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Ductal Budding and Branching Patterns in the Developing Prostate
0022-5347/94/1515-1427$03.00/0 VoL 151, 1427-1432, Printed in
THE JOURNAL OF UROLOGY
Copyright © 1994 by AMERICAN UROLOGICn ASSOCIATION, INC.
DUCTAL BUDDING AND BRANCHING PATTERNS IN THE DEVELOPING PROSTATE BARRY G, TIMMS*, TOM J. MOHS
AND
LIBERATO J. A. DIDIO
From the Department of Anatomy and Structural Biology, School of Medicine, University of South Dakota, Vermillion, South Dakota and the Medical College of Ohio, Toledo, Ohio
ABSTRACT
Development of the prostate was studied by serial section reconstruction and computer-assisted three-dimensional analysis. A comparison of ductal budding in species of rat and mouse and the human revealed patterns consistent with common developmental characteristics. Ventral, lateral and dorsal lines of epithelial buds, which emanated from the urogenital sinus into the surrounding periurethral mesenchyme, followed ventro-dorsal and cranio-caudal axes. Subsequent branching morphogenesis was associated with specific mesenchymal condensations. These patterns of budding were closely related to the adult lobe architecture in the rodent prostate. In the human fetus, prostate ductal budding exhibited patterns compatible with the current concept of zonal anatomy. KEY
WORDS:
prostate; fetal development; image processing, computer-assisted
Over the past 80 years, there have been several classical investigations of prostate morphogenesis in a variety of species. 1 Notable among these are the descriptions of the human and rat prostate by Lowsley 2 and Price. 3 The rat prostate, more than that of any other species, has been subjected to the most thorough light and electron microscopical analysis of embryonic, postnatal and adult stages of growth. 4 - 6 However, owing to the three-dimensional complexity of the developing duct system, these studies provided only a descriptive outline of morphogenesis and adult architecture. Recent investigations on mice, using a whole-mount microdissection technique, have enabled the precise delineation of all the ductal elements to be elucidated in both the early postnatal and adult prostate. 7 Regional differences of ductal arborization in the developing prostate suggested functional heterogeneities both between and within the lobes of the prostate. This type of study, however, by necessity, results in separation of the lobes such that the spatial relationship of the ductal openings into the urethra is lost. Price 3 stated that the relationship of these ductal openings into the urethra remains in the adult in the same relative position as in the fetus. This observation does not appear to have been subjected to further investigation. In contrast, the ensuing studies have placed more emphasis on regional anatomy in the adult and proposed homologies in animal models. Although some investigators have suggested that the lobe concept in the adult remains controversial with respect to the development of benign prostatic hyperplasia,8 the focal nature of human prostatic disease and the anatomical sites in which specific abnormalities are observed currently rely on McNeal's concept of zonal anatomy<9,IO Rat and mouse rarely exhibit spontaneous development of these disorders but are used extensively as animal models for the study of human prostatic disease. The objective of this investigation was to examine the growth patterns of the prostatic primordia, using three-dimensional computer-assisted serial section reconstruction, to shed light on the enigma of homologies between species. More important, the concept of comparative ductal budding patterns and their spatial relationships may prove to be of greater significance than the concept of lobes, lending itself to extrapAccepted for publication December 2, 1993. * Requests for reprints: Department of Anatomy and Structural Biology, School of Medicine, 414 East Clark St., Vermillion, South Dakota 57069. This study was supported in part by the University of South Dakota School of Medicine Faculty Development Fund and Grant DK42609 from the National Institute of Diabetes and Digestive and Kidney Diseases.
olation from animal models and a better understanding of the etiology of human prostatic disease, a major health problem in males. MATERIALS AND METHODS
Animals and tissues. Timed-pregnant female Sprague Dawley rats [Sasco, Omaha, Nebraska] were used to obtain animals of selected fetal ages (vaginal plug equals day 0). Embryos were taken at day 18.5 of gestation, the period just prior to the anlage stage of the prostate. 3 Subsequently, fetuses were taken at 1day intervals until birth, and I-day-old pups postnatally. Swiss Webster mouse fetuses [day 18] and Wistar rats [day 19.5J were also examined. Five animals were taken for each group. The whole pelvic region was placed in cold Bouin's [4CJ fixative for 4 hours. Subsequently the tissue was trimmed, fixed overnight, dehydrated and embedded in TissuePrep®2 (Fisher Scientific, Eden Prairie, Minnesota). Ten-~m. transverse serial sections, perpendicular to the proximal urethra, were collected from the entire urogenital complex and stained with hematoxylin and eosin. Serial sections of the prostatic complex from Praomys (Mastomys) N atalensis were also used from a previous studyY In addition, archived sections were selected from the Hamilton Embryological Slide Collection [Charing Cross and Westminster Medical School, London, United Kingdom] and represented two human fetuses [crown rump length (CR) 70 mm. and 100 mm.]. Serial section reconstruction. Three-dimensional reconstruction of the serial sections was obtained according to the procedures described by Young et aL,12 using an IBM compatible PC equipped with a math coprocessor, dual monitors and a high resolution digitizing tablet [Summagraphics, Fairfield, Connecticut]. The histological sections were traced using a Nikon Alphaphot YS microscope equipped with a camera lucida attachment. Briefly, the outlines of the prostatic buds, together with boundaries of associated structures, including seminal vesicles, coagulating glands, ejaculatory ducts, prostatic utriculus and urethra, along with fiducial marks for each section, were digitized. The reconstructions were rotated about the X, Y and Z axes in order to present a suitable image for photography. Stereo pair images were recorded on Kodak film directly from the color monitor. RESULTS
Sprague-Dawley rat. The earliest evidence of prostate development was observed in the 18.5 day fetal animals and was represented by bilateral ventral buds [fig. 1, A]. Coincidental
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DUCTAL BUDDING AND BRANCHING IN DEVELOPING PROSTATE
FIG. 1. Stereo-pair images of computer assisted serial section reconstructions from fetal and neonatal rat prostate. A, 18.5-day fetal Sprague Dawley rat. Ventral buds [v]; coagulating gland buds [cg]; urogenital sinus [us] ; ductus deferens [dd]; ventral mesenchymal pad [vmp] . Superior dorsolateral view. x90. B, 19.5-day fetal Sprague-Dawley rat [#1]. Seminal vesicle [sv] ; ductus deferens [dd]; coagulating gland bud [cg, blue]; dorsal buds [d, green]; lateral buds [I, grey]; ventral buds [v, right: pale blue, left: dark blue]. Inferior right lateral view. x80. C, 19.5-day fetal Sprague Dawley rat [#2] . Dorsal outgrowths [light and dark green] are associated with prostatic sulcus and lateral buds [light and dark amber] with lateral wall of urogenital sinus [red]. Ventral buds [pale and dark blue] grow towards mesenchymal pad [white outline]. Inferior right dorsal view. x60. D, 19.5-day fetal Sprague Dawley rat [#3]. Bilateral symmetry of dorsal [green], lateral [grey] and coagulating gland buds [blue] . Right seminal vesicle and ductus deferens have been removed for clarity. Prostatic utricle [purple outline] is visible between ejaculatory ducts. Superior dorsal view. x80. E, 19.5-day fetal Sprague Dawley rat [#3]. Direction of growth for dorsal [green], lateral [grey] and ventral [blue] is clearly distinct, as is comparative growth. Superior left lateral view. x80. F, newborn Sprague-Dawley rat. Secondary branching has occurred at tips of ventral ducts [blue & white] . Lateral [pale blue] and dorsal [light and dark green] ducts exhibit minimal branching at this stage of development. Coagulating gland [light blue] has branched and elongated towards contour of ventral surface of seminal vesicle [light purple] . Note that adjacent ventral and lateral ducts on right lateral surface of urogenital sinus originate from same budding line [see figs. 3 and 5] . Inferior right lateral view. X50. G, newborn Sprague-Dawley rat. Right seminal vesicle and ductus deferens have been removed to illustrate pattern of lateral [pale blue] and dorsal [light and dark green] budding. Inferior dorsal view. X50. H, newborn SpragueDawley rat. Right lateral ducts have been removed to show distinctive difference in pattern between ventral [dark blue] and dorsal [light green] growth. Inferior right ventral view. x50. I, 19.5-day fetal Wistar rat. Ventral, lateral and dorsal budding is similar to that of Sprague-Dawley rat. Periurethral smooth muscle [sm, grey] is associated with caudal portion of urogenital sinus and initial urothelial outgrowths of prostatic buds. Inferior right lateral view. x60. J, 19.5-day fetal Wistar rat. Upper contour of smooth muscle [grey] follows cranial-caudal pattern of lateral and dorsal outgrowths. Inferior right dorsal view. x60.
DUCTAL BUDDING AND BRAl',JCHING IN DEVELOPING PROSTATE
with the ventral budding were paired dorsocranial outgrowths of the coagulating gland. During the next 24-hour period there was extensive development of ventral, lateral and dorsal prostate budding from the urogenital sinus epithelium [figs. 1, B and C]. Several features of this stage of development are worth noting. First, solid outgrowths of the urothelium which formed the prostate buds were associated with periurethral mesenchyme [fig. 2]. Surrounding the latter tissue is a sleeve of smooth muscle, the upper boundary of which terminates near the neck of the bladder [see fig. 1, I]. All the prostatic buds exhibit a cranial elongation for the initial stages of growth. However, at the 19.5-day stage of development, the short, 3 to 4 paired ventral buds undergo a caudal curvature, and the distal portion of the cords becomes juxtaposed to a specific ventral pad of mesenchyme [figs. 1, C and 2]. Minimal distal tip branching was observed at this stage in any of the regions. The dorsal buds exhibit the most caudal outgrowth but conform to a distinct budding pattern that follows the contours of the prostatic sulcus and ends contiguous with the paired buds of the coagulating gland [figs. 1, D and E]. This pattern of budding, or line of buds, was repeated on the ventral aspect of the urethra and also enclosed a lateral line, which, during progressive development, sometimes formed an additional line of outgrowth. This pattern is best illustrated in the schematic diagram in which the lines of budding can be clearly demonstrated [fig. 3]. A further significant finding is illustrated in figures 1, Band 1, C with regard to the most anterior (ventral) line of buds. The direction of growth in the most cranial of these buds was towards the ventral mesenchyme. The adjacent, and remaining buds, grew cranially, as did the neighboring buds of the parallel lateral. line. For both the ventral and lateral buds, the predominant outgrowth appeared in the more cranial regions, in contrast to those of the dorsal line, where this feature was reversed. Dorsally, the exception to this rule was seen in the growth ofthe coagulating gland [fig. 1, D]. At birth, the most obvious changes in the growth patterns were reflected in the branching morphogenesis of the distal portions of the ventral ducts and the appearance of distinct lobes [fig. 1, F]. When compared to the ventral, the lateral and dorsal epithelial ducts did not exhibit significant distal branching at this stage of development [figs. 1, G and H]. This observation confirms earlier studies using whole-mount microdissection techniques.? The symmetry of the dorsal ductal outgrowth is also seen in the latter figures. Wistar rats. Ductal budding patterns were similar in the Wistar rat [fig. 1, I]. As illustrated in figure 1, J, the pattern of epithelial outgrowth from the urogenital sinus also conforms to the proximal boundary of the periurethral smooth muscle. On the anterior surface, just caudal to the neck of the bladder, the smooth muscle fibers were discontinuous. This was the region associated with the ventral mesenchymal pad and the distal tips of the ventral ducts [fig. 4, A]. Invasion of this mesenchyme by the tips of the ducts resulted in the induction of branching morphogenesis. Swiss Webster mouse. A comparative study of the development in the mouse revealed that ventral and dorsal budding was most prominent in the is-day fetuses, a time period which approximated the 19.5-day development in the rat. Also, there were two to three well-developed pairs of coagulating buds, in contrast to the single pair in the rat. The most consistent comparative feature was the budding pattern and the growth of the ventral buds towards a ventral mesenchymal pad. Lateral and dorsal views of this stage of development are illustrated in figures 4, Band 4, C. The lateral line of outgrowths is not prominent in this species. Multimammate mouse !Praomys (Mastomys) Natalensis). A similar pattern of budding to that seen in the rat was also observed in the multimammate mouse in that ventral and dorsal buddings were prominent at the earlier stages of prostate growth [fig. 4, D]. In contrast, the paired coagulating buds
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FIG. 2. Transverse section of urogenital sinus from 19.5 day fetal male rat just caudal to openings of ejaculatory ducts. Ventral (V), lateral (L) and dorsal (D) outgrowths are evident. Tips of ventral buds are associated with condensation of ventral urogenital mesenchyme (arrowheads). Initial outgrowths of urothelium are associated with periurethral mesenchyme (*). U: prostatic urethra; SC: seminal colliculus. Hematoxylin and eosin stain. xllO. CRANIAL
microns
Section II
Perimeter (microns) CAUDAL
3. Planimetric representation of prostatic budding pattern in urogenital sinus of 19.5 day fetal male rat. Center line represents midventral axis of urogenital sinus. Relative budding positions, computed at time of digitizing, were extrapolated from caudal-cranial coordinates of section number [left y axis; also shown in micronsright y axis 1and distance from midventral point of urethra. Outgrowths of urogenital epithelium (0) form ventral-dorsal (V -D), lateral dorsal (L-D) and dorsal-dorsal (D-D) lines along cranial-caudal axis. Buds associated with typical anatomical regions are indicated following exhibit key: ventral (e), lateral (0) and dorsal (0). Some buds minimal development. E: ejaculatory duct; U: utricle; CG: coagulating gland. FIG.
developed later and were more apparent at birth. Ductal branching morphogenesis was evident in the ventral prostate lobes at birth and was associated with a similar ventral mesenchymal pad [fig. 4, E]. Human. Two human fetuses, with CR lengths of 70 mm. and 100 mm., were analyzed. In the 70 mm.-fetus, distinct budding lines were apparent, with lateral and dorsal outgrowths being more prevalent [figs. 4, F and G]. In particular, the most dorsal buds were seen in a horseshoe-shaped pattern emanating from the posterior ridge of the prostatic sulcus and surrounding the ejaculatory ducts and prostatic utriculus [fig. 4, G]. Caudal to these buds, a group of outgrowths displayed a direction of growth toward the ventral axis [fig. 4, F]. Many small buds were seen in the dorsocranial region of the urogenital sinus, comparable in position to the coagulating gland buds in the
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DUCTAL BUDDING AND BRANCHING IN DEVELOPING PROSTATE
FIG. 4. Stereo-pair images of computer assisted serial section reconstructions from fetal rat, mouse and human prostate. A, I9.5-day fetal Wistar rat. Smooth muscle is separated on ventral surface of urogenital sinus by condensation of mesenchymal tissue [vmp, white outline, transparent]. Ventral buds grow towards, and ultimately into, this mesenchyme, which induces branching morphogenesis of ventral lobe. Inferior ventral view. x60. B, IS-day fetal Swiss Webster mouse. Ventral [light and pale blue] and dorsal [green] line of budding are most prominent outgrowths in mouse. Lateral buds are not evident at this stage of development. Inferior right lateral view. xSO. C, IS-day fetal Swiss Webster mouse. Two to three pairs of coagulating gland buds [light and dark blue] are typical in early stages of urogenital outgrowths in mouse. Right seminal vesicle has been removed to illustrate dorsal line of budding along prostatic sulcus. Superior dorsal view. x90. D , 20-day fetal multimammate mouse. Paired ventral buds [pale blue and blue] and dorsal outgrowths [green) are evident at this stage of fetal development. Inferior right ventrolateral view. x90. E, newborn multimammate mouse. Similar patterns of budding and branching to those seen in other species are evident in ventral [pale and dark blue], lateral [grey) and dorsal [green) regions of multimammate mouse urogenital sinus. Inferior ventral view. x60. F, 70-mm. CR length human fetus. Dorsal outgrowths [light green) form a horseshoe shaped pattern around ejaculatory ducts and prostatic utricle. Right seminal vesicle has been removed. Lateral buds [blue and grey] are present. Some caudally located dorsal buds [dark green) exhibit anterior growth. Superior right dorsolateral view. x50. G, 70-mm. CR length human fetus. Left and right seminal vesicles have been removed. Superior dorsal view. X50. H, 100-mm. CR length human fetus. At this stage of development dorsal outgrowths [green] are distinct from lateral [grey] buds. Tips of some dorsal buds are also directed anteriorly. Both lateral and dorsal ducts exhibit initial branching at distal tips. Left and right ejaculatory ducts are shown [light purple] . Superior right dorsolateral view. x50. I, 100-mm. CR length human fetus. Right ejaculatory duct has been removed. Superior dorsal view. x50. J, 100-mm CR length human fetus. This ventral reconstruction, with urethra in transparent view [red outline], permits the distinct dorsal [green) and lateral [grey) lines of budding to be observed. Inferior ventral view. x50.
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DUCTAL BUDDING AND BRANCHING IN DEVELOPiNG PROSTATE
rodent prostate. The dorsal line of buds surrounding the ejaculatory ducts appeared to be a separate line. During later development the dorsolateral ducts become elongated and exhibited primary branching [fig. 4, H]. This latter feature was most prominent in the caudal dorsal and lateral ducts. In the specimen analyzed, the dorsocranial ducts were not well developed. Two distinct cranial-caudal lines of buds were apparent [figs. 4, I and 4, J]. One surrounded the ejaculatory ducts [fig. 4, I]; the other formed a lateral line running from the proximal to the distal regions of the urethra [fig. 4, J].
Ductus deferens
Prostatic utricle
Seminal vesicle
Ventral prostatic buds
Ventral mesenchymal pad
DISCUSSION
The question of anatomically defined lobes in the human prostate has been debated periodically since the turn of this century, beginning with a description of the middle, lateral and posterior lobes by Lowsley2 and succeeded by the recent concept proposed by McNeaUo, 13, 14 During the intermediate years, a variety of classifications of the zonal anatomy of the prostate have been proffered or described, based on studies of development and susceptibility to hormones or disease. 8,15-18 Much controversy was engendered as a result of conflicting nomenclature. As McNeal points out, the variety of interspecies differences observed in the prostate gland reflects a diversity which makes it difficult to find a suitable animal model for the study of human prostatic disease. 1o Furthermore, it has also been pointed out that the description of the lobes in the fetal prostate by Lowsley was not compared to the structure in the adult gland. 14 Using a true three-dimensional approach to visualize the microanatomy of prostate development, this study demonstrates, for the first time, a visual interpretation of the spatial relationships of morphogenesis. Whole-mount, microdissection techniques were previously used to reveal ductal branching patterns in mouse 7 and rae 9 from birth to adult. These studies clearly demonstrated the distinctive arborization patterns associated with prostatic lobes. In addition, in the rat, it was shown that the lateral lobe exhibited two patterns of branching. The description by Hayashi et a1.,19 shows that the lateral type 1 ducts are complementary to the cranial ducts of the dorsal line of budding, while the type 2 ducts may represent the caudal ducts developing along the ventral line [see figs. 3 and 5]. The most significant correlation appears to be the association of the distal ducts with specific regions of mesenchymal tissue. This is clearly demonstrated for the ventral budding and is illustrated schematically in figure 5. Also, temporal differences in ductal branching were observed in the newborn rat between ventral and lateral ducts [see fig. 1, F], confirming earlier studies. 7 Through the observations of comparative fetal ductal budding patterns in a variety of species, we have demonstrated a correlation to adult regional anatomy. McNeaFo has described lines of prostate ducts associated with the proximal and distal urethra in the adult human male. They are comparable to those seen in this study. The buds surrounding the ejaculatory ducts [see figs. 4, G and J], associated with Wolffian duct mesenchyme, would compare with the central zone, the most caudal of the adjacent lateral line would form the peripheral zone, and the cranial buds in the proximal portion of the dorsal urethra would develop into the transition zone [see fig. 6 for comparison]. Periurethral glands appear at a later stage of development. It may be necessary to perceive the regional variation of prostate anatomy as a consequence of specific mesenchymal influences along a budding axis rather than to try to anatomically group the ducts. The nature of the budding pattern in rat, mouse and human suggests a symmetrical pattern of urothelium outgrowth, the subsequent development of which is controlled by inductive mechanisms of positional mesenchyme. The importance of stromal-epithelial interactions in prostate development has been well documented. 21 Since marked differences in ductal branching patterns occur within the prostate, it is possible that variations in temporal influences and spatial
FIG. 5. Schematic illustration of typical ductal budding pattern in early stages of development of fetal rodent prostate. Cranial portion of ventral line of budding is associated with growth into ventral pad of mesenchymal tissue. Adjacent buds along this line usually form part of lateral lobe. Mesenchyme surrounding dorsal outgrowths is contiguous with that of W olffian duct and not depicted in diagram. E
UD
FIG. 6. Sagittal diagram of human prostate showing distal urethra (UD), proximal urethra (UP) and ejaculatory duct (E). These structures
are shown in relation to three major glandular regions of prostate, central zone (CZ), peripheral zone (PZ) and transitional zone (TZ). [Redrawn and reproduced, with permission, from J. E. McNeal, Am. J. Surg. Path., 12:619, 1988.]
organization of the mesenchyme may be important modulators of prostate morphogenesis. Furthermore, it has been demonstrated that the regional organization of the male prostatic complex is determined by the regional heterogeneity- of mesenchymal inductiono 22 The early outgrowths of urothelium that form the prostatic buds are associated with periurethral mesenchyme. Subsequent branching morphogenesis, in the ventral buds for example, requires interaction of the distal ducts with the ventral mesenchymal pad. This supports the concept that the process of morphogenesis and cytodifferentiation may be independent but consecutive events of development. 23 Recent work has also demonstrated that the ventral mesenchymal pad is a potent inducer of cytodifferentiation for a lobe-specific secretory protein. 24 Thus, the effects of stromal heterogeneity on growth and differentiation, imprinted during fetal development by in utero hormone actions, would be a more significant relationship to study with respect to zonal growth anomalies and susceptibility to prostatic disease. Acknowledgmentso The authors wish to dedicate this paper in recognition of the career of Ms. Vera Cleland, who has been providing technical assistance to the Department of Anatomy at the University of South Dakota for 38 years. Without her expert help with the serial sectioning for the past four years, this paper would not have been possible. Special thanks also to Dr. Lawrence Garey, Chairman, Department of Anatomy, Westminster and Charing Cross Medical School, London,
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DUCTAL BUDDING AND BRANCHING IN DEVELOPING PROSTATE
United Kingdom, for providing access to archived slides of the human embryos.
12.
REFERENCES 1. Aumiiller, G.: Prostate gland and seminal vesicles. In: Handbuch der mikroskopischen Anatomie des Menschen. Edited by A. Oksche and L. Vollrath. Berlin-Heidelberg-New York: SpringerVerlag, vol. VIl/6, p. 1, 1979. 2. Lowsley, O. S.: The development of the human prostate gland with reference to the development of other structures at the neck of the urinary bladder. Am. J. Anat., 13: 299, 1912. 3. Price, D.: Normal development ofthe prostate and seminal vesicles of the rat with a study of experimental postnatal modifications. Am. J. Anat., 60: 79, 1936. 4. Price, D. and Williams-Ashman, H. G.: The accessory reproductive glands in mammals. In: Sex and Internal Secretions. Edited by W. C. Young. London: Bailliere, Tindall and Cox, vol. 1, pp. 366-448, 1961. 5. Brandes, D.: The fine structure and histochemistry of prostatic glands in relation to sex hormones. Int. Rev. Cytol., 20: 207, 1966. 6. Jesik, C. J., Holland, J. M. and Lee, C.: An anatomic and histologic study of the rat prostate. Prostate, 3: 81, 1982. 7. Sugimura, Y., Cunha, G. Rand Donjacour, A. A.: Morphogenesis of ductal networks in the mouse prostate. Biol. Reprod., 34: 961, 1986. 8. Hiraoka, Y. and Akimoto, M.: Anatomy of the prostate from fetus to adult-origin of benign prostatic hyperplasia. Urol. Res., 15: 177,1987. 9. Lee, F., Torp-Pedersen, S. T., Siders, D. B., Littrup, P. J. and McLeary, RD.: Transrectal ultrasound in the diagnosis and staging of prostatic carcinoma. Radiology, 170: 609, 1989. 10. McNeal, J.: Pathology of benign prostatic hyperplasia. Insight into etiology. Urol. CHn. N. Am., 17: 477, 1990. 11. Gross, S. A. and DiDio, L. J. A.: Prenatal development of the
13. 14. 15. 16. 17. 18. 19. 20. 21.
22. 23.
24.
prostate in male Praomys (Mastomys) Natalensis. Arch. Ital. Anat. Embriol., 91: 21, 1986. Young, S. J., Royer, S. M., Groves, P. M. and Kinnamon, J. C.: Three-dimensional reconstructions from serial micrographs using the IBM PC. J. Electron. Microsc. Tech., 6: 207, 1987. McNeal, J. E.: The prostate and prostatic urethra: a morphologic synthesis. J. Urol., 107: 1008, 1972. McNeal, J. F.: The prostate gland: morphology and pathobiology. Monogr. Urol., 4: 3, 1983. Huggins, C. and Webster, W.O.: Duality of human prostate in response to estrogen. J. Urol., 59: 258, 1948. Franks, L. M.: Benign nodular hyperplasia of the prostate: a review. Ann. R. Coil. Surg. Engl., 14: 92, 1954. Price, D.: Comparative aspects of development and structure in the prostate. Nat. Cancer Inst. Monogr., 12: 1, 1963. Tisell, L.-E., and Salander, H.: The lobes of the human prostate. Scand. J. Urol. Nephrol., 9: 185, 1975. Hayashi, N., Sugimura, Y., Kawamura, J., Donjacour, A. A. and Cunha, G. R: Morphological and functional heterogeneity in the rat prostatic gland. Biol. Reprod., 45: 308, 1991. McNeal, J. E.: Normal histology of the prostate. Am. J. Surg. Path., 12: 619, 1988. Cunha, G. R., Donjacour, A. A., Cooke, P. S., Mee, S., Bigsby, R M., Higgins, S. J. and Sugimura, Y.: The endocrinology and developmental biology of the prostate. Endocr. Rev., 8: 338, 1987. Sugimura, Y., Norman, J. T., Cunha, G. R and Shannon, J. M.: Regional differences in the inductive activity of the mesenchyme ofthe embryonic mouse urogenital sinus. Prostate, 7: 253, 1985. Mizuno, T. and Yasugi, S.: Susceptibility of epithelia to directive influences of mesenchymes during organogenesis: uncoupling of morphogenesis and cytodifferentiation. Cell Differ. Develop., 31: 151,1990. Lee, C. W. and Timms, B. G.: Instructive expression of prostate specific binding protein (PSBP) in heterotypic epithelial-mesenchymal recombinants. Anat. Rec., suppll: 77, 1993.
THE JOURNAL OF UROLOGY
Copyright © 1994 by AMERICAN UROLOGICn ASSOCIATION, INC.
DUCTAL BUDDING AND BRANCHING PATTERNS IN THE DEVELOPING PROSTATE BARRY G, TIMMS*, TOM J. MOHS
AND
LIBERATO J. A. DIDIO
From the Department of Anatomy and Structural Biology, School of Medicine, University of South Dakota, Vermillion, South Dakota and the Medical College of Ohio, Toledo, Ohio
ABSTRACT
Development of the prostate was studied by serial section reconstruction and computer-assisted three-dimensional analysis. A comparison of ductal budding in species of rat and mouse and the human revealed patterns consistent with common developmental characteristics. Ventral, lateral and dorsal lines of epithelial buds, which emanated from the urogenital sinus into the surrounding periurethral mesenchyme, followed ventro-dorsal and cranio-caudal axes. Subsequent branching morphogenesis was associated with specific mesenchymal condensations. These patterns of budding were closely related to the adult lobe architecture in the rodent prostate. In the human fetus, prostate ductal budding exhibited patterns compatible with the current concept of zonal anatomy. KEY
WORDS:
prostate; fetal development; image processing, computer-assisted
Over the past 80 years, there have been several classical investigations of prostate morphogenesis in a variety of species. 1 Notable among these are the descriptions of the human and rat prostate by Lowsley 2 and Price. 3 The rat prostate, more than that of any other species, has been subjected to the most thorough light and electron microscopical analysis of embryonic, postnatal and adult stages of growth. 4 - 6 However, owing to the three-dimensional complexity of the developing duct system, these studies provided only a descriptive outline of morphogenesis and adult architecture. Recent investigations on mice, using a whole-mount microdissection technique, have enabled the precise delineation of all the ductal elements to be elucidated in both the early postnatal and adult prostate. 7 Regional differences of ductal arborization in the developing prostate suggested functional heterogeneities both between and within the lobes of the prostate. This type of study, however, by necessity, results in separation of the lobes such that the spatial relationship of the ductal openings into the urethra is lost. Price 3 stated that the relationship of these ductal openings into the urethra remains in the adult in the same relative position as in the fetus. This observation does not appear to have been subjected to further investigation. In contrast, the ensuing studies have placed more emphasis on regional anatomy in the adult and proposed homologies in animal models. Although some investigators have suggested that the lobe concept in the adult remains controversial with respect to the development of benign prostatic hyperplasia,8 the focal nature of human prostatic disease and the anatomical sites in which specific abnormalities are observed currently rely on McNeal's concept of zonal anatomy<9,IO Rat and mouse rarely exhibit spontaneous development of these disorders but are used extensively as animal models for the study of human prostatic disease. The objective of this investigation was to examine the growth patterns of the prostatic primordia, using three-dimensional computer-assisted serial section reconstruction, to shed light on the enigma of homologies between species. More important, the concept of comparative ductal budding patterns and their spatial relationships may prove to be of greater significance than the concept of lobes, lending itself to extrapAccepted for publication December 2, 1993. * Requests for reprints: Department of Anatomy and Structural Biology, School of Medicine, 414 East Clark St., Vermillion, South Dakota 57069. This study was supported in part by the University of South Dakota School of Medicine Faculty Development Fund and Grant DK42609 from the National Institute of Diabetes and Digestive and Kidney Diseases.
olation from animal models and a better understanding of the etiology of human prostatic disease, a major health problem in males. MATERIALS AND METHODS
Animals and tissues. Timed-pregnant female Sprague Dawley rats [Sasco, Omaha, Nebraska] were used to obtain animals of selected fetal ages (vaginal plug equals day 0). Embryos were taken at day 18.5 of gestation, the period just prior to the anlage stage of the prostate. 3 Subsequently, fetuses were taken at 1day intervals until birth, and I-day-old pups postnatally. Swiss Webster mouse fetuses [day 18] and Wistar rats [day 19.5J were also examined. Five animals were taken for each group. The whole pelvic region was placed in cold Bouin's [4CJ fixative for 4 hours. Subsequently the tissue was trimmed, fixed overnight, dehydrated and embedded in TissuePrep®2 (Fisher Scientific, Eden Prairie, Minnesota). Ten-~m. transverse serial sections, perpendicular to the proximal urethra, were collected from the entire urogenital complex and stained with hematoxylin and eosin. Serial sections of the prostatic complex from Praomys (Mastomys) N atalensis were also used from a previous studyY In addition, archived sections were selected from the Hamilton Embryological Slide Collection [Charing Cross and Westminster Medical School, London, United Kingdom] and represented two human fetuses [crown rump length (CR) 70 mm. and 100 mm.]. Serial section reconstruction. Three-dimensional reconstruction of the serial sections was obtained according to the procedures described by Young et aL,12 using an IBM compatible PC equipped with a math coprocessor, dual monitors and a high resolution digitizing tablet [Summagraphics, Fairfield, Connecticut]. The histological sections were traced using a Nikon Alphaphot YS microscope equipped with a camera lucida attachment. Briefly, the outlines of the prostatic buds, together with boundaries of associated structures, including seminal vesicles, coagulating glands, ejaculatory ducts, prostatic utriculus and urethra, along with fiducial marks for each section, were digitized. The reconstructions were rotated about the X, Y and Z axes in order to present a suitable image for photography. Stereo pair images were recorded on Kodak film directly from the color monitor. RESULTS
Sprague-Dawley rat. The earliest evidence of prostate development was observed in the 18.5 day fetal animals and was represented by bilateral ventral buds [fig. 1, A]. Coincidental
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DUCTAL BUDDING AND BRANCHING IN DEVELOPING PROSTATE
FIG. 1. Stereo-pair images of computer assisted serial section reconstructions from fetal and neonatal rat prostate. A, 18.5-day fetal Sprague Dawley rat. Ventral buds [v]; coagulating gland buds [cg]; urogenital sinus [us] ; ductus deferens [dd]; ventral mesenchymal pad [vmp] . Superior dorsolateral view. x90. B, 19.5-day fetal Sprague-Dawley rat [#1]. Seminal vesicle [sv] ; ductus deferens [dd]; coagulating gland bud [cg, blue]; dorsal buds [d, green]; lateral buds [I, grey]; ventral buds [v, right: pale blue, left: dark blue]. Inferior right lateral view. x80. C, 19.5-day fetal Sprague Dawley rat [#2] . Dorsal outgrowths [light and dark green] are associated with prostatic sulcus and lateral buds [light and dark amber] with lateral wall of urogenital sinus [red]. Ventral buds [pale and dark blue] grow towards mesenchymal pad [white outline]. Inferior right dorsal view. x60. D, 19.5-day fetal Sprague Dawley rat [#3]. Bilateral symmetry of dorsal [green], lateral [grey] and coagulating gland buds [blue] . Right seminal vesicle and ductus deferens have been removed for clarity. Prostatic utricle [purple outline] is visible between ejaculatory ducts. Superior dorsal view. x80. E, 19.5-day fetal Sprague Dawley rat [#3]. Direction of growth for dorsal [green], lateral [grey] and ventral [blue] is clearly distinct, as is comparative growth. Superior left lateral view. x80. F, newborn Sprague-Dawley rat. Secondary branching has occurred at tips of ventral ducts [blue & white] . Lateral [pale blue] and dorsal [light and dark green] ducts exhibit minimal branching at this stage of development. Coagulating gland [light blue] has branched and elongated towards contour of ventral surface of seminal vesicle [light purple] . Note that adjacent ventral and lateral ducts on right lateral surface of urogenital sinus originate from same budding line [see figs. 3 and 5] . Inferior right lateral view. X50. G, newborn Sprague-Dawley rat. Right seminal vesicle and ductus deferens have been removed to illustrate pattern of lateral [pale blue] and dorsal [light and dark green] budding. Inferior dorsal view. X50. H, newborn SpragueDawley rat. Right lateral ducts have been removed to show distinctive difference in pattern between ventral [dark blue] and dorsal [light green] growth. Inferior right ventral view. x50. I, 19.5-day fetal Wistar rat. Ventral, lateral and dorsal budding is similar to that of Sprague-Dawley rat. Periurethral smooth muscle [sm, grey] is associated with caudal portion of urogenital sinus and initial urothelial outgrowths of prostatic buds. Inferior right lateral view. x60. J, 19.5-day fetal Wistar rat. Upper contour of smooth muscle [grey] follows cranial-caudal pattern of lateral and dorsal outgrowths. Inferior right dorsal view. x60.
DUCTAL BUDDING AND BRAl',JCHING IN DEVELOPING PROSTATE
with the ventral budding were paired dorsocranial outgrowths of the coagulating gland. During the next 24-hour period there was extensive development of ventral, lateral and dorsal prostate budding from the urogenital sinus epithelium [figs. 1, B and C]. Several features of this stage of development are worth noting. First, solid outgrowths of the urothelium which formed the prostate buds were associated with periurethral mesenchyme [fig. 2]. Surrounding the latter tissue is a sleeve of smooth muscle, the upper boundary of which terminates near the neck of the bladder [see fig. 1, I]. All the prostatic buds exhibit a cranial elongation for the initial stages of growth. However, at the 19.5-day stage of development, the short, 3 to 4 paired ventral buds undergo a caudal curvature, and the distal portion of the cords becomes juxtaposed to a specific ventral pad of mesenchyme [figs. 1, C and 2]. Minimal distal tip branching was observed at this stage in any of the regions. The dorsal buds exhibit the most caudal outgrowth but conform to a distinct budding pattern that follows the contours of the prostatic sulcus and ends contiguous with the paired buds of the coagulating gland [figs. 1, D and E]. This pattern of budding, or line of buds, was repeated on the ventral aspect of the urethra and also enclosed a lateral line, which, during progressive development, sometimes formed an additional line of outgrowth. This pattern is best illustrated in the schematic diagram in which the lines of budding can be clearly demonstrated [fig. 3]. A further significant finding is illustrated in figures 1, Band 1, C with regard to the most anterior (ventral) line of buds. The direction of growth in the most cranial of these buds was towards the ventral mesenchyme. The adjacent, and remaining buds, grew cranially, as did the neighboring buds of the parallel lateral. line. For both the ventral and lateral buds, the predominant outgrowth appeared in the more cranial regions, in contrast to those of the dorsal line, where this feature was reversed. Dorsally, the exception to this rule was seen in the growth ofthe coagulating gland [fig. 1, D]. At birth, the most obvious changes in the growth patterns were reflected in the branching morphogenesis of the distal portions of the ventral ducts and the appearance of distinct lobes [fig. 1, F]. When compared to the ventral, the lateral and dorsal epithelial ducts did not exhibit significant distal branching at this stage of development [figs. 1, G and H]. This observation confirms earlier studies using whole-mount microdissection techniques.? The symmetry of the dorsal ductal outgrowth is also seen in the latter figures. Wistar rats. Ductal budding patterns were similar in the Wistar rat [fig. 1, I]. As illustrated in figure 1, J, the pattern of epithelial outgrowth from the urogenital sinus also conforms to the proximal boundary of the periurethral smooth muscle. On the anterior surface, just caudal to the neck of the bladder, the smooth muscle fibers were discontinuous. This was the region associated with the ventral mesenchymal pad and the distal tips of the ventral ducts [fig. 4, A]. Invasion of this mesenchyme by the tips of the ducts resulted in the induction of branching morphogenesis. Swiss Webster mouse. A comparative study of the development in the mouse revealed that ventral and dorsal budding was most prominent in the is-day fetuses, a time period which approximated the 19.5-day development in the rat. Also, there were two to three well-developed pairs of coagulating buds, in contrast to the single pair in the rat. The most consistent comparative feature was the budding pattern and the growth of the ventral buds towards a ventral mesenchymal pad. Lateral and dorsal views of this stage of development are illustrated in figures 4, Band 4, C. The lateral line of outgrowths is not prominent in this species. Multimammate mouse !Praomys (Mastomys) Natalensis). A similar pattern of budding to that seen in the rat was also observed in the multimammate mouse in that ventral and dorsal buddings were prominent at the earlier stages of prostate growth [fig. 4, D]. In contrast, the paired coagulating buds
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FIG. 2. Transverse section of urogenital sinus from 19.5 day fetal male rat just caudal to openings of ejaculatory ducts. Ventral (V), lateral (L) and dorsal (D) outgrowths are evident. Tips of ventral buds are associated with condensation of ventral urogenital mesenchyme (arrowheads). Initial outgrowths of urothelium are associated with periurethral mesenchyme (*). U: prostatic urethra; SC: seminal colliculus. Hematoxylin and eosin stain. xllO. CRANIAL
microns
Section II
Perimeter (microns) CAUDAL
3. Planimetric representation of prostatic budding pattern in urogenital sinus of 19.5 day fetal male rat. Center line represents midventral axis of urogenital sinus. Relative budding positions, computed at time of digitizing, were extrapolated from caudal-cranial coordinates of section number [left y axis; also shown in micronsright y axis 1and distance from midventral point of urethra. Outgrowths of urogenital epithelium (0) form ventral-dorsal (V -D), lateral dorsal (L-D) and dorsal-dorsal (D-D) lines along cranial-caudal axis. Buds associated with typical anatomical regions are indicated following exhibit key: ventral (e), lateral (0) and dorsal (0). Some buds minimal development. E: ejaculatory duct; U: utricle; CG: coagulating gland. FIG.
developed later and were more apparent at birth. Ductal branching morphogenesis was evident in the ventral prostate lobes at birth and was associated with a similar ventral mesenchymal pad [fig. 4, E]. Human. Two human fetuses, with CR lengths of 70 mm. and 100 mm., were analyzed. In the 70 mm.-fetus, distinct budding lines were apparent, with lateral and dorsal outgrowths being more prevalent [figs. 4, F and G]. In particular, the most dorsal buds were seen in a horseshoe-shaped pattern emanating from the posterior ridge of the prostatic sulcus and surrounding the ejaculatory ducts and prostatic utriculus [fig. 4, G]. Caudal to these buds, a group of outgrowths displayed a direction of growth toward the ventral axis [fig. 4, F]. Many small buds were seen in the dorsocranial region of the urogenital sinus, comparable in position to the coagulating gland buds in the
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DUCTAL BUDDING AND BRANCHING IN DEVELOPING PROSTATE
FIG. 4. Stereo-pair images of computer assisted serial section reconstructions from fetal rat, mouse and human prostate. A, I9.5-day fetal Wistar rat. Smooth muscle is separated on ventral surface of urogenital sinus by condensation of mesenchymal tissue [vmp, white outline, transparent]. Ventral buds grow towards, and ultimately into, this mesenchyme, which induces branching morphogenesis of ventral lobe. Inferior ventral view. x60. B, IS-day fetal Swiss Webster mouse. Ventral [light and pale blue] and dorsal [green] line of budding are most prominent outgrowths in mouse. Lateral buds are not evident at this stage of development. Inferior right lateral view. xSO. C, IS-day fetal Swiss Webster mouse. Two to three pairs of coagulating gland buds [light and dark blue] are typical in early stages of urogenital outgrowths in mouse. Right seminal vesicle has been removed to illustrate dorsal line of budding along prostatic sulcus. Superior dorsal view. x90. D , 20-day fetal multimammate mouse. Paired ventral buds [pale blue and blue] and dorsal outgrowths [green) are evident at this stage of fetal development. Inferior right ventrolateral view. x90. E, newborn multimammate mouse. Similar patterns of budding and branching to those seen in other species are evident in ventral [pale and dark blue], lateral [grey) and dorsal [green) regions of multimammate mouse urogenital sinus. Inferior ventral view. x60. F, 70-mm. CR length human fetus. Dorsal outgrowths [light green) form a horseshoe shaped pattern around ejaculatory ducts and prostatic utricle. Right seminal vesicle has been removed. Lateral buds [blue and grey] are present. Some caudally located dorsal buds [dark green) exhibit anterior growth. Superior right dorsolateral view. x50. G, 70-mm. CR length human fetus. Left and right seminal vesicles have been removed. Superior dorsal view. X50. H, 100-mm. CR length human fetus. At this stage of development dorsal outgrowths [green] are distinct from lateral [grey] buds. Tips of some dorsal buds are also directed anteriorly. Both lateral and dorsal ducts exhibit initial branching at distal tips. Left and right ejaculatory ducts are shown [light purple] . Superior right dorsolateral view. x50. I, 100-mm. CR length human fetus. Right ejaculatory duct has been removed. Superior dorsal view. x50. J, 100-mm CR length human fetus. This ventral reconstruction, with urethra in transparent view [red outline], permits the distinct dorsal [green) and lateral [grey) lines of budding to be observed. Inferior ventral view. x50.
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DUCTAL BUDDING AND BRANCHING IN DEVELOPiNG PROSTATE
rodent prostate. The dorsal line of buds surrounding the ejaculatory ducts appeared to be a separate line. During later development the dorsolateral ducts become elongated and exhibited primary branching [fig. 4, H]. This latter feature was most prominent in the caudal dorsal and lateral ducts. In the specimen analyzed, the dorsocranial ducts were not well developed. Two distinct cranial-caudal lines of buds were apparent [figs. 4, I and 4, J]. One surrounded the ejaculatory ducts [fig. 4, I]; the other formed a lateral line running from the proximal to the distal regions of the urethra [fig. 4, J].
Ductus deferens
Prostatic utricle
Seminal vesicle
Ventral prostatic buds
Ventral mesenchymal pad
DISCUSSION
The question of anatomically defined lobes in the human prostate has been debated periodically since the turn of this century, beginning with a description of the middle, lateral and posterior lobes by Lowsley2 and succeeded by the recent concept proposed by McNeaUo, 13, 14 During the intermediate years, a variety of classifications of the zonal anatomy of the prostate have been proffered or described, based on studies of development and susceptibility to hormones or disease. 8,15-18 Much controversy was engendered as a result of conflicting nomenclature. As McNeal points out, the variety of interspecies differences observed in the prostate gland reflects a diversity which makes it difficult to find a suitable animal model for the study of human prostatic disease. 1o Furthermore, it has also been pointed out that the description of the lobes in the fetal prostate by Lowsley was not compared to the structure in the adult gland. 14 Using a true three-dimensional approach to visualize the microanatomy of prostate development, this study demonstrates, for the first time, a visual interpretation of the spatial relationships of morphogenesis. Whole-mount, microdissection techniques were previously used to reveal ductal branching patterns in mouse 7 and rae 9 from birth to adult. These studies clearly demonstrated the distinctive arborization patterns associated with prostatic lobes. In addition, in the rat, it was shown that the lateral lobe exhibited two patterns of branching. The description by Hayashi et a1.,19 shows that the lateral type 1 ducts are complementary to the cranial ducts of the dorsal line of budding, while the type 2 ducts may represent the caudal ducts developing along the ventral line [see figs. 3 and 5]. The most significant correlation appears to be the association of the distal ducts with specific regions of mesenchymal tissue. This is clearly demonstrated for the ventral budding and is illustrated schematically in figure 5. Also, temporal differences in ductal branching were observed in the newborn rat between ventral and lateral ducts [see fig. 1, F], confirming earlier studies. 7 Through the observations of comparative fetal ductal budding patterns in a variety of species, we have demonstrated a correlation to adult regional anatomy. McNeaFo has described lines of prostate ducts associated with the proximal and distal urethra in the adult human male. They are comparable to those seen in this study. The buds surrounding the ejaculatory ducts [see figs. 4, G and J], associated with Wolffian duct mesenchyme, would compare with the central zone, the most caudal of the adjacent lateral line would form the peripheral zone, and the cranial buds in the proximal portion of the dorsal urethra would develop into the transition zone [see fig. 6 for comparison]. Periurethral glands appear at a later stage of development. It may be necessary to perceive the regional variation of prostate anatomy as a consequence of specific mesenchymal influences along a budding axis rather than to try to anatomically group the ducts. The nature of the budding pattern in rat, mouse and human suggests a symmetrical pattern of urothelium outgrowth, the subsequent development of which is controlled by inductive mechanisms of positional mesenchyme. The importance of stromal-epithelial interactions in prostate development has been well documented. 21 Since marked differences in ductal branching patterns occur within the prostate, it is possible that variations in temporal influences and spatial
FIG. 5. Schematic illustration of typical ductal budding pattern in early stages of development of fetal rodent prostate. Cranial portion of ventral line of budding is associated with growth into ventral pad of mesenchymal tissue. Adjacent buds along this line usually form part of lateral lobe. Mesenchyme surrounding dorsal outgrowths is contiguous with that of W olffian duct and not depicted in diagram. E
UD
FIG. 6. Sagittal diagram of human prostate showing distal urethra (UD), proximal urethra (UP) and ejaculatory duct (E). These structures
are shown in relation to three major glandular regions of prostate, central zone (CZ), peripheral zone (PZ) and transitional zone (TZ). [Redrawn and reproduced, with permission, from J. E. McNeal, Am. J. Surg. Path., 12:619, 1988.]
organization of the mesenchyme may be important modulators of prostate morphogenesis. Furthermore, it has been demonstrated that the regional organization of the male prostatic complex is determined by the regional heterogeneity- of mesenchymal inductiono 22 The early outgrowths of urothelium that form the prostatic buds are associated with periurethral mesenchyme. Subsequent branching morphogenesis, in the ventral buds for example, requires interaction of the distal ducts with the ventral mesenchymal pad. This supports the concept that the process of morphogenesis and cytodifferentiation may be independent but consecutive events of development. 23 Recent work has also demonstrated that the ventral mesenchymal pad is a potent inducer of cytodifferentiation for a lobe-specific secretory protein. 24 Thus, the effects of stromal heterogeneity on growth and differentiation, imprinted during fetal development by in utero hormone actions, would be a more significant relationship to study with respect to zonal growth anomalies and susceptibility to prostatic disease. Acknowledgmentso The authors wish to dedicate this paper in recognition of the career of Ms. Vera Cleland, who has been providing technical assistance to the Department of Anatomy at the University of South Dakota for 38 years. Without her expert help with the serial sectioning for the past four years, this paper would not have been possible. Special thanks also to Dr. Lawrence Garey, Chairman, Department of Anatomy, Westminster and Charing Cross Medical School, London,
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DUCTAL BUDDING AND BRANCHING IN DEVELOPING PROSTATE
United Kingdom, for providing access to archived slides of the human embryos.
12.
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