J. Anat. (1975), 119, 1, pp. 39-48 With 8 figures Printed in Great Britain
39
Observations on the structure of the dorsal muscle in the bottle-nose dolphin (Tursiops truncatus) R. S. TULSI
Department of Anatomy and Histology, The University of Adelaide, South Australia 5001
(Accepted 5 August 1974) INTRODUCTION
The ability of marine mammals to remain under water for prolonged periods has intrigued biologists for decades. A dolphin may remain submerged for 12 minutes at depths of almost 20 metres (Harrison & Tomlinson, 1963), and is capable of considerable physical activity during this time. The structural and functional adaptations which enable marine mammals to cope with an underwater environment are likely to involve intricate adjustments in most organs. Special adaptations of the skeletal musculature are bound to be important (Ridgway, 1972), and it was therefore decided to study the dorsal muscle of the dolphin. This muscle was chosen because it plays a crucial role in the locomotion of the animal both on and under water (Harrison, personal communication). The muscle was examined by light and electron microscopy. The cytochemistry of the extrafusal muscle fibres and the morphology of the muscle spindles, were studied with the light microscope. The fine structure of the extrafusal fibres and their neuromuscular junctions was examined with the electron microscope. MATERIALS AND METHODS
Five animals were used. They were caught in nets in Spencer Gulf, South Australia, and transported alive to Adelaide. One animal (No. 5 in Table 2) was young and the other four were mature adults. Details of the anaesthetic agents used and the length and sex of animals are given in Tables 1 and 2. Study was made of the dorsal muscle lying immediately lateral to the dorsal fin. Animals 2 and 4 were fixed by perfusion through the aorta at 120 cm of water using 1 % glutaraldehyde and 1 % paraformaldehyde in 0-1 M phosphate buffer at pH 7-3. Prior to removal of pieces of dorsal muscle in all animals the muscle was fixed in situ by pouring on it a small quantity of the chilled fixative. Animals 1, 3, and 5 were not perfused, and blocks of dorsal muscle were removed shortly after induction of anaesthesia and immersed in chilled 6% glutaraldehyde in 0-1 M phosphate buffer at pH 7-3. After 2 hours of fixation in the chilled fixative the specimens were washed in phosphate buffer for 1 hour and then post-fixed in 1 % osmium tetroxide for 11 hours. Blocks of muscle from perfused dolphins were similarly treated. Dehydration was carried out in graded series of ethanol and acetone and embedded in an Epon/Araldite mixture. Semi-thin sections were cut at 0 5-1 micron and stained with 1 % sodium borate
40
R. S. TULSI
Table 1. Anaesthetic agents Ph. H.G.O. P.B. Pe D
Phencyelidine (intravenous injection, as suggested by Ridgway) Halothane, nitrous oxide 80%, oxygen 20% (Ridgway & McCormick, 1971) Pentobarbital sodium (intravenous) Pentothal sodium (intravenous) (Ridgway & McCormick, 1971) Diazepam (intramuscular) 10 mgm/100 kgm as suggested by Ridgway
Table 2. Details of animals used. Animal no.
Length (cm)
Sex
Anaesthetic agent
1 2 3 4 5
202 201 182 201 160
M F M M F
Ph., D., H.G.O. Ph., D., H.G.O. P.B. Ph., D., H.G.O. Pe., H.G.O.
(Richardson, Jarrel & Fink, 1960) for orientation. Silver to grey sections were cut for electron microscopy, collected on uncoated grids, stained with uranyl acetate and lead citrate (Venable & Goggeshall, 1965) and viewed under a Philips 300 transmission microscope. In the preliminary stages of the study a number of cytochemical methods were also applied. These included techniques for demonstrating myosin adenosinetriphosphatase (ATPase), succinic dehydrogenase (SDHase), Sudan Black B (for lipids) and myoglobin (Pearse, 1968). OBSERVATIONS
The dorsal muscle showed three types of fibre as in most mammals. The red fibres, which constituted approximately 30%O of the total, had a small diameter (mean of 26-2 ,tm), a large number of lipid droplets, many mitochondria, and a high myoglobin content (Figs. 3-5). They also showed high SDHase and ATPase activity, and a conspicuous Z-line (Fig. 6). The white fibres, which formed approximately 28 % of the total, had a large diameter (mean of 49-5 ,um), no lipid droplets, few mitochondria, and a low myoglobin content (Figs. 3 and 6). The Z-line was narrow
Fig. 1. Longitudinal section of a white muscle fibre to show the T-system. Parts of four transverse tubules (A) and in some areas the adjacent cisternae of the sarcoplasmic reticulum are also seen. The three upper transverse tubules involving two sarcomeres are joined by longitudinal elements (B) of the T-system at (C). Material from dolphin 1. x 84600. Fig. 2. Longitudinal section of a white muscle fibre to show the T-system and sarcoplasmic reticulum. Two adjacent transverse tubules (A) involving one sarcomere, are linked by a longitudinal element (B) of the T-system which joints the former at (C). Lying adjacent to the lower transverse tubule a cisterna of the sarcoplasmic reticulum is clearly visible which seems to provide an extension (D) along the longitudinal element (B) of the T-system. Other longitudinally oriented tubules (E), forming part of the sarcoplasmic reticulum, are seen extending upwards from the cisterna. Several electron-dense bridges extending between the transverse tubule and adjacent cisterna of the triad are seen in the upper part of the figure. Material from dolphin 1. x 48 350.
41
Skeletal muscle of dolphin
A~~~~A
A7
..: 1
42
R. S. TULSI
(Fig. 6) and there was low SDHase and ATPase activity. The remainder of the fibres could be said to be of 'intermediate' type (Fig. 3 B); however, on the basis of fibre diameter and population of lipid droplets, these intermediate fibres could be divided into two groups. One had smaller fibres (mean 31 8 ,tm) with many lipid droplets and the other had larger fibres (mean 42-8 ,tm) but relatively few lipid droplets. Most features of dolphin muscle, such as its capillaries (Fig. 7), sarcolemma, myofibrils, myofilaments, and sarcomeres (Figs 4 and 6) are essentially similar to those described in other mammals and need not be described here. As there has been some doubt about the existence of muscle spindles in cetacea, some limited observations on spindles and neuromuscular junctions are perhaps worth recording. A total of 45 blocks were taken from five animals in a search for muscle spindles by light microscopy. Three spindles were found with morphology similar to that described in other mammals. Three neuromuscular junctions were seen with the electron microscope, two on white fibres and one on an intermediate fibre. No neuromuscular junction was seen on a red fibre in the present limited study. The neuromuscular junctions on the white fibres showed marked branching of junctional folds which extended deep into the sarcoplasm and there were many subsarcolemmal vesicles features recently described (Padykula & Gauthier, 1970) as typical of white fibres elsewhere. T-system. The T-system was well developed and readily recognized in most muscle fibres. Typical triads were found at the level of the A-I junction, with the result that there were two triads to each sarcomere, a pattern seen in many other mammals. Between the central component of each triad (the transverse tubule) and the two flanking cisternae (of the sarcoplasmic reticulum) there were several evenly spaced electron-dense bridges (Figs 1 and 2). Continuity between the T-system and the sarcolemma could be seen in several places. A feature of particular interest in the dolphin material was the occurrence of longitudinal channels linking adjacent transverse tubules. This linking occurred within a single sarcomere (Fig. 2), as well as between systems of two neighbouring sarcomeres (Fig. 1). In other words the transverse tubules of the T-system were clearly seen to be linked by longitudinal elements when the plane of section passed through both (Fig. 1). It is important to Fig. 3. Transverse section of muscle fibres stained with Sudan Black B to show lipid droplets. Note that there is much lipid in the red (A) fibre, a moderate amount in the intermediate (B) fibre and little or none in the white (C) fibre. Material from dolphin 5. x 960. Fig. 4. Transverse section of part of a red muscle to show several lipid droplets (L), a mitochondrion (M) and muscle filaments. Note that some lipid droplets are partially surrounded by markedly electron-dense granules (arrow). Material from dolphin 3. x 68750. Fig. 5. Longitudinal section of a red muscle fibre to show a large number of lipid droplets (L), many of which are closely associated with mitochondria (M). In some areas closely packed droplets coalesce to form a longitudinal column of lipids. There is also an accumulation of a large number of lipid droplets and mitochondria immediately deep to the plasma membrane producing a convexity on the surface of the fibre. These droplets are not as dense as those deep in the muscle, but parts of the electron-dense walls of some of them are visible in the lower left part of the figure. Material from dolphin 5. x 12000. Fig. 6. Longitudinal section of parts of two adjacent fibres, one a red (lower) and the other a white (upper). The red fibre shows a wider Z-line (Z), more mitochondria (M), many lipid droplets (L), but not so many glycogen granules. Material from dolphin 5. x 16500.
Skeletal muscle of dolphin
ei-
43
44
R. S. TULSI
note that the longitudinal elements of the T-system were themselves flanked by extensions (Fig. 2D) of adjacent cisternae of the sarcoplasmic reticulum, and that such extensions were distinct from the longitudinal tubes (Fig. 2E) of the sarcoplasmic reticulum. The longitudinal elements of the T-system were found only in white fibres: considerable search failed to show similar elements in red fibres. Lipid droplets. Another interesting feature of the dolphin muscle was the large number of lipid droplets in the red muscle fibres. When muscle fibres were stained with Sudan Black B (Fig. 3), the red fibres showed many lipid droplets whereas white fibres showed few or no droplets. These lipid droplets (up to 074 mm in diameter) occurred both in young and fully grown animals and often lay close to mitochondria pressing against them and distorting them (Figs. 4, 5 and 6). The droplets were commonly placed between myofibrils or immediately deep to the sarcolemma, at times so numerous as to produce a convexity on the surface of the muscle (left edge of Fig. 5). When a large number of droplets accumulated between myofibrils they either coalesced or were firmly pressed against one another, forming longitudinal columns extending along several sarcomeres (in the middle of Fig. 5). The lipid droplets were not membrane-bound. Several were partially surrounded by markedly electron-dense granules (Fig. 4). This has also been observed in lipid droplets in other dolphin tissues (Fanning, personal communication) and also in lipid droplets in normal cartilage in other species (Collins, Ghadially & Meachim, 1965). There was considerable variation in the electron density of lipid droplets apparently related to the techniques of fixation and staining used and to the age and physiological state of the animal. In the present study the material from the older animals (Figs. 4, 7 and 8) showed denser droplets than that from the younger animals (Figs. 5 and 6). A further topic of interest was the relationship of lipid droplets and chylomicrons to capillaries and extracellular spaces. Material from animals 1 and 3 (Figs. 7 and 8) suggested that not only is there a large quantity of lipid in red muscle but also that there is a marked movement of lipids across the capillary wall. Figure 7 shows a lipid droplet in a capillary (1), a chylomicron lying free in the lumen (2), another chylomicron in the lumen partially surrounded by an endothelial flap (3) and possibly another four chylomicrons in a vacuole within the capillary endothelium (4). The same figure also shows a lipid droplet (5) in an adjacent muscle fibre. Figure 8 shows pinocytotic vesicles (V1) and a vacuole (V2) within the capillary endothelium and a lipid droplet immediately outside the capillary close to a muscle fibre. Fig. 7. Transverse section of a capillary and part of an adjacent muscle fibre (M). Within the lumen of the capillary is an intensely electron-dense lipid droplet (1), two chylomicrons (2, 3) one of which is partially surrounded by a flap (F) of endothelium. Several other chylomicrons (4) are within a vacuole in the wall of the capillary and another lipid droplet (5) is seen in the adjacent muscle. E, nucleus of an endothelial cell. R, red blood cell. Material from dolphin 3. x 27000. Fig. 8. Oblique section of a capillary and part of an adjacent muscle fibre (M). Note that an intensely electron-dense lipid droplet (L) appears to be wedged between the capillary wall and the muscle fibre. C, lumen of capillary. V1 micropinocytotic vesicles. V2, vacuole within capillary wall. Material from dolphin 1. x 13400.
Skeletal muscle of dolphin ..
TWI,
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. ,s.t
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46
R. S. TULSI DISCUSSION
The present study has shown that the dorsal muscle of the dolphin possesses red, white, and intermediate fibres like most other vertebrate skeletal muscles. The ultrastructure and cytochemistry of the muscle fibres, except for those features noted below, and the morphology of the muscle spindles and neuromuscular junctions, were essentially similar to those in other mammals. The special features of the dolphin material were: (1) the presence in the red fibres of a large number of lipid droplets and (2) the presence in white fibres of longitudinal tubular channels linking adjacent transverse components of the T-system; these links were both inside a given sarcomere and between adjacent sarcomeres. Lipid droplets are normal constituents of ventricular cardiac muscle cells (Maunsbach & Wirsen, 1966) but in skeletal muscle they are only variably present in some species (Bloom & Fawcett, 1969). Lipid droplets in electron micrographs of skeletal muscle show varying density due partly to differences in technique and partly to differences in chemical composition. The evidence supporting the presence of a large amount of lipid in dolphin muscle is at present entirely morphological. However, when electron micrographs from the present study were compared with those published for the normal muscles of many other vertebrates it was clear that none of the latter approached the dolphin in the number of droplets in its red muscle fibres. In fact the lipid droplets shown by Forbes & Sperelakis (1972) in their Plate 2 (Fig. 6) and Plate 3 (Fig. 6) of pathological muscle are far fewer than in normal dolphin muscle (Fig. 5). The significance of the large lipid droplet content of dolphin red muscle is unclear but it is reasonable to suppose that it serves as a readily available source of high energy metabolic fuel. The possible functional implications of one particular observation regarding lipid droplets in the dolphin is worth discussing. The muscle capillaries in the dolphin invariably showed a large number of pinocytotic vesicles as well as several vacuoles on the luminal and basal surfaces of the endothelium. Some of the vesicles contained a single small lipid droplet, but some of the vacuoles had as many as four droplets. None of the droplets was bound by a membrane and they were never seen to be associated with any endothelial organelle. Within the capillary lumen several small droplets were observed, some of which were partially surrounded by endothelial flaps; curiously, however, no droplet of any size was ever seen lying entirely outside the capillary. All this strongly suggests that the movement of at least some of the lipid across capillary endothelium in muscle is by pinocytosis. In a comprehensive review (Page, 1968) the T-system is described as transversely oriented tubular invaginations of the'sarcolemma either at the level of the A-I junction or at the Z-line, along which surface depolarization travels inwards towards the deeply placed filaments during the excitation-contraction process: there is no mention of any connexion between adjacent tubules inside the muscle fibre by means of longitudinal channels. In a more recent review of vertebrate slow muscle fibres (Hess, 1970) it is stated that the system is scanty in avian, frog, and mammalian slow muscle fibres. In the last decade or so, workers studying skeletal muscle of other vertebrate species have occasionally observed what could only be interpreted as parts of longitudinal elements of the T-system. These have been seen in the frog (Page,
Skeletal muscle of dolphin
47
1965; Eisenberg & Eisenberg, 1968), bird (Hikida, 1972; Hoyle, 1973), bat (Revel, 1962) and in the guinea-pig (Ryans, personal communication). In the dolphin, however, the longitudinal element of the T-system seems to be well developed in the white fibres where it links the transverse elements within a single sarcomere and within adjacent sarcomeres. The physiological role of the longitudinal component of the T-system is a matter for speculation. It is tempting to suggest that it has a function similar to the rest of the T-system. This suggestion seems reasonable as the longitudinal tubes appear to be morphologically similar to the transverse tubes and are closely associated with extensions of the terminal cisternae of the triad. The longitudinal component of the T-system should therefore facilitate transmission of impulses within a sarcomere and between adjacent sarcomeres deep inside a muscle fibre: it certainly increases the total surface area of the T-system in contact with myofibrils. SUMMARY
The dorsal muscle of five South Australian bottle-nose dolphins has been studied. The ultrastructure and cytochemistry of the red, white and intermediate fibres were essentially similar to those of other mammalian skeletal muscles. Two features of special interest were: (a) the large number of lipid droplets in the red muscle fibres, and (b) the presence of longitudinal tubes linking the transverse tubes of the Tsystem, both within a sarcomere, and also between adjacent sarcomeres, of the white muscle fibres. The muscle spindles and neuromuscular junctions in the dolphin appeared to be morphologically similar to those of other mammals.
Thanks are due to the State Director of Fisheries and the Director of Wildlife and Fauna Conservation for permission to obtain animals. To Professor R. J. Harrison I am grateful for suggesting the study and for reading the manuscript. I thank Professor Janis Priedkalns, Chairman of the Department, for his interest and suggestions in preparing the manuscript. I acknowledge with gratitude helpful comments and criticism made by Professor V. Navaratnam. The technical assistance of Mrs G. M. Hermanis is gratefully acknowledged. REFERENCES
BLOOM, W. & FAWCETT, D. W. (1969). A Textbook of Histology. London: Saunders Co. COLLINS, D. H., GHADIALLY, F. N. & MEACHIM, G. (1965). Intracellular lipids of cartilage. Annals of the Rheumatic Diseases 24, 123-135. EISENBERG, B. & EISENBERG, R. (1968). A selective disruption of the sarcotubular system in frog sartorius muscle. A quantitative study with exogenous peroxidase as a marker. Journal of Cell Biology 39, 451-467. FORBES, M. S. & SPERELAKIS, N. (1972). Ultrastructure of cardiac muscle from dystrophic mice. American Journal of Anatomy 134, 271-290. HARRISON, R. J. & TOMLINSON, J. D. W. (1963). Anatomical and physiological adaptations in diving mammals. In Viewpoints in Biology (Ed. J. D. Carthy and C. L. Duddlington), pp. 115-162. London: Butterworth. HmS, A. (1970). Vertebrate slow muscle fibres. Physiological Reviews 50, 40-62. HIKIDA, R. S. (1972). The structure of the sarcotubular system in the avian muscle. American Journal of Anatomy 134, 481-496. HOYLE, G. (1973). Ultrastructure of barnacle giant muscle fibres. Journal of Cell Biology 56, 74-91.
48
R. S. TULSI
MAUNSBACH, A. B. & WIRSEN, C. (1966). Ultrastructural changes in the kidney, myocardium, and skeletal muscle of the dog during excessive mobilization of free fatty acids. Journal of Ultrastructural Research 16, 35-54. PADYKULA, H. A. & GAUTHIER, G. F. (1970). The ultrastructure of neuromuscular junctions of mammalian red, white and intermediate skeletal muscle fibres. Journal of Cell Biology 46, 27-41. PAGE, S. (1965). Comparison of the fine structure of frog slow and twitch muscle fibres. Journal of Cell Biology 26, 477-497. PAGE, S. (1968). Structure of the sarcoplasmic reticulum in vertebrate muscle. British Medical Bulletin 24, 170-173. PEARSE, A. G. E. (1968). Histochemistry, Theoretical and Applied. London: J. and A. Churchill. REVEL, J. P. (1962). The sarcoplasmic reticulum of the bat cricothyroid muscle. Journal of Cell Biology 12, 571-588. RICHARDSON, K. C., JARREL, L. & FINK, E. H. (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technology 35, 313-323. RIDGWAY, S. H. (1972). Mammals of the Sea. Biology and Medicine. Springfield, Illinois: C. C. Thomas. RIDGWAY, S. H. & MCCORMICK, J. G. (1971). Anaesthesia of the porpoise. In: Textbook of Veterinary Anaesthesia (Ed. L. R. Soma). Baltimore: The Williams and Wilkins Co. VENABLE, J. H. & COGGESHALL, R. E. (1965). A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology 25, 407-408.
39
Observations on the structure of the dorsal muscle in the bottle-nose dolphin (Tursiops truncatus) R. S. TULSI
Department of Anatomy and Histology, The University of Adelaide, South Australia 5001
(Accepted 5 August 1974) INTRODUCTION
The ability of marine mammals to remain under water for prolonged periods has intrigued biologists for decades. A dolphin may remain submerged for 12 minutes at depths of almost 20 metres (Harrison & Tomlinson, 1963), and is capable of considerable physical activity during this time. The structural and functional adaptations which enable marine mammals to cope with an underwater environment are likely to involve intricate adjustments in most organs. Special adaptations of the skeletal musculature are bound to be important (Ridgway, 1972), and it was therefore decided to study the dorsal muscle of the dolphin. This muscle was chosen because it plays a crucial role in the locomotion of the animal both on and under water (Harrison, personal communication). The muscle was examined by light and electron microscopy. The cytochemistry of the extrafusal muscle fibres and the morphology of the muscle spindles, were studied with the light microscope. The fine structure of the extrafusal fibres and their neuromuscular junctions was examined with the electron microscope. MATERIALS AND METHODS
Five animals were used. They were caught in nets in Spencer Gulf, South Australia, and transported alive to Adelaide. One animal (No. 5 in Table 2) was young and the other four were mature adults. Details of the anaesthetic agents used and the length and sex of animals are given in Tables 1 and 2. Study was made of the dorsal muscle lying immediately lateral to the dorsal fin. Animals 2 and 4 were fixed by perfusion through the aorta at 120 cm of water using 1 % glutaraldehyde and 1 % paraformaldehyde in 0-1 M phosphate buffer at pH 7-3. Prior to removal of pieces of dorsal muscle in all animals the muscle was fixed in situ by pouring on it a small quantity of the chilled fixative. Animals 1, 3, and 5 were not perfused, and blocks of dorsal muscle were removed shortly after induction of anaesthesia and immersed in chilled 6% glutaraldehyde in 0-1 M phosphate buffer at pH 7-3. After 2 hours of fixation in the chilled fixative the specimens were washed in phosphate buffer for 1 hour and then post-fixed in 1 % osmium tetroxide for 11 hours. Blocks of muscle from perfused dolphins were similarly treated. Dehydration was carried out in graded series of ethanol and acetone and embedded in an Epon/Araldite mixture. Semi-thin sections were cut at 0 5-1 micron and stained with 1 % sodium borate
40
R. S. TULSI
Table 1. Anaesthetic agents Ph. H.G.O. P.B. Pe D
Phencyelidine (intravenous injection, as suggested by Ridgway) Halothane, nitrous oxide 80%, oxygen 20% (Ridgway & McCormick, 1971) Pentobarbital sodium (intravenous) Pentothal sodium (intravenous) (Ridgway & McCormick, 1971) Diazepam (intramuscular) 10 mgm/100 kgm as suggested by Ridgway
Table 2. Details of animals used. Animal no.
Length (cm)
Sex
Anaesthetic agent
1 2 3 4 5
202 201 182 201 160
M F M M F
Ph., D., H.G.O. Ph., D., H.G.O. P.B. Ph., D., H.G.O. Pe., H.G.O.
(Richardson, Jarrel & Fink, 1960) for orientation. Silver to grey sections were cut for electron microscopy, collected on uncoated grids, stained with uranyl acetate and lead citrate (Venable & Goggeshall, 1965) and viewed under a Philips 300 transmission microscope. In the preliminary stages of the study a number of cytochemical methods were also applied. These included techniques for demonstrating myosin adenosinetriphosphatase (ATPase), succinic dehydrogenase (SDHase), Sudan Black B (for lipids) and myoglobin (Pearse, 1968). OBSERVATIONS
The dorsal muscle showed three types of fibre as in most mammals. The red fibres, which constituted approximately 30%O of the total, had a small diameter (mean of 26-2 ,tm), a large number of lipid droplets, many mitochondria, and a high myoglobin content (Figs. 3-5). They also showed high SDHase and ATPase activity, and a conspicuous Z-line (Fig. 6). The white fibres, which formed approximately 28 % of the total, had a large diameter (mean of 49-5 ,um), no lipid droplets, few mitochondria, and a low myoglobin content (Figs. 3 and 6). The Z-line was narrow
Fig. 1. Longitudinal section of a white muscle fibre to show the T-system. Parts of four transverse tubules (A) and in some areas the adjacent cisternae of the sarcoplasmic reticulum are also seen. The three upper transverse tubules involving two sarcomeres are joined by longitudinal elements (B) of the T-system at (C). Material from dolphin 1. x 84600. Fig. 2. Longitudinal section of a white muscle fibre to show the T-system and sarcoplasmic reticulum. Two adjacent transverse tubules (A) involving one sarcomere, are linked by a longitudinal element (B) of the T-system which joints the former at (C). Lying adjacent to the lower transverse tubule a cisterna of the sarcoplasmic reticulum is clearly visible which seems to provide an extension (D) along the longitudinal element (B) of the T-system. Other longitudinally oriented tubules (E), forming part of the sarcoplasmic reticulum, are seen extending upwards from the cisterna. Several electron-dense bridges extending between the transverse tubule and adjacent cisterna of the triad are seen in the upper part of the figure. Material from dolphin 1. x 48 350.
41
Skeletal muscle of dolphin
A~~~~A
A7
..: 1
42
R. S. TULSI
(Fig. 6) and there was low SDHase and ATPase activity. The remainder of the fibres could be said to be of 'intermediate' type (Fig. 3 B); however, on the basis of fibre diameter and population of lipid droplets, these intermediate fibres could be divided into two groups. One had smaller fibres (mean 31 8 ,tm) with many lipid droplets and the other had larger fibres (mean 42-8 ,tm) but relatively few lipid droplets. Most features of dolphin muscle, such as its capillaries (Fig. 7), sarcolemma, myofibrils, myofilaments, and sarcomeres (Figs 4 and 6) are essentially similar to those described in other mammals and need not be described here. As there has been some doubt about the existence of muscle spindles in cetacea, some limited observations on spindles and neuromuscular junctions are perhaps worth recording. A total of 45 blocks were taken from five animals in a search for muscle spindles by light microscopy. Three spindles were found with morphology similar to that described in other mammals. Three neuromuscular junctions were seen with the electron microscope, two on white fibres and one on an intermediate fibre. No neuromuscular junction was seen on a red fibre in the present limited study. The neuromuscular junctions on the white fibres showed marked branching of junctional folds which extended deep into the sarcoplasm and there were many subsarcolemmal vesicles features recently described (Padykula & Gauthier, 1970) as typical of white fibres elsewhere. T-system. The T-system was well developed and readily recognized in most muscle fibres. Typical triads were found at the level of the A-I junction, with the result that there were two triads to each sarcomere, a pattern seen in many other mammals. Between the central component of each triad (the transverse tubule) and the two flanking cisternae (of the sarcoplasmic reticulum) there were several evenly spaced electron-dense bridges (Figs 1 and 2). Continuity between the T-system and the sarcolemma could be seen in several places. A feature of particular interest in the dolphin material was the occurrence of longitudinal channels linking adjacent transverse tubules. This linking occurred within a single sarcomere (Fig. 2), as well as between systems of two neighbouring sarcomeres (Fig. 1). In other words the transverse tubules of the T-system were clearly seen to be linked by longitudinal elements when the plane of section passed through both (Fig. 1). It is important to Fig. 3. Transverse section of muscle fibres stained with Sudan Black B to show lipid droplets. Note that there is much lipid in the red (A) fibre, a moderate amount in the intermediate (B) fibre and little or none in the white (C) fibre. Material from dolphin 5. x 960. Fig. 4. Transverse section of part of a red muscle to show several lipid droplets (L), a mitochondrion (M) and muscle filaments. Note that some lipid droplets are partially surrounded by markedly electron-dense granules (arrow). Material from dolphin 3. x 68750. Fig. 5. Longitudinal section of a red muscle fibre to show a large number of lipid droplets (L), many of which are closely associated with mitochondria (M). In some areas closely packed droplets coalesce to form a longitudinal column of lipids. There is also an accumulation of a large number of lipid droplets and mitochondria immediately deep to the plasma membrane producing a convexity on the surface of the fibre. These droplets are not as dense as those deep in the muscle, but parts of the electron-dense walls of some of them are visible in the lower left part of the figure. Material from dolphin 5. x 12000. Fig. 6. Longitudinal section of parts of two adjacent fibres, one a red (lower) and the other a white (upper). The red fibre shows a wider Z-line (Z), more mitochondria (M), many lipid droplets (L), but not so many glycogen granules. Material from dolphin 5. x 16500.
Skeletal muscle of dolphin
ei-
43
44
R. S. TULSI
note that the longitudinal elements of the T-system were themselves flanked by extensions (Fig. 2D) of adjacent cisternae of the sarcoplasmic reticulum, and that such extensions were distinct from the longitudinal tubes (Fig. 2E) of the sarcoplasmic reticulum. The longitudinal elements of the T-system were found only in white fibres: considerable search failed to show similar elements in red fibres. Lipid droplets. Another interesting feature of the dolphin muscle was the large number of lipid droplets in the red muscle fibres. When muscle fibres were stained with Sudan Black B (Fig. 3), the red fibres showed many lipid droplets whereas white fibres showed few or no droplets. These lipid droplets (up to 074 mm in diameter) occurred both in young and fully grown animals and often lay close to mitochondria pressing against them and distorting them (Figs. 4, 5 and 6). The droplets were commonly placed between myofibrils or immediately deep to the sarcolemma, at times so numerous as to produce a convexity on the surface of the muscle (left edge of Fig. 5). When a large number of droplets accumulated between myofibrils they either coalesced or were firmly pressed against one another, forming longitudinal columns extending along several sarcomeres (in the middle of Fig. 5). The lipid droplets were not membrane-bound. Several were partially surrounded by markedly electron-dense granules (Fig. 4). This has also been observed in lipid droplets in other dolphin tissues (Fanning, personal communication) and also in lipid droplets in normal cartilage in other species (Collins, Ghadially & Meachim, 1965). There was considerable variation in the electron density of lipid droplets apparently related to the techniques of fixation and staining used and to the age and physiological state of the animal. In the present study the material from the older animals (Figs. 4, 7 and 8) showed denser droplets than that from the younger animals (Figs. 5 and 6). A further topic of interest was the relationship of lipid droplets and chylomicrons to capillaries and extracellular spaces. Material from animals 1 and 3 (Figs. 7 and 8) suggested that not only is there a large quantity of lipid in red muscle but also that there is a marked movement of lipids across the capillary wall. Figure 7 shows a lipid droplet in a capillary (1), a chylomicron lying free in the lumen (2), another chylomicron in the lumen partially surrounded by an endothelial flap (3) and possibly another four chylomicrons in a vacuole within the capillary endothelium (4). The same figure also shows a lipid droplet (5) in an adjacent muscle fibre. Figure 8 shows pinocytotic vesicles (V1) and a vacuole (V2) within the capillary endothelium and a lipid droplet immediately outside the capillary close to a muscle fibre. Fig. 7. Transverse section of a capillary and part of an adjacent muscle fibre (M). Within the lumen of the capillary is an intensely electron-dense lipid droplet (1), two chylomicrons (2, 3) one of which is partially surrounded by a flap (F) of endothelium. Several other chylomicrons (4) are within a vacuole in the wall of the capillary and another lipid droplet (5) is seen in the adjacent muscle. E, nucleus of an endothelial cell. R, red blood cell. Material from dolphin 3. x 27000. Fig. 8. Oblique section of a capillary and part of an adjacent muscle fibre (M). Note that an intensely electron-dense lipid droplet (L) appears to be wedged between the capillary wall and the muscle fibre. C, lumen of capillary. V1 micropinocytotic vesicles. V2, vacuole within capillary wall. Material from dolphin 1. x 13400.
Skeletal muscle of dolphin ..
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46
R. S. TULSI DISCUSSION
The present study has shown that the dorsal muscle of the dolphin possesses red, white, and intermediate fibres like most other vertebrate skeletal muscles. The ultrastructure and cytochemistry of the muscle fibres, except for those features noted below, and the morphology of the muscle spindles and neuromuscular junctions, were essentially similar to those in other mammals. The special features of the dolphin material were: (1) the presence in the red fibres of a large number of lipid droplets and (2) the presence in white fibres of longitudinal tubular channels linking adjacent transverse components of the T-system; these links were both inside a given sarcomere and between adjacent sarcomeres. Lipid droplets are normal constituents of ventricular cardiac muscle cells (Maunsbach & Wirsen, 1966) but in skeletal muscle they are only variably present in some species (Bloom & Fawcett, 1969). Lipid droplets in electron micrographs of skeletal muscle show varying density due partly to differences in technique and partly to differences in chemical composition. The evidence supporting the presence of a large amount of lipid in dolphin muscle is at present entirely morphological. However, when electron micrographs from the present study were compared with those published for the normal muscles of many other vertebrates it was clear that none of the latter approached the dolphin in the number of droplets in its red muscle fibres. In fact the lipid droplets shown by Forbes & Sperelakis (1972) in their Plate 2 (Fig. 6) and Plate 3 (Fig. 6) of pathological muscle are far fewer than in normal dolphin muscle (Fig. 5). The significance of the large lipid droplet content of dolphin red muscle is unclear but it is reasonable to suppose that it serves as a readily available source of high energy metabolic fuel. The possible functional implications of one particular observation regarding lipid droplets in the dolphin is worth discussing. The muscle capillaries in the dolphin invariably showed a large number of pinocytotic vesicles as well as several vacuoles on the luminal and basal surfaces of the endothelium. Some of the vesicles contained a single small lipid droplet, but some of the vacuoles had as many as four droplets. None of the droplets was bound by a membrane and they were never seen to be associated with any endothelial organelle. Within the capillary lumen several small droplets were observed, some of which were partially surrounded by endothelial flaps; curiously, however, no droplet of any size was ever seen lying entirely outside the capillary. All this strongly suggests that the movement of at least some of the lipid across capillary endothelium in muscle is by pinocytosis. In a comprehensive review (Page, 1968) the T-system is described as transversely oriented tubular invaginations of the'sarcolemma either at the level of the A-I junction or at the Z-line, along which surface depolarization travels inwards towards the deeply placed filaments during the excitation-contraction process: there is no mention of any connexion between adjacent tubules inside the muscle fibre by means of longitudinal channels. In a more recent review of vertebrate slow muscle fibres (Hess, 1970) it is stated that the system is scanty in avian, frog, and mammalian slow muscle fibres. In the last decade or so, workers studying skeletal muscle of other vertebrate species have occasionally observed what could only be interpreted as parts of longitudinal elements of the T-system. These have been seen in the frog (Page,
Skeletal muscle of dolphin
47
1965; Eisenberg & Eisenberg, 1968), bird (Hikida, 1972; Hoyle, 1973), bat (Revel, 1962) and in the guinea-pig (Ryans, personal communication). In the dolphin, however, the longitudinal element of the T-system seems to be well developed in the white fibres where it links the transverse elements within a single sarcomere and within adjacent sarcomeres. The physiological role of the longitudinal component of the T-system is a matter for speculation. It is tempting to suggest that it has a function similar to the rest of the T-system. This suggestion seems reasonable as the longitudinal tubes appear to be morphologically similar to the transverse tubes and are closely associated with extensions of the terminal cisternae of the triad. The longitudinal component of the T-system should therefore facilitate transmission of impulses within a sarcomere and between adjacent sarcomeres deep inside a muscle fibre: it certainly increases the total surface area of the T-system in contact with myofibrils. SUMMARY
The dorsal muscle of five South Australian bottle-nose dolphins has been studied. The ultrastructure and cytochemistry of the red, white and intermediate fibres were essentially similar to those of other mammalian skeletal muscles. Two features of special interest were: (a) the large number of lipid droplets in the red muscle fibres, and (b) the presence of longitudinal tubes linking the transverse tubes of the Tsystem, both within a sarcomere, and also between adjacent sarcomeres, of the white muscle fibres. The muscle spindles and neuromuscular junctions in the dolphin appeared to be morphologically similar to those of other mammals.
Thanks are due to the State Director of Fisheries and the Director of Wildlife and Fauna Conservation for permission to obtain animals. To Professor R. J. Harrison I am grateful for suggesting the study and for reading the manuscript. I thank Professor Janis Priedkalns, Chairman of the Department, for his interest and suggestions in preparing the manuscript. I acknowledge with gratitude helpful comments and criticism made by Professor V. Navaratnam. The technical assistance of Mrs G. M. Hermanis is gratefully acknowledged. REFERENCES
BLOOM, W. & FAWCETT, D. W. (1969). A Textbook of Histology. London: Saunders Co. COLLINS, D. H., GHADIALLY, F. N. & MEACHIM, G. (1965). Intracellular lipids of cartilage. Annals of the Rheumatic Diseases 24, 123-135. EISENBERG, B. & EISENBERG, R. (1968). A selective disruption of the sarcotubular system in frog sartorius muscle. A quantitative study with exogenous peroxidase as a marker. Journal of Cell Biology 39, 451-467. FORBES, M. S. & SPERELAKIS, N. (1972). Ultrastructure of cardiac muscle from dystrophic mice. American Journal of Anatomy 134, 271-290. HARRISON, R. J. & TOMLINSON, J. D. W. (1963). Anatomical and physiological adaptations in diving mammals. In Viewpoints in Biology (Ed. J. D. Carthy and C. L. Duddlington), pp. 115-162. London: Butterworth. HmS, A. (1970). Vertebrate slow muscle fibres. Physiological Reviews 50, 40-62. HIKIDA, R. S. (1972). The structure of the sarcotubular system in the avian muscle. American Journal of Anatomy 134, 481-496. HOYLE, G. (1973). Ultrastructure of barnacle giant muscle fibres. Journal of Cell Biology 56, 74-91.
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MAUNSBACH, A. B. & WIRSEN, C. (1966). Ultrastructural changes in the kidney, myocardium, and skeletal muscle of the dog during excessive mobilization of free fatty acids. Journal of Ultrastructural Research 16, 35-54. PADYKULA, H. A. & GAUTHIER, G. F. (1970). The ultrastructure of neuromuscular junctions of mammalian red, white and intermediate skeletal muscle fibres. Journal of Cell Biology 46, 27-41. PAGE, S. (1965). Comparison of the fine structure of frog slow and twitch muscle fibres. Journal of Cell Biology 26, 477-497. PAGE, S. (1968). Structure of the sarcoplasmic reticulum in vertebrate muscle. British Medical Bulletin 24, 170-173. PEARSE, A. G. E. (1968). Histochemistry, Theoretical and Applied. London: J. and A. Churchill. REVEL, J. P. (1962). The sarcoplasmic reticulum of the bat cricothyroid muscle. Journal of Cell Biology 12, 571-588. RICHARDSON, K. C., JARREL, L. & FINK, E. H. (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technology 35, 313-323. RIDGWAY, S. H. (1972). Mammals of the Sea. Biology and Medicine. Springfield, Illinois: C. C. Thomas. RIDGWAY, S. H. & MCCORMICK, J. G. (1971). Anaesthesia of the porpoise. In: Textbook of Veterinary Anaesthesia (Ed. L. R. Soma). Baltimore: The Williams and Wilkins Co. VENABLE, J. H. & COGGESHALL, R. E. (1965). A simplified lead citrate stain for use in electron microscopy. Journal of Cell Biology 25, 407-408.
