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. 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