Development 102, 815-821 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

815

A critical period for formation of secondary myotubes defined by

prenatal undernourishment in rats

S. J. WILSON, J. J. ROSS and A. J. HARRIS*

The Neuroscience Centre and Department of Physiology, University of Otago Medical School, PO Box 913, Dunedin, New Zealand

* To whom reprint requests should be sent

Summary

Rats fed a restricted diet during gestation and lac-tation gave birth to pups with about 60 % the normalbirth weight. Maintaining the undernutrition afterbirth reduced the rate of growth of the pups so thattheir body weights were only 40 % of control at PN7.Soleus and lumbrical muscles in these animals hadreduced numbers of muscle fibres, and quantitativeexamination of embryonic muscles revealed that thiswas due solely to a decreased formation of secondarymyotubes; the number of primary myotubes remainednormal. Undernutrition did not affect the number ofmotoneurones surviving normal developmental death.Restoration of normal dietary intake on E21, one day

before birth, did not correct the deficit in muscle fibrenumbers in soleus muscles examined when the animalsreached one month of age. Development of the lumbri-cal muscle lags behind the soleus and unrestrictedfeeding from E21 onwards allowed a normal numberof fibres to develop from this time on, although theinitial deficit was never restored. These experimentsdefine a critical period in muscle development duringwhich the potential maximum number of secondarymyotubes is determined.

Key words: prenatal undernutrition, skeletal muscle,secondary myotubes, critical period, lumbrical muscle,soleus muscle, rat embryos.

Introduction

The number of fibres in a muscle depends both ongenetic (Byrne etal. 1973; Stickland & Handel, 1986)and environmental circumstances. Muscle fibre num-bers in young mammals are reduced following under-nutrition of the mother during gestation and lactation(humans: Montgomery, 1962; sheep: Swatland &Cassens, 1973; rats: Bedi et al. 1982) and also in thesmallest animals within a litfer of young (pigs:Hegarty & Allen, 1978; Wigmore & Stickland, 1983).This effect cannot be rectified by subsequent nu-tritional rehabilitation (Bedi etal. 1982). Mammalianmuscle fibre numbers are not permanently affected byperiods of undernutrition after weaning (pigs: Stick-land et al. 1975; mice and hamsters: Goldspink &Ward, 1979; rats: Bedi etal. 1982).

Muscle development is biphasic (Kelly & Zacks,1969; Ontell & Dunn, 1978). Myotubes form byfusion of mononucleate myoblasts, with primarymyotubes forming first and providing a 'cellularskeleton' (Kelly, 1983) for the later development of

secondary myotubes. Secondary myotubes initiallyform near the midpoint of a primary myotube,beneath the basal lamina (Ontell & Dunn, 1978).They then grow longitudinally and eventually connectwith the muscle tendons and separate from theprimary myotube. Secondary myotube numbers aresensitive to environmental variables such as paralysisor denervation (Harris, 1981; McLennan, 1983). Pri-mary myotube formation, by contrast, has so farproved refractory to experimental manipulation(Harris, 1981; Wigmore & Stickland, 1983; Ross etal.1987 b).

In this study, we ask whether the reduction inmuscle fibre number that follows maternal under-nutrition is due solely to modulation of the number ofsecondary myotubes that form. To gain furtherunderstanding of the events involved in initiation offormation of secondary myotubes, we also assess thecapacity of muscles at different stages of developmentto 'catch up' after the undernutrition-imposed slow-ing of the rate of myotube formation. This is done byrestoring a normal diet to the mothers one day prior

816 S. J. Wilson, J. J. Ross and A. J. Harris

to birth, a time when 40 % of the secondary myotubeswould normally have been generated in soleus and20 % in lumbrical muscles. Our results enable us todefine a critical period in muscle development duringwhich secondary myotubes must be determined toform if they are ever to be present in the muscle.

Materials and methods

Adult male and virgin female white Wistar rats (200-250 g)were mated in wire-bottomed cages. The day a copulationplug was found was designated day zero of the pregnancyand the female was caged singly from that time.

Pregnant females were allowed free food for the first 2days of gestation, and then assigned to control, under-nourishment and rehabilitation groups. Control femaleswere allowed food (standard rat pellets) ad lib throughoutgestation and lactation. Undernourished rats were kept ona restricted ration of about 30 % the control food intake.Rats in the rehabilitation group were kept on this regimeuntil day 21 of gestation (E21), one day before parturition,from which time they were allowed food ad lib. All animalshad free access to water. Body weights of mothers andyoung were recorded each morning. Litter sizes werestandardized to eight pups at birth.

Control animals were taken at E17 to count soleus muscleprimary myotubes, at E20 and at postnatal days 7 (PN7)and 28 (PN28). Undernourished animals were examined atE20 and PN7, and rehabilitated animals at PN28. To obtainthe fetuses, the mothers were heavily anaesthetized withether and fetuses from near the bottom of the uterine hornsremoved, placed on ice to maintain anaesthesia and per-fused through the heart with warmed fixative. The fixativecontained 1% paraformaldehyde, 1-25% glutaraldehydeand 0-5mM-CaCl2 in 0-14M-Hepes, with SOi.u.ml"

1

heparin. Two embryos, of undetermined sex, were nor-mally taken from each litter. Postnatal tissue was takenfrom male rats anaesthetized with ether and perfusedthrough the heart with fixative containing 1 % paraformal-dehyde, 1 % glutaraldehyde, 0-4mM-CaCl2, 0-03M-glucosein 0-lM-phosphate buffer with 50i.u. ml"1 heparin. Up tothree siblings were taken from each litter.

Following perfusion, the soleus and FVth lumbricalmuscles and the L4 ventral roots were dissected andimmersed in fixative for a total of 4h. Tissues from bothsides of the animal were normally retained. The tissueswere postfixed in osmium tetroxide and stained en bloc inuranyl acetate, dehydrated and embedded in TAAB epoxyresin. Ultrathin transverse sections (~90nm) were cutthrough E20 soleus and lumbrical and PN7 lumbricalmuscles at the midbelly endplate-containing region andthrough the ventral nerve roots just proximal to the dorsalroot ganglion. The sections were mounted on single-slotFormvar-coated copper grids, stained with uranyl acetateand lead citrate and viewed and photographed with aPhilips 410 electron microscope. Semithin sections (1 ism)from PN7 soleus and PN28 lumbrical and soleus muscleswere mounted on glass slides, stained with methylene blueand azur II, and viewed and photographed with light

microscopy. Photomontages of the entire muscle and nervesections were produced, and all of the fibres counted.

Results

Undernutrition

Body weights

At birth, the undernourished pups had a 42 % deficitin body weight compared to controls. Continuedundernutrition of the mother to PN7 resulted in a61 % deficit in the body weights of the pups comparedto age-matched controls.

Muscle fibre numbers

Soleus muscles from control animals were examinedat E17 to count primary myotubes. There were84 ± 1-7 (n = 4) primary myotubes in this muscle. AtE20 we did not attempt to distinguish primary andsecondary myotubes. At this time, there were914 ±38 (n = 4) muscle fibres in the control soleusand 649 ±25 (n = 7) muscle fibres in the under-nourished soleus muscles (Fig. 1). This is a 29%reduction in soleus muscle fibre number as a result ofprenatal undernutrition.

By E20, the IVth lumbrical muscle normally has itsfull complement of primary myotubes and has beengenerating secondary myotubes for approximately1 day (Ross et al. 1987a), and these two populationsare readily distinguishable with electron microscopy(Fig. 2A). Primary myotubes are large cells contain-ing abundant well-organized myofilaments. Second-ary myotubes are small myofilament-containing cells

3000

2000

1000

10 20 30Gestational age

40 50

Fig. 1. Muscle fibre numbers in soleus muscles fromcontrol ( • ) , underfed (^) and rehabilitated (O) rats.Mothers of undernourished animals were on a restricteddiet from the 3rd day of gestation (E2) continuingthrough lactation; mothers of rehabilitated animals wererestored to a normal diet on E21. Parturition occurred onE22. Error bars, ±S.E.M.

Critical period in skeletal muscle development 817

Fig. 2. Electron micrographs of E20 lumbrical muscles from control and undernourished rat fetuses. (A) Controlmuscle. Clusters of myotubes including a primary myotube (7°), one or two secondary myotubes (2°), andmononucleate cells (mn) are enclosed within a basal lamina. (B) Undernourished muscle. Primary myotubespredominate with only an occasional secondary myotube present. Calibration bars, 2/im.

818 S. J. Wilson, J. J. Ross and A. J. Harris

100-

£ 80-

er o

f m

yotu

l2

| 40-

20-

(i-

rT.(>

\Control Underfed

Treatment

Fig. 3. Histogram to illustrate counts of primary (1°) andsecondary (2°) myotubes, and mononucleate cells(presumed myoblasts) (mn) in E20 control andundernourished lumbrical muscles. Error bars, ±S.E.M.

lying under the same basal lamina and sometimesinterdigitating with primary myotubes. The numbersof primary myotubes were the same in under-nourished (110 ±2 , /i = 7) and control (109 ± 2,n = l) lumbrical muscles (P>0-7). There were,however, very few secondary myotubes in the under-nourished (13 ± 1) as compared to the control lumbri-cals (78 ± 5) (P < 0-001) (Figs 2B, 3), so that the totalnumber of muscle fibres was 34 % less than controls(Fig. 4).

The undernourished E20 lumbrical muscles wereless mature than the controls. In the controls, primarymyotubes were separate and most had one or moreassociated secondary myotubes, in varying stages ofmaturation (Fig. 2A). In the undernourishedmuscles, by contrast, the primary myotubes often hadnot separated and remained in groups of two oroccasionally three, and were sometimes linked by gapjunctions. Secondary myotubes, in addition to beingreduced in number, were usually small and closelyinterdigitated with the primary myotube. There wasrarely more than one secondary myotube associatedwith each primary myotube (Fig. 2B). This level ofmaturity is similar to that of normal lumbrical muscleat E19 (Ross et al. 1987a).

Mononucleate cells lying within the basal lamina,which may include presumptive fibroblasts as well asmyoblasts, were counted in the single midbelly cross-sections of soleus and lumbrical muscles at E20.There was no difference between the number of thesecells in the undernourished and control lumbricalmuscles (Fig. 3). In soleus muscles, there was a 27 %

1000

5 800

I 600-

400-

B3 200

10 20 30Gestational age

40 50

Fig. 4. Muscle fibre numbers in lumbrical muscles fromcontrol ( • ) , underfed ( • ) and rehabilitated « » rats.Mothers of undernourished animals were on a restricteddiet from the 3rd day of gestation (E2) continuingthrough lactation; mothers of rehabilitated animals wererestored to a normal diet on E21. Parturition occurred onE22. Error bars, ±S.E.M.

diminution in mononucleate cell number in themuscles from undernourished animals, but this wasbarely statistically significant (0-05

Critical period in skeletal muscle development 819

this animal were not different from others in theundernourished group.

At PN7, both myelinated and unmyelinated axonswere counted. The number of axons in under-nourished animals (myelinated: 1192 ± 60, unmyelin-ated: 718 ±40, « = 5) was not different (/>>0-25)from that in control animals (myelinated: 1160 ± 13,unmyelinated: 810 ± 28, n = 6).

Nutritional rehabilitationBody weights

When rats were undernourished during pregnancybut given free access to food from E21 their younghad birth weights 31 % less than controls (19 %greater than the continually undernourished group).This deficit persisted through lactation.

Muscle fibre numbersMuscles from animals in the rehabilitation groupwere examined at PN28. Soleus muscles had the samenumber of fibres as in muscles from underfed animalsexamined at PN7, significantly less than controls(Fig. 1). Thus, although rehabilitation was effectivein increasing body weight, it did not increase thenumber of soleus muscle fibres able to be generated.

In PN28 lumbrical muscles, however, the numberof muscle fibres in muscles from rehabilitated ani-mals, while still less than control (863 ±11, n = 8versus 979 ±23, n = 6: P< 0-001), was significantlygreater than in the undernourished animals at PN7(P< 0-001) (Fig. 4). Since the normal number ofprimary myotubes had already been generated at E20in both control and undernourished lumbricalmuscles, these extra fibres must have all been ofsecondary myotube origin. During the period E20 toPN28, the rehabilitated animals generated 740 sec-ondary myotubes and the controls 792. Thus thenumber of secondary myotubes generated during thisperiod was not substantially less in rehabilitated thanin control animals, but the initial fibre deficit was notmade up.

Ventral root axonsAt PN28 the control L4 ventral roots contained1950 ±112 myelinated axons (n = 6) and ventral rootsfrom rehabilitated animals contained 1778 ± 78 axons(n = 10). These numbers are not significantly differ-ent (F>0-2). We also assayed subclasses of axonaccording to fibre size and found no significantdifferences.

Discussion

Our results show that production of secondary myo-tubes, but not of primary myotubes, is sensitive to

prenatal undernutrition. In addition, an experimentwith nutritional rehabilitation revealed that second-ary myotube numbers could be recovered, but successin this manoeuvre depended on the stage of develop-ment of the muscle. From these results, we suggestthat there is a critical period in development duringwhich the maximum number of adult muscle fibres isdetermined.

These results confirm and extend previous obser-vations. Although there have been many studies ofthe effects of nutritional deprivation on musclegrowth and muscle fibre number, few of these haveinvolved undernourishment during the period ofmuscle fibre generation. We severely undernourishedpregnant rats (30 % of normal food intake) from day2 of gestation onwards, and examined their offspring2 days before and one week after birth. The muscleswe studied, the soleus and IVth lumbrical, had19-34 % deficits in fibre number, as determined bycounting every fibre in the muscle cross-section. Thisdeficit is comparable to that found by Bedi et al.(1982) who counted soleus and extensor digitorumlongus muscle fibres (19% and 21% reductions,respectively) in the adult offspring of does under-nourished during gestation and lactation.

By examining lumbrical muscles on E20, we showthat only secondary myotube generation is affectedby prenatal undernutrition. This differential responsewas already suggested by Wigmore & Stickland(1983) who in a semiquantitative light microscopestudy of fetal pigs found that semitendinosus musclesin the smallest animals had fewer secondary myo-tubes but the same number of primary myotubes asthose in their larger littermates. Fetal growth retar-dation is probably a consequence of prenatal malnu-trition due to the position of the animal within theuterine horn (McLaren, 1965).

A critical period for secondary myotube formationOur most interesting finding is that there is a criticalperiod for initiating formation of secondary myotubesand that this period may precede the actual gener-ation of the myotube. Although only about 40 % ofsoleus muscle secondary myotubes would normallyhave formed by E21 (calculated by extrapolation),refeeding undernourished animals from that time didnot correct the depression in their capacity to gener-ate secondary myotubes. The lumbrical muscle isdevelopmentally about 1 day behind the soleusmuscle and only 19% of the secondary myotubeshave developed by E21 (Ross et al. 1987a). Refeedingafter previous undernutrition restored almost the fullmyogenic capacity of this muscle, but there was nocompensation for myotubes not formed prior torefeeding.

820 S. J. Wibon, J. J. Ross and A. J. Harris

Role of innervation in regulating muscle fibrenumbersEvidence from experiments on frogs (Ferns & Lamb,1987) and rats (Ross et al. 1987£>) shows a stoichio-metric relation between muscle innervation and sec-ondary myotube generation. Accordingly, it wasimportant to ask whether undernutrition affectedmuscle innervation. We found no evidence for excessmotoneurone death in undernourished animals. Wecannot, however, comment on whether under-nourished motoneurones formed fewer than theirnormal number of terminals, which in normal devel-opment form in advance of the muscle fibres they willeventually innervate (Harris & McCaig, 1984).

Mechanism of the effect of undernutritionFairly severe undernutrition was required before wesaw any effect on muscle fibre numbers. In a prelimi-nary experiment, holding animals at constant weightduring pregnancy had no effect on muscle fibrenumbers in their offspring. Beerman (1983) found areduction to 50 % of normal food intake from the 8thday of pregnancy onwards in rats did not affectmuscle fibre number in the offspring, although it hada long-lasting effect on muscle growth.

We also did not see the marked reduction inmononucleate cell numbers in the midbelly region ofthe muscle that accompanies the halt in secondarymyotube production produced by fetal denervation(Ross et al. 19876). The reduction we saw after fetalmalnutrition was such that the ratio of mononucleatecells to total fibre number remained constant. Thisimplies that the rates of myoblast proliferation andfusion are both reduced in the muscles of the under-nourished animals, even if the number of myoblastsfusing to form each myotube remained constant(Penney et al. 1983; McLennan, 1987).

As the number of secondary myotubes that form ina muscle is well regulated (Ross et al. 1987a) and verymuch less than the number of fusion-competent cellsthat are present throughout development, it appearsthat secondary myotube formation is initiated by aspecial cell fusion event associated with the presenceof a nerve terminal (Ross et al. 1987i»). Once thenascent secondary myotube exists, it can grow byaccepting fusion from myoblasts that come in contactwith it. It is likely that the effect of undernutrition is,directly or indirectly, to reduce the number of thesubpopulation of myogenic cells which participate inthe initiating event, thereby permanently limitingsecondary myotube numbers.

This work was supported by the New Zealand MedicalResearch Council, the Vernon Willey Trust and the Muscu-lar Dystrophy Association. We thank K. S. Bedi for adviceon the regime for undernourishment.

References

BEDI, K. S., BIRZGALIS, A. R., MAHON, M., SMART, J. L.& WAREHAM, A. C. (1982). Early life undernutrition inrats. 1. Quantitative histology of skeletal muscles fromunderfed young and refed adult animals. Br. J. Nutr.47,417-431.

BEERMAN, D. H. (1983). Effects of maternal dietaryrestriction during gestation and lactation, muscle, sexand age on various indices of skeletal muscle growth inthe rat. J. Anim. Sci. 57, 328-337.

BYRNE, I., HOOPER, J. C. & MCCARTHY, J. C. (1973).Effects of selection for body size on the weight andcellular structure of seven mouse muscles. Anim. Prod.17, 187-1%.

FERNS, M. J. & LAMB, A. H. (1987). Regulation of cellnumbers in the developing neuromuscular system inXenopus laevis. Neurosci. Letts Supplement 27, 572.

GOLDSPINK, G. & WARD, P. S. (1979). Changes in rodentmuscle fibre types during postnatal growth,undernutrition and exercise. J. Physiol. 296, 453-469.

HARRIS, A. J. (1981). Embryonic growth and innervationof rat skeletal muscles. 1. Neural regulation of musclefibre numbers. Phil. Trans. Roy. Soc. Lond. B 293,257-277.

HARRIS, A. J. & MCCAIG, C. D. (1984). Motoneurondeath and motor unit size during embryonicdevelopment in the rat. J. Neurosci. 4, 13-24.

HEGARTY, P. V. J. & ALLEN, C. E. (1978). Effect ofprenatal runting on the postnatal development ofskeletal muscles in swine and rats. J. Anim. Sci. 46,1634-1640.

KELLY, A. M. (1983). Emergence of specialization ofskeletal muscle. In Handbook of Physiology (ed. L. D.Peachey), Section 10, pp. 507-537. Baltimore: Williamsand Wilkins.

KELLY, A. M. & ZACKS, S. I. (1969). The histogenesis ofrat intercostal muscle. /. Cell Biol. 42, 135-153.

MCLAREN, A. (1965). Genetic and environmental effectson foetal and placental growth in mice. J. Reprod.Fertil. 9, 79-98.

MCLENNAN, I. S. (1983). Neural dependence andindependence of myotube production in chickenhindlimb muscles. Devi Biol. 98, 287-294.

MCLENNAN, I. S. (1987). Hormonal regulation ofmyoblast proliferation and myotube production in vivo:Influence of prostaglandins. J. exp. Zool. 241, 237-245.

MONTGOMERY, R. D. (1962). Muscle morphology ininfantile protein malnutrition. J. Clin. Path. 15,511-521.

ONTELL, M. & DUNN, R. F. (1978). Neonatal musclegrowth: a quantitative study. Am. J. Anal. 152,539-556.

PENNEY, R. K., PRENTIS, P. F., MARSHALL, P. A. &

GOLDSPINK, G. (1983). Differentiation of muscle andthe determination of ultimate tissue size. Cell TissueRes. 228, 375-388.

Ross, J. J., DUXSON, M. J. & HARRIS, A. J. (1987a).Formation of primary and secondary myotubes in ratlumbrical muscles. Development 100, 383-394.

Critical period in skeletal muscle development 821

Ross, J. J., DUXSON, M. J. & HARRIS, A. J. (19876).Neural determination of muscle fibre numbers inembryonic rat lumbrical muscles. Development 100,395-409.

STICKLAND, N. C. & HANDEL, S. E. (1986). The numbers

and types of muscle fibres in large and small breeds ofpigs. J. Anat. 147, 181-189.

STICKLAND, N. C , WIDDOWSON, E. M. & GOLDSPINK, G.

(1975). Effects of severe energy and protein

deficiencies on the fibres and nuclei in skeletal muscleof pigs. Brit. J. Nutr. 34, 421-428.

SWATLAND, H. J. & CASSENS, R. G. (1973). Inhibition ofmuscle growth in foetal sheep. /. Agric. Sci., Camb.80, 503-509.

WIGMORE, P. M. C. & STICKLAND, N. C. (1983). Muscledevelopment in large and small pig foetuses. J. Anat.137, 235-245.

{Accepted 19 January 1988)