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Hamster airway at parturitionUltrastructure of the full-term fetal trachea and effects of parturition.

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THE ANATOMICAL RECORD 229:61-72 (1991I
Hamster Airway at Parturition: Ultrastructure of
the Full-Term Fetal Trachea and
Effects of Parturition
RODGER A. COONEY A N D DHARAM P. CHOPRA
Department o f Biochemistry, Southern Research Institute, Birmingham, Alabama
ABSTRACT
Limited studies have described the ultrastructure of trachea of
late fetal and neonatal hamsters but the effects of parturition and the onset of
breathing on structure have not been discussed. This study describes morphological features of ante- and post-partum tracheal mucosa and submucosa and contrasts these features in fetal and neonatal hamster siblings. Significant differences
between these siblings are noted in tracheal cells interfacing the lumen. Such cells
of the fetal animals usually possessed cytoplasm of medium electron density with
cisternal rough endoplasmic reticulum (RER). Surface membranes of these cells
possessed numerous microvilli. In contrast, corresponding cells of post-natal animals often had lucent cytoplasm with mostly tubular or vesicular RER. Surface
membranes of these cells possessed microplicae (microridges).
This study also considers characteristics of fetal and neonatal tracheal development including: lomasome-like structures in secretory cells; dichotomous forms of
oligocilia in mucosal and submucosal cells; intramembranous particles of hemidesmosomes; particles and mitochondria associated with desmosomes; and affiliations
of ciliary basal bodies with the cytoskeleton, cell membrane, and with endoplasmic
reticulum.
The continuity of development of mammalian respiratory systems is interrupted by parturition, an enormous environmental change for the organism. Maternal influences are severed and respiratory tissues are
suddenly flooded with air. The goal of this study was to
establish the morphology of hamster trachea at the end
of embryonic life and to determine whether immediate
ultrastructural metamorphic or cytopathogenic features were discernable in neonatal tracheal tissues
upon parturition. Hamster trachea has been extensively employed as a model for respiratory research
because of similarities with human tracheal architecture (Chopra and Cooney, 1983, 1985; Becci et al.,
1978; Keenan e t al., 1982). Previous developmental
studies of this model (Emura, 1985; Otani et al., 1986;
McDowell et al., 1985a,b),however, have not addressed
the possible effects of parturition. In these studies,
commonly practiced research methods make i t difficult
to recognize morphological changes which may be related to this brief event. Usually, fetal tissues of one
litter have been compared with neonatal tissues of a
different litter. This experimental design has several
limitations. First, the developmental age of the antepartum litter cannot be precisely determined. Second, each of the compared litters has developed in a
separate maternal environment. Third, the postpartum and ante-partum litters are of different parentage. Ideally, a n early developmental study would follow the same young animal in gestation through birth,
but this is not feasible with current technology. Therefore, in the present study, a simple method was developed that not only firmly established the developmenc
1991 WILEY-I,ISS. INC
tal age of the fetuses but also allowed them to be
directly compared with their neonatal siblings.
Since a normal hamster litter ranges from 10 to 12
pups, we were able to compare the tracheal structure of
fetal and neonatal littermates by sacrificing five to six
individual pups a t the moment of birth and subsequently sacrificing the mother and the remaining unborn sibling pups in utero. Further, to reduce genetic
variation, each mother procured from the breeding facility had been allowed to breed with only one male.
Replicate experiments were performed to avoid anomalies peculiar to a single litter.
MATERIALS AND METHODS
Animals
Single-pair bred, pregnant Syrian golden hamsters
were obtained from Charles River Laboratories (Wilmington, MA) a t approximately their 13th day of gestation. The animals were maintained individually in
plastic cages (12”x 14”x 6.5”)a t 22°C with a 12 h-12 h
light-dark regime. They were fed with Purina lab chow
and given water ad libitum. On the 16th gestational
day, dams were inspected hourly until they began to
give birth. Each neonate was removed from its cage a s
soon as the mother severed the umbilical cord (approx-
Received Septeniher 12. 1988: accepted May 16, 1990.
Dr. Dharam 1’. Chopra’s present address IS Wayne S t a t e University.
Institute of Chemical Toxicology. 2727 Second Avenue. Room 4000.
Detroit. M I 48201. Address reprint requests there.
62
R.A. COONEY AND D.P. CHOPRA
imately 10 minutes after birth) and was sacrificed by
intraperitoneal (I.P.) injection of Brevital sodium (Eli
Lilly, Indianapolis, IN). The respiratory tracts were
surgically removed from larynx to lungs and fixed for 2
h a t 22°C in a solution of 4% formaldehyde and 1%
glutaraldehyde in 0.1 M sodium cacodylate buffer pH
7.4 for later histological and ultrastructural processing. A mother was allowed to birth five to six pups
before being sacrificed by I.P. injection of Brevital.
Ante-partum pups were then obtained from the same
mother. The abdomen was opened to expose the uterus
and unborn pups which were injected in situ with
Brevital through the uterine wall. The abdomen of the
mother was then flooded with the aldehyde fixative,
and the pups removed and immediately submerged in
the fixative to preclude exposure to air. The tracheas of
these animals were excised while the animals were
submerged in fixative and further fixed for 2 h at 22°C.
Methods for processing the fixed tissues for scanning
(SEMI and transmission electron microscopy (TEM)
are described below. Six pups (three fetal, three neonatal) from each of four mothers (24 pups total) were used
for the study. Ante-partum and post-partum siblings
were compared to each other and to animals of different
litters.
Scanning and Transmission Electron Microscopy
For TEM and SEM, fixed tissues were further processed a t room temperature. The tissues were washed
in distilled water (DH,O), pH 7.0, post-fixed for 1 h in
1%osmium tetroxide (OsO,) in 0.1 M cacodylate buffer
(pH 7.4), and washed again in DH,O. For standard
TEM, tissues were stained en bloc for 20 min in 4%
aqueous uranyl acetate (UA) and washed three times
with DH,O. [Prolonged washing with buffer between
aldehyde and osmium fixation is known to induce artifacts (Hyatt, 1981) and we have found that washing
with DH,O is superior, possibly because unreacted aldehydes are more effectively removed from the tissue.
Hence, the oxidant-reductant reaction between osmium fixative and aldehyde fixative is diminished.
DH,O is used for osmium fixation not only to remove
excessive OSO, but also to remove excess cacodylate
buffer, which can precipitate UA during en bloc staining (Hyatt, 1981).] After the final wash, samples for
TEM and SEM were dehydrated stepwise to 100% acetone. With CO, a s the transition fluid, SEM samples
were critical point dried, mounted on aluminum stubs,
and sputter coated with gold palladium. Coated specimens were viewed and photographed with a n ETEC
autoscan SEM.
Tissues for TEM were embedded in Polybed 812
(Polysciences, Warrington, PA) epoxy resin and thin
sectioned (900 A). Sections were mounted on unsupported copper grids, stained for 10 min in 4% UA, and
post-stained for 6 min in Reynolds lead citrate (Reynolds, 1963). Thin sections were viewed and photographed with a n Hitachi HU-12 at 75 kV.
in which cartilaginous rings were embedded. Ciliated
cells, identified by tufts of cilia projecting from the mucosal surface, were sparsely distributed in the mucosa.
Examination of a transverse section revealed a layered
organization (Fig. 2). The mucosal layer was separated
from the submucosa by basement membrane. Internal
detail of the mucosal cell types could not be discerned
with SEM. Beneath the basement membrane, a layer of
collagen demarked the upper limit of the submucosa.
Cells of the submucosa appeared to be layered on the
surface of cartilaginous plates in which stellate chondrocytes were embedded. Cells layered on the plates
included vascular and smooth muscle cells (not shown),
fibroblasts, and a semi-stellate cell type which seemed
intermediate between fibroblast and chondrocytes.
Examination of the mucosal surface showed numerous,
regularly spaced microvilli on nonciliated cell luminal
membranes. Cilia were well developed with tall, slender dendritic microvilli (stereocilia) interspersed
among the shafts (Fig. 3).
Transmission electron microscopy
Cross section of the trachea revealed that the mucosa
was pseudostratified, comprised mainly of basal, ciliated, and secretory cells (Fig. 4). Secretory cells were
columnar with numerous digitiform microvilli on their
apical surfaces. They possessed ovoid, centrally located
nuclei, parallel racks of RER, Golgi, and large secretory vesicles. The vesicles (Fig. 5) were filled with flocculent material that usually surrounded electrondense cores. In some vesicles, the flocculent material
appeared to be concentrically layered. Occasionally the
secretory cells contained arrays of tubules resembling
lomasomes (Fig. 6 ) that were generally associated with
Golgi. Some other secretory cells possessed a single cilium (oligocilium) lacking central pair microtubules
(Fig. 7). The proximal end of the basal body of this type
of cilium sometimes projected a striated appendage,
Fig. 1. SEM of section of trachea from full-term anteparturn hamster. Small tufts of cilia (C) are seen sparsely distributed on the surface of the mucosal layer (Mu) upon cartilaginous rings (CR). Bar
equals 100 pm.
Fig. 2.SEM of a transverse section of ante-partum trachea showing
tracheal mucosa (Mu) on basement membrane (BM) overlying the
collagenous lamina propria (LP). A layer of fibroblasts (F) and an
intermediate semistellate cell (I) of the submucosa rest on a cartilage
plate in which stellate chondroblasts (Ch) are embedded. Bar equals
10 pm.
Fig. 3.SEM of tracheal surface epithelium of ante-partum animal.
Numerous digitifnrm microvilli (dm) decorate the field of cells. Cilia
tC1 of a ciliated cell are bordered by dendritic microvilli (Dm). Bar
equals 3 pm.
RESULTS
Morphology of Trachea in the Full-Term Fetus
Fig.4.TEM shows morphology of term fetus trachea. Secretory cells
(SC),ciliated cells (CC), and basal cells (BCI comprise the mucosa.
Secretory cells possess digitiform microvilli (dm) on apical surfaces;
racks of parallel rough endoplasmic reticulum (er),secretory vesicles
(SV), and Golgi (GI are shown. Basal cells possess pools of glycogen
(GL)which flank the nucleus. A fibroblast (F)is seen in the submucosa. Bar equals 3 pm.
Scanning electron microscopy
Samples of the ante-partum trachea (Fig. 1)showed
a thick mucosa layer supported by submucosal tissues
Fig. 5. High-magnification TEM of secretory vesicles tSV) with
dense cores. Content of one vesicle appears layered (arrowhead)and a
vesicle without a core protrudes from the cell surface. Bar equals 0.5
pm.
H A M S T E R T R A C H E A AT PARTURITION
Figs. 1-5.
63
64
R.A. COONEY AND D.P. CHOPRA
CYTOPLASM
BL-
Figs. 6-1 1
. ;.-
-*.*-.
. - . ....
?,.
.
. .
..'5.
.*.
....
.
f
*:
.
*
and the distal end projected basal feet (satellite arms)
that contacted the apical membrane. Secretory cells
were joined at the lumen by junctional complexes (Fig.
8 ) .The desmosomes were sometimes incompletely constructed. Stacks of dense subunits associated with microfilament bundles lined the plaques of cojoined cells.
Intermediate filaments associated with the desmosomes often encompassed mitochondria. A reticulated
network subtending the plasma membrane and comprising the matrix of digitiform microvilli abutted the
top of the desmosomal plaque.
Basal cells (Fig. 9) were pyramidal or cuboidal and
possessed relatively dense cytoplasm and pleomorphic
nuclei commonly flanked by pools of glycogen. Like
secretory cells, basal cells often possessed oligocilia
without central pair microtubules. Basal cells rested on
the basement membrane and were secured to the lamina propria by hemidesmosomes. Hemidesmosomes
(Figs. 10, 11) were composed in part by the basal
plasma membrane of the basal cell. Small particles
were embedded in both leaflets of this membrane and
were frequently connected through the hydrophilic domain of the lipid bilayer. Larger particles in the outer
leaflet encroached into the hydrophilic domain at the
attachment point of intermediate filaments t h a t extended from a dense plaque embedded in the basal lamina. These terminal caps gave a scalloped appearance
Fig. 6. TEM of ante-partum tracheal surface epithelium Lomasome-like profiles (Lo) occupy the apical cytoplasm of a secretory cell
tSC) which abuts a ciliated cell tCC). Bar equals 1 b m .
Fig. 7. TEM of a tapered oligocilium of a secretory cell. Central pair
microtubules are not evident. A striated appendage ( S A )extends from
the basal body into the cytoplasm; basal foot (BF). Bar equals 0.25
I*m.
Flg. 8. TEM of a junctional complex between adjacent secretory
cells. A digitiform microvillus tdm) with finely reticulated matrix
projects from the surface of one cell Stacks of electron-dense subunits
(arrowheads)border t h e desmosomal plaques i D)of both cells. Similar
dense particles a r e seen in congregations of intermediate filaments
(IF) and associated with microfilament bundles ( m f ) . Intermediate
filaments associated with the desmosomal stack encompass a mitochondrion ( M ) on one side. Bar equals 0 1 bm.
Flg. 9. TEM of a basal cell with oligocilium 10)and sister centriole
t GL) surrounds the nucleus and hemidesmosomes !HI
attach the cell to t h e lamina propria (LP).Bar equals 1 pni.
is).Glycogen
Fig. 10. TEM of basal cell plasma membrane and hemidesmosome
t H ) with cytoplasm (CYI above and lamina propria tLPl below. Particles a r e regularly spaced in the hydrophobic leaflets i large black
arrows) t h a t parallel the hydrophilic domain ilarge white arrow) of
the lipid bilayer. Short filaments of'intermediate s i z e Iiiiedium white
arrows) connect the subbasal plaque t o the cell's outer leaflet 1,nrge
particles a t the filaments' termini encroach into the hydrophilic doniain to give a xalloped appearance to the outer leaflet ismall white
arrows) Amorphous material from the lamina propria iarrowheadi
passes through the basal lamina iopen arrows) to connect with the
subbasal plaque. Bar equals 0.1 Fm.
Fig. 11. Illustration of the hemidesmosome and plasma membrane
shown in Figure 10 Particles a r e embedded in both the inner leaflet
( I L ) and outer leaflet ( 0 L l and a r e often connected through the hydrophilic domain iHDI. The dense plaque cDP1 is embedded in the
basal lamina iRL1 with amorphous connections to elements of the
lamina propria iLP) and fibrillar connections ( I F )to the outer leaflet.
These intermediate fibers terminate in dense particulate caps within
the membrane.
to the outer leaflet. Amorphous bridges through the
basal lamina connected collagenous elements of the
lamina propria to the dense plaque. Ciliated cells of the
mucosa were seen in various stages of maturity (Figs.
4, 12), and dendritic microvilli with matrices having
some parallel elements were intercalated among ciliary shafts. Axonemes of the kinocilia had normal 9 + 2
microtubular arrangements. The kinocilium basal
body was proximally associated with smooth endoplasmic reticulum (SER);sometimes the endoplasmic reticulum was studded and appeared like RER. The distal
basal body projected flat basal feet through a firm
dense layer subtending the plasma membrane. Tangential section of the distal basal body (Fig. 13)showed
that the basal feet were triangular and emanated from
triplet microtubules. Termini of the feet were connected to the plasma membrane by amorphous bridges.
Profiles of the basal bodies deeper in the cytoplasm
showed conical rootlets projecting laterally from the
median basal body. Proximally, basal body profiles became spiked. Profiles of dendritic microvilli in tangential section revealed matrices possessing scattered subunits that resembled intermediate filaments in cross
section.
Like basal and secretory cells, cells of the submucosa
frequently possessed oligocilia. The microtubule complement of axonemes of smooth muscle cells and
chondrocytes (not shown) was not established but fibroblasts often possessed oligocilia with central microtubules (Fig. 14). Like kinocilia, basal bodies of fibroblasts projected basal feet t h a t were triangular in cross
section and emanated from triplet microtubules (Fig.
15).
Morphology of Trachea of Immediately
Post-Partum Hamster
Significant differences were observed in the ultrastructure of tracheas derived from ante-partum and
immediately post-partum animals. With SEM,the luminal surfaces of fetal hamster tracheas displayed numerous regularly spaced digitiform microvilli (Fig. 161,
while their neonatal siblings possessed surfaces covered with vermiform microplicae (Fig. 17). In transverse sections, nonciliated luminal cells of the fetal trachea consisted mainly of well-developed secretory cells
with cytoplasm of medium density containing parallel
racks of RER and Golgi (Fig. 18). Basal cells of the
epithelium possessed dense cytoplasm and pools of glycogen. While basal cell morphology of neonates was
essentially similar to t h a t of their fetal siblings, nonciliated luminal cells of neonates were electron-lucent
with poorly organized RER (Fig. 19). Golgi, however.
remained conspicuous. In fetal trachea, microvilli projected from apices of the nonciliated luminal cells. The
microvilli were fingerlike with finely fibrous matrices
that seemed continuous with a shallow layer of similar
composition subtending the apical membrane (Fig. 20).
In contrast, analogous cells of neonatal trachea possessed apical surface elaborations consisting of ruffled
projections with granular matrices. The projection occasionally overlaid fibrous elements (Fig. 21). In some
cells (Fig. 22 1, these elements were prominent and
nearly traversed the cell apex while in other cells, they
were reduced or absent. This may be caused by the
fibrous elements being directional and not in the plane
66
R.A. COONEY A N D D.P. CHOPRA
Fig. 12.TEM of a ciliated cell with immature cilium and a n immature dendritic microvillus (D). Cilium (C) has central microtubules
(black arrows). A firm electron-dense plaque (arrowheads) subtends
the apical membrane. A basal foot (BF) projects from the distal basal
body and is embedded in the plaque. The proximal basal body is associated with smooth endoplasmic reticulum (Ser).Bar equals 0.25
pm.
from profile #4. Triangular basal feet (white arrows) and the plasma
membrane are connected by amorphous bridges (#6).Cross sections of
dendritic microvilli (DM) are seen in the lumen. Bar equals 0.25 pm.
Fig. 13. TEM of a tangential section through a ciliated cell illustrates structural changes of basal bodies (small numbers 1-7) at different levels from the cytoplasm (#1) to the lumen (#7). The dense
plaque extends the length of the bar. A conical rootlet (R)projects
Fig. 15. TEM of oligocilium basal body shows triangular basal foot
(arrowhead) from a triplet. Bar equals 0.1 pm.
of sectioning for all cells. The luminal cells rested on
the lamina propria or upon basal cells which in turn
rested on the lamina propria attached by well-defined
hemidesmosomes. In tangential sections of the mucosa
of neonates, nonciliated cells interfacing the air corridor showed numerous profiles of mostly vesicular and1
or tubular RER (Fig. 2 3 ) .With SEM ciliated cells of the
surface epithelium seemed normal although they were
surrounded by microplicated cells (Fig. 24). In TEM
cross section, the cilia possessed complete and intact
axonemal elements (Fig. 25). Submucosal cells (not
shown) maintained normal morphology and their RER
appeared unaffected.
Fig. 14. TEM of oligocilium of fibroblast. Central microtubules (wedium arrow) and basal foot (BF)are evident. Collagen (CL) surrounds
the cell. Bar equals 0.5 pm.
DISCUSSION
Trachea of Full- Term Hamster
The tracheal mucosa of full-term hamsters included
secretory, ciliated, and basal cells. Ciliated and basal
HAMSTER TRACHEA AT PARTURITION
cells could be easily identified; however, i t is not clear
what type of secretory cells were present. For instance,
it could not be determined that these were Clara cells,
although Plopper e t al. (1983) recognized only Clara,
basal, and ciliated cell types in hamster trachea. The
Clara cell is thought to be a source of pulmonary surfactant (Niden, 1967; Smith et al., 1974; Walker et al.,
19861, and the source of phospholipid precursors of surfactant is thought to be either lamellate (fingerprint)
vesicles (Walker et al., 1986) or SER of the apical cap
(Smith et al., 1974). If the secretory cells seen in the
present study are Clara cells, it is not evident from
their vesicular morphology. The vesicles do not appear
to be surfactant precursors since they are not lamellate. Instead, the flocculent material is sometimes layered on the dense core. These contents may therefore
represent some other secretory product as the occurrence of secretory granules with and without central
cones has been observed in tracheal epithelium of
many species. The absence of a translucent cap in the
secretory cells also deviates from classical characteristics described for Clara cells (Breeze et al., 1976). Diameters of tubules resembling SER in apical caps of
Clara cells reported by Smith et al. (1974), however,
correspond favorably with those comprising the lomasome-like structure (Fig. 6). While lomasomes have not
been previously reported in animal tissues, the same
authors have shown Clara cell mitochondria invested
in tubular profiles. Recently, similar smooth tubular
profiles originating from SER have been reported in
many cell types (Ghadially, 1988).The results of the
present study show that the lomasome-like structures
probably originate from the Golgi.
Secretory cells of the trachea are joined by junctional
complexes t h a t are generally well developed but occasionally seemed to be in the process of being constructed. Jones and Goldman (1985) propose that desmosomes are not polymerization sites of intermediate
filaments but t h a t the filaments are preformed and redistributed to the site of desmosome formation for positioning and incorporation. Figure 8 of the antepartum tracheal epithelium may represent this
process. Strands of microfilaments in the cytoplasm
were associated with intermediate filament cross sections yielding a “beaded necklace” profile. Similar profiles were arranged a s stacks of subunits adjacent to
respective plaque faces of the conjoined cells. Therefore, i t is possible that the migration of intermediate
filaments is orchestrated by microfilaments. The “captured” mitochondrion might be fundamental to the construction process, although the relationship of mitochondria to desmosomes is also noted at junctions that
seem complete. To our knowledge neither association
has been previously reported.
Basal cells are easily identifiable by their position,
dense cytoplasm, small pleomorphic nuclei, and the
ubiquitous presence of lucent pools of glycogen. Similar
condensed cytoplasmic components and accumulated
storage products are typical of encysting cells of algae
and fungi. Perhaps these analogous features provide
the trachea with a resistant pool of cells for the anticipated post-partum environment. The hemidesmosomes that secure the basal cells to the basal lamina
and lamina propria are well developed, and their ultrastructure is consistent with that reported (Tidman
67
and Eady, 1986). Intramembranous terminal caps of
hemidesmosome intermediate filaments, however,
have not been previously reported. These caps may represent the particles that Riddle (1986) has noted in
freeze-fracture studies. There has been some question
whether cytoplasmic basal cell intermediate filaments
traverse the basal plasma membrane to form hemidesmosomes; the results of the present study indicate that
they do not.
A previously unreported association of the kinocilium proximal basal body terminus with SER was noted
in ciliated cells. Whether there is a continuity of the
basal body lumen with this SER remains to be established but the association was common and may represent a method to supply the cilium with intracellular
products. Another new feature was the presence of the
firm plaque subtending the apical membrane in which
the basal bodies were embedded. This structure is apparently fairly rigid because in minced unfixed tissue,
the cilia project from a flat plane (unpublished results).
This rigidity suggests the plaque’s function is cytoskeletal. Figure 13 shows progressive levels of basal body
morphology and illustrates that basal bodies are embedded within the plaque. CChang et al. (1979) have
interpreted a similar figure as stages of basal body development, but the profiles result from the plane of
section.] At the level of the plasma membrane in this
figure the amorphous connections between satellite
arms (basal feet) of basal bodies and the membrane
may provide a n electrical pathway for transmembrane
potentials to control ciliary beat.
Zimmerman (1898) first detected solitary cilia projecting from thyroid epithelial cells and Sorokin’s treatises on fibroblasts and smooth muscle cells (1962) and
on mammalian ciliogenesis (1968) reported the occurrence of a rudimentary 9 + 0 cilium in virtually all cell
types of fetal respiratory mucosa and submucosa
[Wheatley (1967) reports paired cilia in cultured fibroblasts.] In secretory cells of fetal lung, these rudimentary cilia are thought to procede true ciliogenesis and
were therefore named “primary” cilia. Ghadially
(1982) argues that since many of the cell types having
9 + 0 cilia do not produce secondary cilia, Zimmerman’s
term, “oligocilia,” is more proper. In this study, cells of
the trachea produce a single oligocilium that arises
from the diplosome like the flagellum of many lower
eukaryotes, for example, fungal zoospores (Cooney et
al., 1985). Both basal and secretory cells generate oligocilia of the 9 + 0 variety but the central pair of microtubules is often evident in fibroblasts (Fig. 14). The
reason for this dichotomy is unknown. Whether the
other cell types of the submucosa possess 9 + 0 or 9 + 2
axonemes could not be established. It is assumed that
9 + 2 cilia beat but this ability in 9 + 0 cilia is arguable
(Afzelius, 1962; Stubblefield and Brinkley, 1966) and
their possible immotility has promulgated speculation
that they are involved in mitotic regulation (Wheatley,
1971; Rash et al., 1969) and sensory reception (Barnes,
1961). Also, Albrecht-Buehler (1977) and AlbrechtBuehler and Bushnell (1979) suggest that they determine the direction of amoeboid movement during cell
migration. Since many of the hamster tracheal cells
exhibiting oligocilia are probably non-migratory (e.g.,
basal cells, secretory cells, chondrocytes), they may be
residual from a previously migratory predecessor. Al-
68
R.A. COONEY A N D D.P. CHOPRA
Figs. 16-19
HAMSTER TRACHEA AT PARTURITION
69
ternatively, if the oligocilia do beat, they might be gen- vesicular andlor tubular as compared to the well-orerated to move material over the cell’s surface in the dered racks of RER in the corresponding fetal trachea.
Although it is not known which factors of parturition
manner proposed by Coupland (1965).
The striated appendage of the oligocilium basal body contribute to these changes, nor how these changes afin secretory cells (Fig. 7) is also an occasional feature of fect the organism, it is probable that the microvilli desupranuclear centrioles and, rarely, of kinocilia of generate to form microplicae. To begin with, Otani et
hamster trachea (unpublished results). Similar struc- al. (1986) have reported that digitiform microvilli are
tures are ascribed a n anchorage function in many eu- not well developed in the tracheal epithelium of l-daykaryotes (Lungarella et al., 1985; Santander and Cuad- old hamsters. Although our observations of the ultrarado, 1980; Dingemans, 1969) and are sometimes structure of the neonatal trachea agree with their reelaborate, being frequently complexed with other cy- sults, it should be recalled that microvilli were
toskeletal elements. It is doubtful, however, that the conspicuous in the trachea of ante-partum animals.
striated appendages serve to secure basal bodies in Since microvilli are well defined in the immediately
hamster trachea since they are not affiliated with such ante-partum hamster trachea, their absence in neoelements. [This structure should not be confused with nates cannot be related to underdevelopment. Rather,
the lateral rootlet (Fig. 13) which is often associated it is the transformation of microvilli into microplicae a t
with intermediate filaments.] Further, in organ cul- parturition that accounts for the scarcity of microvilli
ture of trachea of adolescent hamsters, we have ob- in post-partum animals. Further, the microvilli posserved centrioles with striated appendages at different sessed matrices composed of actin-like filaments. [The
levels within the cytoplasm of several adjacent cells presence of actin in microvilli has been well estab(unpublished data), which suggests the striated ap- lished (Trier and Madara, 19811.1 In contrast, the mapendage is not a n effective anchor. Rather, the myo- trices of the microplicae were finely granular. Alfibrillar nature of these appendages suggests muscle- though the composition of these granules remains to be
like qualities. Salisbury and Floyd (1978) have determined, it is possible that they are aggregations of
apparently demonstrated calcium-induced contraction globular actin monomers from depolymerized actin filin the green alga Platymonas. We believe that the stri- aments. Such dissolution of the cytoskeletal restraints
ated appendage in the hamster trachea may also be of adjacent microvilli would give the impression that
muscle-like and may play a part in moving the centri- microvilli fuse to form microplicae. [Breipohl et al.
ole from the nucleus to the apical cytoplasm to partic- (1977) have suggested that microplicae in rat laryngeal
ipate somehow in basal body replication during cilio- epithelium result from fusion of microvilli]. The congenesis (unpublished data). This would be consistent cept of degeneration seems more logical since micropliwith the concept of “primary” cilia and, since the cen- cae would be expected to possess filamentous matrices
triole would not be available for mitosis, might also if they were fused microvilli.
explain why ciliated cells do not divide (Boren and ParLewis and Prentice (1980) have suggested t h a t miadise, 1978).
croplicae are a n indication of dying cells. In the immediately post-partum hamster trachea, however, the miEffects of Parturition and Onset of Breathing
croplication involves the majority of the luminal
This study establishes that parturition factors imme- epithelium without any apparent cytopathology or desdiately affect the ultrastructure of the developing res- quamation of cells, at least during the first 2 days folpiratory tract. The morphological differences, before lowing birth (unpublished results). Therefore, the forunreported, between fetal and neonatal trachea were mation of microplicae does not necessarily seem to be
restricted to cells interfacing the air corridor. As noted, associated with cell death. In any case, since microvilli
microvilli, predominant on the surface of secretory might be degraded during degenerative processes precells of ante-partum trachea, were mostly absent from ceding cell death, the hypothesis of these authors is not
the secretory cells, which were instead microplicated. inconsistent with the concept of microplicae ontogeny
In addition, the RER of postpartum secretory cells was presented here.
Because microplication is a n immediate effect of parturition, it may be suspected to result from the increased availability of oxygen with the onset of breathing. In tracheal mucosa of rat, Philpott et al. (1977)
Fig. 16.SEM of surface of nonciliated mucosa cells of ante-partum have demonstrated that microvilli disappeared and
hamster trachea. Numerous digitiform microvilli (dm) decorate the
cells hypertrophied when test animals were exposed to
cells. Bar equals 2.5 pm.
elevated concentrations of oxygen. Further, the scheFig. 17.SEM of tracheal mucosa of a neonatal sibling of Figure 1. matic representation of their study shows granulation
The cells possess numerous microplicae (MP). Bar equals 2.5 pm.
of the apical cytoplasm which agrees with our results
Fig. 18. TEM of nonciliated mucosa of ante-partum hamster tra- with hamster trachea. If atmospheric oxygen does
chea. Digitiform microvilli (dm) project into the lumen (Lu) from the
cause the disappearance of the microvilli in neonatal
Golgi (G) and parallel racks of rough endo- tracheal mucosa of hamster, the question arises a s to
surface mucosal cells (S).
plasmic reticulum (er) inhabit the cytoplasm. Basal cells (BC) with
why microvilli of older animals are not affected by the
pools of glycogen (GL) rest on the basal lamina (BL). Bar equals 2.5
normal aerobic state. A possible answer is that fetal
1Lm.
hemoglobin (at least in humans) sequesters up to 30%
Fig. 19.TEM of mucosa of a neonatal sibling of Figure 3 . Profiles of more oxygen than adult hemoglobin and hemoglobin in
er are few and poorly defined. A Golgi (GI body occupies the cytoplasm
which is more lucent than in the ante-partum sibling (Fig.3 ) .The cells the fetus is 50% more concentrated than in the adult
project irregular surfaces into the lumen (Lu); glycogen pools (GL) mother (Guyton, 1976). The persistence of these factors
persist in the neonatal basal cells. Bar equals 2.5 Fm.
in the neonate may provide sufficient additional oxy-
'70
I1.A. COONEY ANT) D.P. CHOPRA
Figs. 20-22.
HAMSTER TRACHEA AT PARTURITION
Fig. 20. TEM of a nonciliated mucosal cell in trachea of antepartum
animal. A finely reticulated fibrous layer subtends the apical membrane (arrows).The matrices of digitiform microvilli (dm) are composed of and contiguous with the fibrous layer. The cytoplasm is of
medium density and possess Golgi iG) and organized er. Bar equals 1
pm.
Fig. 21. TEM of post-partum nonciliated mucosa cell a t the same
magnification as Figure 5. Microplicae (MP) with granular matrices
project into the lumen. A continuous layer of fibers (arrows) subtends
the apical membrane below the microplicae. The cytoplasm is lucent
and er is inflated and fractionated into vesicles. Golgi (G) are slightly
swollen. Bar equals 1 pm.
Fig. 22. TEM of post-partum tracheal mucosa includes cell of Figure
6. Golgi (G) in lower right corner of Figure 6 is in center of cell in
Figure 7. Microplicae (MP) are seen above the fibrous layer (arrowheads) traversing the left cells’ apices. The cell on the right shows a
few subapical fibrous elements (open arrows). The apical surface is
slightly ruffled and without microvilli: rough endoplasmic reticulum
is disorganized andior vesiculate. Pools of glycogen flank basal cell
nuclei. Basal cells (BC) are attached to the submucosa by hemidesmosomes (small arrows). Fibroblasts (F) inhabit the submucosa. Bar
equals 2.5 km.
gen to induce alteration of the cells of the tracheal
mucosa. Alternatively, severance from maternal hormonal influence might be associated with disappearance of microvilli and formation of microplicae. This
consideration might seem improbable since i t has not
been established that a hormone is required to maintain the structure of microvilli. Also, the absence of
such chemical influence would not be expected to rapidly effect the observed structural changes. I t should be
noted, however, that vasopressin alters microridges to
form microvilli in toad bladder epithelium within 30
min of exposure (Davis et al., 1974). This reverse relationship demonstrates that these two surface membrane modifications are interrelated and establishes
that the influence of hormones on these structures can
be rapid. Since vasopressin not only induces microvilli
formation but also increases membrane permeability
to water (Leaf, 1967), microridge formation may reflect
a structural change which provides resistance to water
loss. Another possibility is that microplication may be
caused by reabsorption of amniotic fluid by the luminal
cells of immediately post-partum trachea. This however, appears unlikely a s there were no significant differences in luminal cell volumes between fetal and neonatal hamsters. Protection against desiccation is
important in the respiratory epithelium of the neonate
as there is a 20% water loss in rabbit lung with the
onset of breathing (Kikkawa et al., 1968). Likewise, in
humans, water comprises 73% of the total neonate body
weight compared to 58% of adult body weight and the
Fig. 23. TEM of neonatal surface mucosal cells in tangential section.
Fractionated er occupies the cytoplasm. Bar equals 2.5 pm.
Fig. 24. SEM of neonatal mucosal surface. Microplicae cover nonciliated cells surrounding a ciliated cell. Cilia (C) appear normal. Bar
equals 2.5 pm.
Fig. 25. TEM of a cilium in trachea of post-partum animal. The
profile displays complete and normal 9 + 2 architecture; nexin links
iN), radial spokes ( S ) ,and inner ( I ) and outer ( 0 )dynein arms. Bar
equals 0.1 pm.
71
72
1t.A. (‘OONEY AN11 11.1’. CHOPRA
post-partum weight loss common in newborns is mostly
water loss (Guyton, 1976). Whether the factors of parturition responsible for microplication affect microvillous matrices alone o r concomitantly affect membrane
characteristics remains to be established. The differences of electron density of cytoplasm observed in the
tracheal cells of ante-partum and post-partum hamsters may be evidence, however, of differences of cell
hydration (Ghadially, 19821, which perhaps result from
changes in membrane permeability. If the factors responsible for microplication do impact the membrane
structure, they may also explain why the RER of the
post-partum secretory cells becomes vesicular.
Ghadially, F.N., ed. 1988 Ultrastructural Pathology of the Cell and
Matrix. Butterworths, London, Val. 1, 3rd Edition pp. 480-481.
Guyton, A.C. 1976 Textbook of Medical Physiology. W.B. Saunders
Company, Philadelphia, pp. 1-1194.
Hyatt, M.A. 1981 Principles and Techniques of Electron Microscopy
Biological Applications. University Park Press, Baltimore, Vol. 1,
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Jones, J.C.R., and R.D. Goldman 1985 Intermediate filaments and the
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Keenan, K.P., J.W. Combs, and E.M. McDowell 1982 Regeneration of
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ACKNOWLEDGMENTS
Lewis, D.J., andD.E.-Prentice 1980 The ultrastructure of r a t larynWe would like to thank Ms. Alissa Beaty for her
geal epithelia. J. Anat., 13Ot617-632.
excellent technical assistance and Ms. Linda Bethune Lungarella, G., M.M. DeSanti, R. Palatresi, and P. Tosi 1985 Ultrastructural observations on basal apparatus of respiratory cilia in
for her secretarial assistance. This study was supported
immotile cilia syndrome. Eur. J. Respir. Dis., 66:165-172.
by USPHS grant number HL 32308 from the National McDowell, E.M., C. Newkirk, and B. Coleman 1985a Development of
Heart Lung and Blood Institute.
hamster tracheal epithelium. I. A quantiative morphologic study
in the fetus. Anat. Rec., 213:429-447.
McDowell, E.M., C. Newkirk, and B. Coleman 198510 Development of
hamster tracheal epithelium: 11. Cell proliferation in the fetus.
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