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Effects of various factors on occurrence of mitochondrial inclusions
Experimental
Cell Research
EFFECTS
32, 379-390
379
(1963)
OF VARIOUS FACTORS ON OCCURRENCE MITOCHONDRIAL INCLUSIONSl
0. A. SCHJEIDE, MARION Laboratory of Nuclear Nuclear Medicine,
OF
RUTH McCANDLESS, MYRNA WILKENS, PETERSON and G. V. ALEXANDER
Medicine and Radiation Biology of the Department and Department of Radiology, School of Medicine, California, Los Angeles, Calif., U.S.A. Received
December
of Biophysics University
and
of
21, 1962
IN most tissues, especially. when fixed with osmium and embedded in Epon, the mitochondria are observed to contain small (0.025 to 0.100 ,B) aggregations of either (or both) densely absorbing or darkly staining materials situated between the cristae. These spherical granules have been described by many electron microscopists [6, 7, 8, 11, 121, several of whom have stressed a frustrating variability in numbers of such inclusions per mitochondrion. Actually, there is also a considerable range in individual sizes. Although the opinion has been ventured that the above named mitochondrial inclusions may consist largely of the cations, potassium or sodium [ 121 no definitive information on their compositions has been forthcoming and their roles, if any, are quite unknown [lo]. It is the purpose of this communication to (a) describe the occurrence of these inclusions during embryonic development in two different tissues, (b) to detail their behavior under various experimental conditions (including direct and indirect X-irradiation) hoping in this way to gain some insight with respect to their metabolic roles and (c) to present some new evidence pertaining to their gross chemical compositions.
MATERIALS
AND
METHODS
For these studies, livers and hearts from anesthetized chicken embryos, chicks and adult chickens were fixed in situ with osmic acid, by dripping into surface incisions. The tissues were immersed in 1 per cent osmicacid (PH. 7.4) for 2-3 hr, were rinsed with isotonic half-strength Verona1buffer (PH. 7.4), were then taken up through 50-, 75-, 95- and 100 per cent ethyl alcohol steps to propylene oxide and were finally embedded in Epon 812 [2]. Polymerization was promoted by adjusting the tempera1 These Commission
studies were supported by Contract and the University of California.
AT(04-l)GEN-12
between
Experimental
the
Atomic
Energy
Cell Research
32
0. A. Schjeide
et al.
ture to 35°C for 12 hr followed by 45°C for 24 hr and 60°C for 48 hr. A four-week curing period was allowed to elapse prior to cutting (60-90 rnp sections) and mounting on copper grids coated with collodion and carbon for examination under the electron microscope (RCA Model EMUSB). Photographs were made at approximately 5000 magnification and enlarged five times. In some cases the sections were destained with 15 per cent H,O, [3]. This provided an index of the extent to which individual structures were made visible by virtue of their uptake of stain. Developmental stages examined included chicken embryos of 7, 10, 15, and 18 days of incubation; chicks newly hatched and 3, 5, and 14 days post-hatching; adult pullets, roosters and rabbits. Various experimental treatments were introduced, including: (a) Whole body Xirradiation (The irradiation factors were 250 KVP, 15 ma, 35 cm TOD, 0, 21 mm Cu inherent, plus 0.5 mm parabolic Cu, plus 1.0 mm Al filters, HVL =1.9 mm Cu, center of parabolic filter. The dose rate was 78 rpm as measured in air. Both chicks and eggswere well centered in the field, eggs standing on their smaller ends in egg boxes and chicks standing in boxes covered by celluloid. In one experiment, irradiation was directed to the liver and immediate tissue only. In another experiment the upper abdomen, including the liver, was shielded.) (b) Thyrozin (0.01 mg of sodium L-thyroxine was administered by oral intubation 16 hr prior to fixation) (c) Cortisone (10 mg of cortisone acetate was injected intraperitoneally 16 hr prior to fixation.) (d) Fasting (Yolk sacswere removed by surgery on the day of hatching. Controls were sham operated. Only water was provided the fasted chicks until 3 days posthatching, when the animals were sacrified.) (e) Estrogen (5 mg of Estrogenic substances (Ayerst Co.) were administered into the allantoic sac or intraperitoneally 3 days prior to sacrifice.) (f) Regeneration (One-third of the left lobe of the liver was removed on the first day post-hatching. Four days later, the regenerated and non-regenerated portions of the sameliver lobe were individually fixed and prepared for examination.) At least three animals were used for each point in these investigations and in several casesmany more than this number were included. Counts of mitochondrial inclusions were made from electron micrographs by three different observers, each of whom employed a rectangular outline on glass of a “standard mitochondrion” measuring one micron in length and one-half micron in width. This was placed over the mitochondrion in the most representative position and only those inclusions falling within the confines of the rectangle were recorded. Good agreement prevailed between all three investigators. Estimates of significance between various developmental stagesand control and experimental animals were obtained.
RESULTS The bar graph shown in Fig. 1 illustrates the trend for liver mitochondria to contain more inclusions as functions of time and degree of development up to 5 days post-hatching. 1 There is, however, one conspicuous point 1 As yet unpublished observations developing egg indicate that within few) granules such as are observed Experimental
Cell Research
32
made in this laboratory on mitochondria occurring in the the newly formed mitochondria there are no (or extremely in the more advanced mitochondria in this same cell.
Mitochondrial
inclusions
381
in an otherwise gradual linear curve. This is a dramatic decrease of inclusions to nearly zero in the mitochondria of newly-hatched chicks, a decrease which lasts less than 24 hr. On the other hand, one of the highest levels attained by liver mitochondria occurs at approximately 5 days posthatching (Fig. 2). Fig. 1 also shows the depressive effect of 558 to 930 rads of whole body
Days port-hatching
Fig. l.-Bar graph illustrating a general increase in liver mitochondrial inclusions as a function of development and the rapidly expressed (2 hr post-irradiation) effect of 558-930 rads in dissolving these inclusions. Note that the stress of hatching is also accompanied by a dramatic decrease in numbers of granules per mitochondrion. An average of 145 mitochondria were counted for each bar on this graph. Probability of a difference between: 7d and 18d: 0.001; 5d and adult: 0.01; 3d and 3d Est.: 0.001; 5d and 5d X: 0.001. (Chi-square comparison of distributions setting the control (or largest group) as “F”.) o, control; w , 558-943 rads; q , estrogen; I, standard error.
X-irradiation on inclusions of the liver mitochondria, which takes place as early as 2 hr post-irradiation (Fig. 3). (A dose of 558 rads is sublethal but will initiate a marked change in lipid metabolism of the liver.) It will be noted that the granules in the mitochondria of 5-day-old chicks are not decreased as much in terms of percentage as are the mitochondrial inclusions of earlier stages but that the actual fall off in inclusions approximates those of stages having fewer such inclusions originally. Liver mitochondria from 3month-old rabbits also responded with decreases in granule content following X-irradiation. In two experiments (Fig. 4) the inclusions were observed to be largely absent from the mitochondria 24 hr post-irradiation. However, they were present in greater numbers again at 3 days post-irradiation. Application of a dose as low as 279 rads appeared to have little effect on the numbers of inclusions. (This dose also fails to affect lipid metabolisms of the liver.) An experiment in which 930 rads were applied directly to the liver (plus Experimental
Cell Research
32
0. A. Schjeide
Fig. 2.-Mitochondria from 5-day-old chick are especially numerous, large and compact.
et af.
liver. The intramitochondrial Approx. 16,000 ): .
granules
of this
stage
those tissues which lie directly over and behind the liver) showed no effect of the irradiation. When, however, all of the body was irradiated, with the exception of a transverse band that included the liver, the usual decrease in population of granules was noted by 2 hr post-irradiation (Fig. 5). This result reveals that the effect of radiations on the granules is indirect. Experimental
Cell Research
32
Mitochondrial
Fig. 3.-Mitochondria diation. Note that mark the original
from a 15-day-old chicken nearly all the intramitochondrial sites of many of these. Approx.
inclusions
embryo fixed 2 hr following 558 rads of X-irrainclusions have disappeared. Diffuse smudges 16,000 x .
A striking effect of thyroxin treatment was to cause the mitochondria to swell and become rounded. There was a concomitant decrease in inclusions per mitochondrion (Fig. 6). The same effects were noted in the case of fasting (Fig. S), but cortisone appeared to cause no decrease in mitochondrial incluExperimental
Cell Research
32
0. A. Schjeide
384
et al.
sions (Fig. 6). Although Estrogenic Substances were found to be very effective in reducing the populations of such inclusions in livers of chicks three days of age, these agents displayed lesser effects in the more resistant S-dayold chicks. In no case did these amounts of estrogens appear to induce a swelling of the mitochondria. However, in the very heavily estrogenized
h-L
L-L2 2472 hr
hr
24
hr
d
10 days incubation
l-day-old
Fig.
4.
chocks
NOX Contrd
(558 radr) Whole body
Fig.
l-i(930 radr) Liver Only
(930 radr) Liver rhielded
5.
Fig. 4.-Bar graph illustrating the duration of effect of a single whole body dose of X-irradiation. At 24 hr the inclusions are still largely decreased in numbers but by 72 hr post-irradiation they are once again well represented. An average of 62 mitochondria were counted for each bar on this graph. Probability of a difference between: 18d control and 18d x 72 hr: 0.20; Id control and Id x 24 hr: 0.001. q , control; n , 558 rads; I, std error. Fig. R.-Bar graph illustrating the lack of effect of direct X-irradiation to the liver of the 5-dayold chick on mitochondrial inclusions and the dissolving effect of whole body X-irradiation although the liver is shielded. The fact that a significant depression of liver granules was obtained by whole body X-irradiation exclusive of the liver attests to an indirect effect of X-irradiation on these bodies. An average of 319 mitochondria were counted for each bar on this graph. Probability of a difference between control and liver shielded: 0.001. I, std error.
chickens, the mitochondria appeared to be reduced in numbers, principally by virtue of a considerable distention of the endoplasmic reticulum as the cells assumed a strongly synthesizing and secretory character. Estrogens appeared to have no effect in reducing numbers of inclusions in heart mitochondria. Mitochondria of regenerating liver contained fewer inclusions than the numbers characteristic of the age group. However, inclusions were even more strongly depressed in the portion of the liver supporting the regenerating cells (Fig. 6). In the regenerating portion of the liver, the mitochondria appeared to be swollen, as in the case of thyroxin treatment. Although data on heart are not as extensive as for liver tissue, it was observed that the numbers of inclusions per mitochondrion in this organ also Experimenfal
Ccl1 Research
32
Mitochondrial
inclusions
385
increase with age (Fig. 7). In contrast to the liver, there is not a sudden decrease in these bodies at the time of hatching. However, the numbers of inclusions per mitochondrion in heart appeared to vary greatly from section to section in the same heart. In some places no inclusions were observed and in other regions the granules averaged as high as 12 per standard mitochond-
--
Days of ,ncubation
Days port-hatching
Fig.
7.
Fig. B.-Bar graph illustrating effect of various agents on liver mitochondrial inclusions of 5-dayold chicks. An average of 190 mitochondria were counted for each bar on this graph. Probability of a difference in granule content between control and regenerated mitochondria: 0.001. q , control; T, thyroxin; C, cortisone; E, estrogen; R, regenerating cells; S, non regenerating portion; F, fasting. Fig. 7.-Bar graph illustrating the general increase in heart muscle mitochondrial inclusions as a function of development and the apparent lack of an effect of either X-irradiation or estrogens on these inclusions. An average of 74 mitochondria were scanned for each bar in this graph. Probability of a difference in granule content between 7d embryo and B-day post-hatching: 0.001. q , control; w 558 rads; q estrogen; I, std error.
rion (Fig. 8). No correlation was obtained between numbers of mitochondrial inclusions and degree of contraction of the heart fibers, nor could any effects of X-irradiation be noted on the inclusions present in heart mitochondria. In rabbit liver cells the mitochondria were sometimes observed to have aggregated in specific sites in the cell (in both control and experimental animals). Such clumping was not observed in chickens until two weeks posthatching. The results of de-staining some of the osmium fixed sections with H,O, [3] indicate that a portion of the mitochondrial granules may be either lipid or metal or both. As shown in Fig. 9, when the osmium is removed from the sections, a definite de-staining of the inclusions takes place to an even greater extent than the lipid-containing mitochondrial membranes and to about the same extent as the lipid borders of fat vacuoles. If most of the reactive sites Experimental
Cell Research
32
0. A. Schjeide
Fig. 8.-Mitochondria and manner in which
from heart mitochondria
et al.
muscle of 14-day-old chick. Xote high are aligned between muscle fibers.
concentration of granules Approx. 23,000 Y .
were occupied by cations, these might be removed by the destaining treatment. However, on the basis of physical considerations it seems unlikely that the cations would be either K+ or Na+ since an unreasonable packing of such atoms would have to be assumed to provide the electron density observed. In an attempt to ultracentrifugally float the granules on an 8.8 per cent NaCl solution of mitochondria that had been disintegrated by ultrasonic vibration, it was observed that these granules displayed a strong tendency to affix to whatever mitochondrial debris might float under such conditions. A crude index of the physical nature of the granules in the fixed condition was provided by sections of incompletely cured tissue. The inclusions appeared to be sufficiently compact so as to be readily pushed about by the glass knife-edge Experimental
Cell
Research
32
Mifochondrial
inclusions
Fig. 9.-Mitochondria from the liver of a 5-day-old chick which have been tion of 15 H,O,. Note that the granules appear as bleached spots that are the external mitochondrial membranes. The critical question is whether this (of lipid?) in the case of the inclusions or extraction of originally occurring Xpprox. 40,000 X .
de-stained by applicasomewhat lighter than is actually de-staining heavy metal, or both.
but soft enough to leave a granular trail in their wake.1 Under the influence of estrogens and X-irradiation the inclusions often present a diffuse periphery surrounding a less-than-normal size solid granule center. In grossly 1 Nass and Nass [5] refer to osmium reactive rods and fibers within the mitochondria chicken embryos. Such tissue is difficult to fix and this fact, together with the lack afforded by a dense matrix of cristae, could lead to the creation of artifacts. Experimental
of early of support
Cell Research
32
388
0. A. Schjeide
et al.
damaged mitochondria they appear to remain intact somewhat longer do the cristae. Supplementary staining with lead does not detectably counts of inclusions.
than alter
DISCUSSION
The intra-mitochondrial granules do not appear to be absolutely essential for mitochondrial integrity or function since they are absent from many early mitochondria and are present in variable numbers in others, being apparently greatly influenced by prevailing metabolic conditions. However, the rapid disappearance of most of these granules as an indirect effect of X-irradiation and during the initial period of hatching, leads to speculation regarding possible vital roles. Ito [ 1] has shown that the intramitochondrial granules of the mitochondria present in parietal cells of the fasting bat disappear as early as 11 min after an initial intake of food. (There is a gradual reappearance of these particles with time.) Although Napolitano [4] observed that when fat cells were depleted of lipid there was actually an accumulation of intramitochondrial bodies, a subsequent increase of lipid in the cells was, as in Ito’s experiment, accompanied by decreases in numbers and sizes of granules to normal values. The behaviors of the mitochondria in these respects are consistent with the idea that factors, elicited by a variety of stimuli, call forth the materials contained within the granules as a homeostatic response. The nature of the inducing agents and the requirements and actions of the mitochondria, as well as the material itself, all remain to be elucidated. However, if the inclusions were simply stockpiles of energy-providing materials it would be expected that they would be largely absent under conditions of accelerated growth or fasting and while they are, indeed, sometimes reduced under such conditions, significant numbers are usually present. An alternative to the above theory of “demand and supply” is suggested by Weiss’s observation that granules within the mitochondria of duodenal absorptive cells, from rats fed large amounts of sodium or potassium, are somewhat increased [12]. If the granules are actually storage sites for excess Na+ or K+, as Weiss has concluded, then the effect of the various degranulating agents discussed may be one of disrupting or damaging the mitochondrion, causing a breakdown of the storage system. In such a case, however, it would be expected that the mitochondria would be further damaged by the release of these high concentrations of cations, which they are not.1 Also, it 1 In neither the heart nor the liver of the embryo to directly change mitochondrial numbers and volumes Experimental
Cell Research
32
fowl does 558 rads of X-irradiation appear or the numbers or geometries of the cristae.
Mifochondrial
inclusions
389
is difficult to explain on such a basis the partial effect of these agents on the liver mitochondria of the five-day-old chick and the complete lack of effect of X-rays or estrogen on mitochondria of heart muscle. More consistent with the available evidence is the notion that the partial degranulation of the mitochondria of 5-day-old chicks following X-irradiation is a reflection of the amount of such material required to carry out a given function under the circumstances. The naturally occurring decrease in intramitochondrial granules of liver mitochondria in the newly hatched chick further suggests that the material in the granules is useful and can be tapped as required. Chemical and physical features.-The conclusion that the granules may contain lipid is based on the observation that treatment of osmium-stained sections with H,O, (which oxidizes the osmium so that it can be rinsed away) results in de-staining of the granules to the same extent as the lipoproteins remaining in the borders of the lipid vacuoles (Fig. 9). As Merriam [3] has pointed out, the ability to react with osmium is greatest in lipids (unpublished results indicate that this is a function of unsaturation). Proteins have a comparatively modest ability to react with osmium and show a lesser change with bleaching. Nucleic acids do not react with osmium to any appreciable extent but have a high intrinsic density. Some metals would also appear to be associated with the granules because they absorb electrons in the absence of treatment with heavy-metaI-containing fixatives or stains. Formalin fixed preparations suggest the presence of granules but less contrast is observed than in osmium fixed preparations. This lesser density may be due to the presence of relatively small amounts,of metals with the additional contrast arising from staining being due to the presence of points of unsaturation in such materials as Na oleate, etc. Better isolation procedures are currently being developed so that more definitive approaches can be employed to interpret the chemistries, roles, and origins of the intramitochondrial granules.
SUMMARY
In general, mitochondrial inclusions increase in numbers with the stage of development of the cell. However, certain metabolic conditions, such as those induced by X-irradiation (indirect effect), hatching, starvation, thyAll cell divisions occur at nearly normal rates during the period of concern and cell destruction is negligible. Although there is a slight decrease in the volumes of mitochondria per unit volume of tissue in the liver as development proceeds, this is due to the increased volume of fatty globules to the point of hatching. X-irradiation causes an additional decrease in mitochondrial volumes, but this is due to a failure of the newly dividing cells to produce as many new mitochondria 191. Experimental
Cell Research
32
0. A. Schjeide
390
et al.
roxin treatment, increase of work load and treatment with estrogenic suhstances, result in rapid dissolution of the granules in the mitochondria of liver (but not in heart mitochondria). These findings suggest a similar response of the liver mitochondrion to a variety of original stimuli. It is considered that this response may be triggered by similar homeostatic signals to the liver cell under conditions which can vaguely be described as “stressful”. The mitochondrial inclusions may consist of substances which are of unique use to the mitochondrion or cell under such conditions.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
ITO, S., Abstract 2nd Annual Meeting Am. Sot. for Cell. Biol. p. 78, 1962. LUFT, J. H., J. Biophys. Biochem. Cyfol. 9, 409 (1961). MERRIAM, R. W., J. Biophys. Biochem. Cyfol. 4, 579 (1958). NAPOLITANO. L., ibid. 8, 129 (1962). NASS, M. M.‘K. ‘and NA& S.; Ezpil Cell Res. 26, 424 (1962). NOVIKOFF, A. B., in The Cell, vol. 2, p. 299. J. BRACHET and A. E. MIRSKY (eds.) Academic Press, New York, 1961. PALADE. G. E.. Subcellular Particles. vol. 64. T. Havashi (ed.) Ronald Press. New York, 1959. RHODIN; J., Correlation of Ultrastructural Organ&ion and Function in Normal and Experimentally Changed Proximal Convoluted Tubule Cells of the Mouse Kidney. Aktiebologet Godvil, Stockholm, 1954. SCHJEIDE, 0. A., RAGAN, N., MCCANDLESS, R. G. and BISHOP, F. C., Radiation Res. 13, 205 (1960). SCH&DE, b. A., MCCANDLESS, R. G., MYERS, JR. L. S., and HENNESSY, T. G., Abstracts International Biophysics Congress, vol. 100, Stockholm, 1961. SJBSTRAND, F. S., Personal communication. WEISS, J. M., J. Expfl Med. 102, 783 (1955).
Erperimenfal
Cell
Research
32
Cell Research
EFFECTS
32, 379-390
379
(1963)
OF VARIOUS FACTORS ON OCCURRENCE MITOCHONDRIAL INCLUSIONSl
0. A. SCHJEIDE, MARION Laboratory of Nuclear Nuclear Medicine,
OF
RUTH McCANDLESS, MYRNA WILKENS, PETERSON and G. V. ALEXANDER
Medicine and Radiation Biology of the Department and Department of Radiology, School of Medicine, California, Los Angeles, Calif., U.S.A. Received
December
of Biophysics University
and
of
21, 1962
IN most tissues, especially. when fixed with osmium and embedded in Epon, the mitochondria are observed to contain small (0.025 to 0.100 ,B) aggregations of either (or both) densely absorbing or darkly staining materials situated between the cristae. These spherical granules have been described by many electron microscopists [6, 7, 8, 11, 121, several of whom have stressed a frustrating variability in numbers of such inclusions per mitochondrion. Actually, there is also a considerable range in individual sizes. Although the opinion has been ventured that the above named mitochondrial inclusions may consist largely of the cations, potassium or sodium [ 121 no definitive information on their compositions has been forthcoming and their roles, if any, are quite unknown [lo]. It is the purpose of this communication to (a) describe the occurrence of these inclusions during embryonic development in two different tissues, (b) to detail their behavior under various experimental conditions (including direct and indirect X-irradiation) hoping in this way to gain some insight with respect to their metabolic roles and (c) to present some new evidence pertaining to their gross chemical compositions.
MATERIALS
AND
METHODS
For these studies, livers and hearts from anesthetized chicken embryos, chicks and adult chickens were fixed in situ with osmic acid, by dripping into surface incisions. The tissues were immersed in 1 per cent osmicacid (PH. 7.4) for 2-3 hr, were rinsed with isotonic half-strength Verona1buffer (PH. 7.4), were then taken up through 50-, 75-, 95- and 100 per cent ethyl alcohol steps to propylene oxide and were finally embedded in Epon 812 [2]. Polymerization was promoted by adjusting the tempera1 These Commission
studies were supported by Contract and the University of California.
AT(04-l)GEN-12
between
Experimental
the
Atomic
Energy
Cell Research
32
0. A. Schjeide
et al.
ture to 35°C for 12 hr followed by 45°C for 24 hr and 60°C for 48 hr. A four-week curing period was allowed to elapse prior to cutting (60-90 rnp sections) and mounting on copper grids coated with collodion and carbon for examination under the electron microscope (RCA Model EMUSB). Photographs were made at approximately 5000 magnification and enlarged five times. In some cases the sections were destained with 15 per cent H,O, [3]. This provided an index of the extent to which individual structures were made visible by virtue of their uptake of stain. Developmental stages examined included chicken embryos of 7, 10, 15, and 18 days of incubation; chicks newly hatched and 3, 5, and 14 days post-hatching; adult pullets, roosters and rabbits. Various experimental treatments were introduced, including: (a) Whole body Xirradiation (The irradiation factors were 250 KVP, 15 ma, 35 cm TOD, 0, 21 mm Cu inherent, plus 0.5 mm parabolic Cu, plus 1.0 mm Al filters, HVL =1.9 mm Cu, center of parabolic filter. The dose rate was 78 rpm as measured in air. Both chicks and eggswere well centered in the field, eggs standing on their smaller ends in egg boxes and chicks standing in boxes covered by celluloid. In one experiment, irradiation was directed to the liver and immediate tissue only. In another experiment the upper abdomen, including the liver, was shielded.) (b) Thyrozin (0.01 mg of sodium L-thyroxine was administered by oral intubation 16 hr prior to fixation) (c) Cortisone (10 mg of cortisone acetate was injected intraperitoneally 16 hr prior to fixation.) (d) Fasting (Yolk sacswere removed by surgery on the day of hatching. Controls were sham operated. Only water was provided the fasted chicks until 3 days posthatching, when the animals were sacrified.) (e) Estrogen (5 mg of Estrogenic substances (Ayerst Co.) were administered into the allantoic sac or intraperitoneally 3 days prior to sacrifice.) (f) Regeneration (One-third of the left lobe of the liver was removed on the first day post-hatching. Four days later, the regenerated and non-regenerated portions of the sameliver lobe were individually fixed and prepared for examination.) At least three animals were used for each point in these investigations and in several casesmany more than this number were included. Counts of mitochondrial inclusions were made from electron micrographs by three different observers, each of whom employed a rectangular outline on glass of a “standard mitochondrion” measuring one micron in length and one-half micron in width. This was placed over the mitochondrion in the most representative position and only those inclusions falling within the confines of the rectangle were recorded. Good agreement prevailed between all three investigators. Estimates of significance between various developmental stagesand control and experimental animals were obtained.
RESULTS The bar graph shown in Fig. 1 illustrates the trend for liver mitochondria to contain more inclusions as functions of time and degree of development up to 5 days post-hatching. 1 There is, however, one conspicuous point 1 As yet unpublished observations developing egg indicate that within few) granules such as are observed Experimental
Cell Research
32
made in this laboratory on mitochondria occurring in the the newly formed mitochondria there are no (or extremely in the more advanced mitochondria in this same cell.
Mitochondrial
inclusions
381
in an otherwise gradual linear curve. This is a dramatic decrease of inclusions to nearly zero in the mitochondria of newly-hatched chicks, a decrease which lasts less than 24 hr. On the other hand, one of the highest levels attained by liver mitochondria occurs at approximately 5 days posthatching (Fig. 2). Fig. 1 also shows the depressive effect of 558 to 930 rads of whole body
Days port-hatching
Fig. l.-Bar graph illustrating a general increase in liver mitochondrial inclusions as a function of development and the rapidly expressed (2 hr post-irradiation) effect of 558-930 rads in dissolving these inclusions. Note that the stress of hatching is also accompanied by a dramatic decrease in numbers of granules per mitochondrion. An average of 145 mitochondria were counted for each bar on this graph. Probability of a difference between: 7d and 18d: 0.001; 5d and adult: 0.01; 3d and 3d Est.: 0.001; 5d and 5d X: 0.001. (Chi-square comparison of distributions setting the control (or largest group) as “F”.) o, control; w , 558-943 rads; q , estrogen; I, standard error.
X-irradiation on inclusions of the liver mitochondria, which takes place as early as 2 hr post-irradiation (Fig. 3). (A dose of 558 rads is sublethal but will initiate a marked change in lipid metabolism of the liver.) It will be noted that the granules in the mitochondria of 5-day-old chicks are not decreased as much in terms of percentage as are the mitochondrial inclusions of earlier stages but that the actual fall off in inclusions approximates those of stages having fewer such inclusions originally. Liver mitochondria from 3month-old rabbits also responded with decreases in granule content following X-irradiation. In two experiments (Fig. 4) the inclusions were observed to be largely absent from the mitochondria 24 hr post-irradiation. However, they were present in greater numbers again at 3 days post-irradiation. Application of a dose as low as 279 rads appeared to have little effect on the numbers of inclusions. (This dose also fails to affect lipid metabolisms of the liver.) An experiment in which 930 rads were applied directly to the liver (plus Experimental
Cell Research
32
0. A. Schjeide
Fig. 2.-Mitochondria from 5-day-old chick are especially numerous, large and compact.
et af.
liver. The intramitochondrial Approx. 16,000 ): .
granules
of this
stage
those tissues which lie directly over and behind the liver) showed no effect of the irradiation. When, however, all of the body was irradiated, with the exception of a transverse band that included the liver, the usual decrease in population of granules was noted by 2 hr post-irradiation (Fig. 5). This result reveals that the effect of radiations on the granules is indirect. Experimental
Cell Research
32
Mitochondrial
Fig. 3.-Mitochondria diation. Note that mark the original
from a 15-day-old chicken nearly all the intramitochondrial sites of many of these. Approx.
inclusions
embryo fixed 2 hr following 558 rads of X-irrainclusions have disappeared. Diffuse smudges 16,000 x .
A striking effect of thyroxin treatment was to cause the mitochondria to swell and become rounded. There was a concomitant decrease in inclusions per mitochondrion (Fig. 6). The same effects were noted in the case of fasting (Fig. S), but cortisone appeared to cause no decrease in mitochondrial incluExperimental
Cell Research
32
0. A. Schjeide
384
et al.
sions (Fig. 6). Although Estrogenic Substances were found to be very effective in reducing the populations of such inclusions in livers of chicks three days of age, these agents displayed lesser effects in the more resistant S-dayold chicks. In no case did these amounts of estrogens appear to induce a swelling of the mitochondria. However, in the very heavily estrogenized
h-L
L-L2 2472 hr
hr
24
hr
d
10 days incubation
l-day-old
Fig.
4.
chocks
NOX Contrd
(558 radr) Whole body
Fig.
l-i(930 radr) Liver Only
(930 radr) Liver rhielded
5.
Fig. 4.-Bar graph illustrating the duration of effect of a single whole body dose of X-irradiation. At 24 hr the inclusions are still largely decreased in numbers but by 72 hr post-irradiation they are once again well represented. An average of 62 mitochondria were counted for each bar on this graph. Probability of a difference between: 18d control and 18d x 72 hr: 0.20; Id control and Id x 24 hr: 0.001. q , control; n , 558 rads; I, std error. Fig. R.-Bar graph illustrating the lack of effect of direct X-irradiation to the liver of the 5-dayold chick on mitochondrial inclusions and the dissolving effect of whole body X-irradiation although the liver is shielded. The fact that a significant depression of liver granules was obtained by whole body X-irradiation exclusive of the liver attests to an indirect effect of X-irradiation on these bodies. An average of 319 mitochondria were counted for each bar on this graph. Probability of a difference between control and liver shielded: 0.001. I, std error.
chickens, the mitochondria appeared to be reduced in numbers, principally by virtue of a considerable distention of the endoplasmic reticulum as the cells assumed a strongly synthesizing and secretory character. Estrogens appeared to have no effect in reducing numbers of inclusions in heart mitochondria. Mitochondria of regenerating liver contained fewer inclusions than the numbers characteristic of the age group. However, inclusions were even more strongly depressed in the portion of the liver supporting the regenerating cells (Fig. 6). In the regenerating portion of the liver, the mitochondria appeared to be swollen, as in the case of thyroxin treatment. Although data on heart are not as extensive as for liver tissue, it was observed that the numbers of inclusions per mitochondrion in this organ also Experimenfal
Ccl1 Research
32
Mitochondrial
inclusions
385
increase with age (Fig. 7). In contrast to the liver, there is not a sudden decrease in these bodies at the time of hatching. However, the numbers of inclusions per mitochondrion in heart appeared to vary greatly from section to section in the same heart. In some places no inclusions were observed and in other regions the granules averaged as high as 12 per standard mitochond-
--
Days of ,ncubation
Days port-hatching
Fig.
7.
Fig. B.-Bar graph illustrating effect of various agents on liver mitochondrial inclusions of 5-dayold chicks. An average of 190 mitochondria were counted for each bar on this graph. Probability of a difference in granule content between control and regenerated mitochondria: 0.001. q , control; T, thyroxin; C, cortisone; E, estrogen; R, regenerating cells; S, non regenerating portion; F, fasting. Fig. 7.-Bar graph illustrating the general increase in heart muscle mitochondrial inclusions as a function of development and the apparent lack of an effect of either X-irradiation or estrogens on these inclusions. An average of 74 mitochondria were scanned for each bar in this graph. Probability of a difference in granule content between 7d embryo and B-day post-hatching: 0.001. q , control; w 558 rads; q estrogen; I, std error.
rion (Fig. 8). No correlation was obtained between numbers of mitochondrial inclusions and degree of contraction of the heart fibers, nor could any effects of X-irradiation be noted on the inclusions present in heart mitochondria. In rabbit liver cells the mitochondria were sometimes observed to have aggregated in specific sites in the cell (in both control and experimental animals). Such clumping was not observed in chickens until two weeks posthatching. The results of de-staining some of the osmium fixed sections with H,O, [3] indicate that a portion of the mitochondrial granules may be either lipid or metal or both. As shown in Fig. 9, when the osmium is removed from the sections, a definite de-staining of the inclusions takes place to an even greater extent than the lipid-containing mitochondrial membranes and to about the same extent as the lipid borders of fat vacuoles. If most of the reactive sites Experimental
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0. A. Schjeide
Fig. 8.-Mitochondria and manner in which
from heart mitochondria
et al.
muscle of 14-day-old chick. Xote high are aligned between muscle fibers.
concentration of granules Approx. 23,000 Y .
were occupied by cations, these might be removed by the destaining treatment. However, on the basis of physical considerations it seems unlikely that the cations would be either K+ or Na+ since an unreasonable packing of such atoms would have to be assumed to provide the electron density observed. In an attempt to ultracentrifugally float the granules on an 8.8 per cent NaCl solution of mitochondria that had been disintegrated by ultrasonic vibration, it was observed that these granules displayed a strong tendency to affix to whatever mitochondrial debris might float under such conditions. A crude index of the physical nature of the granules in the fixed condition was provided by sections of incompletely cured tissue. The inclusions appeared to be sufficiently compact so as to be readily pushed about by the glass knife-edge Experimental
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Mifochondrial
inclusions
Fig. 9.-Mitochondria from the liver of a 5-day-old chick which have been tion of 15 H,O,. Note that the granules appear as bleached spots that are the external mitochondrial membranes. The critical question is whether this (of lipid?) in the case of the inclusions or extraction of originally occurring Xpprox. 40,000 X .
de-stained by applicasomewhat lighter than is actually de-staining heavy metal, or both.
but soft enough to leave a granular trail in their wake.1 Under the influence of estrogens and X-irradiation the inclusions often present a diffuse periphery surrounding a less-than-normal size solid granule center. In grossly 1 Nass and Nass [5] refer to osmium reactive rods and fibers within the mitochondria chicken embryos. Such tissue is difficult to fix and this fact, together with the lack afforded by a dense matrix of cristae, could lead to the creation of artifacts. Experimental
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damaged mitochondria they appear to remain intact somewhat longer do the cristae. Supplementary staining with lead does not detectably counts of inclusions.
than alter
DISCUSSION
The intra-mitochondrial granules do not appear to be absolutely essential for mitochondrial integrity or function since they are absent from many early mitochondria and are present in variable numbers in others, being apparently greatly influenced by prevailing metabolic conditions. However, the rapid disappearance of most of these granules as an indirect effect of X-irradiation and during the initial period of hatching, leads to speculation regarding possible vital roles. Ito [ 1] has shown that the intramitochondrial granules of the mitochondria present in parietal cells of the fasting bat disappear as early as 11 min after an initial intake of food. (There is a gradual reappearance of these particles with time.) Although Napolitano [4] observed that when fat cells were depleted of lipid there was actually an accumulation of intramitochondrial bodies, a subsequent increase of lipid in the cells was, as in Ito’s experiment, accompanied by decreases in numbers and sizes of granules to normal values. The behaviors of the mitochondria in these respects are consistent with the idea that factors, elicited by a variety of stimuli, call forth the materials contained within the granules as a homeostatic response. The nature of the inducing agents and the requirements and actions of the mitochondria, as well as the material itself, all remain to be elucidated. However, if the inclusions were simply stockpiles of energy-providing materials it would be expected that they would be largely absent under conditions of accelerated growth or fasting and while they are, indeed, sometimes reduced under such conditions, significant numbers are usually present. An alternative to the above theory of “demand and supply” is suggested by Weiss’s observation that granules within the mitochondria of duodenal absorptive cells, from rats fed large amounts of sodium or potassium, are somewhat increased [12]. If the granules are actually storage sites for excess Na+ or K+, as Weiss has concluded, then the effect of the various degranulating agents discussed may be one of disrupting or damaging the mitochondrion, causing a breakdown of the storage system. In such a case, however, it would be expected that the mitochondria would be further damaged by the release of these high concentrations of cations, which they are not.1 Also, it 1 In neither the heart nor the liver of the embryo to directly change mitochondrial numbers and volumes Experimental
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fowl does 558 rads of X-irradiation appear or the numbers or geometries of the cristae.
Mifochondrial
inclusions
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is difficult to explain on such a basis the partial effect of these agents on the liver mitochondria of the five-day-old chick and the complete lack of effect of X-rays or estrogen on mitochondria of heart muscle. More consistent with the available evidence is the notion that the partial degranulation of the mitochondria of 5-day-old chicks following X-irradiation is a reflection of the amount of such material required to carry out a given function under the circumstances. The naturally occurring decrease in intramitochondrial granules of liver mitochondria in the newly hatched chick further suggests that the material in the granules is useful and can be tapped as required. Chemical and physical features.-The conclusion that the granules may contain lipid is based on the observation that treatment of osmium-stained sections with H,O, (which oxidizes the osmium so that it can be rinsed away) results in de-staining of the granules to the same extent as the lipoproteins remaining in the borders of the lipid vacuoles (Fig. 9). As Merriam [3] has pointed out, the ability to react with osmium is greatest in lipids (unpublished results indicate that this is a function of unsaturation). Proteins have a comparatively modest ability to react with osmium and show a lesser change with bleaching. Nucleic acids do not react with osmium to any appreciable extent but have a high intrinsic density. Some metals would also appear to be associated with the granules because they absorb electrons in the absence of treatment with heavy-metaI-containing fixatives or stains. Formalin fixed preparations suggest the presence of granules but less contrast is observed than in osmium fixed preparations. This lesser density may be due to the presence of relatively small amounts,of metals with the additional contrast arising from staining being due to the presence of points of unsaturation in such materials as Na oleate, etc. Better isolation procedures are currently being developed so that more definitive approaches can be employed to interpret the chemistries, roles, and origins of the intramitochondrial granules.
SUMMARY
In general, mitochondrial inclusions increase in numbers with the stage of development of the cell. However, certain metabolic conditions, such as those induced by X-irradiation (indirect effect), hatching, starvation, thyAll cell divisions occur at nearly normal rates during the period of concern and cell destruction is negligible. Although there is a slight decrease in the volumes of mitochondria per unit volume of tissue in the liver as development proceeds, this is due to the increased volume of fatty globules to the point of hatching. X-irradiation causes an additional decrease in mitochondrial volumes, but this is due to a failure of the newly dividing cells to produce as many new mitochondria 191. Experimental
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et al.
roxin treatment, increase of work load and treatment with estrogenic suhstances, result in rapid dissolution of the granules in the mitochondria of liver (but not in heart mitochondria). These findings suggest a similar response of the liver mitochondrion to a variety of original stimuli. It is considered that this response may be triggered by similar homeostatic signals to the liver cell under conditions which can vaguely be described as “stressful”. The mitochondrial inclusions may consist of substances which are of unique use to the mitochondrion or cell under such conditions.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12.
ITO, S., Abstract 2nd Annual Meeting Am. Sot. for Cell. Biol. p. 78, 1962. LUFT, J. H., J. Biophys. Biochem. Cyfol. 9, 409 (1961). MERRIAM, R. W., J. Biophys. Biochem. Cyfol. 4, 579 (1958). NAPOLITANO. L., ibid. 8, 129 (1962). NASS, M. M.‘K. ‘and NA& S.; Ezpil Cell Res. 26, 424 (1962). NOVIKOFF, A. B., in The Cell, vol. 2, p. 299. J. BRACHET and A. E. MIRSKY (eds.) Academic Press, New York, 1961. PALADE. G. E.. Subcellular Particles. vol. 64. T. Havashi (ed.) Ronald Press. New York, 1959. RHODIN; J., Correlation of Ultrastructural Organ&ion and Function in Normal and Experimentally Changed Proximal Convoluted Tubule Cells of the Mouse Kidney. Aktiebologet Godvil, Stockholm, 1954. SCHJEIDE, 0. A., RAGAN, N., MCCANDLESS, R. G. and BISHOP, F. C., Radiation Res. 13, 205 (1960). SCH&DE, b. A., MCCANDLESS, R. G., MYERS, JR. L. S., and HENNESSY, T. G., Abstracts International Biophysics Congress, vol. 100, Stockholm, 1961. SJBSTRAND, F. S., Personal communication. WEISS, J. M., J. Expfl Med. 102, 783 (1955).
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