ARTICLE |
Correspondence to: Luis Santamaría, Dept. of Morphology, School of Medicine, Autonomous University, C/ Arzobispo Morcillo, 2, E-28029 Madrid, Spain. E-mail: luis.santamaria@uam.es
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Summary |
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This study was designed to investigate (a) the presence of protein gene product 9.5 (PGP 9.5), ubiquitin, and neuropeptide Y (NPY) in the neuroendocrine and secretory epithelium of the human normal prostate and its secretions, and (b) the changes in immunoreactivity to these proteins in men with benign prostatic hyperplasia. Western blotting and light microscopic immunohistochemistry techniques were used and the numerical density of immunoreactive neuroendocrine cells, and the volume fractions of immunostained secretory epithelium were evaluated. Western blotting revealed the presence of the three antigens in both tissue homogenates and prostate secretion. Some neuroendocrine cells immunoreacted to PGP 9.5 and NPY in all the prostate regions of control specimens. Ubiquitin immunoreactivity was detected in the nuclei from both basal cells and secretory epithelial cells. The cytoplasm of the secretory cells and the glandular lumen also showed immunostaining for the three proteins. The numerical densities of both PGP 9.5 and NPY neuroendocrine cells were lower in hyperplasia than in controls. No differences in the volume fraction occupied by epithelial immunostaining to both proteins was found between hyperplastic and control prostates. We concluded that (a) PGP 9.5 and NPY, but not ubiquitin, are common antigens in both neuroendocrine and secretory prostate cells, (b) the three immunoreactive proteins contribute to the prostate secretions, and (c) the secretion of ubiquitin is markedly diminished in the hyperplastic epithelium.
(J Histochem Cytochem 48:11211130, 2000)
Key Words: prostate, PGP 9.5, neuropeptide Y, ubiquitin, hyperplasia
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Introduction |
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Protein gene product 9.5 (PGP 9.5), an ubiquitin carboxyl-terminal hydrolase (
Neuropeptide Y (NPY) is a small polypeptide expressed in neurons of the central and peripheral nervous systems (
In the present work, we hypothesized that some peptides that have been described to perform only endocrineparacrine activity might also have an exocrine function, and that the secretion of these proteins into the prostate fluid could be relevant for the composition and properties of the seminal plasma (
The aims of this study were (a) to examine the distribution of PGP 9.5, ubiquitin, and NPY immunoreactivities in both neuroendocrine and secretory epithelium of the human prostate and in the prostate secretion and (b) to investigate the changes in immunoreactivity to those proteins in normal prostate and in BPH.
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Materials and Methods |
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Materials
The prostates from 45 men (aged 6080 years) who were clinically and histopathologically diagnosed with BPH were obtained by surgery (adenomectomy). In addition, the prostates of five men (aged 6070 years) with BPH and eight men (aged 2040 years) without BPH (histologically normal prostates) were obtained from autopsies between 8 and 10 hr after death. The condition for selection of these specimens was the absence of reproductive, endocrine, and related diseases in the patient histories. In addition, prostate secretions from the five men with BPH and from eight normal, young men (controls) were obtained during urologic examination. The age ranges of these patients were the same as indicated for histological and immunocytochemical studies. The procedures followed for obtaining all these materials were in accordance with the guidelines of the ethical committee of the La Princesa Hospital.
Processing of Materials
For morphometric and light microscopic immunohistochemical studies, three 2-mm-wide slices sectioned perpendicularly to the prostate urethra were systematic randomly sampled from each specimen (
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Western Blotting Analysis
For Western blotting analysis, prostate secretions, unfixed fragments of the three prostate zones (central, transition, and peripheral zones;
Immunohistochemical Methods
Five-µm-thick serial sections from each slice were immunostained by the avidinbiotinperoxidase method. Before incubation with the primary antibody, endogenous peroxidase activity of the tissues was blocked with 0.3% hydrogen peroxide in PBS. The tissues were incubated overnight at 4C with the same primary antibodies used for Western blotting: rabbit polyclonal antibody against PGP 9.5 at 1:1000 dilution; rabbit polyclonal antibody to ubiquitin at 1:400 dilution; and rabbit polyclonal antibody to NPY at 1:2000 dilution. To compare the immunoreactivity of PGP 9.5 and NPY with that of the neuroendocrine marker chromogranin A (
To assess the immunostaining specificity, negative controls obtained by either omitting primary antibodies or using these antibodies preabsorbed with a tenfold excess (by weight) of purified antigens were also analyzed. The antibodyantigen complexes were incubated in PBS, pH 7.4, overnight at 4C and centrifuged for 15 min before use.
Morphometric Methods
Estimation of the amount of neuroendocrine cells that were chromogranin-, PGP 9.5-, or NPY-immunoreactive was performed using the technique of the optical disector, an unbiased stereological method (
An average of 100 fields per section in each prostate zone from controls and the same number of fields per section in each BPH specimen were systematically randomly sampled (Fig 1) and used to count the number of immunoreactive cells. All measurements were performed using an Olympus microscope with a x100 objective (numerical aperture 1.4) at a final magnification of x1200. The microscope was connected to a videocamera and supplied with a motorized stage connected to a Commodore Amiga 2000 computer. The software used (Stereologic Software Package, GRID; Interactivision, Silkeborg, Denmark) (
where Sd = 1300 µm2 (area of the disector grid), Hd = 10 µm [distance between the two focal planes chosen for determining the disector volume into the tissue section; this distance was measured by means of a microcator (Heidenhain; Traunreut, Germany) connected to the Z displacement of the microscope stage]. Because the total thickness of the section was 15 µm, a safeguard space of 2.5 µm above and below the optical section was maintained to avoid artifacts produced in the physical surface of the section. All these calculations lead to Vdis = 13,000 µm3.
The neuroendocrine cell nuclei eligible to be counted were determined by using Sterio's convention (
where Vdis = total number of disectors applied in each selected section multiplied by Vdis. NV was calculated as number of cells per mm3 of epithelial volume in each prostate zone or BPH specimen.
To quantify the immunostaining of epithelial non-neuroendocrine cells for PGP 9.5, ubiquitin, and NPY, their respective volume fractions were measured, i.e., the ratio between immunostained epithelial area and the reference area of prostate epithelium (excluding the glandular lumen). These volume fractions were estimated in an average of 25 systematically randomly sampled light microscopic fields (an average of 194,000 µm2 per field) in five systematically randomly selected sections of each control prostate area or BPH specimen. These measurements were performed counting the points hitting either immunostained epithelial area or the reference area, using the GRID software package which provides a counting point grid with a point associate area, A(p) = 45 µm2. The final magnification used for these measurements was x500.
Statistical Analysis
For each parameter studied, the means ± SD were calculated. The differences among prostate zones and between transition zone of controls and BPH specimens were evaluated by ANOVA. Comparison between each pair of means was performed using the Fisher and Behren's test.
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Results |
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Comparison of the prostates obtained during surgery with those from autopsies showed neither histological nor immunohistochemical changes.
Western Blotting Results
In the extracts of all prostate specimens studied, either normal or hyperplastic, Western blotting analysis revealed three immunoreactive bands, located at approximately 7 kD (NPY), 27 kD (PGP 9.5), and 60 kD (ubiquitin) (Fig 2a). In the prostate secretions, Western blotting analysis revealed two ubiquitin-immunoreactive bands located at approximately 66 and 97 kD, one band PGP 9.5-immunoreactive at 27 kD, and two NPY-immunoreactive bands located at 7 and 97 kD (Fig 2b).
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Immunohistochemical Qualitative Results
PGP 9.5 immunoreactivity was observed around the nucleus and apical cytoplasm in most epithelial secretory cells throughout all prostate zones (Fig 3). In several tubuloacini, PGP 9.5 immunostaining was also found in the glandular lumen (Fig 3 and Fig 4). Cell debris was occasionally observed in the glandular lumen (Fig 3). Chromogranin-positive neuroendocrine cells were detected scattered among the epithelial secretory cells (Fig 5a). Some of these cells were also immunoreactive for PGP 9.5 (Fig 5b). Many PGP 9.5-immunoreactive nerves were detected in the connective tissue of the capsule, around the prostate vessels, and in the vicinity of tubuloacini (Fig 5b).
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Ubiquitin immunoreactivity was detected in both basal and secretory epithelial cells in all prostate zones (Fig 6). This immunostaining was observed in the nuclei and the cytoplasm in both cell types. In addition, in many areas the glandular lumen and the apical border of the secretory epithelial cells showed intense ubiquitin immunoreaction (Fig 6). Weak ubiquitin immunoreactivity was detected in some cells from the fibromuscular stroma.
NPY immunoreaction was detected in some dispersed areas, mainly in the central and transition prostate zones. This immunoreaction was granular and was localized in the cytoplasm of many secretory epithelial cells (Fig 7 and Fig 8). Occasionally, the glandular lumen also immunoreacted to NPY (Fig 7). In some chromogranin-positive neuroendocrine cells that were scattered throughout the prostate (Fig 9a), NPY immunoreaction was also detected (Fig 9b). NPY-immunoreactive nerve fibers were observed around tubuloacini in all the prostate zones (Fig 8 and Fig 9b). No stromal immunostaining for NPY was seen.
In both control and BPH specimens, the sections incubated either omitting primary antibodies or using each of the antibodies employed in this study preabsorbed with the corresponding antigen showed no immunostaining in any tissue component (Fig 10 Fig 11 Fig 12 Fig 13 Fig 14 Fig 15).
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Neuroendocrine Cells: Quantitative Results
The NV of chromogranin, PGP 9.5, and NPY neuroendocrine-immunoreactive cells was significantly higher (p<0.05) in the transition zone of control prostates than in the prostates of BPH cases. In control cases, the NV of chromogranin-, PGP 9.5-, and NPY-immunostained cells was more elevated (p<0.05) in the transition zone than in both central and peripheral zones. These results are shown in Table 1.
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In transition zone of controls, 67% of the neuroendocrine cells that were immunoreactive to chromogranin also showed immunoreaction to PGP 9.5, whereas 15% of those chromogranin-positive cells were immunostained for NPY. In BPH specimens, 6% of chromogranin-immunoreactive neuroendocrine cells were immunostained for PGP 9.5, and 12% of chromogranin-immunostained cells were also positive for NPY.
Epithelial Immunostaining: Quantitative Results
The quantitative results for epithelial non-neuroendocrine immunostaining are indicated in Table 1. Volume fraction of PGP 9.5 epithelial immunostaining showed no significant differences among prostate zones in controls and between transition zone of controls and BPH cases. Volume fraction for ubiquitin immunostaining did not differ significantly among prostate zones in controls. Nevertheless, this volume fraction was significantly lower in BPH than in the transition zone of control cases (p<0.05).
Volume fraction for NPY immunostaining did not show significant differences among prostate zones in control cases. No significant differences in this fraction were found between BPH and the transition zone of control cases.
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Discussion |
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The specificities of all immunoreactions reported in the present study were confirmed by Western blotting methods. These methods also demonstrated the presence of PGP 9.5, NPY, and ubiquitin immunoreactivities in the prostate secretions.
The estimations of relative amounts of neuroendocrine cells that were immunoreactive for chromogranin, PGP 9.5, and NPY agree with previous observations (
A significant decrease in the global population of chromogranin-immunoreactive neuroendocrine cells and in those cells that co-localized chromogranin and either PGP 9.5 or NPY was found in BPH specimens. This finding agrees with previous studies of large hyperplastic prostate nodules (
Not all neuroendocrine cell subpopulations undergo the same rate of decrease in BPH. Therefore, the percentage of chromogranin-positive cells that expressed NPY shows little change in BPH specimens, whereas the proportion of chromogranin-positive cells that co-localized PGP 9.5 was significantly decreased in BPH.
PGP 9.5 and ubiquitin immunoreactivities were also observed in the cytoplasm of epithelial secretory cells and in the glandular lumen. Cytoplasmic localization of ubiquitin has been reported in other cell types, such as hepatoma cells (
It has been proposed that the family of NPY gene-related peptides might play a role in the physiology of the male reproductive tract (
The localization of NPY immunostaining in the apical cytoplasm of prostate secretory cells and glandular lumen suggests that the proteins synthesized and secreted by these cells also contain NPY-immunoreactive substances. The present finding agrees with the detection in bull semen of a basic protein called seminal plasmin, which has an extensive homology to the NPY family. This peptide has a role in calcium transport regulation in sperm and in modulation of the zona pellucida-induced acrosome reaction (
As was previously hypothesized, the proteins analyzed in this study are present in the epithelial secretory cells and in luminal prostate secretions. The finding of two ubiquitin bands in the Western blot of prostate secretion might be due to the presence of ubiquitinated proteins with diverse molecular weight, and the migration differences detected between tissue extracts and prostate secretion for the NPY band might be explained because the prostasomic NPY may be similar to that detected in the tissue (7-kD migration band) or linked to other proteins (97-kD migration band). It has been observed (
Some of the findings described in the present work could have relevance to normal or pathological function of the prostate. For example, (a) although some authors (
Summarizing, we can conclude that (a) PGP 9.5, and NPY, but not ubiquitin, are antigens common to both neuroendocrine and secretory prostate epithelium, (b) these three immunoreactive proteins appear to have an exocrine function in the prostate, and (c) BPH is associated with a selective impairment of ubiquitin secretion.
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Acknowledgments |
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We are indebted to Prof J.M. Polak (Department of Histochemistry, Hammersmith Hospital; London, UK) who supplied the NPY antibody and the PGP 9.5 antibody manufactured by Ultraclone.
Received for publication November 7, 1999; accepted March 10, 2000.
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