THE AMERICAN JOURNAL OF ANATOMY 165199-209 (1982)

Testosterone Action on the Ventral Prostate Lobe of the Castrated Rat as Assessed With a Stereologic Morphometric Method

EERO HUTTUNEN, TIM0 ROMPPANEN. AND HEIKKI J. HELMINEN Departments of Anatomy (E.H., H.J.H.) and Pathology (T.R.), Uniuersity of Kuopio, 70100 Kuopio 20, Finland

A B S T R A C T The effects of testosterone (1 mg testosterone propionatel100 g body weight for 30 days) on the ventral prostates of rats castrated 30 days prior to the onset of hormone treatment was investigated with a stereologic morphometric method. Testosterone induced rapid growth of the prostate, which was reflected by a 29-fold increase in acinar parenchyma but only a 2.6-fold increase in interaci- nar tissue. There was a 23-fold increase in the total amount of glandular epithelium and a 36-fold increase in total luminal amount. Various volumetric fractions, total surface area, total tubule length, height of the epithelium, mean diameter of the tu- bules, and mean distance between the tubules and between the glandular centers were calculated. Growth of the acinar parenchyma, especially of the glandular lu- men, was the dominant pattern. We could not demonstrate any remarkable re- sponses in the interacinar tissue during any phase of testosterone treatment.

Castration of experimental animals induces prostatic atrophy, which can be reversed with testosterone treatment (Moore et al., 1930; Price and Williams-Ashman, 1961; Mann, 1964; Brandes, 1974). Recently, histoquantita- tive methods have been developed that ensure precise description of the morphologic events in the prostatic tissue. Bartsch and Rohr (1977) have developed a method especially suitable for electron microscopic assessment of cellular alterations in the prostate. DeKlerk and Coffey (1978), by combining biochemical and histolog- ic methods, were able to show the difference be- tween hyperplasia and hypertrophy of the prostate in histologic sections. We have de- signed a histoquantitative method with which several stereologic morphometric parameters can be calculated (Romppanen et al., 1980). We have applied this technique to the analysis of postcastration atrophy of the rat ventral pros- tate (Huttunen et al., 1981).

The present study attempts to clarify the re- sponse of the ventral-prostate tissue compart- ments, the acinar parenchyma, and interacinar tissue to testosterone treatment initiated 30 days after castration of the animals, when the gland was completely atrophied. Volumetric fractions, surface and length densities, and di- ameters of the glandular tubules as well as

mean free distance between the tubules were calculated. This quantitative information was considered to shed more light on the biological quality of the prostatic tissue, especially on the mode of testosterone action.

MATERIALS AND METHODS Animals, hormone administration, tissue preparation, and microscopy

Adult Wistar rats were used in the experi- ments (Table 1). The animals lived in a con- trolled environment with lights on from 0700 to 2100 and lights off from 2100 to 0700 hours (LD 14:lO). The animals were castrated 30 days prior to hormone treatment. Testosterone pro- pionate (Neohombreol, Organon, Oss, The Netherlands) (dose 1.0 mgllOO g body weighti day; vehicle sesame oil, 0.5 ml) was adminis- tered subcutaneously (at 0830 hours) in the in- guinal region. Control animals received only the vehicle. The animals were killed under light ether anesthesia 1/2,1,2,3,5,7,10,15,20, and 30 days after the initiation of testosteronelve- hicle injections (Table l), beginning the sacri-

Address correspondence to Dr. Heikki J. Helminen, ScD. (Med.). Department of Anatomy, University of Kuopio. P.O.B. 138, 70101 Kuopio 10, Finland.

Received November 2. 1981. Accepted June 1. 1982.

OOO2-9106/82/1652-0199$03.50 0 1982 ALAN R. LISS, INC.

200 E. HUTTUNEN, T. ROMPPANEN, AND H.J. HELMINEN

TABLE 1. Weight of rats (in grams) at end of hormone administratiodobseruation period

0 days ( = 30 days after

castration)

Testosterone- treated

Castrated controls

341 + 26 (51

1 2 1 2 3 5 7 1 0 1 5 hours day days days days days days days

314 333 335 334 323 338 363 1 3 2 +44 +25 1 2 5 +10 +22 +28 (5) (5) (5) (4) (4) (4) (4)

323 f 10 (4)

20 30 days days

341 326 1 1 4 +10 (4) (4)

342 + 51 (4)

_ _ _ ~

Values are given as means +- S.D. Numbers in parentheses refer to number of rats per interval.

fice at 0830 hours. The ventral-prostate lobes were dissected free, weighed in a torsion bal- ance, and fixed in 10% buffered (pH 7.4) forma- lin for 48 hours at 4°C. Sections 5 pm thick were cut from paraffin blocks and stained with Van Gieson’s method for collagen fibers (Luna, 1968; cf. Huttunen et al., 1981). Stained sec- tions were studied under a Wild M 501 semiau- tomatic sampling microscope (Wild Heerbrugg Ltd., Heerbrugg, Switzerland) equipped with a projection head and the Weibel multipurpose graticule supplied with 120 points and 60 test lines (Weibel, 1963; Romppanen et al., 1980). The light micrographs were produced with a Leitz Orthoplan light microscope (Ernst Leitz Wetzlar GmbH, Wetzlar, Federal Republic of Germany).

Morphologic method The ventral lobe of the rat prostate consists

of rodlike or cylindrical, folded, and occasional- ly branching tubules embedded in connective tissue (Price and Williams-Ashman, 1961; Au- muller, 1979). Examination of cross, trans- verse, and vertical sections through the ven- tral prostate revealed that the tubules were randomly oriented (Romppanen et al., 1980). The analysis of six different parallel sections from one prostate (300-400 pm apart from each other) showed that different morphometric pa- rameters varied only slightly between the sec- tions (Romppanen et al., 1980). Thus only one section through each of the two lobes is needed for morphometric analysis (Romppanen et al., 1980). On the other hand, the distribution pat- tern of the glandular elements within a section was not taken into account. The method there- fore gives no information about possible changes in the patterns of distribution be- tween different parts of the gland-e.g., be- tween peripheral and central parts of the gland. Details of the morphometric method have been published previously (Romppanen

et al., 1980; Huttunen et al., 1981). In brief, the Weibel multipurpose graticule (120 points, 60 test lines) was utilized in the projection head of the Wild M 501 microscope, and the systemat- ic field sample technique was used. In micro- scopic fields, where a part was outside the tis- sue (“edge” or “end” effects), only those grid areas lying within the tissue section were counted. The analysis was performed at a mag- nification of x 86. The following parameters were determined:

Volume densities (V,). The point-counting method was used (Weibel, 1963). Altogether, 600 points per ventral lobe section were counted. The following tissue compartments were defined: (1) interacinar tissue (VVIT) and (2) acinar parenchyma (Vv~p), which was fur- ther divided into (3) glandular epithelium (VVEP) and (4) glandular lumen (VVLU). Correc- tion for the “Holmes effect” was performed ac- cording to Weibel and Paumgartner (1978; cf. Romppanen et al., 1980).

Surface density (SvJ. The number of inter- sections ( IL~) per unit length of the test line sys- tem at the cut surfaces of the inner border of the glandular epithelium (SVEP) (“inner epithe- lial surface density”), and also at the basement membrane of the glandualar epithelium (SVBM) (“outer epithelial surface density”) were counted. The corresponding surface densities were calculated according to Weibel(l963):

svi = 2 ILi (Eq. 1)

Length density (Lv) of the glandular tubules (length of glandular tubules in 1 mm3 prostatic tissue). The number of tubule profiles per unit section area (QA) was counted in four test screen areas using the unbiased counting rule (Gundersen, 1977). The following equation was used in the calculation of LV (Weibel, 1963):

Lv= 2 QA (Eq. 2)

TESTOSTERONE ACTION ON CASTRATED-RAT PROSTATE 201

Parameters for the whole ventral prostate. Values expressing total amounts of parame- ters were calculated utilizing relative parame- ters (VV, SV, Lv). The volume of the ventral prostate served as the reference volume. For approximation it was assumed that the specif- ic gravity of the prostate tissue was 1.0 (DeKlerk and Coffey, 1978; Huttunen et al., 1981).

Stereologic calculations. The above parame- ters (VV, SV, Lv) defined the spatial and geo- metrical structure of the glandular tubules on the assumption that the tubules are cylindrical.

Using laws of geometry, the following deriv- atives were calculated (for equations see Romppanen et al., 1980; Huttunen et al., 1981; Underwood, 1970):

1. Mean height of the glandular epithelium (h).

2. Mean diameter of the glandular tubules (DAp) was calculated in two ways: D ~ p l is a derivative of the volume density of the glandular lumen (VVLU), surface density of the glandular epithelium (SVEp), and the mean epithelial height (h):

D ~ p z is a derivative of the volume density of the glandular lumen (VVLU), length density of the glandular tubules (Lv), and the mean epi- thelial height (h):

D ~ p 2 = 2 x \i TvzLEv ~~~~ + 2 h (Eq.4)

3. Mean free distance between the glandu- lar tubules (AAp).

4. Mean distance between the glandular centers (u).

RESULTS

In the present study, the weight of the ven- tral prostate increased from 35 mg to 505 mg (Fig. 3a). Growth of the prostate brought about significant histologic changes in the tissue (Fig. la-e). The glandular epithelial cells seemed to increase in height by day 3 (Fig. Ic), and the diameter of the tubules appeared to in- crease by day 10 (Fig. Id). The proportion of ac- inar parenchyma, especially that of the lumen, was strikingly enlarged by day 30 (Fig. le). On the other hand, omission of testosterone ad- ministration aggravated the microscopic signs

of atrophy in the prostate gland (Fig. la,b). During continued atrophy, the proportion of interacinar tissue seemed to increase and of acinar parenchyma to decrease (Fig. lb).

During testosterone administration the vol- umetric fraction of acinar parenchyma (VVAp) increased gradually from 0.51 to 0.91 mm3/mm3, and that of interacinar tissue (VvIT) decreased from 0.49 to 0.09 mm3/mm3 (Fig. 2a,b). The proportion of glandular lumen (VVLU) increased more (from 0.26 to 0.55 mm3/ mm3) than that of the epithelium (VVEP) (from 0.25 to 0.36 mm3/mm3) (Fig. 2c,d). The change was rapid during the first 10 days, after which it slowed.

Although the volumetric fraction of interaci- nar tissue decreased rapidly during testoster- one treatment, its total amount (VITPR) in- creased from 16 mg to 42 mg (about a 2.6-fold increase) (Fig. 3b). The increment in acinar par- enchyma (VAPPR) was, however, far more pro- nounced, increasing from 16 mg to 465 mg- i.e., about 29-fold (Fig. 4a). The corresponding increase in total glandular epithelium (VEP~R; Fig. 4b) was from 8 mg to 180 mg (about 23-fold), and that of lumen (VLUPR; Fig. 4c) from 8 mg to 285 mg (almost 36-fold). The most striking growth of the tissue compartments oc- curred during the first 20 days.

The surface density (SVEP) (“inner epithelial surfaca density”) decreased abruptly during the first 3 days from 17.6 to 12.0 mm2/mm3, but from then on, much more slowly to 11.2 mm2/ mml (Fig. 5a). In contrast, the total epithelial surface at the glandular lumen (SEPPR; Fig. 5b) increased from 600 mm2 to 5,630 mm2 (a 9-fold increase). The surface density (SVBM) (“outer epithelial surface density”), calculated by counting points on the basement membrane, was in general greater than those mentioned above. S V ~ M decreased from 25.0 mm2/ mm3 to 12.4 mm2/mm3 by day 30 (Fig. 6a). The total epithelial surface at the basement membrane (SBMPR) increased from 880 mm2 to 6,180 mm2 (a 7-fold increase) (Fig. 6b). The diagrams in Figures 5 and 6 resemble each other very much.

The length density of the glandular tubules (i.e., length of glandular tubules in 1 mm’ pros- tate tissue) (Lv; Fig. 7a) decreased in 3 days from 102 to 47 mm/mm’; thereafter the reduc- tion was slow. At day 30 the value of length density of the glandular tubules was 13 mm/ mm3. The total length of the glandular tubules (LPR) had doubled by day 20 (from 3,750 mm to 7,600 mm). It then declined to 6,375 mm at day 30 (Fig. 7b). Tubule length appeared to in-

202 E. HU'ITUNEN, T. ROMPPANEN, AND H.J. HELMINEN

Fig. 1. Photographs of the rat ventral prostate: a) 30 and e) 30 days after testosterone administration. Van days after castration, b) 30 days after the onset of vehicle injection (60 days after castration), and c) 3 days, d) 10 days,

Gieson's method for collagen fibers. X 86. Insets, X 258.

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204 E. HUTTUNEN, T. ROMPPANEN, AND H.J. HELMINEN

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206 E. HU’ITUNEN, T. ROMPPANEN, AND H.J. HELMINEN

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prostate of rats castrated 30 days prior to hormone treat- ment. Ordinate: a) mmimm’; b) mm.

crease very rapidly during the first 2 days (from 3,750 to 4,300 mm).

The height of the glandular epithelium (h) in- creased strikingly during the first 3 days (Fig. 8), from 0.014 mm to 0.034 mm (a 2.4-fold in- crease). At day 30 the height of the epithelium was 0.032 mm. There was no significant differ- ence between the two ways of calculating the mean glandular diameter ( D A P ~ estimated from mean intercept length or D A P ~ from vol- ume density of the lumen and length density of glandular tubules). The mean diameter of glan- dular tubules (DAP) doubled during the first 5 days of testosterone treatment (from 0.09 mm to 0.18 mm) (Fig. 9a,b). By day 30 the diameter was 0.263 to 0.298 mm (3.0- to 3.4-fold in- creases). The mean free distance between the glandular tubules (LAP), the “thickness of in- t,eracinar tissue,” declined, after an initial in-

crease at day 3 (0.17 mm), from 0.14 mm to 0.09 mm (Fig. 9c). The mean distance between the glandular centers (a) increased from 0.23 to 0.34 mm by day 3 (Fig. 9d), but did not change thereafter (0.35 mm at day 30).

In castrated control animals that received sesame oil instead of hormone treatment, atro- phy of the prostate continued. At 60 days post- castration (day 30 of the observation period), the weight of the ventral prostate was 24 mg (Fig. 3a). The proportion of interacinar tissue increased to 0.64 (Fig. 2a), whereas the corre- sponding values for glandular epithelium and lumen declined to 0.25 and 0.11, respectively (Fig. 2c,d). The total amount of interacinar tis- sue, however, did not change (Fig. 3b). On the other hand, the total amount of acinar paren- chyma was further reduced from 18 to 9 mg, 60 days after castration (Fig. 4a,c). The total epi-

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Fig. 8. The effect of testosterone administration on the height (h) of the glandular epithelium in the rat ventral pros-

thelial surface ( S E ~ ~ ~ ) declined simultaneous- ly from 600 mm2 to 300 mm2 (Fig. 5b). The total length of glandular tubules (LPR) decreased from 3,750 mm to 2,800 mm (Fig. 7b). The height of the glandular epithelium (h) in- creased slightly, from 0.014 to 0.020 mm (Fig. 8). The mean diameter of the glandular tubules (DAp) decreased from 0.09 to 0.08 mm (Fig. 9a,b) during the observation period. The mean distance between the glandular centers (u) in- creased from 0.23 mm to 0.33 mm (Fig. 9d). The mean free distance between the glandular tu- bules (AAp) almost doubled from 0.14 mm to 0.26 mm (Fig. 9c).

DISCUSSION

The experimental conditions of the present study were chosen to take advantage of the well-known restorative action of testosterone on prostate tissue with castration atrophy (e.g., Moore et al., 1930; Price and Williams- Ashman, 1961; Mann, 1964; Tuohimaa et al., 1973; Aumuller, 1979). Testosterone treat- ment was initiated 30 days after castration when the prostate atrophy was complete (Hut- tunen et al., 1981). In this way we could mini- mize the effect of the early reaction to castra- tion (Lesser and Bruchovsky, 1974). The high dose of testosterone propionate used in this study (1 mgilOO g b.w.) induced rapid growth of the ventral prostate. By selecting this dose we wanted to exert strong androgen action on the prostate with substantial changes of the tissue compartments.

Some previous investigators have used smaller doses of testosterone propionate (0.5-1 mgiday s.c.) (Yamanaka et al., 1975; Tuohimaa et al., 1973), and others have employed larger doses (5 mgiday s.c.) (DeKlerk and Coffey, 1978), even as high as 10 mgi100 g body weight (Brandes, 1974).

tate of rats castrated 30 days prior t o hormone treatment. Ordinate: mm.

We induced striking changes in the tissue compartments of the prostate which could be assessed with the stereologic morphometric method (Romppanen et al., 1980). During the observation period the weight of the ventral lobe increased from 35 to 505 mg, whereas the weight was only 301 mg in noncastrated con- trol animals (Huttunen et al., 1981). This vigor- ous reaction of the ventral prostate can be due to the increased sensitivity of the prostatic ceIIs to androgens after a prolonged interval between castration and testosterone adminis- tration (Tuohimaa, 1980).

Previously published stereologic morpho- metric investigations have not contained as much quantitative data about the action of tes- tosterone on the rat ventral prostate as the present study. Bartsch and Rohr (1977) pub- lished a morphometric method applicable to this task at the electron microscopic level. DeKlerk and Coffey (1978) developed a method called biomorphometrics with which hypertro- phy and hyperplasia of the prostate could be distinguished at the light microscope level. DeKlerk and Coffey (1978) concluded that tes- tosterone treatment (5 mglday for 10 days) completely restored epithelial cell size and stromd cell number and size, but not epithelial cell number. A thorough histoquantitative in- vestigation of rat ventral prostate was carried out by Arvola (1961). He restored the atro- phied prostate with alternate-day testosterone treatment (0.05 to 2.0 mg every second day for 3 weeks). In this study the “weight indices” of “lumen” increased the most. Growth was less striking in the “epithelium,” but even the amount of “stroma” increased. Other stereolog- ic morphometric parameters were not recorded.

Results of the present study confirm those of Arvola (1961), and also extend our insight into

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Fig. 9. The effect of testosterone administration on the mean diameter (DAP) of the glandular tubules in the ventral prostate of rats castrated 30 days prior to hormone adminis- tration. In (a) D ~ p l is estimated from the mean intercept

length, and (b) DApz from V v ~ u and Lv; (c) shows the mean free distance of the glandular tubules ( h ~ p ) ; and (d) the mean distance between the glandular centers (u). Ordinate: mm.

TESTOSTERONE ACTION ON CASTRATED-RAT PROSTATE 209

the alterations of the tissue during testoster- one stimulation. In conclusion, there was a 29-fold increase in the volumetric fraction of the acinar parenchyma (Fig. 2). The proportion of glandular lumen increased more than that of epithelium. The concurrent increase in the amount of interacinar tissue was “only” 2.6-fold (Fig. 3). The surface of the glandular epithelium showed 9- to 7-fold increases (Figs. 5,6). Total length of the glandular tubules al- most doubled (Fig. 7). The height of the glandu- lar epithelium experienced a 2.4-fold increase (Fig. 8). The diameter of the tubules more than tripled (3.0- to 3.4-fold increase), whereas the distance between the glandular tubules was re- duced (Fig. 9). Thus, the prostatic parenchy- mal tissue shows a distinct preponderance over the interacinar tissue after testosterone treatment. However, the quantitative differ- ence does not rule out the possible crucial ini- tial effect, or qualitative significance, of the mesenchymal tissue in the transmission of an- drogenic effect to the acinar parenchyma.

During the last few years increasing atten- tion has been paid to the role of mesenchymal tissue in the development of prostatic epitheli- um (Muntzing et al., 1979; Cunha and Lung, 1980). The cellular details of interstitial tissue have been thoroughly studied (Flickinger, 1972). Connective tissue has been considered to provide a suitable, and necessary, microen- vironment that permits androgenic response (Cunha and Lung, 1980). The mesenchyme of prostate tissue is also regarded as having an in- ductive property. I t is possible that this induc- tive property was activated in the present tes- tosterone-induced restoration of the prostatic tissue, although in quantitative terms we could not demonstrate this mesenchymal par- ticipation. We could find no reaction in the in- teracinar tissue seen 12 hours after castration (Huttunen et al., 1981). The response of the glandular epithelium of the rat ventral pros- tate was much stronger than the reaction in the connective tissue. Whether the role of con- nective tissue was crucial in directing and modifying the hormonal response remains to be shown.

ACKNOWLEDGMENTS

This work was supported by grants from the Medical Research Council of the Academy of Finland. The skillful technical assistance of Mrs. Eij a Voutilainen and Mrs. Maij a Pellikka is gratefully acknowledged.

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