longitudinal growth of striated muscle fibresboth increas ien length and increase in girth of the...

J, Cell Sci. 9, 751-767 (1970 751

Printed in Great Britain

LONGITUDINAL GROWTH OF STRIATED

MUSCLE FIBRES

PAMELAS. WILLIAMS AND G. GOLDSPINKMuscle Research Laboratory, Department of Zoology, University of Hull, Yorkshire,England

SUMMARYThe direct determination, by counting, of the number of sarcomeres in series along the

length of single teased muscle fibres taken from mice of different ages showed that the increasein fibre length during normal growth is accompanied by a large increase in sarcomere number.The greatest increase occurs during the 3 weeks after birth. By counting the number of musclefibre nuclei in single teased fibres it was shown that the number of nuclei per fibre increaseswith age, and that this increase continues beyond the point at which the fibres have ceased togrow in length. It is suggested that post-natal increase in nuclear number is associated withboth increase in length and increase in girth of the muscle fibres.

By injecting tritiated adenosine into young mice, an attempt was made to label newly formedactin filaments and ribosomes and thus to determine the region where new sarcomeres are laiddown during increase in fibre length. Using autoradiography and scintillation counting it wasshown that the radioactive label was incorporated more into the ends than into the middleregions of the muscles. The implication of these findings is that new sarcomeres are added onserially at the ends of the muscle fibres. An investigation, at the ultrastructural level, of musclefibres taken from foetal and newborn mice indicates that the end of the fibre is a region of activedevelopment. This area is characterized by numerous ribosome formations and by myofilamentswhich are not organized into myofibrils. Cells which can occasionally be seen fusing with theend regions of young muscle fibres indicate a possible way in which nuclei are added to thegrowing fibre.

Immobilization experiments have shown that it is possible to alter both the rate and theextent of the post-natal increase in sarcomere number. Immobilization of limb joints, by meansof plaster casts, so that the muscle is held in either the extended or the shortened position resultsin the number of sarcomeres along the fibres falling far short of that in the fibres from controlmuscles. Removal of the restriction is followed by a rapid increase in the number of sarcomeresin series and a return to the normal level within a period of about 4 weeks. These experimentsindicate that for normal growth to occur, it is important for a muscle to be able to contractisotonically.

INTRODUCTION

During post-natal growth, the skeletal muscles of most mammals increase in lengthas a result of the longitudinal growth of their component muscle fibres (Kitiyakara &Angevine, 1963; Goldspink, 1968; Bridge & Allbrook, 1970). There is evidence that,in the mouse, this lengthening of fibres, though due partly to an increase in the lengthof individual sarcomeres, is mainly due to an increase in the number of sarcomeresalong the length of the fibres (Goldspink, 1968). In this investigation further studieshave been made of the post-natal increase in fibre length by directly counting thenumber of sarcomeres along the length of teased single fibres taken from mice of

752 P. E. Williams and G. Goldspink

different ages. It has been shown that the growth of muscle fibres is accompanied byan increase in the number of muscle fibre nuclei (Montgomery, 1962; Enesco & Puddy,1964). Direct counts of nuclei have been made on teased single fibres in order todetermine exactly the rate of increase of muscle fibre nuclei during post-natal growth.

Many workers consider that during the lengthening process the incorporation ofnew sarcomeres into existing myofibrils takes place by serial addition at the ends ofthe muscle fibres (Holtzer, Marshall & Finck, 1957; Kitiyakara & Angevine, 1963;Ishikawa, 1965; Goldspink, 1968; Mackay, Harrop & Muir, 1969). Some authors,however, suggest that new sarcomeres may be inserted at any point along the lengthof the fibres (Ruska & Edwards, 1957; Schmalbruch, 1968). In order to clarify thissituation an attempt has been made to label the newly formed actin filaments andribosomes, using the method of Griffin & Goldspink (in preparation), and thusdetermine the region where new sarcomeres are laid down. Using the electron micro-scope, an investigation has been made into the way in which newly synthesized con-tractile proteins are assembled on to the existing myofibrils.

Experiments have shown that surgical modification of the distance the muscle hasto contract affects the longitudinal growth of the muscle as a whole (Ctawford, 1954;Alder, Crawford & Edwards, 1959). In order to determine whether it is possible toalter the rate and the extent of the increase in sarcomere number, and to ascertain theimportance of movement for normal post-natal growth, some immobilization experi-ments have been carried out.

MATERIALS AND METHODS

Materials

The mice used were normal homozygous males of the i2gjRe strain. The animals were rearedin the Department of Zoology from a colony which was originally obtained from JacksonMemorial Laboratories, Bar Harbor, U.S.A. They were fed on a modified diet formula 41b(Oxoid Ltd.) with food and water available at all times. The biceps brachii and soleus muscleswere the muscles chosen for this study on account of their relatively simple, fusiform structurewith fibres running from tendon to tendon. Also these muscles, in the mouse, are small enoughto be fixed in situ.

Determination of the number of sarcomeres in series and the length of muscle fibres

Mice of ages ranging from newborn to 2 years were used. Young mice were killed with ethervapour and splints were tied to the limbs so that the biceps brachii and soleus muscles werefully extended. Older mice were killed by dislocation of the cervical vertebrae and the limbswere pinned out on a cork board so that the muscles were again in the fully extended position.The biceps brachii and soleus muscles were exposed by removing the overlying skin and othertissues and they were fixed in situ by pipetting fixative on to the muscles for 20 min. Thefixative used was 2#s % glutaraldehyde buffered at pH 7-5 with o-i M phosphate buffer andcontaining 0-5% glucose. The whole limbs were then removed from the animals andplaced in fixative. After 1 h, the biceps brachii and soleus muscles were dissected out andplaced in fresh fixative for a further 90 min. After fixation the muscles were washed in bufferand the distance between the muscle-tendon junctions was measured for each muscle usingmicrometer calipers. The muscles were placed in 30 % (w/v) HNO;, for 2 days to hydrolysemost of the connective tissue. They were then washed and stored in 50 % glycerol to removethe soluble proteins and make the myofibrils more clearly visible. Using electrolytically sharpenedtungsten needles and with the aid of a dissecting microscope, individual whole fibres were

Muscle fibre growth 753

teased out from random positions within the muscles and measured with micrometer calipers.They were then mounted in glycerol jelly with the coverslips supported by strips of glass toavoid compressing the fibre. The fibres were then photographed on 35-mm film, using a LeitzOrtholux microscope with a camera attachment. Photomicrographs were made of 3 fibres fromeach muscle and the number of sarcomeres along the length of the fibres determined bycounting each sarcomere.

Determination of the number of nuclei in single fibres

Mice of ages ranging from 1 day to 2 years were used. Muscles were fixed in Carnoy'sfixative. After 30 min fixation on the bone, the biceps brachii and soleus muscles were removedand fixed for a further 1 h, then placed in absolute ethanol for 90 min. After hydration, theywere immersed for 48 h in gallocyanin stain, adjusted to pH 1-5. The muscles were washed indistilled water and stored in 50 % glycerol. Single fibres were teased out and mounted in glyceroljelly. The fibres were viewed through a binocular microscope and the number of nuclei in eachfibre was determined by counting. Counts were made on 3 fibres taken from random positionswithin each muscle. Care was taken to distinguish between muscle fibre nuclei and the nucleiof the adhering endomysium. The muscle fibre nuclei were found to lie parallel to the axis ofthe fibre and were rod shaped, whereas the connective tissue nuclei were irregularly arranged,fusiform, and stained more darkly.

Autoradiography

Tritiated adenosine was used to locate the region of longitudinal growth in young biceps andsoleus muscles. The adenosine is incorporated into newly formed actin and ribosomes. Freeadenosine and other adenosine-containing compounds are removed by glycerol extraction(Griffin & Goldspink, in preparation).

Mice weighing approximately 5 g were given daily intraperitoneal injections of 25 /idPHJadenosine for periods ranging from 2 days to 3 weeks. The animals were then sacrificed andthe hind and forelimbs removed and pinned to pieces of cork board. They were then immersedfor 2 weeks in a mixture of 50 % glycerol and 50 % 0-2 M phosphate buffer, pH 7-6, and main-tained at — 5°C. During this period the glycerol mixture was changed frequently. Followingglycerol extraction, the limbs were washed twice in phosphate buffer only. The biceps brachiiand soleus muscles were dissected out, fixed in glutaraldehyde and embedded in ester wax. Themuscles were sectioned longitudinally at a thickness of 5 fim and ribbons of sections weremounted on subbed slides and coated with Ilford K2 nuclear emulsion. The dried slides wereplaced in light-tight boxes and exposed at 4 °C. Test slides were developed at intervals of1, 2, 3 and 4 weeks. The slides were stained with haematoxylin and eosin and examined underreflected-light illumination using Leitz Ultropak optics (the silver grains appear as white spots).

Liquid scintillation counting

Young mice weighing approximately 5 g were injected daily with 25 fid PHJadenosine for3 days. The forelimbs were removed and immersed for 2 weeks in 50 % glycerol/buffer mixture.Following this they were washed for 2 days in buffer only. The biceps brachii muscles wereremoved and supported on the microtome chuck in brain tissue taken from a non-injectedanimal. The microtome chuck was then immersed in Freon 12 which had previously beencooled to — 160 CC using liquid nitrogen. Each muscle plus brain tissue was sectioned trans-versly on a Pearse Slee cryostat at a thickness of 20 fim. The first section containing muscle wasplaced on a slide and the following 40 sections were collected in a scintillation vial. The nextsection was put on a slide, the following 40 into a second vial. This procedure was repeated untilthe entire muscle had been sectioned. The sections on slides were fixed and stained and thearea of muscle in each was determined using a slide projector and a planimeter. From thesemeasurements, the volume of muscle tissue in each vial could be estimated.

To each scintillation vial, 0-7 ml of hyamine IOX hydroxide was added and the vials weresealed and rotated for 36 h in a water bath at 37 °C to dissolve the tissue sections. After cleaningthe outside of the vials, 10 ml of PPO in toluene were added to each. The vials were placed in a

754 P- E. Williams and G. Goldspink

Packard automatic scintillation counter and counted for ioo min. To determine the countingefficiency of each sample, 50 /il of tritiated hexadecane was added to each vial and the resultingmixture recounted. The radioactivity of each sample was expressed as dpm/mm3 tissue.

In a second experiment, adult mice were injected daily with 50 /iCi PHJadenosine for 3 days.The limbs were removed and the muscles treated in the same way as the young muscles.

Electron microscopy

Biceps brachii and soleus muscles from foetal and newborn mice were fixed in glutaraldehyde,washed overnight in phosphate buffer at 4 °C, and postfixed for 2 h in OSO4. The musclefibres were teased apart in 70% ethanol and dehydrated in 100% ethanol and 100% acetone.Short lengths of single fibres or small bundles were taken from the middle and end regions ofthe muscles and embedded in Araldite (Ciba). Longitudinal sections were cut using a Reichertultramicrotome. Ribbons of sections showing gold or silver interference colours were collectedon Celloidin-coated grids and double stained with uranyl acetate and lead citrate solutions. Thesections were examined with a JEOL JEM 7 A electron microscope.

Immobilization of the soleus muscle

Soleus muscles of young mice were immobilized in different positions during the growthperiod.

Immobilization in the shortened position. Plaster casts were used to hold the hind limbs of micewith the whole limb in the extended position, that is to say with the soleus in its shortenedposition. Animals weighing approximately 5 g had plaster casts put on one hind limb; thecontralateral leg served as the control. The casts were changed twice weekly throughout theexperiment. The presence of the cast did not usually cause any decrease in the growth of thebones and in those few cases where it did, the animals were discarded from the experiment.

Mice from one group were sacrificed at intervals ranging from 2 to 6 weeks after immobiliza-tion. The number of sarcomeres per fibre in the soleus muscles from experimental and contra-lateral limbs was determined. Mice from a second group had their casts removed when themice weighed between 18 and 20 g. They were left for 3—4 weeks before being sacrificed and thenumber of sarcomeres per fibre was determined in the experimental and contralateral muscles.

Immobilization in an extended position. Hind limbs of young mice were again held in plastercasts but this time with the limb in a flexed position, that is to say with the soleus muscle in thefully extended position. The mice were sacrificed at intervals ranging from 2 to 6 weeks afterimmobilization. The number of sarcomeres per fibre was again determined in soleus musclesfrom experimental and contralateral limbs.

RESULTS

The number of sarcomeres in series and the length of muscle fibres

The number of sarcomeres in single fibres teased from biceps brachii and soleusmuscles is shown in Fig. 1. It will be seen that there was very little variation in thenumber of sarcomeres along the length of different fibres in a muscle at any particularage but that there was a very marked increase in sarcomere number with age (bodyweight). The greatest increase was found to occur during the first 3 weeks after birth.In the soleus muscle the rate of increase in sarcomere number was greater than in thebiceps brachii. When the animal reached a weight of approximately 22 g (6 weeks) nofurther increase in sarcomere number was detected. From the data in Figs. 1 and 2,it will be seen that in both the biceps brachii and the soleus muscles, the rate andextent of increase in fibre length are approximately the same as the rate and extent ofthe increase in sarcomere number.


2500 r-

2000

E 1500

•f 1000

500

EE 6

I I I I I J10 15 20 25 30

Body weight, g

Fig. i

35 40 10 15 20 25

Body weight, g

Fig. 2

30 35

Fig. i. Graph showing the number of sarcomeres along the length of muscle fibres frommice of the following ages and body weights: i day, 2 g; 5 days, 4 g; 2 weeks, 8 g;3"5> ! 3 g-. 4'5> l5 8» 6, 21 g; 7, 24 g; 13-5, 34 g; and 16 weeks, 37 g. 0 , biceps brachii;x , soleus. Each point represents the data from 1 fibre.

Fig. 2. Graph showing muscle belly length (solid line; O» biceps brachii, and • ,soleus) and muscle fibre length (broken line; 0 , biceps brachii, and x , soleus).Note that muscle length measurements were made before and fibre length measure-ments after the nitric acid treatment.

The number of nuclei in muscle fibres

The number of nuclei in single fibres teased from biceps brachii and soleus musclesis shown in Fig. 3. The number of nuclei per fibre increased with body weight, andthis increase continued beyond the point at which the sarcomere increase had ceased.The rate of increase in the number of nuclei per fibre was greater in the soleus thanin the biceps brachii. The nuclei were found to be scattered randomly along the fibresof both muscles. Again the variation between the measurements on different fibres ina muscle at any particular age was quite small.

Autoradiography

Fig. 5 shows an autoradiograph of a longitudinal section of the biceps brachii muscleof a 5-g mouse which had been injected with pHJadenosine. It can be seen that mostof the label was located at the ends of the muscle fibres. These results indicate that


250

200

•S 150

E100

50

0 5 10 15 20 25 30 35 40Body weight, g

Fig. 3. Graph showing the number of nuclei along the length of muscle fibres from miceof different ages. %, biceps brachii; x , soleus.

the newly formed actin and newly formed ribosomes are found mainly in the endregions of growing muscle fibres.

Scintillation counting

It can be seen from Table 1 that in the biceps brachii muscles taken from youngmice the tritiated adenosine was incorporated more into the ends of the muscles thaninto the middle regions. By grouping vials 1 and 5 and vials 2, 3 and 4, and applyinga t test, it was shown that the difference between the counts in the ends and the middleregions of the fibres was significant at the 5% probability level. Very little PH]-adenosine was incorporated into the adult muscles and there was no difference betweenthe ends and the middle regions.

Electron microscopy

Newborn and foetal muscle fibres were seen to contain narrow myofibrils whichwere located around the periphery of the fibres (Fig. 6). Near the ends of the myo-fibrils ribosomes were often found surrounding the myosin filaments (Figs. 7, 8). Atthe muscle-tendon junction the myofibrils appeared to be connected to the plasmamembrane in the region of the sarcolemmal clefts (Fig. 9). Myofilaments which werenot organized into myofibrils were frequently seen in the sarcoplasm at the ends of themuscle fibres near to the plasma membrane (Fig. 10).


Table i. Shows the amount of radioactivity, expressed as dprn/mm3 tissue, that has beenincorporated into the different regions of 3 young and 2 adult biceps brachii muscles. Ineach case the first and last vials contain the end regions of the muscle

Vialnumber

Young vniscles1

2

3451

2

345

1

2

345

Adult vniscles1

2

345671

2

34567

Distancealong muscle,

mm

o-o-8o-8-i-61-6-2-42-4-3-232-39

o-o-8o-8-i-61-6-2-42-4-3-23-2-41

o-o-8o-8-i-61-6-2-42-4-3-23-2-4-1

o-o-8o-8-i-61-6-2-42-4-3-23-2-4-04-0-4-84-8-S-a

o-o-8o-8-i-61-6-2-42-4-3-23-2-4-04-0-4-84-8-5-4

dpm

1 1 4

28

1 0 9

5765

5394242 6 0

47i150

2 0 7

3 1 32332 0 8

593

41

57

26

3688

14

716

61

983 0

Volume of muscletissue in vial,

mm3

0-360-72

o-73o-680-38

0-71i-oo0 9 6o-680-46

0-701-05I'OI0-69o-55

o-530-781 031-47i-55o-860-39

o-45o-550-871-50i-66o-990 4 4

dpm/mm3

tissue

3 i 7

39149

841 7 1

759426272

692326

4462962 3 1

3 0 1

1067

7i - 3

55

174 22 0

i 7

258

11

379969

Immobilization of the soleus muscle

The results of these experiments are shown in Fig. 4. As will be seen from thisfigure, the effect of immobilization during the growth period was to reduce the rateand extent of sarcomere addition. The number of sarcomeres in fibres from muscleswhich had been immobilized in the shortened position was approximately half that infibres from the normal, contralateral muscles. When plaster casts were removed fromthe immobilized limbs the number of sarcomeres rapidly increased, so that after aperiod of 4 weeks the sarcomere number in the experimental and the contralateralmuscle fibres did not differ significantly.

In fibres from muscles which had been immobilized in the extended position, the


2500 r

2000

8 1500

E

1000

500

Control musclesRecovery from

/ immobilization

Immobilization inextended position

— f Z5-8- Immobilization in^* ** shortened position

Casts removed fromsome experimental limbs

Casts put onexperimental limbs

10 15 20

Bod/ weight, g

25 30

Fig. 4. Graph showing the results of the immobilization experiments. (1) The numberof sarcomeres in fibres from soleus muscles which had been immobilized in theshortened position (#) and the number of sarcomeres in fibres from contralateralmuscles (x) . (2) The number of sarcomeres in fibres from soleus muscles whichhad been immobilized in the extended position (O) and the number of sarcomeres infibres from contralateral muscles ( + ). (3) The number of sarcomeres in fibres fromsoleus muscles which had been immobilized in the shortened position until the miceweighed 18 g (€)) and the number of sarcomeres in fibres from contralateral muscles(A). (4) The number of sarcomeres in fibres from soleus muscles which had beenimmobilized in the extended position until the mice weighed 18 g (O) and thenumber of sarcomeres in fibres from contralateral muscles (A)-


number of sarcomeres was reduced to almost the same extent as in those which hadbeen immobilized in the shortened position (Fig. 4). Again, removal of plaster castsafter a period of immobilization was followed by an increase in sarcomere number anda return to the normal level in a period of 4 weeks.

DISCUSSION

Whilst previous work (Goldspink, 1968) has indicated that the main factor in theincrease in fibre length during post-natal growth is an increase in the number ofsarcomeres along the length of the muscle fibres, the number of sarcomeres wasestimated only from measurements of sarcomere length and muscle belly length. Thedirect determination of sarcomere number as reported here proves conclusively thatincrease in fibre length during normal growth is accompanied by a large increase inthe number of sarcomeres in series. This increase in sarcomere number was sufficientto account for the increase in fibre length, but it is possible that the increase in fibrelength is also accompanied by changes in sarcomere length (Goldspink, 1968). Changesin the sarcomere length would not be detected in this study since the nitric acidtreatment causes uneven shrinkage of the sarcomeres.

The apparent discrepancy between the extent of increase in fibre length and musclelength is interesting. After the point at which the fibres cease to grow in length,further increase in muscle belly length must presumably be due to a rearrangementof the fibres. Observations made while teasing the muscles indicated that in the adultmice the points of insertion of the fibres into the tendon were more staggered than inthe young mice.

It is known that different muscles mature at different rates and in mammals themusculature of the front end differentiates first. Since it has been observed that theextent of increase in the number of sarcomeres and the number of nuclei per fibre isgreater in the soleus muscle than in the biceps brachii, it may be assumed that thebiceps brachii is more mature at birth.

Using biochemical methods based on the determination of DNA content andmorphological methods involving tissue sections, some workers (Montgomery, 1962;Enesco & Puddy, 1964) have reported that the number of nuclei increases in musclefibres growing in vivo. Direct determination of the absolute number of muscle fibrenuclei proves conclusively, first, that there is a large increase in the number of nucleiduring the post-natal development of muscle fibres and, secondly, that the number ofnuclei per fibre continues to increase beyond the point at which the fibres have stoppedincreasing in length. This continued increase is probably associated with increase ingirth of the muscle fibres. Moss (1968) found that in growing chick muscle the cross-sectional area of the fibres and the total number of nuclei (estimated from DNAdetermination) maintained a constant ratio. It is known (Rowe & Goldspink, 1968)that mouse muscle fibres continue to increase in girth beyond the 20-g stage at whichsarcomere increase ceases. If a certain number of nuclei are required for a given volumeof cytoplasm, then it is obviously necessary for the nuclei to continue to increase innumber as long as cytoplasm is being added to the fibre, whether it be due to an in-crease in length of the fibre or to an increase in girth.


During the differentiation of muscle tissue, nuclear replication ceases once themyoblasts have fused to form myotubes (Stockdale & Holtzer, 1961). It seems probablethat additional nuclei are provided by the fusion of satellite cells with the musclefibre, as suggested by Shafiq, Gorychi & Mauro (1968). In the electron-microscopestudies reported here some cells were observed in the process of fusion with musclefibres. These cells were located beneath the basal membrane and may, therefore, haveoriginated as satellite cells. It is perhaps significant that such cells were found near theends of the fibres. Kitiyakara & Angevine (1963) noted that during the first few daysafter injection of pHJthymidine, a high proportion of the labelled muscle fibre nucleiwas located near the ends of the muscle, which indicates that the ends of the musclefibres are areas of active development.

The use of tritiated adenosine proved to be very valuable in locating the growthregion of the muscle fibres. (The advantage of using this isotope rather than a labelledamino acid lies in the fact that it is a fairly specific label for actin. Labelled amino acidsare incorporated into all the proteins which are being synthesized by the muscle cells,and, due to their slower rate of synthesis, the myofibrillar proteins do not incorporatemuch of the label. Experiments have shown (Griffin & Goldspink, in preparation)that in myofibrils prepared from young mice which had been injected with PH]-adenosine, approximately two thirds of the label was in the ADP of the actin andapproximately one third was in the RNA of the ribosomes which are bound to themyofibrils.) In young muscle it was found that the isotope was located more in theends than in the middle portions of the fibres, thus strongly suggesting that the newactin filaments and ribosomes, and hence the new sarcomeres, were added to the endsof the existing myofibrils during the process of longitudinal growth. The fact thatsometimes only one end of the muscle was heavily labelled may be attributable tointermittent growth.

In the electron-microscope studies, a variety of evidence was found indicating thatnew sarcomeres are laid down at the ends of the muscle fibres. In the first place, themyofibrils in this region were not well organized; free filaments could be seen,particularly round the periphery of the fibre. Secondly, numerous ribosomes werefound, particularly in foetal muscle. These were often associated with the terminalmyosin filaments in the way described by Larson, Hudson & Walton (1969). Some-times the appearance of these ribosomes suggested that they were helically arrangedround the myosin filaments.

Crawford (1961) has shown that the immobilization of limb joints results in themuscles failing to attain their normal length. The present investigation demonstratesthat this is due to a smaller number of sarcomeres along the length of the constituentfibres. When the lower joints of the hind limbs were immobilized with the soleus ineither the extended or the shortened position, the sarcomere number fell far short ofthat of the controls. Coupled with this, the fact that the bone length was not affectedimplies that the tendon was correspondingly longer. When the plaster casts wereremoved, the total number of sarcomeres in series increased almost to the normal levelduring a period of a few weeks. Moreover, the sarcomere number returned to thenormal level during a stage in development at which the addition of sarcomeres would


normally be almost completed. According to the work of Tabary, Goldspink, Tardieu,Tabary & Tardieu (in preparation), immobilization of muscles by means of plastercasts in adult animals resulted in a loss of sarcomeres in series, followed by a returnto normal levels once the restriction was removed.

The changes found in immobilized muscles may be due mainly to a decrease in theactivity of the muscles. Since, however, immobilized muscles are able to develop someisometric tension (Fischback & Robbins, 1969) it is perhaps necessary that theycontract isotonically for normal fibre growth to occur and be maintained. There isapparently a strict relationship between the length of the limb bone, the degree ofmovement and the number of sarcomeres in series. The nature of this relationshipbetween the function of the muscle and its longitudinal growth is being investigatedfurther.

This work was supported by a research grant from the Agricultural Research Council. Theauthors also wish to acknowledge the advice and help received from Mr G. E. Griffin, Mr S.Waterson and Mrs Lesley Clapison.

REFERENCES

ALDER, A. B., CRAWFORD, G. N. C. & EDWARDS, R. G. (1959). The effect of denervation on thelongitudinal growth of voluntary muscle. Proc. R. Soc. B 150, 554-562.

BRIDGE, D. T. & ALLBROOK, D. (1970). Growth of striated muscle in an Australian marsupial.J. Anat. 106, 2, 285-295.

CRAWFORD, G. N. C. (1954). An experimental study of tendon growth on the rabbit. J. Bonejft Surg. 36 B, 294-303.

CRAWFORD, G. N. C. (1961). Experimentally induced hypertrophy of growing voluntary muscle.Proc. R. Soc. B 154, 130-138.

ENESCO, M. & PUDDY, D. (1964). Increase in the number of nuclei and weight in skeletal musclesof rats of various ages. Am. J. Anat. 114, 235-244.

FISCHBACH, G. D. & ROBBINS, N. (1969). Changes in contractile properties of disused soleusmuscles. J. PhysioL, Land. 201, 305-320.

GOLDSPINK, G. (1968). Sarcomere length during the post-natal growth of mammalian musclefibres. J. Cell Set. 3, 539-548.

HOLTZER, H., MARSHALL, J. & FINCK, H. (1957). An analysis of myogenesis by the use offluorescent antimyosin. J. biophys. biochem. Cytol. 3, 705-723.

ISHIKAWA, H. (1965). The fine structure of the myo-tendon junction in some mammalianskeletal muscles. Archvm histol. jap. 25, 275-296.

KELLY, D. E. (1969). Myofibrillogenesis and Z band differentiation. Anat. Rec. 163, 403-425.KITIYAKARA, A. & ANCEVINE, D. M. (1963). A study of the pattern of post-embryonic growth of

M. gracilis in mice. Devi Biol. 8, 322-340.LARSON, P. F., HUDSON, P. & WALTON, J. N. (1969). Morphological relationships of poly-

ribosomes and myosin filaments in developing and regenerating skeletal muscle. Nature,Lond. 222, 1168-1169.

MACKAY, B., HARROP, T. J. & MUIR, A. R. (1969). The fine structure of the muscle tendonjunction in the rat. Acta anat. 73, 588-604.

MONTGOMERY, R. D. (1962). Growth of human striated muscle. Nature, Lond. 195, 194-195.Moss, F. P. (1968). The relationship between the dimensions of the fibres and the number of

nuclei during normal growth of skeletal muscle in the domestic fowl. J. Anat. 122, 555-564.ROWE, R. W. D. & GOLDSPINK, G. (1968). Muscle fibre growth in five different muscles in both

sexes of mice. 1. Normal mice. J. Anat. 104, 519-530.RUSKA, H. & EDWARDS, G. A. (1957) A new cytoplasmic pattern in striated muscle fibres and

its possible relation to growth. Growth 21, 73-88.


SCHMALBRUCH, H. Z. (1968). Noniusperioden und Langenwachstum der quergestreiftenMuskelfaser. Z. mikrosk.-anat. Forsch. 79, 493-507.

SHAFIQ, S. A., GORYCKI, M. A. & MAURO, A. (1968). Mitosis during post-natal growth inskeletal and cardiac muscles of the rat. J. Anat. 103, 135-141.

STOCKDALE, F. E. & HOLTZER, H. (1961). DNA synthesis and myogenesis. Expl Cell Res. 24,508-520.

(Received 31 March 1971)

Fig. 5. An autoradiograph prepared from a longitudinal section of a biceps brachiimuscle taken from a young mouse of 7 g body weight. (Initial body weight, 5 g.)The mouse had been injected with 75 /tCi of (^HJadenosine over a period of 3days, x 500.Fig. 6. An electron micrograph of a foetal (18 days gestation) biceps brachii muscle.Note the peripherally arranged myofibrils, the central nucleus and numerous glyco-gen granules {g). x 88 250.


49 c EL 9


Figs. 7, 8. Ribosome chains in newborn soleus muscle (Fig. 7) and foetal biceps brachiimuscle (Fig. 8). The ribosomes (r) found in young muscle often appear to be helicallyarranged, particularly round the terminal myosin filaments as in Fig. 8. Fig. 7, x 28750;Fig. 8, x 34375.Fig. 9. An electron micrograph showing the region of the muscle-tendon junction ofa newborn soleus muscle fibre. In this region the sarcolemma forms many clefts (sc).The myofibrils tend to splay out near their points of attachment to the sarcolemmawhich appears denser and thicker in this region. Collagen fibrils (cf) seem to be con-nected to the outer surface of the basement membrane {bm). Again, groups of ribosomes(r) can be clearly seen, x 18750.

Muscle fibre growth 1*5

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Fig. 10. An electron micrograph of the end region of a biceps brachii muscle fibre takenfrom a foetal mouse (18 days gestation). At the periphery of the fibre the myofilamentsare seen to be irregularly arranged. Dense bodies (2) can be seen which may be theprecursors of Z-band material (see Kelley, 1969). Numerous ribosomes can be seenboth round the periphery of the fibre and in the myofibrils. x 16875.Fig. 11. A newborn biceps muscle fibre. The cell located beneath the basal membrane(bnt), which may have originated as a satellite cell, appears to be fusing with the musclefibre. Note that the fusing cell contains myofilaments organized into A- and I-bandsand that these bands are in register with those of the muscle fibre, x 11250.

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longitudinal growth of striated muscle fibresboth increas ien length and increase in girth of the...

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