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Research Article |
Department of Cell Biology,3 State University of Campinas, 13083-863 Campinas, São Paulo, Brazil
Department of Histology and Embryology,4 State University of Rio de Janeiro, 20550-170 Rio de Janeiro, Brazil
Departments of Computational Sciences and Statistics5
Biology,6 State University of São Paulo, 15054-000 São José do Rio Preto, São Paulo, Brazil
ABSTRACT
Theprostate of the female gerbil (Meriones unguiculatus) is similar to the human female prostate (Skene gland) and, despite its reduced size, it is functional and shows secretory activity. However, virtually nothing is known about its physiological regulation. This study was thus undertaken to evaluate the behavior of the gerbil female prostate in a hyperandrogenic condition. Adult females received subcutaneous injections of testosterone cypionate (1 mg/kg body weight every 48 h) up to 21 days. Circulating levels of testosterone and estradiol were monitored, and the prostate and ovaries subjected to structural and immunocytochemical analyses. The treatment resulted in sustained high levels of circulating testosterone, and caused a transient increase in estradiol. There was an increase in epithelial cell proliferation accompanied by significant reorganization of the epithelium and an apparent reduction in secretory activity, followed by a progressive increase in luminal volume density and accumulation of secretory products. Immunocytochemistry identified the expression of androgen receptor and a prostate-specific antigen (PSA)-related antigen in prostatic epithelial cells. A circulating PSA-related antigen was also found, and its concentration showed strong negative correlation with circulating estrogen. Epithelial dysplasia was detected in the prostate of treated females. Analysis of the ovaries showed the occurrence of a polycystic condition and stromal cell hyperplasia. The results indicate that testosterone has a stimulatory effect on the female prostate, inducing epithelial cell proliferation, differentiation, secretory activity, and dysplasia. The results also suggest that prostatic growth and activity, polycystic ovaries, and ovarian stromal cell hyperplasia are related to a hyperandrogenic condition in females.
androgen receptor, female reproductive tract, ovary, prostate, testosterone
INTRODUCTION
The adult gerbil female prostate shows a paraurethral location, exhibiting intimate contact with the urethral wall in the distal and median portions. Two types of secretory cells have been recognized by histology and ultrastructural analysis. The first is a typical prostatic secretory cell. The second is characterized by a unique set of secretory organelles and was named clear secretory cell because of its pale color after HE staining. Although reaching a very limited size in adulthood, the gerbil female prostate has been found to be morphologically similar to the ventral lobe of the prostate of postpubertal males [1]. The histochemical and ultrastructural aspects of the gland permitted us to assume that it secretes glycoproteins functionally and constitutively [1, 2].
Despite an increasing number of reports on the structural characteristics of the female prostate of different species [3–8], including humans [9, 10], reference to this organ is still a matter of controversy among people dealing with women's health. Moreover, its small size led some authors to conclude that the female prostate is a vestigial gland [4, 11, 12].
Androgens regulate the growth, differentiation, and survival of male prostatic cells [13, 14]. Testosterone and its derivative, dihydrotestosterone, along with other hormones, such as estrogen and prolactin, regulate prostate physiology during both development and adulthood [15–18]. Although Zavia
i [9] has suggested that the activity of this gland is regulated by steroid hormones, the influence of androgens on this organ is unclear.
Androgens play an important role in the regulation and development of the female urogenital system. This is supported by the fact that androgen receptors have been identified in various organs, such as ovaries, oviduct, and uterus [19]. Immunohistochemical studies have demonstrated that androgen receptors are also present in the human female prostate (Skene paraurethral gland), suggesting a role for androgens in the maintenance of the structure and functional state of the prostate in the female organism [10, 12].
It seemed important to us to investigate whether androgens regulate the growth and activity of the female prostate gland by comparing the effects of these hormones with those seen in males. Furthermore, this model may have some clinical implications, because 1) women suffer from steroid hormone imbalances after menopause, 2) hyperandrogenism is not unusual in some populations, 3) the use of androgens for adjuvant therapy of female-to-male transsexuals is common [20], and 4) its administration to bodybuilders and female high-performance athletes is a common (although yet illegal) procedure.
Thus, the objective of the present study was to evaluate the response of the adult gerbil female prostate to the experimental administration of testosterone, simulating a hyperandrogenic condition, using a series of structural and immunocytochemical analyses.
The results presented here demonstrate that the epithelial cells of the gerbil female prostate express the androgen receptor and secrete a prostate-specific antigen (PSA)-related antigen. Most importantly, our results show that exogenous testosterone induces prostate growth resulting from the proliferation, differentiation, secretory activity, and dysplasia of epithelial cells, and affects the concentration of circulating estradiol and PSA, which showed a high negative correlation to one another. Moreover, we also observed that testosterone treatment leads to a polycystic ovarian condition, ovarian stromal cell hyperplasia, and urethral wall thickening. Taken together, the results suggest that, in addition to many side effects, the administration of androgens to females may also result in abnormal growth and dysplasia of the prostate gland.
MATERIALS AND METHODS
Animals and Experimental Design
Eighty seven 3-month-old female gerbils (Meriones unguiculatus, Gerbillinae: Muridae) were employed in this study. Seventy-one animals received intradermic testosterone injections (1 mg/kg testosterone cypionate [Novaquímica/Sigma Pharma, Hortolândia, São Paulo, Brazil] in 0.25 ml corn oil) every other day after the beginning of treatment [21] and were divided in to groups that were subjected to the different analyses. The control animals received corn oil injections every other day for 21 days. The treated animals were killed after 3, 7, 14, or 21 days of treatment. After being anesthetized by CO2 inhalation, the animals were weighed and immediately decapitated. Blood samples from some of them were collected for serological analysis, and the prostatic complex (urethra and adjacent tissues) was dissected out, weighed, and fixed according to different protocols, as specified below. Those that were not easily dissected out of adherent tissue were not weighed. The ovaries were also collected to assess histological changes due to testosterone administration. Animal handling and experiments were done according to the ethical guidelines of the State University of Campinas, following the Guide for Care and Use of Laboratory Animals. The large sample size used in this work was justified by the minute size of the organ and the large number of analytical procedures employed.
Plasma Total Testosterone, Estradiol, and PSA Levels
Circulating plasma testosterone, estradiol, and PSA levels were determined by immunochemical assays. Blood was collected by cardiac puncture immediately before death and 24 h after the last injection of testosterone (except for Day 14, the sample for which was collected 48 h after the last injection). Plasma was separated by centrifugation and stored at –20°C for subsequent assays. Measurements were done in duplicate using automated equipment from Vitros-ECi-Johnson & Johnson for ultrasensitive chemiluminescence detection. The sensitivity was 0.1–150 ng/ml for testosterone, 0.1–3 814 pg/ml for estradiol, and 0.1–100 ng/ml for human PSA. The intraassay variations were 1%, 1.1%, and 0.97%, and the interassay variations were 2.1%, 1.5%, and 1.75%, for testosterone, estradiol, and human PSA, respectively.
Morphological Analysis
The urethra and adhering tissues and ovaries were fixed by immersion in Karnovsky solution (5% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2), or in Bouins solution, for 24 h. After fixation, the tissues were washed under running tap water, dehydrated in an ethanol series, cleared in xylene, embedded in paraffin (Histosec; Merck, Darmstadt, Germany) or glycol methacrylate resin (Historesin embedding kit; Leica, Nussloch, Germany), and cut into 3-µm sections with a Leica RM2155 automatic rotatory microtome. Sections from the female prostate and ovaries were stained with hematoxylin-eosin [22]. The Feulgen reaction for DNA was used to count mitotic cells. Neutral carbohydrates were identified by the periodic acid-Schiff (PAS) test [22]. The specimens were analyzed and photographed with a Zeiss Jenaval (Zeiss-Jenaval, Jena, Germany) or an Olympus BX60 light microscope (Olympus, Hamburg, Germany).
Morphometry and Stereology
The relative volume of the tissue compartments was determined according to the procedure of Weibel [23] using a 168-point grid test system, as applied to the rat male prostate by Huttunen et al. [24] and Garcia-Florez et al. [25]. Twenty microscopic fields were chosen at random. The volume density was calculated after counting the number of points that coincided with each of the tissue compartments (epithelium, lumen, or stroma). Absolute volumes could not be determined because it was not possible to separate the female prostate from adhering tissue and to determine its weight.
The number of acinar profiles per prostate was counted after serial sectioning. The relative sectional area of the lumen was measured using the NIH Image J software (available at http://rsb.info.nih.gov/ij/; National Institutes of Health, Bethesda, MD). Briefly, the lumen was artificially stained and its sectional area was measured in square pixels. The number of secretory, clear, and basal epithelial cells per alveolus was counted, and the individual contribution of each cell type is expressed as a percentage. Morphometric analysis also included the determination of epithelial cell height, nucleus/cytoplasm ratio, nuclear area (µm2), nuclear perimeter (µm), and form factor (4
x nuclear area/[nuclear perimeter]2).
For the quantification of mitotic cells, Feulgen-stained sections were examined and 10 acini were digitized. The total number of nuclei per acinus and the number of mitotic figures were counted. Results are reported as the percentage of mitotic cells.
Immunocytochemistry
Sections of Bouin-fixed prostates were subjected to immunocytochemistry for the detection of PSA and androgen receptor (AR), as described elsewhere [26, 27]. Mouse monoclonal immunoglobulin G1 anti-PSA and rabbit polyclonal anti-AR (SC-7316 and SC-816, respectively; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were employed at a dilution of 1:100. Peroxidase-conjugated specific antibodies (Sigma Chemical Co., Saint Louis, MO) and 3,3'-diaminobenzidine were used as secondary antibodies and peroxidase substrate, respectively. Sections were counterstained with Harrys hematoxylin or methyl green.
Three Dimensional Reconstruction
After histological processing, whole prostates (control and testosterone-treated) were serially sectioned at 5 µm and stained with hematoxylin-eosin, as described above. Image capture and processing were carried out using the Analysis 3.2 software (Build 743; Digivision–SIS, San Diego, CA). After image alignment, the urethra and prostatic ducts and alveoli were isolated in each image and then processed to obtain an interface boundary in each section, thus generating a three dimensional (3-D) model [28]. It is important to mention at this point that this software was unable to deal with dead-end structures, such as the prostatic acini, and the images show blunt ends at the tips.
Statistical Analysis
Data were analyzed using Statistica 6.0 software (StarSoft, Inc., Tulsa, OK). The hypothesis tests used to determine statistical significance were the Kruskal-Wallis test, ANOVA, Tukeys multiple comparison test, or the median test, with the level of significance set at 5% (P 
0.05). Values are presented as median or mean ± 1 SD (low number of measurements) or SEM (high number of measurements), or as box-plots, as required for clearer presentation of the data.
RESULTS
Body Weight and Prostatic Complex Weight
Table 1 shows the variation in body and prostatic complex weights. There was a 14.6% increase in body weight after 21 days of testosterone treatment. Significant increase in the prostatic complex weight, including the whole urethra and prostate gland, was observed after 7 days of treatment. The relative weight of the prostatic complex showed an 
52% increase after 21 days of treatment, demonstrating a marked specificity in prostatic complex growth.
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Serum Steroid Hormone and PSA-Related Antigen Levels
Serum steroid hormone and PSA-related antigen levels are shown in Figure 1. As expected, exogenous testosterone administration resulted in sustained high levels of circulating testosterone, reaching as much as 19 ± 2.1 ng/ml after 21 days of treatment. This sustained level was about 12 times the normal circulating testosterone level of female gerbils (Fig. 1A). Testosterone treatment caused a significant rise in circulating estradiol levels, corresponding to double the concentration observed in the control on the third day of treatment. However, this increase was transient, and, despite sustained high levels of testosterone, estradiol returned to control levels after 21 days of treatment, with no significant difference being observed by day 7 of treatment (Fig. 1B).
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Serum PSA-related antigen concentration in control females was 0.156 ng/ml (Fig. 1C). Testosterone treatment caused a subtle decrease in PSA levels, which progressively returned to control levels within the duration of the experiment, with no difference compared to control being observed after 7, 14, or 21 days (Fig. 1C). Although no significant correlation was observed between testosterone and PSA-related antigen concentration, there was a high negative correlation between estradiol and PSA-related antigen levels (r = –0.98). On the other hand, the correlation between estradiol and testosterone levels was very low. This low correlation was due to a marked dissociation between the highest testosterone concentrations, which were associated with a decline in estradiol levels. Lower circulating testosterone concentrations showed a positive correlation with estradiol levels (r = 0.86). This effect was more evident in the testosterone concentration range of 13–17.5 ng/ml, presenting a very high correlation coefficient (r = 0.99).
Morphological Aspects and Alterations Caused by Testosterone Treatment
The gerbil female prostate established a close contact with the median and distal portions of the urethral wall. This organ was formed by a small group of glands and ducts dispersed in a dense stroma. The glands were lined by small cuboidal to moderately tall prismatic cells (Fig. 2, A and B). These glands generally enclosed a voluminous lumen containing PAS-positive secretion (Fig. 2C). Two distinct types of epithelial cells were identified: secretory and basal cells (Fig. 2B). Secretory cells were in contact with the glandular lumen and predominated in the prostatic epithelium. These cells were characterized by large, parabasal nuclei and an acidophilic cytoplasm. PAS-positive secretory material was found to be accumulated close to the apical surface (Fig. 2C). Some chromophobic secretory cells, previously named clear cells [1], were interspersed amongst common secretory cells. Basal cells were located between the secretory cells. These cells were smaller and less frequent than the secretory cells, and had an irregular elliptical shape.
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Many morphological alterations were observed in the epithelial compartment of the female prostate glands after experimental testosterone administration. After 3 days of treatment, a marked increase in prostatic epithelial height was observed, with the epithelium being of the pseudostratified columnar type, with a predominance of tall epithelial cells arranged in frequent epithelial infoldings (Fig. 2, D and E). The secretory cells exhibited a readily observed chromophobic supranuclear area and an apical accumulation of acidophilic material (Fig. 2E), which was not observed in control specimens. One of the most evident epithelial alterations caused by 3 days of testosterone treatment was the appearance of intraepithelial vesicles in the prostatic acini (Fig. 2D, arrow), which were observed between two epithelial layers. In most cases, the apical surface of the lower cell layer was in contact with the content of the vesicle, whereas the basal surface of the upper cell layer delimited the vesicle, and its apical surface was in contact with the main glandular lumen. Basal cells were commonly seen in the epithelium (Fig. 2E), but were not observed in the cell layers that formed the intraepithelial vesicles. PAS staining revealed the loss of apical staining observed in most secretory cells of the vehicle-treated prostate, and the appearance of groups of secretory cells whose cytoplasm was filled with neutral glycoconjugates (Fig. 2F).
Longer exposure to exogenous testosterone resulted in further growth of the gland. The histological aspects described for the third day were more prominent after 7 and 14 days of treatment (Fig. 2, G, H, J, and K, respectively). Secretory cells were even taller by Day 14, and the frequency of epithelial infoldings and epithelial vesicles was higher (Fig. 2, G and J). Also, the number of deeply stained PAS-positive cells was increased (Fig. 2, I and L).
Striking reorganization of the gland was observed after 21 days of treatment. The epithelial cells were considerably shorter (Fig. 2, M and N) despite preservation of the chromophobic supranuclear area. The number of clear cells, which were rare in the normal prostate and had increased by the 7th and 14th days, was also elevated after 21 days of treatment. Intraepithelial vesicles were scarce at this stage. The increased acidophilic apical staining observed in the epithelial secretory cells had almost disappeared (Fig. 2N). Likewise, the disappearance of the apical PAS staining was also transient and, by Day 21, the secretory cells again showed this apical staining. Foci containing PAS-positive cells were smaller but very frequent (Fig. 2O).
Morphometry
The morphometric results are presented in Table 2. There was a significant increase in the mean number of epithelial cells per acinar profile after 7 days of testosterone treatment. The larger number was maintained up to Day 21 of treatment. Clear cells were more frequent among the epithelial cells, and corresponded to 7.2% of the epithelial cells after 21 days of testosterone treatment compared to 1.6% in the control tissues. This increased frequency resulted in a significantly reduced percentage of secretory cells. On the other hand, the number of basal cells remained relatively constant, ranging from 7.9% to 8.5% of the epithelial cells. Epithelial height changed significantly during testosterone treatment, ranging from 14.4 µm in controls to 23.6 µm after 14 days of treatment. After 21 days of treatment, shortening of the epithelium was observed, which became indistinguishable from the controls.
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The cell nucleus/cytoplasm ratio showed a transient reduction by Day 14 of treatment (Table 2). Changes in nuclear structure were also detected. Nuclear area and perimeter showed a progressive increase up to the 14th day of treatment, followed by a small reduction in these nuclear parameters on Day 21. The form factor, an expression of nuclear roundness, was slightly increased after longer periods of testosterone treatment (i.e., 14 and 21 days). Values close to 1 indicate that the cell nucleus is more round and less elliptic, as observed after 14 and 21 days of treatment. The changes in nuclear area, perimeter, and form factor were statistically significant (Table 2).
Volume Density of Tissue Compartments
The stereological data obtained for normal and treated prostates are shown in Table 3. In the prostate of control animals, the stroma occupied most of the prostatic volume ( 40%), and remained relatively constant throughout treatment, showing a small, nonsignificant reduction after 21 days of treatment. The remaining volume was almost equally shared by epithelium and lumen (27.5% and 32%, respectively) in control animals. Testosterone treatment caused an increase in epithelial volume density, which reached about 46% and 43% of the glandular volume after 7 and 14 days of treatment, respectively, returning to control levels by Day 21. The increase in epithelial volume density was counterbalanced by a decrease in luminal volume density which, however, became more prominent than in the control animals after 21 days of treatment. ANOVA revealed that the changes observed in the epithelial compartment during the period of testosterone treatment were significant. Application of Tukeys parametric test demonstrated a significant difference between groups. No significant differences were observed between the group treated for 21 days and the control group, or between the groups treated for 7 and 14 days. As there were no group differences in stromal volume density, no paired test could be applied to compare time points with the control. However, there was an evident variation in luminal volume with treatment.
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Number of Acinar Profiles and Luminal Area
Analysis of the median number of acini per gland revealed that testosterone induced an increase in the number of acini up to Day 7 of treatment. In the normal prostate, the number of acinar profiles ranged from 7 to 14 (median = 13). Prostates treated for 3 days had 21–29 acini (median = 24), and those treated for 7 days had 35–48 acini (median = 37). Thereafter, there was a gradual decline in the number of acinar profiles per gland. Prostates treated with testosterone for 14 days had 19–30 acini (median = 25), whereas those treated for 21 days had 14–20 acini (median = 19). The use of the median was necessary because, for each prostate, only the medial section was counted, and resulted in a very low number of measurements. Although the test of the median showed no marked differences, the application of the Kruskal-Wallis test revealed significant differences between the control and experimental groups. On the other hand, the sectional luminal area showed a marked increase on Days 14 and 21, demonstrating progressive enlargement of the lumen and the accumulation of secretory products (Table 4). The product of the median number of acinar profiles multiplied by the sectional luminal area demonstrated that the decline in the number of acini did not correspond to a decrease in luminal volume (Table 4).
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Cell Proliferation
Mitotic cells were rare in the epithelium of the prostate of control animals, ranging from 0.7% to 1.3% of all epithelial cells (Fig. 3). A large oscillation in the frequency of mitotic cells was observed in the prostates of animals treated for 3 days, with a large deviation to higher figures (up to 3.6%). The frequency of mitotic cells remained constant at about the same values obtained for the control after longer periods of testosterone treatment. No statistically significant differences in the median number of mitotic cells were observed between the different periods of treatment according to the median and Kruskal-Wallis tests. Thus, the differences between groups were due to variation within the group treated with testosterone for 3 days, which showed an 4-fold higher SD (1.1%) than those obtained for the other groups (control SD = 0.3%; 7 days = 0.5%; 14 days = 0.2%; and 21 days = 0.2%).
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3-D Structure
Figure 4 presents the 3-D reconstruction of the prostate and urethra of control and testosterone-treated female gerbils. In both situations, the glandular structures were concentrated in the median portion of the urethra, which was 0.8–1.2 mm long and 0.06–0.1 mm thick. In control animals (Fig. 4, A and C), the prostate was characterized by a ductal system that was inserted into the urethral wall, connecting the organ to the urethra. The alveoli were aligned with the urethra and showed different lengths and thicknesses. Testosterone treatment (Fig. 4, B and D) resulted in an increased number of acini, which seemed to be larger than in control animals. In addition to the growth of the prostate gland, which contributed to the thickening of the urethral wall, it became evident that the wall musculature itself was hypertrophic. Both prostate growth and urethral wall thickening resulted in marked collapse of the urethra.
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Immunolocalization of PSA-Related Antigen and AR
A PSA-related antigen was detected in the female gerbil prostate by immunocytochemistry, regardless of the concentration of circulating testosterone. In control animals, the reaction was diffuse in the secretory material accumulated in the lumen, and was found almost exclusively in the apical cytoplasm. The immunocytochemical reaction was not uniform, with some epithelial cells showing no staining at all. Testosterone treatment did not affect this distribution (Fig. 5A). However, after 14 days of treatment, the concentration of this PSA-related reactive molecule was higher in the vesicles close to the apical portion of the cell (Fig. 5B).
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The immunocytochemical reaction to AR showed that it was concentrated in the nuclei of epithelial cells of the prostate of vehicle-treated control females (Fig. 5C). Basal epithelial cells and stromal cells were also reactive. After 14 days of treatment, the same reaction pattern was identified, except for one in which immunocytochemical staining for AR was detected in the cytoplasm of many, but not all, cells (Fig. 5D). This pattern was maintained for the longer treatment period.
Dysplastic Prostate Growth
Figure 6 shows some aspects of the dysplastic growth of the epithelium in testosterone-treated female gerbils. Abnormal epithelial growth was observed in 100% of the animals treated with testosterone for 21 days. The most common lesions were prostatic intraepithelial neoplasia, atypical hyperplasia, and mucinous adenocarcinomas (Fig. 6).
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Ovarian Structure
Figure 7 presents the histological aspects of the ovaries of control and testosterone-treated female gerbils. Two main aspects became visible with the androgen treatment. First, we observed the structure of a polycystic ovary, which was more evident after 14 days of testosterone treatment. Second, there was marked hyperplasia of stromal cells. Because these animals presented no Reinke crystals, we were unable, at the time, to determine whether they correspond to Leydig cells.
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DISCUSSION
This study demonstrates that the administration of testosterone to female gerbils causes important alterations in the prostate gland. The major alterations involved the epithelial compartment of the gland, which presented a dual behavior during testosterone treatment, represented by an initial proliferative response followed by a secretory burst.
The experimental protocol employed here resulted in circulating levels of testosterone 12 times higher than those measured in untreated females. The resulting circulating testosterone concentration was also about four times the circulating levels determined for male gerbils, corresponding to about 4.8 ng testosterone/ml plasma (unpublished results), and was thus considered hyperandrogenic.
We have followed the effect of high testosterone on estradiol levels to check how the hypothalamus-pituitary-gonad axis was affected. Indeed, it appeared unaffected within the time line of the experiment. The transient increase in estradiol may have resulted from an immediate conversion of testosterone via aromatase. This was reinforced by the high correlation between testosterone levels and estradiol levels in the intermediate concentration range of the former. At the highest testosterone concentration achieved, other mechanisms, such as increased E2 catabolism, seem to be activated.
On the third day of treatment, there was a marked increase in the number of proliferating cells in the prostatic epithelium. The pattern of proliferation was characterized by wide variability, as demonstrated by statistical analysis, which may be attributed to localized cell divisions. As a matter of fact, cell divisions were more frequent in the small, apparently growing acini or buds. The increased proliferation resulted in epithelial hyperplasia, accompanied by a significant increase in the number of acini and the presence of epithelial infoldings and vesicles. The intraepithelial vesicles frequently observed in the epithelium of prostates treated with testosterone for 3, 7, and 14 days are similar to the pseudocribriform pattern of intraepithelial neoplasia found in the gerbil male prostate [29, 30]. In human prostatic neoplasias, this epithelial organization may lead to the development of invasive adenocarcinoma. However, in prostates treated for 21 days, these figures were not as frequent as during earlier periods, suggesting that they represented a transient organization of the epithelial layer.
The epithelial cells became taller and presented a large chromophobic region in the supranuclear cytoplasm on Days 7 and 14 of treatment. This chromophobic area corresponds to the region occupied by the Golgi complex and its vesicular system, and is also seen in the normal male prostate of adult gerbils [1] and other rodents [6, 7]. These aspects were confirmed by transmission electron microscopy (unpublished results). The transition of the prostatic epithelium from small cuboidal or prismatic to tall prismatic or pseudostratified columnar revealed an intense enlargement of the epithelial cells and their engagement in secretory activity, which was maintained up to the 14th day of treatment. Accordingly, statistical analysis revealed a significant increase in the volume density of the epithelial compartment. In contrast to this epithelial development, the relative volume of the lumen was reduced to less than a half during this period.
A second cell type observed at high frequency in the secretory epithelium treated for 3, 7, and 14 days was the basal cell. These cells have been suggested to be responsible for the maintenance of the epithelial cell population in the female prostate [10]. The number of basal cells was relatively constant across testosterone treatments, with a proportion of 1 basal cell to about 12 luminal secretory cells. This aspect more closely resembles the situation found in the rat ventral prostate, and differs from the human male prostate, in which a one-to-one proportion is observed [31]. Immunocytochemical studies on the human male prostate revealed the existence of androgen receptors in these cells [32], a fact that might allow these cells to proliferate in response to testosterone. This was confirmed for the gerbil female prostate. However, we have also observed that luminal cells undergo mitosis when treated with testosterone (data not shown), indicating that proliferation does not exclusively depend on basal cells.
Clear cells, which expressed and accumulated a secretory material mainly consisting of neutral carbohydrates, were rare in the epithelium of the normal female prostate [1], but became gradually more frequent with testosterone treatment, increasing from 1:60 to 1:13 epithelial cells after 21 days of testosterone treatment (see Table 2).
Both morphometric and stereologic analyses revealed a decrease in the total number of acini and an inversion in the relative volume of the epithelial and luminal compartments. This increase in luminal content was more evident when the sectional area of the lumen was taken into account. Epithelium had shorter epithelial cells that still showed a developed Golgi complex and numerous secretory vesicles. Taken together, these results demonstrate that the gland entered a synthetic phase, resulting in the accumulation of secretion in the lumen. These epithelial dynamics led to an enlarged gland after 21 days of treatment, as confirmed by 3-D reconstruction.
On the basis of these results, it is possible to conclude that the experimental administration of testosterone promoted the growth and a greater secretory activity of the female prostate. It is worth mentioning that the dynamics of the tissue compartments analyzed here reproduces in detail the prostate development of male mice [33] and Wistar rats (Vilamaior et al., unpublished data).
We conclude that the lack of androgenic stimuli can contribute to the underdevelopment of the normal adult gerbil female prostate. New questions related to the physiological processes that maintain the active state of the prostate in the female organism can be raised, as there are indications that other hormones, such as estrogen and prolactin, are involved in male prostate homeostasis [6, 13, 17, 18, 34]. Furthermore, the presence of enzymatic complexes, such as 5
-reductase (producing dihydrotestosterone from testosterone) [32] and aromatase (which converts testosterone, but not dihydrotestosterone, into estrogen) [35–38], must be investigated in this system.
The transient decrease in circulating PSA concentration seems to represent a temporary recruitment of epithelial cells for proliferation and differentiation, occurring soon after the beginning of treatment. Surprisingly, there was a strong negative correlation between the decrease in PSA levels and the increase in estrogen levels. At this point, we cannot distinguish a negative effect of the increased estrogen levels on prostatic secretory activity from the recruitment of epithelial cells for proliferation. However, this aspect should be analyzed in the future. It also remains to be determined whether this species expresses PSA or a member of the kallikrein family, which showed cross-reactivity with the antibodies employed in this work, because it is well known that rats and mice do not express PSA [39].
As mentioned previously here, it was very common to find characteristics related to the pseudocribriform structure of the epithelium immediately after the proliferating phase. It was also mentioned that most of these characteristics disappeared after longer testosterone treatment. This pseudocribriform arrangement is indicative of high proliferation, and is usually associated with the development of prostatic dysplasias. Although this pattern was evidently associated with the growth of the epithelium in testosterone-treated females, it may have anticipated a dysplastic growth of the organ. As a matter of fact, in the present study, each prostate analyzed after 21 days of testosterone treatment presented signs of dysplastic growth. Histological aspects of prostatic intraepithelial neoplasia, atypical hyperplasia, and mucinous adenocarcinomas were identified among the epithelial lesions. Although the duration of the experiment was relatively short, we may assume that longer testosterone administration will result in prostate cancer.
Finally, we also suggest that hyperandrogenism or exogenous testosterone intake might result in a complex series of effects (Fig. 8) threatening the health of women, resulting in increased prostatic activity with signs of epithelial dysplasia, a thickened urethral wall, ovarian stromal hyperplasia, and polycystic ovaries. The simple assumption that hyperandrogenism results in polycystic ovaries [40] should be reconsidered, taking into account other organs with a potential to respond to androgens.
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ACKNOWLEDGMENTS
The authors are indebted to Dr. M. Zaviai for providing the bibliographic material. The authors express their gratitude to Mr. L.R. Falleiros, Jr. for technical assistance, to Manuel Garcia-Florez for helping with the measurements of the luminal area, and to Maria Cristina C.G. Marcondes for helpful discussions.
FOOTNOTES
1 Supported by São Paulo State Research Foundation (FAPESP) grants to S.R.T. and H.F.C., and National Research Council (CNPq) grants to H.F.C. F.C.A.S., and A.M.G.C. were recipients of fellowships from CNPq and CAPES, respectively. 
2 Correspondence: Hernandes F. Carvalho, Department of Cell Biology–IB–UNICAMP, CP6109, 13083–863 Campinas SP, Brazil. FAX: 55 19 3788 6111; hern{at}unicamp.br
Received: 17 February 2006.
First decision: 3 March 2006.
Accepted: 15 May 2006.
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F. C.A. Santos, A. M.G. Custodio, S. G.P. Campos, P. S.L. Vilamaior, R. M. Goes, and S. R. Taboga Antiestrogen Therapies Affect Tissue Homeostasis of the Gerbil (Meriones unguiculatus) Female Prostate and Ovaries Biol Reprod, October 1, 2008; 79(4): 674 - 685. [Abstract] [Full Text] [PDF]
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