Ultrastructural Studies of the Myotendonous Junctionof Selenium-Deficient Ducklings

P. R. Sweeny, PhD, and R. G. Brown, PhD

An ultrastructural study was made of the changes occurring within the gastrocnemius in-sertion of normal and selenium-deficient ducklings from 1 to 12 days of age. The cytologiccharacteristics of the fibroblasts, vessels, collagen, and muscle cells are described. Thoseexposed to the selenium deficiency showed major alterations of all components. The fibro-blasts showed changes ranging from collapsed cisternae and degenerating mitochondria torupture. The capillary endothelium was abnormal, as was the smooth muscle of arteriolarwalls. The collagen sizes were altered, and the muscle cell termini showed major patho-logic changes. The above alterations occurred within 4 days of exposure of the deficiency.The muscle cells of the body portion of the gastrocnemius showed no alterations untilDay 8. The observations present evidence that indicates that connective-tissue-vascularabnormalities precede myopathic changes in nutritionally induced dystrophy. The signifi-cance of these findings is discussed with respect to the etiology of nutritionally induceddystrophy. (Am J Pathol 1980, 100:481-496)

NUMEROUS PUBLICATIONS exist describing changes in musclesand nerves following exposure of animals to vitamin E and/or seleniumdeficiencies. These papers deal with biochemical, physiological, and light-microscopic changes in variety of animals; but to our knowledge, no stud-ies are available on the effects of selenium deficiency on the ultrastructureof myotendon insertions of Pekin ducklings. We have found this bird to beextremely sensitive to deficiencies of this element 1,2,3 and indeed haveshown changes in serum levels of fatty acids and ascorbic acid in sele-nium-deprived Pekin ducklings."4 As a result of these observations, de-tailed biochemical and ultrastructural studies on the connective tissuecomponents of these birds were undertaken and revealed a significant de-crease in collagen content as early as 6 days after hatching.3 This findingsuggested that connective tissue alterations leading to subsequent muscleand nerve alterations resulted from the selenium deficiency. At the time ofthe chemical studies, samples of the insertion of the gastrocnemius andtendons were collected for electron-microscopic examination.

This paper deals with the ultrastructure of the developing myo-tendinous junction from 1 to 12 days after the hatching of normal and se-

From the Department of Microbiology, College of Biological Science, University of Guelph,Guelph, Ontario, Canada, and the Department of Food Science and Nutrition. University of Mas-sachusetts, Amherst, Massachusetts.Supported by grants from the National Research Council (Canada), the Medical Research Council

of Canada, and the Ontario Department of Agriculture and Food.Accepted for publication March 18, 1980.Address reprint requests to Dr. P. R. Sweeny, Department of Microbiology, College of Biological

Science, University of Guelph, Guelph, Ontario, Canada NIG 2W1.

0002-9440/80/0806-0481$01.00 481© American Association of Pathologists

482 SWEENY AND BROWN American Journalof Pathology

lenium-deficient ducklings. Observations on changes in the collagen, fi-broblasts, and vascular bed are included, and the relevance of thesechanges will be related to the etiology of the selenium-induced nutritionaldystrophy.

Materials and MethodsMale, 1-day-old Pekin ducklings were obtained from a local hatchery and were main-

tained on control and selenium-deficient diets as described previously.34Tissue samples for this study were taken at 1, 4, 8, 12, 16, 20, and 31 days of treatment.

The gastrocnemius body and insertions were sampled. The ducklings were killed by cer-vical dislocation. Three percent glutaraldehyde in 0.15 M phosphate buffer, pH 7.2, at 4 Cwas immediately placed on the myotendon area; then appropriate samples were dissectedout, placed in fresh fixative at 4 C, cut into small pieces, and fixed for 2 hours. All sampleswere then buffer-washed and postfixed in 1% OS04 in Millonig's phosphate buffer,5 pH 7.2,at 4 C for 2 hours. Samples from all tissues were also fixed in the latter solution only. Alltissue was dehydrated in ethanol, cleared in propylene oxide, and embedded in Epon.fThin sections were cut with glass knives on Porter-Blum Ultramicrotomes I and II,mounted unsupported on copper grids,and poststained with 2% aqueous uranyl acetatefollowed by lead. Samples were examined in a Philips 300 electron microscope.

Results

Untreated Animals

Examination of the myotendinous junction from normal ducklings 1-30days of age showed the characteristic morphologic features described inother publications. This included increasing numbers and prominence ofsarcolemmal invaginations with collagen fibril insertions at the associatedbasal lamina. Immediately subjacent to these invaginations at 1 day of ageis an area of loose connective tissue consisting of random collagen, fibro-blasts, and capillaries. The fibroblasts of this area throughout the periodstudied showed prominent rough endoplasmic reticulum, Golgi com-plexes, and mitochondria (Figure 1). All capillaries appeared normal, andtheir endothelia contained few organelles, some mitochondria, and promi-nent diacytotic vesicles. The major change that occurred during the pe-riod studied was an alteration of collagen fibril diameter. At hatching,85% were 30-50 nm in diameter, whereas at Day 12, 95% were between40 and 70 nm (Text-Figure 1). Also of note is the size distribution, whichchanges from 10 to 50 nm at 1 day of age to a range of 30-100 nm at 12days of age. The density of fibrils appears to increase throughout this timeinterval, but due to the random orientation within this zone, calculationof fibril numbers per square micron could not readily be determined. At30 days of age the loose connective tissue was more compact and resem-bled dense, irregular connective tissue.

Vol. 100, No. 2 SELENIUM-DEFICIENT DUCKLINGS 483August 1980

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Deficient Animals

The selenium deficiency led to major alterations in the myotendinousarea as early as 4 days postinitiation of the diet. The alterations includedmodified fibroblasts, collagen, and vessels, as well as changes in the musclecells themselves.The fibroblasts showed varying degrees of abnormalities, including con-

densed endoplasmic reticulum, which was manifested as collapsed or elec-tron-dense cisternae. Also evident were other major alterations, includingcells whose membranes, mitochondria, and cytoplasm all appeared dis-rupted (Figures 2 and 3). In addition to alterations of the fibroblast popu-lation, collagen fibrils appeared to be absent from the sarcolemmal in-vaginations and were replaced by very fine fibrillar or amorphousmaterial. Indeed, in some areas, no direct continuity is apparent betweenthe basal lamina and the collagen of the insertion area or of the tendon(Figure 3). The capillaries and arterioles contain empty, dilated, endo-thelial cells with few organelles and an apparent lack of diacytotic vesi-cles. These alterations are most evident in the capillaries (Figures 2 and4). Other alterations included dilated endoplasmic reticulum and swollenmitochondria of the smooth muscle cells, and vessels were often seen withaltered basal laminas (Figure 4).

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484 SWEENY AND BROWN American Journalof Pathology

The skeletal muscle cells showed structural alterations at the insertion,principally in the mitochondria, which contained dilated cristae, and thesarcomeres, which were disjointed. The terminal projections of the musclecell often contained no sarcomeres (Figures 3 and 5).

In addition to the major ultrastructural changes, which have been de-scribed, it should be emphasized that perfectly normal insertion areaswere found throughout the muscle at Days 4-12 in the selenium-deficientanimals, indicating that one is dealing with a focal lesion, and that it prog-resses in severity with age. Even at 30 days, normal insertion areas can beseen despite the presence of severe disruptions of many myotendinousareas.

At 8 days, further evidence of myotendinous junctional alterations wasevident, because abnormalities were more frequently seen. The musclecells show more cytologic alterations such as enlarged mitochondria, di-lated sarcoplasmic reticulum, and glycogen accumulation. The collagenfibril arrangement also appears abnormal: Insertion points at the basal la-mina are often nonexistent, and often large fibril-free areas exist betweenthe muscle cell and the tendon.

Further deterioration occurs throughout the progression of the disease,and at 12 days, complete disorganization was evident in certain areas.Commonly observed at this time were muscle cell terminations with obvi-ous complete degeneration of the sarcomeres (Figure 6), swollen electron-lucent mitochondria, and random disorganization of the sarcoplasmic re-ticulum. In the area of the loose connective tissue, areas without collagenfibrils were often seen, and it was evident that the fibroblasts were degen-erating (Figures 6 and 7).The muscle cell itself contains degenerating mitochondria and dilated

terminals with scanty cytoplasm. Also evident in this photograph is a ten-don-associated fibroblast whose mitochondria, endoplasmic reticulum,and Golgi Complex are abnormal and the whole cell and its nucleus arehypertrophic and contain prominent electron-dense granules.

Modifications of the muscle terminations that were bulbous in naturewere frequently seen and these contained numerous normal mitochondriaand some cytoplasmic ribosomes but no contractile elements or sarcoplas-mic reticulum (Figure 8). The fibroblasts and extracellular material ofthese areas appeared normal.Of particular interest through Days 1-12 were the alterations observed

in the size and distribution of collagen fibrils (Text-Figure 1). It is evidentthat control animals showed a progressive increase in fibril diameter fromDay 1 to Day 12, whereas the selenium-deficient group showed a rela-tively constant fibril diameter distribution throughout this time period.

Vol. 1 00, No. 2 SELENIUM-DEFICIENT DUCKLINGS 485August 1980

No description will be given of later stages, because it was apparent atolder ages that degeneration was rampant and no significant alterationsrelevant to a sudy of the pathogenesis of the disease could be seen at thistime.

Discussion

Investigators of nutritionally induced and genetic muscular dystrophieshave concerned themselves primarily with changes in muscle and nerve inattempting to elucidate the pathogenesis of the disease, but recent evi-dence suggests that connective tissues may play a significant part in thesedisorders.37-" Cassato 12 has summarized all the literature relevant to ge-netic dystrophies and suggests that ischemia resulting from connective tis-sue abnormalities can account for the pathogenesis of this condition.

In the nutritional dystrophies, early alterations of the vascular bed havebeen reported.13` 5Workers studying pigs, chickens, and lambs deficient invitamin E or selenium have also reported altered collagen and vesselsearly in the progression of these disorders,7'8 and x-ray diffraction studieshave shown failure to organize in the collagen of tendons.9 It has also beenshown that the addition of vitamin C to selenium-deficient ducklings sig-nificantly prolongs the life of these animals.2 A study by Brown et al ' hasshown that collagen synthesis is significantly altered in the pectoralis andtendon as early as 6 days in ducklings on a selenium-deficient diet. Therewere also alterations in tendon collagen fibril diameters and tenoblasts asearly as 8 days.'The difficulties with assessing the etiologic factors of nutritional muscu-

lar dystrophies are twofold. If one is studying pigs, lambs, or chickens, theearly lesions are focal in nature, and due to restrictions on sampling, onemay miss the affected areas. In addition, one is dealing with a disorderthat occurs during early growth. This means one is confronted with a de-velopmental problem, superimposed on which are the physiological fac-tors of use, which are known to exacerbate the condition. The ducklingappears to be an ideal experimental animal because of its rapid growthand the extensive involvement of muscles in the selenium-induced dys-trophy and therefore a resultant increase in the probability of obtainingaltered areas. Isometric fixation was not utilized in this investigation be-cause preliminary studies had suggested altered insertions, and it wastherefore assumed that any undue stress on this area could conceiveablyalter the ultrastructural characteristics of the myotendonous junction.Consequently, fixative was placed on the insertion area in situ prior to dis-section in an attempt to obtain as near a natural state in this area as pos-sible.

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486 SWEENY AND BROWN American Journalof Pathology

The observations presented in this paper reveal that major alterationsoccur in the ultrastructure of the myotendon junction as early as 4 days.The vascular changes are similar to those described for pigs and chickenson diets deficient in vitamin E and selenium.78 In addition, alterations oc-cur in collagen fibril sizes such that the selenium-deficient animals show asmaller average fibril diameter at 4 days, and this alteration becomes ex-aggerated by Day 12. Similar findings have been reported for tendon byBrown et al.' It should be noted that at 4 days the body of the gastroc-nemius showed no alterations of muscle, nerve, or connective tissue, al-though changes in this area were observed at 8 days.

There is no doubt that at 4 days the connective tissue of the insertionareas of the muscle show focal degeneration, and indeed the muscle ter-mini in these areas are markedly abnormal. The obvious inference is thatmuscle degeneration progresses from the insertion areas into the body ofthe muscle. The bulbous outgrowths occurring at 8-12 days are indicativeof attempts at regeneration, although they could represent the normalgrowth of unaffected fibers, since they were occasionally seen in the con-trol samples of the same ages.

Despite the widespread cellular degeneration (vascular, fibroblast, andmuscle) occurring within this zone by Day 4, no evidence of the charac-teristics of inflammation was evident. Leukocytes and macrophages werenoticeably absent, and only an occasional mast cell was observed. At 16 to30 days, all of the above cells were found throughout the area. These ob-servations suggest one is dealing with an in situ degeneration, perhaps re-lated to anoxia through failure of differentiation of the vascular supply tothis area and/or the connective tissue compartment.The obvious variation that occurs in collagen fibril size and therefore

maturation in the tendon versus the insertion deserves comment. Theformer at hatching has the greatest percentage of fibrils in the 40-60-nmrange, increasing at 12 days of age to 40-140 nm, with the largest being180 nm.3 The insertion, however, has a large percentage of fibrils in the35-45-nm range at hatching, increasing to only 45-55 nm at Day 12, withthe largest being 85-95 nm.The tendons of selenium-deficient and control birds did not show a dif-

ference in fibril size at Days 4 and 8, but by day 12 the selenium-deficientcollagen fibrils were significantly smaller. The insertion, however, differsin that the collagen fibrils in the selenium-deficient birds were smaller atDay 4 than those of the controls. This difference is not apparent at Day 8,but at Day 12 the deficient birds again contained smaller fibrils than thecontrols. Such a change in the insertion area may reflect an attemptedcompensation by the developing bird that cannot be maintained. The ob-

Vol. 1 00, No. 2 SELENIUM-DEFICIENT DUCKLINGS 487August 1980

servations also indicate that the state of differentiation varies betweentendon and insertion. It is possible that the insertion area, being in a moredynamic state of its differentiation at this time, may be more susceptibleto the selenium deficiency.The possible role of collagen in developing systems, particularly

muscle, is becoming well documented.'6-20 Indeed, the findings of Duanceet al 18 and Bailey and Sims 2 that Type III and Type IV collagen is foundprimarily in tendon, in the epimysium, and in small quantities in the peri-mysium are of the interest because it is in these areas that major altera-tions seem to occur first. Therefore, it is probable that selenium deficiencymay alter the developmental factors involved in the production of thesetypes of collagen.The above data strongly support the earlier suggestions by us 1'4' that

in nutritionally induced muscular dystrophies the primary lesion is an al-teration of connective tissue development at the insertion and elsewhere.This alteration would lead, ultimately, to failure of insertions (functionaltentomy) and failure of the development of the vascular bed (ischemia),and both would account for subsequent failure of normal muscle andnerve development or degeneration of existing muscle or neuromuscularjunctions.

References

1. Brown RG, Sweeny PR, George JC, Stanley DW, Moran ET: Selenium deficiencyin the duck: Serum ascorbic acid levels in developing muscular dystrophy. Poult Sci1974, 53:1236-1239

2. Moran ET Jr, Carlson HC, Brown RG, Sweeny PR, George JC, StanelyDW: Alleviating mortality associated with a vitamin E-selenium deficiency bydietary ascorbic acid. Poult Sci 1975, 54:266-268

3. Brown RG, Sweeny PR, Moran ET Jr: Collagen levels in tissues from seleniumdeficient ducks. J Nutr (In press)

4. George JC, John TM, Moran ET Jr, Sweeny PR, Brown RG, Stanley DW: Seleniumdeficiency with the duck: Changes in plasma free fatty acid level from hatching toonset of gross pathology. Can J Zool 1973, 51:383-386

5. Millonig G: Advantages of a phosphate buffer for OSO4 solutions in fixation. J ApplPhysiol 1961, 32:1637,

6. Luft JH: Improvements in epoxy resin embedding methods. J Biophys BiochemCytol 1961, 9:409-414

7. Sweeny PR, Buchanan-Smith JG, deMille F, Pettit JR, Moran ETJr.: Ultrastructure of muscular dystrophy: II. A comparative study in lambs andchickens. Am J Pathol 1972, 68:493-510

8. Sweeny PR, Brown RG: Ultrastructural changes in muscular dystrophy: I. Cardiactissue of piglets deprived of vitamin E and selenium. Am J Pathol 1972, 68:479-492

9. Bartlett MW, Egelstaff PA, Holden TM, Stinson RH, Sweeny PR: Structuralchanges in tendon collagen resulting from muscular dystrophy. Biochim BiophysActa 1973, 338:213-220

10. Bourne GH, Golarz MN: Human muscular dystrophy as an aberration of the con-nective tissue. Nature 1959, 183:1741-1743

488 SWEENY AND BROWN American Journalof Pathology

11. Brown RG, Button GM, Smith JT: Effect of vitamin E deficiency on collagen me-tabolism in the rat's skin. J Nutr 1967, 91:99-106

12. Cazzato G: Considerations about a possible role played by connective tissue prolif-eration and vascular disturbances in the pathogenesis of progressive muscular dys-trophy. Europ Neurol 1968, 1:158-179

13. Cheville NF: The pathology of vitamin E deficiency in the chick. Pathol Vet 1966,3:208-225

14. Carlson CW: Selenium vitamin E interrelationships in poultry, nutrition. Thirty-first Annual Minnesota Nutritional Conference, 1970, pp 21-28

15. Pearce GW: Electron microscopy on the study of muscular dystrophy, MuscularDystrophy in Man and Animals. Edited by GH Bourne, M Golarz New York, HafnerPublishers, 1963, pp 160-191

16. Hauschka SD: Clonal aspects of muscle development and the stability of the differ-entiated state, The Stability of the Differentiated State. Edited by H. Ursprung NewYork, Springer Verlag, 1968, pp 37-57

17. Lipton BH: Collagen synthesis by normal and bromodesoxyuridine-modulated cellsin myogenic culture. Dev Biol 1977, 61:153-165

18. Duance VC, Restall DJ, Beard H, Boume FJ, Bailey HJ: The location of three col-lagen types in skeletal muscle. FEBS (Letters) 1977, 79:248-252

19. Mayne R, Strahs KR: Collagen synthesis by cultures of chick myotubes depleted ofmononucleated cells. J Cell Biol 1974, 63:212a

20. Ketley JN, Orkin RW, Martin GR: Collagen in developing chick muscle in vivoand in vitro. Exp Cell Res 1976, 99:261-268

21. Bailey AJ, Sims TJ: Meat tenderness: Distribution of molecular species of collagenin bovine muscle. J Sci Food Agric 1977, 28:565-570

AcknowledgmentsWe would like to thank Barbara Atkinson, Carol Skene, and Michelle Azan for their technical as-

sistance.

American Journalof Pathology

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[Illustrationsfollow]

489

Figure 1-Electron micrograph of a 4-day-old, normal myotendinous area. The fibroblast in the lowerportion shows Golgi apparatus (G), rough endoplasmic reticulum (R), and mitochondria (M). At thejunctional area, numerous collagenous fibrils can be seen within the saroplasmic invaginations andnear the basal lamina (arrows). (Uranyl acetate and lead, x27,750)

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Figure 2-Electron micrograph of a 4-day myotendinous junction of a selenium-deficient duckling. Notethe abnormal mitochondria (M) of muscle, complete disorganization of the whole junctional area, andfibroblasts (F). Collagen fibrils are not seen at the sarcoplasmic invaginations, and only occasional fi-ber bundles are evident (C). A vessel (V) is evident with an endothelial cell showing pale cytoplasm andpale mitochondria. (Uranyl acetate and lead, x 14,450)

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Figure 3-Electron micrograph of a 4-day selenium-deficient myotendinous junction. Complete dis-organization is evident (S) as are disarrayed collagen (C) and altered fibroblasts. Areas of complete dis-continuity (DC) were often seen. Tendon (T) is visible at the right of the photo. (Uranyl acetate and lead,x 1 2,300)

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Figure 4-Electron micrograph of a 4-day-old, selenium-deficient, small vessel. Note the degeneratingendothelial cell (E) and the peripheral smooth muscle cell with dilated rough endoplasmic reticulum(Er) with electron-dense contents. The basal lamina is discontinuous (arrows), and the extravasculararea contains scattered vesicular elements. (Uranyl acetate and lead, x33,750) Figure 5-Elec-tron micrograph of a 4-day deficient insertion, showing dilation of the muscle terminus. No sarcomeresare evident in this area, and the mitochondria (M) are large; some show dilated cristae (Md). The extra-muscular areas show disorganized collagen (C) and fibroblasts (F). (Uranyl acetate and lead,x 1 2,300)

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Figure 6-Electron micrograph of a 12-day selenium-deficient junction. Note the degenerate fibro-blasts with swollen mitochondria (M) and relatively clear extracellular space (Es). Of particular inter-est also are the prominent degenerative aspects of the muscle cell, ie, degenerate mitochondria (M),terminal disorientation of sarcomeres, and zebralike structure of the sarcomeres (Z). (Uranyl acetateand lead, x 1 2,300)

rigure /-Electron micrograph of a 12-day insertion area. Degenerate mitochondria (M) are evidentthroughout muscle and fibroblasts and at the bottom a hypertrophic cell and nucleus, probably of a te-noblast, is evident. (Uranyl acetate and lead, x 13,600)

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Figure 8-Electron micrograph of a 12-day selenium-deficient insertion. Three bulbous terminations ofthe muscle are evident (B), with normal mitochondria (M). Fibroblasts (F) are also evident and appearreasonably normal. Collagen fibrils (c) are also evident thoughout, though not prominent. Note also thecollapsed capillary (ca). (Uranyl acetate and lead, x9770)