/. Embryol. exp. Morph. Vol. 23, 1, pp. 191-207, 1975
Printed in Great Britain
191
Aspects of tail muscle ultrastructure and its
degeneration in Rana temporaria
By H. FOX 1
From The Department of Zoology,
University College, London
SUMMARY
.1. Tail muscles of larval Rana temporaria and to a lesser extent Xenopus laevis, at prometamorphosis, were investigated by light and electron microscopy. In the case of Rana
their degeneration is described at metamorphic climax.
2. The non-degenerate tail muscles of both genera are generally similar in their ultrastructure and likewise similar to those striated muscles of other adult vertebrates including
amphibians.
3. At climax tail muscles of Rana degenerate by autolysis. It seems likely that lysosomal
enzymes are directly involved in sarcoplasmic degeneration. The relationship between
lysosomes and myofibrillar degradation is difficult to establish. The subject is discussed in
the light of relevant information (mainly mammalian) on muscle dystrophies.
4. Anuran tail muscle is highly degraded before its probable phagocytosis by mesenchymal
macrophages.
INTRODUCTION
Somitic striated muscle is a major component of the anuran larval tail. At
metamorphic climax in vivo, under the influence of a high threshold level of
circulatory thyroid hormones (Etkin, 1964), or in vitro, influenced by a similarly
high ambient concentration of thyroxine, thyroxine analogs or triiodothyronine,
the tail involutes with consequent degeneration and disappearance of its musculature (see among others Kollros, 1961; Shaffer, 1963; Kaltenbach, 1968;
Weber, 1969; Frieden & Just, 1970). As in all vertebrates anuran striated muscle
has a complex ultra-structure (Page 1965; Franzini-Armstrong, 1970; Karlson
& Anderson-Cedergren, 1971; Eichelberg & Schneider, 1973). Features of the
larval axial and tail musculature have likewise been described by electron
microscopy (Porter & Palade, 1957; Weber, 1964; Fox, 1972c), but much is
obscure, particularly causality when anuran tail muscles degrade at climax.
In vertebrates a variety of factors will elicit muscle degeneration (see below)
and at this time a number of structural features are similar whatever the cause
(see Tomanek & Lund, 1973). Information obtained from the study of climactic
1
Author's address: Department of Zoology, University College, Gower Street, London
W.C.I.
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EMB 34
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H. FOX
anuran tail muscle could well be of importance in relation to the wider aspects
of tissue ageing and disease.
The results in the present work lead to the conclusion that at climax the
sarcoplasm degenerates autolytically, probably initiated through the agency of
lysosomal enzymes, though the widespread necrosis expressed visually in terms
of large cytolysomes positive for acid phosphatase is not always recognized
to the same extent as in some other degenerating tail tissues. Myofibrillar
degeneration is described and the vexed question on the possible role played
by lysosomal enzymes is considered.
It is likely that degraded tail muscle is phagocytosed by mesenchymal
macrophages.
MATERIAL AND METHODS
Specimens of Rana temporaria larvae were staged as for Rana dalmatina
(Cambar & Marrot, 1964); Xenopus laevis according to the scheme of Nieuwkoop and Faber (1956). In Rana prometamorphosis includes stages 45-49,
used in the present work, and climax extends from late stage 49-54.
For examination by light microscopy tails from normal Rana temporaria (in
tap water) at various stages from 41 (15 mm) to <54 (14 mm) were utilized.
Larval length is given in brackets. All specimens were fixed in Bouin's fluid,
embedded in paraffin wax, sectioned transversely at 10 ju,m and stained by
haematoxylin and eosin.
For examination by electron microscopy tails at different levels proximodistally, from Rana at stages 45, 45-47, 48, 48-49, 49, 52, 52-53 and 53 were
fixed in osmic acid (Palade, 1952), or glutaraldehyde and post-osmicated (Sabatini, Bensch & Barrnett, 1963), or in a combined mixture of glutaraldehyde and
osmic acid (Hirsch & Fedorko, 1968); each stage was frequently fixed by more
than one method. After dehydration and embedding in Araldite, thick sections
(about 1 fim thick) were examined by phase contrast microscopy for tissue
localization and silver grey sections (about 90-100 nm thick) were stained by
uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963). Tails of
Xenopus laevis at early and late prometamorphic stages 45 and 57 (Nieuwkoop
& Faber, 1956) and fixed by the method of Hirsch & Fedorko (1968), were
likewise investigated mainly from sagittal sections.
Deposition of acid phosphatase in tail muscle was registered for electron
microscopy by the method of Gomori (1952), modified by Barka & Anderson
(1962). Incubated hand-cut transverse sections, about 150/*m thick, were
previously fixed for about 1 h in glutaraldehyde (see Fox, 1974). Controls were
run omitting the sodium /?-glycerophosphate from the incubation medium.
Silver grey sections were stained by uranyl acetate alone. All sections were
viewed under an AEI EM 6B electron microscope and to a lesser extent an
AEI Corinth 275.
infrastructure o/Rana tail muscles
193
OBSERVATIONS
Examination by light microscopy
The tail musculature of well-developed, active larval amphibians comprises
a series of segmentally arranged muscles, separated by connective tissue, the
myocommata, and situated on each side of the tail between the skin and the
nerve cord and notochord. Anterior somites are longer than posterior ones, at
least in Xenopus embryos (Hamilton, 1969), and in Rana larvae individual tail
somite size seems to diminish progressively on proceeding distally (see also
Brown, 1946). Somites disappear just before the tail tip.
Tail muscles of anurans degenerate at metamorphic climax. In Rana at
late stage 49, the tail tip blackens, probably in part due to the accumulation of
pigment and with the exception of macrophages and perhaps some blood cells,
shows features of degeneration.
Initially there is widespread necrosis in the distal tail region. Simultaneously
the tail reduces in length to a tiny sac-like structure, which ultimately disappears. At the height of climax (stages 52-53) the posterior third of one tail
stub (3-4 mm long measured from the front margin of the cloaca) included
degraded tissue, much of it presumably derived from muscle suffering phagocytosis by macrophages. Further forwards the tail includes partially degenerate
blocks of myofibrillar tissue, some dispersed into smaller disorganized units.
The recognizable notochord shows features of degeneration 1-6 mm in
front of the tail tip, though some muscle components are necrotic even further
proximally. The nerve cord is still luminated 0-7 mm from the stub end. On the
whole in Rana at climax, the extent of degeneration is more widespread in
muscle and more localized in the nerve cord than in the case of other tail tissues.
Examination by electron microscopy
(a) Preclimactic tail muscle
As in other vertebrates tail striated muscle cells of larval Rana and Xenopus
typically are made up of a large number of longitudinally arranged, closely
packed (especially in older larvae), parallel myofibrils (about 1 fim thick); each
is cross-striated and of regularly repeating periodicity, due to the overlapping
of constituent thick and thin myofilaments of myosin and actin respectively
(Fig. 1). In longitudinal section the Z lines are usually straight. The occasional
wavy Z lines found in young prometamorphic, non-degenerate tail muscles
may represent either oblique sectional orientation or myofibrillar distortion
owing to incomplete muscle contraction. The dimensions of the I band,
which correspond to the degree of sarcomere contraction, reflect the amount
of mutual sliding of the overlapping myofilaments whose lengths remain
constant (see Price, 1969).
The sarcoplasm includes ribosomes, polyribosomes and a modest granular
13-2
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H. FOX
infrastructure o/Rana tail muscles
195
endoplasmic reticulum. A prominent sarcoplasmic reticulum embraces the
myofibrils throughout their length. Frequently a Golgi complex is recognized.
Occasional large lipid droplets are found at the periphery or between the
myofibrils and there are pigment bodies and numerous glycogen granules.
Mitochondria of varied shape, but usually elongated in the longitudinal plane,
frequently occur in groups at the periphery or singly between the myofibrils
(Fig. 1). Cristae are moderately numerous and of random orientation. Vesicles
may indent the sarcolemma and so-called caviolae often occur - whether they
always open to the extracellular space cannot be decided. Large nuclei often
with a prominent nucleolus are situated usually against the outer regions of the
myofibrils, or terminally in the cell.
The sarcolemma comprises a trilaminar plasma membrane; externally a
DESCRIPTIONS OF PLATE FIGURES OF RANA
TEMPORARIA
Electron micrographs are of sectioned material fixed in a mixture of glutaraldehyde and osmic acid (Hirsch & Fedorko, 1968), except for Fig. 2, which wasfixedin
glutaraldehyde and post-osmicated (Sabatini et al. 1963), and Figs. 11-16, which
were prepared by the method of Gomori (Gomori, 1952; see Barka & Anderson,
1962). All sections were stained by uranyl acetate (Watson, 1958) and lead citrate
(Reynolds, 1963), except for the acid phosphatase sections which were stained by
uranyl acetate alone.
All sections were sagittal except for those used for histochemical examination,
which were sectioned transversely.
A, A band; an, autolytic area; av, autophagic vacuole; ax, axon; cy, cytolysome;
db, dense body; de, dense myofilamentous tissue; dm, dense myofilaments in final
stages of degradation; dse, electron dense striated myofibrils (sarcolytes); es,
extracellular space; g, glycogen; H, H zone; hd, highly degraded myofilamentous
tissue; /, I band; Ip, lipid; ly, lysosome; M, M line; m, mitochondrion; mf,
myofilaments; my, myofibrils; n, nucleus; na, necrotic area; p, pigment body;
rp, reaction product; s, sarcoplasm; ser, smooth endoplasmic reticulum; SM,
sarcomere; sr, sarcoplasmic reticulum; Tt, T tubule of triad; tc, terminal cisterna
of the sarcoplasmic reticulum; va, vacuolation; Z, Z line.
FIGURES 1-5
Fig. 1. Stage 45. Area of myofibrils showing the typical banding pattern in nondegenerate tail muscle.
Fig. 2. Stages 48-49. Myofibril at the Z-I band region. The triad includes the T tubule
at the base of the Z line and the adjacent terminal cisternae of the sarcoplasmic
reticulum. There is communication between the region below the Z line, where the
T tubule is localized, and the extracellular space.
Fig. 3. Stage 52. Myofibrils in the process of losing their striation and merely vestigial
Z lines, 1 bands and M lines are now present. The sarcoplasm includes numerous
round, dense bodies, which are not present in preclimactic, non-degenerate muscle.
Fig. 4. Stage 53. Round, dense bodies in the sarcoplasm between the disorganized
degenerating myofilaments. Such bodies are only found in degenerating muscle at
climax and they are similar to those in degenerating nerve cord of the tail (Fox, 1973 b).
Fig. 5. Stage 52. Myofibrils showing variability in the degree of loss of striation.
Extensive areas of necrosis are seen. Note especially the large cytolysome and the
accumulation of pigment.
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H. FOX
moderately dense basement membrane is intimately associated with collagen
fibrils, which often penetrate between blocks of myofibrils, though they are
always separated from them by the sarcolemmal sheath.
In Rana temporaria tail muscles triads are particularly well represented,
situated at the levels of the Z lines. The T tubular system opens to the cell
exterior, for continuity across the sarcoplasm between the extracellular region
and the base of the Z line, where the T tubule is clearly localized, is occasionally
recognizable (Fig. 2). T tubules encircle the myofibrils at the Z lines (Fig. 1).
They may also extend longitudinally in some places, as in the adult frog sartorius muscle, though still remain separated from the sarcoplasmic reticulum
(Huxley, 1964), a feature likewise recognized in the Rana tail muscle.
In both Rana and Xenopus the diameter of the thick myofilaments is about
16 nm and of the thin myofilaments about 6 nm. The recorded maximum
mitochondrial length and breadth of groups of the organelles, in typical
random muscle profiles, each of Rana and Xenopus, showed those of Rana to
range between 1-3 and 2-7/tm in length and 0-4 and 0-8 jam in breadth. Comparable measurements in Xenopus were 1-5-5-5 /im and 0-6-1-5 jim. Clearly
throughout prometamorphosis mitochondria in Xenopus tail muscles appear
to be of greater area than those of Rana.
(b) Climactic degeneration of tail muscles
At climax ultrastructural profiles of degenerating cells of the anuran tail are
variable, and the intimate relationships of the sub-epidermal cells and associated
macrophages are complex. In different cells degeneration phenomena appear
seemingly at random. Even at the same transverse level tail cells of like tissue
may vary in their degree of degeneration, a variation which may occur within
the same cell. The following description, therefore, provides a generalized
account of muscle necrosis and of its subsequent phagocytosis during tail
involution.
At climax the sarcoplasm normally shows signs of degeneration before the
myofibrils, though the entire muscle is soon affected at any level but mainly
distally. A number of organelles, not usually present in preclimactic stages,
appear in the sarcoplasm. Numerous membrane-bound bodies having a whorled
or mottled appearance occur, often near the nucleus. Other large irregularshaped bodies, frequently appearing fibrous in texture, are often found between
myofibrils or in the sarcoplasm peripherally, and a number of smooth-surfaced,
roundish, dense bodies occur, either in groups at the periphery or singly
between myofibrils (Figs. 3, 4). Similar bodies are found in macrophages and
they are often profuse in degenerating tail nerve cord cells (Fox, 1973 b).
Myelin figures (occasionally seen in earlier stage-45 specimens of Rana) are
present, often near the sarcolemma - the sarcolemmasomes (Bone & Ryan,
1973). Mitochondria degenerate in two ways: either the cristae disorganize
and disappear leaving an empty vesicle, or the outer membranes rupture and
Ultrastructure o/Rana tail muscles
197
the partially disorganized cristae spill out to merge with and subsequently
disperse amid the ambient cytoplasm (see Fox, 1972c, 1973a). Small autophagic vacuoles and somewhat larger cytolysomes are present; these are
occasionally seen between the myofibrils (Figs. 5, 13). Lipid droplets and
pigment bodies increase in number, sometimes the latter are grouped in membrane-bound structures. Usually the Golgi complex, granular endoplasmic
reticulum and ribosomes reduce in amount or have disappeared in highly
degenerate sarcoplasm, but there is an increase in the amount of smooth
endoplasmic reticulum and in the number of glycogen granules.
Areas of necrosis gradually become more widespread and large cytolysomes
are sometimes recognizable (Figs. 5, 7).
The sarcoplasm ultimately loses its typically recognizable form, the sarcolemma ruptures and the remains of the sarcoplasm and the partially or wholly
degraded myofilaments become intimately associated spatially with enveloping
macrophages (see Fox, 1972c, fig. 9; 1973c, fig. 5).
The muscle cell nucleus usually appears normal even when the myofibrils
and sarcoplasm show pronounced features of degeneration. Nuclear resistance
to necrotizing influences is likewise seen in the anuran tail nerve cord and
notochord and in the apical ridge of the limb-buds of chick and mouse embryos
(Jurand, 1965; Fox, 1973 a, b).
The first obvious sign of myofibrillar degeneration is the loss of striation,
when only remains of banding are visible (Figs. 3, 5). The myofilaments become
disorientated (Fig. 4) and lose their parallel, elongate arrangement, seen ideally
in longitudinal sections of preclimactic tail muscles. At climax a sagittal section
often includes profiles of myofilaments orientated in all directions. The clarity
of delineation of the mass of disorganized myofilaments is reduced due to the
blurring of the myofilamentous outlines, which are often still recognizable
in highly degraded myofibrillar tissue.
Vestigial Z lines are occasionally straight but more often wavy in appearance.
The extensive sarcoplasmic reticulum is sometimes expanded amid the degenerating myofibrils. Substantial areas of myofibrillar tissue often become electron
dense and merge with degenerate, often electron-translucent, homogeneous
sarcoplasmic substance (see also Fox, 1972c, figs. 5, 9). In some cases muscle
cells include highly electron dense, fragmented myofibrils, with a recognizable
striation (Fig. 6) - the so-called sarcolytes (Brown, 1946; see also Watanabe &
Sasaki, 1974) - which may occur simultaneously with less degenerate myofilaments within the same cell.
At the height of climax profiles of highly degenerate, tail-stub muscle cells
include roundish areas of varied size and of lightish, homogeneous substance;
frequently they possess a denser occasionally filamentous core (Figs. 7-10).
Disorientated myofilaments are often found alongside, or in spatial continuity
with, such homogeneous degraded tissue (Fig. 9); clearly some at least of the
latter is derived from myofilaments. Degenerating muscle cells thus include
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H. FOX
Ultrastructure o/Rana tail muscles
199
partially and highly degraded myofilamentous (and sarcoplasmic) tissue, which
has degenerated autolytically before it spills out through a ruptured sarcolemma
to suffer phagocytosis.
Macrophages likewise may include similar degraded, homogeneous substance
in heterophagic vacuoles, or phagosomes, presumably the result of previous
engulfment (see also Watanabe & Sasaki, 1974). It is, nevertheless, frequently
difficult to decide between profiles of highly degenerate muscle cells and
macrophages; whether, for example, autophagic or heterophagic vacuoles are
featured. Initially during prometamorphosis mesenchymal macrophages are
spatially associated with muscle and later, at climax, they closely envelop
degraded tail muscle tissue (Fox, 1972c, 1973 c). Yet many cells containing
fragmented, electron-dense, striated myofibrillar tissue (sarcolytes) and classified
as macrophages (Kerr, Harmon & Searle, 1974), may well be degenerate muscle
cells, as earlier implied by Brown (1946). Numerous examples were found of
cells possessing myofilaments showing varying degrees of degradation, including
some which were similar to the so-called heterophagic inclusions (Figs. 6, 10).
Deposition of acid phosphatase represented by reaction product (RP) is not
easily demonstrated in muscle tissue. However, in a number of different specimens of Rana, round or oval-shaped, dense, membrane-bound bodies (3-9 in
each group) and positive for RP, were found in tail muscle sarcoplasm near the
nucleus of late prometamorphic (stage 49) and climax specimens. They varied in
diameter from 0-2 to 0-5 jttm (Fig. 11). RP also occurred in small autophagic
vacuoles (often situated between myofibrils), slightly larger cytolysomes and
occasionally alongside or within the sarcoplasmic reticulum (Figs. 12, 13, 16).
RP was also found deposited in discrete areas against the outermost myofibrillar
FIGURES
6—10
Fig. 6. Stage 53. Degenerate myofibrils (sarcolytes) of varied electron density
showing some striation. Some portions of the myofibrils are isolated within membrane-bound regions and they are degrading autolytically to a homogeneous
substance.
Fig. 7. Stage 53. Heavy necrosis in the sarcoplasm and varied profiles of myofibrillar
degeneration, including disorganized myofilaments, dense myofilaments - all
without striation - and highly degraded homogeneous myofibrillar substance in
which vestigial myofilaments may still be present.
Fig. 8. Stage 53. Highly degraded myofibrillar tissue with vestigial myofilaments
still recognizable. Lipid is abundant. Lesser degenerate myofilaments are seen
nearby.
Fig. 9. Stage 53. Origin of the highly degraded homogeneous substance from
myofilaments during the final stages of muscle degeneration.
Fig. 10. Stage 53. Varied array of myofibrillar degeneration profiles in tail muscle
at climax. They range from striated to unstriated myofibrils, highly electron-dense
sarcolytes, and fully degraded homogeneous tissue of myofibrillar derivation. The
sarcoplasm includes areas of necrosis with abundant lipid, especially near the top
of the illustration between the two nuclei.
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H. FOX
FIGURES
11-16
Fig. 11. Stage 49. Just preceding metamorphic climax. Acid phosphatase-rich organelles near the nucleus. They are not found in younger larval tail muscles and
some of them at least are probably lysosomes.
Fig. 12. Stage 49. Deposition of reaction product (RP) in the sarcoplasm and
within a mitochondrion.
Fig. 13. Stage climax. Autophagic vacuole, positive for RP, between myofibrils.
Fig. 14. Stage climax. Widespread deposition of RP in the degenerating sarcoplasm. The myofilaments show no deposition of RP.
Fig. 15. Higher magnification of a region of Fig. 14 showing the deposition of RP
in the sarcoplasm.
Fig. 16. Stage 49. Cytolysome positive for RP, situated in the sarcoplasm between
the myofibrils.
Ultrastructure o/Rana tail muscles
201
surface in the sarcoplasm, around and sometimes within mitochondria (Fig. 12).
Deposition of RP in the sarcoplasm was sometimes found to be quite extensive
(Figs. 14, 15). RP was never found to impregnate or be freely distributed amid
myofilaments. Controls were always negative for reaction product.
DISCUSSION
Primary lysosomes were not recognized by Weber (1964) in regressing tail
muscles of Xenopus, who concluded that they degenerated in their absence,
perhaps without the intervention of lysosomal enzymes.
Yet it is difficult to imagine lysosomal enzymes not to be involved, in some
way, when vertebrate muscles degenerate. In anurans tail involution depends
upon the de novo synthesis of protein, probably acid hydrolases, near climax
(Tata, 1966; Weber, 1969). A number of them, including cathepsins, acid
phosphatase and DNA-ase, increase in amount (together with an increased
population of macrophages) towards climax (Lehman, 1953; Eeckhout, 1969;
Weber, 1969; Hickey, 1971). Acid hydrolases are probably synthesized by
macrophages (Hassan & Autuori, 1964; Hassan & Marinucci, 1966), though at
climax in autolysing tissues (including muscle; see below) lysosomal enzymes
also substantially increase in amount in the anuran tail (Kaltenbach, 1971;
Fox, 1972a, 1913a, b, 1974; Fry, Leius, Bacher & Kaltenbach, 1973).
Among mammals lysosomes are sparse in normal rat muscle (Gordon, Price
& Blumberg, 1967; Buchanan & Schwartz, 1967; Weinstock & Iodice, 1969)
and like lipofuscin granules (Shafiq, Milhorat & Gorycki, 1967), they increase
in number together with the amount of acid hydrolases (Pennington, 1963;
Tappel et al. 1962; Tappel, Sawant & Shibko, 1963; in ageing, congenital
muscular dystrophy, muscular atrophy from denervation and nutritional deficiency (usually vitamin E) in man, various mammals and the chicken (Pellegrino
& Franzini, 1963; Smith, 1964; Howes, Price & Blumberg, 1964; Milhorat,
Shafiq & Goldstone, 1966; Pearce, 1966; Gordon et al. 1967; Weinstock &
Iodice, 1969; Shafiq, Askanas, Asiedu & Milhorat, 1972). Muscle autolysis in
dystrophic muscles occurs simultaneously with increase in acid hydrolases
(Iodice, Chin, Parker & Weinstock, 1972). The increased activity of acid
hydrolases in dystrophic muscle was considered due to the damage of muscle
lysosomal membranes, permitting the release of enzymes to degrade myofibrillar
and sarcoplasmic tissue, though the main source of enzymes probably arose from
invasive macrophages (Tappel et al. 1962; Tappel et al. 1963; Zalkin et al. 1962).
In contrast, others believed that the main source is from the muscle itself (Weinstock & Lukacs, 1965; Weinstock & Iodice, 1969), especially in later stages of
dystrophy (Shafiq et al. 1972). Lysosomes (Pearce, 1966) and autophagic
vacuoles (Milhorat et al. 1966) occur between myofibrils in human dystrophic
muscles. Furthermore, free reaction product (acid phosphatase) was reported to
occur in the walls of the T tubules and to a lesser extent in the intermyofibrillar
sarcoplasmic reticulum of human dystrophic muscle (Pearce, 1966). Pellegrino
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H. FOX
& Franzini (1963) correlated the appearance of lysosomes with rat muscle
atrophy after denervation.
Lysosome-type bodies (positive for RP) and features of autolysis are rarely
found in tail muscles of young prometamorphic larvae of Rana (stages 45-47),
though occasionally autophagic vacuoles are present at stage 49 (and in Xenopus
at stage 57) just before climax. Organelles and autophagic vacuoles, positive
for RP, are more common at climax and free reaction product was recognizable
in highly degenerate sarcoplasm (see below and Fox, 1972c). In Rana tail
muscle profiles of (sarcoplasmic) degeneration and the acid phosphatase-rich
organelles are generally similar in appearance to those in cells of other degenerating larval tail tissues (Fox, 1972a, 1973a, b, 1974). In terms of location (often
near the nucleus), size and appearance the acid phosphatase-rich bodies are
comparable to the lysosomes illustrated in rat muscle (Gordon et al. 1967).
Furthermore, deposition of RP also occurred in the sarcoplasmic reticulum of
the anuran tail at climax as in humans (Pearce, 1966) and rats (Seiden, 1973),
in addition to its presence in other regions of the sarcoplasm.
It is of interest that in silk moths during the breakdown of the intersegmental
muscles a percentage cross section covered by lysosomes increased 4- to 6-fold;
acid phosphatase likewise is recognized in them and autophagic vacuoles
occur in later stages (Lockshin & Beaulaton 1974a, b). Actinomycin D and
cyclohexamide, which probably act via the genome, inhibit the synthesis of
protein and of lysosomal enzymes (even when thyroxine is administered
exogenously to anuran tadpoles); in consequence tail involution and muscle
degeneration in silk moths are prevented (Tata, 1966; Perriard, 1971; Lockshin
& Beaulaton, 1974a, b).
However, other evidence so far available would seem to emphasize caution
in the assessment of the role of lysosomes in muscle degeneration. In the
sarcoplasm the prominent cytolysomes seen at climax in tail epidermis,
notochord and nerve cord, the pronephros (Fox, 1970, 1971, 1972a, 1973a, b,
1974) and the epidermis of the external gills of Rana (Michaels, Albright &
Patt, 1971), the intestinal epithelium of the larval Discoglossus (Hourdry,
1971a, b) and in the mesonephros of the embryo chick (Salzgeber & Weber,
1966) appear to be less common. Again in the involuting tail of Rana pipiens
alkaline phosphatase, acid phosphatase, esterase and amino peptidase are generally recognized in the epidermis, notochord, nerve cord, spinal ganglia, connective tissue and the endothelium of blood vessels but not in the striated
muscle (Kaltenbach, 1971; Fry et al 1973).
Though acid phosphatase-rich organelles and free RP occur in climactic
sarcoplasm, the extent of the deposition and the amount registered would seem
to be inadequate to attribute to lysosomes sole responsibility for the degradation
of the relatively massive tail muscle. Moreover, little enzyme deposition is found
amid myofilaments and its absence is unlikely to be due to complete failure of
histochemical techniques.
Ultrastructure of Rana tail muscles
203
Lysosomes were not figured, or mentioned, by Webb (1972) in his description
of degenerating skeletal muscle in normal human foetuses. Again, in denervation
atrophy of guinea-pig muscles, though there are fibril and membrane fragments,
degenerate mitochondria, myelin figures, an increase in the amount of lipid,
enlargement of the sarcoplasmic reticulum and accumulation of dense bodies
and lysosomes, the latter would seem to be inadequate in number to account
for these widespread changes (Tomanek & Lund, 1973). Furthermore, after
denervation of rat skeletal muscle intact myofilaments were not found within
autophagic vacuoles, and though the number and size of lysosomes increase
Schiafnno & Hanzlikova (1972) believe that extra-lysosomal enzymes activate
myofibrillar degeneration; those from lysosomes are merely involved in the
final autolysis.
Perhaps lysosomal enzymes activate proteases already present within myofibrils but in an inactive form (Lockshin & Beaulaton, 1974a; Lockshin,
personal communication), which heretofore have not been demonstrated
biochemically.
Fibrils of muscle and collagen of the anuran tail ultimately degrade at climax
to a structureless substance, whose derivation in most cases is not distinguishable before final engulfment (Fox, 1972/?, c). Macrophages ingest these tissues;
their enzyme activity increases dramatically, especially in those near muscle
cells (Kaltenbach, 1971; Watanabe & Sasaki, 1974). They may envelop or
intimately associate with degenerate tail muscle and probably a ruptured
sarcolemma permits release of the latter which stimulates macrophagic
activity (Weber, 1964). Within the degenerating muscle cell, however, such highly
degraded tissue is frequently recognized together with lesser degraded and
recognizable myofilaments, often still retaining Z lines. Within macrophages
small quantities of fibrils usually represent heterophagic inclusions of
partially degraded collagen, seen ideally at climax near the basement lamella
(Fox, 1972a, b). Some profiles of anuran tail cells with substantial quantities
of highly electron-dense, degenerate, yet still recognizable, myofibrillar tissues
are in all probability muscle cells with autolytically degrading myofilaments.
Vacuolar membranes may be the remains of the sarcoplasmic reticulum. Why
degenerating myofibrils should show such varied degrees of electron density is
not known.
There is no unequivocable evidence that macrophages can ingest the highly
dense, yet still recognizable myofibrils (Kerr et al. 1974), though equally it is
difficult to exclude such activity. It is more likely, however, that like nerve
axons of the tail (Brown, 1946), muscle is phagocytosed only when highly
degraded, in most cases when unrecognizable as muscle.
The circulatory level of thyroid hormones rises during anuran prometamorphosis to climax (Etkin, 1964; Fox & Turner, 1967) and in various tail
tissues there is synthesis of orimary lysosomes in the Golgi cisternae (Novikoff,
Essner & Quintana, 1964). At climax lysosomal enzymes are released to
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H. FOX
autolytically degrade tissue organelles in secondary lysosomes or cytolysomes,
subsequently (in some cases) to be phagocytosed by mesenchymal macrophages.
The degree of involvement of lysosomal enzymes in different tail tissues may
well be variable, however. Epidermal cells, for example, do not wholly degrade
as tonofibrils are retained, keratin is laid down and finally the cells slough
(Fox, 1974). Cells of other tissues such as the notochord and nerve cord would
seem to show a greater degree of autolysis. Initially, therefore, myofilaments
may possess an independence to lysosomal enzymes of a kind seen with tonofilaments. Unlike the latter, however, they ultimately fully degrade and are
phagocytosed, influenced it seems by enzymes, whose origin is unknown and
indeed whose presence can only be surmised.
Dr Ruth Beilairs and Dr Sally Page of the Departments of Embryology and Biophysics
respectively, University College, London, kindly read the manuscript and offered valued
advice. Thanks are also due to E. Perry, R. Mahoney and P. Howard of the Department of
Zoology, University College, for technical assistance.
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{Received 28 January 1975, revised 21 March 1975)
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