Surface Differences in Cells of Proximal and Distal Canine Urethra

Vol. 111. .Printed in

THE JOlJRNAL OF UROLOGY

Copyright© 1974 by The Williams & Wilkins Co.

SURFACE DIFFERENCES IN CELLS OF PROXIMAL AND DISTAL CANINE URETHRA JAMES K. MOONEY*

AND

FRANK HINMANt

From the Division of Urologv. University of California School of Medicine, San Francisco, California

exist between the and distal that might explain the differences in colonizabon

The distal urethra is normally colonized with non-pathogenic bacteria but the proximal seg-

note microplicae arranged across individual cells. show clearly unstretched cells. SEM, reduced from x

ments are sterile. Based on this premise our study was designed to determine if structural differences

The female canine urethra was selected for 0tudy because previous experiments in this

for publication 31, 197cl. at annual meeting of n.111e11ca,11 Urological Asso13-17, 1973. ciation, New York, New York, Supported by National of Health Grants. AM10045 and TIAM 5451. * Current address: Squier Urological Clinic, Columbia-

Presbyterian Medical Center, New York, New 10032. t Requests for reprints: Division of University of California School of Medicine, cisco, California 94122.

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fixed in osmium tetroxide for 1 hour and then dehydrated with graded ethanol. The specimens went into 2, 15-minute changes of propylene oxide, followed by an overnight stay in 50: 50 propylene oxide and catalyzed mixture of epon resin. The next morning they were allowed to stand in straight epon resin at room temperature for at least an hour, then embedded in fresh epon resin and left at 60C for 48 hours until polymerization was complete. They were then sectioned and mounted. OBSERVATIONS

FIG. 2. Bladder-microplicae are similar to those of proximal urethra. SEM, reduced from x4,200.

had established information on urethral colonization.' MATERIALS AND METHODS

The urethra, bladder and vagina of female dogs weighing 10 to 25 kg. were excised under anesthesia. Transverse sections from the proximal and distal urethra and from the bladder were obtained. Specimens for light microscopy were placed in a 10 per cent solution of formalin for 48 hours, dehydrated through graded ethanol, embedded in paraffin and sectioned. After being mounted on glass slides the sections were washed with xylene and stained with hematoxylin and eosin. Specimens intended for scanning and transmission electron microscopic study were immersed in a 3 per cent solution of gluteraldehyde with 0.1 M sodium cacodylate buffer at pH 7.4. The solutions were kept at a temperature of 4C. For study by scanning electron microscopy the specimens were left in the gluteraldehyde for 24 hours, in a 16 per cent solution of glycerin for 24 hours and in a 20 per cent solution of ethanol for 24 hours-always at 4C. They were dehydrated in graded ethanol, which was replaced gradually with amyl acetate, and finally submerged in freon 22, cooled in liquid nitrogen and freeze-dried. They were mounted on aluminum stubs with silver paint and coated with gold. For examination by transmission electron microscopy the specimens were immediately minced in the gluteraldehyde solution at 4C and were allowed to remain there for 2 hours. They were rinsed in 3 changes of 0.1 M sodium cacodylate buffer (each rinse of 5-minute duration) and post1 Masih, B. K. and Hinman, F., Jr.: Voiding and intrinsic defenses of the lower urinary tract in the female dog. II. Effect of immunosuppressive drugs on canine urethral and vesical flora. Invest. Urol., 8: 494, 1971.

Proximal urethra. Macroscopically, the mucosa of the collapsed proximal urethra is arranged in longitudinal folds. This is in contrast to the bladder surface on which the folds are randomly arranged but is consistent with its predominantly radial contractility. Sections examined by light microscopy show the well known stellate configuration of the longitudinal folds viewed on end. The little difference in their size is apparent between segments of the urethra. The typical 3 layers of transitional epithelium cover the proximal two-thirds. Viewed by scanning electron microscopy (SEM) the proximal urethral surface (fig. 1) is similar to that of the bladder (fig. 2). The luminal membrane of each cell is arranged in many folds or microplicae, oriented roughly parallel to each other. The more dilated the urethra the less apparent are the folds in the cell membrane and the larger is the exposed surface-just as can be observed for the bladder.2 The mid urethra is little different from the proximal section. Transmission electron microscopy (TEM) shows the luminal membrane of the urethra (fig. 3) to be structured like that of the bladder (fig. 4). It is made up of corrugations which look like villi on section but are revealed by scan to be plicae. The limiting membrane is asymmetric, with a thicker outer layer, and has a thickness of 120 angstrom units. These features are similar to those of bladder cells. Distal urethra. On macroscopic inspection this part of the urethra presents longitudinal folds like those of the more proximal segments but with a somewhat coarser appearance. Under light microscopy the transitional epithelium of the upper segments gives way to a more squamous type in the distal urethra. Two to 4 cell layers are characteristic here, in contrast to the consistent 3 layers of the typical transitional epithelium. Upon examination by ~canning the cells composing the distal urethral surface possess finger-like projections, microvilli (fig. 5). These are in contrast to the microplicae of the proximal urethra. The microvilli are randomly arranged and of various lengths. In the dilated urethra they are shorter and (as with the microplicae of the proximal 2 Mooney, J. K. and Hinman, F.: Mucosa! expansion and contraction. In preparation.

SURFACE DIFFERENCES IN CELLS OF PROXIMAL AND DISTAL CANINE URETHRA

FIG.

3. Proximal urethra-microplicae look like villi on cut section. Note asymmetric limiting unit

TEM. reduced from x30,600.

FIG. 4. Bladder-features are similar to those of proximal urethra. TEM, reduced from x :38.000

urethra) their surfaces of the cell surface. taken up and the does not increase as Transmission electron the distal urethra has a more unit which is units thick (fig. 6). The surface has appearance. The defined as the of the since the urethra is essen in the 7), and the 8) possess cells similar to those of the distal urethra. Microvilii are vu,rn,v,,," and some-

what more distributed than in th,:, ,-,,.,,.,,.._,.,,,-,; cells are found. VV0WJ,V'"""'"'J

DISCUSSION

Bacterial colonization is distal urethra 3 but rises in virulence and tion in worn.en with recurrent 3 Cox, C. E.: The urethra and its rel.ationEJ1lp urinarv tract infection: the flora of the normal fen,ak urethra. Southern Med. 59: 1966. 4 Cox, C. E., and F urethra and its to The urethral !lorn of the infection. J. Urol., 99: 63:2, 1968.

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Fie. 5. Distal urethra-microvilli are prominent over cell surfaces. SEM, reduced from x 25,000.

mechanically washing out bacteria appears to be important 5 • 6 and spontaneous contractions may further clear the urethra. 7 However, intrinsic defenses undoubtedly play a role. 8 They probably consist of several different reactions (from secretions, antigens or phagocytosis). The observation that the cells of the distal urethra have a limiting membrane with characteristics similar to those of the introitus and vagina (such as greater symmetry, lesser thickness and microvilli rather than the microplicae of the proximal segments) is not unexpected since their common embryologic origin and response to hormones are well documented. 9 Similarly, the epithelial surface of the proximal urethra would be expected to resemble that of the bladder. The fact that the 2 types of epithelia play different roles in bacterial defense is suggested by the previous studies of normal and pathologic colonization previously mentioned. The proximal urethra, like the filling bladder but to a lesser degree, is subjected to considerable stretching during micturition. Its surface must adapt to this stretching by losing first its gross folds and then the microscopic folds on the cell surfaces. The distal urethra, structurally comparable to the vagina, is more restricted in its expan5 Schmaelzle, J. F., Cass, A. S. and Hinman, F., Jr.: Effect of disuse and restoration of function on vesical capacity. J. Urol., 101: 700, 1969. 'Hinman, F., Jr.: Dysfunction of the lower segment of the urinary bladder in female children with recurrent infection. Brit. J. Urol., 37: 39, 1965. 7 Mayo, M. E. and Hinman, F., Jr.: Spontaneous myogenic activity and viscoelastic properties of the urethra. Surg. Forum, 23: 524, 1972. 8 Cox, C. E. and Hinman, F., Jr.: Experiments with induced bacteriuria, vesical emptying and bacterial growth on the mechanism of bladder defense to infection. J. Urol., 86: 739, 1961. 9 Cifuentes, D. L.: Cistitis y cistopatias. Madrid: Paz Montalvo, 1947.

FIG. 6. Distal urethra-villi are seen with more symmetric unit membrane. Intercellular juncture with zona occludens is included. TEM, reduced from x48,200.

sion during micturition-a limitation imposed by its greater encasement in collagen and striated muscle 10 and by its hydraulic function 11 and reflected in its configuration, the folds being replaced by villi which contribute much less area to the limiting membrane as the cell is stretched. SUMMARY

The proximal and distal segments of the female urethra differ in bacterial colonization and in configuration during voiding. To explain these differences a detailed study of the distribution and configuration of the urethral luminal cells and, more specifically, of their surface membrane characteristics, was done in dogs with the scanning electron microscope. In the proximal urethra the luminal surface of the superficial cell is limited by a specialized asymmetric unit membrane, 120 angstrom units thick, that is organized in a pattern of fine folds (microplicae) similar to that of the vesical urothelium. On expansion these folds are taken up by the cell surface. In the distal urethra the luminal surface is clearly different, thinner and showing no microplicae but finger-shaped processes instead (micro10 Lyon, R. P. and Tanagho, E. A.: Distal urethral stenosis in little girls. J. Urol., 93: 379, 1965. 11 Gleason, D. M. and Bottaccini, M. R.: The vital role of the distal urethral segment in the control of urinary flow rate. J. Urol., 100: 167, 1968.

SURFACE DIFFERENCES IN CELLS OF PROXIMAL AND DISTAL CANINE URETHRA

Fm. 7. Introitus-villi are more prominent than in distal urethra. Note secretory cells. A, SEl\/i, reduced x2,620. B, SEM, reduced from x9,500.

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FIG. 8. Vagina. A, SEM, reduced from x2,620. B, SEM, reduced from x 19,000

SURFACE DIFFERENCES IN CELLS OF PROXIMAL AND DISTAL CANINE URETHRA

villi). The microvilli are more stable and contribute less to the cell surface during expansion. The distal urethral surface resembles that of the vagina and would be expected to possess similar characteristics for bacterial colonization, while the

proximal urethra is like the bladder which normally sterile. The conclusion is that ultrastructural features are consistent with functional differences in t]ie lower urinary tract.