AN
ELECTRON
MICROSCOPE
STUDY
OF
THE
FETAL
DEVELOPMENT
OF
HUMAN
LUNG
M. A. Campiche, A. Gautier, E. I. Hernandez, and A. Reymond
Clinique Infantile (Professor M. Jaccottet); Centre de Microscopie Electronique (Head: A. Gautier); and Institut d’Anatomie Pathologique (Prof esseur I. L. Nicod),
University of Lausanne, Switzerland
LUNG SI’ECIMENS FROM hUMAN FETUSES
Estimated Fetus Weight length Gestational
Number (gm) (cm) Aye
Fixation b’rucedures 1 4 2 9 3 32 4 45 5 68 6 75 7 125 8 135 9 150 10 200 11 240 12 380 13 1,200 (mu) 5 1
7 2 if
12 2-3 12.5 2-3 15 2-3 1St) 3--I 18 3-4 19.5 3-4 21 3-4
21 4--S if
23 4-5 $
29 4-5
27 5-6
#{149}2% OsO in acetate veronal buffer.” t2% OO in phosphate buffer.’#{176}
1% KMnO< in acetate verona) buffer.#{176} 10% formalin buffered to p11 7428
(10% formalin in phosphate buffer4C followed by buffered 2% OsO,.”
(Submitted May 7, 1963; accepted for publication June 28.)
Dr. E. I. Hernandez is a Fellow of the “Consejo Nacional de Investigaciones Cientificas v Tt.cnicas,”
Argentine Republic.
This work was supported by grants from the Swiss National Foundation for Scientific Research. ADDRESSES: (MAC.) Cardiovascular Research Institute, University of California Medical Center, San Francisco; (AG.) Centre de Microscopie Electronique de l’Universit#{233}, Bugnon 21, Lausanne; (E.I.H.)
In-stituto de Investigaciones M#{233}dicas, Donato Alvarez 3000, Buenos Aires, Argentine Republic; (AR.)
Institut d’Anatomie Pathologique, H#{244}pitalCantonal, Lausanne, Switzerland.
PEDIATRICS, December 1963
T
HE IMPORTANCE of prematurity inperinatal pathology is a well-known
fact as well as the frequent occurrence of
respiratory disorders in premature infants.
Some recent electron microscope
observa-tions of newborn lung3’7’8’16”7’39’42 have
added to the information on this particular
subject.
A morphological study of pulmonary
maturation in the fetus might allow a
better understanding of newborn
respira-tory disorders, specially pulmonary
im-maturity and neonatal atelectasis. In light
microscopy, the fetal development of
ani-mal and human lungs has been extensively
studied.12, 18, 2, 28, 44, 5<), (30, Ol Electron
micro-scopic studies, however, are few and
frag-mentary15’39’54’56-58’#{176}4 although adult lung has
has been frequently investigated
(bibliog-raphy in Schulz5#{176}).
Further information on fetal lung
ultra-structure ought to throw some light on the
following particular problems: (a) the
mor-phological evolution of pulmonary
epithe-hum, from tubular structures to adult
alveolar lining, and the origin of the two
types of alveolar cells described by Karrer2’
and Policard Ct al.#{176}in adult lung;
(b)
thepattern of glycogen distribution in the
epi-thelial cells; (c) the time of appearance
and the formation of structures
correspond-ing to the adult blood-air barrier; (d) the
time of appearance and the origin of
“lamellar inclusions” typical of type II
cells.
After a preliminary study of fetal rat
lung, this study of human fetal lung was
undertaken, using light and electron
micro-scope on sections from the same blocks.
Some early results have been reported
else-where.14
MATERIAL
AND
METHODS
Lung specimens from 13 human fetuses
of about 1 to 6 months gestation were
studied (Table I). The duration of
tion was estimated from the case history
and the length and weight of the fetuses,
taking into account the limited reliability
of these criteria. The pathologist’s
exami-nation of the fetuses showed no
abnormal-ity. Small blocks of tissue of about 1 mm3
were immersed in the various fixatives
(Table I) immediately after therapeutic
in-terruptions of pregnancy by uterotomy.
The tissues were dehydrated in acetone
and embedded in polyester Vestopal
52 In all cases, control sections about
1 i. thick were prepared from the same
blocks for light microscopy. They were
stained usually with silver nitrate,36
occa-sionally with hemalum-erythrosine.’ For
electron microscopy, thin sections were
made using Servall or LKB
ultramicro-tomes fitted with glass knives. Some
sec-tions were stained with silver nitrate,34
potassium permanganate,11 lead hydroxide
using Karnovsky’s method A,2#{176}uranyl
ace-tate or phosphotungstic acid,63 with or
without previous oxidation by hydrogen
peroxide or periodic acid.37’38 These
varia-tions in fixation and staining technique
were used in order to evaluate possible
artef acts and to obtain cytochemical
in-formation. Sections were studied using a
RCA EMU 3 C 1957 electron microscope,
at 50 kv.
Light Microscopy
OBSERVATIONS
Cases 1 to 9 (1-4 months, Figs. 1-4)
showed a tubular structure of the epithelial
ducts. In the youngest cases, the
appear-ance of the tubuli was uniform, with
col-umnar epithehium (Fig. 1). In older ones,
the appearance was more varied: at places
the cells were tall and columnar, at others
flatter and cuboidal (Fig. 2). Terminal
buds were usually compact. Glycogen was
extremely abundant and its distribution was
regular from cell to cell. In some cases,
small glycogen-free lacunae were observed
at the base of the epithehium (Figs. 3 & 4).
During fetal development, the tubuli
ap-peared to grow and constitute a greater
proportion of lung tissue. Ramifications
were more numerous, glycogen tended to
be distributed more irregularly. In all
cases, mesenchymal cells were loosely
scat-tered but showed a tendency to group
themselves round the epithelial tubuhi
(Figs. 2 & 3). Their cytoplasm was meager.
Capillaries were few at first with a very
narrow lumen. Later, they proliferated, the
lumen widened, and they were located
nearer to the tubuli without showing any
contact with the epithehium (Fig. 4). Only
small traces of glycogen were observed in
the mesenchyme and vessels.
In Cases 10 to 12 (4-5 months, Figs.
5 & 6), the structure of the tubuhi became
more irregular. Capillaries were seen to
progressively establish contacts with and to
penetrate wedgelike into the epithelium.
Structures like a blood-air barrier#{176}
ap-peared between the epithehial cells (Fig. 5).
These were frequently cuboidal and
slight-ly flattened. The mesenchyme became less
important. In Case 13 (5-6 months),
alveo-lar configuration was already recognizable
at places (Fig. 6).
Even with the light microscope, it was
often difficult to ascertain the exact
loca-tion of the observed epithelium. In the
younger cases, it was impossible to say if
the investigated epithehial cells would have
developed into alveolar or bronchial cells
because of insufficient differentiation and of
further growth of the tubuli.
Electron Microscopy
EPITHELIAL LINING: The epithelial celLi
were columnar or cuboidal until about the
fourth month (Cases 1-9). In the youngest
cases they showed little differentiation
(Fig. 27), but the development apparently
varied widely within a single case, and
zones of poor differentiation could be
ob-served until the fourth month. The nuclei
were ovoid, homogeneous, oriented
paral-lel to the cell. Interruptions of the nuclear
oThe term “blood-air barrier” is used hereafter
even though air is of course absent in the fetal
978FETAL LUNG
Fics. 1-6. LIGHT MICROSCOPY.
membrane were observed, probably due to
artefacts. Rather few organelles were
pres-ent, but Colgi zones were already well
developed (Fig. 9). The apical cell
mem-brane ran straight or slightly incurvated
(Figs. 8 & 15). The tubular lumen was
never completely collapsed even in the
youngest cases. The membranes of two
contiguous cells generally ran parallel
(Fig. 9). Toward the tubular lumen, they
showed reinforcements which suggested
terminal bars or even desmosomes (Fig. 8).
Near the base of the cell, the membrane
was sometimes interrupted: this was
prob-ably also an artefact due to the high water
content of the tissue.
During the third and fourth months,
better differentiated zones were more
fre-quent. The nucleus/cytoplasm ratio was
lower, the cytoplasmic organelles were
more numerous. The apical cell membrane
was incurvated and bulged into the lumen,
with some small microvilhi (Fig. 15). The
junction of two contiguous cells
pro-truded into the lumen. Desmosomal
struc-tures with cytoplasmic fibrils were
fre-quently observed, specially near the lumen.
Immediately under the subluminal
desmo-some, the cell membranes were infolded
and sinuous (Fig. 14). Further down, they
generally ran parallel and straight, but
separated at places to inclose small
“bal-loon-like” intercellular spaces (Figs. 7, 15,
19). These balloon-like spaces tended to be
more numerous in older cases. No
pino-cytosis vesicles were present.
During the fifth month, the epithehium
changed at places from a columnar or
cuboidal to a “pseudo-cuboidal” pattern.
The zone of close approximation between
two contiguous epithehial cells was smaller
and the cells touched one another only in
the basal region, with infolded cell
mem-branes (Fig. 27). These changes were most
conspicuous at places where direct contact
was first seen to be established between
the capillaries and the epithehium. A few
pinocytosis vesicles were observed in the
epithehial lining. At about six months (Case
13), it was possible in the more
differenti-ated zones to distinguish, as in the
adult,21,49 two different types of epithelial
cells: type I cells showed a small
perinu-clear body with long-attenuated
cyto-plasmic extensions lining a relatively
im-portant part of the alveolar surface; type
II cells were larger and rounder, without
extensions, and contained peculiar
struc-NOTE: All micrographs are from Vestopal W embedded sections. Fixation and staining procedures (cf. Material and Methods and Table I) are abbreviated as follows: F = formalin; F + Os = Formalin followed iy osmium tetroxide; Os = osmium tetroxide; Ag = silver nitrate; Mn = potassium
perman-ganate; Pb = lead hydroxide; PTA = phosphotungstic acid; U = uranyl acetate; AdL = adendothelial
layer; Ad\l = adepithelial membrane; BAB = blood-air barrier; BL balloon-like intercellular space;
C = capillary/capillary lumen; Coll = collagen; End = endothelium/endothelial cell; Epi = epithelium
/epithelial cell; CFS = glycogen-free space; Gly = glycogen; L = epithelial lumen; LI = “lamellar
in-clusion”; Mes = mesenchyme/mesenchymal cell; PV = pinocytosis vesicle.
FIG. 1. Case 1. Epithelial tubule (F + Os/Ag, x400).
Fic. 2. Case 4. Epithelial tubule surrounded by a crown-like arrangement of mesenchvmal cells. Clvcogeii
is illteflselV stained. (Os/Ag, x 1,400).
Fic. 3. Case 5. Epithelial tubuli was glycogen-free lacunes. Capillaries are located in the mesenchvnw. (F + Os/Ag, x400).
Ftc. 4. Case 7. Epithehial tubuli. Glycogen is mostly located in the basal region. (Os/Ag, x400).
FIG. 5. Case 12. Capillaries (arrows) penetrating wedge-like into the epithehium and separating the
flattened epithelial cells. Glycogen deposition is less abundant. (Os/Ag,
x
1,300).Ftc. 6. Case 13. Numerous capillaries bulging into the lumen. The structure is of alveolar type.
980 FETAL LUNG
FIGS. 7-9. EPrmELIurr.
ARTICLES
tures which we call “lamellar inclusions”
on purely descriptive grounds (Figs.
25-27). These inclusions are also known as
ty21 or “characteristic”64 inclusion
bodies, “osmiophihic bodies,”49 or “lamehlar
transformed mitochondria.”5’ 56
Glycogen was strikingly abundant until
the fourth month in the cytoplasm of most
epithehial cells. Large deposits could be
recognized without special staining as clear
areas of mottled appearance (Figs. 7 & 11).
On silver nitrate stained sections, small
dense granules of uniform size were
ob-served (Figs. 14 & 17). Staining with
po-tassium permanganate showed dense
gran-tiles about 50 m in diameter and smaller
15 m.t granuleshl (Figs. 10 & 15). Lead
hydroxide gave nearly the same result, but
not so regularly as potassium
permanga-nate (Fig. 16). Clycogen deposits, specially
in the younger cases, frequently occupied
the greater part of the cytoplasm, pushing
the organelles toward the cell membrane.
This pattern was most noticeable toward
the cell base, where a free 0.3 to 0.5 p.
wide space was seen to separate the
gly-cogen deposit from the cell membrane
(Fig. 10). Inside the glycogen deposits,
glycogen-free zones were very frequent.
The possibility that glycogen-free areas
which were not surrounded by a
mem-brane (Fig. 15) were due to artefacts
can-not yet be eliminated, although these areas
were frequently observed and had the
same appearance with different techniques.
Clycogen-free spaces limited by a
mem-brane (Figs. 7, 14, & 16) could be observed
until about the fifth month. Sometimes
more elaborate membrane patterns
oc-curred with concentric or juxtaposed
mem-branes (Fig. 17). After the fourth month,
glycogen tended to be less abundant,
es-peciahly in the more differentiated zones.
At about 6 months, it was still observed in
a number of epithehial cells, varying widely
in amount from cell to cell.
A few dense homogeneous inclusions of
round or ovoid shape, measuring about
0.2 to 0.5 IL, were present in the epithehial
cells in most cases. The margins of some
of these inclusions showed a relatively high
contrast after treatment of sections by
phosphotungstic acid (Fig. 22). Small
de-posits of neutral fat were seldom seen.
They were dense and homogeneous and of
irregular shape (Fig. 8).
“Larnellar inclusions” were present only
in Case 13 (5-6 months, Figs. 25 & 26).
They were about 1 p. in diameter, and
therefore larger than mitochondria.
Transi-tional forms between mitochondria and
“lamellar inclusions” were never observed,
in contrast with other observations.55,64
Their lamellar structure was quite
compa-rable to that of inclusions found in newborn
or adult lung, with the same variety of
appearances.6 These inclusions are
con-sidered to be typical of type II cells.21,49
Some rare cells of unusual morphology
located within the epithelium appeared to
be very different from the usual epithelial
cells described above and did not contain
glycogen. They could be divided into two
groups: (a) cells containing numerous
round black or gray granules of
homoge-neous texture measuring 0.1 to 0.2 p. in
diameter, limited by a single membrane
(Fig. 18); these cells were usually seen in
areas where the development of cilia and
the presence of smooth muscle cells in the
Ftc. 7. Case 5. Low power view of a basal region. The epithelial cytoplasm contains abundant glycogen
deposits of clear and mottled appearance surrounding glycogen-free spaces limited by a membrane.
(F + Os/U, x7,000).
FIG. 8. Case 2. Apical part of three cells. A terminal bar-like junction (arrow), short microvilli and a lipid droplet are shown. (F + Os/Pb, X 19,000).
982 FETAL LUNG
Fics. 10-13. BASAL REGION OF EPITHELIAL TUBULE AND SURROUNDING MESENCHYME.
vicinity of the epithelium indicated the
beginnings of bronchial differentiation; (b)
cells characterized by numerous
ergasto-plasmic saccules (rough endoplasmic
retic-ulum) were observed in two cases only
(Fig. 19).
A continuous adepithelial membrane,
about 30 mp. thick, separated the
epithe-hum from the mesenchyme, running
parallel to the basal cell membrane of the
epithelium at a distance of about 40 to 60
mp. (Figs. 10 to 13). This narrow
adepi-thelial space seemed to correspond to the
“lamina lucida” and the adepithelial
mem-brane to the “lamina densa,” which in Low’s
description30 together constitute the “outer
boundary membrane of tissue space.” At
places, the adepithelial membrane showed
projections of the same width and density
which penetrated into the tissue space and
sometimes formed network (Fig. 13).
MESENCHYME: The electron microscope
confirmed the light microscope obervations
of a random and loose arrangement of
mes-enchymal cells; with a slight tendency to
concentrate immediately around the
epithel-ial tubuli, forming a crown-like
arrange-ment. Mesenchymal cells were round or
ir-regularly shaped and showed little
differen-tiation. Tiwir cytoplasm was of low density
and contained few organelles. Cell
mem-branes were frequently ruptured, and no
pinocytosis vesicles were seen. Glycogen
was very seldom observed in the
mes-enchymal cells, and only in some of the
older cases.
The intercellular ground substance was
of very low electron density and frequently
contained collagen fibrils, scattered or in
small bundles. In the younger cases the
tissue space side of the adepithelial
mem-brane was frequently coated by a layer of
partly fibrillar electron dense material,
whose fibrils were thinner than collagen,
and in which we were unable to observe a
periodic structure (Fig. 12). This material
resembled that described by Karrer in
chick embryo aorta.23 After further
dif-ferentiation, this layer was replaced by
bundles of collagen fibrils with a
charac-teristic 64 mp. period.
Capilkzries were few and small in the
youngest cases, surrounded by
mesen-chymal tissue, at some distance from the
epithelial tubuli. At the earliest
develop-mental stages, they appeared as round or
ovoid groups of endothelial overlapping
cells, with an onion-like arrangement (Fig.
20). The lumen was extremely small. The’
cytoplasm was clear, with numerous
organ-elles and many pinocytosis vesicles toward
the lumen. The endothelial intercellular
junctions showed terminal bar-like
rein-forcements of cell membranes
characteris-tic of adult endothelium (Fig. 20).
Fur-ther developed capillaries were wider, the
lumen was more open. the endothelium
flatter, and its cytoplasm of slightly higher
density (Fig. 21). Although their
endothe-hum was thicker, capillaries of 3- to 4-month
fetuses no longer had any major
ultra-structural differences from those of adults.
Glycogen was sometimes observed in the
capillaries of older cases and more
fre-quently in the endothelium and smooth
muscle cells of small arteries.
Ftc. 10. Case 1. Abundant glycogen deposits occupying the greatest part of the cytoplasm and leaving a
free margin near the cell membrane. (F + Os/Mn, X30,000).
Ftc. 11. Case 2 Continuous adepithelial membrane. Numerous collagen fibrils in the adjoining tissue space. (F + Os/U, x15,000).
Ftc. 12. Case 4. Layer of partly fibrillar material coating the adepithelial membrane toward the tissue
(Os/unstained, x9,500).
984 FETAL LUNG
Fics. 14-17. DISTRIBUTION OF EPITHELIAL GLYCOCEN.
ARTICLES
An adendothelial layer of amorphous
structure surrounded the capillaries. The
appearance of this layer was very different
from that of the adepithelial membrane:
its density was slightly higher than that of
the intercellular space, its width varied
from 0.1 to 0.5 p., it was not continuous,
and it could either be seen in contact with
the outer cell membrane of the
endothe-hum or at a small distance away (Figs. 20
& 21). This layer seemed to correspond
to the “lamina densa” of Low’s inner
bound-ary membrane of tissue space.3#{176}
CONTACrS BETWEEN CAPILLARIES AND
Eps-THELIUM: Close relationships were seen to
be progressively established between the
capillaries and the epithelium, starting at 4
to 5 months. In Case 10, a few capillaries
were closely approximated to the
epithe-hum which was cuboidal or already
“pseudo-cuboidal” at places. A tissue space
about 0.5 p. wide remained between the
adepithehial membrane and the
adendo-thelial layer; the latter was thinner and
better contrasted than in younger cases
(Fig. 23).
In Case 12, some capillaries penetrated
into the epithelium driving back the
adepithelial membrane which always
re-mained continuous, and were separated
from the epithelial lumen by extensions of
the epithelial cells (Fig. 27). These
exten-sions were short and thick, and epithelial
cells could not yet be classified in two
dif-ferent groups. The blood-air barrier
struc-tures created by the junction between
the capillaries and the epithelium were
com-parable to, although thicker (about 1-1.5 p.)
than, blood-air barriers observed in adult
lung, which SchulzSG found to be 0.28 to
0.64 p. thick. Pinocytosis vesicles were
nu-merous in the endothelial lining. A few
vesicles were observed in the epithelial
lin-ing, whereas pinocytosis vesicles were
ex-tremely rare or nonexistent in the
epithe-hum of younger cases. The two cytoplasmic
linings of the barrier were separated by the
adepithelial membrane and the
adendothe-hal layer. These usually remained distinct
(Fig. 24).
In Case 13 (5-6 months), blood-air
bar-rier structures were more numerous, the
capillaries showed a wider lumen and
fre-quently bulged into the alveolar lumen
(Fig. 25). The barriers were longer and
thinner. Their epithelial lining belonged to
type I cells and contained more numerous
pinocytosis vesicles. The adepithelial and
adendothelial membranes frequently joined
to form an apparently single dense layer.3#{176}
The intermediate tissue space then
com-pletely disappeared.
The main features of our observations
are schematically summarized on Fig. 27.
DISCUSSION
The Origin of Adult Alveolar Cells
The human alveolus, in children and in
adults as in other mammals, is known to be
lined by two cellular types of different
morphology. The thin cytoplasmic lining
covering the greater part of the alveolar
surface, including blood-air barriers,
be-longs to type I cells.29 “Lamellar
inclu-sions” are characteristic of type II
cehhs.21,40 These findings have settled some
of the old controversies about the existence
of a cellular alveolar lining, which have
FIG. 14. Case 2. Intracellular glycogen-free space limited by a membrane. Infolded cell membranes
(arrow). (F + Os/Ag, x 18,000).
Ftc. 15. Case 6. Apical region. Glycogen-free spaces without limiting membranes. (F + Os/Mn,
x
18,000).FIG. 16. Case 10. Glycogen-free space limited by a membrane. (F + Os/Pb,
x
19,000).FIG. 17. Case 10. Elaborate membrane systems associated with glycogen-free spaces. (F + Os/Ag,
986 FETAL LUNG
Fics. 18-19. INFREQUENT TYPES OF EPITHELIAL CELLS WITHOUT GLYCOGEN.
ARTICLES
987
been reviewed by von Hayek19 and
Pot-ter.5#{176}However, new differences of opinion
have appeared concerning the origin of
alveolar epithelium. Both types of alveolar
cells have been claimed to be of
mesen-chymal origin by Policard et Type II
cells have been thought to be mesenchymal
by Marinozzi,33 who observed that their
supporting basement membrane was
dis-continuous. On the other hand, Low and
Sampaio31 ascribe an endodermal origin to
the alveolar epithehium.
Our observations seem to point toward
an endodermal origin of all alveolar cells.
The first line of evidence is the occurrence
of transitional cell forms
(“pseudo-cu-boidal” epithehium) which seem to establish
a link between the columnar epithehial cells
of the youngest fetuses and the alveolar cells
of types I and II of fully developed lung.
Secondly, the adepithelial membrane, which
is a general feature of the ultrastructure of
epithelia, is always observed in our
ma-terial as a continuous line separating the
epithelium, including type II cells, from
deeper mesenchymal tissue.
The occurrence of morphohogically
dif-ferent cells in the epithehium of some
cases may be explained in the following
way. Glycogen-free cells with dense
gran-ules perhaps represent the precursors of
bronchial mucous cells as they can often be
observed in areas showing evidence of
bronchial differentiation. It must,
how-ever, again be stressed that it often is
im-possible to distinguish potential alveolar
from potential bronchial cells, especially in
young fetuses. Glycogen-free cells with
numerous ergastoplasmic saccules are very
infrequent and have been observed in two
cases only. They may either be the
expres-sion of a particular functional state of the
usual type of epithelial cell, or a different
cell type, but still of epithelial character.
The Epithelial Glycogen
The presence of glycogen in fetal lung
epithelium is well established. Its
distribu-tion in developing mammalian lung has
been recently studied by Sorokin et
061 Electron microscope studies have
not yet been published to our knowledge.
On unstained sections, the appearance of
glycogen deposits is in agreement with
ob-servations of glycogen in chick embryo
2 Woodside and Dalton had
inter-preted this appearance in the pulmonary
epithelium of embryo mice as suggesting
degeneration (see Fig. 3 of their
publica-tion). Granules observed in stained sections
of our material are quite comparable with
those demonstrated as glycogen in other
tissues with the same staining procedures:
potassium permanganate,11 silver nitrate,35
or lead hydroxide.5’ This seems sufficient
evidence for the identification of glycogen
as such in our material. The significance of
glycogen-free areas inside tile glycogen
deposits is as yet unknown.
The Prenatal Alveolar Differentiation
Potter5#{176}has reviewed the numerous
the-ories expressed about the fetal
develop-ment of human lung and its structure
be-fore and after birth, and has shown that
during the fifth month of gestation
capil-laries begin to penetrate the hitherto
con-tinuous epithehium, and that uncovered
capillary loops come to be directly
ex-posed to the lumen. Our findings are
en-tirely consistent with her conclusions
ex-cept for the fact that capillary ioops are
always shown by the electron microscope
to be covered by a continuous epithelial
lining. In our material, the first blood-air
barrier structures were observed at 4 to 5
months, contemporary with the
“pseudo-cuboidal” transformation of the epithehium.
At 5 to 6 months, differentiation of two
Fic. 18. Case 3. Cells containing numerous granules. (F + Os/U, x30,000).
988 FETAL LUNG
FIGS. 20-21. CAPILLARIES.
ARTICLES
distinct types of epithelial cells was
com-pleted with the first appearance of
“lamel-lar inclusions,” and capillary loops bulging
into the lumen were fairly numerous.
Al-though fetal barriers are thicker, these
ul-trastructural features are quite comparable
to those of the alveolar lining of adults.
The “Lamellar Inclusions”
“Lamellar inclusions” were first
de-scribed in alveolar cells by Schlipkoter.53
They have been observed in the lungs of
all mammals studied with the electron
microscope. For morphological reasons,
lipids21 or phospholipids6 have been
sug-gested to be their main components.
“Lam-ellar inclusions” have been interpreted as a
transformation of alveolar cell
mitochon-dria’’54 due to effects of various noxious
agents. On the other hand, Treciokasl2 did
not find, after oxygen poisoning, any
evi-dence supporting the mitochondrial origin
hypothesis. Nor do our observations
pre-sent any such evidence, and it is probable
that lamellar inclusions originate from
sources other than directly transformed
mitochondria. Considering the problem
from this point of view, the decrease of
epithelial glycogen and the simultaneous
appearance of “lamellar inclusions” may be
of interest, together with the peculiar
morphology of some membrane systems
surrounding glycogen-free areas.
Studies on the alveolar lining layer have
been recently reviewed.t Von Neergard4’
had already stressed the importance of
sur-face tension in pulmonary mechanics. In
recent years, a substance which lowers
sur-face tension has been demonstrated in lung
extracts.’#{176}’4’ This substance or “surfactant”
has been shown to be probably a
lipopro-tein containing abundant
phosphohip-ids.4’25’48 The surfactant is assumed to line
the internal surface of alveoli. The alveolar
surface membrane, observed by Chase
us-ing freezing-drying techniques and electron
microscopy,9 has been thought to represent
its morphological equivalent.
More recently, it has been suggested
that “lamellar inclusions” contain the
sur-factant or its ‘#{176}This hypothesis
is supported by the following evidence. In
the lungs of fetal mice, the first “lamellar
inclusions” are observed the eighteenth day
of gestation,64 and the surfactant appears
at the same time.5’46 Pigeon lung does not
possess “lamellar inclusions.”’ and Klaus et
al.26 could not demonstrate a low surface
tension in pigeon lung extracts. These
workers further showed in guinea pig lung
a decrease of “lamellar inclusions” and a
simultaneous loss of surface activity after
bilateral vagotomy. They also found that
surface activity was located in the washed
mitochondrial fraction of rabbit lung, and
observed lamellar structures in surface
ac-tive material isolated from beef lung. In
lungs of infants with hyaline membrane
disease, surface activity is not foundl47
and the mean number of “lamellar
inclu-sions” per thin section of alveolar cell is
usually low;7 however, the value of this
last observation is somewhat diminished by
the possible influence of the duration of
extra-uterine life on the number of
“lamel-lar inclusions.” Surfactant cannot be
dem-onstrated in the lungs of human fetuses
weighing less than 1,000 gm,2 which
cor-relates well with the fact that “lamellar
inclusions,” in our material, were not
ob-served before 5-6 months.
Although the origin of “lamellar
990 FETAL LUNG
FIGs. 22-26. FURTHER DIFFERENTIATION OF EPITHELIUM AND CAPILLARIES.
ARTICLES
sions” and the mechanism of their
pro-posed action on the alveolar surface are
still not clear, the evidence reviewed here
strongly suggests the existence of a
rela-tionship between these organelles and the
surface properties of the lung lining.
SUMMARY
Results of a study of lung ultrastructure
in 13 human fetuses of about 1 to 6 months
of gestation are reported. Light microscope
controls were obtained in all cases. The
columnar epithelial cells of young fetuses
contained large deposits of glycogen which
tended to become less abundant between
the fourth and the sixth month. The fine
structure of epithehial glycogen is
de-scribed. A gradual transition between the
columnar or cuboidal epithelium and the
attenuated cytoplasmic lining of fully
de-veloped alveoli started during the fifth
month, when the first contacts between
capillaries and epithelium were
estab-lished. Further differentiation of the
epithe-hal cells in two morphologically different
types, as observed in adult lung, took place
during the sixth month. Both types
ap-peared to have the same endodermal
ori-gin. Lamellar inclusions were first
ob-served during the sixth month. Their
sig-nificance is discussed in the light of recent
work on alveolar surface activity.
REFERENCES
1. Avery, M. E.: The alveolar lining layer: a review of studies on its role in pulmonary mechanics and in pathogenesis of atelectasis.
PEDIATRICS, 30:324, 1962.
2. Avery, NI. E., and Mead, J.: Surface
proper-ties in relation to atelectasis and hyahine membrane disease. J. Dis. Child., 97:517,
1959.
:3. Van Breemen, V. L., Neustein, H. B., and
Bruns, P. D. : Pulmonary hyahine membranes studied with the electron microscope. Amer.
J. Path., 33:769, 1957.
4. Buckingham, S. : Studies on the identification
of an antiatelectasis factor in normal sheep
lung (Abstract). Amer. J. Dis. Child., 102:
521, 1961.
5. Buckingham, S., and Avery, M. E. : Time of
appearance of lung surfactant in the foetal
mouse. Nature, 193:688, 1962.
6. Campiche, M.: Les inclusions lamellaires des
cellules alv#{233}olaires dans le poumon du Raton. Relations entre l’ultrastructure et ha fixation.
J. Ultrastructure Res., 3:302, 1960.
7. Campiche, M., Jaccottet, M., and Juillard, E.: La pneumonose
a
membranes hyalines.Ob-servations au microscope #{233}lectronique. Ann.
Paediat., 199:74, 1962.
8. Campiche, M., Prod’hom, S., and Gautier, A.: Etude au microscope #{233}lectronique du
pou-mon de pr#{233}matur#{233}smorts en
d#{233}tresserespi-ratoire. Ann. Paediat., 196:81, 1961.
9. Chase, M.: The surface membrane of
pulmo-nary alveolar walls. Exp. Cell Res., 18:15, 1959.
10. Clements, J. A.: Surface tension of lung
ex-tracts. Proc. Soc. Exp. Biol. Med., 95:170,
1957.
11. Drochmans, P.: Mise en evidence du glycog#{232}ne. Proc. Europ. Reg. Conf. Electron Micr., I)elft 1960, vol. 2. Delft, Neederlandse Ver.
voor Elektronenmicr., 1961, pp. 645-649.
12. Dubreuil, G., Lacoste, A., and Raymond, R.: Observations sur le d#{233}veloppement du pou-mon humain. Bull. Histol. AppI. Physiol., 13:235, 1936.
13. Cautier, A.: Techniques de coloration his-tologique de tissus inclus dans des
polyes-ters. Experientia, 16:124, 1960.
14. Gautier, A., Campiche, M., Bozic, C.,
Hernan-(leE, E., Reymond, A., and Verdan C.: Pul-monary epithehium in the human fetus and
Ftc. 22. Case 6. Some dense inclusions (arrows) in epithelial cells stained by PTA. (F + Os/PTA, x 14,000).
Fic. 23, Case 10. Distance between the endothehial and the epithehial cell reduced to about 0.5i (Os/Pb,
x 14,000).
Ftc. 24. Case 12. “Blood-air barrier” structure. The adepithelial membrane and adenothelial layer are clearly distinct at places. (Os/nnstained, x34,000).
Ftc. 25. Case 13. Types I and II epithelial cells here clearly differentiated. (Os/unstained, X4,000).
992 FETAL LUNG
Ftc. 27. DIAGRAM SUMMARFZING THE OBSERVATIONS.
(a) Columnar epithelium with large glycogen deposits. Capillaries show overlapping endothelial cells.
(b) “Pseudo-cuboidal” epithelium. Capillaries are closer to the epithelium.
(c) Differentiation of epithelial cells in two types. Clvcogen deposition is iess abundant. “Lamellar
inclusions” are present in type II cells. “Blood-air barrier” structures are constituted.
newborn. Electron Microscopy. Proc. 5th Internat. Congr. Electron Micr. Philadel-phia, 1962, vol. 2. New York, Academic
Press, 1962, pp. WW-6:
4ten Internat. Kongr. Elektronenmikr., Ber-un, 1958, vol. 2. Berlin, Springer, 1960, pp. 404-409.
16. Groniowski, J., und Djaczenko, W. : Die
Fein-struktur des Lungengewebes nach dem
Beginn der Atmung. Z. Zellforsch. Mikr. Anat., 53:639, 1961.
17. Groniowski, J., and Biczyskowa, W. : Ultra-structure of the blood-air barrier of the neonatal human lungs. Electron Microscopy.
Proc. 5th Internat. Congr. Electron Micr., Philadelphia, 1962, vol. 2. New York, Aca-demic Press, 1962, pp. WW-5.
18. Ham, A. W., and Baldwin, K. W. : A histo-logical study of tile development of the
lung with particular reference to the nature
of alveoli. Anat. Rec., 81 :363, 1941. 19. von Hayek, H. : Die menschliche Lunge.
Ber-un, Springer, 1953.
20. Kamovsky, M. J.: Simple methods for “stain-ing with lead” at high pH in electron mi-croscopy. J. Biophys. Biochem. Cytol., 11: 729, 1961.
21. Karrer, H. E. : The ultrastructure of mouse lung. General architecture of capillary and alveolar walls. J. Biophys. Biochem. Cytol., 2:241, 1956.
22. Karrer, H. E., and Cox, J.: Electron micro-scopic study of glycogen in chick embryo liver. J. Ultrastructure Res., 4:191, 1960. 23. Karrer, H. E.: Electron microscope study of
developing chick embryo aorta. J. Ultra-structure Res., 4:420, 1960.
24. Kellenberger, E., Schwab, W., and Ryter, A.: L’utilisation des polyesters comme materiel d’inclusion en ultramicrotomie. Experientia, 12:421, 1956.
25. Klaus, M. H., Clements, J. A., and Havel, R. J.: Composition of surface-active material isolated from beef lung. Proc. U.S. Nat. Acad. Sci., 47: 1858, 1961.
26. Klaus, M., et al.: Alveolar epithelial cell mito-chondria as source of the surface-active lung lining. Science, 137:750, 1962.
27. Lelong, M., and Laumonier, R.: Histological and histochemical evolution of the foetal lung; in Anoxia of the Newborn Infant, a Symposium; edited by J. F. Delafresnaye and T. E. Opp#{233}. Springfield, Illinois: Thomas, 1954, pp. 61-84.
28. Loosli, C. C., and Potter, E. L.: Pre- and post-natal development of the respiratory por-tion of the human lung. Amer. Rev. Resp. Dis., 80 (Suppl.): 5, 1959.
29. Low, F. N.: The pulmonary alveolar epithe-hum of laboratory mammals and man. Anat.
Rec., 117:241, 1953.
30. Low, F. N.: The extracellular portion of the
human blood-air barrier and its relation to tissue space. Anat. Rec., 139:105, 1961.
31. Low, F. N., and Sampaio, M. M. : The pul-monary alveolar epithehium as an entodermal derivative. Anat. Dec., 127:51, 1957. 32. Luft, J. H. : Permanganate-a new fixative for
electron microscopy. J. Biophys. Biochem.
Cytol., 2:799, 1956.
33. Marinozzi, V. : La structure de l’alv#{233}olepul-monaire #{233}tudi#{233}eau moyen de l’impr#{233}gnation
a
l’argent. Verh. 4ten Internat. Kongr.Elek-tronenmikr., Berlin, 1958, vol. 2. Berlin,
Springer, 1960, pp. 412-415.
34. Marinozzi, V.: Silver impregnation of ultra-thin sections for electron microscopy. J.
Bio-phys. Biochem. Cytol., 9: 121, 1961. 35. Marinozzi, V. : Cytochimie ultrastructurale.
Fixations et colorations. Experientia, 17:429,
1961.
36. Marinozzi, V.: Techniques de coloration de
tissus inclus dans des mati#{234}res plastiques pour l’#{233}tudeau microscope optique
a
hauteresolution.
z.
wiss. Mikr. 65:219, 1963.37. Marinozzi, V., and Gautier, A. : Essais de
cyto-chimie ultrastructurale. Du role de l’osmium
r#{233}duitdans les “colorations” #{233}lectroniques. C. R. Acad. Sci. Paris, 253:1180, 1961. 38. Marinozzi, V., and Gautier, A. : Fixations et
colorations. Etude des affinit#{233}s des com-posants nucl#{233}oprot#{233}iniquespour l’hydroxyde de plomb et l’ac#{233}tate d’uranyhe. J. Ultra-structure Res., 7:436, 1962.
39. Matsumura, T., et al.: Electron microscopic ob-servations on the development of alveolar structures with special references to the
cause of respiratory distress in premature in-fants. Ann. Paediat. Japon., 8:72, 1962. 40. Millonig, G.: Advantages of a phosphate buffer
for OsO4 solutions in fixation. (Abstract).
J. Appl. Phys., 32:1637, 1961.
41. von Neergard, K.: Neue Auffassungen #{252}ber einen Grundbegriff der Atemmechanik. Die Retraktionskraft der Lunge, abhangig von der Oberflachenspannung in den Alveo-len. Z. Ges. Exp. Med., 66:373, 1929. 42. Newsom, W. T.: Electron microscopy in the
premature infant lung. Bull. Tulane Med. Fac., 16:61, 1957.
43. Palade, G. E.: A study of fixation for electron
microscopy. J. Exp. Med., 95:285, 1952. 44. Parmentier, R: L’a#{233}ration n#{233}onatale du
pou-mon. Contribution exp#{233}rimentale et anatomo-clinique. Rev. Belge Path. Med. Exp., 29: 121, 1962.
45. Pattle, R. E.: Properties, function and origin of the alveolar lining layer. Nature, 175: 1125, 1955.
46. Pattle, R. E.: The formation of a lining film
by foetal lungs. J. Path. Bact., 82:333, 1961.
47. Pattle, R. E., et al.: Inability to form a
respiratory-994
distress syndrome in the newborn. Lancet,
2:469, 1962.
48. Pattle, R. E., and Thomas, L. C.: Lipoprotein composition of the film lining the lung.
Nature, 189:844, 1961.
49. Policard, A., Collet, A., and Pr#{233}germain, S.: Electron microscope studies on alveolar cells from mammals. Proc. Europ. Reg. Conf. Elec-tron Micr. Stockholm 1956, Stockholm, Almqvist & Wiksell, and New York, Academic
Press, 1957, p. 244.
50. Potter, E. L.: Pulmonary pathology in the newborn. Advanc. Pediat., vol. 4. Chicago, illinois, Year Book Publishers, 1953, pp. 157-189.
51. Revel, J. P., Napolitano, L., and Fawcett, D. W.: Identification of glycogen in electron
micrographies of thin tissue sections. J.
Biophys. Biochem. Cytol., 8:575, 1960.
52. Ryter, A., and Kellenberger, E.: L’inclusion
au polyester pour l’ultramicrotomie. J. Ui-trastructure Res., 2:200, 1958.
53. Schlipk#{246}ter, H. W.: Elektronenoptische Unter-suchungen ultrad#{252}nner Lungenschnitte. Deutsche Metd. Wschr., 45:1658, 1954. 54. Schulz, H.: Elektronenmikroskopische
Unter-suchungen der Lunge des Siebenschl#{228}fers im Winterschlaf und der embryonalen Ratten-lunge. Verh. I)eutsche Ges. Path., 41:343, 1957.
55. Schulz, H.: Die Pathologic der Mitochondrien im Alveolarepithel der Lunge. Beitr. Path.
Anat., 119:45, 1958.
56. Schulz, H.: Die submikroskopiche Anatomic
und Pathologie der Lunge. Berlin, Springer,
1959.
57. Schwarz, W.: Die Entstehung der elastischen Fasern in der embryonalen Lunge des
Men-schen. Verh. 4ten Internat. Kongr.
Elek-tronenmikr., Berlin 1958, vol. 2. Berlin,
Springer, 1960, pp. 409-412.
58. Schwarz, W., und Merker, H. J.:
Lichtmikro-FETAL LUNG
skopische and elektronenoptische
Unter-suchungen #{252}berdie Entwicklung der ento-dermalen und mesenchymalen Lungenaniage
(Interstitium) des Menschen. Beitr.
Silikose-Forsch., Sonderband, 3: 103, 1960.
59. Selby, C. C.: An electron microscope study of the epidermis of mammalian skin in thin sections. I. Dermo-epidermal junction and basal cell layer. J. Biophys. Biochem. Cytol., 1:429, 1955.
60. Sorokin, S.: Histochemical evetlts in develop-ing human lungs. Acta Anat., 40:105, 1960. 61. Sorokin, S., Padykula, H. A., and Herman, E.: Comparative histochemical patterns in de-veloping mammalian lungs. Developmental Biol., 1:125, 1959.
62. Treciokas, L. J.: The effect of “oxygen
poison-ing” on the alveolar cell mitochondria as
revealed by electron microscopy. Aerospace Med., 30:674, 1959.
63. Watson, M. L.: Staining of tissue sections for electron microscopy with heavy metals.
J.
Biophys. Biochem. Cytol., 4:475, 1958. 64. Woodside, G. L., and Dalton, A. J.: Theultra-structure of lung tissue from newborn and embryo mice. J. Ultrastructure Res., 2:28, 1958.
Acknowledgment
We wish to express our thanks to Professor V. R. Merz, Director of the Department of Obstetrics and Gynaecology, Lausanne University Hospital, who kindly made available the fetuses used in this study. We are indebted to Dr. C. Bozic of the Department of Pathology for his help in the exam-ination of the fetuses, to Dr. L. S. Prod’hom of the Department of Pediatrics for his stimulating
criticism and to Miss A. Wansbrough who
effi-ciently corrected the manuscript. The technical
