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The Musculus Complexus of Normal and Dystrophic Chicken Embryos
The Musculus Complexus of Normal and Dystrophic Chicken Embryos E. RAWORTH ALLEN
Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana 70119 (Received for publication October 31, 1983)
INTRODUCTION A pure strain of New Hampshire Red chickens has been isolated and selectively bred for an early onset of inherited muscular dystrophy. The progressive myopathy was shown to primarily affect the fast twitch pectoral musculature (Asmundson and Julian, 1956), and was similar to certain forms of human dystrophies (Hadlow, 1973). Twitch fibers of the pectoralis major muscle taken from embryos were shown to be diseased in vivo (Allen and Murphy, 1979) and when cultured in vitro (Allen and May, 1979). Murphy and Allen (1981) found a defect in sarcomere organization within myofibrils of brachial somites (precursors of the pectoral muscles) from 3- to 5-day-old embryos. During prior experimentation, it was noted that approximately 60% fewer dystrophic chick embryos hatch. On further investigation, the unhatched embryos were found to be term but unable to pip, break, and escape from their egg shells. The musculus complexus (hatching muscle) is believed to play an important role in the hatching process (Bock and Hikida, 1969; Brooks and Garrett, 1970). There is a scarcity of electron microscopical investigations of this muscle (Hayes and Hikida, 1976) and only one primarily biochemical study in chickens affected with inherited muscular dystrophy (Ashmore et al, 1973). Therefore, it was decided to look for ultrastructural abnormalities that show the myopathy of this muscle.
MATERIALS AND METHODS
Fertile New Hampshire chickens of Line 413 were purchased from the Department of Avian Sciences, University of California at Davis. Control eggs were obtained both from Davis (412) and from a mixed flock of New Hampshire Reds raised at Louisiana State University Medical Center. The eggs were hatched at 37 C with a relative humidity of 50%. The musculus complexus was removed from embryos after intervals of 15, 17, 19, and 21 (just prior to hatching) days. The muscle was also removed from chicks 2 and 4 days after hatching. A minimum of 3 control and 5 diseased birds were examined at each time period. The birds were perfused with a mixture of 4.0% glutaraldehyde and 2.0% paraformaldehyde in .1 M cacodylate buffer (pH 7.2) (Karnovsky, 1965). The left ventricles were opened, and the perfusate was injected directly into the ascending aortas. The hatching muscle was then removed from each bird, cut into smaller pieces, and kept overnight in cacodylate buffer prior to postfixing with 1.0% osmium tetroxide. Uranyl acetate staining was accomplished overnight in bloc with a 5% aqueous solution. Thick (approx. 1 /im) sections were mounted on glass slides and stained with a 1% solution of toluidine blue in order to ensure proper content and orientation before obtaining thin (silver) sections with a Reichert OM-U3 ultramicrotome. These sections were stained with lead citrate (Reynolds, 1963), and electron micro-
2087
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
ABSTRACT Abnormalities have previously been reported in the pectoral muscle of embryos and young chicks from a pure strain of New Hampshire Red chickens homozygous for inherited muscular dystrophy. Fine structural studies of the musculus complexus in normal and dystrophic embryos were undertaken because of a sharp decrease in hatching by the diseased birds. Ultrastructural differences found between the normal and dystrophic embryos included a leached sarcoplasm, swollen and distorted mitochondria and tubular components, a lack of polyribosomes (myosin synthesis), and the formation of pseudostraps during differentiation of the myopathic hatching muscle. These differences may curtail differentiation until a point after the critical hatching time. (Key words: musculus complexes, muscular dystrophy, ultrastructure) 1984 Poultry Science 63:2087-2093
2088
ALLEN
graphs were obtained with a Philips 300 electron microscope. RESULTS
DISCUSSION The large size increase of the musculus complexus prior to hatching is due to a massive water inhibition that allows the muscle tissue to act as a cushion to brace the chick's head during hatching (Bock and Hikida, 1969). The previous authors also suggested no interference by the size of the complex with muscle contraction. Bock and Hikida (1968) postulated a disintegration of specialized tonus fibers to release proteins into the interstitial fluid and raise the colloidal osmotic pressure in order to retain fluid and thus assume a much larger size. Ishikawa (1968) postulated that T-system formation occurs by caveolae formation from the sarcolemma and appears as elaborate vesicular membrane system areas. These may be selectively accelerated or inhibited in muscular tissue by abrupt changes in the outside medium. The membranous system units he described are identical to those found in the present investigation. Day 19 is the approximate time of the largest size increase of the musculus complexus, and this is the only age, in these studies, when the random, highly organized patches of membrane are observed. According to Asmundson and Julian (1956), only fast twitch fibers are affected by this progressive myopathy. Until the present investigation, the author has only seen pectoral muscle (fast twitch) affected by this disease in experiments contrasting normal and dystrophic muscle in embryos of the same strain (Allen and Murphy, 1978; 1979; Allen and May, 1979; Murphy et al, 1981;Murphy and Allen, 1981). Swollen mitochondria and a dilated tubular
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
There is a considerable size increase in the musculus complexus from 17 to 19 days of incubation in normal embryos (Figs. 1, 2). The gross structure of the hatching muscles from dystrophic and normal embryos appeared similar. At about 15 to 16 days of incubation the musculus complexus increases in size until hatching (21 days) and then decreases until 2 days after hatching when it is again the size of a 15-day embryo (Fisher, 1958). A section for electron microscopy through normal hatching muscle tissue often showed two or more nuclei sharing the sarcoplasm in one skeletal muscle myofiber. In dystrophic tissue from 15-day material (Fig. 3), a separation was detected between cells indicating an incomplete fusion of the myoblasts. This was not always, but often, the case in myopathic tissue. The separation was of such a small size that without visualization with the electron microscope the two cells would appear as a single myotube or muscle strap. In 17-day normal and dystrophic muscle (Figs. 4 and 5) a sharp contrast was readily observed. The myofibrils, although containing normal appearing sarcomeres with typical banding patterns, were more sparsely oriented within the sarcoplasm of the diseased tissue. There was more space between fibrils that often appeared leached and contained swollen or rounded mitochondria as well as dilated tubular components (Fig. 5). The sarcoplasm also appeared washed out in diseased embryos, but it was very granular in muscle from normal embryos. Extra large polyribosomes indicative of active myosin synthesis in developing muscle (Allen and Pepe, 1965; Allen and Terrence, 1968) were present in both diseased and normal muscular tissue at each embryonic age. A much larger and progressive increase in number of the large polyribosomes were present in muscle from the normal embryos from Days 15 to 19. Muscle from 19-day embryos contained networks of membranes as patches of well organized units randomly dispersed throughout the sarcoplasm (Fig. 6). Muscle tissue from the embryos at 21 days of age (day of hatching) appeared similar to the muscle from 19-day embryos, but there were
no membranous networks and fewer large polyribosomes observed. Figures 7 and 8 show normal and diseased hatching muscle from chickens 2 days after hatching. Myofibrils from the dystrophic tissue were more widely separated from one another but, like earlier embryonic stages, had banding patterns similar to those from normal embryos. The sarcoplasm contained some granularity in both types of tissue due to the presence of small polyribosomes and free ribosomes, but none of the extra large polyribosomes were observed in either normal or dystrophic tissue. Again, although there were some granules, the sarcoplasm appeared much clearer or leached out in the diseased tissue.
NORMAL AND DYSTROPHIC HATCHING MUSCLE
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Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015 FIG. 1. The paired musculus complexus removed from the anterodorsal portion of the neck from a normal chick embryo incubated 17 days. X250. FIG. 2. The paired musculus complexus from a chicken embryo incubated for 19 days. Lymphatic tissue can be seen surrounding the hatching muscles. X250. FIG. 3. Micrograph of diseased 15-day musculus complexus tissue. A separation is detected (arrow) between cells of a myofiber indicating pseudostrap formation. X12.560.
ALLEN
s Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015 FIG. 4. Micrograph of musculus complexus tissue from a 17-day normal chicken embryo, p = Polyribosomes. Granularity is prominent. X 12,500. FIG. 5. Micrograph of musculus complexus tissue from a 17-day dystrophic chick embryo, p = Polyribosomes, t = tubules. X 17,100.
NORMAL AND DYSTROPHIC HATCHING MUSCLE
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/ Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
%
%
/
V "*fc
13 FIG. 6. Micrograph of musculus complexus tissue from a 19-day normal chick embryo. Randompolyribosomes and patches of membranous units (arrows) are seen in the sarcoplasm. p = Polyribosomes. X27.10O.
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ALLEN seriously impair t h e c o n d u c t i o n of action potentials and t h u s contraction. As in o t h e r comparative studies of n o r m a l and d y s t r o p h i c muscle, t h e sarcoplasm of t h e diseased cells often appears clear or leached o u t . This p h e n o m e n o n m a y be a result of lipid a c c u m u l a t i o n , since these e m b r y o s are characterized b y having a high muscle fat c o n t e n t (Ash m o r e ct al., 1 9 7 3 ) . Perhaps t h e fat remains partially b r o k e n d o w n b u t still within t h e tissue and is n o t stained b y o s m i u m t e t r o x i d e during postfixation. O s m i u m salts blacken certain u n s a t u r a t e d lipids. A n o t h e r , m o r e plausible
FIG. 7. Micrograph of musculus complexus from a 2-day posthatched normal chick. X5,700. FIG. 8. Micrograph of musculus complexus from a 2-day posthatched dystrophic chick. Note the washed-out appearance. X 5,700.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
system, as were seen in t h e musculus complexus of diseased chickens, were also observed in t h e dystrophic muscle from all of t h e previous experiments. A lack of c o m p l e t e fusion, often n o t e d in t h e diseased musculus c o m p l e x u s agrees with previous in vitro studies of d y s t r o p h i c m o u s e muscle cells (Pearce, 1 9 6 3 ; Moore, 1975) and chicken pectoralis muscle from t h e same strain (Allen and May, 1 9 7 9 ) . A p r e d o m i n a n c e of pseudostraps over normal fusion to form muscle straps in d y s t r o p h i c tissue was r e p o r t e d b y all of t h e previous investigators. This would
NORMAL AND DYSTROPHIC HATCHING MUSCLE
REFERENCES Allen, E. R., and J. F. May, 1979. Aspects of normal and dystrophic chicken muscle grown in vitro. Virchows Arch. Cell Pathol. 31:243-250. Allen, E. R., and B. J. Murphy, 1978. Sarcotubular development in dystrophic skeletal muscle cells. Cell Tissue Res. 194:125-130. Allen, E. R., and B. J. Murphy, 1979. Early detection of inherited muscular dystrophy in chickens. Cell Tissue Res. 197:165-167. Allen, E. R., and F. A. Pepe, 1965. Ultrastructure of
developing muscle cells in the chicken embryo. Am. J.Anat. 116:115-148. Allen, E. R., and C. F. Terrence, 1968. Immunochemical and ultrastructural studies of myosin synthesis. Proc. Natl. Acad. Sci. 60:1209-1215. Ashmore, C. R., P. B. Addis, L. Doerr, and H. Stokes, 1973. Development of muscle fibers in the complexus muscle of normal and dystrophic chicks. J. Histochem. Cytochem. 21:266—278. Asmundson, V. S., and L. M. Julian, 1956. Inherited muscle abnormality in the domestic fowl. J. Hered. 47:248-252. Bock, W. J., and R.S. Hikida, 1968. An analysis of twitch and tonus fibers in the hatching muscle. Condor 70:211-222. Brooks, W. S., and S. E. Garrett, 1970. The mechanism of pipping in birds. Auk 87:458—466. Fisher, H. I., 1958. The hatching muscle in the chick. Auk 75:391-398. Hadlow, W. J., 1973. Myopathies of animals. Pages 3 6 4 - 4 0 4 in The Straited Muscle. C. F. Pearson and F. K. Mostofi, ed. Williams and Wilkins Co., Baltimore, MD. Hayes, V. E., and R. S. Hikida, 1976. Naturally occurring degeneration in chick muscle development: ultrastructure of the M. complexus. J. Anat. 122:67-76. Ishikawa, H., 1968. Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation. J. Cell Biol. 38:51-66. Karnovsky, M. J., 1965. A formaldehyde glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 2 7 : 1 3 7 A 138A. Moore, M. J., 1975. A myogenic maliformation in adult dystrophic mice muscle studied in vitro. J. Neurol. Sci. 2 4 : 7 7 - 9 3 . Murphy, B. J., and E. R. Allen, 1981. Sarcomere formation in dystrophic skeletal muscle. Expl. Cell Biol. 49:285-293. Murphy, B. J., E. R. Allen, and C. H. Narayanan, 1981. An analysis of the integrity of the brachial motor unit in the dystrophic chick embryo. Cell Tiss. Res. 215:537-545. Pearce, G. W., 1963. Tissue culture in the study of muscular dystrophy. Pages 177—191 in Muscular Dystrophy in Man and Animals. G. H. Bourne and N. Gularz, ed. Hafner Publ. Co., New York, NY. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Smail, J. R., 1964. A possible role of the musculus complexus in pipping the chicken egg. Am. Midi. Nat. 72:499-506.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
explanation is t h a t t h e lipid is removed b y p r o p y l e n e oxide, a fat solvent used in embedding p r o c e d u r e s . T h e use of a water-soluble e m b e d d i n g m e d i u m m a y answer this question. Muscle from n o r m a l chickens shows a progressive increase in t h e n u m b e r of e x t r a large p o l y r i b o s o m e s (indicative of myosin synthesis) in 15-, 17-, and 19-day embryos. Very few p o l y r i b o s o m e s are observed in D a y 21 e m b r y o s and n o n e after hatching. In d y s t r o p h i c e m b r y o s , although a few of t h e large polyribosomes are seen in muscle from each emb r y o n i c age, no increase in n u m b e r was det e c t e d . Allen and May ( 1 9 7 9 ) and Allen and M u r p h y ( 1 9 7 9 ) f o u n d a delay in t h e appearance of t h i c k myosin filaments as well as large p o l y r i b o s o m e s synthesizing t h e m in developing pectoral muscle from diseased chickens. This same inactivity or possible delay in myosin synthesis appears t o b e prevalent in t h e dyst r o p h i c chicken hatching muscle. T h e rapid increase in t h e n u m b e r of extra large polyribosomes in t h e hatching muscle from normal" chickens suggests a rapid m y o s i n synthesis and, t h u s , assemblage in a brief t i m e interval p r i o r t o hatching. A lack or delay of an increased activity of myosin synthesis m a y leave t o o little t i m e for t h e hatching muscle t o differentiate into a functional state, and m a n y of t h e t e r m e m b r y o s affected with inherited muscular d y s t r o p h y c a n n o t hatch. These d a t a strongly s u p p o r t t h e t h e o r y t h a t cushioning and cont r a c t i o n of muscle fibers (Bock and Hikida, 1 9 6 9 ) , rather t h a n hydraulic pressure (Smail, 1 9 6 4 ) , are responsible for pipping.
2093
Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana 70119 (Received for publication October 31, 1983)
INTRODUCTION A pure strain of New Hampshire Red chickens has been isolated and selectively bred for an early onset of inherited muscular dystrophy. The progressive myopathy was shown to primarily affect the fast twitch pectoral musculature (Asmundson and Julian, 1956), and was similar to certain forms of human dystrophies (Hadlow, 1973). Twitch fibers of the pectoralis major muscle taken from embryos were shown to be diseased in vivo (Allen and Murphy, 1979) and when cultured in vitro (Allen and May, 1979). Murphy and Allen (1981) found a defect in sarcomere organization within myofibrils of brachial somites (precursors of the pectoral muscles) from 3- to 5-day-old embryos. During prior experimentation, it was noted that approximately 60% fewer dystrophic chick embryos hatch. On further investigation, the unhatched embryos were found to be term but unable to pip, break, and escape from their egg shells. The musculus complexus (hatching muscle) is believed to play an important role in the hatching process (Bock and Hikida, 1969; Brooks and Garrett, 1970). There is a scarcity of electron microscopical investigations of this muscle (Hayes and Hikida, 1976) and only one primarily biochemical study in chickens affected with inherited muscular dystrophy (Ashmore et al, 1973). Therefore, it was decided to look for ultrastructural abnormalities that show the myopathy of this muscle.
MATERIALS AND METHODS
Fertile New Hampshire chickens of Line 413 were purchased from the Department of Avian Sciences, University of California at Davis. Control eggs were obtained both from Davis (412) and from a mixed flock of New Hampshire Reds raised at Louisiana State University Medical Center. The eggs were hatched at 37 C with a relative humidity of 50%. The musculus complexus was removed from embryos after intervals of 15, 17, 19, and 21 (just prior to hatching) days. The muscle was also removed from chicks 2 and 4 days after hatching. A minimum of 3 control and 5 diseased birds were examined at each time period. The birds were perfused with a mixture of 4.0% glutaraldehyde and 2.0% paraformaldehyde in .1 M cacodylate buffer (pH 7.2) (Karnovsky, 1965). The left ventricles were opened, and the perfusate was injected directly into the ascending aortas. The hatching muscle was then removed from each bird, cut into smaller pieces, and kept overnight in cacodylate buffer prior to postfixing with 1.0% osmium tetroxide. Uranyl acetate staining was accomplished overnight in bloc with a 5% aqueous solution. Thick (approx. 1 /im) sections were mounted on glass slides and stained with a 1% solution of toluidine blue in order to ensure proper content and orientation before obtaining thin (silver) sections with a Reichert OM-U3 ultramicrotome. These sections were stained with lead citrate (Reynolds, 1963), and electron micro-
2087
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
ABSTRACT Abnormalities have previously been reported in the pectoral muscle of embryos and young chicks from a pure strain of New Hampshire Red chickens homozygous for inherited muscular dystrophy. Fine structural studies of the musculus complexus in normal and dystrophic embryos were undertaken because of a sharp decrease in hatching by the diseased birds. Ultrastructural differences found between the normal and dystrophic embryos included a leached sarcoplasm, swollen and distorted mitochondria and tubular components, a lack of polyribosomes (myosin synthesis), and the formation of pseudostraps during differentiation of the myopathic hatching muscle. These differences may curtail differentiation until a point after the critical hatching time. (Key words: musculus complexes, muscular dystrophy, ultrastructure) 1984 Poultry Science 63:2087-2093
2088
ALLEN
graphs were obtained with a Philips 300 electron microscope. RESULTS
DISCUSSION The large size increase of the musculus complexus prior to hatching is due to a massive water inhibition that allows the muscle tissue to act as a cushion to brace the chick's head during hatching (Bock and Hikida, 1969). The previous authors also suggested no interference by the size of the complex with muscle contraction. Bock and Hikida (1968) postulated a disintegration of specialized tonus fibers to release proteins into the interstitial fluid and raise the colloidal osmotic pressure in order to retain fluid and thus assume a much larger size. Ishikawa (1968) postulated that T-system formation occurs by caveolae formation from the sarcolemma and appears as elaborate vesicular membrane system areas. These may be selectively accelerated or inhibited in muscular tissue by abrupt changes in the outside medium. The membranous system units he described are identical to those found in the present investigation. Day 19 is the approximate time of the largest size increase of the musculus complexus, and this is the only age, in these studies, when the random, highly organized patches of membrane are observed. According to Asmundson and Julian (1956), only fast twitch fibers are affected by this progressive myopathy. Until the present investigation, the author has only seen pectoral muscle (fast twitch) affected by this disease in experiments contrasting normal and dystrophic muscle in embryos of the same strain (Allen and Murphy, 1978; 1979; Allen and May, 1979; Murphy et al, 1981;Murphy and Allen, 1981). Swollen mitochondria and a dilated tubular
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
There is a considerable size increase in the musculus complexus from 17 to 19 days of incubation in normal embryos (Figs. 1, 2). The gross structure of the hatching muscles from dystrophic and normal embryos appeared similar. At about 15 to 16 days of incubation the musculus complexus increases in size until hatching (21 days) and then decreases until 2 days after hatching when it is again the size of a 15-day embryo (Fisher, 1958). A section for electron microscopy through normal hatching muscle tissue often showed two or more nuclei sharing the sarcoplasm in one skeletal muscle myofiber. In dystrophic tissue from 15-day material (Fig. 3), a separation was detected between cells indicating an incomplete fusion of the myoblasts. This was not always, but often, the case in myopathic tissue. The separation was of such a small size that without visualization with the electron microscope the two cells would appear as a single myotube or muscle strap. In 17-day normal and dystrophic muscle (Figs. 4 and 5) a sharp contrast was readily observed. The myofibrils, although containing normal appearing sarcomeres with typical banding patterns, were more sparsely oriented within the sarcoplasm of the diseased tissue. There was more space between fibrils that often appeared leached and contained swollen or rounded mitochondria as well as dilated tubular components (Fig. 5). The sarcoplasm also appeared washed out in diseased embryos, but it was very granular in muscle from normal embryos. Extra large polyribosomes indicative of active myosin synthesis in developing muscle (Allen and Pepe, 1965; Allen and Terrence, 1968) were present in both diseased and normal muscular tissue at each embryonic age. A much larger and progressive increase in number of the large polyribosomes were present in muscle from the normal embryos from Days 15 to 19. Muscle from 19-day embryos contained networks of membranes as patches of well organized units randomly dispersed throughout the sarcoplasm (Fig. 6). Muscle tissue from the embryos at 21 days of age (day of hatching) appeared similar to the muscle from 19-day embryos, but there were
no membranous networks and fewer large polyribosomes observed. Figures 7 and 8 show normal and diseased hatching muscle from chickens 2 days after hatching. Myofibrils from the dystrophic tissue were more widely separated from one another but, like earlier embryonic stages, had banding patterns similar to those from normal embryos. The sarcoplasm contained some granularity in both types of tissue due to the presence of small polyribosomes and free ribosomes, but none of the extra large polyribosomes were observed in either normal or dystrophic tissue. Again, although there were some granules, the sarcoplasm appeared much clearer or leached out in the diseased tissue.
NORMAL AND DYSTROPHIC HATCHING MUSCLE
2089
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015 FIG. 1. The paired musculus complexus removed from the anterodorsal portion of the neck from a normal chick embryo incubated 17 days. X250. FIG. 2. The paired musculus complexus from a chicken embryo incubated for 19 days. Lymphatic tissue can be seen surrounding the hatching muscles. X250. FIG. 3. Micrograph of diseased 15-day musculus complexus tissue. A separation is detected (arrow) between cells of a myofiber indicating pseudostrap formation. X12.560.
ALLEN
s Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015 FIG. 4. Micrograph of musculus complexus tissue from a 17-day normal chicken embryo, p = Polyribosomes. Granularity is prominent. X 12,500. FIG. 5. Micrograph of musculus complexus tissue from a 17-day dystrophic chick embryo, p = Polyribosomes, t = tubules. X 17,100.
NORMAL AND DYSTROPHIC HATCHING MUSCLE
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/ Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
%
%
/
V "*fc
13 FIG. 6. Micrograph of musculus complexus tissue from a 19-day normal chick embryo. Randompolyribosomes and patches of membranous units (arrows) are seen in the sarcoplasm. p = Polyribosomes. X27.10O.
2092
ALLEN seriously impair t h e c o n d u c t i o n of action potentials and t h u s contraction. As in o t h e r comparative studies of n o r m a l and d y s t r o p h i c muscle, t h e sarcoplasm of t h e diseased cells often appears clear or leached o u t . This p h e n o m e n o n m a y be a result of lipid a c c u m u l a t i o n , since these e m b r y o s are characterized b y having a high muscle fat c o n t e n t (Ash m o r e ct al., 1 9 7 3 ) . Perhaps t h e fat remains partially b r o k e n d o w n b u t still within t h e tissue and is n o t stained b y o s m i u m t e t r o x i d e during postfixation. O s m i u m salts blacken certain u n s a t u r a t e d lipids. A n o t h e r , m o r e plausible
FIG. 7. Micrograph of musculus complexus from a 2-day posthatched normal chick. X5,700. FIG. 8. Micrograph of musculus complexus from a 2-day posthatched dystrophic chick. Note the washed-out appearance. X 5,700.
Downloaded from http://ps.oxfordjournals.org/ at Karolinska Institutet University Library on June 2, 2015
system, as were seen in t h e musculus complexus of diseased chickens, were also observed in t h e dystrophic muscle from all of t h e previous experiments. A lack of c o m p l e t e fusion, often n o t e d in t h e diseased musculus c o m p l e x u s agrees with previous in vitro studies of d y s t r o p h i c m o u s e muscle cells (Pearce, 1 9 6 3 ; Moore, 1975) and chicken pectoralis muscle from t h e same strain (Allen and May, 1 9 7 9 ) . A p r e d o m i n a n c e of pseudostraps over normal fusion to form muscle straps in d y s t r o p h i c tissue was r e p o r t e d b y all of t h e previous investigators. This would
NORMAL AND DYSTROPHIC HATCHING MUSCLE
REFERENCES Allen, E. R., and J. F. May, 1979. Aspects of normal and dystrophic chicken muscle grown in vitro. Virchows Arch. Cell Pathol. 31:243-250. Allen, E. R., and B. J. Murphy, 1978. Sarcotubular development in dystrophic skeletal muscle cells. Cell Tissue Res. 194:125-130. Allen, E. R., and B. J. Murphy, 1979. Early detection of inherited muscular dystrophy in chickens. Cell Tissue Res. 197:165-167. Allen, E. R., and F. A. Pepe, 1965. Ultrastructure of
developing muscle cells in the chicken embryo. Am. J.Anat. 116:115-148. Allen, E. R., and C. F. Terrence, 1968. Immunochemical and ultrastructural studies of myosin synthesis. Proc. Natl. Acad. Sci. 60:1209-1215. Ashmore, C. R., P. B. Addis, L. Doerr, and H. Stokes, 1973. Development of muscle fibers in the complexus muscle of normal and dystrophic chicks. J. Histochem. Cytochem. 21:266—278. Asmundson, V. S., and L. M. Julian, 1956. Inherited muscle abnormality in the domestic fowl. J. Hered. 47:248-252. Bock, W. J., and R.S. Hikida, 1968. An analysis of twitch and tonus fibers in the hatching muscle. Condor 70:211-222. Brooks, W. S., and S. E. Garrett, 1970. The mechanism of pipping in birds. Auk 87:458—466. Fisher, H. I., 1958. The hatching muscle in the chick. Auk 75:391-398. Hadlow, W. J., 1973. Myopathies of animals. Pages 3 6 4 - 4 0 4 in The Straited Muscle. C. F. Pearson and F. K. Mostofi, ed. Williams and Wilkins Co., Baltimore, MD. Hayes, V. E., and R. S. Hikida, 1976. Naturally occurring degeneration in chick muscle development: ultrastructure of the M. complexus. J. Anat. 122:67-76. Ishikawa, H., 1968. Formation of elaborate networks of T-system tubules in cultured skeletal muscle with special reference to the T-system formation. J. Cell Biol. 38:51-66. Karnovsky, M. J., 1965. A formaldehyde glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 2 7 : 1 3 7 A 138A. Moore, M. J., 1975. A myogenic maliformation in adult dystrophic mice muscle studied in vitro. J. Neurol. Sci. 2 4 : 7 7 - 9 3 . Murphy, B. J., and E. R. Allen, 1981. Sarcomere formation in dystrophic skeletal muscle. Expl. Cell Biol. 49:285-293. Murphy, B. J., E. R. Allen, and C. H. Narayanan, 1981. An analysis of the integrity of the brachial motor unit in the dystrophic chick embryo. Cell Tiss. Res. 215:537-545. Pearce, G. W., 1963. Tissue culture in the study of muscular dystrophy. Pages 177—191 in Muscular Dystrophy in Man and Animals. G. H. Bourne and N. Gularz, ed. Hafner Publ. Co., New York, NY. Reynolds, E. S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Smail, J. R., 1964. A possible role of the musculus complexus in pipping the chicken egg. Am. Midi. Nat. 72:499-506.
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explanation is t h a t t h e lipid is removed b y p r o p y l e n e oxide, a fat solvent used in embedding p r o c e d u r e s . T h e use of a water-soluble e m b e d d i n g m e d i u m m a y answer this question. Muscle from n o r m a l chickens shows a progressive increase in t h e n u m b e r of e x t r a large p o l y r i b o s o m e s (indicative of myosin synthesis) in 15-, 17-, and 19-day embryos. Very few p o l y r i b o s o m e s are observed in D a y 21 e m b r y o s and n o n e after hatching. In d y s t r o p h i c e m b r y o s , although a few of t h e large polyribosomes are seen in muscle from each emb r y o n i c age, no increase in n u m b e r was det e c t e d . Allen and May ( 1 9 7 9 ) and Allen and M u r p h y ( 1 9 7 9 ) f o u n d a delay in t h e appearance of t h i c k myosin filaments as well as large p o l y r i b o s o m e s synthesizing t h e m in developing pectoral muscle from diseased chickens. This same inactivity or possible delay in myosin synthesis appears t o b e prevalent in t h e dyst r o p h i c chicken hatching muscle. T h e rapid increase in t h e n u m b e r of extra large polyribosomes in t h e hatching muscle from normal" chickens suggests a rapid m y o s i n synthesis and, t h u s , assemblage in a brief t i m e interval p r i o r t o hatching. A lack or delay of an increased activity of myosin synthesis m a y leave t o o little t i m e for t h e hatching muscle t o differentiate into a functional state, and m a n y of t h e t e r m e m b r y o s affected with inherited muscular d y s t r o p h y c a n n o t hatch. These d a t a strongly s u p p o r t t h e t h e o r y t h a t cushioning and cont r a c t i o n of muscle fibers (Bock and Hikida, 1 9 6 9 ) , rather t h a n hydraulic pressure (Smail, 1 9 6 4 ) , are responsible for pipping.
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