Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.     

A comparison of the effects of castration and 6-methylene progesterone, a 5 alpha-reductase inhibitor, on the rat ventral prostate

6-Methylene progesterone (6MP) is an irreversible in vitro kcat inhibitor of rat prostate 5 alpha-reductase, the enzyme which converts testosterone (T) to dihydrotestosterone (DHT). Treatment of adult rats with 6MP or diethylstilbestrol (DES) decreased the weight of the ventral prostate (VP) by 45%, while castration reduced it by 86%. Histologically, the 6MP-treated VP were indistinguishable from those of controls, while the VP from DES-treated rats showed fibrous stromal hypertrophy as in castrated rats. The prostatic hydroxyproline content, an index of collagen levels, was enhanced by castration or DES, but was not significantly increased by 6MP. Within 2 days of 6MP treatment, the 5 alpha-reductase activity was reduced by 46% and ornithine decarboxylase (ODC) activity was lowered by 27%. During this time the prostatic acid phosphatase activity increased 42% and remained elevated with continued exposure to 6MP up to 13 days. The castration-induced involution of the VP was accompanied by a reduction in serum T and an increase in serum luteinizing hormone (LH). 6MP had no effect on T and LH serum levels but reduced the DHT content within the VP by 64%. Our results indicate that the structure and secretory acid phosphatase activity of the VP are less sensitive to changes in the ratio of T:DHT than is cell proliferation. Thus, the relative amounts of DHT and T within the VP may prove to be more significant than the absolute amount of either androgen in controlling prostate growth or its attendant neoplasms.     

Intravesicular Fas localization in epithelial cells of castrated rat prostate glands

Androgenic steroids regulate the development and size of mammalian prostate epithelial cells. To evaluate the relationship between Fas-Fas ligand system and apoptosis in prostate epithelial cells of the castrated rats, we have examined immunocytochemical localization of Fas antigen in the castrated rat prostate glands at a series of different times. We used a rabbit polyclonal anti-Fas antibody with a streptavidin-biotin method and confocal laser scanning method or an immunogold method. Fas immunolocalization was examined in ventral lobes of prostate glands taken from intact or castrated adult male Wistar rats on day 1, 2, 3, 4 and 5 by light or electron microscopy. At a light microscopic level, the castrated prostate epithelial cells showed mostly Fas immunolocalization in their apical parts of cytoplasm on day 2 after the castration. In addition, their extent of the Fas expression was expanded throughout the cytoplasm in proportion to the androgen ablation periods, and later the Fas expression was detected at luminar or basolateral sides of the epithelial cells. Both immunogold labeling with ultrathin sections and immunoperoxidase technique with cryostat sections demonstrated that Fas was localized mainly in secretory granules of the castrated prostate epithelial cells and some parts of their cell membranes at later stages. Our immunocytochemical findings showed that Fas expression was time-dependently induced in most of the prostatic epithelial cells after castration of rats. The rate of Fas-expressing epithelial cells was too high and inconsistent with the previously reported rate of TUNEL-positive ones. The membrane-associated Fas may have little effect on the apoptosis in the present case, bacause a lot of soluble Fas was secreted from the prostatic epithelial cells. A further study is needed to clarify some significance of the secretory Fas in the prostatic epithelium after the rat castration.     

Intracellular localization of Prostatic Binding Protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry

Extra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by "western blotting" and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl2 in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.     

MMP-2 and TIMP-2 in the prostates of male and female mongolian gerbils: effects of hormonal manipulation

TIMPs in the prostates of male and female gerbils and evaluated the effects of testosterone on the expression of these enzymes. Ventral prostates from male gerbils that were either intact or had been castrated for 7 or 21 days, along with prostates from female gerbils that were either intact or had been treated with testosterone for 7 or 21 days, were submitted to histological, stereological and immunohistochemical analyses. Stereology of prostatic components showed significant alterations of tissue compartments in the ventral male prostate after castration, especially after 21 days, with a significant increase in stroma. Administration of testosterone led to disorganization in the female prostate, with a significant increase in collagen fibers and smooth muscle cells after 21 days, along with the development of epithelial lesions such as PINs. MMP-2 increased after 21 days of castration in males; however, the TIMP-2 immunoreaction for this group was weak or absent. In females, the expression of MMP-2 appeared to decrease after 7 days of treatment with testosterone, but after 21 days, both epithelium and stroma showed a stronger reaction for MMP-2 than the controls. The expression of TIMP-2 in the treated females was similar to its expression in the castrated males. We conclude that the distribution of MMPs and TIMPs in both male and female prostates is altered by androgen manipulation, but the mechanism of stromal regulation appears to be distinct between genders because both the lack of T in castrated males and the excess levels of T in treated females lead to the same effect.     

Intracellular localization of prostatic binding protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry

Summary Extra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by western blotting and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl₂ in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.Supported by a grant from the Deutsche Forschungsgemeinschaft (Au 48/7-6) and a grant from the Cystic Fibrosis Foundation (G 261 A)     

Gene expression profiling identifies a unique androgen-mediated inflammatory/immune signature and a PTEN (phosphatase and tensin homolog deleted on chromosome 10)-mediated apoptotic response specific to the rat ventral prostate

Understanding androgen regulation of gene expression is critical for deciphering mechanisms responsible for the transition from androgen-responsive (AR) to androgen-independent (AI) prostate cancer (PCa). To identify genes differentially regulated by androgens in each prostate lobe, the rat castration model was used. Microarray analysis was performed to compare dorsolateral (DLP) and ventral prostate (VP) samples from sham-castrated, castrated, and testosterone-replenished castrated rats. Our data demonstrate that, after castration, the VP and the DLP differed in the number of genes with altered expression (1496 in VP vs. 256 in DLP) and the nature of pathways modulated. Gene signatures related to apoptosis and immune response specific to the ventral prostate were identified. Microarray and RT-PCR analyses demonstrated the androgen repression of IGF binding protein-3 and -5, CCAAT-enhancer binding protein-delta, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) genes, previously implicated in apoptosis. We show that PTEN protein was increased only in the luminal epithelial cells of the VP, suggesting that it may be a key mediator of VP apoptosis in the absence of androgens. The castration-induced immune/inflammatory gene cluster observed specifically in the VP included IL-15 and IL-18. Immunostaining of the VP, but not the DLP, showed an influx of T cells, macrophages, and mast cells, suggesting that these cells may be the source of the immune signature genes. Interestingly, IL-18 was localized mainly to the basal epithelial cells and the infiltrating macrophages in the regressing VP, whereas IL-15 was induced in the luminal epithelium. The VP castration model exhibits immune cell infiltration and loss of PTEN that is often observed in progressive PCa, thereby making this model useful for further delineation of androgen-regulated gene expression with relevance to PCa.     

Immunocytochemical localization and lectin-binding properties of the 22 kDa secretory protein from rat ventral prostate

The distribution of the 22 kDa secretory protein from rat ventral prostate was studied by light and electron microscopic immunocytochemistry. An anti-22 kDa protein antiserum was raised in rabbits and its specificity was tested by Western blotting. With the immunofluorescence technique, the 22 kDa protein was detected in the luminal secretions and intracellular apical granules of the ventral prostate. No reaction was observed in the seminal vesicle or dorsolateral prostate. After castration, no intracellular immunoreactivity was detected in ventral prostate, although positively labeled secretory material was retained within the acinar lumen. Restoration of normal intracellular staining pattern was incomplete after 5 daily testosterone injections. At the ultrastructural level, labeling was confined to apical secretory granules and condensing vacuoles. The 22 kDa protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose was shown to bind intensely to wheat germ agglutinin (WGA) but only faintly to Concanavalin A. This protein was thus demonstrated to contain N-acetylglucosamine residues. Accordingly, on tissue sections, WGA reacted intensely with condensing vacuoles and secretory granules.     

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.     

Immunochemical characterization of the androgen-dependent spermine-binding protein of the rat ventral prostate.

A solid-phase radioimmunoassay was developed to measure the level of the androgen-dependent spermine-binding protein (SBP) in the cytosol fraction of the rat ventral prostate during endocrine manipulation. The concentration of SBP and immunologically cross-reacting material (CRM) in the ventral prostate was at least 5000 times higher than the level of CRM detected in rat serum or cytosol from other rat tissues. Cytosol from the ventral prostate of intact rats was separated by DEAE-cellulose chromatography into three major fractions of CRM. One of these fractions corresponded to the elution position of SBP. Cytosol prepared from rats 48 h after castration lacked SBP and one of the two other fractions of CRM. This loss coincided with an increase in CRM in the remaining fraction. No significant difference was detected in the total level of CRM when intact and 48 h-castrated rats were compared. Injection of rats with 5 alpha-dihydrotestosterone (DHT) immediately after castration prevented these changes in the profile of CRM. Several proteins cross-reacting with antibodies to purified SBP were detected in cytosol by using an immunoblot procedure. The highest-Mr band corresponded to SBP. The effect of short- and long-term castration and subsequent DHT treatment on CRM was studied by using the immunoblot technique. Short-term castration (2 days) led to the disappearance of CRM coinciding with SBP (Mr 35 000-38 000) and an increase in smaller forms of CRM (Mr 24 000 and 22 000). Injection of rats with DHT 2 days after castration led to the reappearance of CRM corresponding to SBP, which returned to normal levels within 4 to 5 days of treatment. Long-term castration (up to 14 days) led to a gradual disappearance of all CRM; subsequent DHT treatment led to the reappearance of all forms of CRM and normal levels were attained within 5 days. We have identified SBP and the various forms of CRM as a secretory product of the rat ventral prostate by immunohistochemical staining and by DEAE-cellulose fractionation of prostatic fluid. Prostatic fluid is rich in proteolytic activity and these proteinases may be responsible for processing SBP to small forms of CRM.     

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of ∼20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role. This work was funded by FAPESP (Sao Paulo State Research Foundation; grants 04/13261-8, to S.L.F.), by FUNDUNESP (Foundation for Development of Sao Paulo State University), and by CNPq (Brazilian National Research and Development Council; fellowship to L.A.J. Jr). This paper represents part of the PhD thesis presented by L.A.J. Jr to the State University of Campinas - UNICAMP, Brazil.     

Modulation of smooth muscle cell function: morphological evidence for a contractile to synthetic transition in the rat ventral prostate after castration

In this study, we evaluated the involvement of rat ventral prostate smooth muscle cells (SMC) in secretory activity and whether this function is modulated after castration. Cell morphology was examined at both light and electron microscopy levels and the organelles involved in secretory function were labeled by the zinc-iodide-osmium (ZIO) method at the ultrastructural level and their volume density was determined by stereology. Castration resulted in marked changes of the SMC, which adopted a spinous aspect and abandoned the layered arrangement observed in the prostates of non-castrated rats. The volume density of ZIO reactive organelles increased progressively after castration, reaching significantly higher levels 21 days after castration. Since previous studies have demonstrated that SMC express SMC markers (even 21 days after castration) and are able to respond to adrenergic stimulation, we concluded that differentiated SMC are able to shift from a predominantly contractile to a more synthetic phenotype without changing their differentiation status.     

Early changes in the dog prostate after castration. An ultrastructural study

Using electron microscopic techniques the prostate glands of male Beagle dogs were studied 3 days after castration. At this time marked differences in the extent of alterations of the glandular epithelium were observed: Whereas several acini showed only minor changes with reduction of epithelial height and diminution of secretory granules, many acini were severely affected with pronounced alteration of cellular structure and accumulation of large lipid droplets. A constant feature was the stimulation of the basal cells of the grandular epithelium. Additionally, in some areas of the gland aggregations of stimulated basal cells forming an acinus-like structure with a slit-like lumen were found. Our study shows that castration leads to marked alterations of prostatic epithelium within a short time. Androgen deprivation causes regressive changes of secretory epithelial cells, but clearly stimulates the basal cell population.     

Histochemical determination of carbohydrates in the prostate of male and female Praomys (Mastomys) natalensis

The ventral lobes of the prostate in the female and male Praomys (Mastomys) natalensis were studied using light microscopic techniques for the demonstration and localization of carbohydrates. A weakly PAS-positive material appeared in the secretory product and secretory granules in epithelial cells of both female and male ventral lobes. This reaction is unaffected by diastase and is completely blocked by acetylation. Alcian blue, toluidine blue and methylene blue stains demonstrate metachromatic changes only after sulphation. All reactions indicate the presence of neutral mucosubstances in the secretory product and secretory granules of epithelial cells of the ventral lobes in either sex.     

Production of monoclonal antibody against the C1 component of rat estramustine-binding protein: Immunohistochemical study of rat prostate

The monoclonal antibody against estramustine-binding protein (EMBP) was produced by immunizing a mouse with EMBP antigen purified from rat ventral prostate. On western blotting analysis this antibody recognized the EMBP C₁ component, and in an absorption test it recognized the EMBP antigen. Immunohistochemically, this antibody revealed positive staining for the ventral and dorsolateral prostate. In the epithelium of the ventral prostate, intense immunostaining was observed in the intraluminal secretory product; however, in the epithelium of the dorsolateral prostate, the staining was intense in the cytoplasm of the epithelial cells. These findings suggested differences of EMBP localization in the ventral and the dorsolateral prostate. Since the intensity of immunostaining for EMBP was decreased in the prostate of castrated rats, we considered that this antibody reflected the androgen dependency of EMBP.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.