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Morphological Observations on the Epithelium of the Developing Urinary Bladder of the Mouse and Rat
0022-534 7/85/1333-0506$02.00/0 Vol. 133, March
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright © 1985 by The Williams & Wilkins Co.
MORPHOLOGICAL OBSERVATIONS ON THE EPITHELIUM OF THE DEVELOPING URINARY BLADDER OF THE MOUSE AND RAT PAUL H. AYRES,t YOSHITAKA SHINOHARA,+
AND
CHARLES H. FRITH*
From the Departments of Pathology and Interdisciplinary Toxicology and Pharmacology, University of Arkansas for Medical Sciences, and Intox Laboratories, Little Rock, Arkansas
ABSTRACT
Bladders from fetal and neonatal BALB/cStCrlfC3H/Nctr mice and Sprague-Dawley rats were studied to establish the sequence of events in their morphological development by using scanning electron, transmission electron and light microscopy. On fetal day 18 or 19 the epithelium from the mouse and the rat displayed 2 or 3 distinct cell layers. With transmission electron microscopy a star-like contraction of the cell surface of the mouse· bladder occurred which was not seen in the developing rat bladder. In both the mouse and the rat, some of the superficial cells sloughed between fetal day 18 or 19 and the day of birth. On the day of birth, the epithelium was composed of only 2 layers. The nuclei of both the superficial and hasal layers contained prominent euchromatin, and mitotic figures were often present in the basal layer. By the 5th postnatal day, some of the superficial cells contained autophagic vacuoles, and the epithelium was still 2 cell layers thick. One week after birth the epithelium consisted of 2 to 3 cell layers. Three weeks after birth the epithelium was 3 cell layers thick and appeared as the adult pattern with both transmission and scanning electron microscopy. The study demonstrated that the fetal and neonatal mouse and rat urinary bladders undergo a series of rapid developmental changes and suggest that the fetal and neonatal urinary bladder may be a target organ susceptible to toxic insult. Positive results in 2-generation carcinogenesis studies with saccharin have suggested an increased sensitivity of the immature rodent bladder epithelium to carcinogenic insult. Continued interest in the carcinogenicity of a compound of such economic significance has made investigators aware that little data is available in the current literature on the morphological development of the rodent urinary blaq.der. The literature is limited to light and electron microscopy and is not in complete agreement. 1- 3 Utilizing light microscopy, Walker3 observed that in 14 ½day-old mouse fetuses the bladder epithelium was multilayered, with all the cells and their nuclei being approximately the same size. The epithelium of 16 2/a-day-old fetuses displayed polyploid mitotic figures in squash preparations, and the average diameter of nuclei and cells showed a pronounced increase with binucleate cells occurring with a high frequency over a considerable portion of the epithelium. W alker3 reported that no other significant changes occurred throughout the remainder of fetal development: Shortly after birth, however, a 2nd increase in the average diameter of nuclei and cells occurred, which brought the surface cell to its final stage of differentiation regarding nuclear and cell size. In studies utilizing light and electron microscopy1• 2 it was reported that until the 15th day of gestation in the rat the epithelium of the urinary bladder was lined by a single layer of morphologically undifferentiated cuboidal cells, many of which were in mitosis. Between the 15th and 18th day of gestation, the epithelium developed into 2 cell layers with many binucleate superficial cells that showed some differentiation. Between the 19th day of gestation and the 2nd postnatal day, some of the superficial cells sloughed into the lumen. Other cells sloughed between the 5th and 10th day after birth causing the epithelium
to be 1 cell layer thick. The basal cell layer proliferated to form a new superficial layer by the middle of the 2nd week after birth. A 3rd cell layer was established in the 3rd week to form a mature type of epithelium. To investigate the potential of the urinary bladder as a target organ for transplacental carcinogenesis, the present study was undertaken to determine if the bladder epithelium was in a state of rapid change and proliferation or whether it was simply a qµiescent tissue during the late fetal period. Scanning electron microscopy has been widely used in studies of the adult urinary bladder and should prove useful in studies of the fetal and neonatal bladder. The present study utilized both scanning and transmission electron microscopy, as well as light microscopy, to study the development of the uripary bladder epithelium to establish a basis for morphological evaluation of control and carcinogen-treated fetal and neonatal hlaq.ders and to provide a comparison between the mouse and the rat.
The Mouse Female BALB/cStCrlfC3H/Nctr mice 6 to 8 weeks of age were mated overnight with BALBcStCrlfC3H/Nctr males. The morning a vaginal plug was found was designated day zero of gestation. Maternal animals were maintained on lab chow throughout the experiment in conventional animal rooms at 50 ± 10 per cent humidity and 22 ± 2C. Three litters were used at each time point with at least 2 offspring from each litter examined for morphological features. Urinary blaq.ders were studied at gestational days 18, 19 and 20 and postnatal days zero (day of birth), 1, 3, 5, 7, 10, 14, 21 and 28. Both male and female offspring were studied.
Accepted for publication November 6, 1984. * Requests for reprints: Intox Laboratories, Inc., # 1 Intox Drive, P.O. Box 250, Redfield, Arkansas 72132. t Current address: Lovelace Inhalation Toxicology Research Institute, Albuquerque, New Mexico. t Current address: Department of Pathology, Nagoya City University Medical School, Nagoya, Japan.
The Rat Twelve to 14-week-old virgin female Nctr:S23(SD)BR Sprague-Dawley rats were mated overnight with males of the same strain. If a vaginal plug was fognd the next morning, the female was designated pregnant at day z!)ro of gestation. Maternal animals were fed lab chow ad libitum throughout the
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experiment and maintained in conventional animal rooms at 50 ± 10 per cent humidity, 22 ± 2C and a 12 hour light-dark cycle. Offspring were studied at gestational days 18, 19, 20 and 21 and postnatal day zero (day of birth), 1, 3, 5, 7, 11, 14 and 21. Three litters were used at each time point, and at least 2 offspring from each litter were examined for morphological features. Male and female offspring were studied.
Mice and Rats The pregnant mice or rats were anesthetized with carbon dioxide gas and the fetuses were removed by caesarean section. The fetuses were anesthetized with carbon dioxide and decapitated. A midline incision was made on the ventral surface of the fetus to expose the urinary bladder. A linen string was looped around the neck of the bladder and a loose knot was tied around the urethra. The bladder was removed by a gentle tug on the string and a 31 gauge needle was passed into the urethra through the knot. The bladder was inflated with fixative and then the knot was tightened. Bladders that were normally distended and did not require inflation were fixed in situ after the urethra was ligated. Brief initial fixation in situ or distension with fixative provided adequate presentation of morphological features with no difference seen between the 2 fixative methods. Initially, either 2 per cent or 4 per cent glutaraldehyde or Trump's fixative 4 in a cacodylate buffer was used for fixation. Trump's fixative was used for the duration of the experiment after no readily apparent differences between fixatives were noticed. The bladder was then removed and placed in fresh fixative for 15 minutes. The bladder was then divided sagitally and one half was processed for transmission electron microscopy. The other half was processed for scanning electron microscopy. Tissues for transmission electron microscopy (TEM) were rinsed in 0.2 M cacodylate buffer (pH 7.2 to 7.4) at room temperature 3 times for 10 minutes each. They were then postfixed in 1 per cent osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2 to 7.4) at room temperature for 1 hour and rinsed in 0.2 M cacodylate buffer 3 times for 10 minutes each. The tissues were routinely dehydrated in ethanol, cleared in acetone on an automatic tissue processor, and then infiltrated and embedded in an Epon/ Araldite mixture. Sections were cut with glass or diamond knives on LKB Ultrotome III ultramicrotomes and examined with a Philips EM201 electron microscope at 60 kv. Tissues for scanning electron microscopy (SEM) were rinsed in 0.2 M cacodylate buffer (pH 7.2 to 7.4) at room temperature for 10 minutes 3 times and postfixed for 15 minutes at room temperature in 1 per cent osmium tetroxide buffered with 0.2 M cacodylate buffer (pH 7.2 to 7.4). The tissues were dehydrated through graded strengths of ethanol to absolute ethanol. The tissues were then critical point dried from liquid CO 2 • The tissues were mounted on specimen holders with conductive silver paint and coated in a sputter coater with approximately 200A of gold/palladium. The tissues were examined with a JEOL JSM-35 scanning electron microscope at 25 kv at 15 mm. working distance. RESULTS
Fetal Day 18 or 19 The epithelium of the urinary bladder of the 18 or 19-day fetus of both the mouse and the rat displayed 2 or 3 distinct cell layers. In the mouse dark cells were sometimes seen in the superficial layer. These dark cells had an angular profile bordering the lumen and contained many fusiform vesicles (fig. 1). The elongated nucleus with several distinct nucleoli and prominent heterochromatin was electron dense, as was the adjacent cytoplasm. Bundles of tonofilaments were present in most of the dark superficial cells (fig. 1 inset). Other superficial cells were found that were of normal cytoplasmic and nuclear density
FIG. 1. Fetal Day 18, Mouse. Transmission electron micrograph of bladder epithelium from untreated 18-day mouse fetus. Superficial cell is dark and contains numerous fusiform vesicles. Uranyl acetate and lead citrate stained thin section, X6,608. Inset. High magnification of bundles of tonofilaments in superficial cell cytoplasm. Uranyl acetate and lead citrate stained thin section, X34,320.
FIG. 2. Fetal Day 18, Rat. Epithelium of rat consists of 3 or 4 cell layers. Superficial cells appear to be degenerating and contain semicircular convex areas. Nuclei of basal and intermediate cells are oval in shape and predominantly euchromatic with single nucleolus with prominent fibrillar component. Uranyl acetate and lead citrate stained thin section, x5,048.
and contained fevv fusiform vesicles. The nuclei of these cells were composed o:,u,uuuuau.c,y of euchromatin and often contained multiple nucleoli. The cytoplasm of basal cells was of normal density and contained amounts of free ribosomes. The nuclei of basal cells were oval in shape and contained multiple nucleoli. In the rat, the epithelium was multilayered as in the mouse but revealed some morphological features which were dissimilar. Primarily, the epithelial superficial cells showed a loss of cytoplasmic organization with a lack of fusiform vesicles (fig. 2) possibly due to cytoplasmic degeneration. The lumenal surface of the urinary bladder from the 18 or 19-day mouse fetus displayed features that were not seen in normal adult animals. In the fetal bladder epithelium, the predominant cell had coarse ridges and globular projections primarily in the central portion of the cell. These globular projections and ridges seemed to coalesce in the central portion of the cell to form a star-like contraction of the cell surface. Maturing cells were occasionally seen on the lumenal surface of the bladder (fig. 3). Small patches of cell surfaces that
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contained very short uniform microvilli were seen. Other maturing cells often contained microridges with a foliate appearance (fig. 3 inset). Fetal Day 20
FIG. 3. Fetal Day 19, Mouse. SEM of maturing bladder cell protruding into lumen of bladder from control 19-day mouse fetus, Xl,630. Inset. Maturing bladder cell with foliate microridges that does not protrude into lumen, from control 19-day fetus, Xl,278.
FIG. 4. Fetal Day 20, Mouse. TEM of bladder epithelium from control 20-day mouse fetus. Superficial cell layer covered several underlying cells and had bulbous projections on superficial border. Uranyl acetate and lead citrate stained thin section, X4,554.
FIG. 5. Fetal Day 20, Rat. TEM demonstrates widening of intercellular gaps. Superficial cell of rat bladder shows evidence of degeneration. Intermediate and basal layer cells contain large amounts of free ribosomes. Uranyl acetate and lead citrate stained thin section, X7,323.
On the 20th day of gestation the bladder epithelium of both the mouse and the rat consisted of 2 to 3 cell layers. In the mouse the superficial cells covered several of the underlying cells and were not dark, unlike the epithelium on day 18 or 19. The superficial cells contained many free ribosomes and few fusiform vesicles. The lumenal border was thrown into bulbous projections which may be the coarse ridges that were seen with SEM (fig. 4). The SEM of the lumenal surface of the bladder revealed that maturing cells were still present and that they projected into the lumen. The star-like formation was readily observed on most of the other cells on the surface. The bulbous projections on the surface of the cells seem to coalesce to form the coarse ridges. Other maturing intermediate cells were present that were flat and contained the foliate type of microridge and bulbous projections. On gestational day 20 in the rat the superficial epithelial cells still showed signs of cytoplasmic degeneration without the
FIG. 6. Fetal Day 20, Rat. Surface cells from rat bladder show irregularity in size and shape and are slightly convex. Smooth, fine microridges and sparse small microvilli-like projections are present. Cell borders are thick and seem to protrude from surface, X2,900.
FIG. 7. Postnatal Day Zero (Day of Birth), Mouse. TEM of bladder epithelium from mouse on day of birth. Epithelium is 2 cell layers thick with angular profile on superficial border. Bundles of tonofilaments and fusiform vesicles are present overlying nucleus. Nuclei of basal and superficial layers contained prominent euchromatin. Uranyl acetate and lead citrate stained thin section, X6,608. Inset. Mitotic figure in basal layer.
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evidence bulbous projections or coarse that was seen in the mouse bladder (fig. 5). In addition, the epithelium demonstrated a widening of the intercellular junctions. The SEM of the lumenal surface of the rat bladder was remarkably different from that of the mouse. The superficial cells varied in size and shape and displayed small microvilli and fine ridges on the cell surface. Cell junctions were very distinct (fig. 6). No maturing cells were seen projecting into the lumen, unlike the mouse.
0 (day of birth) The epithelium of the urinary bladder of the mouse on the day of birth appeared to be composed of only 2 cell layers (fig. 7). The superficial cells were large and covered several of the underlying cells. The superficial cell surface had an angular profile and sparse amounts of fusiform vesicles appeared in the central portion of the cell to produce the elevated area seen as the star-like formation on SEM (fig. 8). Tonofilaments were abundant within the cytoplasm and usually appeared over the nucleus in the elevated area. The nuclei of both the superficial and basal layers contained prominent euchromatin and often contained multiple nucleoli. The cytoplasm contained many free ribosomes, and mitotic figures were often present in the basal cell layer (fig. 7 inset), as they also were in the epithelium from the fetal period.
FIG. 10. Postnatal Day 3, Mouse. TEM of bladder epithelium from 3-day-old control mouse. Epithelium contains 2 cell layers and superficial cell covers several underlying cells. Superficial layer had angular profile with fusiform vesicles present in cytoplasm. Uranyl acetate and lead citrate stained thin section, X7,000.
FIG. 11. Postnatal Day 1, Mouse. SEM view of bladder epithelium for 1-day-old control mouse. Superficial cells often contain 2 separate foci of star-like formation on same cell, >cl,700. FIG. 8. Postnatal Day Zero (Day of Birth), Mouse. SEM of lumenal surface of mouse bladder on day of birth. Almost all superficial cells have star-like constriction on cell surface, x750. Inset. High magnification of constricted area which is composed of coarse ridges and globular projections, X2,400.
FIG. 9. Day Zero, Rat. Convex areas on surface cells of rat bladder are prominent, Xl,500.
With SEM, almost all of the superficial cells of the epithelium displayed the characteristic star-like formation (fig. 8}. In the central portion of the formation there was a tightly constricted area made up of coarse ridges and globular projections (fig. 8 The globular projections were not as prominent on the of birth as they were in the late fetal period. Some of the immature types of cells that projected into the lumen were microridges. Others leafy dome-shaped and had microridges and globular projections. After examination of many specimens, it was found that the maturing types of cells that projected into the lumen were not seen as frequently on the day of birth as they were in the late fetal period. In the rat on the day of birth the urothelium was generally composed of 2 to 3 layers. Evidence of cytoplasmic degeneration was not noted in the superficial cells as it was in the late fetal period. The superficial cells as viewed by SEM were radically different from those of the mouse. The cells were roughly hexagonal in shape (fig. 9) and none of the star-like formations characteristic of the mouse bladder were seen. Most of the cells displayed 1 or 2 circular convex projectives on the cell surface.
Postnatal Day 1-3 The epithelium of the urinary bladder from mice 1 to 3 days of age was composed of 2 cell layers (fig. 10). The basal cells
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contained elongated nuclei which were predominantly euchromatin and often contained multiple nucleoli. The superficial cells were larger and covered several of the underlying cells. The lumenal membrane was thrown into a scalloped appearance in the central portion of the cell. Fusiform vesicles and bundles of tonofilaments commonly appeared over the nucleus in the elevated region. With SEM, the star-like formation on the mouse bladder continued to be present and 2 separate foci often appeared on the same cell (fig. 11). The majority of the cell surface was thrown into small folds as radiating arms of the star-like shape. Globular projections were rarely seen, but they were situated near the center of the cell when they occurred. Several types of maturing cells were seen. These immature cells commonly were dome-shaped, protruded from the surface of the bladder and were covered with small ridges. Occasionally, maturing cells were covered with a lacy network of ridges (fig. 12). They did not protrude from the surface. Small globular microvilli were seen on some of the maturing cells (fig. 12 inset left), and sometimes these microvilli appeared to fuse together to form the lacy ridges (fig. 12 inset right). By the 3rd postnatal day, some of the cells contained the adult form of microridges (fig. 13) characteristic of the asymmetric membrane of the fusiform vesicles. The epithelium from the rat bladder on postnatal days 1 to 3 was 2 to 3 cell layers thick. Cells of all cell layers contained large nuclei that were predominantly euchromatic. Some fusiform vesicles were noted in the superficial cells as they were in the mouse bladder. The SEM view was very similar to that on the day of birth with 1 or 2 small convex projections being seen on the superficial cells. The star-like formation, small microvilli and lacy ridges seen on the mouse were not seen on the rat bladder.
FIG. 13. Postnatal Day 3, Mouse. High magnification SEM view of superficial surface of bladder epithelium from 3-day-old control mouse. This magnification reveals characteristic microridges which make up asymmetric membrane of fusiform vesicles, X20,000.
Postnatal Day 5 By the 5th postnatal day the epithelium in the mouse was still 2 layers thick and the superficial cells contained fewer bundles of tonofilaments than they did in previous days. The star-like formation was still seen with SEM but it did not seem to be as tightly constricted as it was on the epithelium from earlier sampling times. Maturing cells displayed a lacy network of ridges and some displayed leafy microridges. No immature cells were seen that protruded from the surface of the bladder.
FIG. 14. Postnatal day 7, Rat. Urothelium of rat consists of only 2 cell layers. Fusiform vesicles are increased in number and cytoplasm of underlying cells is dark. Uranyl acetate and lead citrate stained thin section, X9,040.
The rat bladder on postnatal day 5 was essentially the same as it was on postnatal day 3.
Postnatal Day 7 One week after birth in both the mouse and rat the bladder consisted of 2 to 3 cell layers in which the epithelium was almost adult-like in appearance. The basal cells were predominantly oval or round in shape and the nuclei r,ontained prominent amounts of euchromatin. The superficial layer had the characteristic fusiform vesicles and angular membrane and was replete with cellular organelles. The nuclei in all the epithelial cell layers displayed multiple nucleoli with prominent fibrillar regions. The nuclei were predominantly euchromatin with sparse amounts of heterochromatin (fig. 14). Even though the bladder was multilayered and appeared much like the adult form, the bladder may have been immature in the sense that the nuclei had an immature chromatin pattern. FIG. 12. Postnatal Day 1, Mouse. SEM view of maturing bladder epithelium cells with lacy network of microridges from 1-day-old control mouse. Some small patches of short uniform microvilli are also seen, X2,140. Inset left. Higher magnification of area of small uniform microvilli bordering area of lacy microridges on superficial cell surface, X6,000. Inset right. Higher magnification of area with lacy microridges reveals that microridges may develop as fusion of small microvilli to form lacy network, X6,000.
Postnatal Day 10-14 Ten to 14 days after birth the bladder epithelium of both the mouse and the rat consisted of 2 to 3 cell layers with very prominent angular membranes and fusiform vesicles in the superficial layer. The nuclei were predominantly euchromatin
DEVELOPfl\JG ";JRL.\JARY BLADDZR
:V.lOUSE Ar\~D RAT
~,~,::,,•-,•;;•,c.:;;',v,,,, dev:3lopn1errt a11d maturation of the bladder in the mouse and rat be an extremely dynamic process. The most rapid changes occurred during the late fetal and early neonatal periods. Many of the features observed in the mouse appeared similar to those observed in the de-differentiated state of bladder carcinogenesis. No sexrelated morphological differences were observed. The short uniform microvilli observed in these normal mouse bladders in the present study appeared much like those observed by other investigators 5 on bladder cells from rats fed FANFT. Fusion of the small surface blebs led to the formation of ridges. Formation of ridges in this manner has also been noted by other authors on both normal and carcinogen treated rat bladders. 5- 7 The leafy microridges that appeared at approximately postnatal day 5 have been seen on hyperplastic bladders in rats fed FANFT. 5 Pleomorphic microvilli were not observed in this study. Less importance is placed on the discovery of pleomorphic microvilli now than has been in the recent past. 8 The absence of pleomorphic microvilli in the present study and in mice fed 2-AAF 9 may suggest that this form of morphological alteration occurs as a result of acute toxicity to the bladder and may not represent a de-differentiated state in the mouse. The bundles of tonofilaments that were observed in the superficial cells of the mouse bladder from the day of birth until the 5th postnatal day have been reported by other investigators on transitional cell tumors from rats fed 2-AAF. 10 These tonofilaments appear to play an important role in the formation of the star-like configuration. This star-like formation may be a phenomenon that is peculiar to the mouse since it has not been reported by other investigators 1' 2 and was not seen in the rat, The dark cells observed in the late fetal mouse bladder that were electron dense and contained many fusiform vesicles, prominent heterochromatin and multiple nucleoli, were replaced in the early postnatal period by cells that had normal cytoplasmic and nuclear density and a paucity of fusiform vesicles. In the rat, degenerative changes in the cytoplasm of the superficial cells were also noted. These superficial cells in the early postnatal period contained nuclei which were predominantly euchromatin and often contained multiple nucleoli. Numerous free ribosomes were present in the cytoplasm. These superficial cells of normal cytoplasmic and nuclear density, instead of dark cells noticed in the late fetal period in the mouse or cytoplasmic degradation in the rat, may reflect a physiological change in the neonatal bladder due to the
FIG. 15. Postnatal Day 28, Mouse. SEM of superficial surface of bladder epithelium from 28-day-old control mouse. Superficial cells are pentagonal or hexagonal in shape and all are roughly same size. Slight cobblestone appearance is noted but it is within normal limits, X900.
but contained patches of heterochromatin along with multiple nucleoli. The SEM revealed that the superficial cells were more regular in size and shape than on the previous and appeared almost like the adult with the mouse and rat bladders being almost
Postnatal
21-28
Three weeks after birth the epithelium in both the mouse and rat appeared as the adult pattern on TEM, with a slight irregularity in cell size and shape being detected with SEM. Four weeks after birth the epithelium had a mature pattern except for some areas of cobblestone appearance on SEM, which are considered to be within the normal limits (fig. 15). Characteristic adult microridges were present, and the cells were all 5 to 6-sided and similar in size and shape. By 4 weeks of age the epithelium was essentially mature in all respects. DISCUSSION
The present morphological of the developing mouse and rat urinary bladder epithelium began with the late fetal period. An added advantage over previous studies was that both transmission and "uuu""'" electron ,,,rncr•,m,,, as well as uc,cmmt the surface late fetal and neonatal mouse bladder was unusual and believed to be 8.11 artifact of prOC!c!S!'ilngo trials with different fixatives and fixation difference in surface ;,uvic,,;l'cct1 appearance a role in the characteristic was that the bladders from neonatal mice were markedly urine. The configuration of the surface cells could be a reaction to stress caused by stretching of the bladder muscular wall. The rat urinary bladder epithelium revealed a totally different morphological appearance during the early neonatal period, even though the same phenomenom of distension was noted. The view as seen by SEM was considered to represent the true biological status. This was in fact supported by TEM with the presence of bundles of tonofilaments in superficial cells which could have resulted in the contracted appearance on the surface of the cells. (Mouse pups which received an intraperitoneal injection of 0.1 mg. of cylophosphamide per gram body weight on the day of birth did not demonstrate the star-like formation when sacrificed on postnatal day l or 3).
511
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These rapid developmental changes, along with the mitotic activity noted by TEM, indicate the dynamic nature of the fetal and neonatal bladder epithelium of the mouse and rat. The active state of the developing bladder epithelium suggests that it may be a susceptible target organ to toxic insult.
Acknowledgements. We would like to thank Mr. Alan Warbritton for his photographic assistance and Ms. Annette Andrews and Ms. Yvonne White for electron microscopic assistance. REFERENCES
1. Firth, J. A. and Hicks, R. M.: Differentiation and cell death in transitional epithelium of the urinary bladder of fetal and suckling rats. J. Anat., 107: 192, 1970. 2. Firth, J. A. and Hicks, R. M.: Membrane specialization and synchronized cell death in developing rat transitional epithelium. J. Anat., 113: 95, 1972. 3. Walker, B. E.: Polyploidy and differentiation in the transitional epithelium of mouse urinary bladder. Chromosoma, 9: 105, 1958. 4. McDowell, E. M. and Trump, B. F.: Practical fixation techniques for light and electron microscopy. Comparative Path. Bulletin,
9: 3, 1977. 5. Jacobs, J. B., Arai, M., Cohen, S. M. and Friedell, G. H.: Early lesions in experimental bladder cancer: scanning electron microscopy of cell surface markers. Cancer Res., 36: 2512, 1976. 6. Hicks, R. M. and Chowaniec, J.: Experimental induction, histology, and ultrastructure of hyperplasia and neoplasia of the urinary bladder epithelium. Int. Rev. Exp. Pathol., 18: 199, 1978. 7. Hodges, G. M., Hicks, R. M. and Spacey, G.D.: Scanning electron microscopy of cell-surface changes in methylnitrosourea (MNU)treated rat bladders in vivo and in vitro. Differentiation, 6: 143, 1976. 8. Fukushima, S. and Cohen, S. M.: Saccharin-induces hyperplasia of the rat urinary bladder. Cancer Res., 40: 734, 1980. 9. Frith, C. H., Ayres, P. H. and Shinohara, Y.: A scanning and transmission electron microscopic study of urinary bladders of mice treated with 2-acetylaminofluorene. Invest. Urol., 19: 17, 1981. 10. Battifora, H. A., Eisenstein, R., Sky-Peck, H. H. and McDonald, J. H.: Electron microscopy and tritiated thymidine in gradation of malignancy of human bladder carcinoma. J. Urol., 93: 217, 1965. 11. Koss, L. G.: The asymmetric unit membranes of the epithelium of the urinary bladder of the rat. Lab. Invest., 21: 154, 1969.
THE JOURNAL OF UROLOGY
Printed in U.S.A.
Copyright © 1985 by The Williams & Wilkins Co.
MORPHOLOGICAL OBSERVATIONS ON THE EPITHELIUM OF THE DEVELOPING URINARY BLADDER OF THE MOUSE AND RAT PAUL H. AYRES,t YOSHITAKA SHINOHARA,+
AND
CHARLES H. FRITH*
From the Departments of Pathology and Interdisciplinary Toxicology and Pharmacology, University of Arkansas for Medical Sciences, and Intox Laboratories, Little Rock, Arkansas
ABSTRACT
Bladders from fetal and neonatal BALB/cStCrlfC3H/Nctr mice and Sprague-Dawley rats were studied to establish the sequence of events in their morphological development by using scanning electron, transmission electron and light microscopy. On fetal day 18 or 19 the epithelium from the mouse and the rat displayed 2 or 3 distinct cell layers. With transmission electron microscopy a star-like contraction of the cell surface of the mouse· bladder occurred which was not seen in the developing rat bladder. In both the mouse and the rat, some of the superficial cells sloughed between fetal day 18 or 19 and the day of birth. On the day of birth, the epithelium was composed of only 2 layers. The nuclei of both the superficial and hasal layers contained prominent euchromatin, and mitotic figures were often present in the basal layer. By the 5th postnatal day, some of the superficial cells contained autophagic vacuoles, and the epithelium was still 2 cell layers thick. One week after birth the epithelium consisted of 2 to 3 cell layers. Three weeks after birth the epithelium was 3 cell layers thick and appeared as the adult pattern with both transmission and scanning electron microscopy. The study demonstrated that the fetal and neonatal mouse and rat urinary bladders undergo a series of rapid developmental changes and suggest that the fetal and neonatal urinary bladder may be a target organ susceptible to toxic insult. Positive results in 2-generation carcinogenesis studies with saccharin have suggested an increased sensitivity of the immature rodent bladder epithelium to carcinogenic insult. Continued interest in the carcinogenicity of a compound of such economic significance has made investigators aware that little data is available in the current literature on the morphological development of the rodent urinary blaq.der. The literature is limited to light and electron microscopy and is not in complete agreement. 1- 3 Utilizing light microscopy, Walker3 observed that in 14 ½day-old mouse fetuses the bladder epithelium was multilayered, with all the cells and their nuclei being approximately the same size. The epithelium of 16 2/a-day-old fetuses displayed polyploid mitotic figures in squash preparations, and the average diameter of nuclei and cells showed a pronounced increase with binucleate cells occurring with a high frequency over a considerable portion of the epithelium. W alker3 reported that no other significant changes occurred throughout the remainder of fetal development: Shortly after birth, however, a 2nd increase in the average diameter of nuclei and cells occurred, which brought the surface cell to its final stage of differentiation regarding nuclear and cell size. In studies utilizing light and electron microscopy1• 2 it was reported that until the 15th day of gestation in the rat the epithelium of the urinary bladder was lined by a single layer of morphologically undifferentiated cuboidal cells, many of which were in mitosis. Between the 15th and 18th day of gestation, the epithelium developed into 2 cell layers with many binucleate superficial cells that showed some differentiation. Between the 19th day of gestation and the 2nd postnatal day, some of the superficial cells sloughed into the lumen. Other cells sloughed between the 5th and 10th day after birth causing the epithelium
to be 1 cell layer thick. The basal cell layer proliferated to form a new superficial layer by the middle of the 2nd week after birth. A 3rd cell layer was established in the 3rd week to form a mature type of epithelium. To investigate the potential of the urinary bladder as a target organ for transplacental carcinogenesis, the present study was undertaken to determine if the bladder epithelium was in a state of rapid change and proliferation or whether it was simply a qµiescent tissue during the late fetal period. Scanning electron microscopy has been widely used in studies of the adult urinary bladder and should prove useful in studies of the fetal and neonatal bladder. The present study utilized both scanning and transmission electron microscopy, as well as light microscopy, to study the development of the uripary bladder epithelium to establish a basis for morphological evaluation of control and carcinogen-treated fetal and neonatal hlaq.ders and to provide a comparison between the mouse and the rat.
The Mouse Female BALB/cStCrlfC3H/Nctr mice 6 to 8 weeks of age were mated overnight with BALBcStCrlfC3H/Nctr males. The morning a vaginal plug was found was designated day zero of gestation. Maternal animals were maintained on lab chow throughout the experiment in conventional animal rooms at 50 ± 10 per cent humidity and 22 ± 2C. Three litters were used at each time point with at least 2 offspring from each litter examined for morphological features. Urinary blaq.ders were studied at gestational days 18, 19 and 20 and postnatal days zero (day of birth), 1, 3, 5, 7, 10, 14, 21 and 28. Both male and female offspring were studied.
Accepted for publication November 6, 1984. * Requests for reprints: Intox Laboratories, Inc., # 1 Intox Drive, P.O. Box 250, Redfield, Arkansas 72132. t Current address: Lovelace Inhalation Toxicology Research Institute, Albuquerque, New Mexico. t Current address: Department of Pathology, Nagoya City University Medical School, Nagoya, Japan.
The Rat Twelve to 14-week-old virgin female Nctr:S23(SD)BR Sprague-Dawley rats were mated overnight with males of the same strain. If a vaginal plug was fognd the next morning, the female was designated pregnant at day z!)ro of gestation. Maternal animals were fed lab chow ad libitum throughout the
MATERIALS AND METHODS
506
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experiment and maintained in conventional animal rooms at 50 ± 10 per cent humidity, 22 ± 2C and a 12 hour light-dark cycle. Offspring were studied at gestational days 18, 19, 20 and 21 and postnatal day zero (day of birth), 1, 3, 5, 7, 11, 14 and 21. Three litters were used at each time point, and at least 2 offspring from each litter were examined for morphological features. Male and female offspring were studied.
Mice and Rats The pregnant mice or rats were anesthetized with carbon dioxide gas and the fetuses were removed by caesarean section. The fetuses were anesthetized with carbon dioxide and decapitated. A midline incision was made on the ventral surface of the fetus to expose the urinary bladder. A linen string was looped around the neck of the bladder and a loose knot was tied around the urethra. The bladder was removed by a gentle tug on the string and a 31 gauge needle was passed into the urethra through the knot. The bladder was inflated with fixative and then the knot was tightened. Bladders that were normally distended and did not require inflation were fixed in situ after the urethra was ligated. Brief initial fixation in situ or distension with fixative provided adequate presentation of morphological features with no difference seen between the 2 fixative methods. Initially, either 2 per cent or 4 per cent glutaraldehyde or Trump's fixative 4 in a cacodylate buffer was used for fixation. Trump's fixative was used for the duration of the experiment after no readily apparent differences between fixatives were noticed. The bladder was then removed and placed in fresh fixative for 15 minutes. The bladder was then divided sagitally and one half was processed for transmission electron microscopy. The other half was processed for scanning electron microscopy. Tissues for transmission electron microscopy (TEM) were rinsed in 0.2 M cacodylate buffer (pH 7.2 to 7.4) at room temperature 3 times for 10 minutes each. They were then postfixed in 1 per cent osmium tetroxide in 0.1 M cacodylate buffer (pH 7.2 to 7.4) at room temperature for 1 hour and rinsed in 0.2 M cacodylate buffer 3 times for 10 minutes each. The tissues were routinely dehydrated in ethanol, cleared in acetone on an automatic tissue processor, and then infiltrated and embedded in an Epon/ Araldite mixture. Sections were cut with glass or diamond knives on LKB Ultrotome III ultramicrotomes and examined with a Philips EM201 electron microscope at 60 kv. Tissues for scanning electron microscopy (SEM) were rinsed in 0.2 M cacodylate buffer (pH 7.2 to 7.4) at room temperature for 10 minutes 3 times and postfixed for 15 minutes at room temperature in 1 per cent osmium tetroxide buffered with 0.2 M cacodylate buffer (pH 7.2 to 7.4). The tissues were dehydrated through graded strengths of ethanol to absolute ethanol. The tissues were then critical point dried from liquid CO 2 • The tissues were mounted on specimen holders with conductive silver paint and coated in a sputter coater with approximately 200A of gold/palladium. The tissues were examined with a JEOL JSM-35 scanning electron microscope at 25 kv at 15 mm. working distance. RESULTS
Fetal Day 18 or 19 The epithelium of the urinary bladder of the 18 or 19-day fetus of both the mouse and the rat displayed 2 or 3 distinct cell layers. In the mouse dark cells were sometimes seen in the superficial layer. These dark cells had an angular profile bordering the lumen and contained many fusiform vesicles (fig. 1). The elongated nucleus with several distinct nucleoli and prominent heterochromatin was electron dense, as was the adjacent cytoplasm. Bundles of tonofilaments were present in most of the dark superficial cells (fig. 1 inset). Other superficial cells were found that were of normal cytoplasmic and nuclear density
FIG. 1. Fetal Day 18, Mouse. Transmission electron micrograph of bladder epithelium from untreated 18-day mouse fetus. Superficial cell is dark and contains numerous fusiform vesicles. Uranyl acetate and lead citrate stained thin section, X6,608. Inset. High magnification of bundles of tonofilaments in superficial cell cytoplasm. Uranyl acetate and lead citrate stained thin section, X34,320.
FIG. 2. Fetal Day 18, Rat. Epithelium of rat consists of 3 or 4 cell layers. Superficial cells appear to be degenerating and contain semicircular convex areas. Nuclei of basal and intermediate cells are oval in shape and predominantly euchromatic with single nucleolus with prominent fibrillar component. Uranyl acetate and lead citrate stained thin section, x5,048.
and contained fevv fusiform vesicles. The nuclei of these cells were composed o:,u,uuuuau.c,y of euchromatin and often contained multiple nucleoli. The cytoplasm of basal cells was of normal density and contained amounts of free ribosomes. The nuclei of basal cells were oval in shape and contained multiple nucleoli. In the rat, the epithelium was multilayered as in the mouse but revealed some morphological features which were dissimilar. Primarily, the epithelial superficial cells showed a loss of cytoplasmic organization with a lack of fusiform vesicles (fig. 2) possibly due to cytoplasmic degeneration. The lumenal surface of the urinary bladder from the 18 or 19-day mouse fetus displayed features that were not seen in normal adult animals. In the fetal bladder epithelium, the predominant cell had coarse ridges and globular projections primarily in the central portion of the cell. These globular projections and ridges seemed to coalesce in the central portion of the cell to form a star-like contraction of the cell surface. Maturing cells were occasionally seen on the lumenal surface of the bladder (fig. 3). Small patches of cell surfaces that
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contained very short uniform microvilli were seen. Other maturing cells often contained microridges with a foliate appearance (fig. 3 inset). Fetal Day 20
FIG. 3. Fetal Day 19, Mouse. SEM of maturing bladder cell protruding into lumen of bladder from control 19-day mouse fetus, Xl,630. Inset. Maturing bladder cell with foliate microridges that does not protrude into lumen, from control 19-day fetus, Xl,278.
FIG. 4. Fetal Day 20, Mouse. TEM of bladder epithelium from control 20-day mouse fetus. Superficial cell layer covered several underlying cells and had bulbous projections on superficial border. Uranyl acetate and lead citrate stained thin section, X4,554.
FIG. 5. Fetal Day 20, Rat. TEM demonstrates widening of intercellular gaps. Superficial cell of rat bladder shows evidence of degeneration. Intermediate and basal layer cells contain large amounts of free ribosomes. Uranyl acetate and lead citrate stained thin section, X7,323.
On the 20th day of gestation the bladder epithelium of both the mouse and the rat consisted of 2 to 3 cell layers. In the mouse the superficial cells covered several of the underlying cells and were not dark, unlike the epithelium on day 18 or 19. The superficial cells contained many free ribosomes and few fusiform vesicles. The lumenal border was thrown into bulbous projections which may be the coarse ridges that were seen with SEM (fig. 4). The SEM of the lumenal surface of the bladder revealed that maturing cells were still present and that they projected into the lumen. The star-like formation was readily observed on most of the other cells on the surface. The bulbous projections on the surface of the cells seem to coalesce to form the coarse ridges. Other maturing intermediate cells were present that were flat and contained the foliate type of microridge and bulbous projections. On gestational day 20 in the rat the superficial epithelial cells still showed signs of cytoplasmic degeneration without the
FIG. 6. Fetal Day 20, Rat. Surface cells from rat bladder show irregularity in size and shape and are slightly convex. Smooth, fine microridges and sparse small microvilli-like projections are present. Cell borders are thick and seem to protrude from surface, X2,900.
FIG. 7. Postnatal Day Zero (Day of Birth), Mouse. TEM of bladder epithelium from mouse on day of birth. Epithelium is 2 cell layers thick with angular profile on superficial border. Bundles of tonofilaments and fusiform vesicles are present overlying nucleus. Nuclei of basal and superficial layers contained prominent euchromatin. Uranyl acetate and lead citrate stained thin section, X6,608. Inset. Mitotic figure in basal layer.
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evidence bulbous projections or coarse that was seen in the mouse bladder (fig. 5). In addition, the epithelium demonstrated a widening of the intercellular junctions. The SEM of the lumenal surface of the rat bladder was remarkably different from that of the mouse. The superficial cells varied in size and shape and displayed small microvilli and fine ridges on the cell surface. Cell junctions were very distinct (fig. 6). No maturing cells were seen projecting into the lumen, unlike the mouse.
0 (day of birth) The epithelium of the urinary bladder of the mouse on the day of birth appeared to be composed of only 2 cell layers (fig. 7). The superficial cells were large and covered several of the underlying cells. The superficial cell surface had an angular profile and sparse amounts of fusiform vesicles appeared in the central portion of the cell to produce the elevated area seen as the star-like formation on SEM (fig. 8). Tonofilaments were abundant within the cytoplasm and usually appeared over the nucleus in the elevated area. The nuclei of both the superficial and basal layers contained prominent euchromatin and often contained multiple nucleoli. The cytoplasm contained many free ribosomes, and mitotic figures were often present in the basal cell layer (fig. 7 inset), as they also were in the epithelium from the fetal period.
FIG. 10. Postnatal Day 3, Mouse. TEM of bladder epithelium from 3-day-old control mouse. Epithelium contains 2 cell layers and superficial cell covers several underlying cells. Superficial layer had angular profile with fusiform vesicles present in cytoplasm. Uranyl acetate and lead citrate stained thin section, X7,000.
FIG. 11. Postnatal Day 1, Mouse. SEM view of bladder epithelium for 1-day-old control mouse. Superficial cells often contain 2 separate foci of star-like formation on same cell, >cl,700. FIG. 8. Postnatal Day Zero (Day of Birth), Mouse. SEM of lumenal surface of mouse bladder on day of birth. Almost all superficial cells have star-like constriction on cell surface, x750. Inset. High magnification of constricted area which is composed of coarse ridges and globular projections, X2,400.
FIG. 9. Day Zero, Rat. Convex areas on surface cells of rat bladder are prominent, Xl,500.
With SEM, almost all of the superficial cells of the epithelium displayed the characteristic star-like formation (fig. 8}. In the central portion of the formation there was a tightly constricted area made up of coarse ridges and globular projections (fig. 8 The globular projections were not as prominent on the of birth as they were in the late fetal period. Some of the immature types of cells that projected into the lumen were microridges. Others leafy dome-shaped and had microridges and globular projections. After examination of many specimens, it was found that the maturing types of cells that projected into the lumen were not seen as frequently on the day of birth as they were in the late fetal period. In the rat on the day of birth the urothelium was generally composed of 2 to 3 layers. Evidence of cytoplasmic degeneration was not noted in the superficial cells as it was in the late fetal period. The superficial cells as viewed by SEM were radically different from those of the mouse. The cells were roughly hexagonal in shape (fig. 9) and none of the star-like formations characteristic of the mouse bladder were seen. Most of the cells displayed 1 or 2 circular convex projectives on the cell surface.
Postnatal Day 1-3 The epithelium of the urinary bladder from mice 1 to 3 days of age was composed of 2 cell layers (fig. 10). The basal cells
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contained elongated nuclei which were predominantly euchromatin and often contained multiple nucleoli. The superficial cells were larger and covered several of the underlying cells. The lumenal membrane was thrown into a scalloped appearance in the central portion of the cell. Fusiform vesicles and bundles of tonofilaments commonly appeared over the nucleus in the elevated region. With SEM, the star-like formation on the mouse bladder continued to be present and 2 separate foci often appeared on the same cell (fig. 11). The majority of the cell surface was thrown into small folds as radiating arms of the star-like shape. Globular projections were rarely seen, but they were situated near the center of the cell when they occurred. Several types of maturing cells were seen. These immature cells commonly were dome-shaped, protruded from the surface of the bladder and were covered with small ridges. Occasionally, maturing cells were covered with a lacy network of ridges (fig. 12). They did not protrude from the surface. Small globular microvilli were seen on some of the maturing cells (fig. 12 inset left), and sometimes these microvilli appeared to fuse together to form the lacy ridges (fig. 12 inset right). By the 3rd postnatal day, some of the cells contained the adult form of microridges (fig. 13) characteristic of the asymmetric membrane of the fusiform vesicles. The epithelium from the rat bladder on postnatal days 1 to 3 was 2 to 3 cell layers thick. Cells of all cell layers contained large nuclei that were predominantly euchromatic. Some fusiform vesicles were noted in the superficial cells as they were in the mouse bladder. The SEM view was very similar to that on the day of birth with 1 or 2 small convex projections being seen on the superficial cells. The star-like formation, small microvilli and lacy ridges seen on the mouse were not seen on the rat bladder.
FIG. 13. Postnatal Day 3, Mouse. High magnification SEM view of superficial surface of bladder epithelium from 3-day-old control mouse. This magnification reveals characteristic microridges which make up asymmetric membrane of fusiform vesicles, X20,000.
Postnatal Day 5 By the 5th postnatal day the epithelium in the mouse was still 2 layers thick and the superficial cells contained fewer bundles of tonofilaments than they did in previous days. The star-like formation was still seen with SEM but it did not seem to be as tightly constricted as it was on the epithelium from earlier sampling times. Maturing cells displayed a lacy network of ridges and some displayed leafy microridges. No immature cells were seen that protruded from the surface of the bladder.
FIG. 14. Postnatal day 7, Rat. Urothelium of rat consists of only 2 cell layers. Fusiform vesicles are increased in number and cytoplasm of underlying cells is dark. Uranyl acetate and lead citrate stained thin section, X9,040.
The rat bladder on postnatal day 5 was essentially the same as it was on postnatal day 3.
Postnatal Day 7 One week after birth in both the mouse and rat the bladder consisted of 2 to 3 cell layers in which the epithelium was almost adult-like in appearance. The basal cells were predominantly oval or round in shape and the nuclei r,ontained prominent amounts of euchromatin. The superficial layer had the characteristic fusiform vesicles and angular membrane and was replete with cellular organelles. The nuclei in all the epithelial cell layers displayed multiple nucleoli with prominent fibrillar regions. The nuclei were predominantly euchromatin with sparse amounts of heterochromatin (fig. 14). Even though the bladder was multilayered and appeared much like the adult form, the bladder may have been immature in the sense that the nuclei had an immature chromatin pattern. FIG. 12. Postnatal Day 1, Mouse. SEM view of maturing bladder epithelium cells with lacy network of microridges from 1-day-old control mouse. Some small patches of short uniform microvilli are also seen, X2,140. Inset left. Higher magnification of area of small uniform microvilli bordering area of lacy microridges on superficial cell surface, X6,000. Inset right. Higher magnification of area with lacy microridges reveals that microridges may develop as fusion of small microvilli to form lacy network, X6,000.
Postnatal Day 10-14 Ten to 14 days after birth the bladder epithelium of both the mouse and the rat consisted of 2 to 3 cell layers with very prominent angular membranes and fusiform vesicles in the superficial layer. The nuclei were predominantly euchromatin
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~,~,::,,•-,•;;•,c.:;;',v,,,, dev:3lopn1errt a11d maturation of the bladder in the mouse and rat be an extremely dynamic process. The most rapid changes occurred during the late fetal and early neonatal periods. Many of the features observed in the mouse appeared similar to those observed in the de-differentiated state of bladder carcinogenesis. No sexrelated morphological differences were observed. The short uniform microvilli observed in these normal mouse bladders in the present study appeared much like those observed by other investigators 5 on bladder cells from rats fed FANFT. Fusion of the small surface blebs led to the formation of ridges. Formation of ridges in this manner has also been noted by other authors on both normal and carcinogen treated rat bladders. 5- 7 The leafy microridges that appeared at approximately postnatal day 5 have been seen on hyperplastic bladders in rats fed FANFT. 5 Pleomorphic microvilli were not observed in this study. Less importance is placed on the discovery of pleomorphic microvilli now than has been in the recent past. 8 The absence of pleomorphic microvilli in the present study and in mice fed 2-AAF 9 may suggest that this form of morphological alteration occurs as a result of acute toxicity to the bladder and may not represent a de-differentiated state in the mouse. The bundles of tonofilaments that were observed in the superficial cells of the mouse bladder from the day of birth until the 5th postnatal day have been reported by other investigators on transitional cell tumors from rats fed 2-AAF. 10 These tonofilaments appear to play an important role in the formation of the star-like configuration. This star-like formation may be a phenomenon that is peculiar to the mouse since it has not been reported by other investigators 1' 2 and was not seen in the rat, The dark cells observed in the late fetal mouse bladder that were electron dense and contained many fusiform vesicles, prominent heterochromatin and multiple nucleoli, were replaced in the early postnatal period by cells that had normal cytoplasmic and nuclear density and a paucity of fusiform vesicles. In the rat, degenerative changes in the cytoplasm of the superficial cells were also noted. These superficial cells in the early postnatal period contained nuclei which were predominantly euchromatin and often contained multiple nucleoli. Numerous free ribosomes were present in the cytoplasm. These superficial cells of normal cytoplasmic and nuclear density, instead of dark cells noticed in the late fetal period in the mouse or cytoplasmic degradation in the rat, may reflect a physiological change in the neonatal bladder due to the
FIG. 15. Postnatal Day 28, Mouse. SEM of superficial surface of bladder epithelium from 28-day-old control mouse. Superficial cells are pentagonal or hexagonal in shape and all are roughly same size. Slight cobblestone appearance is noted but it is within normal limits, X900.
but contained patches of heterochromatin along with multiple nucleoli. The SEM revealed that the superficial cells were more regular in size and shape than on the previous and appeared almost like the adult with the mouse and rat bladders being almost
Postnatal
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Three weeks after birth the epithelium in both the mouse and rat appeared as the adult pattern on TEM, with a slight irregularity in cell size and shape being detected with SEM. Four weeks after birth the epithelium had a mature pattern except for some areas of cobblestone appearance on SEM, which are considered to be within the normal limits (fig. 15). Characteristic adult microridges were present, and the cells were all 5 to 6-sided and similar in size and shape. By 4 weeks of age the epithelium was essentially mature in all respects. DISCUSSION
The present morphological of the developing mouse and rat urinary bladder epithelium began with the late fetal period. An added advantage over previous studies was that both transmission and "uuu""'" electron ,,,rncr•,m,,, as well as uc,cmmt the surface late fetal and neonatal mouse bladder was unusual and believed to be 8.11 artifact of prOC!c!S!'ilngo trials with different fixatives and fixation difference in surface ;,uvic,,;l'cct1 appearance a role in the characteristic was that the bladders from neonatal mice were markedly urine. The configuration of the surface cells could be a reaction to stress caused by stretching of the bladder muscular wall. The rat urinary bladder epithelium revealed a totally different morphological appearance during the early neonatal period, even though the same phenomenom of distension was noted. The view as seen by SEM was considered to represent the true biological status. This was in fact supported by TEM with the presence of bundles of tonofilaments in superficial cells which could have resulted in the contracted appearance on the surface of the cells. (Mouse pups which received an intraperitoneal injection of 0.1 mg. of cylophosphamide per gram body weight on the day of birth did not demonstrate the star-like formation when sacrificed on postnatal day l or 3).
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These rapid developmental changes, along with the mitotic activity noted by TEM, indicate the dynamic nature of the fetal and neonatal bladder epithelium of the mouse and rat. The active state of the developing bladder epithelium suggests that it may be a susceptible target organ to toxic insult.
Acknowledgements. We would like to thank Mr. Alan Warbritton for his photographic assistance and Ms. Annette Andrews and Ms. Yvonne White for electron microscopic assistance. REFERENCES
1. Firth, J. A. and Hicks, R. M.: Differentiation and cell death in transitional epithelium of the urinary bladder of fetal and suckling rats. J. Anat., 107: 192, 1970. 2. Firth, J. A. and Hicks, R. M.: Membrane specialization and synchronized cell death in developing rat transitional epithelium. J. Anat., 113: 95, 1972. 3. Walker, B. E.: Polyploidy and differentiation in the transitional epithelium of mouse urinary bladder. Chromosoma, 9: 105, 1958. 4. McDowell, E. M. and Trump, B. F.: Practical fixation techniques for light and electron microscopy. Comparative Path. Bulletin,
9: 3, 1977. 5. Jacobs, J. B., Arai, M., Cohen, S. M. and Friedell, G. H.: Early lesions in experimental bladder cancer: scanning electron microscopy of cell surface markers. Cancer Res., 36: 2512, 1976. 6. Hicks, R. M. and Chowaniec, J.: Experimental induction, histology, and ultrastructure of hyperplasia and neoplasia of the urinary bladder epithelium. Int. Rev. Exp. Pathol., 18: 199, 1978. 7. Hodges, G. M., Hicks, R. M. and Spacey, G.D.: Scanning electron microscopy of cell-surface changes in methylnitrosourea (MNU)treated rat bladders in vivo and in vitro. Differentiation, 6: 143, 1976. 8. Fukushima, S. and Cohen, S. M.: Saccharin-induces hyperplasia of the rat urinary bladder. Cancer Res., 40: 734, 1980. 9. Frith, C. H., Ayres, P. H. and Shinohara, Y.: A scanning and transmission electron microscopic study of urinary bladders of mice treated with 2-acetylaminofluorene. Invest. Urol., 19: 17, 1981. 10. Battifora, H. A., Eisenstein, R., Sky-Peck, H. H. and McDonald, J. H.: Electron microscopy and tritiated thymidine in gradation of malignancy of human bladder carcinoma. J. Urol., 93: 217, 1965. 11. Koss, L. G.: The asymmetric unit membranes of the epithelium of the urinary bladder of the rat. Lab. Invest., 21: 154, 1969.