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A Possible Mechanism for Canine Prostatic Secretion
Vol. 102, Dec. Printed in U.S.A.
THE JOURNAL OF UROLOGY
Copyright
© 1969 by The Williams & Wilkins Co.
A POSSIBLE MECHANISM FOR CANINE PROSTATIC SECRETION MORTON D. MASER, WARD A. SOANES
AND
MAURICE J. GONDER
From the Millard Fillmore Hospital Research institute, Bujj'alo, New York
'\Ve have observed electron microscope images of secretory cells in the normal canine prostate. These images suggest to us a sequence of events in the elaboration of secretion from these cells into the glandular lumen. Microscopic studies by other investigators of prostatic epithelium, in various physiological conditions produced by hormones or other experimental treatment, have demonstrated the changes in potential for secretion of the epithelial cells or their appearance before and after secretion. However, because of the apparently rapid release of the secretion and because of the difficulty of temporally following a process in preserved material, the mechanism of cellular secretion has not been established. 1 The only previous electron microscope study of canine prostate of which we are aware describes the general morphology of the epithelial cells. 2 Our results do not contradict these findings. The nature of secretory release in the prostate is interesting in that relatively large quantities of secretion are emitted by the gland in a short period. In addition, remnants of portions of cells or the discrete granular bodies in which the secretion is present intracellularly have not been observed in expelled prostatic secretion. By studying mature normal dogs which were sexually constrained for relatively long periods, we have observed with the electron microscope several stages in the cellular release of secretion. ~IATERIALS AND METHODS
The mongrel dogs, which ,vere obtained from the city pound and ranged in age from 2 to 6 Accepted for publication December 3, 1968. Presented in outline form as an exhibit at the annual meeting of the American Urological Association, ::viiami Beach, Florida, May 13-16, 1968. This study was supported by a grant from the John A. Hartford Foundation, Inc. 1 Price, D. and Williams-Ashman, H. G.: The accessory reproductive glands of mammals. In: Sex and Internal Secretions, 3rd ed. Edited by W. C. Young. Baltimore: The Williams and Wilkins Co., vol. 1, chap. 6, pp. 366-488, 1961. 2 Seaman, A.R. and "\Vinell, M.: The ultrafine structure of the normal prostate gland of the dog. Acta Anat., 51: 1, 1962.
years, were all in good general health as evidenced by observation for at least 1 week. Specimens from the prostates of 12 dogs were examined. The clogs were anesthetized with sodium pentothal and the prostates were surgically exposed by extensive dissection through abdominal (supra.pubic) incisions. Biopsies of the prostate were obtained either with a coring device3 or with a scalpel. All biopsies included tissue extending along a radius near the maximum diameter of the gland from the capsule to the prostatic urethra. Some animals were subsequently sacrificed and others were surgically closed for other studies not discussed in this report. Immediately after removal, the biopsies were placed in the fixative, which was either 3 per cent glutaraldehyde in O.lM phosphate buffer, pH 7.2, containing 0.00llYI CaCh, or 2 per cent osmium tetroxide in the same buffer. The core biopsies, which had a diameter of about 2 mm., were not additionally divided. The slice biopsies were cut into pieces about 1 mm. cubed. Primary fixation was for 2 to 4 hours, except in 3 cases in which the specimens were left in the glutaralclehycle for 3 days. The tissues fixed in glutaraldehycle were washed in buffer for 90 minutes and then post-fixed for 2 hours in the osmium tetroxide fixative. All tissues were dehydrated with ethanol and embedded in epon 812. 4 For light microscopical examination, sections were cut at Ui microns thickness and stained by Richardson's method for Mallory's methylene blue-azure II5 or with this stain in combination with the periodic acid-Schiff procedure. 6 For electron microscopy, silver-reflecting sections were cut with a diamond knife on a Porter-Blum J\IT-2 ultramicrotome, stained with uranyl 3 Jagodzinski, R. V. and Tanski, W.: A new biopsy coring device. J. Lab. Clin. Med., 72 832, 1968. 4 Luft, J. H.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409, 1961. 5 Richardson, K. C., Jarett, L. and Finke, E. H.: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Techn., 35: 313, 1960. 6 Barka, T. and Anderson, P. J.: Histochemistrv Theory, Practice, Bibliography. New York:: Harper & Row Publisher, pp. 73-74, 1965.
734
CANINE PROSTATIC SECRETION
735
Fm. 1. A, low power photomicrograph shows general architecture of normal canine prostate. Densely staining secretory material is concentrated in apical portions of secretory cells but is absent from lumens. Scale represents 10,u. Reduced from X370. B, photomicrograph of part of single acinus of normal canine prostate. Particulate nature of secretory material (SG) is clearly shown. Outlined area is similar to that shown in figure 2. N = nucleus, L = lumen. Scale represents 10,u. Reduced from X 1,470.
acetate and lead citrate7 and examined and photographed in an RCA-EMU-3H electron microscope operated at 100 kilovolts. RESULTS
Figure 1 shows light micrographs of sections of normal dog prostate. Figure 1, A demonstrates the general architecture of the tissue. The acini are closely apposed, separated by narrow, but 7 Reynolds, E. S.: Use oflead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208, 1963.
well-defined, bands of connective tissue stroma. The nuclei are generally in the basal portions of the epithelial cells. There is a high concentration of dark-staining secretory material in the apical portions of the cells. Figure 1, B shows part of a single acinus at higher magnification. The particulate nature of the intracellular secretion substance is evident. Figure 2 is a low magnification electron micrograph of an area similar to that outlined in figure I, B. The apical end is at the top of the picture and the basal end is at the bottom. Several struc-
736
MASER, SOANES AND GONDER
Fm. 2. Electron micrograph of ultrathin section of normal canine prostate. Area shown corresponds in size to that outlined in figure 1, B. Two of numerous apical secretory granules (SG) are indicated by arrows. Other identified structures are nuclei (N), mitochondria (M), and lipid bodies (LB) which are often associated with glycogen granules. Scale represents lµ. Reduced from X 12,400.
tund features are labeled and will be elaborated upon in future reports. The structures of interest for our purposes are the densely staining secretory granules concentrated at the apical ends. Figures 3 aml 4 are electron micrographs at higher magnification of the apical portions of secretory epithelial cells. They represent what we interpret to be events in the sequence of secretion. In figure 3, A, the earliest stage, a line or zone of slight cytoplasmic condensation extends completely across the cell as indicated by the arrows.
In figure 3, B definite plasma membranes have formed in the location of this condensation zone, separating the apical region of the cell from the basal portion. Figure 4, A shows a stage in which the walled-off apical portion has separated from the mother cell. ::\Iicrovilli appear on all surfaces of both the mother and daughter cells. All cytoplasmic organelles and components of both portions appear to be intact and unaltered. In figure 4, B the appearance of the lumen after the apical portion has disintegrated is demonstrated. The
C-"-NI1':E PTlOSTATfC SECRETIOX
:Fm,
electron micrograph shows
737
stage io prostatic
foecretory grnnules are highly coneentrated, beginning to be band of cytoplasm extending between arrows. ~ = nucleus, L lumen. Scale represents Reduced from X 13,800. B, apical secretory package beco1nes isolated from mother cell hr formation of plasma memb1a.nes (arrows) along condensation band. L = lumen. Scale represents lf,. Rednced fror:n Xl9,100.
secreton- ma1.erial has losi it, appeDrauce and bas coalesced into large ::,rnorphom- masses. There are no indicatiou.c, of intact organelles in the lumen. The mother cells all appear to be as the.1· ,Yere before rlle portions separated, except that 1.hr enncen tratic,n •Jf secretor.1· granules i,s not RO great. Figure 3 is t, series of diagrams showing in outlille form on:· .interpretation of the events depicted HJ figmes :3 and 4, re8pectivcly
DTSCUSSIOX
The result6 herein described ,vere ent on the ,.,ariouR preparntion L and 2 were 8electcd for their nrntic qualitie~, and the arcltitec:tnral appearance ~hould not be construecl as being: uniformly typical of ll(Jl'lnal cani1lc J;rostate \Ye emphasize that figures :3 and 4., wbich described the 3ecretion proces~, were not all taken from the same ,;pecimen, but represent our
738
MASER, SOANES AND GONDER
Fm. 4. A, isolated apical package migrates into lumen. Microvilli appear on newly formed surfaces. L = lumen. Scale represents lµ.Reduced from XlS,600. B, package and its contents disintegrate, filling lumen with amorphous mass. Scale represents lµ. Reduced from Xll,800.
interpretive synthesis of the morphological mechanism of secretion. Previous light microscope studies of canine prostate tissue by other investigators used more or less standard methods of fixation and embedding.1 The higher quality of preservation in light microscope specimens prepared as for electron microscopy is demonstrated by figure 1. The subcellular detail, particularly with respect to the discreteness of the secretory granules, is not readily apparent in specimens prepared by standard methods. We have never observed, in canine prostate specimens, extracellular, free-standing secretory
granules nor have we seen evidence of intracellular disintegration of the granules. We have not observed the breaking of apical plasma membranes or the passage of granules through the plasma membrane. However, the concentration of secretory granules within the cells is very high and frequently we see large aggregates of amorphous secretory material in the lumens. The sequence of events we suggest in this report can explain these phenomena. It appears that the secretory cells release into the lumen an intact portion of their apical ends. These discrete apical secretory packages, after separation from the mother cells, rapidly disintegrate with respect
739
CANINE PROSTATTC SECRETI0l''f
l
L
Fm. 5. Diagram cf sequence of events in secretion. Parts A to D correspond to figures 3 and 4, respec-
buth to the patency of the package itself and to the discrete of the secretory granules contained within the package. The in this 1-L a faint band across the cell of zone or· bfancl of condensation form~ 2 distinct '·""'w·""""'" continuous with the lateral boundaries of the cell These r,ell isolate the secretory c.he 1emaining basal portion of the The paelrnge contains a large number of secretory granules and smaller quantities of organelles and ground sub.stance. That these such as in figure 3, B, :Lre newly formed is supported their
lack of microvillar poorly developed in
which, although are numerous
from distanees of several microns or
age 11nd the individual secretory it disintegrate into an 4, B). We think that this cuc,,u,,c,,, occurs since we have never observed a cell stopped by fixation during disintegration . Such a series of events -'",.,,,"-~ the apparnnt
740
MASER, SOANES AND GONDER
anomaly of the transformation of secretion contained in granular form within the cell to an amorphous condition outside the cell, without the occurrence of rupture or invagination of the apical surface. 1¥ e have examined enough specimens in different orientations and in serial steps to be sure that images such as those in figures 3, B and 4, A are not the results of the plane of section intercepting portions of adjacent cells. What we call secretory packages are, in fact, isolated portions of epithelial cells. To our knowledge, this mode of secretion, if it is as we think, can explain some of the puzzling properties of ejaculation. It may be, for instance, that in a dog deprived of sexual opportunity, the prostatic epithelium produces apical secretion in a quantity that saturates its storage capability. Synthesis of secretion may continue past this point (although more slowly) and may produce a continual overflow or residual secretion from the cells. This event is probably the one we have reported here, since the animals we examined were without opportunity for sexual release for at least a week previous to biopsy. Upon proper nervous or hormonal stimulation preceding ejaculation, the packages may be released en
masse, rapidly disintegrate and be available for expulsion by muscular contraction from the gland. These hypotheses are currently being tested in our laboratories. SUMMARY
Electron microscopy and high resolution light microscopy have been used to demonstrate what the authors interpret as several stages in the release of secretion from epithelial cells of normal canine prostates. The suggested secretion mechanism consists of the following stages: 1) the apical end of the cell, containing a high concentration of secretion-filled bodies, is delineated by a band of condensed cytoplasm; 2) plasma membranes form along the condensation band, isolating the apical secretory package from the mother cell; 3) the package migrates into the glandular lumen; 4) the package and its included secretory granules disintegrate into an amorphous pool. This suggested mechanism may explain some of the puzzling aspects of ejaculation. Mr. R.. V. Jagodzinski assisted in much of the operative procedure and made available to us the coring biopsy device.
THE JOURNAL OF UROLOGY
Copyright
© 1969 by The Williams & Wilkins Co.
A POSSIBLE MECHANISM FOR CANINE PROSTATIC SECRETION MORTON D. MASER, WARD A. SOANES
AND
MAURICE J. GONDER
From the Millard Fillmore Hospital Research institute, Bujj'alo, New York
'\Ve have observed electron microscope images of secretory cells in the normal canine prostate. These images suggest to us a sequence of events in the elaboration of secretion from these cells into the glandular lumen. Microscopic studies by other investigators of prostatic epithelium, in various physiological conditions produced by hormones or other experimental treatment, have demonstrated the changes in potential for secretion of the epithelial cells or their appearance before and after secretion. However, because of the apparently rapid release of the secretion and because of the difficulty of temporally following a process in preserved material, the mechanism of cellular secretion has not been established. 1 The only previous electron microscope study of canine prostate of which we are aware describes the general morphology of the epithelial cells. 2 Our results do not contradict these findings. The nature of secretory release in the prostate is interesting in that relatively large quantities of secretion are emitted by the gland in a short period. In addition, remnants of portions of cells or the discrete granular bodies in which the secretion is present intracellularly have not been observed in expelled prostatic secretion. By studying mature normal dogs which were sexually constrained for relatively long periods, we have observed with the electron microscope several stages in the cellular release of secretion. ~IATERIALS AND METHODS
The mongrel dogs, which ,vere obtained from the city pound and ranged in age from 2 to 6 Accepted for publication December 3, 1968. Presented in outline form as an exhibit at the annual meeting of the American Urological Association, ::viiami Beach, Florida, May 13-16, 1968. This study was supported by a grant from the John A. Hartford Foundation, Inc. 1 Price, D. and Williams-Ashman, H. G.: The accessory reproductive glands of mammals. In: Sex and Internal Secretions, 3rd ed. Edited by W. C. Young. Baltimore: The Williams and Wilkins Co., vol. 1, chap. 6, pp. 366-488, 1961. 2 Seaman, A.R. and "\Vinell, M.: The ultrafine structure of the normal prostate gland of the dog. Acta Anat., 51: 1, 1962.
years, were all in good general health as evidenced by observation for at least 1 week. Specimens from the prostates of 12 dogs were examined. The clogs were anesthetized with sodium pentothal and the prostates were surgically exposed by extensive dissection through abdominal (supra.pubic) incisions. Biopsies of the prostate were obtained either with a coring device3 or with a scalpel. All biopsies included tissue extending along a radius near the maximum diameter of the gland from the capsule to the prostatic urethra. Some animals were subsequently sacrificed and others were surgically closed for other studies not discussed in this report. Immediately after removal, the biopsies were placed in the fixative, which was either 3 per cent glutaraldehyde in O.lM phosphate buffer, pH 7.2, containing 0.00llYI CaCh, or 2 per cent osmium tetroxide in the same buffer. The core biopsies, which had a diameter of about 2 mm., were not additionally divided. The slice biopsies were cut into pieces about 1 mm. cubed. Primary fixation was for 2 to 4 hours, except in 3 cases in which the specimens were left in the glutaralclehycle for 3 days. The tissues fixed in glutaraldehycle were washed in buffer for 90 minutes and then post-fixed for 2 hours in the osmium tetroxide fixative. All tissues were dehydrated with ethanol and embedded in epon 812. 4 For light microscopical examination, sections were cut at Ui microns thickness and stained by Richardson's method for Mallory's methylene blue-azure II5 or with this stain in combination with the periodic acid-Schiff procedure. 6 For electron microscopy, silver-reflecting sections were cut with a diamond knife on a Porter-Blum J\IT-2 ultramicrotome, stained with uranyl 3 Jagodzinski, R. V. and Tanski, W.: A new biopsy coring device. J. Lab. Clin. Med., 72 832, 1968. 4 Luft, J. H.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409, 1961. 5 Richardson, K. C., Jarett, L. and Finke, E. H.: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Techn., 35: 313, 1960. 6 Barka, T. and Anderson, P. J.: Histochemistrv Theory, Practice, Bibliography. New York:: Harper & Row Publisher, pp. 73-74, 1965.
734
CANINE PROSTATIC SECRETION
735
Fm. 1. A, low power photomicrograph shows general architecture of normal canine prostate. Densely staining secretory material is concentrated in apical portions of secretory cells but is absent from lumens. Scale represents 10,u. Reduced from X370. B, photomicrograph of part of single acinus of normal canine prostate. Particulate nature of secretory material (SG) is clearly shown. Outlined area is similar to that shown in figure 2. N = nucleus, L = lumen. Scale represents 10,u. Reduced from X 1,470.
acetate and lead citrate7 and examined and photographed in an RCA-EMU-3H electron microscope operated at 100 kilovolts. RESULTS
Figure 1 shows light micrographs of sections of normal dog prostate. Figure 1, A demonstrates the general architecture of the tissue. The acini are closely apposed, separated by narrow, but 7 Reynolds, E. S.: Use oflead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17: 208, 1963.
well-defined, bands of connective tissue stroma. The nuclei are generally in the basal portions of the epithelial cells. There is a high concentration of dark-staining secretory material in the apical portions of the cells. Figure 1, B shows part of a single acinus at higher magnification. The particulate nature of the intracellular secretion substance is evident. Figure 2 is a low magnification electron micrograph of an area similar to that outlined in figure I, B. The apical end is at the top of the picture and the basal end is at the bottom. Several struc-
736
MASER, SOANES AND GONDER
Fm. 2. Electron micrograph of ultrathin section of normal canine prostate. Area shown corresponds in size to that outlined in figure 1, B. Two of numerous apical secretory granules (SG) are indicated by arrows. Other identified structures are nuclei (N), mitochondria (M), and lipid bodies (LB) which are often associated with glycogen granules. Scale represents lµ. Reduced from X 12,400.
tund features are labeled and will be elaborated upon in future reports. The structures of interest for our purposes are the densely staining secretory granules concentrated at the apical ends. Figures 3 aml 4 are electron micrographs at higher magnification of the apical portions of secretory epithelial cells. They represent what we interpret to be events in the sequence of secretion. In figure 3, A, the earliest stage, a line or zone of slight cytoplasmic condensation extends completely across the cell as indicated by the arrows.
In figure 3, B definite plasma membranes have formed in the location of this condensation zone, separating the apical region of the cell from the basal portion. Figure 4, A shows a stage in which the walled-off apical portion has separated from the mother cell. ::\Iicrovilli appear on all surfaces of both the mother and daughter cells. All cytoplasmic organelles and components of both portions appear to be intact and unaltered. In figure 4, B the appearance of the lumen after the apical portion has disintegrated is demonstrated. The
C-"-NI1':E PTlOSTATfC SECRETIOX
:Fm,
electron micrograph shows
737
stage io prostatic
foecretory grnnules are highly coneentrated, beginning to be band of cytoplasm extending between arrows. ~ = nucleus, L lumen. Scale represents Reduced from X 13,800. B, apical secretory package beco1nes isolated from mother cell hr formation of plasma memb1a.nes (arrows) along condensation band. L = lumen. Scale represents lf,. Rednced fror:n Xl9,100.
secreton- ma1.erial has losi it, appeDrauce and bas coalesced into large ::,rnorphom- masses. There are no indicatiou.c, of intact organelles in the lumen. The mother cells all appear to be as the.1· ,Yere before rlle portions separated, except that 1.hr enncen tratic,n •Jf secretor.1· granules i,s not RO great. Figure 3 is t, series of diagrams showing in outlille form on:· .interpretation of the events depicted HJ figmes :3 and 4, re8pectivcly
DTSCUSSIOX
The result6 herein described ,vere ent on the ,.,ariouR preparntion L and 2 were 8electcd for their nrntic qualitie~, and the arcltitec:tnral appearance ~hould not be construecl as being: uniformly typical of ll(Jl'lnal cani1lc J;rostate \Ye emphasize that figures :3 and 4., wbich described the 3ecretion proces~, were not all taken from the same ,;pecimen, but represent our
738
MASER, SOANES AND GONDER
Fm. 4. A, isolated apical package migrates into lumen. Microvilli appear on newly formed surfaces. L = lumen. Scale represents lµ.Reduced from XlS,600. B, package and its contents disintegrate, filling lumen with amorphous mass. Scale represents lµ. Reduced from Xll,800.
interpretive synthesis of the morphological mechanism of secretion. Previous light microscope studies of canine prostate tissue by other investigators used more or less standard methods of fixation and embedding.1 The higher quality of preservation in light microscope specimens prepared as for electron microscopy is demonstrated by figure 1. The subcellular detail, particularly with respect to the discreteness of the secretory granules, is not readily apparent in specimens prepared by standard methods. We have never observed, in canine prostate specimens, extracellular, free-standing secretory
granules nor have we seen evidence of intracellular disintegration of the granules. We have not observed the breaking of apical plasma membranes or the passage of granules through the plasma membrane. However, the concentration of secretory granules within the cells is very high and frequently we see large aggregates of amorphous secretory material in the lumens. The sequence of events we suggest in this report can explain these phenomena. It appears that the secretory cells release into the lumen an intact portion of their apical ends. These discrete apical secretory packages, after separation from the mother cells, rapidly disintegrate with respect
739
CANINE PROSTATTC SECRETI0l''f
l
L
Fm. 5. Diagram cf sequence of events in secretion. Parts A to D correspond to figures 3 and 4, respec-
buth to the patency of the package itself and to the discrete of the secretory granules contained within the package. The in this 1-L a faint band across the cell of zone or· bfancl of condensation form~ 2 distinct '·""'w·""""'" continuous with the lateral boundaries of the cell These r,ell isolate the secretory c.he 1emaining basal portion of the The paelrnge contains a large number of secretory granules and smaller quantities of organelles and ground sub.stance. That these such as in figure 3, B, :Lre newly formed is supported their
lack of microvillar poorly developed in
which, although are numerous
from distanees of several microns or
age 11nd the individual secretory it disintegrate into an 4, B). We think that this cuc,,u,,c,,, occurs since we have never observed a cell stopped by fixation during disintegration . Such a series of events -'",.,,,"-~ the apparnnt
740
MASER, SOANES AND GONDER
anomaly of the transformation of secretion contained in granular form within the cell to an amorphous condition outside the cell, without the occurrence of rupture or invagination of the apical surface. 1¥ e have examined enough specimens in different orientations and in serial steps to be sure that images such as those in figures 3, B and 4, A are not the results of the plane of section intercepting portions of adjacent cells. What we call secretory packages are, in fact, isolated portions of epithelial cells. To our knowledge, this mode of secretion, if it is as we think, can explain some of the puzzling properties of ejaculation. It may be, for instance, that in a dog deprived of sexual opportunity, the prostatic epithelium produces apical secretion in a quantity that saturates its storage capability. Synthesis of secretion may continue past this point (although more slowly) and may produce a continual overflow or residual secretion from the cells. This event is probably the one we have reported here, since the animals we examined were without opportunity for sexual release for at least a week previous to biopsy. Upon proper nervous or hormonal stimulation preceding ejaculation, the packages may be released en
masse, rapidly disintegrate and be available for expulsion by muscular contraction from the gland. These hypotheses are currently being tested in our laboratories. SUMMARY
Electron microscopy and high resolution light microscopy have been used to demonstrate what the authors interpret as several stages in the release of secretion from epithelial cells of normal canine prostates. The suggested secretion mechanism consists of the following stages: 1) the apical end of the cell, containing a high concentration of secretion-filled bodies, is delineated by a band of condensed cytoplasm; 2) plasma membranes form along the condensation band, isolating the apical secretory package from the mother cell; 3) the package migrates into the glandular lumen; 4) the package and its included secretory granules disintegrate into an amorphous pool. This suggested mechanism may explain some of the puzzling aspects of ejaculation. Mr. R.. V. Jagodzinski assisted in much of the operative procedure and made available to us the coring biopsy device.