THE ANATOMICAL RECORD 233:121-134 (1992) Postnatal Development of the Cervical Epithelium in the Mongolian Gerbil Department of A. KRESS AND L. MARDI Anatomy, University of Basel, Switzerland ABSTRACT This study analyzes the postnatal development of the Mongolian gerbil’s cervical epithelium, in relation to its future functions. In the newborn gerbil the outline of the cervical canal is smooth, showing hardly any signs of folding. The epithelium consists of 1to 3 layers. The cervical cells have rounded apices of regular outline and contain a large amount of glycogen. The first secretory products of specific mucus type appear about day 23 postnatally (pm.). Initially two types of vesicles can be identified, as compared with only one type in sexually mature animals. The process of mucification begins in the vagina and the external 0s of the cervix and spreads towards the cervical horns. The cervical canal, besides growing longer, becomes increasingly folded during development. At about day 50 p a . , with the onset of sexual maturity, an upper endocervix and a lower ectocervix can be distinguished within the cervical canal. In the fully mature animal, the endocervix consists of 4 to 5 layers, in which apical cells mucify and exfoliate. In the ectocervix, the epithelium can be divided into 4 to 5 basal layers and 5 to 7 upper layers which mucify, keratinize, and exfoliate, according to the cyclic phases of the vagina. Diapedesis of leucocytes through the epithelium starts around day 45 p.n. o 1992 Wiley-Liss, Inc. The Mongolian gerbil belongs to the family of Crice- anisms for controlling the uterine luminal environtidae. It is one of the more recently introduced labora- ment, the blood-uterine barrier, seem to be of tory animals and has become a popular biomedical re- importance, and these mechanisms depend on horsearch model. Several studies of its reproductive monal stimulation during estrous cycle and pregnancy behavior have been published (Salzmann, 1963; Norris (McRae, 1984). and Adams, 1972a,b, 1979, 1981a,b, 1982; Norris and The postnatal development of the uterus and its Rall, 1983; Barfield and Beeman, 1968; Bagwell and glands has been described in two previous papers Leavitt, 1974; Wu, 1974, 1975; Clark et al., 19861, but (Kress and Mardi, 1990a,b) which examined the role of surprisingly little is known about the structure of the those epithelial structures which might especially be female genital tract and about its postnatal develop- concerned with the future functions of the animal’s ment (Kress and Mardi, 1990a,b; Kress et al., 1989). uterus. This present investigation analyzes the strucTherefore, basic studies are needed to guide future ex- tural changes of the luminal cervical epithelium from perimental work. the time of birth to sexual maturity. There are remarkable differences among mammaMATERIALS AND METHODS lian species in the anatomical and histological archiAnimals tecture of the cervical canal. Differences have been The Mongolian gerbils (Meriones unguiculatus) used noted in the anatomy of external orifices, the complexity of cervical foldings, the possible existence of glands, in this study were housed under controlled light for the the transition from uterus to cervix, and the division of period from 0600 to 1800 h r daily. They were allowed the cervical canal into endocervix and ecto- or exo- free access to food. Counting the day of birth a s the first postnatal day (1p.n.), 23 postnatal (p.n.1 stages up to cervix (El-Banna and Hafez, 1972; Mossman, 1987). Essentially the uterus consists of two portions with sexual maturity have been analyzed. For purposes of different functions: 1)the gestational portion, which is comparison, several multi- and nulli-parous females lined by a mucosa capable of special differentiation were also studied. during estrous cycle and during pregnancy; and 2) the Tissue Processing cervix, lined by a mucosa which is dominated either by Transmission electron microscopy (TEM) mucous glands or by mucous epithelium, having a n Animals were anesthetized with ether. The body caveffect on sperm migration and storage. The epithelium ity was opened and flooded with ice-cold solution of 2% is related to the surrounding connective tissue and smooth muscles in such a way that the whole acts as a sphincter during gestation (Mossman, 1987). For the Received November 9, 1990; accepted September 19, 1991 preimplantation stages and for ensuing embryonic deAddress reprint requests to Professor Dr. Annetrudi Kress, Departvelopment, the uterine milieu is of importance. Mech- ment of Anatomy, Pestalozzistrasse 20, CH-4056 Basel, Switzerland. 0 1992 WILEY-LISS, INC 122 A. KRESS AND L. MARDI glutaraldehyde in 0.1 M Millonig or sodium cacodylate buffer (pH 7.4). The whole genital tract was then removed. Ovaries, tubae uterinae, uterus including cervix, and vagina were dissected and stored separately in the fixative for about 2 hr. After fixation, the samples were rinsed in the according buffer and postfixed in 1% OsO, in 0.1M Millonig or sodium cacodylate buffer. The tissue was dehydrated in acetone and embedded in Epon 812. In parallel to the method described above, specimens were fixed directly in 1%OsO, in either 0.1 Millonig or sodium cacodylate. The adult animals were perfused, via the aorta descendens, with a mixture of 2% glutaraldehyde in the above-mentioned buffers. Scanning electron microscopy Dissection and fixation procedures followed the course already described above. Tissues were then dehydrated in a n ascending series of acetones, dried with CO, in a critical-point dryer (Balzers), mounted on metal studs with silver paint, and coated with 15 nm gold in a Polaron E 5150 sputter coater. The specimens were examined in a Jeol-JSM 255 111. Light microscopy For all the stages described, serial sections were also provided. The genital tracts were embedded in Paraplast, cut in serial sections, and stained according to Pasini and PAS. To identify cells producing neutral, moderately acid, and sulfated (highly acid) mucous glycoproteins, serial sections were stained with alcian blue (8GX Sigma A 3157) in 3% acetic acid for 30 min and counterstained in nuclear fast red or PAS. RESULTS Cervic Morphology Fig. 1. Opening of the H-shaped cervical canal (external 0s) into the vagina (day 20 p.n.). The opening is enclosed by four lips, which form the portio. The dorsal bulge (P) is the most prominent. Arrows point to the vaginal fornices. SEM, x 110. outline of the cervical canal is straight; no folding of the wall can yet be seen (Fig. 2). Cell apices are mostly rounded but are sometimes flat (Figs. 4, 6). There are hardly any of the heteromorphic protrusions as described by Kress and Mardi (1990a) for the uterine luminal cells (Fig. 5). The apices carry sparse, short, and irregular microvilli (Figs. 4,7). The luminal cell coat or glycocalyx is well expressed (Fig. 7). In many cells, a pair of centrioles are situated a t right angles to each other just beneath the luminal membrane, where the closer of the two constitutes the base of a solitary cilium (Fig. 6). Cell nuclei often contain 1 to 3 nucleoli; nuclear bodies exist but are rare. Continuity of the nuclear envelope with the ER (Fig. 9) and blebbing activities are regular features. Between their apices, adjacent cells form typical junctional complexes (Fig. 7). The filamentous material connected with these junctions and the cytoskeleton of the microvilli are of a delicate nature. The intercellular spaces are narrow. Some dilatations, connected with interdigitations, are a t this stage confined mainly to the basal cell areas (Fig. 8). Desmosomes are small and difficult to see. The basal lamina Day 1 p.n. is thin and closely follows the contour of the epithelium In the newborn gerbil, the lining of the entire cervi- (Fig. 8). cal canal consists mainly of a 1 to 2 layered, pseudoA striking feature of the cervical epithelial cells of stratified epithelium 25-36 bm in height (Figs. 2, 3). the newborn gerbil is their content of glycogen, which Around the external 0s it is occasionally 2 to 3 layered. occurs as huge deposits in supranuclear and in basal The transition between the mainly single layered positions (Figs. 3 , 4 , 8). Mitochondria are numerous in uterine and the pseudostratified cervical epithelium the apical as well as in the basal cytoplasm. Golgi comoccurs in the upper third of the cervical horns. The plexes, found mainly in supranuclear and lateral posi- In comparative anatomy, three main types of uterus are recognized, depending on the extent of fusion of the Mullerian ducts within the uterine area: 1)The uterus duplex which has two separate uteri and cervices, each with an independent opening into the vagina; 2 ) The uterus bicornis which develops as a result of fusion of longer parts of the uterus and cervix, forming a common corpus uteri with only a single cervical opening into the vagina; and 3) The uterus simplex which results from a complete fusion of the uterus anlagen (ElBanna and Hafez, 1972; Mossman, 1987; Starck, 1975). The uterus bicornis has many intermediate types, depending on the extent of fusion. Where only the most caudal ends of the uterus lumina fuse, to form a very short cervix communis, the outcome is called a uterus bipartitus. The Mongolian gerbil uterus fulfills these criteria. It has two separate uterine horns leading into separate cervical horns which form a very short, Hshaped cervix communis. The external 0s opens between four cervical lips which protrude into the vagina (Fig. 1). POSTNATAL DEVELOPMENT OF THE CERVICAL EPITHELIUM 123 Fig. 2. Day 1 p.n. Section through the area of the cervix communis. The cervical canal has smooth outlines; no folds or clefts are present. The dorsal part of the H-shaped cervix is rather more pronounced. After parafin embedding, glycogen is largely lost, resulting in conspicuous vacuoles in apical and basal cell areas. Alcian blueinuclear fast red; x 400. Fig. 3.Semithin section through the cervix communis. After fixation with glutaraldehyde and OsO,, glycogen appears as dark areas in the apical and basal cell cytoplasm. x 560. tions, are accompanied by many vesicles. In the apical area, profiles of granular ER and also free ribosomes are located. Along the luminal membrane, vesicles of pinocytotic and coated nature are formed. These vesicles, together with multivesicular bodies (mvbs), indicate that metabolic exchange must take place. Mitotic activities can be noted. rows, first from 2 to 3 (Fig. 10) and then mostly from 3 to 5, takes place. The overall height of the epithelium remains between 25-36 pm. The change in type from uterine epithelium to cervical epithelium becomes apparent in the light microscope. The uterus has only one cell layer (Figs. 13, 14), but the cervical epithelium changes towards day 13 p.n. to about 3 to 4 layers (Fig. 15). The luminal cervical cells are characterized by vacuoles; the adjacent cells of the uterine epithelium, however, are generally of homogeneous density (Fig. 13). Where the cervical lumen is very narrow, opposite cell apices are flat and often devoid of microvilli. In more spacious areas, cell apices are rounded and bulge into the lumen, they are covered with rather stubby Days 2-25 p.n. The cervical canal takes an irregular outline, invaginations or folds appear and increase in size (Fig. 10). A considerable growth in cervical length takes place during this period. Between individual epithelial cells, clefts become noticeable (Fig. 12). During differentiation of the cervix an increase in the number of cell 124 A. KRESS AND L. MARDI Fig. 4. Day 1p.n. Section through luminal cervical cells. The apical cell membrane carries short microvilli. The cytoplasm is dominated by a large amount of glycogen (Gl). Besides mitochondria and rERcisternae, different vesicles and multivesicular bodies (arrows) form part of a regular organelle pattern. x 14,200. small amount of glycogen in the apical cytoplasm. Junctional complexes along the lateral membranes of the apices seem more prominent in the uterus than in the cervix. x 17,100. Fig. 6. Day 1p.n. Section through four cervical cell apices. A solitary cilium (arrowhead) protrudes into the cervical lumen (L). x 20,400. Fig. 5. Day 1 p.n. Luminal uterine epithelium shown for comparison. Typical are the longer microvilli, apical protrusions, and the often forked microvilli and a fuzzy glycocalyx (Fig. 12). Solitary cilia are still present and can occasionally be detected in intermediate cell layers also, where they protrude into the intercellular space. The junctional complexes are connected with fine filaments, but none of the thick filamentous belts a s seen in uterine cells are visible, nor are the dominant apical domes perceptible (Kress and Mardi, 1990a,b). Interdigitations between adjacent cells become more pronounced in depth, and density and size of desmosomes increase. Hemidesmosomes between the plasmalemma of the basal cell layer and the basal lamina develop around days 2 to 8 p.n. A more undulating borderline evolves between epithelium and lamina propria (Fig. 11). Mitotic and apoptotic processes are regular features. Until about day 5 p.n., the amount of glycogen is similar to that found in the newborn animal. After this date, a decrease in glycogen content seems to occur but not in all the cells to the same extent. Apical cell areas contain small patches or dispersed glycogen, while in basal cell areas more prominent accumulations persist. The Mongolian gerbil develops no real cervical glands. The cervical epithelium of the lining folds and POSTNATAL DEVELOPMENT OF THE CERVICAL EPITHELIUM Figs. 7-9.TEM of cervix epithelium, day 1 p.n. Fig. 7. The plasmalemma of the luminal cell apices is covered with a distinct, fuzzy glycocalyx. The junctional complexes between adjacent cells are well developed (arrows). Typical are the accumulations of glycogen (GI) and mitochondria. x 30,000. Fig. 8. Basal portion of luminal cervical cells. Interdigitations and intercellular space between neighboring cells (arrow) are more pro- 125 nounced than in apical regions. Desmosomes are rare a t this age. The basal lamina closely follows the smooth basal plasmalemma (arrowhead) GI, Glycogen. x 14,200. Fig. 9. The nuclear (N) envelope exhibits blebbing and sites where it extends into ER cisternae (arrows). GI, Glycogen; arrowheads, cell membranes of neighboring cells. x 45,500. 126 A. KRESS AND L. MARDI Figs. 10-12. POSTNATAL DEVELOPMENT OF THE CERVICAL EPITHELIUM clefts differentiates into individual mucous cells. In the sections stained with alcian blue and PAS, the first cervical mucous cells, showing blue or purple secretory products, appear in the luminal layer and are first observed around the external 0s of the cervix about day 17 p.n. In the cervix communis and in the cervix horns, however, only a faint blue border along the cell apices can be seen, similar to the findings in the uterine horn. From day 23 p.n. on, individual, distinct, blue- or purple-stained mucous cells appear in the epithelium of the cervix communis, and this process is spreading into the cervical horns. In the uterine and glandular luminal cells, only minute blue dots and a bluish border along the cell apices can be seen. These findings indicate that epithelial differentiation is of different nature in the uterus and cervix. EM pictures corroborate the development of secretory products in the cervix. In luminal cells especially, the Golgi complexes have extended and are budding off a variety of vesicles. ER profiles have increased in length and below the apical plasma membrane, vesicles of different natures and mvbs accumulate. From day 23 p.n. on, many cells show distinct secretory granules. There seem to be two different types of granules (Fig. 16). One is electron lucent (diameter 0.5 pm), and the other, small type has some electron-dense material (diameter 0.12-0.16 pm). Around this time the cervical lumen becomes filled with increasingly dense material (Fig. 16). Days 30-52 p.n. Around day 30 p.n., secretory mucous cells are visible predominantly in the lower part of the cervix. The mucification process is in a more advanced stage in the vagina luminal cell layers than in the cervix. First signs of keratinization appear in the vagina below the mucified cells, and the desquamation of some surface cells occur, a process not observed before day 45 p.n. in the cervical canal. At day 45 p.n. the keratinization process, already taking place in the vagina, expands towards the external 0s of the cervix, where faint signs of these activities can be noted (Fig. 17). Leucocytes begin to appear within the cervical epithelium. A different behavior of the cervical epithelium adjacent to the uterine lining and to the lower parts of the cervix, adjacent to the vagina, is noticeable for the first time. In the vagina an intensive process of mucification, keratinization, and exfoliation takes place. This process seems to stretch from the vagina via the external 0s into the cervix and is first seen shortly before the animal reaches sexual maturity. The activity declines 127 farther up the cervical horns. The areas near the uterus have fewer mucified cells and do not keratinize. These variations in differentiation lead to the formation of an endo- and ecto-cervix. Adult The adult cervix exhibits an extensive pattern of folds and small irregular invaginations or clefts (Figs. 20, 21). No real cervical glands develop. Figs. 10-1 2. Cervix epithelium, day 8 p.n. Fig. 10. Semithin section through part of the cervical horn where the formation of folds is in progress. Dark-stained areas within the epithelial cells indicate fields of glycogen. x 480. Fig. 11. Borderline area between epithelial cells and connective tissue. The arrowheads point to the basal lamina and the developing hemidesmosornes. The presence of a simple nuclear body, as found in all developmental stages, is seen in the nucleoplasm (arrow). X 17,100. Fig. 12. Besides fold formation, the growth of clefts (arrows) between individual cervical cells can be noted. Golgi complexes appear active. The amount of glycogen (Gl) diminishes. X 17,100. Fig. 13. Day 23 p.n, This paraffin section demonstrates the transition (arrows) from the uterus epithelium (without vacuoles) to the cervical epithelium (with vacuoles). No mucous granules have yet become stained in the cervical area close to the uterine epithelium. Alcian blueinuclear fast red; x 580. Figs. 14, 15. Day 22 p.n. Semithin sections of the single-layered uterine epithelium (Fig. 14) and the 3 to 5 layers of the thick cervical epithelium (Fig. 15). Arrow points to the zone of the basal lamina. x 640. 128 A. KRESS AND L. MARDI Fig. 16. Day 24 p.n. A section through apical areas of two adjacent cervical cells. Secretory vesicles have accumulated below the plasmalemma. Some of the vesicles seem to be partly empty, as in previous stages of development. In the cell to the right, vesicles of homogenous appearance dominate (arrows). In the cell to the left, smaller vesicles or granules with electron-dense areas are present (arrowheads). Note the flocculent content of the cervical lumen (L). Some of the cellular material may have been pinched off from apical protrusions. Inset: Detail of secretory granules containing electron-dense areas. x 23,000, inset; x 54,600. The endometrial lining of the uterus horn, which is 18) and distinct desmosomes. Glycogen is still present, predominantly single-layered and 15-30 pm high, disseminated as well as in patches, and in greater changes at the uterocervical junction into a stratified quantity than in neighboring uterine cells. In the nuepithelium of 4 to 6 layers, about 30-45 pm in height, clei, nuclear bodies may be present (Fig. 18). In the sexually mature cyclic animal, a sharp demarindicating the beginning of the cervical area (Fig. 19). The number of mucified cells increases visibly from the cation line between a n upper endocervical and a lower cervical horns downward, through the cervix commu- ectocervical region is conspicuous especially during esnis and towards the external 0s. In the uterus and the trus (Fig. 22). The transition lies in the lower regions of uterine glands, no distinct mucous cells can be de- the cervical horns. Histologically, therefore, the Montected. The apices of the lining cells show only faintly golian gerbil cervix is composed of two regions; a lower bluish tinged structures and a distinct blue borderline segment or ectocervix that resembles the vagina, and along the apices, including the glycocalyx. Cervical a n upper segment or endocervix that is transitional cells exhibit a n accumulation of secretory substance between the lower segment and the uterus. In the 4 to 6 cell layers of the endocervix, mucified (Figs. 18, 19). In some cervical areas, secretory cells lie tightly packed, but other patches are practically devoid cells are less numerous. Diapedesis of leucocytes through the epithelium seems just as intensive as in of them. No ciliated cells exist. Since not all the cervical cells examined contain the the ectocervix. Desquamation of surface cells during same amount of secretory products, the area of active the estrous cycle occurs, but no keratinization process Golgi complexes and dilated ER cisternae varies con- can be noticed. The stratified epithelium of the ectocersiderably from cell to cell. A distinct increase of secre- vix, however, resembles more closely the vaginal epitory vesicles in the cell apices can generally be noted, thelium. Four to five cell layers seem to form basal compared with cells of day 45 or 52 p.n. But only one cells for renewal; 5 to 7 more apical cell layers either type of secretory vesicle seems to differentiate, namely differentiate into mucous cells or keratinize and exfothe granule of larger diameter and homogenous char- liate (Fig. 23), according to the cyclic processes which acter (Fig. 18). The apices of luminal cervical cells are take place in the vagina. flat or rounded, sometimes forming protrusions. IsoDISCUSSION lated apical parts in the lumen give the impression that some of the protrusions have been pinched off. The anatomy of the cervix and its histological strucMicrovilli are mainly stubby and the glycocalyx is dis- tures varies considerably in different mammalian spetinct (Fig. 18). The junctional complexes between cell cies (El-Banna and Hafez, 1972; Kanagawa and Hafez, apices are well developed, and the plasma membrane of 1973). The meat majority of descriptions deal with the lateral cell walls shows intensive interdigitations (Fig. structure ofthe aduit cervix; only few papers adduce a POSTNATAL DEVELOPMENT O F THE CERVICAL EPITHELIUM 129 Fig. 17. Day 45 p.n. Semithin section through the cervix communis (Cc) near the external os, which partially opens into the vagina (Va). Note the exfoliation of mucified cells (arrow) and the beginning of keratinization in the vaginal area (arrowhead). In the cervix commu- nis and the two cervical horns, mucified cells are less numerous and only individual cells have been shed. No signs of keratinization can be detected. P, Portio. x 160. data about the regular postnatal cervical development (Lamb et al., 1977). These data, moreover, are often only side products from studies where neonatal mice have been treated hormonally and where the interest has been focussed on how different hormonal conditions inf h e n c e the epithelium (Eide and Mellgren, 1973; Eide, 1975; Doskeland et al., 1976; Forsberg and Kalland, 1981; Eroschenko, 1982). The identification of the true cervical region may be difficult. Often i t is not made clear where the true endometrium meets the cervical mucosa or where the junction between cervical and vaginal epithelium lies. Often, too, the terms “endo- and ectocervix” or “upper and lower cervix” are not clearly defined or their descriptions are misleading (Allen, 1992; Hamilton, 1947; Graham, 1966; Mossman, 1987). Endocervical epithelium in rodents may be described as different from the uterine lining with respect to the shape, and often to the number of cell layers and to the presence of typical mucous cells. These are shed during the cycle, but no keratinization takes place. The ectocervix, however, is characterized by stratified squamous epithelium taking part in a n alternating process of mucification, keratinization, and exfoliation similar to the activities observed during the cycle in the vagina (Jurow, 1943; Leppi, 1964). The location of the zone of transition between the columnar or few-layered epithelium of the endocervix into the stratified squamous epithelium of the ectocervix is described for several species by El-Banna and Hafez (1972) and Hafez and Jaszcak (1972). The differences between the two segments of the cervix in r a t are more apparent a t the late proestrous or early estrous phase (Hamilton, 1947). This observation is also true for the gerbil’s cervix. The Mongolian gerbil develops a distinct division of the cervical canal into upper and lower parts, a n endoand a n ecto-cervix, respectively. The division can be observed for the first time about day 50 p.n. The two segments differ in the number of cell layers and in their cyclic behavior. In the endocervix, 4 to 6 layers are generally found. In the ectocervix, there are 4 to 5 layers forming a basal zone, with 5 to 7 cell layers on top of these basal cells. Exfoliation of surface cells takes place in the endocervix, but there is no keratinization process. In the ectocervix, on the other hand, the apical layers mucify, keratinize, and exfoliate according to the state of the cycle in the vagina. Many species differ from each other by the presence or absence of cervical glands. In many animals the cervical lining consists of a highly complicated system of deep folds and clefts which are sometimes difficult to distinguish from true tubular glands, e.g., in ewes or goats (El-Banna and Hafez, 1972; Heydon and Adams, 1979). Folds and clefts enlarge the mucus-producing surface enormously and are typical for cattle (ElBanna and Hafez, 1972; Heydon and Adams, 19791, the rabbit (Odor and Blandau, 19881, and in the present case for the gerbil. True cervical glands have been described for human, many monkeys and dogs (El-Banna and Hafez, 1972), and for the guinea pig (Jurow, 1943). The epithelial lining of the cervix consists of secretory cells and, in some species, of ciliated cells also. The latter have been described in rabbits (Odor and Blandau, 1988; Odor e t al., 1989), in sheep (Wergin, 1979; More, 1984), and in monkeys (Hafez and Jaszcak, 1972). In rodents, however, only non-ciliated secretory cells have been noted, as is the case in Meriones. Cervical mucus has been analyzed in a number of species, 130 A. KRESS AND L. MARDI Fig. 18-23. Cervix, adult. Fig. 18. TEM of apical areas of mucus-secreting cells as depicted in Figs. 19-22. The mucous granules in cycling animals seem to be of a homogeneous nature. The amount of mucous granules per cell varies. Nuclear bodies (arrow) are regularly found. x 17,100. Fig. 19. The transition of the single-layered uterine epithelium into a stratified, 4 to 5 layered cervical epithelium of the endocervix can be clearly seen (arrow). The cervical cells, which contain a variable amount of mucous granules exhibit either distinct blue-stained apices or apical rims (arrowheads). Alcian bluehuclear fast red, x 400. POSTNATAL DEVELOPMENT OF THE CERVICAL EPITHELIUM Fig. 20. Cross-section through the cervix communis (ectocervix) a t low magnification, demonstrating the intensive fold formation of a sexually mature animal. Alcian blue/PAS, x 40. Fig. 21. Detail of cervical folds near the cervix communis. The clefts between individual cells have widened into secondary invaginations. 131 The epithelium has increased in thickness and can be divided into 4 to 5 basal layers and 5 to 7 more apical layers, where the darkly stained mucus forming cells differentiate. The arrow points t o the borderline between the basal and apical layers, where later in the cycle the keratinization process takes place. Alcian blue/PAS, x 252. especially in rabbits and in ruminants (Hafez et al., secretory granules. This heterogeneity among the mu1971; Chilton et al., 1980, 1986; Heydon and Adams, cus-producing cells has been very conspicuous in the 1979). In these cases the mucus consists mainly of acid developing and adult Mongolian gerbil cervix. (sulfated and non-sulfated) and neutral glycoproteins. In the cervical canal of the gerbil, specific mucusThese components form a variation of mucus granule producing cells develop at about day 23 p.n. in the lucontent which varies between species and is affected by minal cell layers. These first secretory products appear the mucous cell location within the cervix and the in two different types of granules. One is electron-luphase of estrous cycle (Heydon and Adams, 1979). cent and about 0.5 p,m in diameter, the other is smaller Hafez et al. (1971) described 6 and Chilton et al. and electron-lucent with some electron-dense cores. In (1980, 1986) 3 different types of secretory cells in the the mature animal, only the electron-lucent type has cervix. Odor and Blandau (1988) and Odor et al., (1989) been found. According to Chilton et al. (1980), acid glyin their careful study of the rabbit cervix, discussed coproteins correspond to large electron-lucent granules only one such type. They observed, however, that the while neutral glycoproteins correspond to similar elecapical and supranuclear structures vary considerably tron-dense granules. Whether this holds true for the from cell to cell, e.g., the amount and the texture of the gerbil secretory granules also has still to be tested. 132 A. KRESS AND L. MARDI Fig. 22. Semithin section showing the transition (arrow) from the upper endocervix (En) into the lower ectocervix (Ec) epithelium. The endocervix is lined by numerous mucus-producing cells. The ectocervix shows signs of keratinization (arrowhead). X 640. Fig. 23. Keratinization and exfoliation of cell layers in the ectocervix region, a process not taking place in the endocervical area. In the upper right corner of the picture, some migrated leucocytes have accumulated. Pasini, x 640. Lysozyme is a known constituent of cervical secretion and has been identified within the secretory granules in the rabbit cervical cells (Nicosia et al., 1984). The amount of glycogen accumulated in the apices and basally to the nucleus in gerbil cervical cells at birth is striking. The same situation has been described for cervical cells in newborn mice (Abrg, 1974; Abrg and Lingaas, 1977). The deposits of glycogen are thought t o represent a quickly mobilized source of energy for the newborn in the first crucial period of extrauterine life and to play an important role for the proliferation and the differentiation of the lining cervical epithelium during cervical development (Abrg, 1974).In mice the glycogen-depletionprocess starts immediately after birth with a rapid diminution. At day 12 p.n., only small glycogen patches are left. In mice, this glycogen depletion involves autophagic processes with aggregated glycogen bodies in membrane-limited vacuoles, resembling the glycogenosomes found in the liver of newborn rats (Abrg, 1974). In the Mongolian gerbil, the glycogen depletion starts about day 5 p.n. with some glycogen patches still left at day 15 pm. Only in a few exceptional cases could autophagic vacuoles be observed; the glycogen content just diminishes. Epithelial cells during airway development in the golden hamster are depleted of glycogen within two days after birth, soon thereafter the cells become presecretory and show an increase in ER and complex folding of lateral membranes (It0 et al., 1990). A similar succession of events accompaniesthe loss of glycogen in the gerbil cervical cells. It is doubtful whether a rise of estrogen level is responsible for the glycogen decrease during development although this has been suggested by some authors (Abrg and Kvinnsland, 1972). Diapedesis of lymphocytes and eosinophil and neutrophil granulocytes through the cervical epithelium is a well-known fact during the estrous cycle (Odor, 1974). The migration of leucocytes in the gerbil cervix has first been detected a t day 45 p.n., shortly before the onset of sexual maturity and cyclic behavior in the cervical epithelium. Recent research has focussed interest on the effect of the migratory cells, not only in the epithelium but also in the cervical stroma where at term widespread collagenolysis occurs. This seems to soften and relax the cervical neck for delivery (Ito et al., 1987; Luque and Montes, 1989). After birth the gerbil epithelium seems still to be under the influence of maternal hormones, reflected in the intensive activities to be seen especially in the uterine luminal cells. This observation has been corroborated by Dohler and Wuttke (1975), who studied the hormonal development of postnatal rats and found elevated levels of estrogen just after birth. If steroid hormones are to exert their influence on the target cells, the latter must develop receptors to bind the hormones. There has been a change in opinion about the localization of estrogen receptors. Many authors have differentiated between cytoplasmic and nuclear receptors (Clark and Gorski, 1970; Somjen et al., 1973; Katzenellenbogen and Greger, 1974; Nguyen et al., 1986). In more recent studies, the authors conclude that the estrogen receptor is a nuclear protein. It is synthesized in the cytoplasm but rapidly enters the nucleus where it is tightly bound to the chromatin; therefore, it does not reside in the cytoplasm for a long period (Stumpf, 1983; King and Greene, 1984; Brenner et al., 1990). Considerable attention has been paid to the behavior of uterine estrogen receptors in some rodents during the postnatal period (Clark and Gorski, 1970; Somjen et al., 1973; Katzenellenbogen and Greger, 1974; Nguyen et al., 1986). Receptors are present at birth, and their number increases during the first 10 days to decline somewhat after 15-20 days (Brenner and West, 1975). The amount of receptors found corresponds to the rising levels of estrogen during early postnatal de- POSTNATAL DEVELOPMENT OF THE CERVICAL EPITHELIUM velopment (Somjen et al., 1973; Katzenellenbogen and Greger, 1974; Brenner and West, 1975; Dohler and Wuttke, 1975; Eide and Forsberg, 1976). One indicator for the presence of receptors is the nuclear bodies. Nuclear bodies are found in the uterus, cervix, and vagina in many animals (Le Goasgogne and Baulieu, 1977; Clark et al., 1978; Padykula et al., 1981; Padykula and Pockwinse, 1983). They are round physiological organelles, 0.2 to 0.1 km in diameter; in most cases they are observed in uncondensed chromatin regions, not necessarily close to the nucleolus. In the development in the Mongolian gerbil cervix, therefore, the presence of nuclear bodies at birth indicates the presence of estrogen and its receptors. In rats, various types of nuclear bodies have been described, some simple, some with more complex structures. The nuclear bodies seen in the nuclei of gerbil cervical cells resemble rat nuclear bodies at the corresponding developmental age (Le Goasgogne and Baulieu, 1977). In the rat, the nuclear bodies change in appearance into a more complex structure as the animal reaches sexual maturity, but the cervical cell nuclear bodies of the gerbil do not change visibly at all. The developmental pattern of nuclear bodies may be related to hormonal factors. Hyperestrogenization in adult rats leads to increased numbers and greater complexity of the nuclear bodies in the uterus epithelium (Clark et al., 1978). Their function, however, remains obscure. ACKNOWLEDGMENTS This study was partly supported by the Ciba Foundation. The authors appreciate the skillful technical assistance of H. Schaller, M. Zulliger, G. Morson, and I. Bartuskova and the secretarial help of P. Krause. The authors are also grateful to H.J. 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