Cell Tiss. Res. 192, 39-51 (1978) Cell and Tissue Research �9 by Springer-Verlag 1978

The Ultrastructure of the Accessory Sex Organs of the Male Rat

XII. Nuclear Alterations of Prostatic Epithelial Cells Induced by Anti-Androgens

Erik Dahl

University of Oslo, Department of Anatomy, Dental Faculty, Blindern, Oslo, Norway

Summary. The fine structure of the nuclei of epithelial cells of the dorsal lobe of the rat prostate were studied after administration of three different anti- androgenic compounds.

The nucleolus appears to undergo a progressive disorganisation with partial fragmentation and dispersion of its normal components. Different types of intranuclear inclusions were found.

The various alterations observed were often encountered within the same section. This may indicate that the nuclear alterations occur in the same compartment of the cell, and represent a dysfunction of integrated biochemical events occurring within this compartment.

The findings support a view that the stimulatory secretory effect of androgens is mediated via a 'secretory center', located within the nucleolus- associated chromatin. Within this 'secretory center', the initial steps of the secretory process, the binding of the DHT receptor complex to D N A is assumed to occur.

Key words: Prostate - Rat-Anti-Androgens - Nuclear alterations - Electron microscopy.

Introduction

Administration of anti-androgens to rats results in a general involution of the accessory sex organs, with macroscopic atrophy of the different organs, reduced amounts of secretory material and marked ultrastructural alterations of the epithelial cells (Dahl and Hars, 1975). Although the whole prostatic complex consists of androgen-dependent organs, there seem to be differences in the effect of testosterone on the different prostatic lobes (Dahl, 1976). In previous studies, it has been demonstrated that the dorsal lobe, when compared with the other lobes, seems

Send offprint requests to. Dr. Erik Dahl, University of Oslo, Dept. of Anatomy, Dental Faculty, 1052 Blindern, Oslo 3, Norway

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40 E. Dahl

to react differently both to the withdrawel of testosterone (Dahl, 1976) and to the administration of anti-androgens (Dahl and Hars, 1975). This was reflected ultrastructurally by nuclear alterations. Since it was obvious that these nuclear changes were confined to the dorsal lobe, and since their origin, function and significance were unclear, they required further investigation. In the present study, the structural alterations found in the nuclei of the dorsal lobe cells as seen after administration of three different potent anti-androgens are described in detail.

Materials and Methods

Twenty-two albino male rats (derived from the Charles River CDF strain) 4~6 months old, were used in this study. The anti-androgenic compounds used were cyproterone acetate, which will be referred to as Cyp A; S K an F22340 (Smith Kline and French Laboratorie; Philadelphia, Penn.) which will be referred to as SKF, and chlormadinon acetate which will be referred to as chlormadinon. Of the 26 rats used in these experiments, 10 rats served as controls. The anti-androgenic compounds were administered as follows: four rats received 15 mg Cyp A as subcutaneous injections daily for 18 days (total 270 mg Cyp A). Four rats received 15 mg of SKF as daily subcutaneous injections for 18 days (total 270 mg SKF). Four rats were given 15mg chlormadinon as subcutaneous injections for 20 days (total 300mg chlormadinon). The control animals received 1 ml of the solvent as subcutaneous injections over the same experimental periods.

All the animals were sacrificed two days after the last injection. Under ether anesthesia, the aorta was cannulated, and intra-aortic fixation perfusion was carried out as described (Dahl et al., 1973). The dissection of the prostate, the preparation of the samples and the electron microscopic techniques were those already reported (Dahl et al., 1973).

Observations

All the observations made in the present investigation were compared with the results obtained from normal, castrated and testosterone treated rats. Although almost every epithelial cell of the dorsal lobe showed some sort of alteration of the fine structure after administration of the different anti-androgenic compounds, there were quantitative and qualitative differences from cell to cell within a single acinus, and also between various acini, varying from deep nuclear indentations and alterations in the chromatin pattern, to large intranuclear inclusions. As to the effect of the different anti-androgens used, the qualitative alterations seemed to be quite uniform. As to the quantity of the alterations, chlormadinon generally seemed to induce intranuclear inclusions of larger sizes than the two other anti-androgens. On the other hand, the largest intranuclear inclusions seen after administration of chlormadinon were not bigger than the largest inclusions produced by the other components. An attempt to quantify the alterations observed in relation to the number of nuclei examined was cancelled for two main reasons: 1) Repeated screening revealed that almost every nucleus showed some sort of alteration when compared with normal controls. 2) Serial sections of numerous nuclei revealed that their configuration and morphology changed, depending upon the part of the cell to be examined (Figs. 10-13, 18, 19). An attempt to quantify the different types of alterations also had to be cancelled for the same reasons. To avoid a repetitive, detailed description of the various anti-androgens used, the effect of the three compounds is described jointly.

Prostate and Anti-Androgens 41

Fig. 1. Dorsal lobe epithelial cell of control rat. The nucleus is roughly elongated with a rather even nuclear envelope. The nucleolus (Nu) is compact with a smooth contour. Chromatin is indistinct, but slightly more visible along the nuclear envelope, x 15,600

Fig. 2. Dorsal lobe cell after treatment with cyproterone acetate. The nucleolus (Nu) is located peripherally, while the central part of the nucleus is occupied by electron-opaque aggregations (open arrows), intermingled with fibrillar material. The amount of condensed chromatin is increased throughout the whole nucleoplasm, including the nucleolus-associated area (arrow). A small indentation (Id) is seen adjacent to the nuclear alteration, x 17,000

42 E. Dahl

Controls. The nucleus of the epithelial cell of the dorsal lobe is roughly elongated, and shows a distinct, rather even nuclear envelope (Fig. 1). The nucleolus is compact with a smooth contour, Chromatin is rather indistinct, evenly dispersed throughout the nucleus, and shows no focal aggregations. Usually chromatin is slightly more prominent along the nuclear envelope.

Experimental Animals. The Nuclear Envelope. Irregularities of the contour of the nuclear envelope were always present. They appeared as indentations of different sizes and configurations (Figs. 2-4, 6, 14, 18, 19). In some sections they penetrated deeply into the nucleus, being narrow and of the same width throughout their whole length (Fig. 6). In other nuclei, they shoved a narrow base at the periphery, ending in a bulbous expansion near the center of the nucleus (Figs. 14, 18, 19). An attempt to count the number of ribosomes attached to the nuclear envelope within the indentations did not reveal any significant differences when compared with the number of ribosomes associated with the smooth nuclear envelope of the controls, except in the bottom of the indentations. However, since the indentations usually are cut at an oblique angle, the membranes tend to appear fuzzy in this region. For this reason, the number of ribosomes counted may not be correct; a visual impression was that a certain reduction in the number of ribosomes occurred in this area. In some of the larger indentations, typical autophagic vacuoles within the cytoplasm were observed (Figs. 18, 19). Indentations cut transversely appeared as regular intranuclear pseudo-inclusions (Fig. 6).

The Nucleolus. This organelle showed different types of alterations, varying from cell to cell, and, also within different parts of a single nucleolus (Figs. 10-13). In some cells the nucleolus appeared elongated and irregular in shape, being partly disorganized and fragmented (Figs. 10-13). In other cells, areas of electron-opaque masses adjacent to a peripherally located nucleolus were found (Fig. 2). It was not possible, even by studying serial sections, to establish whether these alterations (as seen in Fig. 2) represented a disorganized fragmented elongated nucleolus or alterations of the nucleolus-associated chromatin. While the nucleolus in the controls was located in the central region of the nucleus (Fig. 1), it was very often found more peripherally placed in the animals treated with anti-androgens (Figs. 2, 8).

The Nucleoplasm. When compared with the controls, the chromatin was more prominent adjacent to the nuclear membrane (Figs. 3, 5, 6, 14) and within the peripheral region of the nucleolus (Fig. 2). Aggregates of interchromatin granules (Figs. 5, 10, 18) and circular, pale areas without any membranous demarcation from the rest of the nucleoplasm were regularly encountered (Fig. 3). Although there was an increased amount of condensed chromatin, the nucleoplasm in general appeared pale and more empty-looking than normal (Figs. 3, 5, 8) indicating a possible rarification of the constituents of the nucleoplasm.

Nuclear Inclusions. Several types of inclusions were regularly encountered. They varied, both in size and configuration, and serial sections revealed inclusions of different types within a single cell. Although their form and size seemed to indicate

Fig. 3. Part of a nucleus (N) with a circumscribed, pale area (arrows) without any membranous demarcation from the surrounding nucleoplasm. Adjacent to the pale area an indentation (Id) is seen. Cyproterone. x 13,600

Fig. 4. Intranuclear inclusion of the fibrillar type which occupies a large area of the central part of the nucleus where the nucleolus usually is observed. The inclusion is surrounded by a smooth, single membrane (open arrow), and contains fibrillar material (FM) and concentric membranous configurations (arrow). Several small indentations are also seen. (SKF-treated animals), x 10,200

Fig. 5. Part of a nucleus (N) with a large intranuclear inclusion which consists of a fibrillar (FM) and a flocculent (FL) part located in the vicinity of the area where the nucleolus usually is observed. The single membrane which surrounds the inclusion is disrupted (large arrow). Small, dense, electron-opaque structures (D) are scattered within the nucleoplasm in addition to aggregations of condensed chromatin (arrows). The nucleoplasm appears rather pale and empty showing increased amount of condensed chromatin peripherally. A deep indentation (ld) divides the nucleus in two parts. Chlormadinon. x 17.000

Fig. 6. A nucleus (N), almost completely divided by a deep, narrow indentation with two typical pseudoinelusions (P/) surrounded by condensed chromatin. Chlormadinon. x 13,000

Fig. 7. Two nuclei (N) with different types of inclusions. In the lower nucleus, a pseudoinclusion surrounded by heterochromatin is seen (arrow). This inclusion has formations of membranous configurations surrounded by a single membrane. Note that it contains more heterogenous material than seen in Figure 6. In the upper nucleus a more complex inclusion is seen, composed of a fibrillar material (FM) surrounded by a single membrane, condensed chromatin and alterations within the adjacent nucleoplasm (open arrow). Chlormadinon. x 9100

Figs. $ and 9 demonstrate what is assumed to be an advanced evolution of an inclusion similar to that seen in Figure 5. In Figure 8, aggregations of condensed chromatin supposed to be the remainder of the nucleolus is labelled by arrow, indicating that the alterations observed are located in the nucleolus- associated area. In Figure 9, the inclusion which is almost empty appears as an "intranuclear autophagic vacuole". Contrary to the true cytoplasmic autophagic vacuole seen in Figures 18 and 19, no brim of cytoplasm surrounds the inclusion. FM fibrillar material; FL flocculent material. Chlormadinon. Fig. 8: x 10,400; Fig. 9: • 13,000

Prostate and Anti-Androgens 45

Figs. 10-13. Serial sections of a single nucleus demonstrate the close structural relationship between the deep indentation (Id), nucleolus (Nu), nucleolus-associated chromatin which is assumed to have been converted into visible condensed chromatin (arrows), and the fibrillar alterations (FM) within the nucleoplasm. Fig. 12 illustrates the confluence of nucleolus, nucleolus-associated chromatin and condensed chromatin which surrounds the fibrillar alterations. Note the elongation of the nucleolus (Figs. 11 and 12), the increased amount of condensed nucleolus-associated chromatin (see Figs. 11-12) and the increase in size of the fibrillar alterations (Figs. 11-13). Chlormadinon. Fig. 10:13,000, Fig. 11 : x 17,000, Fig. 12: • 19,500, Fig. 13: x 17,000

Fig. 14. The spatial relationship between a nuclear body (Nb) and the nucleolus-associated condensed chromatin (open arrow). The nuclear body is cut longitudinally and consists of a fibrillar part and a granular part which apparently seem to be extensions of the nucleolus-associated condensed chromatin. Aggregations of electron-opaque masses supposed to be the nucleolus are labelled by arrows. Note the deep, narrow indentation (Id) ending in a bulb, surrounded by condensed chromatin. Chlormadinon. x 17,000

Figs. 15-17. Transversal sections of nuclear bodies similar to that seen in Figure 14, but cut at different levels. Generally they are demarcated from the surrounding nucleoplasm by fuzzy, fibrillar material. In Figure 15 the nuclear body consists of granules only, so densely packed that it appears almost totally electron-opaque, while in Figure 16 the granules are seen as distinct entities. Finally (Fig. 17) the nuclear body is composed of a granular(G) and a fibrillar (FM) part. Chlormadinon. Fig. 15: x 52,000; Fig. 16: x 52,000; Fig. 17: x 52,000

Prostate and Anti-Androgens 47

Figs. 18 and 19. Serial sections of a nucleus (N) with a deep narrow indentation (Id) which divides the nucleus in two parts. From this indentation, a smaller one extends to the right, ending in a bulb containing cytoplasmic debris (arrows). In serial sections a large typical autophagic vacuole (A V) was disclosed (Fig. 19), containing unidentified cytoplasmic material, demarcated from the surrounding cytoplasm by a delicate, distinct membrane (arrows) (see Fig. 9) NB nuclear body. Chlormadinon. Fig. 18: x 10,400, Fig. 19: • 13,000

that they have a rather miscellaneous, structure an analysis based on their localization and appearance revealed that they could be classified into the following three groups: 1) Regular pseudoinclusions, 2) Fibrillar inclusions, apparently originating de novo within the nucleus, 3) Complex inclusions, intermediate in appearance to the two others.

The regular psedoinclusions were easily recognized as circular, double membrane-bound structures, containing cytoplasmic matrix, surrounded by a variable amount of condensed chromatin (Figs. 6, 7).

The fibrillar inclusions which are assumed to originate de novo within the nucleus varied both in size and configuration. Often they were found in the central area of the nucleus (Figs. 4, 5, 7), completely or partly surrounded by a smooth single membrane. The fibrillar matrix varied from a rather dense consistence to a more flocculent appearance. Some of the largest inclusions of this type were almost empty with only remnants of the original content, giving them the appearance of intranuclear autophagic vacuoles (Figs. 8, 9). Serial sections did not reveal any structural communication between the intranuclear inclusions and the cytoplasm.

The complex type of inclusions was recognized by its mingled appearance. Often they were of similar size as the pseudoinclusions, contained concentric

48 E. Dahl

membranous whorls and fibrillar material and was surrounded by an apparently single membrane. Noteworthy is that adjacent to this type of inclusions, alterations within the nucleoplasm were regularly encountered (Fig. 7). Serial sections revealed a very close spatial relationship between the different types of inclusions (Figs. 10- 13).

Nuclear Bodies. Different types of nuclear bodies were regularly observed (Figs. 14-17). They were clearly demarcated from the nuclear chromatin by a zone of fluffy membranous material. Usually the matrix consisted of rather large, distinct granules of the same size as heterochromatin (Fig. 16). The nuclear bodies sometimes were so densely packed with granules that they formed electron-opaque masses (Fig. 15). In others, the content consisted of a inner granular part and a outer fibrillar part (Fig. 17). Serial sections of a single cell, in which a nuclear body was cut in longitudinal direction, revealed that the body seemed to be in direct continuation with the nucleolus-associated condensed chromatin (Fig. 14), at least in this cell. Furthermore, it was disclosed that the nuclear bodies apparently consist of a granular and a fibrillar part which may be completely separated (Fig. 14).

Discussion

Testosterone induces the same basic physiological responses in rodents and man (Barry et al., 1952). Several studies have established that 5-dihydrotestosterone (DHT) is the active principle of the androgenic stimulation of the accessory sex organs, rather than testosterone itself (Andersson and Liao; Baulieu et al., 1968; Bruchovsky and Wilson, 1968; Tveter and Aakvaag, 1969; Tveter et al., 1971). Autoradiographic investigations have demonstrated that the greater part of the labelled androgen seems to be associated with the nuclei. The selective accumulation of testosterone in the prostate is due to an association of DHT with specific cytoplasmic (Mainwaring, 1969) and nuclear (Mainwaring, 1969) proteins, designated as androgen receptors. In the nucleus, the DHT receptor complex is primarily bound to DNA, which subsequently results in a stimulation of RNA synthesis (Mainwaring and Mangan, 1971). Since the androgen receptors have not been isolated in a pure state, the definite association of any cytoplasmic steroid receptor with a particular intracellular structure has yet to be demonstrated. Distribution studies at the cellular level strongly suggest that the receptors are confined to specific cell compartments (Dahl, 1976; Wagner and Hughes, 1974).

It is generally accepted that anti-androgens inhibit the formation of DHT complexes by competitive interference with cytoplasmic and nuclear binding of DHT (Neumann and Steinbeck, 1974). In previous communications, the cytoplasmic alterations following administration of antiandrogens have been described. The major changes included loss of cytoplasm and reduction of Golgi area, rough endoplasmic reticulum and number of free ribosomes (Dahl and Hars, 1975; Dahl and Kjaerheim, 1974).

In the present study, it has been established that administration of anti- androgens to male rats also induces a series of ultrastructural alterations within the nuclei of the epithelial cells of the prostate dorsal lobe. These changes comprise

Prostate and Anti-Androgens 49

disorganization of the nucleolus, alterations within the nucleolus-associated chromatin and the nuclear envelope. Different types of intranuclear inclusions and alterations within the nucleoplasm are also regularly encountered. While some of the inclusions are typical pseudoinclusions, others more likely seem to originate de novo within the nucleus. By studying serial sections it was disclosed that the various alterations observed occurred almost constantly on the same or subsequent sections. This indicates that the different alterations encountered occur within the same cell compartment and are a result of a dysfunction of biochemical events within functionally integrated structure, viz. the nuclear envelope, the nucleoplasm, the nucleolus-associated chromatin and the nucleolus.

Disorganization of nucleoli has previously been demonstrated in rat liver cells after injections of D-galactosamine (Shinozuka et al., 1973). Fibrillar nucleoli have been observed in animal cells after treatment with actinomycin D (Simard and Bernhard, 1966) and D-galactosamine (Shinozuka et al., 1973). There is strong evidence to suggest that fibrillar nucleoli like those induced by galactosamine may represent a common morphological manifestation of the absence of ribosomal RNA synthesis (Shinozuka et al., 1973).

Actinomycin D in low dose is known to selectively suppress RNA synthesis in the nucleolar region where RNA is synthesized (Wagner and Hughes, 1974). From their experiments with actinomycin D given to testosteron-treated castrated animals Liao et al. (1966) concluded that androgen administration enhances RNA synthesis.

When these previous morphological and biochemical results are compared with, and correlated to, the observations made in the present investigation, it seems reasonable to assume that the morphological changes seen in the present study represent the effect of antiandrogens within specific areas of the nucleus, i.e. where the androgen receptors are located. Furthermore, it seems conceivable that the morphological alterations are due to the inhibition of RNA synthesis in this area.

Previously it has been demonstrated that castration induces marked nuclear alterations in the epithelial cells of the dorsal lobe of the prostate (Dahl, 1976). It was proposed that the secretory function of the epithelial cells may be regulated by an intranuclear 'secretory center' located within the nucleolus-associated chro- matin. When micrographs from castrated and anti-androgen treated animals were compared, it was striking that the nuclear alterations found in the two investigations were located within corresponding areas of the nuclear structures, viz. the nuclear envelope, the nucleolus-associated chromatin and within the nucleolus. Furthermore, it was evident that some of the fibrillar nuclear alterations found in the present and previous studies (Dahl, 1976; Dahl and Kjaerheim, 1974; Tveter and Dahl, 1975) originated within the nucleolus-associated chromatin. As mentioned above, fibrillar nucleoli are a morphological manifestation of a suppressed RNA synthesis (Shinozuka et al., 1973). In our study, the cessation of RNA synthesis is supposed to be due to the interference of the anti-androgens with the binding process of the DHT receptor complex to DNA. The present study supports the view (Dahl, 1976) that the stimulatory effect of androgens is mediated via a 'secretory center', located within the nucleolus-associated chromatin. Within this 'secretory center', the initial steps of the secretory process, the binding of the DHT receptor complex to DNA is assumed to occur. It should be emphasized that

50 E. Dahl

although the morphological alterations found in the castrated and the anti- androgenic treated animals were found within corresponding areas of the nuclear structures, they were not identical. This suggests that the alterations found in the present study are most likely due to the specific effects of the anti-androgens. That they should be a result of nonspecific degenerative events in the process of involution triggered by the drugs seems less likely, although it has to be taken into consideration.

Since the animals did not seem to be physically, affected by the drugs during the whole experimental period and since the effects of castration and anti-androgens were confined to the dorsal lobe nuclei, cytotoxic effects of the drugs can be excluded. Should this have been the case, then at least some alterations in the nuclei of the other lobes of the prostatic complex could be expected.

The function and significance of the nuclear bodies are unclear. Since their structure apparently changes in relation to the metabolic state of the nucleus (Dahl, 1970) they may be involved in the secretory process of the cell. Krishan et al. (1967), on the basis of histochemical and enzymatic extractions, suggested that fibrous nuclear bodies do not contain any DNA or RNA. Noteworthy is that in our study, the fibrillar material in the nuclear bodies seems to increase with decreased function of the cell. So far, however, evidence has not been brought forward for the possibility that the nuclear alterations observed start primarily in the nuclear bodies. On the contrary, the changes observed within the nuclear bodies seem to be secondary to the other alterations occurring in the nucleolus associated chromatin.

It should be emphasized that the nuclear changes observed are confined to the epithelial cells of the dorsal lobe. This is most likely due to differences in the metabolic transformation of androgens within the dorsal lobe when compared with the other lobes (Dahl, 1976). Although there are only quantitative morphological differences in the general effects of the anti-androgens used, it should be borne in mind that chlormadinon is both antiandrogenic and antigonadotrophic. However, no really significant quantitative or qualitative differences in their effects on the ultrastructure of the cells have been observed. This seems to indicate that all three compounds exert their effects through similar mechanisms.

References

Anderson, K.M., Liao, S.: Selective retention of dihydrotestosterone by prostatic nuclei. Nature (Lond.) 219, 277-279 (1968)

Barry, M.C., Eidinoff, M.L., Dobriner, K., Gallagher, T.F.: Fate of C14-testosterone and C 14- progesterone in mice and rats. Endocrinology 50, 587-599 (1952)

Baulieu, E.E., Lasnitzki, I., Robel, P. : Metabolism of testosterone and action of metabolism on prostate glands grown in organ culture. Nature (Lond.) 219, 1155-1156 (1968)

Bruchovsky, N., Wilson, J.D.: The conversion of testosterone to 5a-androstan-17B-013-one by rat prostate in vivo and in vitro. J. biol. Chem. 243, 2012-2021 (1968)

Dahl, E.: The fine structure of nuclear inclusions. J. Anat. (Lond.) 106, 255-262 (1970) Dahl, E.: The ultrastructure of the accessory sex organs of the male rat. XI. Nuclear alterations of

prostatic epithelial cells induced by castration. Cell Tiss. Res. 171, 285-296 (1976) Dahl, E., Hars, R.: The ultrastructure of the accessory sex organs of the male rat. 10. Effect of

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Prostate and Anti-Androgens 51

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Accepted March 16, 1978