ON THE PRESENCE OF A CILIATED COLUMNAR EPITHELIAL CELL TYPE WITHIN THE BOVINE CERVICAL MUCOSA 1

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1 ON THE PRESENCE OF A CILIATED COLUMNAR EPITHELIAL CELL TYPE WITHIN THE BOVINE CERVICAL MUCOSA 1 R. I. Wordinger, 2 J. B. Ramsey, I. F. Dickey and I. R. Hill, Jr. Clemson University, Clemson, South Carolina Summary ERVICAL tissue samples were obtained at C slaughter from the mid-cervix of three heifers at 4 days postestrus and from the anterior, middle and posterior cervix of two cows at 14 days postestrus. Tissues were processed for carbohydrate histochemistry and electron microscopy. Both ciliated and non-ciliated columnar epithelial cells were present in all tissues examined. At 4 days postestrus the non-ciliated cells contained a large amount of stored mucin in the supranuclear region; nuclei were located in the extreme basal region of the cell. Sulfated and carboxylic acidic mucosubstances were present in both the stored mucin and mucin secreted into the cervical lumen. At 14 days postestrus less mucin was observed in the non-ciliated cells. The ciliated epithelial cells from animals 4 days postestrus exhibited a more intense eosinophilia than the non-ciliated cells and were constricted between two adjacent non-ciliated cells. This constriction was less prominent at 14 days postestrus. The nucleus of the ciliated cell was more centrally located than in the non-ciliated cell. Numerous cilia containing the 9+2 filament arrangement were observed. Introduction There have been numerous reports that the bovine cervical mucosa consists of a simple columnar epithelial cell type (Hammond, 1927; Cole, 1930; Roark and Herman, 1950; Herrick, 1951). However, in 1967, Marinov and Lovell reported the presence of ciliatedcolumnar epithelial ceils within the bovine x Published with approval of the Director of the South Carolina Agricultural Experiment Station as Technical Contribution No g Present address: Laboratory of Reproductive Physiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, 936 cervical mucosa. Recently, Hafez, EI-Banna and Yamashita (197I) reported on the histochemistry of the bovine cervical mucosa and concluded that there were no ciliated columnar cells present. A similar conclusion was put forth in a review article (EI-Banna and Hafez, 1972). These recent reports again raise the question as to whether ciliatedcolumnar cells do exist within the bovine cervical mucosa. The objective of this study was to determine by carbohydrate histochemistry and electron microscopy whether the bovine cervical mucosa contains ciliated columnar epithelial cells. Experimental Procedure Cervical tissue samples were collected at slaughter from the mid-cervix of three heifers at 4 days postestrus and from the anterior, middle, and posterior cervix of two cows at 14 days postestrus. Tissue samples were fixed for 12 to 18 hr. in 10% buffered formalin for light microscopy. Subsequently, the tissue samples were embedded in paraffin and sectioned at 7~. Sections from each tissue sample were stained with each of the following procedures: 1. hematoxylin and eosin; 2. diastaseperiodic acid-schiff; 3. aldehyde fuchsin-alcian blue; 4. diastase-alcian blue (ph 2.5)-periodic acid-schiff; 5. high iron diamine. The exact procedure for these stains can be obtained from the review of Spicer, Horn and Lippi (1967). Cervical tissues were fixed by immersion for 2 hr. in cold (4 C) 3% glutaraldehyde in s- collidine buffer (Bennet and Luft, 1959) for electron microscopy and high resolution light microscopy. Following glutaraldehyde fixation, the tissue samples were rinsed in cold buffer for 30 min., postfixed in 2% osmium JOURNAL OF ANIMAL SCIENCE, vol, 36, no. 5, 19;'3

2 CELL TYPES IN BOVINE CERVICAL MUCOSA 937 tetroxide for 2 hr., dehydrated in graded series of alcohols and embedded in Epon 812 (Luft, 1961). Thick Epon sections ( t~) were stained with azure II and examined with light microscopy. Also, Epon was partially removed from thick sections by the method of Paiz and Hennigar (1970) and the sections stained by the periodic acid-schiff-azure II procedure. Ultrathin sections were stained with 3% uranyl acetate (20 rain.) followed by 1% lead citrate (20 sec.) and examined with a Hitachi HS-8-1 electron microscope. Light Microscopy Results Routine histological examination and carbohydrate histochemistry revealed that the bovine cervical mucosa consisted of both ciliated and non-ciliated columnar epithelial cells. Paraffin Embedded Material. With hematoxylin and eosin staining the cytoplasm of the ciliated-columnar epithelial cells was devoid of mucus and exhibited a more intense eosinophilia (figure 1A). The non-ciliated columnar cells contained a large amount of stored mucin in the supranuclear region (figure 1A). The nuclei of the non-ciliated cells were located in the extreme basal region of the cell while nuclei of the ciliated cells were more centrally located within the cytoplasm. At 4 days postestrus the ciliated ceils appeared to be constricted by the large amounts of mucin in adjoining non-ciliated cells. Carbohydrate histochemistry of paraffin embedded material indicated that the nonciliated cells contained both sulfated and carboxylic acidic mucosubstances but the ciliated columnar cells did not contain stored mucin (figure I B). By 14 days postestrus the ciliated cells were more prominent, yet contained no stored mucin; the amount of stored rnucin in non-ciliated epithelial cells was greatly reduced. Epon Embedded Material. Because light microscopy of paraffin embedded material did not offer adequate differentiation between ciliated and non-ciliated columnar epithelial cells in the bovine cervical mucosa, light microscopy of thick Epon sections was undertaken. Azure II staining (figure 1C and 1E) and periodic acid-schiff-azure II staining (figure 1D) confirmed the results obtained with paraffin sections but afforded better differentiation of the cell types. Electron Microscopy The results of the electron microscopic exination supported the results obtained with light microscopy. The ciliated cell at 4 days postestrus was usually constricted between mucin containing simple columnar cells (figure 2A). The simple columnar epithelial cell contained abundant mucin in the supranuclear region at 4 days postestrus. The material appeared relatively electron translucent in micrographs but contained some dense staining material within the mucin (figure 2B). Rough endoplasmic reticulum was frequently seen in close proximity to the stored mucin (figure 2B). A Golgi complex was usually in close proximity to the stored mucin. By I4 days postestrus the amount of stored mucin was greatly reduced but similar in density. The constriction of the ciliated cell which was observed at 4 days postestrus was not as evident. Numerous cilia with basal bodies within the cell were obvious and the nuclei of these cells were centrally located (figure 2C). Cytoplasmic projections (figure 2C) were usually evident among the cilia. Cross sections of cilia (figure 2D) dearly revealed the presence of the typical 9+2 arrangement of filaments associated with a motile cilium. Discussion The results of this study confirms the work of Marinov and LoveU (1967) who first described the presence of ciliated cells within the bovine cervical mucosa. It should be noted that the reports that ciliated epithelial cells could not be seen in the bovine cervical mucosa had resulted from studies conducted on paraffin embedded material. However, when light microscopy was used with Epon embedded material superior morphological differentiation was obtained and the ciliated columnar epithelial cells were rather obvious. The presence of basal bodies and the characteristic 9+2 arrangement of filaments in electron micrographs confirm the presence of cillated columnar cells in the epithelium of the bovine cervix. In paraffin sections cilia could easily be misinterpreted as thin strands of mucus on the tips of the epithelial celis. This study also revealed that there were no apparent regional differences as to the occurrence of ciliated and non-ciliated columnar epithelial cells in the bovine cervical mucosa.

3 938 W O R D I N G E R E T AL. Figure 1. Figure A. Section from paraffin embedded bovine cervical mucosa at 4 days postestrus showing ciliated columnar (CC) and non-ciliated columnar (NCC) epithelial cells. Non-ciliated columnar cells contain a large amount of stored mucin in the supranuclear region. Ciliated cells appear constricted and exhibit an intense eosinophilia. Hemotoxylin and eosin stain (335X). Figure B. Section from paraffin embedded bovine cervical mucosa at 4 days postestrus showing the presence of sulfated acid mucosubstances within the non-ciliated epithelial cells. Ciliated cells (CC) are devoid of mucin. High Iron Diamine stain (355X). Figure C. Epon section of bovine cervical mucosa at 4 days postestrus showing non-ciliated columnar cells. Clearly defined granules of stored mucin can be differentiated. Azure I I stain (516X). Figure D. Epon section of bovine cervical mucosa 4 days postestrus showing the lack of staining of the ciliated columnar ceu and the abundant amount of stored mucin (periodate positive) in the non-ciliated columnar cell (NCC). Periodic acid-schiff-azure I I stain after partial removal of Epon (516X). Figure E. Epon section of bovine cervical mucosa 14 days postestrus showing the presence of numerous ciliated columnar cells. Non-ciliated columnar cells contain small amounts of stored mucin (M) in the very apical part of the cell. Azure I I stain (516X).

4 CELL TYPES IN BOVINE CERVICAL MUCOSA 939 Figure 2, Figure A. Electron micrograph of the bovine cervical mucosa at 4 days postestrus which illustrates the ciliated and non-ciliated columnar cell types. Ciliated cell appears constricted between 2 non-ciliated cells containing stored mucin in the apical part of the cell. Uranyl aceate-lead citrate stain (I0,584X). Figure B. The stored mucin found in the simple columnar cel! appears as secretory granules with some denser staining material present within the granules. Rough endoplasmic reticulum in close proximity to the mucin can be seen, Uranyl aceate-lead citrate stain (26,928X). Figure C. Higher magnification of a ciliated cell found within the bovine cervical mucosa. Numerous basal bodies (BB) are present and cytoplasmic projections (arrows) can be seen among the cilia, Desmosome (D). Uranyl aceate-lead citrate stain (14,994X). Figure D. Typical cross section of a cilium showing the presence of the 9q-2 filament arrangement. Uranyl aceate-lead citrate stain (39,072X).

5 940 WORDINGER ET AL. No differences were seen among tissue samples from the anterior, middle, or posterior cervical mucosa. Also, there appears to be some cyclic variation in the amount of mucin in the non-ciliated columnar cells because there was abundant storage at 4 days postestrus but very little storage at 14 days postestrus. This probably reflects the changing ratio of estrogen to progesterone during the bovine reproductive cycle and the subsequent effect on the cervix as previously reported by Herrick (1951) and Marinov and Lovell (1967). Our investigation did not reveal possible functions for the ciliated cell within the bovine cervical mucosa. These cells may be vestigial components resulting from an embryological origin common to the uterus and may assist in the movement of mucus secreted by the non-ciliated cells of the cervical crypts and folds. Literature Cited Bennet, H. S. and J. H. Luft S-Collidine as a basis for buffering fixatives. J. Biophys. Bioehem. Cytol. 6:113. Cole, H. H Study of the mucosa of the genital tract of the cow, with special reference to cyclic changes. Amer. J. Anat. 46:261. E1-Banna, A. A. and E. S. E. Hafez The uterine cervix in mammals. Amer. J. Obster and Gynecol. 112:145. Hafez, E. S. E., A. A. EI-Banna and T. Yamashita Histochemical characteristics of cervical epithelia in rabbit and cattle. Acta Histochem. 39:195. Hammond, J The physiology of reproduction in the cow. Cambridge University Press. London. Herrick, J Cytological changes in cervical mucosa of the cow. Amer. J. Vet. Res. 12:276. Luft, H. L Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9:409. Marinov, V. and J. E. Lovell Secretary and ciliated cells of the bovine cervix. Amer. J. Vet. Res. 28:1763. Paiz, C. and C. R. Hennigar Electron microscopy and histochemical correlation of human anterior pituitary cells. Amer. ~. Path. 59:43. Roark, D. B. and H. A. Herman Physiological and histochemical phenomena of the bovine estrous cycle with special reference to vaginal-cervical secretions. Missouri Agr. Exp. Sta. Res. Bull Spicer, S. S., R. G. Horn and J. T. Lippi Histochemistry of connective tissue mucopolysaccharides. In The Connective Tissue. Int. Acad. Path. Monogr. Williams and Wilkins Company, Baltimore, Md.