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Effects of estrogen on the transition zone of the mouse uterine cervix.

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Effects of Estrogen on the Transition Zone of the
Mouse Uterine Cervix '
CAROLYN F. BRADLEY AND CHARLES E . GRAHAM
Yerkes Regional Primate Research Center, Emory University,
Atlanta, Georgia 30322
ABSTRACT
Histologic changes in the region of the squamocolumnar junction of the uterine cervix in ovariectomized C3H/HeJ mice were studied at 6-12
hour intervals after the injection of 2.3 pg estradiol benzoate. Mitotic activity,
especially in the basal layer of stratified squamous epithelium, increased as time
elapsed after estrogen administration and produced a concomitant increase in
stratification of squamous epithelium; some mitotic activity was present in the
uterine columnar epithelium throughout the period of observation. The histologic
character of the squamocolumnar junction changed from gradual transition to
an abrupt demarcation, as in intact mice during the estrous cycle. Subcolumnar
cells in the transition zone of untreated animals formed a unilayered reticulum
which after estrogen injection developed into a reticulum of stratified squamous
epithelium. Extensions of stratified epithelium which develop during chronic
estrogen treatment apparently develop from the stratified reticulum. There was
no evidence to indicate any contribution of metaplasia to estrogen-induced epidermization.
In a previous study of epidermization
(j.e., the spread of stratified squamous epithelium) in the cervix and uterine horns
of mice during chronic estrogen treatment,
it was found that most of the newlyformed stratified squamous epithelium
originated by the proliferation and extension of the existing stratified squamous
epithelium (Graham, '67b, '68a,b). At the
earliest interval studied after the commencement of treatment (2 weeks), small
atpparently isolated islands (or nodules) of
stratified squamous epithelium were noted
in the cervix. It was shown by a study of
serial sections that most of these nodules
of squamous cells actually formed a network continuous with the main mass of
stratified squamous epithelium.
It appears possible that this reticulum
arose by multifocal squamous metaplasia
(of the columnar epithelium, followed by
secondary proliferation and coalescence of
many foci. If so, squamous metaplasia
must contribute to estrogen-induced epidermization. (Metaplasia is the formation
of stratified squamous epithelium by the
proliferation and rearrangement of columnar cells.) Resolution of the possible role
ANAT.REC., 173: 235-248.
of metaplasia in the mouse cervix is of
considerable theoretical importance, since
the mouse is a valuable model for the
study of cervical carcinoma (Graham,
'71b), and some workers have proposed
that cervical carcinoma in women is associated with areas of metaplasia (see review by Baggish and Woodruff, '67).
The cervical epithelium of the mouse,
studied under natural and experimental
conditions, has been described by Graham
('66, '67b, '68a,b) and Leppi ('64). Two
basic types of interface between the stratified squamous and columnar epithelia may
be recognized (Graham, '66). ( 1 ) transitional junctions, characterized by a gradual decrease in cell layers between the
vaginal stratified squamous and uterine
columnar epithelia; the area of intergradation of the two epithelia is referred to as
the transition zone, and the point at which
the epithelium first becomes single-layered
Received Sept. 7, '71. Accepted Jan. 3, '72.
1 Supported by PHS Research grant RR-00165from
NIH.
2 Present address : Zoology Department, University
of Georgia, Athens, Georgia 30601.
3 Submit reprint requests to: C. E. Graham, Yerkes
Regional Primate Research Center, Emory University,
Atlanta, Georgia 30322.
235
236
CAROLYN F. BRADLEY AND CHARLES E . GRAHAM
is called the squamocolumnar junction; columnar epithelium. The columnar cells
( 2 ) abrupt junctions, characterized by an appear to overlap the adjacent stratified
immediate, distinct change from several squamous epithelium, and this relationlayers of squamous epithelium to a single ship has been described as epithelial overcolumnar layer. Transitional junctions lap (fig. 1 ) ; however, i t is important to
predominate when the estrogenic stimula- remember that the stratified squamous epition is slight or absent, as in intact mice thelium itself produces this surface layer
in proestrus and early estrus, and in un- of columnar cells. Epithelial overlap can
treated, ovariectomized mice. The transi- be associated with both transitional and
tion zones become shorter and the inci- abrupt junctions; thus, squamocolumnar
dence of abrupt junctions increases as the junctions may be classified as one of the
estrous cycle progresses, or as time ad- following types : transitional with overlap,
vances after the injection of estrogen into transitional without overlap, abrupt with
ovariectomized animals. During the first overlap or abrupt without overlap (fig. 1).
half of the estrous cycle the surface layer
MATERIALS AND METHODS
of the stratified squamous epithelium of
Thirteen-week-old C3H/HeJ mice (The
the transition zone differentiates into columnar cells morphologically identical to, Jackson Laboratory, Bar Harbor, Maine)
and continuous with, the adjacent uterine were bilaterally ovariectomized under
A
B
C
D
Fig. 1 Diagram illustrating changes i n the mouse squamocolumnar junction area during the
estrous cycle; uterus at left, vagina at right. A, Diestrus: epithelium is atrophic, squamocolumnar
junction is transitional. €3, Proestrus: hyperplasia of squamous epithelium and differentiation of
columnar surface layer begin; junction is transitional with overlap. C, Early estrus: proliferation of
stratified squamous epithelium has led to formation of a n abrupt junction; keratinization is leading
to desquamation of some of the columnar surface-layer cells. D, Metestrus: loss of columnar surface
cells is complete; junction is abrupt without overlap. (after Graham, '67a)
ESTROGEN EFFECTS ON MOUSE CERVICAL EPITHELIUM
sodium pentobarbital anesthesia (Pilgrim
and DeOme, ’ 5 5 ) , and one week later each
animal received a single subcutaneous injection of 2.3 pg estradiol benzoate in 0.1
ml cottonseed oil. (We have found that
this dose induces a genital response resembling that seen in the intact mouse at
estrus.) Groups of mice were sacrificed at
each of the following intervals after injection: 0, 6, 12, 18, 24, 36, 48, 60, 72, 84
and 96 hours. Each genital tract was removed, fixed in Bouin’s solution, embedded in paraffin, and sectioned serially
in the frontal plane at 6 p. In order to
demonstrate mucin and the basement
membrane, which are PAS-positive, the
sections were stained in periodic acidSchiff (PAS); alternate slides were first
exposed to digestion in malt disastase
(PASD). The nuclei were stained with
Erlichs hematoxylin (Pearse, ’61).
The following analyses were performed
on the serially sectioned material from
each group:
Classification of junctions. All of the
squamocolumnar junctions visible in
every tenth section were classified according to type.
Epithelial proliferation analysis. Figure
2 shows the essential anatomical features
of the mouse genital tract. Two regions
each of columnar epithelium and stratified
squamous epithelium (fig. 2) were chosen
for mitotic index determinations: region 1
is a Iuminal surface (i.e., non-glandular)
area of columnar epithelium at least 0.2
cm above the squamocolumnar junction;
region 2 is the columnar epithelium at the
j,unction; regions 3 and 4 are stratified
squamous epithelium at the squamocolumliar junction, and low in the common cervical canal, respectively.
The mitotic index for each region of a
(cervix was found by determining the percentage of a sample of 300 cells which
were in any recognizable phase of mitosis.
The sample of 300 cells for each region
was attained by counting 50 cells in every
tenth section until 300 cells per region had
been counted in the cervix. This method
insured that representative areas throughout the cervix were evaluated. In the
stratified squamous regions (fig. 2), distinct mitotic figures were seen in the layer
237
Fig. 2 Schematic frontal section through
mouse uterus, showing location of areas subjected to detailed study. Columnar epithelium,
thin line; stratified squamous epithelium, thick
line.
of “epithelial overlap” as well as in the
basal layer, so separate mitotic indices
were calculated for these two layers. (The
basal layer is the deepest layer of squamous epithelium, i.e., the row of cells immediately contiguous to the basement
membrane.)
The number of layers of squamous epithelium were counted in regions 3 and 4.
Stratified and columnar epithelia. By
means of a microprojector, the distribution
of the epithelial components of a frontal,
approximately median section passing
through the squamocolumnar junction was
plotted. The procedure was repeated for
adjacent serial sections until a precise twodimensional map representing a view of
the distribution of the epithelia over a portion of the luminal surface of the cervix
was obtained (fig. 3).
The number of apparently isolated
nodules of stratified squamous cells cranial
to the main squamocolumnar junction was
238
CAROLYN F. BRADLEY AND CHARLES E. GRAHAM
0
RESULTS
Histologic changes in the cervical canal.
The stratified cervical epithelium of untreated, ovariectomized mice presented an
atrophic appearance and typically consisted of two layers of cells (fig. 4 ) , a
superficial layer of low cuboidal cells, and
a basal layer of cells with very little cytoplasm, so that the nuclei were crowded
close together. These two layers merged
very gradually into the single layer of
cuboidal-to-columnar cells of the uterine
horns, located cranially; the area of intergradation is the transition zone. After
estrogen injection, the two layers of the
stratified epithelium became more easily
distinguishable. The nuclei of the basal
cells in the transition zone became round
and larger, and several prominent nucleoli
appeared. The surface layer of cells differentiated into cells closely resembling the
columnar epithelium of the adjacent
uterine horns, having marked cytoplasmic
basophilia, and often distal regions of
clear cytoplasm, the nuclei being located
towards the base of the cells. Later, proliferation and keratinization of the stratified epithelium took place, leading to
Fig. 3 Illustration of the location of area sub- desquamation of the surface layer of
jected to mapping of epithelial distribution. The
cuboid-to-columnar cells, and its replacecylinder represents the lumen of one of the cervial
canals. The plane ABCD represents a frontal ment by layers of keratinized cells. Kerahistologic section passing through the canal. The
tinization first appeared in the vaginal
squamocolumnar junction is represented by the fornices at 36 hours after the injection of
circumferential ring at the center of the cylinder.
estrogen (we did not examine more caudal
The small area EFGH outlined on the wall of the
lumen astride the squamocolumnar junction rep- portions of the vagina). As time after
resents an area which was mapped, using ABCD
estrogen injection increased, progressively
and adjacent sections. The portion of the mapped
more cranial areas of the stratified epiarea caudal to the squamocolumnar junction is
thelium commenced to cornify, and conshown in black (stratified epithelium) and the
sequently desquamation of the surface
cranial portion white (columnar epithelium).
area layer occurred earlier in the common
cervical canal (48 hours post injection)
counted in every tenth section of the serial than near the squamocolumnar junction
sections from all animals sacrified 48 (desquamation was incomplete at 92
through 96 hours after estrogen injection. hours post injection in portions of some
In addition to the material prepared specimens). Leucocytic infiltration of the
specifically for this study, the histological cervical epithelium commenced 84 hours
material previously described in Graham's after estrogen injection suggesting a wanstudy ('66) of the normal cyclic changes ing of estrogenic effect.
in the intact mouse cervix was reviewed in
T h e squamocolumnar junction. Table
order to obtain comparative information 1 summarizes the classification of juncfrom intact animals. This material con- tions and shows that the character of the
sisted of representative non-serial sections, squamocolumnar junction changed as
stained with hematoxylin and eosin, from time increased after estrogen injection.
several strains of mice killed at appropri- Each type of junction showed a peak freate stages of the estrous cycle.
quency at a different time after the initia-
239
ESTROGEN EFFECTS ON MOUSE CERVICAL EPITHELIUM
tion of estrogen treatment. The majority
of junctions were transitional without
overlap until 24 hours after the injection
of estrogen. At 36 hours, all the junctions
were transitional with overlap, reflecting
the tendency of the surface layer of the
transition zone to differentiate and acquire
characteristics similar to the adjacent
columnar epithelium of the uterine horn.
The first abrupt junctions were observed
at 48 hours post injection, at the same
time that a marked increase in the number of cell layers appeared in this area
(table 2); these junctions were still predominantly associated with overlap. By 96
hours, a significant proportion of the predominantly abrupt junctions were without
overlap, due to partial desquamation of
the cuboidal-to-columnar surface layer of
the transition zone.
Analysis of mitotic indices. Table 2 is
a summary of mitotic counts and the
number of layers of squamous epithelium
in the cervix at various intervals after
estrogen injection. Mitotic activity was
absent in the basal layer of the cervical
slratified epithelium of untreated ovariectomized animals, but surprisingly, mitoses were present in the surface layer of
tlhis epithelium (fig. 4). The mitotic index
of the surface layer close to the squamocolumnar junction (area 3 , table 2, fig. 2 )
was three times the index of the surface
1,ayerlow in the common canal (area 4).
A few mitoses also occurred in the columnar epithelium of untreated animals.
After estrogen injection, the mitotic index of both areas of the columnar epithe-
lium showed a progressive, but erratic increase until 24 hours post injection, and
then a decrease. The frequency of mitosis
was generally higher in area 1 high in the
uterine horn, compared with area 2 close
to the squamocolumnar junction.
Mitotic figures were present in the stratified epithelium of the cervix after estrogen
injection (figs. 5, 6). The stratified epithelium close to the squamocolumnar junction (area 3 ) showed a similar pattern of
changes to the columnar epithelium of the
uterine horns, with an initial rise in the
mitotic index which ended 24 hours post
injection, and was followed by a decline.
However, unlike the columnar epithelium
of the uterine horns, the frequency of
mitoses in area 3 eventually fell to zero
(at 84 hours post injection). The mitotic
index of the surface layer in area 4 (low
in the cervical canal) did not show evidence of stimulation by estrogen, except
possibly at 24 hours : thereafter mitoses
were absent, and in this area the superficial layer was completely desquamated
by 60 hours.
The mitotic response of the basal layer
of the cervical stratified epithelium differed
strikingly from the surface layer. Mitoses
were absent until 18 hours post injection
in area 4. At this time a gradual rise commenced which persisted until 84 hours. At
92 hours a slight fall in the mitotic index
was noted. Close to the squamocolumnar
junction, the mitotic index remained at
zero until 36 hours post injection, when
a progressive rise commenced and con-
TABLE 1
Frequency of various types of cervical squamocolumnar junction after a sirzgle injection
of e8stradiol benzoate in C 3 H / H e J mice ( 2 . 3 p g )
Number
of
animals
Hours
after
injection
Total
number of
determinations
3
4
5
4
2
0
6
12
18
24
36
48
60
72
84
96
59
83
125
114
70
83
123
90
106
109
152
4
4
2
2
2
3
Type of junction and frequency ( % )
Transitional
w/out overlap
Transitional
w/overlap
100
96.4
96
95.6
28.6
0
3.6
4
4.4
71.4
100
74
51.1
28.3
16.5
2.6
0
0
0
0
0
0
Abrupt
w/overlap
Abrupt
w/out overlap
0
0
0
0
0
0
26
48.9
71.7
81.7
75
0
0
0
0
1.8
22.4
1.08
(13/1200)
6.25
(50/800)
4.60
(55/1200)
1.40
(17/1200)
2.30
(14/600)
1.70
(10/600)
0.50
(3/600)
1.10
(10/900)
18
24
36
48
60
72
84
96
4
2-3
4
4
2
2
2
3
~
0.80
( 12/1500)
12
5
-3
-2
-3
3.75
(45/1200)
3.1
(37/1200 )
3.7
(22/600)
5.5
(33/600)
6.0
(36/600)
4.2
(38/900)
0.3
(4/ 1200)
0.4
(4/1055)
0.2
(1/495)
0.2
(1/430)
0
(0/340)
0
(0/145)
0
( 0/600 )
2.5
(30/ 1200)
2.5
(30/1200)
3.3
(20/600)
3.5
(21/600)
5.7
(34/600)
6.7
(60/900)
2.75
(33/1200)
1.60
(19/1200)
3.00
(18/600)
2.30
(14/600)
1.20
(7/600)
3.10
(28/900)
2.2
(13/600)
1
2
2.7
(16/600)
(4/1200)
0.33
3.80
(23/600)
1.58
(19/1200)
0
(0/1200)
1.33
(16/1200)
-3
0
(0/400)
0
(0/1200)
1.2
(7/600)
0.33
(4/1200)
0.13
(2/1500)
0
(0/1500)
1.33
(20/1500)
0
(0/1500)
2.0
(30/1500)
0.25
(3/1200)
0
(0/1200)
0.22
(2/900)
0.75
(9/1200)
0
of
surface
layer
(O/1200)
0
(0/900)
0.67
(6/900)
basal
layer
Low in common canal
(area 4 )
0.42
( 5/ 1200)
0
(0/900)
0.56
(5/900)
surf ace
layer
Squamo-columnar junction.
One of the specimens was sectioned so that the area of the SCJ could not be seen.
3 T h i s layer was no longer present in these specimens.
0.42
( 5/ 1200)
basal
layer
At SCJ (area 3)
At SCJ 1
(area 2 )
Columnar epithelium
Highin
uterus
(area 1 )
Squamous epithelium
Mitotic index
(number of mitoses/number of cells counted)
squamous cell layers in the genital tract of C$H/HeJmice after a single injection
6
~~
Hours
after
injection
of
4
3
No.
of
animals
Mitotic counts and number
TABLE 2
4-8
3-5
3
2-5
2-5
2-3
2-3
2
2
2
2
A t SCJ
(area3)
~~
11-14
10-12
8-11
8-11
6-9
3-5
2 4
2
2
2
2
Common
can a1
(area4)
No. of squamous layers
estradiol benzoate (2.3 p g )
0
A
ta
ESTROGEN EFFECTS ON MOUSE CERVICAL EPITHELIUM
24 1
was suggested by the presence of mitotic
figures in the subcolumnar layer (fig. 7).
No evidence of the origin of subcolumnar
cells from columnar cells was encountered.
The distribution of apparently isolated
subcolumnar cells from the 12- to 36-hour
intervals was determined in representative
specimens by the serial mapping technique. In these specimens the subcolumnar cells were found to form a simple
reticulum which was continuous with the
Fig. 4 Photomicrograph showing part of the basal layer of the two-layered squamous
two-layered transition zone from the cervix of an epithelium of the squamocolumnar juncuntreated, ovariectomized mouse. The basement
tion transition zone (fig. 8). The distribumembrane is not clearly defined. Note mitosis
tion of subcolumnar cells was rather simi(arrow) in surface layer. (PAS. x 800).
lar to that of the nodules of stratified
squamous epithelium described below, altinued until the end of the period of ob- though not quite so complex (compare
servation.
figs. 8, 9).
Distribution of stratified and columnar
Subcolumnar cells were no longer recogepithelia. In untreated mice the two- nizable 48 hours after estrogen injection;
1,ayered transition zone was extensive, and instead, examination of longitudinal secthe cervical epithelium was essentially tions of cervices from animals killed at
atrophic, as described above. An occasional this time revealed the first presence of
oval-to-spherical cell that was morphologi- small, discrete, oval-to-spherical clusters of
cally identical to the basal-layer cells of the stratified squamous cells (nodules) among
two-layered epithelium was distinguish- the uterine columnar cells just cranial to
able under the uterine columnar cells the squamocolumnar junction. These nodcranial to the squamocolumnar junction. ules of squamous cells were clearly de'These cells, which we designated as sub- marcated from the overlying columnar epi(columnar cells became larger and more thelial cells and the underlying basement
easily recognized at later intervals after membrane, and were separated from each
estrogen injection. At 36 hours after estro- other by varying numbers of columnar
gen administration, the subcolumnar cells cells. They were always located between
occurred not only singly, but also in short the columnar cell layer and the basement
rows, and the source of the additional cells membrane. The individual cells in these
Fig. 5 Photomicrograph showing transitional junction from a mouse cervix 18 hours
after estrogen injection; uterus to the left, vagina to the right. Surface-layer mitosses in the
two-layered epithelium at right, also mitotic figure (arrow) in the columnar epithelium at
left. The basal-layer cells contain distinct nucleoli, in contrast to basal cells in untreated
animals (compare fig. 4 ) .
242
CAROLYN F. BRADLEY AND CHARLES E . GRAHAM
Fig. 6 Photomicrographs of surface-layer mitoses in mouse cervical stratified squamous epithelium 24 hours after estrogen injection. a, is low
in the cervical canal (area 4); b, is near the
squamocolumnar junction (area 3 ) . (both PASD.
a, x 740; b, x 6 0 0 ) .
nodules were not always clearly delineated
from each other; their nuclei were identical in appearance with the nuclei of the
individual subcolumnar cells described
above. Mitotic figures were frequently seen
in these nodules at all stages of treatment
from 48 hours onward. After the sudden
occurrence of the nodules at 48 hours ( a
finding that coincided with the appearance
of the first abrupt squamocolumnar junctions), they reached a peak frequency at
60 hours (table 3), then showed a marked
decrease to a relatively stable frequency
for the remaining period of observation.
In individual histologic sections these
nodules of squamous cell appeared to be
isolated, discrete entities (fig. 10). However, a thorough study of serial sections
from all specimens in which they occurred
showed that, in fact, in all cases the
nodules coalesced to form a network of
stratified squamous epithelium continuous with the main area of cervico-vaginal
stratified squamous epithelium. Figure 9
shows a representative map of the distribution of stratified squamous epithelium
near the end of the period of observation.
Re-examination of the histological material previously described by Graham
('66) revealed the presence of similar nodules of squamous cells in mice at estrus
or metestrus I or 11. Since serial sections
of these specimens were not available,
no assessment of the continuity of the apparently isolated nodules with the main
mass of stratified squamous epithelium
was possible. No mitoses were noted in any
of these nodules in the intact animals, and
mitotic figures were infrequent in the
stratified squamous epithelium throughout the genital tract, although occasional
mitoses were noted in the columnar-cell
surface layer of epithelial overlap near
the squamocolumnar junction in some
specimens.
DISCUSSION
Location and incidence of mitoses.
Basal-layer mitoses in both regions of
stratified squamous epithelium were absent until 18-24 hours after estrogen administration, and then showed a steady
Fig. 7 Photomicrograph of subcolumnar cells in transition zone, 36 hours after estrogen
injection; columnar epithelium on the right. Arrows indicate two subcolumnar cells that
appear isolated from the subcolumnar cells of the transition zone. (PASD. x 520).
243
ESTROGEN EFFECTS ON MOUSE CERVICAL EPITHELIUM
jection of 5 pg estrone into a small number of castrate mice. The lack of a comparable peak in our material may be due
in part to our use of estradiol benzoate,
which is relatively long acting.
Fig. 8 Map of distribution of subcolumnar
cells (stippled area) o n part of the luminal surface of the cervical canal of a mouse 24 hours
after estrogen injection. The subcolumnar cells
are continuous caudally with the stratified squamous epithelium (solid black area) of the lower
cervix and vagina. Cranially, the subcolumnar
cells abut the columnar cells (white areas), penetrate among the columnar cells and completely
slurround two small groups of columnar cells,
thus presenting a primitive reticulum of subcolumnar cells.
increase in frequency throughout the
period of study, without any definite peak
being reached. This mitotic activity produced a concomitant increase in the number of cell layers of squamous epithelium
in both regions (table 2). Biggers and
Claringbold ('55) found a similar increase
jn the number of cell layers in mouse
vaginal epithelium through 54 hours (the
extent of their observations) after a single
dose of 0.0064 pg estrone, and noted a
maximum mitotic rate at 30 to 36 hours.
Allen, Smith and Gardner ('37) found a
similarly-timed mitotic peak after the in-
Fig. 9 Map showing distribution of stratified
squamous epithelium (solid black) on part of
the luminal surface of the cervical canal of a
mouse 72 hours after estrogen injection. The
solid white areas correspond to columnar epithelium. The hatched line indicates the plane
of one of many histologic sections in the frontal
axis used to compile the map; this particular section would have shown four apparently isolated
nodules of squamous epithelium. The stratified
squamous epithelium penetrates into the columnar epithelium in a complex reticulate manner,
and the squamocolumnar junction (the border
of adjacent black and white areas) is thus convoluted and discontinuous.
TABLE 3
Frequency of apparently isolated nodules of stratified squamous epithelium in the uterus of
C&I/HeJ mice after a single injection of estradiol benzoate ( 2 . 3 f i g )
Hours after injection
Frequency of nodules
0-36
0
48
4.0
60
5.2
72
2.6
84
2.58
96
2.69
244
CAROLYN F. BRADLEY AND CHARLES E. GRAHAM
Fig. 10 Photomicrograph of abrupt squamocolumnar junction in the cervix of a mouse
60 hours after estrogen injection; the cervico-vaginal epithelium is to the right, uterine
columnar epithelium is to the left. A nodule of stratified squamous epithelium is seen
among columnar cells to the left of (i.e., cranial to) the junction. The basement membrane
is quite distinct. Note mitotic figure in the surface of the stratified squamous layer near
the junction. (PAS. X 321)).
The frequent occurrence, in our estradiol-treated animals, of mitoses in the surface layer of cervical stratified squamous
epithelium was unexpected; it is usually
assumed that the cells of this layer have
completed their differentiation and, consequently, have lost the capacity to divide.
Barker and Walker ( ' 6 6 ) illustrated a
mitotic figure in one of the Schiff-positive
surface cells of vaginal epithelium from
an ovariectomized mouse treated for several days with both estradiol and progesterone.
Mitotic activity was present in both regions of the uterine columnar epithelium
throughout the period of observation. The
fluctuations in the mitotic index which
occurred might be due in part to diurnal
effects since animals were sacrified at 6to 12-hour intervals, but no corresponding
pattern of variation was seen in either of
the stratified squamous regions analyzed.
There was some similarity between the
mitotic index in the surface layer of squamous epithelium at the squamocolumnar
junction and in the uterine columnar epithelium near the junction. A distinct peak
of mitotic activity was seen at 24 hours in
the columnar epithelium well cranial to
the squamocolumnar junction. (Tice, '61,
found a similar mitotic peak in ovariectomized 24-27 hours after a single dose
of either 1 ?g or 100 pg estradiol).
Distributzon of subcolumnar cells and
sbatified squamous epithelium. The primary objective of our investigation was to
determine the mode of origin of the reticulum of multi-layered squamous epithelium
that appears in the region of the squamocolumnar junction after appropriate
estrogenic stimulation.
Scattered subcolumnar cells could be
recognized in the region of the squamocolumnar junction at zero and six hours.
However, as a result of the undifferentiated state of the epithelium at these times,
it was not possible to determine their exact
distribution. The cytoplasmic differentiation which was noted 12 hours after estrogen injection renders the subcolumnar
cells more readily recognizable, especially
because the superficial cells assume
cuboid-to-columnar characteristics. It was
possible at this time to show that the subcolumnar cells were arranged in a reticulate pattern. Since mitoses did not appear
in the subcolumnar cells until 36 hours
after estrogen injection, it may be concluded that the reticulum was not formed
as a result of estrogen injection, but that
it is a component of the untreated cervix.
The subcolumnar cell reticulum and the
overlying columnar cells must be considered as a histological unit which is an
extension of, and identical with, the twolayered epithelium that forms the transition zone; this conclusion is based upon
the morphological similarities of the constituent cells and especially upon the continuity of the subcolumnar cell reticulum
with the epithelium of the transition zone.
An approximately similar time of onset of
:
-
ESTROGEN EFFECTS ON MOUSE CERVICAL EPITHELIUM
mitosis after estrogen injection, and time
of (occurrenceof stratification, corroborate
this conclusion.
Forty-eight hours after estrogen administration, the unilayered reticulum of subcolumnar cells had been replaced by a
reticulum of stratified squamous cells.
There is no doubt that this change was
due to the participation of the two-layered
epithelium in the generalized proliferative
response shown by the stratified squamous
epithelium of the transition zone at this
time. This conclusion is based upon the
fact that the subcolumnar cells disappeared at the time that the multi-layered
reticulum appeared; additional evidence is
provided by the fact that the two types of
reticulum were similar in distribution, and
also by the presence of mitoses in the subcolumnar cell reticulum at 36 hours after
estrogen injection just before the multilalyered reticulum appeared.
After 60 hours, the reticulum appeared
to become less complex, as evidenced by
the reduced nodule counts. This finding
indicates that continued proliferation resulted in the coalescence of some adjacent
nodules.
Since the reticulum of squamous epithelium is derived from the cervical stratified squamous epithelium, we found no
evidence of any contribution of metaplasia
to estrogen-induced epidermization.
The squamocolumnar junction. This
study shows that the demarcation between
the cervico-vaginal epithelium and the unilayered uterine cuboidal or columnar epithelium (which we defined as the squamocolumnar junction) does not follow a
simple circumferential course around the
cervical lumen; instead, the two-layered
cervico-vaginal epithelium projects cranially in reticulate fashion into the unilayered uterine cuboidal or columnar epithelium.
Four different types of squamocolumnar
junction were recognizable after estrogen
injection (table l), and the fact that each
type reached a peak frequency at a different time after estrogen injection, in an
order that corresponded to changes seen in
the intact mouse during the estrous cycle
(Graham, '66), indicated that estrogen injection is capable of inducing a similar
sequence of changes.
245
The transitional junction with overlap
is characterized by the continuity of the
surface layer of the stratified squamous
epithelium with the zdjacent uterine columnar epithelium. A morphological gradient is present between the cells of these
areas, with the surface cells of the transition zone having a definite tendency toward uterine characteristics, i.e., especially
toward a columnar or cuboidal habit, with
a basal nucleus and clear distal cytoplasm
and presence of cell divisions. The similarity between the mitotic index of this layer
and of the adjacent columnar cells of the
uterine horn was striking. The retention
of the ability by the surface layer of cells
of the cervical stratified epithelium to
undergo mitotic division may perhaps be
understood as another uterine characteristic, since it is well known that the differentiated cells of the uterine columnar epithelium in the mouse, as well as in many
other mammals, retain the capacity to
undergo mitosis (e.g., Allen, '22; Allen,
Smith and Gardner, '37; Asdell, '64;
Bensley, '51; Eckstein and Zuckerman,
'56; Leroy, Galand and Chrbtien, '69; Martin and Finn, '68; Tice, '61).
A complete discontinuity between the
two types of cervical epithelium became
evident after adequate estrogen stimulation: all of the cervico-vaginal and reticular
epithelium became multi-layered, resulting in the formation of an abrupt squamocolumnar junction. The transition-zone
surface layer of cells that resembled the
adjacent columnar cells of the uni-layered
uterine epithelium was shed as the result
of the appearance of a different type of
differentiation (keratinization) in the
cells beneath them.
Subcolumnar cells in man. Previous
investigators have repeatedly noted the
presence of subcolumnar cells in the cervical region of several species, including
man (e.g., Carmichael and Jeaffreson,
'39; Fluhmann, '53); these cells have been
referred to variously as basal cells, reserve
cells, infraepithelial cells or indifferent
cells (see review by Baggish and Woodruff, '67). Although the relationship of the
subcolumnar cells in the human cervix to
the subcolumnar cells we have described
in the mouse is unknown, their strikingly
similar morphology and location strongly
246
CAROLYN F. BRADLEY AND CHARLES E. GRAHAM
suggest identity. However, detailed studies
on the distribution of these subcolumnar
cells, using techniques as complete and
precise as ours, have not been made in
other species; consequently, i t is not
known if the subcolumnar cells in the
human cervix form a reticulum that is an
extension of the cervical stratified squamous epithelium.
A number of workers proposed that subcolumnar, or reserve cells in the human
cervix are capable of proliferating to produce differentiated stratified squamous
epithelium (e.g., Auerbach and Pund, '45;
C armich ael and Je aff reson, '39; Fluhmann,
'53; Howard, Erickson and Stoddard, '51).
The basis for this hypothesis would be
strengthened if it could be demonstrated
that reserve cells in the human cervix are
actually a part of the cervico-vaginal stratified squamous epithelium, as are the subcolumnar cells in the mouse.
Other authorities have considered that
reserve cells are derived from columnar
cells, and that they represent an intermediate stage in the transformation of columnar cells into stratified squamous epithelium (see Baggish and Woodruff, '67).
Such a process is known as metaplasia, or
prosoplasia (Fluhmann, '53). However, a
metaplastic origin of reserve cells seems
unlikely in view of the fact that in none
of the models in which epidermization
could be induced experimentally, was it
possible to demonstrate that metaplasia
made any significant contribution to the
newly formed stratified squamous epithelium; on the contrary, the new stratified squamous epithelium was formed only
by the proliferation and extension of previously existing stratified squamous epithelium (Graham, '67b, '68a,b, '69a,b, '70,
'71a,b; Graham and Manocha, '71).
Some workers have proposed that human cervical carcinoma arises in areas of
reserve cells, which are held to be derived
from the columnar epithelium (e.g., Johnson, Easterday, Gore and Hertig, '64; Old,
Wielenga and von Haam, '65); according
to this view, squamous carcinoma is derived from columnar epithelium (metaplastic origin). All of a group of induced
squamous carcinomas located in the cervix
and uterine horn of C3H/HeJ mice were
ultimately derived from the cervico-vaginal
stratified squamous epithelium (Graham,
'71b). If it can be shown that human reserve cells, or subcolumnar cells, originate
from the stratified squamous epithelium
as was originally proposed by Eichholz
('02) and Meyer ('lo, '23), then it follows that squamous carcinoma, if in reality
it arises from such areas, is ultimately derived from the stratified squamous epithelium, as in mice, a conclusion of considerable potential theoretical importance.
ACKNOWLEDGMENT
The authors thank Mrs. Carolyn Floyd
for competent technical assistance.
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