THE AMERICAN JOURNAL OF ANATOMY 188:269-281 (1990)

Giant Mitochondria Distinct From Enlarged Mitochondria in Secretory and Ciliated Cells of Gerbil Trachea and Bronchioles S.S.SPICER, R.T. PARMLEY, L. BOYD,

AND

B.A. SCHULTE

Department of Pathology and Laboratory MedLcine, Medical University of South Carolina, Charleston, South Carolina 29425 (S.S.S., B.A.S.); Department of Pediatrics, University of Texas Health Science Center at S u n Antonio, S u n Antonio, Texas 78284 (R.T.P., L.B.)

ABSTRACT Numerous mitochondria ranging from slightly larger than normal to several micrometers in diameter (giant) were found in about one-half the serous secretory cells in the surface epithelium of the normal gerbil trachea and proximal bronchi. Tracheal serous cells of mice also were found to contain numerous giant mitochondria. Clara cells of gerbil bronchioles contained abundant giant mitochondria in addition to normal tubular mitochondria and the second population of enlarged spherical mitochondria that have been described in Clara cells of several genera. In contrast, mouse Clara cells revealed the normal tubular and the enlarged spherical mitochondria but no giant mitochondria. A survey of a number of cell types in gerbils failed to disclose hypertrophied mitochondria outside tracheobronchial surface epithelium and bronchioles. The mitochondrial enlargement resulted from an increase of matrix but not cristae. The expansion of matrix displaced the relatively sparse cristae into small collections compressed against the outer membrane. The prevalence of giant mitochondria and of granular endoplasmic reticulum is similar among cells, and these two organelles are codistributed within cells. The megamitochondria and granular reticulum occupy a central stratum, whereas normal mitochondria occur in the apical and basal regions. The giant mitochondria are considered related to a normal biologic activity that is characteristic of respiratory tract epithelium of mice and gerbils selectively and is more prominent in secretory cells than in ciliated cells.

chondrial enlargement has been ascribed to a reaction to drugs such as cymetidine (Schwartz et al., 1986), propylthiouracil (Aguas et al., 19811, cuprizone (Suzuki, 19691, and other electron-donating ammonia derivatives (Wakabayashi et al., 1984). Mitochondrial gigantism has been recorded also as a manifestation of aging (Murakoshi et al., 1985) and of several diseases, including acute lymphocytic leukemia (Iwama and Eguchi, 1986; Eguchi et al., 19871, thyrotoxicosis (Korenyi-Both et al., 1981), radiation carditis (Maeda, 1982), hyperlipoproteinemia (Gariot et al., 1987), and submandibular gland adenoma (Tandler and Erlandson, 1983). On the other hand, the occurrence of giant mitochondria has been observed in apparently normal cells, such as the A cells of lizard pancreas (Mattei et al., 1985), postovulatory (Cornillie et al., 1985) and progesteronephase (Secchi and Lecaque, 1984) endometrial epithelium, bone-marrow macrophages (Enomoto and Watanabe, 1985), and certain retinal cones (Kuhne, 1983). Mitochondrial dimorphism exists in the bronchiolar Clara cells, which in some species but not in others contain a population of normal-sized tubular mitochondria and a second population of larger, more spherical mitochondria apparently unique to these cells (Karrer, 1956; Petrik and Collet, 1970; Stinson and Loosli, 1978; Plopper e t al., 198Oa-c). The larger variant often predominates in Clara cells and reaches 0.5-1.0 pm in diameter depending on the animal, but it does not attain the several times greater size characteristic of giant mitochondria. The present study adds to knowledge of giant mitochondria in normal cells. These mitochondria show a n unusual increase of matrix that leads to extreme mitochondria1 enlargement in secretory and, to a lesser extent, also in ciliated cells of surface epithelium in gerbil trachea and bronchioles.

INTRODUCTION

MATERIALS AND METHODS

Mitochondria display wide variations in structure under diverse physiologic or pathologic conditions (for reviews, see Munn, 1974; Carafoli and Roman, 1980; Ghadially, 1988). One type of structural variation entails a n increase in size, yielding giant mitochondria with profiles several times larger than normal. Mitochondrial enlargement has been described in a range of abnormal conditions. Among these are experimentally induced states such a s deficiency of riboflavin or essential fatty acids (Wilson and Leduc, 1963; Tandler et al., 1968; Kobayashi et al., 1980), consumption of a high-protein diet (Zarasoza et al., 1987), and hypopituitarism (Nickerson, 1987). In addition, mito-

Five adult male gerbils (Meriones unguiculatus) were purchased from Tumble Brook Farms (West Brookfield, MA) and housed under sanitary conditions a t the University of Texas School of Medicine, San Antonio. Six young-adult gerbils and two C57 M u s domesiticus mice were obtained from colonies bred and housed in animal facilities a t the Medical University of South Carolina.

G 1990 WILEY-LISS, INC.

Received J u n e 6, 1989. Accepted January 29, 1990

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The animals were sacrificed by pentobarbital injection intraperitoneally. Tracheas were removed promptly and fixed for 1h r in neutral buffered 1%glutaraldehyde solution followed by 1 h r in 1%osmium tetroxide. For cytochemical demonstration of cytochrome oxidase, the osmium tetroxide fixation was omitted, and finely minced specimens or cryosections were incubated overnight in substrate medium lacking H202and then for 30 min in medium containing H202. The Graham and Karnovsky (1966) pH 7.6 substrate medium was employed except for substituting 5 mg of diaminobenzidine (DAB) tetrahydrochloride for 3 mg DABilO ml solution. All specimens were then dehydrated routinely through graded alcohols and embedded in Epon 812. To demonstrate glycoconjugates cytochemically, a n aliquot of some specimens was fixed with glutaraldehyde, dehydrated, and embedded in Epon, and thin sections were stained with the periodic acid-thiocarbohydrazide-silverproteinate (PA-TCHSP) sequence (Thiery, 1967). Ultrathin sections of morphologic preparations only were stained with uranyl acetate-lead citrate. Thin sections were examined in a Jeol 100s electron microscope. To estimate approximately the proportion of cells possessing enlarged mitochondria in gerbil trachea, the total numbers of secretory and of ciliated cells in ultrathin sections were counted, as were the numbers of each type possessing large mitochondria. Several grids were examined to acquire a cell population exceeding 100. For assessing the prevalence of enlarged mitochondria in gerbil bronchi and mouse trachea, toluidine blue-stained thick sections were examined, and the proportion of cells with the clearly discernible large mitochondria was determined. RESULTS Gerbil Trachea and Bronchi

Serous secretory cells in the surface epithelium of the gerbil trachea revealed mitochondria of increased size. Cell counts testified to the presence of such altered mitochondria in about one-half of the serous cells (Table 1).The prevalence of cells with atypical mitochondria differed little between animals or regions of the trachea, but such cells appeared unevenly distributed within a section. The mitochondrial alteration was clearly visible in the toluidine blue-stained thick sections, and its prevalence in these sections was comparable to that estimated ultrastructurally. The megamitochondria generally appeared grouped together in a midregion of the cell near, and more often above than below, the nucleus (Fig. 1).Thus the distribution of megamitochondria in the cell differed from

TABLE 1. Distribution of giant mitochondria in the gerbil’s trachea

Organ Upper and lower trachea

Histologic site Surface epithelium

Cell type Nonciliated Ciliated

Approximate percent of cells with enlarged mitochondria 45 < 10

that of the more apically or basally located and more numerous normal mitochondria. The size of altered mitochondria varied from slightly enlarged up to 6 pm in greatest dimension (Figs. 1-6). Profiles of giant mitochondria differed in contour from round to oval to irregular, often with rounded or sharp protrusions, but showed no indication of mitochondrial fusion or division. In cells with large mitochondria, the prevalence of those that were increased in size ranged from one or two per cell profile to approximately onefourth of the population. The atypical mitochondria were mainly of intermediate size in some cell profiles and were mainly giant-sized in others, but they covered a continuous range from normal to gigantic in occasional cells. Expansion of the matrix compartment but not the intermembrane space accounted for the mitochondrial enlargement. In the less affected organelles, increased matrix separated apparently normal cristae that protruded into the center (Figs. 3, 5). The greatly increased matrix of giant mitochondria, however, compressed a few membranous structures interpreted as cristae into the accumulations located along the outer membrane (Figs. 4,6). The expanded matrix consisted of uniform finely fibrillar material that was somewhat denser than t h a t of normal-sized mitochondria and often enclosed widely scattered, dense matrix granules (Fig. 6). An abundance of rough endoplasmic reticulum characterizcd the serous secretory cells containing enlarged mitochondria (Figs. 1-6). Profiles of these cells ranged from the ones filled with granular reticulum and reminiscent of plasma cells, to those containing abundant reticulum centrally, to others with sparse rough endoplasmic reticulum. Cells with a great prevalence of this organelle commonly possessed larger than normal mitochondria. However, some cells with megamitochondria, particularly those with only one or two such organelles, contained little rough reticulum. Within a cell profile, the granular reticulum lay congregated mostly in the region enclosing variably enlarged mitochondria and closely bordered or encircled them (Figs. 1-6). Distribution of the reticulum did not correspond to that of apically or basally distributed normal mitochondria. Nonciliated cells with megamitochondria frequently possessed a few secretory granules measuring 0.4-0.6 pm in diameter and lying under the apical plasmalemma (Figs. 1-3, 5, 6). Some profiles of nonciliated tracheal surface cells lacked granules, whereas others enclosed numerous granules, generally in apical cytoplasm. Secretory granules in the latter cells often appeared large, reaching 0.7-1.0 pm in diameter. A few bodies interpreted as lysosomes from their heterogeneous content were observed in the periphery of some secretory cells (Figs. 3 , 5). Occasional ciliated cells disclosed several 0.20.5-pm-wide mitochondria surrounded by more numerous normal mitochondria in the supranuclear area. Infrequently, ciliated cells enclosed a megamitochondrion in the range of 2.5 pm bordered by less enlarged or normal neighbors (Fig. 7). Tracheal ciliated cells contained fewer by far of the megamitochondria than did nonciliated cells (Tables 1, 2), and the degree of enlargement was less in ciliated cells. Accumulated

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Figs. 1-7. Surface epithelium of gerbil trachea processed for routine electron microscopy. Thin sections were stained with uranyl acetatelead citrate.

of the secretory cells. The secretory cells contain a moderate number of apical secretory granules and abundant granular reticulum. Cytoplasm of one deep cell with large mitochondria consists mainly of rough endoplasmic reticulum. X 12,500.

Fig. 1. Ciliated cells with clear cytoplasm flank dense secretory cells in gerbil trachea. Numerous moderately to extremely enlarged mitochondria (arrows) with abundant dense matrix lie close above, and less often beside or below, the nucleus in the secretory cells. Normalsized mitochondria populate ciliated cells in apical and basal regions

Fig. 2. An enlarged mitochondrion invested with granular reticulum lies between mitochondria on the left and numerous secretory granules on the right. x 22,000.

dense matrix compressed sparse cristae against the outer membrane in ciliated as in secretory cells. Cytochemical localization of cytochrome oxidase helped to identify the giant bodies in the tracheal epithelium a s mitochondria. The membranous profiles that lay compressed against the outer border and that were interpreted a s vestigial cristae evidenced strong cytochrome oxidase activity (Figs. 8, 9). The unstained

matrix appeared to extend to the unreactive outer membrane between collections of cristae in the megamitochondria. Isolated compartments of inner mitochondrial membranes rich in cytochrome oxidase thus gave the impression of lying unconnected by inner membrane at intervals along the outer membrane. This perception differed, however, from that gained from morphologic examination, which evidenced inner

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Fig. 3. Granular reticulum crowds cytoplasm in secretory cells containing some mitochondria of normal or slightly increased size (M) and others that are moderately to extremely enlarged (arrows).Norma1 mitochondria lie in the apical cytoplasm and interspersed with those showing expanded matrix deeper in the cell. One cell contains a central Golgi complex (G)and sparse secretory granules ( S )under the apical plasmalemma. Mitochondria appear normal in the ciliated cell a t upper left. x 15,000.

Fig. 4. Cisternae of granular reticulum intervene between and border closely three hypertrophied mitochondria. That in the upper right shows peripheral membranous profiles interpreted as cristae. x 56,050.

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273

Fig. 5. Secretory cells contain mitochondria that are expanded to varying degrees by dense matrix, which compresses sparse cristae against the outer membrane. The cell a t the bottom consists mainly of granular reticulum except for the variably sized mitochondria, a Golgi complex (G),and a few secretory granules (S). In another secre-

tory cell, megamitochondria (arrows)are codistributed with the rough endoplasmic reticulum. Normal appearing mitochondria in the apical cytoplasm are not associated with an abundance of reticulum. Sparse secretory granules border the apical plasmalemma. A ciliated cell (upper right) contains normal-sized mitochondria. x 15,000.

and outer membranes together encompassing the mitochondria’s full circumference, possibly reflecting the absence of the oxidase from inner membrane between

cristal infoldings. In support of the latter explanation, occasional mitochondria disclosed light oxidase activity in some but not other segments of inner mem-

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Fig. 6. Pleomorphic mitochondria exhibiting expanded matrix; sparse, peripherally displaced cristae (arrows);and scattered dense matrix granules occupy a region of a secretory cell that is also rich in rough endoplasmic reticulum. A secretory granule (S) underlies the apical plasmalemma. x 30,000.

brane between cristae (Fig. 10). Reactivity in the cytochrome oxidase preparations was confined to cristae of normal or atypical mitochondria except for staining of infrequent peroxisomes (Figs. 8, 9). The PA-TCH-SP method demonstrated glycoconjugate-containing sugars with periodate-reactive uic glycols in secretory granules and Golgi cisternae of secretory cells. The matrices of normal and enlarged mitochondria failed to stain, however (Fig. 11). In the gerbil’s major bronchi, serous secretory cells exhibited giant mitochondria (Table 2). The degree of alteration of the organelles was judged to be somewhat less than in trachea. Some smaller, apparently more distal bronchial profiles lacked the atypical mitochondria. Gerbil Bronchioles

Clara cells in the gerbil’s distal airway contained both the normal-sized tubular and the larger spherical mitochondria found in a number of species (Table 2) (Plopper et al., 1980a-c). In many Clara cell profiles, the majority of the mitochondria were moderately en-

TABLE 2. Incidence of enlarged mitochondria

Animal Gerbil

Cell type Tracheal surface serous cells Tracheal ciliated cells Bronchial surface serous cells Bronchiolar Clara cells Bronchiolar ciliated cells

Enlarged Giant spherical mitochondria mitochondria Frequent Infrequent

Absent Absent

Frequent

Absent

Frequent

Predominant

Infrequent

Absent

Frequent Absent

Absent Absent

Absent

Predominant

Infrequent

Absent

Mouse Tracheal surface serous cells Tracheal ciliated cells Bronchiolar Clara cells Bronchiolar ciliated cells

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275

Fig. 7.A tracheal ciliated cell contains normal mitochondria in the most apical cytoplasm. Beneath them are a megamitochondrion and mitochondria with slight expansion of matrix and peripheral displacement of cristae. Ciliogenesis (arrow) is evident somewhat below the cilia’s basal bodies. x 25,000.

Fig. 8. The intermembrane space stains selectively for cytochrome oxidase in both normal and expanded mitochondria in surface epithelium of gerbil trachea. Membranous structures a t the periphery of two enlarged mitochondria (arrows) are identified by their staining as remnants of cristae. Secretory granules (S) are electron lucent and devoid of enzyme activity. x 20,000.

larged and approximately spherical. These mitochondria appeared somewhat larger than in other species, measuring up to 2 pm in diameter. Gerbil Clara cells, however, frequently contained a number of larger mitochondria measuring from 2-3 pm across. Other commonly encountered cells exhibited usually only one, but occasionally two or more, giant mitochondria whose greatest dimension reached 4-7 pm (Fig. 12). From analysis of stained thick sections, the prevalence of cells in the latter two categories approximated 50%. A profile of a telophase mitotic cell contained a megamitochondrion, demonstrating persistence of such organelles through more than one cell generation (Fig. 12). Ciliated cells in gerbil bronchioles as a rule enclosed mitochondria of normal size but infrequently contained a megamitochondrion (Fig. 13, Table 2). The latter

commonly lay close above the nucleus and had a diameter of about 2.5 pm. Common morphologic characteristics of the cell type were observed in some but not all of the gerbil’s Clara cells. These included protrusion at the apex into the lumen, abundance of smooth endoplasmic reticulum, and a content of small dense secretory granules that in this species measured 0.25-0.5 pm in diameter and occasionally revealed a relatively large eccentric core. The granules were numerous and widely disseminated in the apical half of some Clara cells (Fig. 12) and were sparse and confined to cytoplasm under the apical plasmalemma in others. Notably, some Clara cell profiles in the gerbil contained abundant elongated cisternae of granular reticulum, often associated with large mitochondria, whereas others revealed numerous free polysomes or short segments of rough endoplasmic reticu-

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Figs. 9-1 1.

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Figs. 12-14. Uranyl acetate-lead citrate stained thin sections of routinely processed specimens.

telophase (arrowhead). The dense cytoplasmic granules (thin arrows) widely distributed in these gerbil bronchiolar nonciliated cells differ from those in tracheal serous cells (cf. Figs. 1-3, 5, 6 ) . x 4,500.

Fig. 12. Two Clara cells at the left enclose megamitochondria (thick arrows), as does one of the nonprotruding daughter Clara cells in

lum in cytoplasmic areas infiltrated with mitochondria. Cells Outside the Respiratory Tract in Gerbils

A wide range of cells in various gerbil tissues provided no evidence on ultrastructural examination for a n increase in the size of mitochondria. Cells found not to show such mitochondrial alteration included fibroblasts, histiocytes, mast cells, endothelial cells, hepatocytes, central nervous system neurons and glia, and cells in proximal tubules of the kidney and serous acini of the submandibular gland. Mouse Trachea and Bronchioles

Variably sized megamitochondria, measuring up to 6 pm in diameter, occurred in the serous cells of mouse trachea (Fig. 14, Table 2). The enlarged organelles re-

Fig. 9,lO. Cytochrome oxidase activity in surface epithelium of gerbil trachea. Fig. 9. Dense reaction product localizes cytochrome 0x1dase to the outer compartment of mitochondria disclosing rearranged cristae and variably expanded matrix. The matrix accumulation appears to extend to the outer mitochondrial membrane and disrupt the inner membrane, separating cristal elements into separate compartments. More likely, inner membrane connects such compartments as seen in morphologic preparations but often lacks the oxidase (cf. Fig. 11).Microperoxisomes (arrows)stain intensely. x 40,000. Fig. 10. Isolated cristal elements in a megamitochondrion evidence cytochrome oxidase, as does inner membrane connecting some (arrow) but not other cristae. x 42,000. Fig. 11. Secretory granules tS) and Golgi lamellae tG) reveal PATCH-SP staining demonstrative of glycoconjugate in the surface epithelium of gerbil trachea. Normal mitochondria (M)and a giant mitochondrion (arrow) fail to stain. x 40,000.

sembled those in lhe gerbil in lhe increase of rrialrix and paucity of cristae. The prevalence of megamitochondria, estimated from examining thick sections, appeared roughly comparable in the mouse to that determined in the gerbil trachea. Abundant granular reticulum was co-distributed with enlarging mitochondria in most of the serous cells that possessed giant mitochondria. Clara cells observed in epoxy sections of mouse bronchioles lacked giant mitochondria (Table 2). At the ultrastructural level, the Clara cells’ mitochondria varied in size in accordance with the described tubular and larger spherical populations but did not reach the giant size, greater than 2 pm, commonly observed in mouse tracheal secretory cells. DISCUSSION

A predominant population of mitochondria in Clara cells of mouse, guinea pig, rabbit, and cat enlarges to 0.2-1.0 pm across and becomes spherical from excess accumulation of matrix (Karrer, 1956; Plopper, 1980ac). However, Clara cells possessing organelles that measure several micrometers in diameter and qualifying as giant mitochondria have not been reported in the above-mentioned animals and were not found here in mice. Because of their greater size and a distribution restricted to gerbils and mice, the giant mitochondria are judged to represent a form of atypism different from that underlying the larger mitochondria in Clara cells of several genera. Moreover, the absence of the predominant population of enlarged spherical mitochondria from the megamitochondria-laden tracheal serous cells of mice and gerbils and ciliated cells of gerbils further indicates that two types of alteration affect this organelle. Gerbil Clara cells differ from those of other

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Fig. 13. A ciliated cell in a gerbil bronchiole encloses a giant mitochondrion lying close above the nucleus. Some neighboring mitochondria show an increased matrix: crista ratio. x 14.000.

animals in containing both the enlarged spherical pop- tracheal and bronchiolar epithelium of all gerbils exulation and the giant mitochondria. The biologic sig- amined and the absence of light or electron microscopic nificance presumably differs then for the giant mito- changes indicative of pathology in the animals’ respichondria of gerbils and mice and for the moderately ratory tract justify interpreting the increased mitoenlarged mitochondria in Clara cells of several animal chondrial size a s a normal event in the biology of the affected cells. Development of these distinctive strucclasses. The megamitochondria of gerbil trachea differ mor- tures therefore relates not to abnormal genesis as in phologically from those referred to in the Introduction pathologic conditions but to a nonpathologic adaptaa s occurring in various physiologic, experimental, or tion unique for the respiratory tract of gerbils and pathologic conditions (for reviews, see Munn, 1974; Ca- mice. The megamitochondria appear not to perform the rafoli and Roman, 1980; Ghadially, 1988). An increased matrix compartment, which typifies the alter- conventional role of mitochondria in respiration. An ation described here, has been reported for some abundance of cristae correlates with increased adenomegamitochondria in abnormal but not in normal cells sine triphosphate (ATP) production or heat release a s (Wilson and Leduc, 1963; Thoenes, 1966; Suzuki, 1969; exemplified by mitochondria of cells in thyrotoxic aniGhadially and Skinnider, 1974; Aguas et al., 1981; mals (Korenyi-Both et al., 1981)and of brown fat cells Tandler and Erlandson, 1983). Moreover, the occur- in the newborn rodent (Suter, 1969). The paucity of rence of greatly enlarged mitochondria throughout the cristae and excess of dense matrix in the tracheal

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which, in general, are devoid of other Ca2+-transporting organelles. Mitochondria influence free cytosolic Ca2 by setting a point at which to maintain its level (Murphy, 1986). Mitochondria of secretory cells differ from those of many other cells in mediating Ca2 efflux via a n N a + - H + exchanger and Na’ efflux via a n Na /H antiporter (Carafoli and Roman, 1980). The mechanisms in mitochondria for sequestering cation suggest a connection between expansion of the matrix component in giant mitochondria and a n influence on cytosolic cation levels. A role of the large mitochondria in affecting the serous cell’s secretory mechanism, in addition to whatever part they play in ciliated cells, would be consistent with the greater mitochondrial change in the secretory than in the ciliated cells. Whether the mitochondrial alteration has the same physiologic connotation for the gerbil’s tracheal serous cells a s for Clara cells remains a question. Because the volume density of Clara cells in a n animal exceeds that of tracheal secretory cells, the mitochondrial change in Clara cells perhaps makes a greater functional contribution to the animal. The biologic basis for the mitochondrial hypertrophy in gerbils apparently relates to physiologic activity characteristic of the tracheal and bronchiolar cells, which alone contained the altered organelles. Although merocrine secretion presumably constitutes the predominant function of tracheal serous cells and Clara cells, profiles of these cells frequently display few or sometimes no stored secretory granules. Many of the cells accordingly appeared inactive in secretion of macromolecules, and conceivably they perform other functions. Since ciliated cells occasionally exhibited mitochondrial hypertrophy, this phenomenon is, in part at least, unrelated to merocrine secretion and is assumed to concern a n activity common to ciliated and serous cells but more prominent in the latter. Transport of fluid and ions across the epithelium is shared by these ciliated and secretory and most other epithelial cells. Fluid transport by the respiratory tract epithelium counters the drying effects of the inspired air and maintains mucociliary flow. Increased mitochondrial matrix in these cells possibly concerns storage of a component associated with fluctuations in such transport. Tracheal epithelium of man and rat (unpublished observations), on the other hand, and Clara cells of several species so far examined (Plopper et al., 1980a-c) lack megamitochondria. The giant mitochondria of the gerbil’s respiratory tract possibly arise then in connection with a n exceptional biologic activity related to the habits and environment of this desert animal. Nocturnal foraging and daytime underground habitat expose the gerbil to cycles that periodically require a n increased capacity to warm, moisten, and filter inspired air. Uncoupling of oxidative phosphorylation in mitochondria could provide a thermogenic mechanism serving the host animal in its adaptation to cold night air. Brown fat cells in neonatal or cold-adapted rodents specialize in nonshivering thermogenesis (Nicholls and Locke, 1984; Trayburn and Nicholls, 1986). However, mitochondrial atypism in gerbil lung appears unrelated to heat generation, because the altered mitochondria in the gerbil pulmonary cells differ dramatically from the uniform mitochondria containing numerous, +

+

+

Fig. 14. A serous cell in mouse trachea contains a giant mitochondrion (M) above the nucleus. X 8,400.

megamitochondria conversely reflect diminished respiration and a lowered level of oxidative metabolism. The dearth of cristae in these organelles indicates that the matrix components act other than to provide respiratory substrates, a s would be the case if they consisted of Krebs cycle enzymes. The accumulation of matrix material suggests rather that the altered organelles serve in storing components in the matrix. Storage of a matrix constituent, analogous to that of secretory product in a zymogen cell granule, would facilitate fluctuation of a cell activity, but storage has not been considered a mitochondrial function. Information concerning the substances accreting in the matrix could provide insight into what cell function mitochondrial enlargement serves. The membrane interactions mediating exocrine secretion along with many other cell activities depend on Ca2+ fluxes (Carafoli, 1976). Storage of calcium, possibly facilitated by calcium binding protein, therefore, comes to mind. Mitochondrial matrix is known to sequester cations a s evidenced by the heavy iron deposits demonstrable ultrastructurally a t this site in reticulocytes of patients with sideroblastic anemia (Ghadially, 1988).Moreover, mitochondria accumulate and retain calcium (Rasmussen e t al., 19651, and calcium (Nickerson, 1987) and calmodulin (Hatase et al., 1985) have been localized in mitochondria. Mitochondria are considered the main Ca2+ sink in epithelial cells (Carafoli, 1976)

+

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long cristae and sparse matrix in brown fat cells (Suter, 1969). Some other cyclic cellular adaptation presumably underlies the mitochondrial alteration. Growth of mitochondria through synthesis of protein coded by nuclear or mitochondrial DNA results in mitochondrial division (Tandler et al., 1970) or in increased size when division is impaired, as in experimental fatty-acid deficiency (Wilson and Leduc, 1963). Fusion also underlies formation of giant mitochondria in some pathologic conditions (Kraus and Cain, 1980), such as riboflavin deficiency (Tandler and Hoppel, 1980). The extremely large mitochondria shown here apparently occur from continued acquisition of matrix, without a n increase of cristae. Such a n imbalance is assumed to preclude mitochondrial division. A mechanism for the progressive accretion of matrix protein underlying the hypertrophy is indicated by frequent intimate association with granular reticulum and perhaps free polysomes. A close spatial arrangement between rough endoplasmic reticulum and mitochondria exists in many cells where a contribution of mitochondrial ATP to energy requirement for protein synthesis explains the association. Paucity of cristae in the gerbil's enlarging mitochondria casts doubt, however, on the importance of ATP generation for their codistribution with rough endoplasmic reticulum. Most serous cell profiles showing growing mitochondria, moreover, possess scant secretory granules and sparse Golgi cisternae, and they appear not to be forming export protein with all the abundant granular reticulum investing the enlarging mitochondria. Prevalence of the ergastoplasm correlates strikingly with that, of megamitochondria among cells, and the two types of organelles are distributed together within the cell. Such a structural relationship implicates the rough endoplasmic reticulum in producing the matrix protein that expands the neighboring mitochondria. The majority of mitochondrial proteins and apparently most of those in the matrix are coded by nuclear DNA, only 20% or so depending on mitochondrial DNA. The extrinsic proteins are thought to be translated on free cytoplasmic polysomes and have been shown to possess N terminus precursor peptides that signal penetration of mitochondrial membranes (see Attardi and Schatz, 1988; Hartl et al., 1989). The present unorthodox suggestion that bound ribosomes produce mitochondrial proteins requires a n additional mechanism for nascent protein to exit the lumen of the granular reticulum. If the precursor sequence was not cleaved by a protease following its release into the lumen of the cisterna, the sequence conceivably could mediate exit a s it did entry of the protein across the reticulum membrane. The present observations shed little light on the fate of the giant mitochondria. Their disposal cannot readily be ascribed to autophagy, because the hyperplasia of lysosomes that would be expected for digestion of such prevalent organelles was not observed, and the few lysosomes present revealed no structural relationship to mitochondria. The observation of a megamitochondrion in a mitotic Clara cell demonstrates that large mitochondria survive from one cell generation to the next and attests to their lonaevitv. The sharing of mitochondrial gigantism exclusively by the tracheobronchial serous cells and Clara cells in I

"

gerbils prompts the consideration that these are the same or closely related cell types. In gerbils, Clara cells resembled the tracheal serous secretory cells with large mitochondria in many morphologic features but differed in others. Clara cells often bulged into the lumen. They generally contained less granular and more smooth endoplasmic reticulum than the tracheal cells and more free polysomes and short segments of granular reticulum. Some Clara cells differed from the tracheal cells in enclosing numerous dense, 0.25-0.5 km granules widely dispersed in the apical half of the cell. Nevertheless, such morphologic variability existed among the Clara and among the tracheal serous cells that these two cell types cannot be judged identical or dissimilar by morphologic criteria alone. Cytochemical evidence against a common identity for these cell types derives from the failure of human tracheal epithelium to immunostain with a n antiserum that localizes a 10 kD antigen isolated from human lung lavage fluid specifically to Clara cells (Singh et al., 1988). The demonstration of giant mitochondria in tracheal but not bronchiolar cells of mice in this study further differentiates the two cell types. ACKNOWLEDGMENTS

We thank Mrs. Christina Smith and Mrs. Torie Hasher for skilled technical assistance and Mrs. Leslie Harrelson for valued editorial contributions. LITERATURE CITED Aguas, A.P., M. Carlota-Proenca, and J. Martins e Silva 1981 Giant mhrhondria in rat liver cells after short-term administration of propylthioracil. J . Submicrosc. Cytol., 13t385-390. Attardi, G., and G. Schatz 1988 Biogenesis of mitochondria. Annu. Rev. Cell Biol., 4.289-333. Carafoli, E. 1976 Mitochondria1 calcium transport and calcium binding proteins. In: Mitochondria: Bioenergetics, Biogenesis and Membrane Structure. L. Packer and A. Gomez-Puyou, eds. Academic Press, New York, pp. 47-60. Carafoli, E., and I. Roman 1980 Mitochondria and disease. Mol. Aspects Med., 3t295-429. Cornillie, F.J., J.M. Lauweryns, and LA. Brosens 1985 Normal human endometrium. An ultrastructural survey. Gynecol. Obstet. Invest., 20: 113-129. Eguchi, M., Y. Iwama, F. Ochiai, K. Ishikawa, H. Sakakibara, H. Sakamaki, and T. Furukawa 1987 Giant mitochondria in acute lymphocytic leukemia. Exp. Mol. Pathol., 47t69-75. Enomoto, Y., and Y. Watanabe 1985 Giant mitochondria with filamentous structures in bone marrow macrophages. J. Electron Microsc. [Tokyo], 34t25-32. Gariot, P., E. Barrat, P. Drovin, P. Genton, J.P. Pointel, B. Folisuet, M. Kolopp, and G. Debry 1987 Morphometric study of human heDatic cell modifications induced by fenofibrate. Metabolism, 36: 203-210. Ghadially, F.N. 1988 Ultrastructural Pathology of the Cell and Matrix. Butterworths, London. Ghadially, F.N., and L.F. Skinnider 1974 Giant mitochondria in erythroleukemia. J. Pathol., 114.113-117. Graham, R.C., Jr., and M.J. Karnovsky 1966 The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14t291-302. Hartl, F-U., N. Pfanner, D.W. Nicholson, and W. Neupert 1989 Mitochondria] protein import. Biochim. Biophys. Acta, 988t1-45. Hatase, O., A. Doi, T. Itano, H. Matsui, and Y. Ohmura 1985 A direct evidence of the localization of mitochondrial calmodulin. Biochem. Biophys. Res. Commun., 132t63-66. Iwama, Y., and M. Eguchi 1986 Quantitative evaluation of leukemic mitochondria with a computer-controlled image analyzer. Virchows Arch. (Cell Pathol.), 51t375-384. Karrer, H.E. 1956 Electron microscopic study of bronchiolar epithelium of normal mouse lung. Exp. Cell Res., IOt237-241. Kobavashi. K.. T. Yamamoto. and T. Omae 1980 Ultrastructural

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