JOURNAL OF ULTRASTRUCTURE RESEARCH 60, 1-11 (1977)

Ultrastructural Studies on the Effect of Testosterone, 5o~- Dihydrotestosterone, and 5~-Androstane-3c~,17~x-diol on the Canine

Prostate Cultured In Vitro

F, SINOWATZ, J. A. CHANDLER, AND C. G. PIERREPOINT

Tenovus Institute for Cancer Research, Welsh National School of Medicine, Heath Park, Cardiff, CF4 4XX, Wales

Received July 20, 1976

Organ culture of canine prostate was performed with testosterone, 5a-dihydrotestosterone and 5a-androstane-3~,17a-diol in a defined medium (199) in concentrations of 10 -~ to 10 -5 M. Addition of 5a-androstane-3a,17a-diol to the medium was found to mair/ tain the epithelial cells in the i r functional polarity and secretory processes. In the presence of testosterone or 5a- dihydrotestosterone degenerat ive changes took place progressively up to a period of 5 days. Failure to ma in t a in the cells was accompanied by interference with the secretory processes within the endoplasmic ret iculum and Golgi apparatus. Prolonged culture with 5a-androstane- 3a,17a-diol produced s t imulat ion of basal cell growth with consequent hyperplasia. The observations confirm biochemical evidence tha t 5a-androstane-3a,17a-diol is the principal active androgen in the dog prostate.

In the dog, as in some other carnivores, the prostate is the only male accessory sex gland (29). In addition, the canine pros- tate, as in man, displays an age-related tendency to develop benign hyperplasia and adenocarcinoma (15, 20, 21). For this reason dogs are widely used for the study of prostatic disease.

The growth and function of the prostate gland has been demonstrated by many in- vestigators to be controlled by testicular androgens, while more recent experimen- tal evidence suggests that many of the apparent androgenic effects of testosterone are mediated by intracellular metabolites, such as 5a-dihydrotestosterone (1, 5, 23); 5a-androstane-3a,17fi-diol and 5a-andros- tane-3B,17fl-diol (2, 3, 26); and 5a-andros- tane-3a,17a-diol (7, 9-12, 16).

In organ culture the prostate gland re- mains androgen-dependent and responsive to both testosterone and its metabolites (19), thus providing a convenient tech- nique for studying the action of various androgens on the prostatic epithelium.

The effects of testosterone and its main metabolites on canine prostate in organ culture have been examined at the light

microscope level (32) but to further eluci- date the action of these hormones on the epithelial cells, electron microscopical ex- aminations were considered essential. In the study reported here, the effects of tes- tosterone, 5~-dihydrotestosterone and 5~- androstane-3a,17a-diol have been ex- tended to the ultrastructural level

MATERIALS AND METHODS

The prostates of four mature dogs were removed under aseptic conditions after pentobarbitone so- dium (Euthatal , May & Baker Ltd, Dagenham, Es- sex) euthanasia . Fa t and connective tissue were re- moved and the g landular t issue was cut into pieces approximately 1-2 mm 3. Some tissues were fixed immediately for electron microscopy while others were prepared for organ culture s imilar to the method of Trowell (35).

In each culture dish the tissue explants were supported on strips of moistened lens paper rest ing on stainless steel rafts and par t ly submerged in the medium (1 ml). Cultures were incubated at 37.5°C in an atmosphere of 95% oxygen, 5% COs. A defined medium (30) was employed, using medium 199 (Bio- cult Laboratories, Paisley, Scotland) containing 200 IU penicillin and 100 ttg/ml streptomycin. Serum and other nonexper imental growth promoters were avoided. Testosterone and 5a-dihydrotestosterone were supplied by Sigma, London. 5a-Androstane- 3a,17a-diol was a generous gift of Schering Chemi- cals Ltd, U.K. The steroids were added to the cul-

Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 0022-5320

SINOWATZ, CHANDLER AND PIERREPOINT

ture dishes in I t~l ethanol at final concentrations varying from 10 _7 to 10 5 M. Control cultures re- ceived the vehicle alone.

Uncultured controls, and explants after 1, 3 and 5 days in culture were fixed in 3% glutaraldehyde in 0.01 M phosphate buffer (pH 7.4) for 3 hr. The speci- mens were subsequently washed for 2 hr in 0.1 M phosphate buffer (pH 7.4), then postfixed in 1% os- mium tetroxide (Millonig's pH 7.4) for 1 hr and further washed in buffer. After dehydration in a graded series of alcohols and immersion in propyl- erie oxide, the tissue was embedded in Araldite.

Semithin (1 ~m) sections were cut, mounted on glass slides and stained in toluidine blue for histo- logical examination. Ultrathin sections were then cut on an LKB ultramicrotome, collected on unsup- ported grids and stained with uranyl acetate and lead citrate. They were examined in an AEI EM6B electron microscope.

RESULTS

a. Noncultured Control Tissue

The u l t ras t ruc tu re of the normal canine prostate is shown in Fig. 1, and has been described by a number of invest igators (4, 22, 24, 31). The prostat ic ep i the l ium con- sists of two types of cells: h igh columnar secretory cells containing large numbers of dense secretory granules , and small, flat- tened or t r i l a te ra l basal cells which are character ized by few cytoplasmic organ- elles and large amounts of glycogen (34). The nuclei of the secretory cells occupy an ex t remely basal position and often exhibi t large nucleoli, while in the basal cells the nuclei are f requent ly indented (Fig. 2).

Rough endoplasmic re t icu lum in the se- cretory cells varies in extent but is gener- ally only modera te ly developed. The basal cells, however, contain only a few scat- tered profiles of rough endoplasmic reticu- lum. In both cell types, there are m a n y free ribosomes in the cytoplasm.

The secretory cell mi tochondr ia are usu- ally oval or e longated wi th a modera te ly dense ma t r ix and plate-l ike cristae, while those in the basal cells are smal ler and oval or round in shape. The supranuc lear cytoplasm of the secretory cells contains a well-developed Golgi complex from which a large number of dense, spherical secre- tory granules arise and which almost com-

pletely fill the apical par t of the cell. Nu- merous short microvilli can be seen on the luminal cell apex.

b. Cultured in the Absence of Hormones

In prostat ic explants, grown in a me- dium wi thout hormones for I day, the number of secretory granules was mark- edly decreased and confined to the apical region which had become reduced in size and exhibited fewer microvilli (Fig. 3). The endoplasmic re t icu lum of m a n y epi- thelial cells appeared enlarged and filled with an electron-dense mater ia l , while there was an increase in the n u m b er of lipid droplets, lysosomes and autophagic vacuoles in the cytoplasm. The nuclei of the secretory cells r emained re la t ively un- changed.

After 3 days, degenera t ive changes were more pronounced, the cells had lost the i r polari ty and there were m a n y bizarre types. Secretory granules and microvilli had almost completely disappeared, while there was a fu r the r increase in the amoun t of endoplasmic re t iculum, lysosomes and autophagic vacuoles. There were m a n y de- genera ted cells in the lumen. Some nuclei appeared pyknotic, with i r regu la r shapes and condensed chromatin . The basal lain- ina had become loose, extensive, and in- vaded the s troma.

After 5 days, degenera t ion of the cells was almost complete wi th just a few non- polarized cells surviving.

c. Cultures with Testosterone and 5a-Di- hydrotestostero ne

Ult ras t ruc tu ra l features of prostat ic epi- the l ium cul tured wi th tes tos terone or 5a- dihydrotes tosterone were very similar and are therefore described together .

After 1 day the height of the epithel ial cells was drast ical ly reduced, especially in the apical region (Fig. 4) and the cells had par t ia l ly lost the i r polari ty. A modera te number of secretory granules remained , vary ing in size and dis t r ibuted th roughou t the cells. In m a n y cells the endoplasmic

FIo. 1. Normal dog prostate. The epithelial cells (E) are tall and columnar. Note the abundance of dense, apical, spherical secretory granules and the underlying basal cell (B). Evidence of apocrine secretion is found in the lumen (L). x 6000.

FIG. 2. Basal cell in normal prostate showing marked indentat ions of the nucleus and cytoplasmic glycogen (GL). x 6000.

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FIG. 3. Epithelium after i day control culture. The ceils begin to lose polarity, the endoplasmic reticulum~ is increased and filled with electron-dense material (ER), and there is an increase in the lipid content, Cellular debris fills the lumen (L). LIP, lipid droplets, x 12000.

FIG. 4. One day after culture with testosterone (similar to 5a-dihydrotestosterone). The cells are flat- tened and have lost polarity. The greatly increased endoplasmic reticulum (ER) is again filled with electron- dense material and there are abundant autophagic processes, lysosomes (LYS) and lipid droplets (LIP). The basal lamina (BL) has become loose and penetrates the stroma. × 6000.

FIG. 5. The endoplasmic reticulum in epithelial ceils is filled and fragmented after 3 days culture with testosterone or 5a-dihydrotestosterone. In some regions the change from normal appearance can be seen (arrow). x 30 000.

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reticulum had become extensive and filled with electron-dense material, the cister- nae pinching off into short segments. There were many large lipid droplets and autophagic vacuoles, several elongated mitochondria, but little development of the Golgi apparatus. The nuclei mostly appeared quite normal except for occa- sional pyknotic cells.

After 3 days culture the outstanding characteristic of each cell was the great increase in amount of endoplasmic reticu- lum. This exhibited a fragmented, par- tially dilated form with decreased num- bers of attached ribosomes, most cisternae being filled with electron-dense material (Fig. 5). Again the Golgi apparatus was not maintained. Many microfibrils were found scattered throughout the cells which had become very flattened in profile. The nuclei showed pronounced irregularity, shrinkage and peripheral distribution of condensed heterochromatin. Degenerative changes were also evident in the stroma and the lumen was filled with cellular de- bris. The amount of collagen had mark- edly increased compared with the uncul- tured controls. After 5 days there was even greater degeneration and most acini were devoid of surviving cells while some had the appearance of those described after 3 days. The density of collagen in the stroma had further increased.

d. Cultures with 5a-Androstane-3a,17a- diol

In the explants cultured in medium con- taining 5a-androstane-3a,17a-diol the early structural changes compared to the controls before explantation were small. After 1 day the high columnar appearance of the secretory cells was maintained and many secretory granules were observed in the apical region of the cells (Fig. 6). The microvilli were well maintained as were most cytoplasmic organelles, although some mitochondria were elongated. The epithelial cells had retained their polarity and there was an increased infolding of the

SINOWATZ, CHANDLER AND PIERREPOINT

plasma membrane in the lateral regions with a greater number of microfibrils in the cytoplasm. Mitoses could be observed in a number of cells particularly those lo- cated basally (Fig. 7). The endoplasmic reticulum did not appear to be greatly in- creased or to take on the form shown in the previous cultures although several secre- tory granules were apparent developments from the fragmented endoplasmic reticu- lum (Fig. 8).

After 3 days of culture the epithelial cells in many acini still appeared normal (Fig. 9). An outstanding feature of this tissue, however, was the stimulation of the basal cells which had increased greatly in size. The nuclei were quite pale and more spherical compared with control cells, while there was frequently more than one prominent nucleolus. The endoplasmic re- ticulum was more developed and there was a greater number of mitochondria but a lowered quantity of glycogen in each cell. Attachment between basal and epithelial cells were less cohesive than in control tissue, but the basal lamina was well maintained.

After 5 days many acini appeared simi- lar to those seen after 3 days culture, with secretory epithelial cells being maintained in their normal polarity and height. I n some acini, however, there were clusters of poorly differentiated basal cells in appar- ently hyperplastic acini (Fig. 10) where the epithelial cells had been replaced. These basal cells had no polarity and the plasma membrane was extensively inter- digitated on all sides. The nuclei w e r e rounded and often had prominent nucleoli. In the cytoplasm there were few organelles besides small mitochondria, free ribo- somes, and small autophagic products. The Golgi apparatus was moderately well developed and microfibrils encircled the nuclei in many cells.

DISCUSSION

The advantages of organ culture meth- ods in studying the maintenance and

Fia. 6. Maintenance of epithelial cells after I day culture with 5 a-androstane-3 a, 17a-diol. The secretory mechanism is maintained but fewer granules are spherical (SG). Polarity is not lost. Mitochondria are elongated (MIT), the lateral plasma membrane is increased (M) and there are abundant microvilli (MV). × 9000.

FIG. 7. Mitosis is observed in some basal cells after 1 day culture with 5~-androstane-3a,17a-diol. × 25OO.

Fro. 8. Secretion is maintained after 1 day culture with 5a-androstane-3a,17a-diol but the spherical granules are not fully developed (arrow). x 15 000.

FIG. 9. After 3 days culture with 5a-androstane-3a,17a-diol the polarity and differentiation are still maintained in many epithelial cells (E) but basal cells (B) are stimulated and many have more than one nucleolus, x 4500.

FIG, 10. Hyperplasia occurs after 5 days culture with 5~-androstane-3a,17a-diol due to replacement of epithelial cells with basal cells. These cells remain undifferentiated, however, with plasma membrane (M) being extensively produced along all sides. Microfibrils (F) surround the nucleus, x 6000.

CANINE PROSTATE IN VITRO

growth of prostatic tissue of various spe- cies has been shown on many occasions (19, 36). Compared with other in vitro methods organ culture permits tissue maintenance with a minimum disturbance of the normal histopathology. This is of special importance for the investigation of hormonal effects on prostatic tissue in which the epithelial and stromal compo- nents are both influenced by hormones (19).

Furthermore, it was considered neces- sary when studying the influence of hor- mones on prostatic tissue in culture to use a defined medium, i.e., one without added serum, since serum contains unknown amounts of various endogenous hormones which could compete or act synergistically with the test substances.

Degenerative changes in the epithelium of prostatic explants grown in a non-hor- mone-supplemented medium have been described for several species, e.g., rat (14), man (36) and dog (32), and resemble the alterations observed following castration. In the experiments described, degenera- tive changes in dog prostate explants were seen after I day of culture in the absence of hormones. In particular the ribosomal pro- tein synthesizing and secretory processes appeared greatly altered.

In the rat (14) testosterone and 5a-dihy- drotestosterone fully maintained the nor- mal prostatic ultrastructure with numer- ous secretory granules and microvilli, well-developed Golgi apparatus and endo- plasmic reticulum. With canine prostate, however, both hormones in concentration from 10 -7 to 10 -5 M failed to prevent severe morphological changes. In addition to a marked decrease in the number of secre- tory granules and microvilli, considered indicators of good maintenance, the changes in the rough endoplasmic reticu- lum were again outstanding.

In contrast to these observations, the explants grown in the presence of 5a-an- drostane-3a,17a-diol showed compara- tively little ultrastructural changes in the

first 3 days although displaying a tendency to basal cell hyperplasia with prolonged culture time.

These results reinforce those previously reported from this laboratory (9-13) that 5a-androstane-3a,17a-diol is the active androgen in the canine prostate. Apart from the demonstration of the uptake of this steroid by a cytoplasmic protein recep- tor in the dog prostate (10) and its trans- ference to the nucleus (13) in the presence of prostatic cytosol, it induces an enhance- ment of RNA polymerase (7, 12) in vitro and maintains polysome structure and polypeptide synthesis in vivo in the cas- trate animal (8). The regulation of the synthesis, methylation and maturat ion of rRNA precursors appears also to be under its control (8).

An outstanding feature of the cultures with 5a-androstane-3a,17a-diol was the apparent stimulation of the basal cells which clearly differ in their hormonal re- sponse compared to epithelial cells. The proliferation of the basal cells started on Day 1 and led to hyperplasia in a number of acini on Day 5.

Basal cells have been previously de- scribed in detail in the prostate of both dog and rat (34) and have been shown to be stimulated, in the rat at least, under endo- crine conditions adverse to the mainte- nance of normal secretory epithelium dur- ing experimentation both in vivo (33) and in vitro (6). In the culture experiments reported here it would seem that during prolonged culture with 5a-androstane- 3a,17a-diol the eventual failure of epithe- lial cell maintenance stimulates the re- placement of these cells by basal cells which, under the defined culture condi- tions, fail to fully differentiate. Possibly the absence of other factors (e.g., protein hormones) within the medium provided this stimulus.

The observation in the 3-5 daY cultures with 5~-androstane-3~,17a-diol that epi- thelial cells become separated from the basal cell layer and from the basement

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membrane (Fig. 9) suggests that stimula- tion of the basal cells is associated with isolation of the epithelial cells from the normal and beneficial epithelial-stromal interaction. Such isolation may also be caused by a thickening of the basal lamina or by an increase in the connective tissue. The basal lamina is synthesized by the epithelial (27, 28) and, possibly, the basal cells and acts as a diffusion barrier to rapid ion or molecular exchanges.

The outstanding characteristic of the ep- ithelial cells cultured in medium with tes- tosterone, 5a-dihydrotestosterone or with- out added steroids, was the interruption of the secretory mechanism at the endoplas- mic reticulum. Protein was still appar- ently being synthesized and stored in the cisternae of the endoplasmic reticulum but was not passing to the Golgi apparatus for eventual secretion. Consequently the en- doplasmic reticulum became increased and pinched off into small fragments (Fig. 5). The focal detachment of ribosomes from the rough endoplasmic reticulum was sim- ilar to that described by Helminen and Ericsson (18) in rat prostate after castra- tion. With the 5a-androstane-3a,17a-diol added to the culture, the secretory and packaging processes were maintained over the short time, but even then the secretory granules had a somewhat immature ap- pearance (Fig. 8).

It fs well established that the cellular synthesis of protein is controlled by steroid hormones (37) and that removal of these hormones by castration interferes with the endoplasmic reticulum and Golgi appara- tus (17, 18). Castration of the dog, how- ever, leads to degenerative cellular changes quite different to those observed in these cultured explants (25) in which disruption of cellular function appeared to occur much later in the synthesis and se- cretory process.

The failure of the Golgi apparatus to maintain the formation of secretory prod- ucts is paralleled by the loss of orientation in those cells cultured without the 5a-an-

SINOWATZ, CHANDLER AND PIERREPOINT

drostane-3~,17~-diol. The Golgi apparatus may therefore be ceasing to produce and distribute heterogenously those molecules necessary to maintain the functional po- larity. The same result is found in the long-term culture with 5a-androstane- 3~, 17~-diol.

The observations made here confirm previous biochemical results that 5c~-an- drostane-3~,17~-diol, rather than testos- terone or 5~-dihydrotestosterone is the principal active androgen in the dog pros- tate.

The authors are grateful to the Tenovus Organi- zation for f inancial assistance and laboratory facili- ties. One of us (F.S.) wishes to t h a n k the Bri t ish Council for the receipt of a Bri t ish Council Scholar- ship.

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