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J. Anat. (1978), 127, 1, pp. 117-123 Printed in Great Britain
Muscles and bones of large and small mice compared at equal body weights A. C. B. HOOPER
Department of Anatomy, University College, Dublin
(Accepted 18 August 1977) INTRODUCTION
Although muscle and bone account for a large proportion of body mass, and determine the characteristic shape of the body, surprisingly little is known about the quantitative genetic control of the growth of these tissues. Genetic factors influence bone dimensions in a number of species (Clark, 1941; Crary & Sawin, 1949; Fabian, 1959), while the length and weight of the long bones in mice are altered by selection for an index based on the length of the bones (Dawson, Stephenson & Fredline, 1972). Muscle fibre number is genetically determined (Luff & Goldspink, 1970), and there is evidence to suggest that fibre diameter is also affected by genetic influences (Joubert, 1956; Smith, 1963; Staun, 1968). Hooper (1976) has shown that changes in fibre length, resulting from differences in sarcomere number, also contribute to genetically determined alterations in muscle mass. The muscles and bones of mice of particular ages have been studied in lines selected for high and low body weight. Seven muscles in particular were studied in the two lines and it was found that there were alterations in their overall weights because of consistent changes in both fibre number and fibre diameter (Byrne, Hooper & McCarthy, 1973), although additive genetic influences were of more importance in the determination of fibre number than of fibre diameter. Four bones, the humerus, ulna, femur and tibia, were investigated and, although the quantitative genetic control of the two parameters appears to differ, it is clear that alterations in the length and diameter of the long bones contribute to genetically determined alterations in body weight (Hooper, 1977). These components of muscle and bone mass contribute unequally to genetically determined alterations in body weight. It was therefore decided to study mice from the high and low body weight selection lines at approximately the same body weight regardless of age. Such an investigation ought to reveal the presence of any associations which may exist between the quantitative genetic controls of muscle growth and bone growth. MATERIALS AND METHODS
Two lines of mice (termed 'high line' and 'low line'), consisting respectively of animals bred for high and low body weight when they are 10 weeks old, were used. At the time of the experiment the lines had undergone 15 generations of withinlitter selection. An unselected control line was also studied, all the lines being derived from Q strain mice (Falconer, 1973).
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A. C. B. HOOPER
Table 1. Points used to measure bone length Humerus Ulna Femur Tibia
Proximal point Highest point of head Tip of olecranon process Highest point of greater trochanter Centre of condyles
Distal point Lowest point of trochlea Tip of styloid process Lowest point of condyles Tip of medial malleolus
Male mice of both lines were killed by ether overdosage when they had attained a body weight of approximately 25 g, which is close to the mature weight of low line mice, but is reached by high line mice early in the fourth week of life. Muscle fibre differentiation is known to be complete in mice of this age (Rowe & Goldspink, 1969). The carcasses were pinned out on a polystyrene slab with the limbs fully extended, and were left in this position for 4 hours to allow the onset of rigor mortis. The right and left biceps brachii and tibialis anterior muscles were then excised. Muscles from the right side were weighed on a torsion balance, and a suspension of separated fibre fragments was prepared in calcium-free Ringer Locke solution using the technique described by Hegarty & Naude (1970). A portion of this suspension was placed in the well of a hanging-drop slide with a Pasteur pipette and a coverslip was placed over it. The diameters of 25 randomly selected fibres were measured with an image-splitting eye-piece micrometer. Short fragments were excluded as were fragments whose entire length was not in focus and which therefore were not in the plane of focus. Muscles from the left side were fixed in formol saline and embedded in paraffin wax. The regions of the muscles which contain all their constituent fibres have been identified (Goldspink, 1962; Rowe, 1967), and a complete transverse section was cut at a thickness of 5 ,m from this region of each muscle. Sections were stained with haematoxylin and eosin, mounted in Canada balsam, and after projection of their images on to white card at a magnification of x 250, the number of fibres per muscle was recorded with an electric pen counter. Several muscles were lost during histological processing. The carcasses were placed in a supine position on the outside of envelope-packed X-ray film, and were taped down with the limbs flat against the surface in full extension. The films were then exposed for 20 mA seconds at 40 kV with an anode di,stance of 12 inches. The length and diameter of the right humerus, ulna, femur atid tibia were measured on the developed plates with a micrometer callipers. Because of the difficulty in defining the ends of some bones, the length of a bone was defined as the distance between readily identifiable points at its proximal and distal ends (Table 1). The diameter of each bone was measured midway along its
length. Least squares analysis of the data was carried out, and the line effects were partitioned to allow single degree of freedom comparisons for deviations from the control line. RESULTS
Body weight and age Line means for body weight and age at assay are shown in Table 2, together with their standard errors and numbers of mice per line. The mean age of the high and low lines at assay differed significantly from the control line (P< 0 001). The mean
119
Muscle and bone growth Table 2. Numbers of mice per line, line means and standard errors of the means for body weight and age at assay Numbers per linet
Assay weight (g)
Assay age (day)
35 (29, 31) 25 4±0 3 29±1 40 (35, 30) 24-0±0-2 36±1 33 (29, 24) 73±1 24-1±0-3 in refer to the number of mice t Figures parentheses represented in fibre number counts of m. biceps brachii and m. tibialis anterior respectively. High line Controls Lowline
Table 3. Least squares means and average standard errors for muscle weight, fibre number and mean fibre diameter Muscle weight (mg)
Fibre diameter (>m)
Fibre number
Tibialis Tibialis Biceps Biceps Tibialis Biceps anterior anterior anterior brachii brachii brachii 50.1* High line 13-3 NS 1396** 1952** 43 0 NS 31-7 NS 52 2 32-2 Controls 1172 1694 41V7 13X8 53.9** Low line 43.9** 13-5 NS 36-3* 978** 1443** 0-6 Standard error 0-6 05 03 48 59 Probability levels versus control: * P< 0-05; ** P< 0-01; NS, not significant (P> 0 05).
Table 4. Least squares means and average standard errors for bone length and diameter Humerus
Ulna
Femur
Tibia
Bone length (,um) 151 NS 12-3 NS 13-0 NS 15 2 12 4 13-1 15.7** 13-9** 12-5 NS 0.1 0.1 03 Bone diameter (/,tm) 1P23 NS 105 NS 1P23 NS 1V48 NS High line 1P00 1.19 1P19 1-38 Controls 1-28NS 1-18NS 1 15** 1-14NS Lowline 0-03 0-03 Standard error 004 005 Probability levels versus control: * P 0 05), but the difficulty encountered in choosing a suitable assay weight is emphasized by the small but significant (P < 001) -increase in the weight of high line mice. Muscle weight, fibre number and fibre diameter Line means for the weight, fibre number and mean fibre diameter of the two muscles studied are shown in Table 3, together with the appropriate standard errors. With the exception of tibialis anterior in the low line there was no significant difference in muscle weight (P < 005). The fibre number of both muscles was
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A. C. B. HOOPER
significantly increased in the high line and decreased in the low line (P < 00 1). The mean fibre diameter of both muscles was significantly increased in low line mice (P
J. Anat. (1978), 127, 1, pp. 117-123 Printed in Great Britain
Muscles and bones of large and small mice compared at equal body weights A. C. B. HOOPER
Department of Anatomy, University College, Dublin
(Accepted 18 August 1977) INTRODUCTION
Although muscle and bone account for a large proportion of body mass, and determine the characteristic shape of the body, surprisingly little is known about the quantitative genetic control of the growth of these tissues. Genetic factors influence bone dimensions in a number of species (Clark, 1941; Crary & Sawin, 1949; Fabian, 1959), while the length and weight of the long bones in mice are altered by selection for an index based on the length of the bones (Dawson, Stephenson & Fredline, 1972). Muscle fibre number is genetically determined (Luff & Goldspink, 1970), and there is evidence to suggest that fibre diameter is also affected by genetic influences (Joubert, 1956; Smith, 1963; Staun, 1968). Hooper (1976) has shown that changes in fibre length, resulting from differences in sarcomere number, also contribute to genetically determined alterations in muscle mass. The muscles and bones of mice of particular ages have been studied in lines selected for high and low body weight. Seven muscles in particular were studied in the two lines and it was found that there were alterations in their overall weights because of consistent changes in both fibre number and fibre diameter (Byrne, Hooper & McCarthy, 1973), although additive genetic influences were of more importance in the determination of fibre number than of fibre diameter. Four bones, the humerus, ulna, femur and tibia, were investigated and, although the quantitative genetic control of the two parameters appears to differ, it is clear that alterations in the length and diameter of the long bones contribute to genetically determined alterations in body weight (Hooper, 1977). These components of muscle and bone mass contribute unequally to genetically determined alterations in body weight. It was therefore decided to study mice from the high and low body weight selection lines at approximately the same body weight regardless of age. Such an investigation ought to reveal the presence of any associations which may exist between the quantitative genetic controls of muscle growth and bone growth. MATERIALS AND METHODS
Two lines of mice (termed 'high line' and 'low line'), consisting respectively of animals bred for high and low body weight when they are 10 weeks old, were used. At the time of the experiment the lines had undergone 15 generations of withinlitter selection. An unselected control line was also studied, all the lines being derived from Q strain mice (Falconer, 1973).
118
A. C. B. HOOPER
Table 1. Points used to measure bone length Humerus Ulna Femur Tibia
Proximal point Highest point of head Tip of olecranon process Highest point of greater trochanter Centre of condyles
Distal point Lowest point of trochlea Tip of styloid process Lowest point of condyles Tip of medial malleolus
Male mice of both lines were killed by ether overdosage when they had attained a body weight of approximately 25 g, which is close to the mature weight of low line mice, but is reached by high line mice early in the fourth week of life. Muscle fibre differentiation is known to be complete in mice of this age (Rowe & Goldspink, 1969). The carcasses were pinned out on a polystyrene slab with the limbs fully extended, and were left in this position for 4 hours to allow the onset of rigor mortis. The right and left biceps brachii and tibialis anterior muscles were then excised. Muscles from the right side were weighed on a torsion balance, and a suspension of separated fibre fragments was prepared in calcium-free Ringer Locke solution using the technique described by Hegarty & Naude (1970). A portion of this suspension was placed in the well of a hanging-drop slide with a Pasteur pipette and a coverslip was placed over it. The diameters of 25 randomly selected fibres were measured with an image-splitting eye-piece micrometer. Short fragments were excluded as were fragments whose entire length was not in focus and which therefore were not in the plane of focus. Muscles from the left side were fixed in formol saline and embedded in paraffin wax. The regions of the muscles which contain all their constituent fibres have been identified (Goldspink, 1962; Rowe, 1967), and a complete transverse section was cut at a thickness of 5 ,m from this region of each muscle. Sections were stained with haematoxylin and eosin, mounted in Canada balsam, and after projection of their images on to white card at a magnification of x 250, the number of fibres per muscle was recorded with an electric pen counter. Several muscles were lost during histological processing. The carcasses were placed in a supine position on the outside of envelope-packed X-ray film, and were taped down with the limbs flat against the surface in full extension. The films were then exposed for 20 mA seconds at 40 kV with an anode di,stance of 12 inches. The length and diameter of the right humerus, ulna, femur atid tibia were measured on the developed plates with a micrometer callipers. Because of the difficulty in defining the ends of some bones, the length of a bone was defined as the distance between readily identifiable points at its proximal and distal ends (Table 1). The diameter of each bone was measured midway along its
length. Least squares analysis of the data was carried out, and the line effects were partitioned to allow single degree of freedom comparisons for deviations from the control line. RESULTS
Body weight and age Line means for body weight and age at assay are shown in Table 2, together with their standard errors and numbers of mice per line. The mean age of the high and low lines at assay differed significantly from the control line (P< 0 001). The mean
119
Muscle and bone growth Table 2. Numbers of mice per line, line means and standard errors of the means for body weight and age at assay Numbers per linet
Assay weight (g)
Assay age (day)
35 (29, 31) 25 4±0 3 29±1 40 (35, 30) 24-0±0-2 36±1 33 (29, 24) 73±1 24-1±0-3 in refer to the number of mice t Figures parentheses represented in fibre number counts of m. biceps brachii and m. tibialis anterior respectively. High line Controls Lowline
Table 3. Least squares means and average standard errors for muscle weight, fibre number and mean fibre diameter Muscle weight (mg)
Fibre diameter (>m)
Fibre number
Tibialis Tibialis Biceps Biceps Tibialis Biceps anterior anterior anterior brachii brachii brachii 50.1* High line 13-3 NS 1396** 1952** 43 0 NS 31-7 NS 52 2 32-2 Controls 1172 1694 41V7 13X8 53.9** Low line 43.9** 13-5 NS 36-3* 978** 1443** 0-6 Standard error 0-6 05 03 48 59 Probability levels versus control: * P< 0-05; ** P< 0-01; NS, not significant (P> 0 05).
Table 4. Least squares means and average standard errors for bone length and diameter Humerus
Ulna
Femur
Tibia
Bone length (,um) 151 NS 12-3 NS 13-0 NS 15 2 12 4 13-1 15.7** 13-9** 12-5 NS 0.1 0.1 03 Bone diameter (/,tm) 1P23 NS 105 NS 1P23 NS 1V48 NS High line 1P00 1.19 1P19 1-38 Controls 1-28NS 1-18NS 1 15** 1-14NS Lowline 0-03 0-03 Standard error 004 005 Probability levels versus control: * P 0 05), but the difficulty encountered in choosing a suitable assay weight is emphasized by the small but significant (P < 001) -increase in the weight of high line mice. Muscle weight, fibre number and fibre diameter Line means for the weight, fibre number and mean fibre diameter of the two muscles studied are shown in Table 3, together with the appropriate standard errors. With the exception of tibialis anterior in the low line there was no significant difference in muscle weight (P < 005). The fibre number of both muscles was
120
A. C. B. HOOPER
significantly increased in the high line and decreased in the low line (P < 00 1). The mean fibre diameter of both muscles was significantly increased in low line mice (P
