139
J. Anat. (1990), 170, pp. 139-149 With 14 figures Printed in Great Britain
An enzyme histochemical study of large muscle fibres in the neonatal mouse K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI
Department of Anatomy and Physiology, The University, Dundee DD1 4HN,
Scotland (Accepted 23 November 1989) INTRODUCTION
In 1937 Wohlfart described the presence of large extrafusal muscle fibres in the sartorius of human fetuses and neonates. He designated the fibres b-Fasern or B fibres as distinct from the smaller predominant a-Fasern or A fibres. B fibres in the human fetus at 22 weeks can have a cross-sectional area five times greater than typical A fibres (Fenichel, 1963). Wohlfart noted that these fibres were not routinely present in all fetal and neonatal muscle samples, a finding confirmed in later studies by Fenichel (1963) and Colling-Saltin (1978). The proportion of muscles exhibiting B fibres after 20 weeks gestation is about three quarters, with a frequency not higher than 4-5 %; they are rare at birth and in the neonate, have low ATPase activity and are classified Type 1 or slow-twitch fibres (Fenichel, 1963; Dubowitz, 1965; Colling-Saltin, 1978). B fibres do not appear to be exclusive to human muscles. Davies (1972) reported similar fibres in the neonatal pig, and Stewart (1984) found large oxidative fibres in normal and myopathic neonatal mice. As a result of Stewart's work, we decided to look more closely at the histochemistry of these fibres in the mouse. In addition to sizing and characterising them with conventional enzyme techniques (ATPase EC 3.6.1.3; phosphorylase a via phosphorylase kinase EC 2.7.1.38 and NADH-tetrazolium reductase, probably NADH dehydrogenase EC 1.6.99.3) qualitative and quantitative estimates were made of the activities of the following hydrolases and proteases: acid phosphatase (EC 3. 1.3.2), ,-glucuronidase (EC 3.2. 1 .31), N-acetylglucosaminidase (EC 3.2.1.30), dipeptidyl peptidase II (EC 3.4.14.2) and microsomal aminopeptidase (mAAP) (EC 3.4.11.2). The results presented cover the first 40 days postnatal and reveal unique activity profiles for several enzymes in the large fibres. MATERIALS AND METHODS
Homozygous male and female C57BL/ lOScSn mice bred in the Department animal unit were used throughout the study. They were permitted food (Rat & Mouse Diet No. 1, S.D.S., Witham, Essex) and water ad libitum. Four animals were killed by stunning and exsanguination at each of the following ages postpartum: 2, 7, 12, 25 and 40 days. A 3 mm thick transection of the complete left hindlimb was taken at mid-calf, mounted in 10% gum tragacanth on a cork disc and quenched in isopentane cooled with liquid nitrogen. Ten micrometre thick transverse sections were cut on a motorised cryostat run at constant speed and either mounted on clean, grease-free coverslips or, in the case of DPP II, microsomal aminopeptidase and the glycosidases, mounted on washed, dry semipermeable membranes (Visking Tubing, Medicell International, Liverpool) over gelled substrate media (Lojda, Gossrau & Schiebler, 1979).
140
K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI
Histology andfibre-typing Fresh cryostat sections on coverslips were fixed in Clarke's fluid (Hopwood, 1977) for 10 minutes and then stained with Harris's haematoxylin and eosin. Fibres were typed in fresh cryostat sections by the tetrazolium reductase method, ATPase method (conventional and following acid pre-incubation) and by the phosphorylase technique (Dubowitz & Brooke, 1973). Fibre area determination Representative normal fibre areas were obtained by measuring slow-twitch intermediate fibres (red Type 1) and fast-twitch white fibres (Type 2B). Soleus and extensor digitorum longus (EDL) were chosen as the sources of red and white fibres respectively. Fibre types were not distinguishable until 12 days. Photomicrographs were taken with a x 24 objective and prints enlarged to the magnification at which 1 mm2 represented 1 ,um2. Areas were determined using a MOP Digiplan graphics tablet (Kontron, Messgerate, GMBH). Approximately 10% of the number of fibres in a muscle were measured; in the case of soleus this was 80, and in extensor digitorum longus, 130. In very young mice where differentiation was incomplete, the areas of individual myotubes were measured. In general, a discrete extrafusal structure containing myofibrils was regarded as a fibre. Enzyme histochemistry Acid phosphatase activity was determined against naphthol AS-BI phosphate (Sigma Chemical Co., Poole, Dorset) in fresh cryostat sections. The sections were incubated for one hour at 37 °C in the substrate medium containing freshly hexazotised pararosaniline and 20 mM-MnCl2 (Lodja et al. 1979). They were then fixed overnight in 10% formalin, washed for 10 minutes in running tap water and mounted in Kaiser's glycerine gelatin (Lojda et al. 1979). The identity of acid phosphatase was confirmed by the exclusion of naphthol AS-BI phosphate from the substrate medium, or the inclusion of 5 mM-NaF in the complete medium. ,/-Glucuronidase and N-acetylglucosaminidase were detected in fresh cryostat sections by the semipermeable membrane technique using respectively naphthol ASBI-/3-D-glucuronic acid and N-acetyl-fi-D-glucosamine naphthol AS-LC (both from Sigma) (techniques after Lojda et al. 1979). Post-incubation treatment was as for acid phosphatase. Specific inhibitors used to identify the enzymes were, for ,I-glucuronidase, D-saccharic acid 1,4-lactone (0-48% w/v) and for N-acetylglucosaminidase, N-acetylglucosamine (10 and 100 mM). Dipeptidyl peptidase II (DPP II) and microsomal aminopeptidase (mAAP) were also demonstrated using variants of the semipermeable technique (Stoward, Christie & Thomson, 1988; Christie & Stoward, 1988). DPP II activity was detected by hydrolysis of H-Lys-Pro-4MNA . 2HCl and mAAP by H-Ala-4MNA. HCl. A variety of inhibitors was used to characterise both proteases, including for DPPII, phenylmethylsulphonyl fluoride (1 mM) and Tris (1 M) and for mAAP, actinonin (10 ,ug/ml) (Umezawa et al. 1985). Actinonin was purchased from the Peptide Institute, Osaka, Japan and the substrates from Bachem Feinchemikalien, Budendorf, Switzerland. Glycogen staining was demonstrated by the periodic acid-Schiff method following fixation Glycogen of fresh cryostat sections in Clarke's fluid. Specificity of the reaction was confirmed by
Large muscle fibres in neonatal mice 141 pre-Schiff treatment of the fixed sections with 0-5 % w/v a-amylase in 0-004 M acetate buffer, pH 5-5, for 3 hours at 37 'C. Microdensitometry of enzyme reaction product Densitometric measurements of enzyme final reaction product were made on a Vickers M86 scanning microdensitometer. The machine settings, with a x 40 objective, for acid phosphatase, mAAP and DPPII, were scanning spot no. 2 and 530 nm (reaction product absorption maxima) and for NADH-tetrazolium reductase, scanning spot no. 2 and 550 nm. The mean reaction product absorbance of 24 Type 1 fibres in soleus and 24 Type 2B fibres in EDL was determined (this number was sufficient to give a constant cumulative mean absorbance and standard deviation) and the grouped mean absorbance of the same muscle in four age-matched animals derived. The absorbance of as many large fibres as could be found was measured and treated similarly. Enzyme activity was expressed as integrated optical density, derived by multiplying mean fibre area by mean absorbance at the appropriate wavelength (Christie & Thomson, 1987). RESULTS
Histology andfibre sizes Haematoxylin and eosin staining and the NADH-tetrazolium reductase method were used first to screen the sections for large fibres, which were always more eosinophilic than their normal counterparts and yielded an extra strong NADHtetrazolium reductase final reaction product. After 12 days of age eosinophilia declined markedly, and identification was based on the intensity of the NADH final reaction product and an area more than 15 % greater than the mean of the largest 'normal' fibres present. The large fibres usually occurred in isolated fascicles of between 5 and 10 fibres within and at the periphery of intermediate and deep muscles such as soleus and flexor hallucis longus. Isolated single large fibres were rare, but occasionally present in flexor and extensor compartments. The difference in size was most apparent at two days, when the mean area of the large fibres was three times greater than typical fibres (P < 0.01, Student's t test) (Figs. 1, 2). The growth rates of normal-sized and large fibres were similar until about 21 days, when the large fibres slowed to approximate with the faster growing 2B fibres. By 25 days it was difficult to identify the large fibres as a separate population, morphologically or histochemically.
Histochemistry Up to 12 days old the NADH-tetrazolium reductase method did not distinguish between fibre types; nevertheless, from 2 to 12 days and beyond there was considerably more reaction product in the large fibres than in typical Type 1 fibres (Fig. 3). The large fibres gave a strong ATPase reaction up to 12 days of age at pH 9-7 and after pre-incubation at pH 4-4 (Figs. 4, 5). Beyond this age acid pre-incubation produced differentiation. From 2 to 25 days the large fibres exhibited a strong granular and diffuse acid phosphatase reaction product (Fig. 6). Over the same period typical fibres displayed a similarly distributed but much weaker reaction. In normal fibres, up to 21 days, DPPII reaction product was present as finely dispersed particles. By contrast the large fibres exhibited a concentrated, coarse, granular product with overall diffuse sarcoplasmic staining (Fig. 7). fl-Glucuronidase
142
K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI 2000
1200
400
0
I
I
2
7
12
21 Age (days)
25
40
Fig. 1. Muscle fibre areas versus age. The large fibres are only significantly greater in area at two days old. A, undifferentiated fibres; 0, large fibres; *, white fibres; A, red fibres (means±S.D.).
activity was weak or absent in most normal sized fibres at all ages. A weak granular and sarcoplasmic reaction product was, however, present in the large fibres. No Nacetylglucosaminidase activity was detected in any fibres. The activity and distribution of microsomal aminopeptidase was similar to DPPII, with greatest activity again in the large fibres (Fig. 8). The large fibres gave a strong phosphorylase and glycogen reaction up to 12 days; beyond this age differentiation was apparent (Figs. 9, 10). Integrated densitometry (enzyme activity) Integrated optical density measurements showed NADH-tetrazolium reductase activity to be significantly greater in the large fibres during the first 21 days (P < 0-001) (Fig. 11). At 2 days, activity was fourfold higher, increasing to a probable maximum by 21 days. Acid phosphatase activity in the large fibres was fivefold higher than in normal sized fibres at 2 days (Fig. 12). Activity was again significantly increased for the first 21 days (P < 0-001). DPPII was considerably more active in the large fibres at 2 days, remaining significantly so for up to 12 days (P < 0-001) (Fig. 13). Microsomal aminopeptidase activity was also higher in the large fibres during the first 25 days, but not to any degree of significance (Fig. 14). Fig. 2. Two days old mouse. Large eosinophilic fibres are evident in the region of flexor hallucis longus. H & E. x 250. Fig. 3. Twelve days old mouse. This section, reacted for NADH-tetrazolium reductase, shows the massive deposition of formazan in the large fibres. x 140. Fig. 4. Seven days old mouse. Section reacted for ATPase at pH 9.7. The large fibres are strongly reactive. x 100. Fig. 5. Serial section to that shown in Fig. 4 reacted for ATPase at pH 4-4. The large fibres remain highly reactive. x 100.
143
Large muscle fibres in neonatal mice
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J. Anat. (1990), 170, pp. 139-149 With 14 figures Printed in Great Britain
An enzyme histochemical study of large muscle fibres in the neonatal mouse K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI
Department of Anatomy and Physiology, The University, Dundee DD1 4HN,
Scotland (Accepted 23 November 1989) INTRODUCTION
In 1937 Wohlfart described the presence of large extrafusal muscle fibres in the sartorius of human fetuses and neonates. He designated the fibres b-Fasern or B fibres as distinct from the smaller predominant a-Fasern or A fibres. B fibres in the human fetus at 22 weeks can have a cross-sectional area five times greater than typical A fibres (Fenichel, 1963). Wohlfart noted that these fibres were not routinely present in all fetal and neonatal muscle samples, a finding confirmed in later studies by Fenichel (1963) and Colling-Saltin (1978). The proportion of muscles exhibiting B fibres after 20 weeks gestation is about three quarters, with a frequency not higher than 4-5 %; they are rare at birth and in the neonate, have low ATPase activity and are classified Type 1 or slow-twitch fibres (Fenichel, 1963; Dubowitz, 1965; Colling-Saltin, 1978). B fibres do not appear to be exclusive to human muscles. Davies (1972) reported similar fibres in the neonatal pig, and Stewart (1984) found large oxidative fibres in normal and myopathic neonatal mice. As a result of Stewart's work, we decided to look more closely at the histochemistry of these fibres in the mouse. In addition to sizing and characterising them with conventional enzyme techniques (ATPase EC 3.6.1.3; phosphorylase a via phosphorylase kinase EC 2.7.1.38 and NADH-tetrazolium reductase, probably NADH dehydrogenase EC 1.6.99.3) qualitative and quantitative estimates were made of the activities of the following hydrolases and proteases: acid phosphatase (EC 3. 1.3.2), ,-glucuronidase (EC 3.2. 1 .31), N-acetylglucosaminidase (EC 3.2.1.30), dipeptidyl peptidase II (EC 3.4.14.2) and microsomal aminopeptidase (mAAP) (EC 3.4.11.2). The results presented cover the first 40 days postnatal and reveal unique activity profiles for several enzymes in the large fibres. MATERIALS AND METHODS
Homozygous male and female C57BL/ lOScSn mice bred in the Department animal unit were used throughout the study. They were permitted food (Rat & Mouse Diet No. 1, S.D.S., Witham, Essex) and water ad libitum. Four animals were killed by stunning and exsanguination at each of the following ages postpartum: 2, 7, 12, 25 and 40 days. A 3 mm thick transection of the complete left hindlimb was taken at mid-calf, mounted in 10% gum tragacanth on a cork disc and quenched in isopentane cooled with liquid nitrogen. Ten micrometre thick transverse sections were cut on a motorised cryostat run at constant speed and either mounted on clean, grease-free coverslips or, in the case of DPP II, microsomal aminopeptidase and the glycosidases, mounted on washed, dry semipermeable membranes (Visking Tubing, Medicell International, Liverpool) over gelled substrate media (Lojda, Gossrau & Schiebler, 1979).
140
K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI
Histology andfibre-typing Fresh cryostat sections on coverslips were fixed in Clarke's fluid (Hopwood, 1977) for 10 minutes and then stained with Harris's haematoxylin and eosin. Fibres were typed in fresh cryostat sections by the tetrazolium reductase method, ATPase method (conventional and following acid pre-incubation) and by the phosphorylase technique (Dubowitz & Brooke, 1973). Fibre area determination Representative normal fibre areas were obtained by measuring slow-twitch intermediate fibres (red Type 1) and fast-twitch white fibres (Type 2B). Soleus and extensor digitorum longus (EDL) were chosen as the sources of red and white fibres respectively. Fibre types were not distinguishable until 12 days. Photomicrographs were taken with a x 24 objective and prints enlarged to the magnification at which 1 mm2 represented 1 ,um2. Areas were determined using a MOP Digiplan graphics tablet (Kontron, Messgerate, GMBH). Approximately 10% of the number of fibres in a muscle were measured; in the case of soleus this was 80, and in extensor digitorum longus, 130. In very young mice where differentiation was incomplete, the areas of individual myotubes were measured. In general, a discrete extrafusal structure containing myofibrils was regarded as a fibre. Enzyme histochemistry Acid phosphatase activity was determined against naphthol AS-BI phosphate (Sigma Chemical Co., Poole, Dorset) in fresh cryostat sections. The sections were incubated for one hour at 37 °C in the substrate medium containing freshly hexazotised pararosaniline and 20 mM-MnCl2 (Lodja et al. 1979). They were then fixed overnight in 10% formalin, washed for 10 minutes in running tap water and mounted in Kaiser's glycerine gelatin (Lojda et al. 1979). The identity of acid phosphatase was confirmed by the exclusion of naphthol AS-BI phosphate from the substrate medium, or the inclusion of 5 mM-NaF in the complete medium. ,/-Glucuronidase and N-acetylglucosaminidase were detected in fresh cryostat sections by the semipermeable membrane technique using respectively naphthol ASBI-/3-D-glucuronic acid and N-acetyl-fi-D-glucosamine naphthol AS-LC (both from Sigma) (techniques after Lojda et al. 1979). Post-incubation treatment was as for acid phosphatase. Specific inhibitors used to identify the enzymes were, for ,I-glucuronidase, D-saccharic acid 1,4-lactone (0-48% w/v) and for N-acetylglucosaminidase, N-acetylglucosamine (10 and 100 mM). Dipeptidyl peptidase II (DPP II) and microsomal aminopeptidase (mAAP) were also demonstrated using variants of the semipermeable technique (Stoward, Christie & Thomson, 1988; Christie & Stoward, 1988). DPP II activity was detected by hydrolysis of H-Lys-Pro-4MNA . 2HCl and mAAP by H-Ala-4MNA. HCl. A variety of inhibitors was used to characterise both proteases, including for DPPII, phenylmethylsulphonyl fluoride (1 mM) and Tris (1 M) and for mAAP, actinonin (10 ,ug/ml) (Umezawa et al. 1985). Actinonin was purchased from the Peptide Institute, Osaka, Japan and the substrates from Bachem Feinchemikalien, Budendorf, Switzerland. Glycogen staining was demonstrated by the periodic acid-Schiff method following fixation Glycogen of fresh cryostat sections in Clarke's fluid. Specificity of the reaction was confirmed by
Large muscle fibres in neonatal mice 141 pre-Schiff treatment of the fixed sections with 0-5 % w/v a-amylase in 0-004 M acetate buffer, pH 5-5, for 3 hours at 37 'C. Microdensitometry of enzyme reaction product Densitometric measurements of enzyme final reaction product were made on a Vickers M86 scanning microdensitometer. The machine settings, with a x 40 objective, for acid phosphatase, mAAP and DPPII, were scanning spot no. 2 and 530 nm (reaction product absorption maxima) and for NADH-tetrazolium reductase, scanning spot no. 2 and 550 nm. The mean reaction product absorbance of 24 Type 1 fibres in soleus and 24 Type 2B fibres in EDL was determined (this number was sufficient to give a constant cumulative mean absorbance and standard deviation) and the grouped mean absorbance of the same muscle in four age-matched animals derived. The absorbance of as many large fibres as could be found was measured and treated similarly. Enzyme activity was expressed as integrated optical density, derived by multiplying mean fibre area by mean absorbance at the appropriate wavelength (Christie & Thomson, 1987). RESULTS
Histology andfibre sizes Haematoxylin and eosin staining and the NADH-tetrazolium reductase method were used first to screen the sections for large fibres, which were always more eosinophilic than their normal counterparts and yielded an extra strong NADHtetrazolium reductase final reaction product. After 12 days of age eosinophilia declined markedly, and identification was based on the intensity of the NADH final reaction product and an area more than 15 % greater than the mean of the largest 'normal' fibres present. The large fibres usually occurred in isolated fascicles of between 5 and 10 fibres within and at the periphery of intermediate and deep muscles such as soleus and flexor hallucis longus. Isolated single large fibres were rare, but occasionally present in flexor and extensor compartments. The difference in size was most apparent at two days, when the mean area of the large fibres was three times greater than typical fibres (P < 0.01, Student's t test) (Figs. 1, 2). The growth rates of normal-sized and large fibres were similar until about 21 days, when the large fibres slowed to approximate with the faster growing 2B fibres. By 25 days it was difficult to identify the large fibres as a separate population, morphologically or histochemically.
Histochemistry Up to 12 days old the NADH-tetrazolium reductase method did not distinguish between fibre types; nevertheless, from 2 to 12 days and beyond there was considerably more reaction product in the large fibres than in typical Type 1 fibres (Fig. 3). The large fibres gave a strong ATPase reaction up to 12 days of age at pH 9-7 and after pre-incubation at pH 4-4 (Figs. 4, 5). Beyond this age acid pre-incubation produced differentiation. From 2 to 25 days the large fibres exhibited a strong granular and diffuse acid phosphatase reaction product (Fig. 6). Over the same period typical fibres displayed a similarly distributed but much weaker reaction. In normal fibres, up to 21 days, DPPII reaction product was present as finely dispersed particles. By contrast the large fibres exhibited a concentrated, coarse, granular product with overall diffuse sarcoplasmic staining (Fig. 7). fl-Glucuronidase
142
K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI 2000
1200
400
0
I
I
2
7
12
21 Age (days)
25
40
Fig. 1. Muscle fibre areas versus age. The large fibres are only significantly greater in area at two days old. A, undifferentiated fibres; 0, large fibres; *, white fibres; A, red fibres (means±S.D.).
activity was weak or absent in most normal sized fibres at all ages. A weak granular and sarcoplasmic reaction product was, however, present in the large fibres. No Nacetylglucosaminidase activity was detected in any fibres. The activity and distribution of microsomal aminopeptidase was similar to DPPII, with greatest activity again in the large fibres (Fig. 8). The large fibres gave a strong phosphorylase and glycogen reaction up to 12 days; beyond this age differentiation was apparent (Figs. 9, 10). Integrated densitometry (enzyme activity) Integrated optical density measurements showed NADH-tetrazolium reductase activity to be significantly greater in the large fibres during the first 21 days (P < 0-001) (Fig. 11). At 2 days, activity was fourfold higher, increasing to a probable maximum by 21 days. Acid phosphatase activity in the large fibres was fivefold higher than in normal sized fibres at 2 days (Fig. 12). Activity was again significantly increased for the first 21 days (P < 0-001). DPPII was considerably more active in the large fibres at 2 days, remaining significantly so for up to 12 days (P < 0-001) (Fig. 13). Microsomal aminopeptidase activity was also higher in the large fibres during the first 25 days, but not to any degree of significance (Fig. 14). Fig. 2. Two days old mouse. Large eosinophilic fibres are evident in the region of flexor hallucis longus. H & E. x 250. Fig. 3. Twelve days old mouse. This section, reacted for NADH-tetrazolium reductase, shows the massive deposition of formazan in the large fibres. x 140. Fig. 4. Seven days old mouse. Section reacted for ATPase at pH 9.7. The large fibres are strongly reactive. x 100. Fig. 5. Serial section to that shown in Fig. 4 reacted for ATPase at pH 4-4. The large fibres remain highly reactive. x 100.
143
Large muscle fibres in neonatal mice
Al,~~~~~~,. * F*,b % .. .;vA V. *~~~~~~~~~~~~~~~~~~~~~~~~~~~ ok8
w ..... .
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144
K. N. CHRISTIE, R. J. G. STEWART AND G. BACCIOCCHI
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V W
