The ultrastructure of human cervical epithelial cells during pregnancy

OBSTETRICS

The ultrastructure

of human cervical

epithelial cells during pregnancy GEORGE

B.

EDWARD

C.

CHAPMAN, MANN,

ROBERT

WEGRYN,

CHARLES

HULL,

New

York,

New

PH.D M.D. A.B.

B.S.

York

Materials

F E w studies have as yet been published which accurately describe the fine structure of the human cervical epithelium. So far as is known, no one has demonstrated, by electron microscopy, the cellular morphology of this area during pregnancy. The present study, therefore, was planned specifically to determine whether any fine structural changes occur in human cervical epithelial cells related to the striking gross changes that occur in the cervix during pregnancy. In addition, because previous reports have uniformly listed methacrylate as the embedding medium, it was deemed advisable to determine whether better preservation might be obtained with the use of the Epon embedding. technique which has been described by Luft.l From the Departments of Anatomy Obstetrics and Gynecology, Cornell University Medical College.

and

methods

Biopsies were taken from the squamocolumnar junction of 38 randomly selected patients, between the ages of 20 and 44 years. Fourteen of the women were pregnant; 3 were in the first trimester, 7 in the second, and 4 in the third. A specially built jaw-toothed biopsy forceps was used which yielded 1 mm.3 blocks of tissue. Bleeding proved to be no serious problem and was easily controlled with negatol and pressure. This report is primarily based on the observations of 5 of the pregnant patients. Each of the women had a normal healthy cervix and pregnancy, as determined by history, repeated physical examinations, and light microscopy of the tissue obtained. Subsequently each had a normal full-term spontaneous delivery without fetal complication. Tissues were fixed for 2 to 4 hours at 5” C. in a 1 per cent osmium tetroxide solution buffered with acetate-barbital at pH 7.4.2 They were then washed in buffer solution, dehydrated by passage through a

and

Supported by United States Public Health Service Research Grants (E-3517) and (M-2340), and by Graduate Training Grant [2G-895 (C2)]. 3

4

Chapman

et al.

graded ethanol series, and embedded in Epon as noted previously. One micron sections were cut and stained, according to the method of Richardson and co-workers,” for examination in the light microscope for purposes of orientation. These sections, as well as the ultrathin sections, were cut with glass knives on a Servall, Porter-Blum ultramicrotome. Ultrathin sections were stained with uranyl acetate, following the method of Watson4 and were examined with an RCA type EMU-2D electron microscope. Review

of literature

Normal changes in the human cervical epithelium associated with pregnancy have been extensively studied histologically with the light microscope, especially by O’Leary,” Nesbitt and Hellman,” Murphy and Herbut,’ Epperson and associates,’ Carrow and Greene,” Danforth,l’ and Glass and Rosenthal.ll Notable among the changes reported was an increase in the number of cervical glands, a hyperplasia of the glandular epithelium leading to the replacement of the original single cell layer by three or more cell layers, an adenomatous hyperplasia represented by the formation within the larger glands of many small glandular spaces, epidermization indicated by the growing beneath the columnar epithelium of the glands of stratified squamous epithelium, hyperactivity of the basal layer of the epithelium, and epithelial thickening. Carrow and Greene” point out that many of these changes have been regarded as characteristic of and specific to pregnancy but that, as they are seen in the nonpregnant cervix, they should no longer be so regarded. These authors also state that recent efforts have failed to demonstrate constant cyclic changes in cervical epithelium, which lend themselves to precise correlation with endometrial cyclic changes. This is somewhat surprising in the light of Murphy and Herbut’s comment that markedly increased and sustained levels of estrogen and progesterone provide the essential physiological basis for the tissue alterations observed in their own study.

Cyclical cytological changes occurring in the endometrial epitheliurrr during the hrlman menstrual cycle have been studied with the electron microscope by Bore11 and coand Nilsson.14. Ii Bore11 workers,” Gompel,‘” and co-workers found that the glandular epithelial cells were short and possessed short (0.2 p) microvilli in the inactive phase, became taller and exhibited 1.0 p microvilli in the proliferative phase, and remained tall but showed shorter microvilli in the secretory phase. Cells in the last two phases also contained numerous granules, vesicles and vacuoles, but the only evidence of secretory activity was provided by the occurrence of irregular cytoplasmic processes, attaining a length of 1 to 2 p. The secretory phase also showed the presence of 1 ,u low density amorphous areas, which were thought to be glycogen because of their resemblance to similar zones in liver cells that contain much glycogen. Gompel’s’” findings are in essential agreement with those of Bore11 and associates, but he states quite c!early that, although glycogen practically fills the cells in the late secretory phase, its excretion into the lumen has not been clearly demonstrated. Nilssonl~is 15 has added to the above descriptions by mentioning the occurrence of 1 p granulated bodies that may contain 200 to 500 A particles and small groups of parallel membranes. These bodies are presumed to be lysosomes. Nilsson has also further elucidated the subject of glycogen secretion by describing two cytoplasmic components rich in this material. One of these components is lightly staining and has a mottled appearance; the other is composed of 300 to 9001 A particles. Moricard and co-workersI” have described changes in mucosal cell structure induced by the action of estradiol and progesterone on the ovariectomized human being. Estrogen produces a differentiation of the endoplasmic reticulum and mitochondria without a deposition of glycogen. Successive and associated action of estrogen and progesterone produces ultrastructural modifications of the endoplasmic reticulum and mitochondria,

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with the appearance of glycogen near the basal pole of the nucleus.

Cervical

deposition

Observations and comments For purposes of orientation, the appearance in the light microscope of the general area under study is indicated (Fig. 1). A single layer of cervical columnar epithelial cells may be seen at the top of the figure. The nuclei are located basally and many reveal one or more prominent nucleoli. Beneath the epithelium (at the bottom of the figure) is the stroma, which in this area

epithelial

cells

during

pregnancy

5

contains two projections from the stratified squamous epithelium. Only the basal layer and spinous layer of this epithelium are illustrated. However, it should be noted that this epithelium showed an appreciable amount of cornification. The fine structural aspects of this epithelium will be the subject of a subsequent report. Fig. 2 shows a portion of the field of Fig. 1 at a higher magnification. The secretory product (6’) of the epithelial cells, which is the main subject of interest in this communication, is illustrated in this light micrograph. Note

Fig. 1. Low magnification light micrograph of a 1 p section of the second trimester squamocolumnar junction. Columnar epithelial cells, squamous

stroma, epithelium

and cells of the spiny are included. (x300.)

layer

of the

stratified

6

Chapman

et al.

that this product occupies most of the apical cytoplasm of these cells. The nucleus I,,.V1 shows three prominent nucleoli. A plasma cell (P) may be seen in the stroma. In a relatively low magnification electron micrograph (Fig. 3) ) much more cellular detail may be seen. The numerous microvilli (1-i) which project from the apical surface of the cells, appear in the lumen as circular or oval profiles. Several secretory granules (S,, S,) give the impression that they were in the process of being extruded from the cells at fixation. The very slight damage to the cells suggests that this might be considered a case of merocrine secretion. Several desmosomes (0) may be clearly seen. Several secretory granules (S,, Sk 1 demonstrate the presence of a limiting membrane. Golgi zones (G ) and granular endoplasmic reticulum (ER) are seen dispersed among the secretory granules. To the left of the nucleus (N), with its prominent nucleolus (NL) , may be seen a region of extensive plasma membrane interdigitation. The basement membrane (BM) , beneath the epithelial layer, is inconspicuous because of the orientation of the section and to the local state of its preservation. A portion of the nucleus (SN) of a stromal cell may be seen at the bottom of the figure. At higher magnification (Fig. 4) ~ the cellular detail of the basal area may be seen

to advantage. The linliting membranc~ oi ttlc. secretory granule (S is oh\ious and scvvr;l! mitochondria (M! with cristac are \.isiblr. The region of extensive plasma mcmbranc. interdigitation, to the left of the nucleus (N), is very well defined. Such an arrangement would be particularly advantageous in maintaining the integrity of an epithelial sheet. Such maintenance could become FStremely important in a simple epithelium, with very few cells evidencing mitosis. These cells are apparently dedicated to secretory activity. The granular cndoplasmic reticulum (ER) is readily discernible. It is pertinent to future discussion to note the narrow intracisternal space (i.e., the space enclosed by the nearly parallel membranes of the endoplasmic reticulum) studded with ribonucleoprotein particles. An unidentified inclusion (X) also appears in the cytoplasm. The basement membrane (BM) consists of a small amount of amorphous material and an occasional collagen fibril (CF). A stromal cell with obliquely sectioned nuclear envelope (SN) is just visible. There appear to be numerous free ribonucleoprotein particles in the cytoplasm of this cell. The apical portion of each of these cells (Fig. 5 I appears to be occupied mainly by the secretory granules (S). However. Golgi zones (G) . granular endoplasmic reticulum and an occasional mitochondrion (ER),

Fig. 2. Higher magnification light micrograph of a portion of Fig. 1. Secretory granules (S), nucleus (N), with three prominent nucleoli, and a plasma cell (P). (x970.)

Volume Number

88 1

Cervical

epithelial

cells

during

pregnancy

Fig. 3. Low magnification electron micrograph of second trimester columnar epithelial cells. Microvilli (V) are seen at the top of the figure. Secretory granules (S, SI) appear about to be extruded; granules (Sd, S1) clearly show a limiting membrane. Golgi zones (G) and granular endoplasmic reticulum (ER) are among the granules. Nucleus (N), nucleolus (NL), basement membrane (BM), stromal cell nucleus (SN). (~7,700.)

7

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Chapman

Fiq, 4. High

et al.

magnification electron micrograph of basal portion of second trimester columnar epithelial cell. Serretory granule with obvious limiting membrane (A’), mitochondrion with rristac (M), nucleus (N), granular endoplasmic reticulum (ER), unidentified inclusion (X), basement membrane (BM), interdigitated lateral cell membranes (LM), collagen fibril (CF), stromal cell nucleus (SN). (~17,300.)

Vohme Number

88 1

Fig. 5. Electron Secretory granule reticulum (ER), G,. (~10,500.)

Cervical

micrograph (S), Go& mitochondrion

epithelial

of apical portion of second trimester zone (G), nucleus (N), nucleolus (M). Golgi cavities may expand

cells during

pregnancy

columnar epithelial cells. granular endoplasmic into secretory granules at

(NL),

9

(M) may also be seen. There is a suggestion (G, ) that the secretory granules result from an expansion of the Golgi cavities. Similar Golgi configurations may be seen (G,, G, ) in Fig. 8. Serial sections of more stages in gesta-

tion would be rquiwd to confir] m tllis sibility, but the similarity in If‘stirre density of the material included within Golai \.esicles, vacuoles1 and pa ired II branes to that in the secretory gr anules

Fig. 6. Electron

epithelial cells. (BM). (~7.900.)

granule

(S),

micrograph of first trimester extracellular areas (F), basement

columnar membrane

Cilia

(CZ).

secretory

Vohmle Number

88 1

Cervical

Fig. 7. Electron micrograph mosome (D), mitochondrion (BM). (x15,700.)

of basal portion (M), secretory

of first granule

ports the possibility. The situation here resembles closely that described by Palay’? in the jejunal goblet cell, where the secretory material is considered to be collecting in the vesicles of the Golgi complex. Similar findings in other secretory epithelia by Palay17 have led him to suggest that the Golgi material generally serves a segregating function. The similarity in the appearance of the secretory material of the present study and that histochemically identified and reported by Palay, plus the fact that the cervical epithelium is known to be secreting mucus during pregnancy, leave little doubt that the secretory

protein. cedure

material

of

these

cells

is a muco-

A modified P. A. S. staining proresulted in a positive reaction in the

epithelial

trimester columnar (S), nucleus (N),

cells

epithelial basement

during

pregnancy

11

cells. Desmembrane

apical areas of these cells. Such a reaction indicates the presence of mucopolysaccharide. Only glycogen and mucus are to be expected in these cells. As glycogen has never been described in the form taken by the secretory material of these cells, further evidence is provided which indicates that the secretory material is mucus. The electron micrographs accompanying this report appear to be the first demonstrating such material in the human pregnant uterus at different gestational stages. The cells discussed until now were derived from a specimen taken during the second trimester of pregnancy. Specimens from a first trimester uterus reveal the same features as those described above (Fig. 6). The

12

Chapman

et al.

ciliated cell in the center of lhe illustration deser\:es particular attention because such However, cells are rather uncommon. Gompel” includes one in his paper and

Fig. 8. Electron

micrograph of central zone (G), desmosome (D), unidentified tory granules at G,, G,. (~15,700.)

notes that the endoplasmic ret :iculurn in these cells is rather poorly de\ Ieloped. A particularly interesting feature of sI1cil (:ells is the fact that secretory granule are

portion of first trimester inrlusion (X). Golgi

cobmar cavities

epithelial may expand

cells. Golgi into secre-

Volume Number

88 1

Cervical

Fig. 9. Electron micrograph of basal portion of third trimester inent granular endoplasmic reticulum (ER) with expanded secretory granule (S), nuclei (iV), and basement membrane

very rare. Thus, it seems as though the cell, in becoming specialized to form cilia, has suffered a retardation in its secretory ability. It is also possible that the ability to form cilia is more primitive than that to secrete, and that such a cell is one which has persisted from earlier ontogenetic stages, perhaps a descendant from a ciliated cell of the Milllerian duct. In this particular specimen, several rather extensive extracellular areas of accumulation of a flocculent material (F) are noted. The significance of these areas cannot now be determined. The secretory cells on each side of the ciliated cell appear to be as heavily laden as any from the second trimester. Amorphous basement membrane (BM) material may be seen at the bottom of the illustration.

epithelial

coIumnar cisternae.

cells

during

epithelial cells. Mitochondrion

pregnancy

13

Prom(M),

(BM). (~15,700.)

The basal cytoplasm of the first trimester cell contains abundant granular endoplasmic reticulum and free ribonucleoprotein particles (Fig. 7) . Several prominent desmosomes mitochondria (M) , and secretory CD), granules (5’) may be seen. A well-defined amorphous basement membrane (BM) lies just beneath the epithelial cells. A portion of centrally located cytoplasm from a first trimester cell (Fig. 8) contains all of the organelles found in the cytoplasm generally. Several Golgi zones (G) and desmosomes (D) are noteworthy. This particular cell contains a number of the unidentified inclusions (X) mentioned previously. No significant difference in fine structure of the epithelial cells can be noted between the first and second trimesters.

cclis, it seemed worthwhile

The basal region of cells of the third trimester contains a granular endoplasmic reticulum (ER) which differs somewhat from that of the other trimesters in having more expanded cisternae (Fig. 9) . As data rclating the rate of mucus production to trimester of pregnancy are not available, there is no basis on which to rest the obvious possible conclusion, viz., that expanded cisternae are related to increased rate of secretion. Mitochondria (M) , secretory granules (5’) , nuclei (N) , and a prominent basement membrane (BM) are readily visible. As centrioles were rarely observed in these

Fig. 10. Electron Proximal centriole

micrograph (C,), distal

of basal portion of centriole (CA), nucleus

to include in this report an electron micrograph of a cell demonstrating a proximal (C,) and distal (C, ) centriole (Fig. 10). As is frequentIy the case, these centrioles are oriented at 90’ to each other. The apical region of a third trimester cell shows an abundance of Golgi material (G I and secretory granules (S) (Fig. 11) . Mitochondria (M) and desmosomes (D) are also clearly seen. It should be noted that the present study has shown no glycogen deposits or granulated bodies, both of which were reported by and Nilsson.‘“, I5 However, none Compel’”

third (N).

trimester (x15.700.)

columnar

epithelial

cells.

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Number

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Fig. 11. Electron micrograph Golgi material (G), secretory

Cervical

of apical portion of third granule (S), mitochondrion

of the previous electron microscope investigations of this material has included micrographs showing centrioles or mucous secretory granules. These differences, with the exception of the finding of centrioles, probably are caused by the fact that the pregnant cervix was the subject of the present study, while the earlier studies were of nonpregnant cervical tissue. It remains, however, somewhat puzzling that no mucous secretory cells were encountered by the previous investigators. Summary

Normal human cervical epithelial cells have been studied during the three trimesters of pregnancy for the first time with the electron microscope. The cells revealed strik-

epithelial

trimester columnar (M), desmosome

cells

during

pregnancy

15

epithelial cells. (D). (~15,700.)

ingly few changes, the only conspicuous fine structural change noted was an expansion of the cisternae of the endoplasmic reticulum during the third trimester. Quite surprisingly, although a tremendous (in an electron microscope frame of reference) amount of tissue was surveyed, not a single mitotic figure was encountered. The secretion of mucus apparently involves the same segregating role of the Golgi apparatus as has been described in other secretory cells. Centrioles and an unidentified inclusion were described. Epon appears to be a more effective embedding medium than methacrylate for this material. We wish to thank Dr. R. Gordon his critical reading of this report knowledge the technical assistance Greminger.

Douglas for and to acof Mrs. Joan

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et al.

REFERENCES

10.

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Hellman, L. M., Galvin, AM. J. OBST. & GYNEC. Greene, R. 237, 1951.

R.:

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York York

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York