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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2008 Feb;89(1):13–24. doi: 10.1111/j.1365-2613.2007.00550.x

Age-related histopathological lesions in the Mongolian gerbil ventral prostate as a good model for studies of spontaneous hormone-related disorders

Silvana Gisele Pegorin Campos *, Cristiani Zanetoni *, Wellerson Rodrigo Scarano *, Patrícia Simone Leite Vilamaior †, Sebastião Roberto Taboga †
PMCID: PMC2525758  PMID: 18197870

Abstract

The Meriones unguiculatus (Mongolian) gerbil has demonstrated significant prostatic responses to hormonal treatments, and to drugs against human prostatic hyperplasia Spontaneous neoplasia develops in the older animals. Thirty gerbils (age 18 months) were divided into non-affected and prostatic lesion bearers and the prostate lesions were evaluated morphologically, immunohistochemically and quantitatively. The most frequent changes were in epithelial sites and, namely prostatic intraepithelial neoplasias, microinvasive carcinomas and adenocarcinomas. In the stromal compartment, cellular hyperplasia, when verified, was always associated with the sites of anomalous epithelium. Additionally, larger deposition of collagen fibrils, generating stromal fibrosis, was found in all the old gerbils analysed. The quantitative analysis showed that prostatic tissue proportions differed in altered areas, being specific for each lesion type. Isolated nuclear and nucleolar parameters were not effective in diagnosing the malign potential of lesions. However, the cellular proliferation and death indexes indicated larger cellular turnover in invasive lesions such as carcinomas. With these analyses, it could be verified that old gerbils present high propensity to develop spontaneous prostate changes and this may aid in a better understanding of the biological behaviour of human prostate cancer.

Keywords: ageing, cellular proliferation, gerbil, prostate cancer, prostatic intraepithelial neoplasia, ventral prostate

The prostate is an accessory gland of the male urogenital system that contributes to the production of nutrients for seminal fluid and promotes the maintenance of the ionic gradient and appropriate pH in this secretion (Untergasser et al. 2005). At the present time, great clinicopathological importance has been attributed to this gland since prostate adenocarcinoma emerged as the main neoplasia affecting men (Taplin & Ho 2001; Schulz et al. 2003). Despite the progress in understanding prostate carcinogenesis in recent years, it remains a complex disease with a poorly understood natural history (Bonkhoff & Remberger 1998; Huss et al. 2001).

Hormonal imbalances related to the ageing process, involving accentuated decrease in the proportions between androgenic and oestrogenic hormones, can contribute to the evolution of pathological prostate alterations (Banerjee et al. 2001; Schulz et al. 2003). In addition to a hormonal imbalance there is a progressive loss of homeostasis between the processes of cellular proliferation and apoptosis in prostatic tissue during ageing (Bostwick et al. 1996; Tang & Porter 1997).

Existent anatomical similarities between the human and rodent prostate have helped sustain the application of murine models for studies of molecular alterations that accompany the normal development of the organ, as well as the progression of prostate cancer (Pollard & Luckert 1986, 1987, 1992; Huss et al. 2001). In the two species, prostate gland development starts from the urogenital sinus and in both it is an androgen-sensitive organ. The cellular populations are similar and probably perform the same physiological functions. The main differences between human and rodent prostates regard the macro- and microscopic glandular anatomy; and these differences can impair the interpretation of pathological alterations in rodents (Abate-Shen & Shen 2000;Cunha et al. 2004).

Our research group adopted as an experimental model for prostate study the Meriones unguiculatus gerbil. The prostate of this animal presents lobes very close to each other and, therefore, anatomically the gland is more compact in relation to other rodent species (Zanetoni & Taboga 2001; Pegorin de Campos et al. 2006). Additionally, the model has presented significant responses to hormonal treatments (Santos et al. 2006; Scarano et al. 2006; Oliveira et al. 2007), to drugs against human prostatic hyperplasia (Corradi et al. 2004) and to development of spontaneous neoplasias associated with ageing, in which the latter arise autochthonously (Zanetoni & Taboga, 2001; Pegorin de Campos et al. 2006).

From these previous studies concerning the gerbil prostate, the objective of this work was the characterization and classification of spontaneous lesions in the ventral prostate of the old gerbil to evaluate its potential as a new model for prostatic microenvironmental studies of hormone-related spontaneous proliferative diseases.

Materials and methods

Animals, sample preparation and structural analysis

Thirty old gerbils, aged 18 months, were experimented on accordance with institutional guidelines for animal treatment, housed under conventional conditions (25 °C, 40–70% relative humidity, 12 light/12 dark) with a supply of water and balanced chow ad libitum. The lesion-type classification of the groups could be executed only after necropsy, histological processing of the samples and light microscopy analysis. The animals were divided into two groups: non-affected (controls) and prostatic lesion bearers. The latter were subdivided into four groups according to the type of the most representative prostatic alteration among the following: prostatic intraepithelial neoplasia (PIN), microinvasive carcinoma, adenocarcinoma and hyperplastic stromal areas (HSAs).

Before necropsy, the animals were placed in a chamber containing CO2 and, immediately after, sacrificed and dissected. The entire prostatic complex was removed, and the ventral prostate separated and fragmented.

For light microscopy, some prostatic fragments were fixed for 24 h in 10% neutral buffered formalin and embedded in paraffin (Histosec™, Merck, Darmstadt, Germany), and the others in Karnovisky fixative (0.1 m Sörensen phosphate buffer, pH 7.2, containing 5% paraformaldehyde and 2.5% glutaraldehyde) and embedded in glycol methacriylate resin (Leica™ Historesin Embedding Kit, Nussloch, Germany).

Historesin sections were cut at 2 μm thickness and submitted to cytochemical staining: Haematoxylin–Eosin (H&E, general tissue analysis), Gömori reticulin (selective for collagen and reticular fibres), Feulgen Reaction (nuclear phenotypes) and AgNOR (nucleolar phenotypes) techniques. The paraffin sections (5 μm) were submitted to immunohistochemical evaluation and detection of fragmentation of apoptosis-associated DNA.

The tissue sections were analysed under an Olympus photomicroscope (Olympus, Hamburg, Germany) and the image digitizations were accomplished in image-pro-plus software version 4.5 for Windows (Media Cybernetics Inc., Bethesda, MD, USA).

Detection of cell proliferation in prostate epithelium

For immunohistochemical analysis, the sections were deparaffinized, rehydrated through graded alcohol, and antigen retrieval was performed in 10 mm citrate buffer pH 6.0, at 100 °C for 15 min. The blockade of endogenous peroxidases was obtained by covering the slides with H2O2 (3% in methanol) for 5 min. After pretreatment, the sections were incubated overnight at 4 °C with mouse anti-human Ki-67 antibody (1:100 Santa Cruz Biotech, Santa Cruz, CA, USA) diluted in 1% bovine serum albumen in phosphate buffered saline (PBS). After that, they were washed in PBS and incubated with a corresponding biotinylated IgG in a humidified chamber at 37 °C for 30 min, followed by another 30 min of incubation with streptavidin peroxidase complex (Novocastra Laboratories, New Castle, UK). After more washing in PBS, the sections were visualized by diaminobenzidine (DAB) solution and then conterstained with routine haematoxylin.

Detection of DNA fragmentation associated with apoptosis in prostate epithelium

The sections were deparaffinized, rehydrated and immersed in Tris base buffered saline (TBS, 20 mm Tris pH 7.6, 140 mm NaCl). Subsequently, the specimens were submitted to digestion by Proteinase K (1:100 in 10 mm Tris pH 8.0) for tissue permeabilization, at room temperature for 20 min. The sections were processed according to the instructions supplied by the manufacturer of the Apoptosis kit (TdT-FragEl-DNA fragmentation detection kit; Calbiochem & Oncogene, Darmstadt, Germany), which is based on the TUNEL reaction. The positive staining was revealed by DAB/H2O2 (0.5 mg/ml tap faucet H2O) substrate, in the dark for 13 min. Thus, the sections were washed in water, counterstained with haematoxylin and mounted using Canadian balsam.

Transmission electron microscopy

Ventral prostatic fragments were fixed by immersion with 3% glutaraldehyde, plus 0.25% tannic acid solution in Millonig’s buffer, pH 7.3 containing 0.54% glucose for 24 h (Cotta-Pereira et al. 1976). After washing with the same buffer, they were postfixed with 1% osmium tetroxide for 2 h, washed again, dehydrated in graded acetone series and embedded in Araldite resin. Ultrathin sections were cut using a diamond knife and contrasted with 2% uranil acetate for 30 min, followed by 2% lead citrate for 10 min. The samples were observed and evaluated with a LEO – Zeiss 906 transmission electron microscope (Zeiss, Cambridge, UK).

Quantitative analysis

Stereological analysis

Thirty random prostatic areas (stained by H&E) of each group were analysed: non-affected, PIN and HSA. The stereological analyses were carried out using the method of Weibel (1978), which employs a graticulate system with 120 points and 60 lines. Starting from this test, relative proportions were obtained (%) for the glandular and stromal prostate portions, as well as for each prostatic tissue constituent (epithelium, lumen, non-muscular subepithelial stroma, non-muscular interacinar stroma and smooth muscle cells) in the groups.

Karyometric analysis

This evaluation was carried out using Feulgen Reaction-stained sections. Nuclear areas, perimeters and the form factor [=4π.nuclear area/(nuclear perimeter)2] parameter were determined for 200 nuclei of the epithelial secretory cells in each group, that is: non-affected, PIN, microinvasive carcinoma and HSA. The form factor parameter measures nuclear roundness and values <1 are associated with nuclei which are less round (Taboga et al. 2003).

Nucleolar analysis

Nuclei with the following numbers of nucleoli: 0 (non-evident), 1, 2 and more than 2 were counted in 25 fields selected by animal group. The absolute values found were converted into percentages. Subsequently, the nucleolus number/nucleus ratio was calculated. Additionally, 30 area and perimeter measurements of nuclei and nucleoli were also taken simultaneously in each group. This parameter was adopted to verify whether the quantitative alterations experienced by the areas and nucleolar perimeters of the secretory cells were proportional to those suffered by the nuclei.

Cell proliferation and programmed cell death frequency in prostate epithelium

Immunohistochemically stained proliferation cells were counted in 2000 nuclei of epithelial cells for animal group (at a magnification of ×400). For the calculation of proliferation and apoptotic indexes, the total number of epithelial cells per analysed field was determined, and the positive staining indexes were expressed as a proportion of the total cells.

Statistical analysis

All statistical analyses were performed with statistica 6.0 software (StatSoft, StatSoft, Tulsa, OK, USA). The anova and Tukey honest significant difference (HSD) tests were applied and P ≤ 0.05 was considered statistically significant.

Results

Old gerbil prostate – structure and ultrastructure

The ventral prostate profile of an old gerbil, in morphologically and functionally normal conditions, corresponds to a group of tubuloacinar structures formed by simple epithelium surrounded by fibromuscular stroma (Figure 1a). In the present study, such prostatic areas were considered controls for comparative studies with those ageing-related lesions.

Figure 1.

Figure 1

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Old gerbil prostates stained by Haematoxylin–Eosin. a: General view of prostatic acini (a). The epithelial layer (ep) and the smooth muscle cells (SM) are separated by a large subepithelial collagen deposit (arrowhead). In the epithelium there is maintenance of synthetic activity, with the presence of secretion vesicles (arrow) inside the acinar lumen. Stroma (S). b: Prostatic intraepithelial neoplasia (PIN) in prostatic acinus. There is very thick epithelium (ep), irregular distribution of SM and larger collagen deposit (co) at the epithelium base. Note that the epithelium presents hypertrophied cells (arrowheads) inside a proliferative aggregate. Normal epithelium area (arrow). c: PIN with epithelial folds inside the acinar lumen. Some PIN components present atypical phenotypes, such as cellular hypertrophy, pale cytoplasm and voluminous nucleus (arrowhead). Muscle layer (SM). d: Detail of anomalous acinus with altered distribution of epithelial cells (arrowhead) and SM (arrow). Normal epithelium (ep); blood vessel (V). e: PIN composed of cells exhibiting atypical phenotypes (cellular pleomorphism). Inside the acinar lumen there is deposition of crystalline structures (arrow). f: Detail of PIN with cellular stratification and nuclear pleomorphism. Smooth muscle layer (SM). g: Gömöri´s reticulin method. Prostatic stroma (S) with accentuated increase of reticular fibres (arrows) at the PIN base. Normal epithelium (arrowhead). h: Adenocarcinoma. The microacini can be observed due to proliferation and rearrangement of epithelial cells (arrows) inside a larger acinus (a). Stroma (S); blood vessels (V); smooth muscle cells (SM); normal epithelium area (arrowhead); hypertrophied cells (double arrowheads). i: Microinvasive carcinoma (MC). Acinus (a); epithelium (arrow); blood vessel (V); smooth muscle cells (SM). j: Detail of the cellular components of a microinvasive carcinoma.

Prostatic histopathological alterations compromising mainly the epithelial compartment (Figure 1b–i) were observed in 80% of the analysed animals. Most of these alterations were of proliferative order, involving homeostatic disturbances in the secretory cell population (Figure 1b–h).

The most frequent epithelial alterations were the PINs, which appeared in 46.67% (14/30) of the analysed prostates. Microinvasive lesions were less representative, 26.67% (8 of 30), and they were characterized by carcinomas and adenocarcinomas. In the stroma, cellular hyperplasia occurred in 20% (6 of 30) of the specimens and it was always associated with anomalous epithelial sites. The histopathological classification of prostate lesions presented in the old gerbil was accomplished according to the Bar Harbor Classification System for the mouse prostate, developed by National Cancer Institute Mouse Models of the Human Cancer Consortium Prostate Steering Committee (Shappell et al. 2004).

The ultrastructural analysis confirmed the verifications obtained by conventional light microscopy. In normal prostatic areas, the epithelium exhibited columnar cells with elongated nuclei accompanying their form (Figure 2a). At these sites, there was retention of cellular polarity and intact basement membranes were confirmed by electron microscopy (Figure 2a).

Figure 2.

Figure 2

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Ultrastructure of old gerbil prostatic epithelium. a: Normal secretory epithelium (ep) exhibiting typical columnar cells, with their nuclei matching the cellular form. Epithelium is surrounded by smooth muscle layer (SM). Basement membrane (arrow); Stroma (S); blood vessel (V). Bar: 15.12 μm. b: The epithelial cell cytoplasm is full of lipid droplets (ld). Close to these lipid deposits are several mitochondria (black arrows). Note the condensed chromatin masses deposited mainly in the nuclear periphery (N) (white arrows). Bar: 1.5 μm. c: Apical portion of a secretory epithelial cell with vesicles (sv) being released in the acinar lumen. The cytoplasm is full of synthetic organelles (arrowheads, RER) and lipid droplets (ld). Structures similar to lipofuscin (arrows) can be seen in the cytoplasm and acinar lumen. Bar: 1.95 μm. d: Higher magnification of an epithelial cell with abundant endoplasmic reticulum (RER) and a large lipofuscin-like deposit (arrow). Nucleus (N). Bar: 1.16 μm. e: Detail of lipid droplets (ld) and abundant endoplasmic reticulum (arrow) in the epithelial cell cytoplasm. Bar: 1.16 μm. f: Atypical epithelial cell (ep) components of proliferative aggregate detached from each other. The cytoplasmic and nuclear outlines are irregular (arrows). Nucleus (N). Bar: 3.25 μm. g: Cell in degeneration process exhibiting condensed chromatin (arrow). Nucleus (N). Bar: 3.25 μm.

Additionally, the majority of epithelial cells presented densely condensed nuclear chromatin, mainly distributed in masses in the nuclear periphery (Figure 2b,d). The cellular cytoplasm commonly presented numerous lipid droplets (Figure 2b–e), which were dispersed throughout the cell. The peculiar aspects of the secretory epithelial cells, with an abundance of synthesis organelles and secretion vesicles in their apices, were maintained (Figure 2c–e). Frequently, osmiophilic deposits resembling lipofuscin were found in epithelial cells (Figure 2c,d).

The PINs (Figure 1b–g) were characterized by variable cell types and morphological organization, as well as accentuated epithelial invaginations inside the lumen of the acini (Figure 1b,c) and/or cellular stratification (Figure 1e–g). This last aspect was observed to be eventually associated with portions of simple epithelium (Figure 1b,d,g). The cellular components of PIN exhibited different phenotypes in a same proliferative aggregate (Figure 1b–e) and at distinct proliferative sites. Some PINs were also composed of hypertrophied cells with evident nuclei and pale cytoplasm, seeming to contain mucine deposits (Figure 1b–e). The secretory activity was maintained in PIN component cells, although the exact type of product liberated by such cells is still unknown.

In microinvasive carcinomas (Figure 1i,j), the cellular aggregates invaded the surrounding stroma, by probable rupture of basement membrane. The majority of carcinomatous cells presented hyperchromatic nuclei with scarce cytoplasm and absence of glandular formation in the invaded stroma (Figure 1j). Ultrastructurally, these cells presented nuclei with bizarre forms (Figure 2f) and some could be observed undergoing a degeneration process (Figure 2g).

In adenocarcinomas, there was a diffuse acinar expansion (Figure 1h) where atypical epithelial cells and an increase of stromal cells were evident adjacent to the lesion. The formation of microacini was also observed inside larger acini. This event could be observed due to a proliferation and rearrangement of the secretory epithelial cells (Figure 3a–c). Their secretory products were released in small lumen formed by these new cellular arrangements (Figure 3a–c).

Figure 3.

Figure 3

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Ultrastructure of old gerbil prostatic adenocarcinoma. Microacinar arrangement with a lumen (l) delimited by epithelial cells (ep). Note that these cells present evident synthetic organelles (arrowhead) represented mainly by endoplasmic reticulum. Microvilli (arrows) are common in the cellular apices. In the microacinar lumen (l) can be observed ceramide-like structures (asterisks). Acinar lumen (L). Bars, a: 2.51 μm; b: 1.16 μm; c: 1.16 μm.

A thicker basement membrane was common in old animal acini and, at proliferation sites, this structure underwent permanent compression by cytoplasmic projections of the atypical cells (Figure 4a–d). These altered epithelial cells showed focal adhesions in such expansions, which aid cellular fixation, implying the possible migratory capacity of such cells (Figure 4d). The invasive potential of anomalous cells was confirmed through basement membrane rupture points (Figure 4e,g), visualized by electron microscopy.

Figure 4.

Figure 4

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Ultrastructure of old gerbil prostatic stroma. a: General view of stroma and (S) below the prostatic epithelium (ep). Bunches of collagen fibrils are distributed at the epithelium base and interspersed with smooth muscle cells (SMC). The nucleus (N) of these cells is prominent and usually matches their form. Basement membrane (arrowhead); Fibroblast (F). Bar: 1.95 μm. b: Detail of the epithelial-stromal interface with a thick basement membrane (bm). The latter presents undulations (arrowheads) provoked by the compression realized by cytoplasmic projections of epithelial cells (ep). Bar: 0.9 μm. c: Detail of atypical epithelial cell (ep) with cytoplasmic expansions (arrows) compressing the basement membrane (arrowhead). Nucleus (N); blood vessel (V). Bar: 1.95 μm. d: Cytoplasmic projection of epithelial cell (ep) where focal adhesions can be observed (arrowheads). A large collagen deposit (co) localized at the epithelium base. Elastic fibre (arrow); basement membrane (bm). Bar: 0.54 μm. e: Collagen fibres (co) arrayed in different directions and associated with basement membrane components (arrowhead). Epithelium (ep); lipid droplets (ld). Bar: 1.5 μm. f and g: Areas with probable rupture (arrows) of basal membrane structure (bm), favouring the invasion of epithelial cells (ep) inside prostatic stroma. Collagen (co). Bars, f: 0.42 μm; g: 0.42 μm. h: Smooth muscle cell (SMC) with irregular cytoplasmic and nuclear outlines (arrow). Collagen (co). Bar: 1.95 μm.

Old gerbil prostatic stroma underwent remodelling associated as much with normal epithelium as with altered areas (Figure 1a–i), and this event was common in most of the analysed animals.

A great increase in the deposition of collagen fibrils could be verified at the glandular epithelium base (Figure 1g) as being distributed among smooth muscle cells, compressing the latter. Such fibrils were organized in thick bunches and they promoted an accentuated stromal fibrosis (Figure 4a,d–f). Their distribution became irregular when associated with epithelial lesions (Figure 1b–d) and, in some acini, there were amorphous collagen deposits at the epithelium base (Figure 1b,d,g). Smooth muscle cells underwent an increase and formed loose and irregular arrangements when associated with lesions (Figure 1b,d,h,i). Ultrastructurally, in some cases, smooth muscle cells presented external and irregular nuclear outlines (Figure 4a).

Quantitative analyses

The data regarding quantitative analyses are displayed in Tables 1 and 2, and in Figure 5 (a,b). The comparative analyses were made between non-affected old animals (controls) and those with histopathological prostate alterations, with the latter divided into two subgroups according to the prevalent lesion type (PIN and HSA). In the present analysis, prostatic fragments carrying adenocarcinoma were not included because they were found in a number under the necessary minimum to proceed to a reliable statistical analysis.

Table 1.

Values of the morphometric-stereological data in prolifetative lesions of the old gerbil ventral prostate

Groups
Prostatic measurements Non-affected Hyperplastic stromal area Prostatic intraepithelial neoplasia Microinvasive carcinoma
Density of compartment (%)
    Glandular epithelial** 55.74 ± 13.52a 51.26 ± 13.05a,b 63.90 ± 13.40a,c NV
    Stromal* 44.26 ± 13.52a 47.20 ± 14.51a,b 36.10 ± 13.40a,c NV
Volume of tissue components (%)
    Epithelium** 17.33 ± 5.53a 27.72 ± 6.99b 35.77 ± 9.48c NV
    Lumen** 38.41 ± 13.36a 23.54 ± 13.67b 28.13 ± 14.46b NV
    Smooth muscle cells*** 14.28 ± 7.15a 29.43 ± 6.75b 15.28 ± 6.23a NV
    Colagen subepithelial layer* 7.61 ± 5.36a 0.51 ± 1.74b 4.28 ± 4.28c NV
    Non-muscular interacinar stroma 18.22 ± 11.10 17.26 ± 13.39 16.54 ± 10.50 NV
Karyometric data
    Nuclear area (μm2)* 24.58 ± 5.47a 19.00 ± 3.41b 22.11 ± 6.45c 20.19 ± 6.19b
    Nuclear perimeter (μm)*** 20.12 ± 2.53a 18.18 ± 2.10b 18.78 ± 2.82b,c 17.64 ± 2.88b,d
    Nuclear form factor* 0.76 ± 0.1a 0.73 ± 0.09b 0.78 ± 0.08a 0.80 ± 0.08c
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NV, non verified.

Values are represented as mean ± SD.

Statistical analysis based on the anova and Tukey tests.

*

P ≤ 0.01;

**

P ≤ 0.001;

***

P ≤ 0.0001.

a

Different superindices indicate inter-group significant differences.

b

Different superindices indicate inter-group significant differences.

c

Different superindices indicate inter-group significant differences.

d

Different superindices indicate inter-group significant differences.

Table 2.

Values of indices of proliferation and cell death (apoptosis) in epithelial prolifetative lesions of the old gerbil ventral prostate

Groups Proliferation index (PI) Apoptotic index (AI) AI/PI
Non-affected 0.021 ± 0.004a 0.062 ± 0.019a 0.034
Prostatic intraepithelial neoplasia 0.085 ± 0.018b 0.089 ± 0.021a 0.095
Microinvasive carcinoma 0.162 ± 0.036c 0.100 ± 0.040b 1.620
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Values are represented as mean ± SD.

Statistical analysis based on the anova and Tukey tests.

P ≤ 0.05.

a

Different superindices indicate inter-group significant differences.

b

Different superindices indicate inter-group significant differences.

c

Different superindices indicate inter-group significant differences.

Figure 5.

Figure 5

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a: Percentage distribution of the nucleoli number per nucleus in prostatic epithelial lesions of old gerbil prostate. b: Ratio between nucleolar and nuclear areas in epithelial cells. a,bDifferent superindices indicate inter-group significant differences. HSA, hyperplastic stromal area; PIN, prostatic intraepithelial neoplasia.

The mean proportion of glandular epithelial compartment of two altered groups did not differ significantly from those not affected (55.74 ± 13.52%) (Table 1). Among the lesions, that compartment became larger in prostate areas with PIN, a fact justified by an increase in epithelial tissue that characterizes this lesion type. The areas with prostatic stromal hyperplasia were always associated with epithelial alterations, and its epithelium also was increased in relation to non-affected areas.

The acinar lumen decreases in altered groups, although the secretory activity apparently remained stable, independently of the lesion type (Table 1).

The volume density of prostatic stroma proportions did not differ between altered and non-affected groups (Table 1); however, this compartment increased in hyperplastic stromal sites in relation to PIN. The stromal expansion was directly related to the mean increase of smooth muscle cells, which practically doubled in frequency in relation to those not affected. The non-muscular subepithelial stroma was larger in control animals, whereas in the altered groups, the fibre components of this tissue lost the typical disposition around the epithelium base. They were distributed randomly among the cellular arrangements. The non-muscular interacinar stroma did not undergo significant oscillation among groups, maintaining values close to those of the non-affected ones (18.22 ± 11.10%).

In relation to karyometric data, the lesions presented measures of nuclear area and a perimeter significantly smaller than at normal sites (Table 1). Among lesions, PIN maintained larger nuclear area in relation to the other groups and the carcinomas presented the smallest perimeter measures. These verifications may indicate that secretory epithelial cells of prostate lesions reduce their transcriptional nuclear activities. The form factor, although not significantly different among the groups, presented values larger than 0.7, indicating near uniformity of nuclei in all situations.

The analyses of nucleolar behaviour indicated that the predominant phenotype in the old animals was of non-evident nucleolus in epithelial cell nuclei (Figure 5a), a phenotype statistically more accentuated in HSAs.

Old controls and the PIN group presented larger measures of nucleolar areas, while perimeter measures did not vary significantly between lesions and the control. The nucleolar/nuclear area ratio (Figure 5b) was larger in PIN compared to other groups, whose values oscillated close to those of the control.

Indices of proliferation and cellular death increased significantly with the aggressiveness of the lesion (Table 2; Figures 6a–c and 7a–c). In PIN, the indices between the two processes remained in equilibrium, although in microinvasive carcinomas the apoptosis decreased in relation to cellular proliferation, indicating homeostatic imbalance between these opposed phenomena, resulting in abnormal prostate growth.

Figure 6.

Figure 6

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Anti-Ki-67 immunocytochemistry. Counterstained: haematoxylin. a: Demarcation of nuclear proliferation is very low in normal epithelium (ep); immunoreactive nucleus (arrow and brown nuclei). b: Prostatic intraepithelial neoplasia (PIN) (arrow). c: There is increase of cell proliferation in hyperplastic epithelium (ep).

Figure 7.

Figure 7

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TUNEL/haematoxylin. The arrows are indicative of in situ detection of fragmented DNA (apoptotic nuclei) in the prostatic epithelium (ep). a: Normal epithelium. b: Prostatic intraepithelial neoplasia (PIN). c: Carcinoma.

Discussion

The behaviour of spontaneous proliferative lesions in the ventral prostate of the old gerbil was evaluated and it was verified that the epithelial compartment is the main histopathological alteration site, being able to attack an individual acinus or groups of acini. In the prostatic stroma, ageing was associated with the increase and redistribution of cellular components and of extracellular matrix.

The most common epithelial alterations were PIN, followed by microinvasive carcinomas and, in a much smaller percentage, adenocarcinomas. The secretory epithelial cells in PINs underwent a great increase, making it possible that the epithelial compartment reached a relative frequency of more than 60%. At 18 months of age, some degree of prostate lesion was found in 46% of the animals analysed. ACI/Seg rats provide another experimental model that spontaneously develop microscopic cancer in the ventral prostate, and only after 33 months of age does invasive carcinoma appear (Ward et al. 1980). Apparently, the gerbil needs a shorter time to develop invasive spontaneous lesions since 26% of the analysed animals presented microinvasive foci in the period of time studied. Additionally, long-term administration of testosterone in adult gerbils reduces the time for appearance of neoplastic lesions in this model (Zanetoni et al. 2005; Scarano et al. 2006).

There is great contradiction among researchers about the exact region in which prostate carcinogenesis begins in rodents. In the gerbil, as well as in ACI/Seg rats, the ventral lobe corresponded to the main site of origin and establishment of proliferative lesions (Ward et al. 1980). Pollard and Luckert (1992) indicate the anterior and dorsolateral lobes as the sites where prostate alterations begin in mice, while Cohen et al. (1994) hold that the tumour propagation starts from the seminal vesicle of those animals. Recently, genetic data showed that the dorsolateral lobe of the mouse prostate is homologous to the peripheral zone of the human prostate, where cancer is most prevalent (Berquin et al. 2005). The features of this lobe in the gerbil have been evaluated in our laboratory and its potential in developing proliferative lesions will be better studied in the future.

Prostatic intraepithelial neoplasias were characterized by agglomerations of heterogeneous epithelial cells with probable atypical function. Ultrastructurally, anomalous cells presented nuclei with bizarre forms and marginal distribution of chromatin while their cytoplasm was replete with lipid droplets. Cytoplasmic projections extended towards the extracellular matrix, compressing the basement membrane. The latter presented rupture in some regions, indicating the invasive potential of the anomalous epithelial cells. Thus, it could be confirmed that the proliferative agglomerations installed in the prostatic stroma really were cells of epithelial origin. Additionally, data still unpublished from our laboratory showed that adult gerbils with prostatic invasive lesions induced by a chemical carcinogen present expression of the enzyme α-methylacyl-CoA racemase (P405S), a new marker of prostate cancer cells.

The lipid droplets found in epithelial cells of the gerbil prostate are also common in old mice and lineages of human prostate cancer cells (Cohen et al. 1994; Swinnen et al. 2004). The increase of lipogenesis is one of the main markers of cancerous cells whereas the gene encoders of lipogenic enzymes are regulated by androgens. This event is found as much in the initial stages of neoplastic transformation (PIN) as in invasive carcinomas, persisting even in androgen-independent cells (Swinnen et al. 2004).

Osmiophilic structures, frequently observed in epithelial and stromal cells of old gerbils, resembled lipofuscin and/or ceramide deposits. Increase in the intracellular levels of these seems to be a common event in senescence and in cellular response to a stressor factor (Hannun 1996; Venable & Obeid 1999). In human LNCaP prostate cancer cells submitted to radiation, the production of ceramides is involved in the activation of apoptotic cellular death (Kimura et al. 1999).

Differently than occurs in other models of prostate cancer and in humans, measures of area, perimeter and nuclear form factors of secretory cells did not increase in altered groups (PIN, microinvasive carcinoma and prostatic stromal hyperplasia), indicating a distinct nuclear behaviour of proliferative lesions in old gerbils. Additionally, this type of isolated quantitative analysis had not been effective in aiding the diagnosis of prostate lesions in old gerbils. In human prostate diseases, nuclear parameters were also ineffective in differentiating adenocarcinomas of different Gleason degrees, but they were efficient in comparing BPH and adenocarcinoma (Martínez-Jabaloyas et al. 2002; Taboga et al. 2003).

The nucleolar DNA content is an indicator of functional condition and of cellular proliferation degree (Trére 2000; Karalyan et al. 2004). In the old gerbil prostate, the number of nucleolar corpuscles increased slightly in PINs and carcinoma, while in prostatic stromal hyperplasia almost 70% of the nuclei did not present evident nucleoli. As for the area and perimeter parameters of this organelle, PINs presented a higher nucleolus/nucleus ratio, indicating an increase of the total nucleolar size and, possibly, greater cellular metabolic activity in those regions. It is presupposed that, after the acquisition of invasive potential, the cells reduce their transcriptional activities, restricting their metabolism to the maintenance of the replication process. The minimal variable behaviour of nucleoli in most of the epithelial lesions can still imply a less aggressive aspect of these neoplasias. In agreement with these data, no analysed old animal presented a lesion that was metastatic or that would compromise totally the prostate function.

Additionally, the indices of proliferation and cellular death indicated a larger cellular turnover as the histopathological alterations became more severe in the old gerbils. In humans and mice, similar events of cellular kinetics were also found (Berges et al. 1995; Xie et al. 2000).

In PIN, there was a balance between the processes of proliferation and cellular death, while in carcinoma, the cellular proliferation index surpassed that of apoptosis. The reduction or larger resistance to apoptotic cellular death appears to be a common event in prostate cancer, and this factor accelerates tumour growth (O‘Neill et al. 2001;Schulz et al. 2003).

Among the events that contribute to homeostatic imbalance in prostate tissue during ageing are alterations in the levels of androgenic hormones. Although few studies have truly demonstrated a significant relationship between the serum testosterone levels and prostate cancer, most of the studies suggest an association between the two factors (Hsing et al., 2002), including in gerbils. In these rodents, the mean serum testosterone level was 4.82 ng/ml in adult animals, while in old animals, those values decreased to 2.80 ng/ml (Pegorin de Campos et al. 2006). Androgen receptors are expressed in all histological types of prostate cancer, whereas somatic mutations in the gene for these receptors are common in human tumours; and those mutations are probably involved in the tumoural progression and aggressiveness (Scher et al. 2004).

Gerbil prostatic stroma also underwent alterations associated with ageing, mainly in areas adjacent to abnormal epithelium. In this, there was an accentuated increase of collagen fibres, characterizing a desmoplastic process. This type of stromal response is frequent in many cancer types (Robert 2002). The disintegration and remodelling of extracellular matrix were events that probably favoured the dispersion of epithelial cells in stroma. Additionally, the malignant transformation is intimately dependent on the composition and structure of the extracellular matrix and, it is believed that tumourous cells can modify the production and degradation of matrix constituents, favouring the migratory process (Robert 2002).

The development of spontaneous prostate cancer is a relatively rare event in most of the known rodent models. As this is a disease related to ageing, it is speculated that the life expectancy of experimental animals may be too short for effective installation and evolution of this disease type (Pollard & Luckert 1986; Banerjee et al. 1998). However, model systems are primordial for acquisition of greater knowledge on pathogenesis, progression and therapy in prostate cancer (Shirai et al. 2000). Although the old gerbil does not present high frequencies of invasive prostate alterations, PIN is common in a significant portion of these animals. As these lesions are considered precursory to prostate cancer, a more detailed analysis of their cellular and molecular components would be sufficient to justify the use of the gerbil as a good experimental model in the study of premalignant prostatic lesions. Additionally, the ageing gerbil tumour system with the characteristics analysed above appears useful for investigating its pathogenesis and developing therapies.

Acknowledgments

The authors thank Mr Luiz Roberto Falleiros, Jr, and Ms Rosana S. Sousa for technical assistance, as well as all other researchers at the Microscopy and Microanalysis Laboratory. Acknowledgement is also due Ms Lara Silvia Corradi and Ms Sabrina Santos Rochel for English-language revision of this article. This article is part of the thesis presented by S.G.P.C. to the Institute of Biology, UNICAMP, in partial fulfillment of the requirement for a PhD degree.

Funding

This study was funded by Brazilian National Research and Development Council (CNPq, Fellowship to SRT – Proc. Nr. 301111/05-7), Coordinating Body for Training University-Level Personnel (CAPES) and São Paulo State Research Foundation (FAPESP).

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