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Comparative ultrastructure of chick fibroblasts in vitro at early and late stages during their growth span
© 1971 by Academic Press, Inc.
J. ULTRASTRUCTURERESEARCH36, 291--311 (1971)
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Comparative U ltrastructure of Chick Fibroblasts in Vitro at Early and Late Stages during Their Growth Span M. A. BROCK and R. J. HAY
Laboratory of Cellular and Comparative Physiology, Gerontology Research Center, National Institute of Child Health and Human Development, PHS, U.S. Department of Health, Education, and Welfare, Bethesda, and the Baltimore City Hospitals, Baltimore, Maryland 21224, and Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210 Received April 24, 1970, and in revised form December 28, 1970 Chick fibroblasts were cultured through 4.5 generations (early passage) and 18 generations (late passage). The ultrastructure of early passage, rapidly dividing cells was compared with that of late passage cells showing a reduced proliferation rate and degeneration. In late passage cells, the marked lobation of the nucleus, the absence of chromatin adjacent to the nuclear envelope and ellipsoidal mitochondria, generally bent into a shallow " U " with longitudinally oriented cristae, contrasted with the nearly oval nuclei containing peripheral chromatin and oval mitochondria with transversely oriented cristae seen in early passage cells. Highly developed RER and Golgi complexes were observed at both passage levels. The most striking change was the presence of conspicuous secondary lysosomes and residual bodies in over 90 % of the late passge cells. The possible relation of these results to altered metabolic activities in an in vitro environment and to in viva aging is discussed. The possibility that ultrastructural changes may be correlated with the aging of diploid cells in culture has received little attention. It is clear that primary cell strains isolated from chick or human tissues have a limited growth potential in vitro. This is reflected either in a finite number of potential doublings or in a limited time of survival (I8, 22, 23, 41). The observations of cellular mortality in vitro have led to the hypothesis that cultured diploid cells undergo senescent changes analogous to those which occur during cellular aging in viva (23). Changes in the shape of chick and human cells, with continued subcultivation, have been noted at the light microscope level. Harris (18) reported that fibroblasts from embryonic chick muscle gradually lost the uniform appearance of primary cultures and became heterogeneous. An increase in the number of cells with very large, irregular nuclei was also noted. These observations were confirmed by H a y and 19 -
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Strehler (21), w h o further stated t h a t the changes were a c c o m p a n i e d b y a decrease in the t e n d e n c y to f o r m multilayers. H a y f l i c k a n d M o o r h e a d (23) r e p o r t e d that late p a s sage, degenerating fibroblasts f r o m h u m a n e m b r y o s were m u c h less p o l a r i z e d a n d m o r e s p r e a d out t h a n their early passage, r a p i d l y dividing c o u n t e r p a r t s . Simons (40) m e a s u r e d the diameters of dissociated cells f r o m strains of h u m a n fibroblasts isolated originally f r o m a d u l t skin. Late passage cells were m u c h less u n i f o r m in d i a m e t e r t h a n actively proliferating cells. M e t a b o l i c a n d b i o c h e m i c a l studies of early a n d late passage cells have i n d i c a t e d t h a t R N A , lipid a n d l y s o s o m a l e n z y m a t i c activity increase with age (7-9, 21). Nevertheless, the basic m e c h a n i s m s responsible for b o t h the m o r p h o l o g i c a l changes a n d for the finite life span of cells in vitro r e m a i n obscure. Because the age-associated m o r p h o l o g i c a l changes o b s e r v e d in light m i c r o g r a p h s a p p e a r to be so m a r k e d , it seems plausible to expect that cell organelles m a y d i s p l a y u l t r a s t r u c t u r a l changes with time in culture a n d that these changes c o u l d be related to the observed cellular mortality. Therefore, in this study, the fine structure of early passage chick fibroblasts is c o m p a r e d with t h a t of late passage cells.
MATERIALS AND METHODS
Tissure culture. Chick cell strains were isolated and subcultivated as described previously (21). Eagle's minimum essential medium supplemented with 10 % calf serum and 5 % embryo extract was used throughout. Cells for early passage material had been passed once in vitro and were inoculated in 7.5 ml of growth medium to 60 mm glass petri plates at 106 cells per plate. Late passage cultures were derived from cells which had been stored in liquid nitrogen after completing fifteen generations and seven passages in vitro. These were reconstituted and were subcultivated twice before being used as source material. It has been shown for both chick (21) and human (23) cell strains that this storage procedure has no detectable effect on growth span. Therefore, no lasting structural change would b e expected following resumption of normal growth. Late passage cells were inoculated to glass petri plates at the same time and in the same manner as described for early passage cells. After 4 days incubation, the fibroblasts had passed through 4.5 generations (early passage cells) and 18 generations (late passage cells). Those having completed 18 generations were considered senescent since the maximum number of doublings is about 25 for this strain (21). The following abbreviations will be used to indicate the type of preliminary fixation of material illustrated in each figure. GCS: 3 % glutaraldehyde in 0.075 M cacodylate buffer made 0.33 M in sucrose. GPS: 3 % glutaraldehyde in 0.06 M phosphate buffer made 0.14 M in sucrose. F1o. 1. Portions of three early passage chick fibroblasts show the cellular structure usually seen. Blocks of chromatin are both at the periphery and scattered throughout the karyoplasm of the two nuclei. A nuclear pore (P) is sectioned transversely in one nucleus that also shows a prominent nucleolus. Microtubules (MT) are seen in the cellular cortex. Distended cisternae of the rough endoplasmic reticulum (RER), free ribosomes, and abundant Golgi complexes (G) fill the cytoplasm. Two mitochondria are seen in near-longitudinal and -cross sections. A centriole (C) lies near the nucleus of one cell. GPS, 4 °. x 24 000.
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Each culture was washed three times with Hanks' saline before being treated for ultrastrucrural examination. Electron microscopy. The cells were detached from the petri plates by gentle scraping with rubber policemen. The fragmented monolayers were then prepared for electron microscopy by initial fixation for (a) 2 hours in a solution containing 3 To glutaraldehyde, 0.075 M cacodylate buffer, pH 7.4 (38), 0.33 M sucrose, at either 4 ° or 25°C; (b) 2 hours in a solution containing 3 % glutaraldehyde, 0.06 M phosphate buffer, pH 7.3, 0.14 M sucrose, at either 4 ° or 25°C; or (c) 1 hour in a salution containing 1% OsO,, 0.06 M phosphate buffer, pH 7.3, 0.016 M sucrose, at either 4 ° or 25°C (29). The glutaraldehyde-fixed material, after thorough washing at 4°C in (a) 0.33 M sucrose in 0.075 M cacodylate buffer, pH 7.4, or (b) 0.16 M sucrose in 0.06 M phosphate buffer, pH 7.3, was postfixed for 1 hour at 4°C with 1% OsO4 in 0.06 M phosphate buffer, pH 7.3, containing 0.016 M sucrose (29). The ceils were washed in 0.33 M sucrose, dehydrated through a graded series of alcohols and propylene oxide and fiat-embedded in Epon 812. Thin sections placed on 300- or 400-mesh uncoated copper grids were stained with lead citrate (35) prior to observation with an RCA-EMU-3G electron microscope. At least 100 cells were examined from both early and late passage cultures. Those fixed initially in glutaraldehyde followed by osmium tetroxide yielded the best preserved material.
OBSERVATIONS
Early Passage Cells Figure 1 illustrates fibroblasts at early passage. The cell nuclei, lying in the expanded central portion of the cells, had fairly regular oval contours. Dense blocks of chromatin were scattered centrally in the karyoplasm as well as adjacent to the nuclear envelope, which was interrupted at intervals by pores. Invaginations of the cell membrane and micropinocytotic vesicles just within the plasma membrane were commonly seen (Fig. 2). The fusion of two or more micropinocytotic vesicles resulted in larger vesicles whose fate could not be followed definitely without the use of appropriate markers. However, a few images suggested that micropinocytotic vesicles fused with larger single-membrane bounded bodies whose homogeneous contents were similar in density (Fig. 3). Just adjacent to the plasma membrane, the cortical cytoplasm was devoid of cellular organelles other than free ribosomes, occasional cisternae of the rough endoplasmic reticulum (RER) and microtubules (Fig. 1). Mitochondria, free ribosomes, RER, Golgi complexes and large single-membranebounded droplets were scattered from the cortex throughout the cytoplasmic matrix (Figs. 1 and 7). In the mitochondria of early passage cells, the double limiting membranes and transversely oriented cristae were accentuated against the somewhat flocculent-appearing, clearer matrix (Figs. 1 and 6). Intramitochondrial granules were common. All the mitochondrial images seen in young cells could be accounted for by an ellipsoid mitochondrial shape with platelike cristae traversing the short axis.
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FIG. 2. Micropinocytotic vesicles, formed by invaginations of the cell membrane in this early passage cell, lie in the cortical cytoplasm where many vesicles may fuse (arrows). Free ribosomes fill most of the cytoplasm in this area along with sparse cisternae of the RER. Part of a homogeneous droplet is sectioned obliquely at A. GCS, 4 °. × 23 400. FIo. 3. Two vesicles are apparently coalescing with a large clear body (A) in this early passage cell. The contents of one vesicle (arrow) are similar in density to that of micropinocytotic vesicles (Fig. 2) and of the larger body. In contrast, the contents of the second vesicle (unmarked) are similar in density to the material in the adjacent cisternae of the RER and many Golgi vesicles (Figs. 4, 5, 7, 8). Two large droplets (D) are also seen. GPS, 25 °. × 25 400. F~G. 4. A single-membrane bounded body (A) contains in its clear matrix complete vesicles similar in appearance to the Golgi vesicles close to its limiting membrane (arrows) and associated with a nearby Golgi complex (G). GPS, 25 °. × 27 400. F~o. 5. In this early passage cell, the single-membrane bounded structure at A appears to result from the fusion of a clear body with a homogeneous droplet or possibly from the rupture of vesicles within the body (as seen in Fig. 4). GPS, 25 °. x 24 200.
T h e R E R , a l t h o u g h u s u a l l y a p p e a r i n g to be r a n d o m l y o r i e n t e d , was s o m e t i m e s a r r a n g e d in s h o r t p a r a l l e l a r r a y s in t h e s e y o u n g cells. C i s t e r n a e w e r e o f t e n d i s t e n d e d a n d filled w i t h a h o m o g e n e o u s m a t e r i a l of m e d i u m d e n s i t y (Figs. 1, 3, 6, a n d 7). T h e s a c c u l a r d i l a t a t i o n s of t h e c i s t e r n a e a n d t h e a s s o c i a t e d vesicles of t h e h i g h l y develo p e d G o l g i c o m p l e x e s w e r e also filled w i t h a h o m o g e n e o u s m a t e r i a l s i m i l a r in app e a r a n c e to t h a t in t h e c i s t e r n a e of the R E R (Figs. 7 a n d 8).
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Single-membrane-bounded droplets were conspicuous in young cells. Their contents either appeared completely homogeneous and similar in density to that within the cisternae of the R E R and Golgi complexes (Figs. 3 and 7) or contained a number of small vesicles (Figs. 9 and 10). Clusters of Golgi vesicles were seen in close proximity to the homogeneous droplets with which they apparently coalesced (Fig. 8). It is suggested that some Golgi vesicles may also be incorporated into the droplets but retain their limiting membrane for a time. These images (Figs. 6-9) further suggest a sequence of synthetic activity from the R E R through the Golgi to eventual formation of the large droplets. Vesicles similar to the Golgi vesicles apparently also coalesced with or were incorporated into large, less dense bodies bounded by a single membrane (Figs. 3 and 4). Some structures could be interpreted as either a stage following the fusion of a homogeneous droplet with a large, less dense body or a stage in the transformation of the contents of a body (Fig. 5). It would appear that in these cells the Golgi vesicles are closely asseciated with both the homogeneous droplets and the less dense bodies that probably resulted from the fusion of micropinocytotic vesicles. Approximately 15 % of the cells examined showed what may be initial stages in the reorganization and degradation of material within the droplets. In Fig. 10, three droplets are closely apposed to a very large membrane-bounded homogeneous area, and at some points there is discontinuity of the membranes separating the droplets from the larger area. The continuity between these structures, provided by the lack of limiting membranes, and the presence throughout the area of small vesicles like those in the droplet, suggests that the large area devoid of cytoplasmic components was formed through fusion of several droplets. Apparent transitional changes in the appearance of the droplet contents from flocculent materials (Figs. 10 and 11) to the inclusion of cellular components and membranous residues (Fig. 11) suggests that digestive activity has ensued and these bodies are secondary lysosomes. FIG. 6-10 illustrate portions of early passage cells.
FIG. 6. The mitochondrial shape and orientation of the cristae are seen in this near-longitudinal section (M). Distended cisternae of the RER and ribosomes fill the cytoplasm. GPS, 4°. x 23 200. FIG. 7. Several highly developed Golgi complexes are often grouped in areas where Golgi vesicles are apparently coalescing with intermediate sized droplets (arrow). The homogeneous material of medium density fills the distended cisternae of the RER, many Golgi saccules and vesicles, and intermediate-sized and large droplets. GPS, 4°. × 21 100. FIG. 8. Golgi vesicles surround intermediate-sized droplets and are apparently coalescing with them (arrows). GPS, 4°. × 26 300. FIG. 9. The homogeneous, moderately dense matrix of these droplets contains vesicles similar in appearance to Golgi vesicles. GPS, 25°. x 21 100. F~G. 10. Three droplets (A, B, C) are apparently coalescingwith a large, membrane-bounded vacuolelike area (D) that fills much of the cytoplasm. Although the limiting membranes of the droplets are partially lost where they are in contact with the large area, the droplet contents are more homogeneous and dense than the somewhat flocculent matrix of the large area. Vesicles similar to Golgi vesicles are randomly distributed in both the droplets and the large area. GPS, 4 °. x 24 500.
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Late Passage Cells The irregularly rounded shape of late passage cells as well as many of their fine structural features are shown in Fig. 12. Marked changes, independent of the fixation regimen, were visualized in certain cellular components: (a) the nucleus and mitochondria were appreciably altered; (b) most cells contained conspicuous secondary lysosomes and residual bodies; and (c) lipid droplets were sometimes present. Other cellular components appeared morphologically similar to those of early passage cells. Nucleus. In contrast to the more-or-less oval nuclei of early passage cells, the late passage nuclei always presented a lobed appearance and were often bent so that portions of the same nucleus appeared as separate entities in one plane of section (Figs. 12 and 14). Blocks of chromatin were scattered centrally in these nuclei and were rarely seen at the periphery. As in early passage cells, the nuclear envelope was interrupted at intervals by pores, and its outermost membrane was ribosome studded in part. Mitochondria. The mitochondrial profiles and the orientation of the cristae were markedly changed in late passage cells. A variety of bizarre mitochondrial shapes in addition to those that were elliptical or nearly circular were seen (Figs. 12, 13a-d, and 17). Some appeared to be contorted, passing in and out of the plane of section several times, and bent at intervals along their length (Figs. 12, 13a-c). These images seen in late passage cells could be accounted for by a narrow ellipsoid usually shaped into a shallow " U " with either or both arms of the " U " sometimes twisted or bent into another plane of section. The cristae traversed the long axis of the mitochondria. Occasionally, the cristae appeared to traverse the short axis, as was the case in early passage cells. However, this orientation could be obtained in oblique sections through the ellipsoidal-shaped mitochondria (Figs. 13 a and c). In contrast to early passage cells, the mitochondrial matrix appeared homogeneous and more condensed as judged by its density. Granules were commonly present, as in the early passage cells. Lipid droplets. About 20 % of the late passage cells contained a few lipid droplets (Figs. 12 and 18). Most were relatively inconspicuous in the heterogeneous mixture of other cellular components. RER, Golgi complexes. Interestingly, the cellular organelles implicated in synthetic activity appeared as highly developed in late passage cells as in the early passage Fie. 11. Vacuole-like areas, apparently derived from droplets that have been modified by digestion of their contents, fill a cellular process in this early passage cell. Some contain only flocculent material (A); in others membranous residues lie in the flocculent matrix (B). Occasionally, portions of cytoplasm appear to have been incorporated into these areas (C). The cytoplasmic matrix resembles other early passage cells with abundant ribosomes, and distended cisternae of the RER, more clearly seen in the cell on the left, and mitochondria. GPS, 4°. × 28 800.
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fibroblasts. Cisternae of the R E R and Golgi complexes were randomly situated interior to the cortical area (Figs. 12 and 14). The cisternae of the RER, sometimes arranged in snort parallel arrays, were often distended with a homogeneous material of medium density (Fig. 12). Material similar in appearance usually filled the saccular dilatations of the Golgi cisternae, the Golgi vesicles, and droplets of intermediate size close to the inner cisternae of the Golgi complex (Fig. 14). The presence in these cellular components of a homogeneous material similar in density to that seen in early passage ceils suggests that the synthetic activity might also result in the formation of homogeneous droplets like those of early passage cells. However, the droplets were rarely seen in late passage cells, and, when observed, their ultrastructural appearance was modified appreciably as is evident in Figs. 15-17. Secondary lysosomes: modified droplets, autolysosomes, residual bodies. It was clear that in late passage cells the contents of the few homogeneous droplets present were being degraded in a manner similar to that seen in a small fraction of the early passage cells. The contents of some droplets were only slightly modified, while others contained flocculent material, partially degraded cellular components, and both myelin figures and vesicles that were unlike cellular organelles (Figs. 15-17). Because of the heterogeneous nature of the contents of these vacuole-like areas and of the evidence of autolysis, they are interpreted to be secondary lysosomes. The periphery of some cells (Fig. 17) was packed with these vacuole-like areas or secondary lysosomes. At A in Fig. 14, the configuration of a limiting membrane suggests that small areas of cellular components may be pinched off from the cytoplasm and incorporated into these bodies in a process similar to pinocytosis. Autolysosomes that resembled those in a variety of mammalian cells were filled with cellular components, some of which were apparently being lysed. The more dense appearance of areas filled with free ribosomes and of the mitochondria in comparison to these structures lying in the cytoplasm as well as the presence of membranous reresidues, lipid like material, and structures unlike cell organelles suggest that degradation has begun (Fig. 18). Although the mechanism for the segregation of cellular components was not studied in detail, it was clear, from m a n y micrographs, that paired, smooth membranes surrounded the cellular components and could be traced from the areas of degradation into morphologically unaltered cytoplasmic areas (Figs. 19 and 20).
FIG. 12. In this rounded late passage cell, two lobes of the nucleus are sectioned so that they appear to be separate entities. The karyoplasm is devoid of condensed chromatin in this plane of section; nuclear pores are common (arrows). The highly contorted mitochondria, modified droplets (D) and lipid droplets (L) are characteristic of late passage ceils. In addition, distended cisternae of the RER, smooth ER segments, and ribosomes fill the cytoplasmic matrix except for the cortex, where only ribosomes and occasional cisternae of rough and smooth ER are seen. GCS, 25 °. × 15 900.
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The most consistent and striking change in over 90 % of the late passage cells was the presence of opaque material often heterogeneous in appearance in single membrane-bounded bodies, residual bodies (Figs. 13d, 16, 17 and 20). Some of these residual bodies were filled only with myelin figures (Fig. 21); occasionally they were massed near the periphery of the cell (Fig. 17). DISCUSSION The ultrastructural alterations in fibroblasts that have been cultured for 18 generations undoubtedly reflect marked changes in cellular metabolic activity. As indicated by the appearance of the RER and Golgi complexes, synthetic functions are apparently carried out actively in both early and late passage cells. It seems reasonable to speculate, however, that the synthetic activities of these cells change as a function of passage level. This is suggested by the conspicuous absence in late passage cells of homogeneous droplets surmised to be one end product of synthetic activity. Furthermore, the nuclear and mitochondrial changes as well as the clear evidence for extensive lysosomal activity in late passage cells indicate that cellular functions must be appreciably different. In the consideration of the ultrastructure of these cells, one must question (a) whether these morphological alterations are a consequence of the length of cellular life in vitro and could be considered analogous to age-associated cellular changes observed in vivo; (b) whether the cellular organelles reflect, by their altered morphology, compensatory changes to meet the demands for maintaining cellular homeostasis in an artificial medium; or (c) whether both processes occur. Regardless of the direction of biosynthetic events, however, there is eventual loss of replicative ability and cellular death. Increased irregularity or lobation has characterized nuclei in a variety of cells from old animals. In the dog myocardium, it was regarded as a mechanism for increasing the surface area of the nuclear membrane in an attempt to maintain an optimal nuclear cytoplasmic ratio in cells that increased in size with age (30). Such changes were also observed in anterior pituitary cells of old mice (44), astrocytes of old chinchillas (5), and Purkinje and hepatic cells of old mice (1, 2). The macronucleus of the unicellular protozoan, Tokophrya infusionum, was distinctly irregular, with many invaginations, and markedly larger in old individuals that were about twice the size of the young protozoans (36, 37). In the Purkinje cells and Tokophrya the extreme nuclear invagination and lobation were thought to be prerequisite to amitotic division, another compensatory mechanism for maintaining an optimal nucleocytoplasmic ratio. The amount of stainable chromatin is thought to be an indicator of cellular metabolic activity, those cells with abundant euchromatin or nonstained chromatin being
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FIG. 13. Some of the unusual mitochondrial shapes as well as the orientation of their cristae and the more dense matrix are seen in portions of late passage ceils. The mitochondria are bent in areas where the double limiting membranes and the cristae are sectioned obliquely (arrows), and portions of some pass out of the plane of section (crossed arrows). An extremely contorted mitochondrion lies near a residual body and an autolysosome in 13d. GPS, 4°. 13a, 13d, x 25 800; 13b, 13c. x 23 300.
m o r e active t h a n those with blocks of stainable h e t e r o c h r o m a t i n (13). A l t h o u g h there was a reduction with age in the a m o u n t of h e t e r o c h r o m a t i n adjacent to the nuclear m e m b r a n e s of r a b b i t a n d chinchilla cerebellar granule ceils viewed by C a m m e r m e y e r (5), he n o t e d that this was n o t a consistent change reported by other authors. Therefore, it may be that the p r e d o m i n a n c e of e u c h r o m a t i n in the late passage cell nuclei reflects a c o m p e n s a t o r y increase in synthetic activity. This could result f r o m a decline in the accuracy of p r o t e i n synthesis, a n hypothesis suggested by Orgel (33) to ac-
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count, at least in part, for cellular senescence in the whole animal. Furthermore, the evidence for autophagy suggests an increased synthesis of hydrolytic enzymes. H r u b a n et al. (24) have provided suggestive evidence that the induction of aberrant proteins in m a m m a l i a n cells m a y be a stimulus for autolysosome formation. The intramitochondrial structural complexity and the intracellular distribution of mitochondria have been shown to depend on the energy requirements of the cell (13). F o r example, mitochondria with well-developed cisternal networks are generally seen in cell types with a high oxidative metabolism; where lipid components serve as important substrates, the mitochondria often surround lipid droplets. The sparse evidence f r o m aging studies regarding mitochondrial structure in cells of old individuals is contradictory. In early studies of Tokophrya infusionum and mouse anterior pituitary cells, there was a reduction in the length of the cristae (37, 44). On the other hand, the internal structure of h u m a n hepatic cell mitochondria in old individuals was not appreciably different f r o m that in y o u n g humans. However, irregularly shaped, giant mitochondria were often seen in hepatic cells f r o m older people, and this size increase was thought to be a compensation for the reduced n u m b e r of mitochondria in these cells (42). In general, the present studies also show changes in the shape of mitochondria and in their cristae. Interestingly, a remarkable correlation is seen with the structural changes in mitochondria that occurred in frog proximal tubule ceils either seasonally or with starvation (25). In this study, the cristae of the mitochondria f r o m summer frogs were oriented at right angles to the long axis of the mitochondria. The modified mitochondria of starved summer frogs and winter frogs had cristae parallel to the long axis, and intermediate structures showing the characteristics of b o t h mitochondrial types were observed. In these cells, there was little or no cytochrome oxidase activity when the modified mitochondria were present. It was suggested that the structural and biochemical changes might be adaptive responses of the cells as well as indicative of altered mitochondrial metabolism, an hypothesis which could apply equally well to the early and late passage fibroblasts. That starvation results in changes in mitochondrial configuration was evident also in studies of
FIG. 14. This portion of a late passage cell is especially rich in highly developed Golgi complexes. Golgi vesicles are abundant and at some sites appear to fuse with forming droplets (arrow). Segments of the RER are filled with a homogeneous material similar in density to that in some Golgi cisternae and vesicles. A droplet (D) with unevenly stained contents lies near a large Golgi area. An area of degradation is seen at A. The karyoplasm of the lobed nucleus is devoid of condensed chromatin in this plane of section. GPS, 4 °. x 22 800. FI6. 15. An altered droplet in a late passage cell contains membranous whorls in the somewhat flocculent matrix. This droplet and part of another (arrow) are similar to those in early passage cells (Fig. 11) where autolysis has evidently ensued. GPS, 25°. x 40 500. F~G. 16. Further digestion within droplets results in the membranous residues and vesicles seen in a more heter0genous matrix. A large, amorphous, dense residual body is at the right in this portion of a late passage cell. GPS, 4 °. × 29 200.
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c a r b o n - d e p r i v e d Euglena gracilis (3, 28). A b n o r m a l l y long a n d b i z a r r e - s h a p e d m i t o c h o n d r i a were characteristic of starved animals. However, it is n o t clear f r o m the p u b l i s h e d m i c r o g r a p h s whether there were c o n c o m i t a n t changes in the o r i e n t a t i o n of the cristae. There is no r e a s o n to suspect that extensive s t a r v a t i o n occurred u n d e r the g r o w t h c o n d i t i o n s e m p l o y e d in this investigation. However, the possibility t h a t the p h e n o m e n o n of limited cell survival in vitro is a response of n o r m a l cells to general environm e n t a l deficiencies m u s t be considered. H a y (20) reviewed the evidence f r o m studies with cells of various species indicating t h a t changes in the culture conditions affect the g r o w t h span very dramatically. A d d i t i o n of serum a l b u m i n to the culture m e d i u m greatly extends the g r o w t h span of h u m a n fibroblasts (43). Similar increases in the longevity of chick cell strains were n o t e d when the g r o w t h m e d i u m was m o d i f i e d by r e d u c i n g the serum c o n c e n t r a t i o n and substituting an h y d r o l y z e d p r o t e i n p r e p a r a t i o n for e m b r y o extract (20). Interestingly, two i n d e p e n d e n t studies with r a t cell strains suggest t h a t euploid cells f r o m this species m a y be m a i n t a i n e d indefinitely if an enriched m e d i u m is used (26, 34). W h i l e no p a r t i c u l a r nutrient except serum a l b u m i n has been definitely i m p l i c a t e d as effecting extended g r o w t h of cell strains, recent w o r k of F r e e d a n d Schatz (16) m a y be of special interest. They r e p o r t e d that c h r o m o s o m a l a b e r r a t i o n s can be i n d u c e d in Chinese h a m s t e r cells in culture b y omission of a n y single essential a m i n o acid f r o m the m e d i u m . A n increased incidence of such a n o m a l i e s has been n o t e d in late passage h u m a n f i b r o b l a s t s (39) as well as in cells f r o m several other species (19). One could speculate, therefore, t h a t c o n t i n u a l or r e p e a t e d exposure of n o r m a l cells to n o n o p t i m a l levels of a m i n o acids or other growth factors could induce the a b n o r m a l i t i e s observed. Such c o n d i t i o n s are difficult or impossible to a v o i d in culture except in c o n t i n u o u s flow a p p a r a t u s e s . The a b u n d a n t a n d highly d e v e l o p e d e n d o p l a s m i c reticulum in chick f i b r o b l a s t s at b o t h passage levels contrasts with the d i m i n u t i o n with age in Tokophrya infusionum FIGS. 17 and 18. Portions of late passage cells. FIG. 17. Secondary lysosomes and residual bodies occupy a large part of this cellular area. What is interpreted as successive stages in the autolysis of the contents of droplets are numbered I through 3. At A in droplet 2, it appears that a portion of the cytoplasm is being pinched off and incorporated into the droplet. Residual bodies are filled with membranous residues and dense, amorphous material. The bent mitochondrion passes out of this plane of section at either end. GPS, 4°. x 25 900. FIG. 18. Three autolysosomes show progressive stages in the lysis of cellular components. Much of 1 is filled with ribosomes, cisternae of the RER and mitochondria (M), and the segregated cytoplasm is more compacted and dense than comparable cellular areas. This is even more in evidence in 2 (arrows). A lipid droplet (L) is recognizable, and large portions of 2 are filled with homogeneous material. Except for a small, very dense area filled with ribosomes, 3 contains membranous residues and dense material unidentifiable with cellular components. The nucleus (N) of this cell was one of only five of the late passage cells viewed in which blocks of chromatin were adjacent to the nuclear envelope. GCS, 25 °. × 19 800.
~ 0 -- 7 1 1 8 2 2 J .
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and mouse anterior pituitary cells (37, 44). No change in synthetic activity of chick fibroblasts could be detected biochemically by the observations that total protein concentrations as well as amino acid uptake and exchange apparently remained constant (21). The transport of extracellular materials into the fibroblasts via micropinocytotic vesicles was evident at both passage levels. However, large digestive vacuoles containing the imbibed material, like those described in cultured macrophages (6) and strain L fibroblasts (17) were rare or absent in the chick fibroblasts. There appeared to be a close structural association between the micropinocytotic vesicles, Golgi vesicles, and droplets. This suggested that bodies containing engulfed extracellular materials might rapidly coalesce with cellular components and thereby become indistinguishable. However, their fate must remain speculative until identifying markers can be used. The criteria used in defining the lysosomes visualized in chick fibroblasts were those proposed by de Duve and Wattiaux (10). These authors classed all lysosomes either as primary lysosomes which contain hydrolytic enzymes but have not yet been engaged in a digestive event or as secondary lysosomes in which digestion has ensued. The secondary lysosomes may include either extracellular materials or cellular components, thus subdividing them as heterolysosomes or autolysosomes. The latter separation is less clear in cases where there is an interrelationship of the heterophagic and autophagic pathways (I7). Residual bodies that contain undigested materials are the late forms of either heterolysosomes or autolysosomes. Of the cellular organelles studied, lysosomes have been most frequently implicated as associated with the aging process. It is generally accepted that residual bodies are prominent features of nondividing and presumably old cells such as neurons, cardiac muscle, and others. However, the secondary lysosomes which display what can be interpreted as successive stages in digestive events resulting in residual bodies were shown to be an age-associated characteristic only in the marine coelenterate, Carnpanularia flexuosa (4). The late passage chick fibroblasts, if they are indeed "old cells," may represent another system in which variable and successive stages of degradation in lysosomes can be seen with age. However, in evaluating the presence of lysosomes as a useful index to the age of cells, other physiological events which may be responsible for activation of the lysosomal system must also be considered. Autophagy occurs in cells throughout growth and developmental sequences, and in stressed cells (10), and it is possible that environmental conditions currently available for cell culture are inadequate for the maintenance of optimal cell function. Such an inadequacy or environmental stress might well be reflected in the fine structural changes of cellular organelles in late passage cultures. The induction of lysosomal activity has occurred with rather drastic changes in the in vitro cellular environment. In cultured KB cells deprived of arginine or treated with ultraviolet radiation or X-radiation (27) and in
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FIGS. 19-21. Portions of late passage cells. FIGS. 19 and 20. Many sections show paired, smooth membranes surrounding areas of degradation and extending into morphologically unaltered cytoplasm, which suggests that the smooth ER functioned in the segregation of cellular components. The more dense ribosome-studded area of degradation in Fig. 19 is limited by two sets of paired, smooth membranes (arrow). Two additional sets of paired membranes lie a short distance away and appear to be in the process of sequestering the larger cytoplasmic area (C). In Fig. 20, a single membrane encloses the residual material (R). In addition, paired smooth membranes surround this residual body and adhere closely to it at some points (arrows). The two membranes of this pair are not as closely apposed to each other as those in the two sets of paired membranes lying some distance away from the residual body and surrounding both the residual body and apparently unaltered cytoplasm. GPS, 4 °. Fig. 19, × 24 200; Fig. 20, × 30 100. FIG. 21. Some areas of degradation were occupied only by myelin figures (arrows) in late passage cells GPS, 4 °. × 21 700.
c u l t u r e d m a c r o p h a g e s and L-strain fibroblasts treated with c h l o r o q u i n e condary
(14, 15),
se-
lysosomes were p r o m i n e n t . Stationary phase L-strain fibroblasts showed
n u m e r o u s a u t o l y s o s o m e s and telolysosomes and were called " a g i n g cultures"
(17).
A l t h o u g h the L-strain cell type does n o t exhibit a limited g r o w t h span, it is clear that their cellular m e t a b o l i s m does r e s p o n d to the change in g r o w t h and replication during each individual passage in culture.
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The topographical relationship of paired smooth membranes to morphologically unaltered cytoplasmic components, that in chick fibroblasts frequently appeared to be segregated by these membranes, are of interest because of the attention focused on the membrane systems implicated in autolysosome formation. In early papers, Novikoff and others suggested from morphological evidence alone that smooth ER components in rat hepatocytes appeared to envelop organelles prior to their recognizable lysis (31, 32). Ericsson (11, 12) in a series of elegant experiments, confirmed this assumption by showing ER-associated enzymatic activity within the cisternae of paired membranes wrapped around intact organelles. The morphological evidence suggests that this is also one of the mechanisms for the segregation process in chick fibroblasts. The induction of autophagy may depend either on the intrinsic properties of the membranes or on other cellular factors. It could be suggested that membranes of senescent cells are altered in structure either during synthesis or due to accumulation of as yet undefined factors with time. The present studies showing changes in the shape and internal organization of cellular components, which are also seen in autolysosomes, may reflect the presence of aberrant molecules acting as a stimulus for the segregation process. The evidence discussed above indicates that environment can affect the growth span as well as induce changes in cellular structure. One source of such alteration could be effected through an induced decrease in the efficiency of protein synthesis. Cristofalo (7) has suggested a more specific mechanism involving only proteins associated with D N A synthesis without implicating environment. That some of the ultrastructural changes in late passage cells resemble those seen in cells of senescent animals argues for the hypothesis that a relationship exists between the in vitro phenomenon and in vivo aging. It remains to be determined what specific factors both in vivo and in vitro are involved in inducing the observed changes. The authors gratefully acknowledge the constructive criticism of Drs Dorothy F. Travis and James D. Ebert in the preparation of the manuscript. The prints used in this publication were made by Mr William Fisher. REFERENCES 1. ANDREW, W., J. Gerontol. 10, 1 (1955). Amer. J. Anat. 110, 1 (1962). 3. BRANDES,D., BUETOW, D. E., BERTIM, F. and MALKOF;, D. B., Exp. Mol. Pathol. 3, 583 (1964). 4. BROCK, M. A., J. Ultrastruct. Res. 32, 118 (1970). 5. CAMMERMEYER,J., J. Gerontol. 18, 41 (1963). 6. COrlN, Z. A., FEDORKO,M. E. and HIRSCH, J. B., J. Exp. Med. 123, 757 (1966). 7. CmSTOEALO,V. J., Aging in Cell and Tissue Culture, p. 83. Plenum Press, New York, 1970. 2. - -
ULTRASTRUCTUREOF CULTUREDCHICKFIBROBLASTS 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
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CRISTOFALO,V. J. and KR1TCHEVSKY,D., J. Cell Physiol. 67, 125 (1966). CRISTOFALO,V. J., PARRIS,N. and KRITCHEVSKY,D., J. Cell Physiol. 69, 263 (1967). DEDUVE,C. and WATTIAUX,R., Annu. Rev. Physiol. 28, 435 (1966). ERICSSON,J. L. E., Exp. Cell Res. 55, 95 (1969). -ibid. 56, 393 (1969). FAWCETT,D., The Cell. Saunders, Philadelphia, 1966. FEDORKO, M. E., HIRSCH, J. G. and COHN, Z. A., J. Ceil Biol. 38, 377 (1968). -ibid. 38, 392 (1968). FREED, J. J. and SCHATZ, S. A., Exp. Cell Res. 55, 393 (1969). GORDON, G. B.,oMILLER, L. R. and BENSCH, K. G., J. Cell Biol. 25, 41 (1965). HARRIS, M., Growth 21, 149 (1957). -Cell Culture and Somatic Variation, p. 162. Holt, Rinehart and Winston, New York, 1964. HAY, R. J., Aging in Cell and Tissue Culture, p. 7. Plenum Press, New York, 1970. HAY, R. J. and STREHLER,B. L., Exp. Gerontol. 2, 123 (1967). HAY, R. J., MENZIES, R. A., MORGAN, H. P. and STREHLER,B. L., Exp. Gerontol. 3, 35 (1968). HAYFLICK,L. and MOORrtEAD, P. S., Exp. Cell Res. 25, 585 (1961). HRUBAN, L., SWIFT, H. and WISSLER, R. W., J. Ultrastruct. Res. 7, 273 (1962). KARNOVSKY,M. J., Exp. Mol. Pathol. 2, 347 (1963). KROOTH,R. S., SHAW, M. W. and CAMVBELL,B. K., J. Nat. Cancer Inst. 32, 1031 (1964). LANE, N. J. and NovmoFF, A. B., J. Cell Biol. 27, 603 (1965). MALKOFE,D. B. and BUETOW, D. E., Exp. Cell Res. 35, 58 (1964). MILLONIG,G., Proc. Int. Congr. Electron Microsc. 5th, Vol. 2, P-8 (1962). MtSNNELL,J. F. and GETTY, R., J. Gerontol. 23, 363 (1968). NOVIKOFr,A. B. and SHIN, W.-Y., J. Microsc. (Paris) 3, 187 (1964). NOVlKOEF,A. B., ESSNER, E. and QUINTANA,N., Fed. Proc. Fed. Amer. Soc. Exp. Biol. 23, 1010 (1964). ORGEL, L. E., Proc. Nat. Acad. Sci. U.S. 49, 517 (1963). PETtmSSON,G., COUGHLIN,J. I. and MEYLAN, C., Exp. Cell Res. 33, 60 (1964). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). RUDZlNSKA, M. A., J. Gerontol. 16, 213 (1961). -ibid. 16, 326 (1961). SABATINI,D. D., BENSCH,K., and BARRNETT,R. J., J. Cell Biol. 17, 19 (1963). SAKSELA,E. and MOORHEAD,P. S. Proe. Nat. Acad. Sci. U.S. 50, 390 (1963). SIMONS,J. W. I. M., Exp. Ceil Res. 45, 336 (1967). SWIM, H. E. and PARKER,R. F., Amer. J. Hyg. 66, 235 (1957). TAUCHI,H. and SATO, T., J. Gerontol. 23, 454 (1968). TODARO,G. J. and GREEN, H., Proc. Soc. Exp. Biol. Med. 116, 688 (1964). WEISS,J. and LANSING,A. I., Proc. Soc. Exp. Biol. Med. 82, 460 (1953).
J. ULTRASTRUCTURERESEARCH36, 291--311 (1971)
291
Comparative U ltrastructure of Chick Fibroblasts in Vitro at Early and Late Stages during Their Growth Span M. A. BROCK and R. J. HAY
Laboratory of Cellular and Comparative Physiology, Gerontology Research Center, National Institute of Child Health and Human Development, PHS, U.S. Department of Health, Education, and Welfare, Bethesda, and the Baltimore City Hospitals, Baltimore, Maryland 21224, and Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210 Received April 24, 1970, and in revised form December 28, 1970 Chick fibroblasts were cultured through 4.5 generations (early passage) and 18 generations (late passage). The ultrastructure of early passage, rapidly dividing cells was compared with that of late passage cells showing a reduced proliferation rate and degeneration. In late passage cells, the marked lobation of the nucleus, the absence of chromatin adjacent to the nuclear envelope and ellipsoidal mitochondria, generally bent into a shallow " U " with longitudinally oriented cristae, contrasted with the nearly oval nuclei containing peripheral chromatin and oval mitochondria with transversely oriented cristae seen in early passage cells. Highly developed RER and Golgi complexes were observed at both passage levels. The most striking change was the presence of conspicuous secondary lysosomes and residual bodies in over 90 % of the late passge cells. The possible relation of these results to altered metabolic activities in an in vitro environment and to in viva aging is discussed. The possibility that ultrastructural changes may be correlated with the aging of diploid cells in culture has received little attention. It is clear that primary cell strains isolated from chick or human tissues have a limited growth potential in vitro. This is reflected either in a finite number of potential doublings or in a limited time of survival (I8, 22, 23, 41). The observations of cellular mortality in vitro have led to the hypothesis that cultured diploid cells undergo senescent changes analogous to those which occur during cellular aging in viva (23). Changes in the shape of chick and human cells, with continued subcultivation, have been noted at the light microscope level. Harris (18) reported that fibroblasts from embryonic chick muscle gradually lost the uniform appearance of primary cultures and became heterogeneous. An increase in the number of cells with very large, irregular nuclei was also noted. These observations were confirmed by H a y and 19 -
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Strehler (21), w h o further stated t h a t the changes were a c c o m p a n i e d b y a decrease in the t e n d e n c y to f o r m multilayers. H a y f l i c k a n d M o o r h e a d (23) r e p o r t e d that late p a s sage, degenerating fibroblasts f r o m h u m a n e m b r y o s were m u c h less p o l a r i z e d a n d m o r e s p r e a d out t h a n their early passage, r a p i d l y dividing c o u n t e r p a r t s . Simons (40) m e a s u r e d the diameters of dissociated cells f r o m strains of h u m a n fibroblasts isolated originally f r o m a d u l t skin. Late passage cells were m u c h less u n i f o r m in d i a m e t e r t h a n actively proliferating cells. M e t a b o l i c a n d b i o c h e m i c a l studies of early a n d late passage cells have i n d i c a t e d t h a t R N A , lipid a n d l y s o s o m a l e n z y m a t i c activity increase with age (7-9, 21). Nevertheless, the basic m e c h a n i s m s responsible for b o t h the m o r p h o l o g i c a l changes a n d for the finite life span of cells in vitro r e m a i n obscure. Because the age-associated m o r p h o l o g i c a l changes o b s e r v e d in light m i c r o g r a p h s a p p e a r to be so m a r k e d , it seems plausible to expect that cell organelles m a y d i s p l a y u l t r a s t r u c t u r a l changes with time in culture a n d that these changes c o u l d be related to the observed cellular mortality. Therefore, in this study, the fine structure of early passage chick fibroblasts is c o m p a r e d with t h a t of late passage cells.
MATERIALS AND METHODS
Tissure culture. Chick cell strains were isolated and subcultivated as described previously (21). Eagle's minimum essential medium supplemented with 10 % calf serum and 5 % embryo extract was used throughout. Cells for early passage material had been passed once in vitro and were inoculated in 7.5 ml of growth medium to 60 mm glass petri plates at 106 cells per plate. Late passage cultures were derived from cells which had been stored in liquid nitrogen after completing fifteen generations and seven passages in vitro. These were reconstituted and were subcultivated twice before being used as source material. It has been shown for both chick (21) and human (23) cell strains that this storage procedure has no detectable effect on growth span. Therefore, no lasting structural change would b e expected following resumption of normal growth. Late passage cells were inoculated to glass petri plates at the same time and in the same manner as described for early passage cells. After 4 days incubation, the fibroblasts had passed through 4.5 generations (early passage cells) and 18 generations (late passage cells). Those having completed 18 generations were considered senescent since the maximum number of doublings is about 25 for this strain (21). The following abbreviations will be used to indicate the type of preliminary fixation of material illustrated in each figure. GCS: 3 % glutaraldehyde in 0.075 M cacodylate buffer made 0.33 M in sucrose. GPS: 3 % glutaraldehyde in 0.06 M phosphate buffer made 0.14 M in sucrose. F1o. 1. Portions of three early passage chick fibroblasts show the cellular structure usually seen. Blocks of chromatin are both at the periphery and scattered throughout the karyoplasm of the two nuclei. A nuclear pore (P) is sectioned transversely in one nucleus that also shows a prominent nucleolus. Microtubules (MT) are seen in the cellular cortex. Distended cisternae of the rough endoplasmic reticulum (RER), free ribosomes, and abundant Golgi complexes (G) fill the cytoplasm. Two mitochondria are seen in near-longitudinal and -cross sections. A centriole (C) lies near the nucleus of one cell. GPS, 4 °. x 24 000.
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Each culture was washed three times with Hanks' saline before being treated for ultrastrucrural examination. Electron microscopy. The cells were detached from the petri plates by gentle scraping with rubber policemen. The fragmented monolayers were then prepared for electron microscopy by initial fixation for (a) 2 hours in a solution containing 3 To glutaraldehyde, 0.075 M cacodylate buffer, pH 7.4 (38), 0.33 M sucrose, at either 4 ° or 25°C; (b) 2 hours in a solution containing 3 % glutaraldehyde, 0.06 M phosphate buffer, pH 7.3, 0.14 M sucrose, at either 4 ° or 25°C; or (c) 1 hour in a salution containing 1% OsO,, 0.06 M phosphate buffer, pH 7.3, 0.016 M sucrose, at either 4 ° or 25°C (29). The glutaraldehyde-fixed material, after thorough washing at 4°C in (a) 0.33 M sucrose in 0.075 M cacodylate buffer, pH 7.4, or (b) 0.16 M sucrose in 0.06 M phosphate buffer, pH 7.3, was postfixed for 1 hour at 4°C with 1% OsO4 in 0.06 M phosphate buffer, pH 7.3, containing 0.016 M sucrose (29). The ceils were washed in 0.33 M sucrose, dehydrated through a graded series of alcohols and propylene oxide and fiat-embedded in Epon 812. Thin sections placed on 300- or 400-mesh uncoated copper grids were stained with lead citrate (35) prior to observation with an RCA-EMU-3G electron microscope. At least 100 cells were examined from both early and late passage cultures. Those fixed initially in glutaraldehyde followed by osmium tetroxide yielded the best preserved material.
OBSERVATIONS
Early Passage Cells Figure 1 illustrates fibroblasts at early passage. The cell nuclei, lying in the expanded central portion of the cells, had fairly regular oval contours. Dense blocks of chromatin were scattered centrally in the karyoplasm as well as adjacent to the nuclear envelope, which was interrupted at intervals by pores. Invaginations of the cell membrane and micropinocytotic vesicles just within the plasma membrane were commonly seen (Fig. 2). The fusion of two or more micropinocytotic vesicles resulted in larger vesicles whose fate could not be followed definitely without the use of appropriate markers. However, a few images suggested that micropinocytotic vesicles fused with larger single-membrane bounded bodies whose homogeneous contents were similar in density (Fig. 3). Just adjacent to the plasma membrane, the cortical cytoplasm was devoid of cellular organelles other than free ribosomes, occasional cisternae of the rough endoplasmic reticulum (RER) and microtubules (Fig. 1). Mitochondria, free ribosomes, RER, Golgi complexes and large single-membranebounded droplets were scattered from the cortex throughout the cytoplasmic matrix (Figs. 1 and 7). In the mitochondria of early passage cells, the double limiting membranes and transversely oriented cristae were accentuated against the somewhat flocculent-appearing, clearer matrix (Figs. 1 and 6). Intramitochondrial granules were common. All the mitochondrial images seen in young cells could be accounted for by an ellipsoid mitochondrial shape with platelike cristae traversing the short axis.
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FIG. 2. Micropinocytotic vesicles, formed by invaginations of the cell membrane in this early passage cell, lie in the cortical cytoplasm where many vesicles may fuse (arrows). Free ribosomes fill most of the cytoplasm in this area along with sparse cisternae of the RER. Part of a homogeneous droplet is sectioned obliquely at A. GCS, 4 °. × 23 400. FIo. 3. Two vesicles are apparently coalescing with a large clear body (A) in this early passage cell. The contents of one vesicle (arrow) are similar in density to that of micropinocytotic vesicles (Fig. 2) and of the larger body. In contrast, the contents of the second vesicle (unmarked) are similar in density to the material in the adjacent cisternae of the RER and many Golgi vesicles (Figs. 4, 5, 7, 8). Two large droplets (D) are also seen. GPS, 25 °. × 25 400. F~G. 4. A single-membrane bounded body (A) contains in its clear matrix complete vesicles similar in appearance to the Golgi vesicles close to its limiting membrane (arrows) and associated with a nearby Golgi complex (G). GPS, 25 °. × 27 400. F~o. 5. In this early passage cell, the single-membrane bounded structure at A appears to result from the fusion of a clear body with a homogeneous droplet or possibly from the rupture of vesicles within the body (as seen in Fig. 4). GPS, 25 °. x 24 200.
T h e R E R , a l t h o u g h u s u a l l y a p p e a r i n g to be r a n d o m l y o r i e n t e d , was s o m e t i m e s a r r a n g e d in s h o r t p a r a l l e l a r r a y s in t h e s e y o u n g cells. C i s t e r n a e w e r e o f t e n d i s t e n d e d a n d filled w i t h a h o m o g e n e o u s m a t e r i a l of m e d i u m d e n s i t y (Figs. 1, 3, 6, a n d 7). T h e s a c c u l a r d i l a t a t i o n s of t h e c i s t e r n a e a n d t h e a s s o c i a t e d vesicles of t h e h i g h l y develo p e d G o l g i c o m p l e x e s w e r e also filled w i t h a h o m o g e n e o u s m a t e r i a l s i m i l a r in app e a r a n c e to t h a t in t h e c i s t e r n a e of the R E R (Figs. 7 a n d 8).
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Single-membrane-bounded droplets were conspicuous in young cells. Their contents either appeared completely homogeneous and similar in density to that within the cisternae of the R E R and Golgi complexes (Figs. 3 and 7) or contained a number of small vesicles (Figs. 9 and 10). Clusters of Golgi vesicles were seen in close proximity to the homogeneous droplets with which they apparently coalesced (Fig. 8). It is suggested that some Golgi vesicles may also be incorporated into the droplets but retain their limiting membrane for a time. These images (Figs. 6-9) further suggest a sequence of synthetic activity from the R E R through the Golgi to eventual formation of the large droplets. Vesicles similar to the Golgi vesicles apparently also coalesced with or were incorporated into large, less dense bodies bounded by a single membrane (Figs. 3 and 4). Some structures could be interpreted as either a stage following the fusion of a homogeneous droplet with a large, less dense body or a stage in the transformation of the contents of a body (Fig. 5). It would appear that in these cells the Golgi vesicles are closely asseciated with both the homogeneous droplets and the less dense bodies that probably resulted from the fusion of micropinocytotic vesicles. Approximately 15 % of the cells examined showed what may be initial stages in the reorganization and degradation of material within the droplets. In Fig. 10, three droplets are closely apposed to a very large membrane-bounded homogeneous area, and at some points there is discontinuity of the membranes separating the droplets from the larger area. The continuity between these structures, provided by the lack of limiting membranes, and the presence throughout the area of small vesicles like those in the droplet, suggests that the large area devoid of cytoplasmic components was formed through fusion of several droplets. Apparent transitional changes in the appearance of the droplet contents from flocculent materials (Figs. 10 and 11) to the inclusion of cellular components and membranous residues (Fig. 11) suggests that digestive activity has ensued and these bodies are secondary lysosomes. FIG. 6-10 illustrate portions of early passage cells.
FIG. 6. The mitochondrial shape and orientation of the cristae are seen in this near-longitudinal section (M). Distended cisternae of the RER and ribosomes fill the cytoplasm. GPS, 4°. x 23 200. FIG. 7. Several highly developed Golgi complexes are often grouped in areas where Golgi vesicles are apparently coalescing with intermediate sized droplets (arrow). The homogeneous material of medium density fills the distended cisternae of the RER, many Golgi saccules and vesicles, and intermediate-sized and large droplets. GPS, 4°. × 21 100. FIG. 8. Golgi vesicles surround intermediate-sized droplets and are apparently coalescing with them (arrows). GPS, 4°. × 26 300. FIG. 9. The homogeneous, moderately dense matrix of these droplets contains vesicles similar in appearance to Golgi vesicles. GPS, 25°. x 21 100. F~G. 10. Three droplets (A, B, C) are apparently coalescingwith a large, membrane-bounded vacuolelike area (D) that fills much of the cytoplasm. Although the limiting membranes of the droplets are partially lost where they are in contact with the large area, the droplet contents are more homogeneous and dense than the somewhat flocculent matrix of the large area. Vesicles similar to Golgi vesicles are randomly distributed in both the droplets and the large area. GPS, 4 °. x 24 500.
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Late Passage Cells The irregularly rounded shape of late passage cells as well as many of their fine structural features are shown in Fig. 12. Marked changes, independent of the fixation regimen, were visualized in certain cellular components: (a) the nucleus and mitochondria were appreciably altered; (b) most cells contained conspicuous secondary lysosomes and residual bodies; and (c) lipid droplets were sometimes present. Other cellular components appeared morphologically similar to those of early passage cells. Nucleus. In contrast to the more-or-less oval nuclei of early passage cells, the late passage nuclei always presented a lobed appearance and were often bent so that portions of the same nucleus appeared as separate entities in one plane of section (Figs. 12 and 14). Blocks of chromatin were scattered centrally in these nuclei and were rarely seen at the periphery. As in early passage cells, the nuclear envelope was interrupted at intervals by pores, and its outermost membrane was ribosome studded in part. Mitochondria. The mitochondrial profiles and the orientation of the cristae were markedly changed in late passage cells. A variety of bizarre mitochondrial shapes in addition to those that were elliptical or nearly circular were seen (Figs. 12, 13a-d, and 17). Some appeared to be contorted, passing in and out of the plane of section several times, and bent at intervals along their length (Figs. 12, 13a-c). These images seen in late passage cells could be accounted for by a narrow ellipsoid usually shaped into a shallow " U " with either or both arms of the " U " sometimes twisted or bent into another plane of section. The cristae traversed the long axis of the mitochondria. Occasionally, the cristae appeared to traverse the short axis, as was the case in early passage cells. However, this orientation could be obtained in oblique sections through the ellipsoidal-shaped mitochondria (Figs. 13 a and c). In contrast to early passage cells, the mitochondrial matrix appeared homogeneous and more condensed as judged by its density. Granules were commonly present, as in the early passage cells. Lipid droplets. About 20 % of the late passage cells contained a few lipid droplets (Figs. 12 and 18). Most were relatively inconspicuous in the heterogeneous mixture of other cellular components. RER, Golgi complexes. Interestingly, the cellular organelles implicated in synthetic activity appeared as highly developed in late passage cells as in the early passage Fie. 11. Vacuole-like areas, apparently derived from droplets that have been modified by digestion of their contents, fill a cellular process in this early passage cell. Some contain only flocculent material (A); in others membranous residues lie in the flocculent matrix (B). Occasionally, portions of cytoplasm appear to have been incorporated into these areas (C). The cytoplasmic matrix resembles other early passage cells with abundant ribosomes, and distended cisternae of the RER, more clearly seen in the cell on the left, and mitochondria. GPS, 4°. × 28 800.
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fibroblasts. Cisternae of the R E R and Golgi complexes were randomly situated interior to the cortical area (Figs. 12 and 14). The cisternae of the RER, sometimes arranged in snort parallel arrays, were often distended with a homogeneous material of medium density (Fig. 12). Material similar in appearance usually filled the saccular dilatations of the Golgi cisternae, the Golgi vesicles, and droplets of intermediate size close to the inner cisternae of the Golgi complex (Fig. 14). The presence in these cellular components of a homogeneous material similar in density to that seen in early passage ceils suggests that the synthetic activity might also result in the formation of homogeneous droplets like those of early passage cells. However, the droplets were rarely seen in late passage cells, and, when observed, their ultrastructural appearance was modified appreciably as is evident in Figs. 15-17. Secondary lysosomes: modified droplets, autolysosomes, residual bodies. It was clear that in late passage cells the contents of the few homogeneous droplets present were being degraded in a manner similar to that seen in a small fraction of the early passage cells. The contents of some droplets were only slightly modified, while others contained flocculent material, partially degraded cellular components, and both myelin figures and vesicles that were unlike cellular organelles (Figs. 15-17). Because of the heterogeneous nature of the contents of these vacuole-like areas and of the evidence of autolysis, they are interpreted to be secondary lysosomes. The periphery of some cells (Fig. 17) was packed with these vacuole-like areas or secondary lysosomes. At A in Fig. 14, the configuration of a limiting membrane suggests that small areas of cellular components may be pinched off from the cytoplasm and incorporated into these bodies in a process similar to pinocytosis. Autolysosomes that resembled those in a variety of mammalian cells were filled with cellular components, some of which were apparently being lysed. The more dense appearance of areas filled with free ribosomes and of the mitochondria in comparison to these structures lying in the cytoplasm as well as the presence of membranous reresidues, lipid like material, and structures unlike cell organelles suggest that degradation has begun (Fig. 18). Although the mechanism for the segregation of cellular components was not studied in detail, it was clear, from m a n y micrographs, that paired, smooth membranes surrounded the cellular components and could be traced from the areas of degradation into morphologically unaltered cytoplasmic areas (Figs. 19 and 20).
FIG. 12. In this rounded late passage cell, two lobes of the nucleus are sectioned so that they appear to be separate entities. The karyoplasm is devoid of condensed chromatin in this plane of section; nuclear pores are common (arrows). The highly contorted mitochondria, modified droplets (D) and lipid droplets (L) are characteristic of late passage ceils. In addition, distended cisternae of the RER, smooth ER segments, and ribosomes fill the cytoplasmic matrix except for the cortex, where only ribosomes and occasional cisternae of rough and smooth ER are seen. GCS, 25 °. × 15 900.
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The most consistent and striking change in over 90 % of the late passage cells was the presence of opaque material often heterogeneous in appearance in single membrane-bounded bodies, residual bodies (Figs. 13d, 16, 17 and 20). Some of these residual bodies were filled only with myelin figures (Fig. 21); occasionally they were massed near the periphery of the cell (Fig. 17). DISCUSSION The ultrastructural alterations in fibroblasts that have been cultured for 18 generations undoubtedly reflect marked changes in cellular metabolic activity. As indicated by the appearance of the RER and Golgi complexes, synthetic functions are apparently carried out actively in both early and late passage cells. It seems reasonable to speculate, however, that the synthetic activities of these cells change as a function of passage level. This is suggested by the conspicuous absence in late passage cells of homogeneous droplets surmised to be one end product of synthetic activity. Furthermore, the nuclear and mitochondrial changes as well as the clear evidence for extensive lysosomal activity in late passage cells indicate that cellular functions must be appreciably different. In the consideration of the ultrastructure of these cells, one must question (a) whether these morphological alterations are a consequence of the length of cellular life in vitro and could be considered analogous to age-associated cellular changes observed in vivo; (b) whether the cellular organelles reflect, by their altered morphology, compensatory changes to meet the demands for maintaining cellular homeostasis in an artificial medium; or (c) whether both processes occur. Regardless of the direction of biosynthetic events, however, there is eventual loss of replicative ability and cellular death. Increased irregularity or lobation has characterized nuclei in a variety of cells from old animals. In the dog myocardium, it was regarded as a mechanism for increasing the surface area of the nuclear membrane in an attempt to maintain an optimal nuclear cytoplasmic ratio in cells that increased in size with age (30). Such changes were also observed in anterior pituitary cells of old mice (44), astrocytes of old chinchillas (5), and Purkinje and hepatic cells of old mice (1, 2). The macronucleus of the unicellular protozoan, Tokophrya infusionum, was distinctly irregular, with many invaginations, and markedly larger in old individuals that were about twice the size of the young protozoans (36, 37). In the Purkinje cells and Tokophrya the extreme nuclear invagination and lobation were thought to be prerequisite to amitotic division, another compensatory mechanism for maintaining an optimal nucleocytoplasmic ratio. The amount of stainable chromatin is thought to be an indicator of cellular metabolic activity, those cells with abundant euchromatin or nonstained chromatin being
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FIG. 13. Some of the unusual mitochondrial shapes as well as the orientation of their cristae and the more dense matrix are seen in portions of late passage ceils. The mitochondria are bent in areas where the double limiting membranes and the cristae are sectioned obliquely (arrows), and portions of some pass out of the plane of section (crossed arrows). An extremely contorted mitochondrion lies near a residual body and an autolysosome in 13d. GPS, 4°. 13a, 13d, x 25 800; 13b, 13c. x 23 300.
m o r e active t h a n those with blocks of stainable h e t e r o c h r o m a t i n (13). A l t h o u g h there was a reduction with age in the a m o u n t of h e t e r o c h r o m a t i n adjacent to the nuclear m e m b r a n e s of r a b b i t a n d chinchilla cerebellar granule ceils viewed by C a m m e r m e y e r (5), he n o t e d that this was n o t a consistent change reported by other authors. Therefore, it may be that the p r e d o m i n a n c e of e u c h r o m a t i n in the late passage cell nuclei reflects a c o m p e n s a t o r y increase in synthetic activity. This could result f r o m a decline in the accuracy of p r o t e i n synthesis, a n hypothesis suggested by Orgel (33) to ac-
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count, at least in part, for cellular senescence in the whole animal. Furthermore, the evidence for autophagy suggests an increased synthesis of hydrolytic enzymes. H r u b a n et al. (24) have provided suggestive evidence that the induction of aberrant proteins in m a m m a l i a n cells m a y be a stimulus for autolysosome formation. The intramitochondrial structural complexity and the intracellular distribution of mitochondria have been shown to depend on the energy requirements of the cell (13). F o r example, mitochondria with well-developed cisternal networks are generally seen in cell types with a high oxidative metabolism; where lipid components serve as important substrates, the mitochondria often surround lipid droplets. The sparse evidence f r o m aging studies regarding mitochondrial structure in cells of old individuals is contradictory. In early studies of Tokophrya infusionum and mouse anterior pituitary cells, there was a reduction in the length of the cristae (37, 44). On the other hand, the internal structure of h u m a n hepatic cell mitochondria in old individuals was not appreciably different f r o m that in y o u n g humans. However, irregularly shaped, giant mitochondria were often seen in hepatic cells f r o m older people, and this size increase was thought to be a compensation for the reduced n u m b e r of mitochondria in these cells (42). In general, the present studies also show changes in the shape of mitochondria and in their cristae. Interestingly, a remarkable correlation is seen with the structural changes in mitochondria that occurred in frog proximal tubule ceils either seasonally or with starvation (25). In this study, the cristae of the mitochondria f r o m summer frogs were oriented at right angles to the long axis of the mitochondria. The modified mitochondria of starved summer frogs and winter frogs had cristae parallel to the long axis, and intermediate structures showing the characteristics of b o t h mitochondrial types were observed. In these cells, there was little or no cytochrome oxidase activity when the modified mitochondria were present. It was suggested that the structural and biochemical changes might be adaptive responses of the cells as well as indicative of altered mitochondrial metabolism, an hypothesis which could apply equally well to the early and late passage fibroblasts. That starvation results in changes in mitochondrial configuration was evident also in studies of
FIG. 14. This portion of a late passage cell is especially rich in highly developed Golgi complexes. Golgi vesicles are abundant and at some sites appear to fuse with forming droplets (arrow). Segments of the RER are filled with a homogeneous material similar in density to that in some Golgi cisternae and vesicles. A droplet (D) with unevenly stained contents lies near a large Golgi area. An area of degradation is seen at A. The karyoplasm of the lobed nucleus is devoid of condensed chromatin in this plane of section. GPS, 4 °. x 22 800. FI6. 15. An altered droplet in a late passage cell contains membranous whorls in the somewhat flocculent matrix. This droplet and part of another (arrow) are similar to those in early passage cells (Fig. 11) where autolysis has evidently ensued. GPS, 25°. x 40 500. F~G. 16. Further digestion within droplets results in the membranous residues and vesicles seen in a more heter0genous matrix. A large, amorphous, dense residual body is at the right in this portion of a late passage cell. GPS, 4 °. × 29 200.
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c a r b o n - d e p r i v e d Euglena gracilis (3, 28). A b n o r m a l l y long a n d b i z a r r e - s h a p e d m i t o c h o n d r i a were characteristic of starved animals. However, it is n o t clear f r o m the p u b l i s h e d m i c r o g r a p h s whether there were c o n c o m i t a n t changes in the o r i e n t a t i o n of the cristae. There is no r e a s o n to suspect that extensive s t a r v a t i o n occurred u n d e r the g r o w t h c o n d i t i o n s e m p l o y e d in this investigation. However, the possibility t h a t the p h e n o m e n o n of limited cell survival in vitro is a response of n o r m a l cells to general environm e n t a l deficiencies m u s t be considered. H a y (20) reviewed the evidence f r o m studies with cells of various species indicating t h a t changes in the culture conditions affect the g r o w t h span very dramatically. A d d i t i o n of serum a l b u m i n to the culture m e d i u m greatly extends the g r o w t h span of h u m a n fibroblasts (43). Similar increases in the longevity of chick cell strains were n o t e d when the g r o w t h m e d i u m was m o d i f i e d by r e d u c i n g the serum c o n c e n t r a t i o n and substituting an h y d r o l y z e d p r o t e i n p r e p a r a t i o n for e m b r y o extract (20). Interestingly, two i n d e p e n d e n t studies with r a t cell strains suggest t h a t euploid cells f r o m this species m a y be m a i n t a i n e d indefinitely if an enriched m e d i u m is used (26, 34). W h i l e no p a r t i c u l a r nutrient except serum a l b u m i n has been definitely i m p l i c a t e d as effecting extended g r o w t h of cell strains, recent w o r k of F r e e d a n d Schatz (16) m a y be of special interest. They r e p o r t e d that c h r o m o s o m a l a b e r r a t i o n s can be i n d u c e d in Chinese h a m s t e r cells in culture b y omission of a n y single essential a m i n o acid f r o m the m e d i u m . A n increased incidence of such a n o m a l i e s has been n o t e d in late passage h u m a n f i b r o b l a s t s (39) as well as in cells f r o m several other species (19). One could speculate, therefore, t h a t c o n t i n u a l or r e p e a t e d exposure of n o r m a l cells to n o n o p t i m a l levels of a m i n o acids or other growth factors could induce the a b n o r m a l i t i e s observed. Such c o n d i t i o n s are difficult or impossible to a v o i d in culture except in c o n t i n u o u s flow a p p a r a t u s e s . The a b u n d a n t a n d highly d e v e l o p e d e n d o p l a s m i c reticulum in chick f i b r o b l a s t s at b o t h passage levels contrasts with the d i m i n u t i o n with age in Tokophrya infusionum FIGS. 17 and 18. Portions of late passage cells. FIG. 17. Secondary lysosomes and residual bodies occupy a large part of this cellular area. What is interpreted as successive stages in the autolysis of the contents of droplets are numbered I through 3. At A in droplet 2, it appears that a portion of the cytoplasm is being pinched off and incorporated into the droplet. Residual bodies are filled with membranous residues and dense, amorphous material. The bent mitochondrion passes out of this plane of section at either end. GPS, 4°. x 25 900. FIG. 18. Three autolysosomes show progressive stages in the lysis of cellular components. Much of 1 is filled with ribosomes, cisternae of the RER and mitochondria (M), and the segregated cytoplasm is more compacted and dense than comparable cellular areas. This is even more in evidence in 2 (arrows). A lipid droplet (L) is recognizable, and large portions of 2 are filled with homogeneous material. Except for a small, very dense area filled with ribosomes, 3 contains membranous residues and dense material unidentifiable with cellular components. The nucleus (N) of this cell was one of only five of the late passage cells viewed in which blocks of chromatin were adjacent to the nuclear envelope. GCS, 25 °. × 19 800.
~ 0 -- 7 1 1 8 2 2 J .
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and mouse anterior pituitary cells (37, 44). No change in synthetic activity of chick fibroblasts could be detected biochemically by the observations that total protein concentrations as well as amino acid uptake and exchange apparently remained constant (21). The transport of extracellular materials into the fibroblasts via micropinocytotic vesicles was evident at both passage levels. However, large digestive vacuoles containing the imbibed material, like those described in cultured macrophages (6) and strain L fibroblasts (17) were rare or absent in the chick fibroblasts. There appeared to be a close structural association between the micropinocytotic vesicles, Golgi vesicles, and droplets. This suggested that bodies containing engulfed extracellular materials might rapidly coalesce with cellular components and thereby become indistinguishable. However, their fate must remain speculative until identifying markers can be used. The criteria used in defining the lysosomes visualized in chick fibroblasts were those proposed by de Duve and Wattiaux (10). These authors classed all lysosomes either as primary lysosomes which contain hydrolytic enzymes but have not yet been engaged in a digestive event or as secondary lysosomes in which digestion has ensued. The secondary lysosomes may include either extracellular materials or cellular components, thus subdividing them as heterolysosomes or autolysosomes. The latter separation is less clear in cases where there is an interrelationship of the heterophagic and autophagic pathways (I7). Residual bodies that contain undigested materials are the late forms of either heterolysosomes or autolysosomes. Of the cellular organelles studied, lysosomes have been most frequently implicated as associated with the aging process. It is generally accepted that residual bodies are prominent features of nondividing and presumably old cells such as neurons, cardiac muscle, and others. However, the secondary lysosomes which display what can be interpreted as successive stages in digestive events resulting in residual bodies were shown to be an age-associated characteristic only in the marine coelenterate, Carnpanularia flexuosa (4). The late passage chick fibroblasts, if they are indeed "old cells," may represent another system in which variable and successive stages of degradation in lysosomes can be seen with age. However, in evaluating the presence of lysosomes as a useful index to the age of cells, other physiological events which may be responsible for activation of the lysosomal system must also be considered. Autophagy occurs in cells throughout growth and developmental sequences, and in stressed cells (10), and it is possible that environmental conditions currently available for cell culture are inadequate for the maintenance of optimal cell function. Such an inadequacy or environmental stress might well be reflected in the fine structural changes of cellular organelles in late passage cultures. The induction of lysosomal activity has occurred with rather drastic changes in the in vitro cellular environment. In cultured KB cells deprived of arginine or treated with ultraviolet radiation or X-radiation (27) and in
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FIGS. 19-21. Portions of late passage cells. FIGS. 19 and 20. Many sections show paired, smooth membranes surrounding areas of degradation and extending into morphologically unaltered cytoplasm, which suggests that the smooth ER functioned in the segregation of cellular components. The more dense ribosome-studded area of degradation in Fig. 19 is limited by two sets of paired, smooth membranes (arrow). Two additional sets of paired membranes lie a short distance away and appear to be in the process of sequestering the larger cytoplasmic area (C). In Fig. 20, a single membrane encloses the residual material (R). In addition, paired smooth membranes surround this residual body and adhere closely to it at some points (arrows). The two membranes of this pair are not as closely apposed to each other as those in the two sets of paired membranes lying some distance away from the residual body and surrounding both the residual body and apparently unaltered cytoplasm. GPS, 4 °. Fig. 19, × 24 200; Fig. 20, × 30 100. FIG. 21. Some areas of degradation were occupied only by myelin figures (arrows) in late passage cells GPS, 4 °. × 21 700.
c u l t u r e d m a c r o p h a g e s and L-strain fibroblasts treated with c h l o r o q u i n e condary
(14, 15),
se-
lysosomes were p r o m i n e n t . Stationary phase L-strain fibroblasts showed
n u m e r o u s a u t o l y s o s o m e s and telolysosomes and were called " a g i n g cultures"
(17).
A l t h o u g h the L-strain cell type does n o t exhibit a limited g r o w t h span, it is clear that their cellular m e t a b o l i s m does r e s p o n d to the change in g r o w t h and replication during each individual passage in culture.
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The topographical relationship of paired smooth membranes to morphologically unaltered cytoplasmic components, that in chick fibroblasts frequently appeared to be segregated by these membranes, are of interest because of the attention focused on the membrane systems implicated in autolysosome formation. In early papers, Novikoff and others suggested from morphological evidence alone that smooth ER components in rat hepatocytes appeared to envelop organelles prior to their recognizable lysis (31, 32). Ericsson (11, 12) in a series of elegant experiments, confirmed this assumption by showing ER-associated enzymatic activity within the cisternae of paired membranes wrapped around intact organelles. The morphological evidence suggests that this is also one of the mechanisms for the segregation process in chick fibroblasts. The induction of autophagy may depend either on the intrinsic properties of the membranes or on other cellular factors. It could be suggested that membranes of senescent cells are altered in structure either during synthesis or due to accumulation of as yet undefined factors with time. The present studies showing changes in the shape and internal organization of cellular components, which are also seen in autolysosomes, may reflect the presence of aberrant molecules acting as a stimulus for the segregation process. The evidence discussed above indicates that environment can affect the growth span as well as induce changes in cellular structure. One source of such alteration could be effected through an induced decrease in the efficiency of protein synthesis. Cristofalo (7) has suggested a more specific mechanism involving only proteins associated with D N A synthesis without implicating environment. That some of the ultrastructural changes in late passage cells resemble those seen in cells of senescent animals argues for the hypothesis that a relationship exists between the in vitro phenomenon and in vivo aging. It remains to be determined what specific factors both in vivo and in vitro are involved in inducing the observed changes. The authors gratefully acknowledge the constructive criticism of Drs Dorothy F. Travis and James D. Ebert in the preparation of the manuscript. The prints used in this publication were made by Mr William Fisher. REFERENCES 1. ANDREW, W., J. Gerontol. 10, 1 (1955). Amer. J. Anat. 110, 1 (1962). 3. BRANDES,D., BUETOW, D. E., BERTIM, F. and MALKOF;, D. B., Exp. Mol. Pathol. 3, 583 (1964). 4. BROCK, M. A., J. Ultrastruct. Res. 32, 118 (1970). 5. CAMMERMEYER,J., J. Gerontol. 18, 41 (1963). 6. COrlN, Z. A., FEDORKO,M. E. and HIRSCH, J. B., J. Exp. Med. 123, 757 (1966). 7. CmSTOEALO,V. J., Aging in Cell and Tissue Culture, p. 83. Plenum Press, New York, 1970. 2. - -
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