M. Hanamoto and T. Inoué

supranuclear region, usually representing a “U”
shape as a whole (Fig.3). Most of the Golgi
vacuoles and vesicles having been unconnected
to membranous structures were generally removed during the specimen preparation procedure. However, Golgi vesicles firmly attachedto the Golgi cisternae were preserved. The
mitochondria, surrounded by the meshwork ofthe ER, were rod-shaped, branching and anastomosed to each other (Figs. 2 and 4).
The tubular smooth ER appeared to be continuous all over the interior of the ciliated cells.
The rough ER with ribosomes was sometimes
observed around the Golgi apparatus and nucleus.

ciliated cells (Fig. 2a). The Golgi apparatus
was highly developed and many Golgi vesicles
were attached to the lateral margin of the Golgi
cisternae (Fig. 3a). The Golgi stack was composed of five to eight flattened cisternae whichwere piled up in close parallel array.
The plate-like rough ER was observed
around the nucleus (Fig. 4a). Many ribosomes
were attached to the surface, some of themforming polysomes. The rough ER had a few
fenestrations about 0.02 to 0.2 µm in diameter.
The tubular smooth ER extended from themargin of the rough ER, linking to the adjacentrough ER.

SPF rats
Conventional rats

The surface of the trachea was composed ofciliated cells and non-ciliated cells. The ciliated cells were less frequently observed in SPF
rats than in conventional rats (Fig. 1b). Mucous
cells were also less frequently visible than inconventional rats. The apical surface of thenon-ciliated cells was covered with short micro-

Ciliated cells were a major component of the epithelium. Mucous cells were often seen between
the ciliated cells (Fig. 1a), and non-ciliated cells
were scarcely visible.
Many cilia, together with a few microvilli,
were distributed on the apical surface of thea

1 µmb

1 µmFig. 3. The Golgi apparatus of tracheal ciliated cells in a conventional rat (a) and a SPF rat (b). a: The Golgi
stack is composed of five to eight flattened cisternae which are piled up in a close parallel array. Notenumerous Golgi vesicles (arrows). × 23,000. b: The Golgi stack is composed of four to seven fenestratedcisternae. × 28,000.

22

SEM of tracheal ciliated cella1 µmb1 µmFig. 4. The perinuclear intracellular structures exposed by the removal of the nucleus. a: A conventional rat.
The tubular smooth endoplasmic reticulum extends from the margin of a plate-like rough endoplasmicreticulum. The rod-shaped mitochondria run parallel to the long axis of the cell. × 14,000. b: A SPF rat. A
network of the tubular smooth endoplasmic reticulum is observed in the perinuclear region; the roughendoplasmic reticulum is not as highly developed as in conventional rats. × 17,000.

villi, bulging into the tracheal lumen. The cilia
were not so highly developed as in conventionalrats. Instead, numerous microvilli were visible(Fig. 2b). The Golgi apparatus was not so highly
developed either. The Golgi stack was composedof four to seven cisternae. Golgi vesicles
attached to the Golgi cisternae were fewer thanthose in conventional rats.
The ER in the perinuclear space was mostly
smooth and tubular. The rough ER was scarcely visible (Fig. 4b).

opment of specimen preparation for SEM madeit possible to demonstrate intracellular structures three-dimensionally (Tanaka, 1980;
Tanaka and Naguro, 1981; Inoué, 1982; Tanakaand Mitsushima, 1984; Inoué, 1985; Inoué andOsatake, 1989), intracellular structures of thetracheal ciliated epithelium have not been sufficiently studied by SEM. The osmic maceration procedure of the A-O-D-O method (Tanakaand Mitsusima, 1984) was effective to remove
excess cytoplasmic matrices, thus intracellularstructures such as the mitochondria, ER andGolgi apparatus were three-dimensionally observed as shown in Figs. 1 to 4.
The tracheal surface of conventional rats
was densely covered with cilia as reported byAlexander et al. (1975). In contrast, there werefewer ciliated cells in SPF rats than in conventional rats. Non-ciliated cells of SPF rats weremore often visible than those of conventionalrats. The mucous cells were more frequently
observed in conventional rats than in SPF rats.
This apparently indicates that ciliated cells andDiscussion
The intracellular structures of tracheal ciliatedcells have been precisely examined by transmission electron microscopy (TEM) (Rhodinand Dalhamn, 1956; Jefferry and Reid, 1975;
Marin et al., 1979). However, it has been difficult to understand the three-dimensional
configuration of intracellular structures by twodimensional TEM images. Although the devel23

M. Hanamoto and T. Inoué

mucous cells are closely related to microorganisms in the air.
The most interesting finding obtained in thisstudy was a three-dimensional network of thesmooth ER. This network appeared to becontinuous all over the interior of the ciliatedcells. Such continuity of the ER has been shown
three-dimensionally in rat spermatids by SEM(Inoué, 1982). An intensive network was demonstrated beneath the basal bodies, partly
enclosing the underlying mitochondria (Fig.2).
An amorphous layer, referred to as the hypobasal hyaline zone, was described between thebasal bodies and underlying mitochondria bylight microscopy (Hioki, 1942). Later, electron
microscopy proved that the hypobasal hyaline
zone contains the smooth ER (Graf andStockinger, 1966), which is identical to theintensive ER network demonstrated in thisstudy.
Kanamura (1975) considered that trachealciliated cells probably require a large amount ofglucose-6-phosphate for ciliary movement. Inaddition, acetylcholine has been proven to havea close relationship with this movement(Kordik et al., 1952; Burn, 1954; Salathe andBookman, 1995). Graf and Stockinger (1966)
proved acetylcholinesterase activity in the ERof rat respiratory ciliated cells using histochemical techniques. Rhodin (1960) speculated
that mitochondria under the basal bodies furnish the energy required for the ciliary beat.
The close relationship between the smooth ER,
basal bodies and mitochondria indicates thatthese intracellular organelles under the basal
bodies are engaged in the supplementation ofenergy for ciliary movement.
This study showed that the Golgi apparatus
and rough ER were highly developed in conventional rats (Fig. 3), but less so in SPF rats.
The development of the Golgi apparatus can be
estimated by the increased number of the Golgi
cisternae and Golgi vesicles. Although no significant morphological differences were notedin the smooth ER under the basal bodies (Fig.
2), the plate-like rough ER was seen in theperinuclear region only in conventional rats
(Fig. 4a). Since proteins, including enzymes,
are synthesized and processed in the Golgi

apparatus and rough ER, it is reasonable thatsuch intracellular structures are highly developed for ciliary formation and movement. Probably, protein synthesis can be activated in conventional rats, thus the tubular smooth ER
transformed into a plate-like rough ER.
Aoki et al. (1986) demonstrated that peroxidase activity in the rat tracheal epithelium washigher in conventional rats than in SPF rats.
Kinbara et al. (1992) also showed similarfindings in peroxidase activity, suggesting thatperoxidase plays a role in mucosal antimicrobial defense mechanisms. However, the relationship between the peroxidase activity andintracellular morphology has not been clarified.
The present morphological study on intracellular structures has been proven useful as ananother approach when considering the defense
mechanism of tracheal ciliated cells.

Acknowledgments: The authors are grateful to Prof.
H. Ikoma from the Dept. of Otorhinolaryngology
and Prof. E. Shimizu from the Third Dept. of Internal
Medicine, Faculty of Medicine, Tottori University,for their helpful discussions. The authors also express sincere thanks to Mr. H. Osatake and Mr. S.
Morino for their technical assistance.

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(Received December 20, 1999, Accepted January 4, 2000)

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