and depended on the state of contraction of each single fibre. Contracted cells showed a higher density of gold particles than relaxed cells. Unmyelinated nerve fibres, found adjacent to smooth muscle cells from the muscularis mucosae and the lamina propria of the stomach, were unstained or poorly stained.     

The presynaptic active zones in three different types of fibres in frog muscle

Presynaptic active zones were studied in slow, fast and intermediate types of frog muscle fibres in freeze-fracture replicas. In fast fibres, the double rows of paired particles are present on active zone ridges perperdicular to the longitudinal axis of the nerve whereas in slow fibres active zone ridges are rudimentary or absent and double rows of particles occur in all directions, mostly paired, sometimes single. In the intermediate type of muscle fibres both types of active zone deployment coexist on a single muscle fibre.     

The properties of the extraocular muscles of the frog. I. Mechanical properties of the isolated superior oblique and superior rectus muscles.

The mechanical properties of two extraocular muscles (superior oblique and superior rectus muscles) of the frog were studied and compared with those of a frog's skeletal muscle (iliofibularis muscle) which contains the same types of muscle fibres as the oculorotatory muscles. The extraocular muscles are very fast twitching muscles. They exhibit a smaller contraction time, a smaller half-relaxation time, a higher fusion frequency, and a lower twitch-tetanus ratio than the skeletal muscles. The maximum isometric tetanic tension produced per unit cross-sectional area is lower in the extraocular muscles than in skeletal muscles. However, the extraocular muscles show a higher fatigue resistance than the skeletal muscles. With respect to the dynamic properties there are some differences between the various oculorotatory muscles of the frog. The superior rectus muscle exhibits a faster time-course of the contraction, a higher fusion frequency, and a higher fatigability than the superior oblique muscle. An increase of the extracellular K+-concentration evokes sustained contractures not only in the extraocular muscles but also in the iliofibularis muscle; between these muscles there are no striking differences in the mechanical threshold of the whole muscle preparation. The mechanical threshold depends on the Ca++-concentration of the bathing solution and it is found in a range between 12.5 and 17.5 mM K+ in a normal Ringer solution containing 1.8 mM Ca++. The static-mechanical properties of the extraocular muscles of the frog and the dependence of the active developed tension on the muscle extension are very similar to those which are known to exist in the extraocular muscles of other vertebrates. In tetanic activated frog's oculorotatory muscles a linear relationship exists between length and tension. A variation of the stimulation frequency does not change the slope of this curve but causes parallel shifts of the curve. The peculiar properties of the extraocular muscles of the frog are discussed with respect to the muscle fibre types in these muscles and to the diameter of the muscle fibres.     

Myosin Heavy Chain Profiles in Regenerated Fast and Slow Muscles Innervated by the Same Motor Nerve Become Nearly Identical

Plasticity of mature muscles exposed to different activation patterns is limited, probably due to restricted adaptive range of their muscle fibres. In this study, we tested whether satellite cells derived from slow muscles can give rise to a normal fast muscle, if transplanted to the fast muscle bed. Marcaine-treated rat soleus and extensor digitorum longus (EDL) muscles were transplanted to the EDL muscle bed and innervated by the EDL nerve. Six months later expression of myosin heavy chain isoforms was analysed by areal densities of fibres, binding specific monoclonal antibodies, and by SDS gel electrophoresis. Both regenerated muscles closely resembled each other. Their myosin heavy chain profiles were similar to those in fast muscles although they were not identical to that in the control EDL muscle. Since not even regenerated EDL was able to reach the myosin heavy chain isoform profile of mature EDL muscle, our experimental model did not permit studying the adaptive capacity of satellite cells in different muscles in its whole extent. However, the results favour the multipotential myoblast stem cell population in rat muscles and underline the importance of the extrinsic regulation of muscle phenotype.     

On the character of the monolayer outgrowth and the fate of the peritubular myoid cells in cultured mouse testis

Postnatal differentiation of the peritubular myoid cells in mouse testis is hormone dependent. In order to analyse the differentiation of the peritubular tissue, an attempt was made to develop an experimental model system utilizing an in vitro method. Fragments obtained from adult, 7- or 10-day-old mice, were cultured in McCoy's modified 5a medium for 9–19 days. The fragments and monolayers that grew from them were examined with the electron microscope at the end of the culture period. Monolayers originating from either mature or immature testicular expiants were comparable in appearance. They were composed of spindle-shaped cells that contained abundant profiles of granular endoplasmic reticulum and free ribosomes, as well as arrays of 40–60 Å thick filaments and associated dense bodies. In these respects they resembled smooth muscle cells in culture, in developmental, and in pathological conditions. Examination of the peritubular tissue in the testicular explants indicated that the monolayer of myoid cells originated from the fibroblasts rather than the peritubular myoid cells. Peritubular cells in explants from mature rats retained their myoid features at the end of the culture period but myoid cell differentiation failed to progress in expiants obtained from immature animals. Additional work is necessary in order to establish the suitability of these preliminary culture attempts to support normal development before conclusions may be drawn concerning the role of hormones in myoid cell differentiation. The role of microfilaments as a contractile organelle of cells is discussed.     

Satellite cells in movement disorders of various etiologies

The ultrastructural analysis of the muscle biopsy samples from patients with different neuro-muscular diseases revealed; both degeneration and regeneration of the muscle fibres. The number of satellite cells was increased due to their division and myonuclear segregation. Activated satellite cells were converted into myoblasts which probably could replace the injured fragments of the muscle fibres or form the myotubes. The process of muscle fibre regeneration is restricted not only by the damage of the muscle itself, but by the dystrophic process affecting satellite cells.     

The chloride conductance of intermediate fibres from frog muscles

Sheets of muscle fibres dissected from surface portions of frog ileofibularis and semitendinosus muscles were soaked in solutions with elevated K and Cl concentrations. The KCl-loaded muscles were then bathed in low [Cl-] solutions, whereby the membrane potential became transiently inside positive. The repolarization of the twitch fibres from the tonus bundle ("intermediate fibres") was faster than that of the fibres adjacent to it ("fast fibres") when the preceding exposure to high KCl was brief (7-15 min), and it was slower than that of the fast fibres when KCl was applied for 4 hours. Measurements of the voltage displacement at constant current and of the current in a point voltage clamp showed that inwardly rectifying K channels were present in the membranes of both types of fibres. The ionic conductance ratio, gK/gCl, was 191/523 in fast fibres and 335/230 in "intermediate" fibres. The different repolarization rates may thus be explained by differences in the chloride conductance of the fast and intermediate fibre membranes. The smaller diameter of the latter fibres may be another factor.     

Interpretation of the cross sectional structure of skeletal muscle fibers. 2. Light and electron microscopic studies of m. rectus abdominis in Rana esculenta]

In both longitudinal and cross sections of rectus abdominis muscle of Rana esculenta three types of muscle fibres are identified by means of light and electron microscopy. A comparison is made between these fibre types in homologous muscles of frog and mammals (rat and mouse). In longitudinal sections of mammalian and frog muscle the Z-line can be used for discrimination of the fibre types A, B and C because that line is of different thickness in each type. The proportions of the thickness in frog and mammalian muscles are relatively the same, but the absolute values are different. In cross sections there are no differences between frog and mammalian muscle fibres concerning the typical form of myofibrils in type A- and B-fibres, whereas in type C-fibres the arrangement of the filaments in the Z- and H-layer is different in the members of both animal classes. The amount of mitochondria and lipid droplets is different as well. In the species examined the distribution of A-, B- and C-fibres changes within the whole muscle. In frog, this pattern depends on the level in which the muscle has been sectioned. This is not true for mammalian muscle. On the other hand both ends of the rectus abdominis muscle in frog, rat and mouse show an accumulation of B- and C-type fibres.     

Myofibril breakdown during spreading damage. II. A calcium-free medium]

Peculiarities of Zenker's degeneration (ZD) have been investigated in fast muscle fibres of the frog incubated in a Ringer solution free of Ca++ (R--Ca) with a normal or increased (by 100 mM) concentration of KCl. ZD in these solutions is distinguished by a 10--90 minutes delay of the appearance of the primary contraction knot and cessation of ZD development in the majority of fibres after formation of several (1--5) contraction knots. In the presence of 0.5 mM EDTA in R--Ca, after a few typical contraction knots are formed, fibres commonly fall into large fragments that retain cross-striation. Contracted or super-contracted state of sarcomeres in detached contraction knots and at the necrosis boundary, as well as an increasing lysis of contactile material and proliferation of fibre membrane structures in the region of ZD arrested boundary, are characteristic of ultrastructural changes during ZD in calcium-free solutions.     

Quantitative histochemistry of three mouse hind-limb muscles: the relationship between calcium-stimulated myofibrillar ATPase and succinate dehydrogenase activities

Summary A quantitative modification of Meijer's calcium-lead capture method, for the demonstration of calcium-stimulated myofibrillar ATPase activity at physiological pH, is described. A range of myofibrillar ATPase activities has been found among fast muscle fibres in two mouse hind-limb muscles. The myofibrillar ATPase activity of fast muscle fibres is 1.5–3 times higher than the myofibrillar ATPase activity of slow muscle fibres.Myofibrillar ATPase activities and succinate dehydrogenase activities of individual muscle fibres have been determined in serial sections. Activities of the two enzymes are correlated positively in soleus (fast and slow fibres), and negatively in plantaris (almost all fast) and extensor digitorum longus muscle (all fast). However, this correlation is not significant among the oxidative fibres in the extensor digitorum longus. The fibres of the latter muscle cannot be classified satisfactorily into two sub-types.     

The fine structure of the boundary tissue of the seminiferous tubule of the camel (Camelus dromedarius)

An ultrastructural study of the boundary tissue of the seminiferous tubule of the camel reveals that it consists of three lamellae; inner fibrous, inner cellular and outer cellular. The inner lamella is subdivided into two homogeneous layers enclosing a third one that contains collagenous fibres and fine filaments. The inner cellular lamella consists of several layers of myoid cells; each layer is separated from the adjacent layer by homogeneous material and varying amounts of collagen. The outer cellular lamella consists predominantly of fibrocytes together with some fibroblasts and scattered collagen.     

Slowly contracting muscles power the rapid jumping of planthopper insects (Hemiptera, Issidae)

The planthopper insect Issus produces one of the fastest and most powerful jumps of any insect. The jump is powered by large muscles that are found in its thorax and that, in other insects, contribute to both flying and walking movements. These muscles were therefore analysed by transmission electron microscopy to determine whether they have the properties of fast-acting muscle used in flying or those of more slowly acting muscle used in walking. The muscle fibres are arranged in a parallel bundle that inserts onto an umbrella-shaped tendon. The individual fibres have a diameter of about 70 μm and are subdivided into myofibrils a few micrometres in diameter. No variation in ultrastructure was observed in various fibres taken from different parts of the muscle. The sarcomeres are about 15 μm long and the A bands about 10 μm long. The Z lines are poorly aligned within a myofibril. Mitochondrial profiles are sparse and are close to the Z lines. Each thick filament is surrounded by 10–12 thin filaments and the registration of these arrays of filaments is irregular. Synaptic boutons from the two excitatory motor neurons to the muscle fibres are characterised by accumulations of ~60 translucent 40-nm-diameter vesicle profiles per section, corresponding to an estimated 220 vesicles, within a 0.5-μm hemisphere at a presynaptic density. All ultrastructural features conform to those of slow muscle and thus suggest that the muscle is capable of slow sustained contractions in keeping with its known actions during jumping. A fast and powerful movement is thus generated by a slow muscle.     

Collagen of slow twitch and fast twitch muscle fibres in different types of rat skeletal muscle

The appearance of collagen around individual fast twitch (FT) and slow twitch (ST) muscle fibres was investigated in skeletal muscles with different contractile properties using endurance trained and untrained rats as experimental animals. The collagenous connective tissue was analyzed by measuring hydroxyproline biochemically and by staining collagenous material histochemically in M. soleus (MS), M. rectus femoris (MRF), and M. gastrocnemius (MG). The concentration of hydroxyproline in the ST fibres dissected from MS (2.72 +/- 0.35 micrograms X mg-1 d.w.) was significantly higher than that of the FT fibres dissected from MRF (1.52 +/- 0.33 micrograms X mg-1 d.w.). Similarly, the concentration of hydroxyproline was higher in ST (2.54 +/- 0.51 micrograms X mg-1 d.w.) than in FT fibres (1.60 +/- 0.43 micrograms X mg-1 d.w.), when the fibres were dissected from the same muscle, MG. Histochemical staining of collagenous material agreed with the biochemical evidence that MS and the slow twitch area of MG are more collagenous than MRF and the fast twitch area of MG both at the level of perimysium and endomysium. The variables were not affected by endurance training. When discussing the role of collagen in the function of skeletal muscle it is suggested that the different functional demands of different skeletal muscles are also reflected in the structure of intramuscular connective tissue, even at the level of endomysial collagen. It is supposed that the known differences in the elastic properties of fast tetanic muscle compared to slow tonic muscle as, e.g., the higher compliance of fast muscle could at least partly be explained in terms of the amount, type, and structure of intramuscular collagen.