Scanning Electron Microscopy of the Rabbit Cervix Epithelium

Transcription

1 BIOLOGY OF REPRODUCTION 12, (1975) Scanning Electron Microscopy of the Rabbit Cervix Epithelium W. G. RICHES, R. E. RUMERY AND E. M. EDDY Department of Biological Structure. University of Washington School of Medicine. Seattle. Washington 98/95 Accepted February 7, 1975 The cervical epithelium of intact postovulatory rabbits. ovariectomized rabbits, and ovariectomized rabbits which had received estrogen were studied with the scanning electron microscope. The cervical epithelium of postovulatory rabbits consists of ciliated cells and nonciliated cells with bulbous apical processes. In rabbits spayed for 7 months, there appear to be slightly fewer cilia than in intact rabbits and the bulbous apices of nonciliated cells are not seen. In rabbits spayed for months, patches of nonciliated cells are interspersed throughout the epithelium. The ciliated cells have both long and short cilia and microvilli on their apical surface. The cervical epithelium of rabbits spayed for 16 months and then given estrogen for 10 days, closely resembles that of intact postovulatory rabbits. Estrogen appears to play a modest role in the maintenance of the ciliated cells in the cervical epithelium, but has a somewhat more important role in the maintenance of the nonciliated secretory cells. The transport of sperm and eggs through the female reproductive tract has been an area of intensive investigation in recent years. The importance of the ciliated epithelium of the oviduct in egg transport and the role of estrogens in its maintenance have been clearly established for the rabbit (Blandau, 1969; Odor and Blandau, 1973). It is unfortunate that the mechanisms of sperm transport through the cervix have not been equally well resolved (Blandau, 1973). The epithelium of the cervix is similar to that of the oviduct, and its physiologic state no doubt does have some effect upon sperm transport. A scanning electron microscopic study recently demonstrated the effect of ovariectomy upon the oviductal epithelium of the rabbit (Rumery and Eddy, 1974). The cervices of these same animals were investigated by the present authors in an attempt to determine the effect of ovariectomy upon the rabbit cervix. This report deals with scanning electron microscopic studies of the cervical epithelium of intact rabbits, spayed rabbits and spayed rabbits which had received estrogen. This investigation was supported by USPHS Grants HD and HD and Contract from the National Institutes of Health. MATERIALS AND METHODS The cervices utilized for this study were obtained from sexually mature New Zealand White rabbits. The control group consisted of 4 intact females injected with 100 IU human chorionic gonadotropin (Follutein, E. K. Squibb) 12 h prior to being sacrificed. The 7 female rabbits, serving as the experimental group, were ovariectomized bilaterally under a dissecting microscope, using electrosurgical technique. The surgical procedures were conducted under sterile conditions. Five of the 7 experimental rabbits were spayed for 7 to 18 months, while the other 2. spayed for 8 months and 16 months. were injected with 5 zg estradiol benzoate in I ml sesame oil every 12 h for 10 days, and then killed 12 h after the last injection. The cervices were quickly removed from each rabbit and placed in Hank s solution. The two cervices were separated and trimmed of fat and other extraneous tissue. Each cervix was opened and several of the longitudinal ridges (Blandau, 1973: Hafez, 1973: Kanagawa et al., 1972) were dissected free. During this procedure, the Hank s solution was changed frequently in order to ensure a specimen devoid of surface obscuring secretions. The ridges were divided into sections approximately 3 mm in length, and then placed in a fresh solution of 1.66 percent osmic acid, 2.4 percent glutaraldehyde and 0.2 M phosphate buffer for I h. Following fixation, the tissues were dehydrated in successive grades of ethanol. The ethanol was replaced with amyl acetate and the specimens were critical point dried (Anderson. 1951) using carbon dioxide as the transitional fluid. They were coated subsequently with approximately 200 A of carbon and gold in a vacuum evaporator. The central 573 Copyright #{174} 1975 by The Society for the Study of Reproduction. All rights of reproduction in any form reserved.

2 574 RICHES, RUMERY AND EDDY region of the cervical specimens was examined with an ETEC scanning electron microscope operating at 20 KV in the secondary electron mode. Some specimens were prepared for light microscopy following their examination by scanning electron microscopy. They were placed in propylene oxide and then infiltrated and subsequently embedded in Epon epoxy resin. Sections I Mm thick were cut on a Sorvall PB-2 ultramicrotome, stained with I percent toluidine blue and photographed on a Zeiss Universal microscope with an orange filter (Wrattan 22) in the light path. Postovu!aiorv RESULTS Rabbits The mucosa of each cervical ridge is thrown into a series of longitudinal folds (Fig. 1). Both ciliated and nonciliated cells form the epithelial covering of the ridges with the ciliated cells being by far the more prominent cell type. Their long slender cilia obscure nearly all other cell surface details, but in a favorable view, it is seen that the ciliated cells also possess microvilli (Fig. 2). The cilia do not demonstrate any directional organization, appearing instead as a randomly tangled network (Fig. 3). Frequently, the cilia possess a distal, acute hook-like bend (Fig. 4). Scattered randomly among the cilia are the apices of nonciliated cells (Figs. 3,4). These are probably secretory cells. The apices of some of these cells lie beneath and are partially obscured by the layer of cilia (Fig. 4). Microvilli densely cover the cell surface and are occasionally interconnected by delicate threads of secretory material. On some of these cells a portion of the cell apex balloons upward to the level of the tips of the cilia (Fig. 4). Microvilli are sparse on these cytoplasmic blebs. The apical process occasionally protrudes well above the cilia, in which case microvilli are not present (Fig. 5). When the epithelium is examined by light microscopy, it is seen that it consists of tall, columnar ciliated and nonciliated cells. The nonciliated cells have dense cytoplasm in their basal portion while their apical portion appears to be engorged with secretory material (Fig. 15). Spayed Rabbiis The cervical surface epithelium of rabbits spayed for 7 months exhibits subtle alterations in both ciliated and nonciliated cells as compared to the epithelium from intact rabbits. Upon a cursory examination, the surface appears to resemble the control epithelium (compare Figs. 3 and 6). However, the nonciliated cells do not have apical blebs and their apices are flat and densely covered with short microvilli (Fig. 7). It appears that the ciliated cells are affected also in that they seem to possess fewer cilia per cell (Fig. 7). While the majority of the cilia are long and slender, clusters of very short cilia are commonly noted (Fig. 7). These short cilia appear to arise from the same cells as do the long well-formed cilia; often the short cilia can be distinguished from microvilli only by their greater diameter (Fig. 8). Clumps of secretory material frequently adhere to the distal ends of the cilia (Figs. 7,8). Light microscopy Intact Postovulatory Fto. I. Each cervical ridge is thrown into a series of longitudinal folds and the columnar surface epithelium faithfully conforms to these inpocketings. x240. Ftc. 2. The simple columnar cervical epithelium is composed of both ciliated and nonciliated cells. The apical surfaces of both cell types possess numerous microvilli. A short cilium is occasionally seen (arrow). x12,000. FIG. 3. The surface epithelium of the cervix is densely ciliated in the normal postovulatory state. The cilia are long, slender, and erect, and they exhibit no directional organization. Secretory blebs extend from the apical surface of the nonciliated cells and protrude into the layer of cilia (arrows). x2,250. FIG. 4. The bulbous apices of the nonciliated cells are seen scattered throughout the layer of cilia. The microvilli appear to be less numerous on the surface of the taller nonciliated cells than on the other nonciliated cells. x6,000. Fio. 5. Nonciliated cells are seen occasionally to have apical blebs which extend on stalks above the cilia. Few or no microvilli are present on these blebs. x6,000.

3 RABBIT CERVIX EPITHELIUM 575

4 576 RICHES, RUMERY AND EDDY reveals that the epithelial cells are shorter than those in intact postovulatory rabbits. Although cells are present with densely staining cytoplasm like that seen in nonciliated cells in intact postovulatory rabbits, their apical portions lack the apparent secretory material and bulbous processes (Fig. 16). In rabbits spayed for IS to 18 months, the morphologic alterations in the cervical epithelium are clearly evident. Although ciliated cells remain the dominant surface feature, patches of nonciliated cells are a frequent occurrence (Fig. 9). The nonciliated cells have flat apical surfaces covered with numerous stubby microvilli (Fig. 10). Cells with bulbous apices are rarely encountered. The ciliated cells appear to possess fewer cilia per cell than do those of control rabbits, but the majority of the cilia are long, slender and well formed (Fig. II). However, short cilia do occur frequently on the ciliated cells (Fig. 10). By light microscopy, this epithelium is seen to be composed of cuboidal cells, some of which lack cilia (Fig. 17). It is not possible to tell here whether these nonciliated cells are derived from the ciliated cells or from the secretory cells present in the intact postovulatory rabbit. Estrogen- Treated Spayed Rabbits Exogenous estrogen administered to rabbits spayed for 8 and 16 months leads to a dramatic restitution of the cervical surface epithelium. The large patches of nonciliated cells disappear (Fig. 12) and cilia again obscure the other cell surface details (Fig. 13). The cilia are long and slender, and only rarely are clusters of short cilia encountered. The numerous bulging apices of nonciliated cells are seen throughout the cervical epithelium (Fig. 12). Microvilli densely covering the surface of these cells are often joined together by delicate threads of secretory material (Fig. 14). It is apparent that the cervical epithelium of the estrogen-treated spayed rabbits resembles greatly that of the control animals (compare Figs. 4 and 14). This is verified at the light microscopic level, where the epithelium is seen to be composed of tall columnar ciliated and nonciliated cells (Fig. 18). The nonciliated cells contain densely staining cytoplasm and their bulbous apical processes are apparent. DISCUSSION Our description of the surface morphology of the cervical epithelium of the intact postovulatory rabbits confirms the findings of previous SEM investigators (Hafez, l972a,b; Kanagawa et a!., 1972; Kanagawa and Hafez, 1973; Ferenczy and Richart, 1974). While the present observations also demonstrate that long-term ovariectomy does result in some degree of deciliation and in a cessation of secretory activity, the cervical epithelium does not appear to be as sensitive to the loss of estrogens as does the oviductal epithelium. In the menopausal human (Ferenczy ci a!., 1972) and the castrate rhesus monkey (Brenner, 1969,1970) the oviductal epithelium responds with nearly complete deciliation to the estrogen deficiency. Also, the oviducts of the same rabbits used in this study were previously examined by SEM (Rumery and Eddy, 1974) and found to have undergone a widespread loss of cilia following long-term ovari- Seven Month Spayed FIG. 6. The general appearance of the cervical epithelium appears to be little changed 7 months after ovariectomy. The surface is still densely ciliated; however, the bulbous processes of nonciliated cells are not seen. x2,250. FIG. 7. When examined at higher magnification, the nonciliated cells are seen to be present but to have a flat apical surface. Also, the cilia appear to be less numerous and clusters of short cilia (arrow) are occasionally encountered. x9,600. FIG. 8. The short cilia may occur either individually or in clusters among the longer ciliary shafts. These cilia (arrows) can often only be distinguished from the numerous surrounding microvilli by their greater diameter. Secretory material adheres to some of the cilia. x 18,000.

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9 RABBIT CERVIX EPITI-IELIUM 581 Eighteen Month Spayed FIG. 9. Following 18 months of ovariectomy, the cervical surface epithelium demonstrates a patchy deciliation. Ciliated cells still cover much of the surface, but now large areas are devoid of cilia. x500. FIG. 10. The nonciliated cells present with a flat apex, covered by numerous short microvilli. Occasionally, clusters of short, developing cilia are noted. x9,600. FIG. II. The nonciliated cells possess only a slight bulge to their apex, giving the deciliated regions a cobblestone-like appearance. No single, central cilium is present on their apex. Cytoplasmic blebs or other secretory activity are rarely encountered. The majority of the cells are ciliated, however, and the cilia are long and erect. x2,250. Estrogen Treated Sixteen Month Spayed FIG. 12. Following 10 days of estrogen treatment, the cervical epithelium of a spayed rabbit closely resembles that of an intact rabbit. Deciliated patches are no longer observed and the surface once again has the characteristic appearance of that seen in the postovulatory cervix. x600. FiG. 13. Cilia densely cover the epithelial surface, obscuring the cell apices. The cilia are long and well formed, and only rarely are short cilia encountered. Microvilli can no longer be observed on the surfaces of ciliated cells because of the dense layer of cilia. The bulbous apices of nonciliated cells extend above the layer of cilia. x 2,100. FIG. 14. The bulbous apices of the nonciliated cells are covered with numerous microvilli. Delicate threads of secretory material often extend between adjacent microvilli and between adjacent cells. The cilia are long, slender, and erect, much like those in the cervical epithelium of an intact preovulatory animal (compare to Fig. 4). x 1,400. FIG. 15. The cervical epithelium of an intact postovulatory rabbit consists of tall columnar ciliated cells and nonciliated cells. The bulbous apices of three nonciliated cells are indicated by the arrows. These cells have dense cytoplasm and their supranuclear portion appears to be engorged with secretory material. x 1,200. FIG. 16. Seven months following ovariectomy the cervical epithelium remains heavily ciliated, but nonciliated cells are infrequently seen. The cilia are especially prominent in this specimen because they were coated with gold for the SEM prior to processing for light microscopy. x 1,200. FIG. 17. Eighteen months following ovariectomy, the cervical epithelium is composed of cuboidal ciliated cells and nonciliated cells. However, the nonciliated cells (arrows) lack the bulbous apical processes of the nonciliated cells in intact postovulatory rabbits. x 1,200. FIG. 18. The cervical epithelium of a rabbit, ovariectomized 16 months earlier and then receiving estrogen for 10 days, is composed of tall columnar ciliated cells and nonciliated cells with dense cytoplasm and bulbous apical processes (arrows). The surface features of this cervical epithelium are much like those in the intact rabbit. x 1,200.

10 582 RICHES, RUMERY AND EDDY ectomy. However, when examined by SEM the cervices of these same animals were seen to have sustained only a patchy loss of cilia. These observations apparently agree with the transmission electron microscopic findings of Odor (1973). She reported that approximately half of the cervical epithelial cells in the rabbit are ciliated in estrous, ovulatory, long-term ovariectomized and ovariectomized animals treated with estradiol. Despite the patchy deciliation observed in the cervix of long-term spayed rabbits, no obviously degenerating cells were noted. The very short cilia, which were frequently present, do not resemble degenerating organelles. On the contrary, they do seem to resemble the immature or developing cilia reported by other investigators (Andrews, 1974; Ferenczy and Richart, 1973). In the deciliation which normally occurs in the oviduct of rhesus monkeys on day 25 of the menstrual cycle, the entire top third of the cell pinches off in a process similar to apocrine secretion (Brenner, 1970). Such a process or the loss of entire cells might account for the patches of nonciliated cells. However, no observations are available in this instance to support either of these possibilities. Rather, the apparent decrease in the number of cilia per cell, nottd in both the short- and long-term spayed animals, would tend to indicate that there had been a loss of individual cilia. The mechanism of the observed deciliation remains to be clarified, however. The cervical secretory cells appear to be more dependent upon estrogens than do the ciliated cells. In both humans (Fand, 1973) and rabbits (EI-Banna and Hafez, 1972), estrogens are known to increase the production and secretion of cervical mucus. As was seen here, the cell apex normally bulges prominently into the layer of cilia, but after ovariectomy, the cell apex flattens, assuming a low profile. Light and transmission electron microscopy have demonstrated that the number, location, and structure of the secretory granules of cervical epithelial cells are determined by the hormonal status of the animal, and that these granules disappear following ovariectomy (Odor, 1973). The secretory cells of the oviduct undergo a similar cessation of activity and frequently develop a single stubby cilium in the center of their apical surface following castration (Rumery and Eddy, 1974). However, no such cilium is produced in cervical epithelial cells after long-term ovariectomy. Treatment with estrogens completely and rapidly restores the cervical epithelium. The surface is again thickly ciliated and the secretory cells have prominent apical bulges. Within three days after estradiol treatment, the secretory granules are again reported to be present in considerable numbers in the apical cytoplasm (Odor, 1973). A similar regeneration occurs in the oviductal epithehum of castrate monkeys (Anderson and Brenner, 1971; Brenner, 1970) and rabbits (Rumery and Eddy, 1974). These scanning electron microscopic observations demonstrate the modest role that estrogens play in the maintenance of ciliated cells in the cervical epithelium. Ovariectomy results in only a partial deciliation as compared to that which occurs in the oviductal epithehium. However, secretory activity in both the cervical and the oviductal epithelium is apparently considerably depressed following castration. ACKNOWLEDGM ENTS The authors wish to thank Dr. Richard Blandau for his support and encouragement during this study. They also wish to thank Dorothy Patton for her helpfulness with some of the special methods used in this study and they appreciate greatly the excellent technical assistance of Mrs. Jeanne Blaser. REFERENCES ANDERSON. R. AND BRENNER, R. (1971). The formation of basal bodies (centrioles) in the Rhesus monkey oviduct. J. Cell Biol. 50, ANDERSON, T. (1951). Techniques for the preservation of three-dimensional structures in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. 13, ANDREWS, P. (1974). A scanning electron microscopic study of the extrapulmonary respiratory tract. Am. J. Anat. 139,

11 RABBIT CERVIX EPITHELIUM 583 BRENNER, R. (1970). Hormonal control of cilia renewal in the primate oviduct: ultrastructural studies. In Progress in Gynecology (S. Sturgis and M. Taymor, ads.), pp Grune & Stratton, New York. BRENNER, R. (1969). Renewal of oviduct cilia during the menstrual cycle of the Rhesus monkey. Fertil. Steril. 20, BLANDAIJ, R. (1969). Gamete transport-comparative aspects. In The Mammalian Oviduct (E. Hafez and R. Blandau, ads). pp University of Chicago Press, Chicago. BLANDAU, R. (1973). Sperm transport through the mammalian cervix: comparative aspects. In The Biology of the Cervix (R. Blandau and K. Moghissi, ads.), pp University of Chicago Press, Chicago. EL-BANNA, A. AND HAFEZ, E. (1972). The uterine cervix in mammals. Am. J. Obstet. Gynec. 112, FAND, S. (1973). The histochemistry of human cervical epithelium. In The Biology of the Cervix (R. Blandau and K. Moghissi, eds.), pp University of Chicago Press, Chicago. FERENCZY, A. AND RICHART, R. (1973). Scanning and transmission electron microscopy of the human endometrial surface epithelium. J. Clin. Endocrinol Metab. 36, FERENCZY, A. AND RICHART, R. (1974). Female Reproductive System: Dynamics of Scan and Transmission Electron Microscopy. John Wiley and Sons, New York. FERENCZY, A., RICHART, R., AGATE, F., PURKERSON, M. AND DEMPSEY, E. (1972). Scanning electron microscopy of the human Fallopian tube. Science 175, HAEEZ, E. (1972a). Scanning electron microscopy of female reproductive tract. J. Reprod. Med. 9, HAFEZ, E. (1972b). Scanning electron microscopy of rabbit and monkey female reproductive tract epithehum. J. Reprod. Fertil. 30, HAFEZ, E. (1973). The comparative anatomy of the mammalian cervix. In The Biology of the Cervix (R. Blandau and K. Moghissi, ads.). pp University of Chicago Press, Chicago. HAFEZ. E. AND KANAGAWA, H. (1972). Ciliated epithehum in the uterine cervix of the Macaque and rabbit. J. Reprod. Fertil. 28, KANAGAWA, H. AND HAFEZ, E. (1973). Kinocilia and sperm dynamics in the cervix uteri of the rabbit. J. Reprod. Med. 10, KANAGAWA, H., HAFEZ, E.. PITCHFORD. W., BAECHLER, C. AND BARNHART, M. (1972). Surface patterns in the reproductive tracts of the rabbit observed by scanning electron microscopy. Anat. Rec ODOR, D. (1973). Ultrastructure of the cervix of the rabbit. Anat. Rec. 175, 401. (Abstract) ODOR, D. AND BLANDAU, R. (1973). Egg transport over the fimbrial surface of the rabbit oviduct under experimental conditions. Fertil. Steril. 24, RUMERY, R. AND EDDY, E. (1974). Scanning electron microscopy of the fimbriae and ampullae of rabbit oviducts. Anat. Rec. 178,