Bisphenol-A analogs induce lower urinary tract dysfunction in male mice.

Bisphenol-A (BPA), an estrogenic endocrine disrupting chemical, significantly impacts numerous diseases and abnormalities in mammals. Estrogens are known to play an important role in the biology of the prostate; however, little is known about the role of bisphenols in the etiology of prostate pathologies, including benign prostate hyperplasia (BPH) and associated lower urinary tract dysfunction (LUTD). Bisphenol-F (BPF) and bisphenol-S (BPS) are analogs often used as substitutes for BPA; they are both reported to have in vitro and in vivo estrogenic effects similar to or more potent than BPA. The objective of this study was to assess the role of these bisphenols in the development of LUTD in adult male mice. In adult mice exposed to BPA, BPS or BPF, we examined urinary tract histopathology and physiological events associated with urinary dysfunction. Mice treated with bisphenols displayed increased bladder (p < 0.005) and prostate (p < 0.0001) mass, and there was an increased number of prostatic ducts in the prostatic urethra (p < 0.05) and decreased size of the urethra lumen (p < 0.05) compared to negative controls. After two months of bisphenol exposure, mice displayed notable differences in cystometric tracings compared to controls, consistent with LUTD. Treatment of male mice with all bisphenols also induced voiding dysfunction manifested by detrusor instability and histologic changes in the prostatic urethra of male rodents, consistent with LUTD. Our results implicate BPA and its replacements in the development and progression LUTD in mice and provide insights into the development and progression of BPH/LUTS in men.

Novel expression patterns of the myc/max/mad transcription factor network in developing murine prostate gland.

PURPOSE: Expression of myc proto-oncogenes and myc-antagonizing mad/mxi genes typically predominate in proliferating versus differentiating cells, respectively. C-myc expression in prostate cells is well established but to our knowledge that of several recently discovered mad/mxi genes is completely uncharacterized. Such characterization is particularly relevant because mxi1 is lost or mutated in some human prostate tumors and mouse mxi1-null mutants show prostatic hyperplasia. MATERIALS AND METHODS: Developing murine prostatic lobes at select postnatal days 1 to 28 were analyzed by in situ immunohistochemical and in vitro RNA analysis. The expression patterns of the 3 myc genes c-, L- and N-myc, and the mad1, mxi1 and mad4 genes were studied in most detail with nonradioactive in situ and immunohistochemical analyses. RESULTS: We describe what is to our knowledge previously unreported expression of N- and L-myc in the prostate with particularly the latter strongly expressed throughout development. High c-myc expression was lost at day 7 with re-elevation at day 14, followed by subsequent low expression, representing a unique in vivo confirmation of c-myc expression changes seen previously in several in vitro differentiation systems. The alternatively spliced weak and strong repressor mxi1 isoforms showed distinct, partially overlapping expression patterns. Of particular interest were continual mad1 and mad4 expression during the proliferative and differentiative phases. Similarly mad1 was evident in proliferating normal prostate cell cultures but not in tumor cell lines, suggesting that mad1 expression in prostate may be clinically relevant. CONCLUSIONS: Myc network expression in developing mouse prostate is novel and does not completely fit previous simpler models of Myc versus Mad expression based on other cell types.

Prostate gland growth during development is stimulated in both male and female rat fetuses by intrauterine proximity to female fetuses.

In rodents, steroid hormones are transported between adjacent fetuses, and male or female fetuses that develop in utero between female fetuses (2F males or 2F females) have higher serum levels of estradiol and lower serum levels of testosterone relative to siblings of the same sex that develop between two male fetuses (2M males or 2M females). The present study was prompted by the prior unexpected finding that as adults, 2F male mice have an enlarged prostate, and increased numbers of prostatic androgen receptors relative to 2M males. We examined prostate development in both male and female rat fetuses from different intrauterine positions using computer-assisted, 3-dimensional reconstruction of the urogenital complex. In males, this included the prostate, seminal vesicles and utricle (a remnant of the Müllerian ducts), while in females it included development of prostatic glandular buds. The mean cross-sectional area of developing prostatic epithelial buds, utricle and seminal vesicles was significantly increased in 2F male relative to 2M male fetuses. In female fetuses, prostatic bud development was significantly more likely to occur in 2F (67%) than in 2M (29%) animals. These findings suggest that the transport of a small supplement of estrogen from adjacent female fetuses enhances androgen-dependent accessory organ development. We also found that mRNAs encoding receptors for both estrogen and androgen were located in the mesenchyme of the developing male prostate. The localization of estrogen and androgen receptor mRNA in this region further suggests that the mesenchymal induction of prostatic epithelial growth involves both hormones. The cranial dorsolateral prostatic buds exhibited the greatest enlargement in 2F males. This region of the developing prostate in rats is comparable (that is the embryonic homologue) to the region exhibiting benign prostatic hyperplasia (BPH) during aging in men. We propose that the potential for pathological regrowth of the prostate during aging is imprinted by estradiol during fetal development.

In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development. 2. Effects on growth and cytodifferentiation.

In the male Holtzman rat, in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases prostate weight without inhibiting testicular androgen production or decreasing circulating androgen concentrations. Therefore, the present study sought to characterize effects of TCDD exposure on prostate development, from very early outgrowth from the urogenital sinus (Gestation Day [GD] 20) until rapid growth and differentiation are essentially complete (Postnatal Day [PND] 32). Pregnant Holtzman rats were administered a single dose of TCDD (1.0 microgram/kg po) or vehicle on GD 15 and offspring were exposed via placental transfer (GD 20 euthanasia) or placental and subsequent lactational transfer until euthanasia (if before PND 21) or weaning. Results show that the prostatic epithelial budding process was impaired by in utero TCDD exposure, as evidence by significant decreases in the number of buds emerging from dorsal, lateral, and ventral aspects of the GD 20 urogenital sinus. Ventral prostate cell proliferation index was significantly decreased on PND 1 but was similar to or higher than control at later times, whereas apoptosis was an extremely rare event in ventral prostates from both control and TCDD-exposed animals. Delays were noted in the differentiation of pericordal smooth muscle cells and luminal epithelial cells. In addition, ventral prostates from approximately 40% of TCDD-exposed animals examined on PNDs 21 and 32 exhibited alterations in the histological arrangement of cell types that could not be explained by a developmental delay. Compared to controls, these ventral prostates exhibited a disorganized, hyperplastic epithelium containing fewer luminal epithelial cells and an increased density or continuous layer of basal epithelial cells, as well as thicker periductal smooth muscle sheaths. In addition, in ventral prostates from TCDD-exposed animals, the intensity of androgen receptor staining was relatively low in the central and distal epithelium, and the number of androgen receptor-positive cells was relatively high in the periductal stroma. These data suggest that in utero and lactational TCDD exposure interferes with prostate development by decreasing very early epithelial growth, delaying cytodifferentiation, and, in the most severely affected animals, producing alterations in epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression that may be of permanent consequence.

Prostate enlargement in mice due to fetal exposure to low doses of estradiol or diethylstilbestrol and opposite effects at high doses.

On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship.

Localization of androgen receptor and cell-specific cytokeratins in basal cells of rat ventral prostate.

Immunocytochemical techniques labeled androgen receptor and cell-specific cytokeratins in the basal cells of rat ventral prostate. In addition, nonradioactive in situ hybridization verified the production of androgen receptor transcripts in the basal cells. Androgen receptors and transcripts were localized in the nuclei and cytoplasm of the adult basal cells using these two techniques. Monoclonal anti-cytokeratin antibodies identified temporal changes in the expression of basal cell-specific intermediate filaments of fetal, neonatal, normal adult, orchidectomized adult, and testosterone-treated orchidectomized adult prostates. Labeling intensity of the basal cells was elevated during development when compared to the staining in normal adult tissue. Orchidectomized adults exhibited the greatest intensity of labeling, which decreased after testosterone treatment. The detection of androgen receptor and its transcripts in the basal cells supports the hypothesis that these cells are androgen responsive. The observed change in the anti-cytokeratin staining patterns of these cells during development, growth, and regression is an indirect measure of androgenic influence. The androgen-repressed cytokeratin expression in basal cells is similar to that found in prostatic luminal cells.

Instructive induction of prostate growth and differentiation by a defined urogenital sinus mesenchyme.

Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium.

Ductal budding and branching patterns in the developing prostate.

Development of the prostate was studied by serial section reconstruction and computer-assisted three-dimensional analysis. A comparison of ductal budding in species of rat and mouse and the human revealed patterns consistent with common developmental characteristics. Ventral, lateral and dorsal lines of epithelial buds, which emanated from the urogenital sinus into the surrounding periurethral mesenchyme, followed ventro-dorsal and cranio-caudal axes. Subsequent branching morphogenesis was associated with specific mesenchymal condensations. These patterns of budding were closely related to the adult lobe architecture in the rodent prostate. In the human fetus, prostate ductal budding exhibited patterns compatible with the current concept of zonal anatomy.

Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (sarcalumenin)-dalton glycoproteins of the sarcoplasmic reticulum.

The 53-kDa glycoprotein and sarcalumenin (160-kDa glycoprotein) were extracted from rabbit skeletal muscle sarcoplasmic reticulum with EGTA and purified by fractionation on DEAE-Sephadex A-25 and lentil lectin-Sepharose 4B. Sarcalumenin was shown to bind up to 400 nmol of Ca2+/mg of protein at pH 7.5, which is equivalent to binding of approximately 35 mol of Ca2+/mol of protein. The apparent dissociation constant was 300 microM in the presence of 20 mM KCl and 600 microM in 150 mM KCl. The 53-kDa glycoprotein did not bind any Ca2+ under the conditions examined. Immunoblot analysis of isolated sarcoplasmic reticulum subfractions demonstrated the presence of the two glycoproteins in both the longitudinal sarcoplasmic reticulum and the terminal cisternae. Their concentrations were higher, however, in the longitudinal sarcoplasmic reticulum vesicles. Comparative immunoelectron microscopic studies using monoclonal antibodies revealed a codistribution of the 53-kDa glycoprotein with the Ca2(+)-ATPase in all regions of the free sarcoplasmic reticulum. A similar distribution was found for sarcalumenin, although immunolabeling was much weaker. The colocalization of the 53-kDa glycoprotein and sarcalumenin with the Ca2(+)-ATPase and the Ca2+ binding properties of sarcalumenin suggest that the glycoproteins may be involved in the sequestration of Ca2+ in the nonjunctional regions of the sarcoplasmic reticulum.

Isolation and characterization of rabbit ovarian surface epithelium, granulosa cells, and peritoneal mesothelium in primary culture.

Mammalian ovarian surface epithelial (OSE) cells and peritoneal mesothelial (PM) cells have a common embryologic origin, yet certain morphologic and histochemical characteristics are different in the adult. In this study, a two-step culture method was developed to examine the characteristics of these two cell types in vitro. OSE, PM, and ovarian granulosa (GC) cells were isolated from estrous rabbits and cultured for 6 d in 5% serum-supplemented D-valine medium (to inhibit fibroblast growth), then incubated for a further 2 d in serum-free McCoy's 5A medium. This study showed that rabbit OSE and PM cells in vitro maintained certain in vivo morphologic characteristics; OSE cells exhibited distinct cell borders and abundant microvilli of homogeneous size and shape, whereas PM cells were characterized by obscure cell borders and abundant microvilli of heterogeneous form. GC in vitro exhibited overlapping cell borders and sparse microvilli of homogeneous structure. This study showed for the first time that cultured rabbit OSE and PM cells, but not GC, contain distinct filaments of cytokeratin 18. In addition, rabbit OSE cells and GC, but not PM cells, contained 17 beta-hydroxysteroid dehydrogenase. However, only GC contained delta 5-3 beta hydroxysteroid dehydrogenase. OSE, PM, and GC maintained their ultrastructural and histochemical characteristics in serum-free medium. These results suggest that rabbit OSE cells in vitro could be distinguished from PM cells by histochemical and ultrastructural differences. Furthermore, because these characteristics were not altered in serum-free medium, the two-step culture method will be valuable in further hormonal studies of these cells in vitro.

Immunohistochemical localization of metallothionein in the rat prostate gland during postnatal development.

Immunocytochemical and electrophoretic techniques were used to investigate the presence of metallothionein, a metal-binding protein, in the dorsolateral and ventral lobes of the developing rat prostate. Male rats aged 7 and 14 days were injected subcutaneously with 6 and 20 mg/kg body weight of cadmium and zinc, respectively, or with saline for controls, 24 h prior to tissue sampling. Immunohistochemical localization of metallothionein was observed in the epithelial tissues of the dorsolateral prostate from 7 and 14 day-old animals and in 1 day-old untreated rats. This staining pattern did not appear to be significantly affected by cadmium or zinc treatment. In contrast, metallothionein localization in the ventral prostate decreased with age but demonstrated a slight response to metal-ion treatment in the 7 day-old animals. Electrophoretic and immunoblot analysis confirmed the presence of metallothionein in the control and metal-induced prostate samples from neonatal rats. Lobe-specific differences in localization suggest a functional significance for metallothionein, independent of inducible protein.

Postembedding immunogold labeling for electron microscopy using "LR White" resin.

A method is described for performing postembedding immunogold immunocytochemistry on sections of LR White-embedded tissues. Fixation of tissue in a combination of paraformaldehyde and glutaraldehyde, or with low concentrations of glutaraldehyde followed by partial dehydration, resulted in preservation of antigenicity for a variety of proteins in different tissue samples. Good structural preservation facilitated high-resolution immunolabeling when coupled with the use of purified monoclonal antibodies. The technique is straightforward and versatile, offering the potential for many immunocytochemical applications with minimal modifications.

Immunocytochemical localization of metallothionein in the rat prostate gland.

Metallothionein (MT), which binds zinc and other metals, was localized within the rat prostatic complex at light and electron microscopic levels utilizing immunocytochemistry. Two groups of mature, male Sprague-Dawley rats were injected with cadmium chloride subcutaneously (2 mg/kg body weight) to induce the synthesis of MT, and were sacrificed 48 h postinjection. From the first group, prostatic tissue from the three lobes was prepared for light microscopy. The indirect peroxidase antiperoxidase procedure was used for MT localization using rabbit anti-MT as the primary antibody. The three lobes of the rat prostate demonstrated a positive reaction for MT, but among the lobes variations in the intensity and localization of the reaction product occurred. Tissues from the second group of animals were prepared for electron microscopy. Using the indirect immunogold labeling technique with anti-MT, MT localization was confirmed in the rough endoplasmic reticulum, secretory vesicles, secretory products, and subepithelial connective tissue. These localizations suggest that MT binds zinc both intra- and extracellularly, where it may function in zinc storage and metabolism.

The effects of estradiol-17 beta on the ultrastructure and subcellular distribution of zinc in the prostatic epithelium of castrated rats.

Male Sprague-Dawley rats, previously castrated for a 7-day period, were maintained on either a low or high dose of estradiol-17 beta for 3 days. Some areas of the prostatic epithelium in the lateral lobe exhibited the ultrastructural characteristics of the untreated, intact animals in response to the small dose of estrogen. The ventral lobe by comparison was not similarly affected. This stimulation in the lateral prostate was not reflected by comparable changes in the subcellular distribution of zinc.

Endogenous elements in the prostate. An X-ray microanalytical study of freeze-dried frozen sections and histochemical localization of zinc by potassium pyroantimonate.

Freeze-dried frozen sections prepared from unfixed rat lateral prostate were examined by X-ray microanalysis in an attempt to establish the in vivo distribution of endogenous ions. Poor morphological resolution was a limiting factor in the analysis of subcellular regions of the tissue. Glutaraldehyde fixation prior to cryo-sectioning resulted in considerable loss of elements. The results are discussed and compared with those obtained from ultrathin sections of tissue treated with potassium pyroantimonate. Using the latter method, it was possible to demonstrate a subcellular distribution pattern for the element zinc and to correlate the metal with specific organelles. It is considered that, unlike a number of other tissues, the rat prostate does not lend itself readily to cryoultramicrotomy as a preparative regime.

Ultrastructural and analytical studies on the prostate of castrated rats.

Rat lateral and ventral prostate tissue was studied using ultrastructural and analytical techniques in adult animals castrated for periods of 3 to 20 days. As in previous reports involution of the prostatic epithelium following testosterone deprivation resulted in alterations of the amount, distribution, and conformation of the endoplasmic reticulum, Golgi, and lysosomes in addition to some nuclear changes. Although the fundamental changes were similar in both lobes there were specific effects on the individual lobes. Reductions in the subcellular levels of zinc were more pronounced in the lateral prostate, particularly with respect to the secretory and stromal concentrations of the metal. Despite low concentrations of circulating testosterone, secretory activity was still evident after a 20-day castration period and would appear to reflect a different sensitivity in the lateral lobe to the lowered testosterone level or stimulation by other hormones when compared to the ventral lobe. The significance of the subcellular distribution pattern of zinc during the experimental periods is discussed in relation to the possible functional significance of the metal in prostatic tissue.

Effects of androgens on the ultrastructure and subcellular zinc distribution in the prostatic epithelium of castrated rats.

Sprague Dawley rats were maintained on testosterone propionate or 5 alpha-dihydrotestosterone for 3 days following bilateral orchidectomy for a 7-day period. Ultrastructural examination showed only partial recovery of the prostatic epithelium with testosterone propionate while 5 alpha-dihydrotestosterone caused the lateral and ventral lobes to revert to the appearance of control tissues. The latter metabolite induced greater stimulation of the prostate evidenced by increased mitotic division of the epithelial cells and an increased number of basal cells exhibiting ciliary formation was observed. Zinc concentrations in subcellular regions of both lateral and ventral prostate lobes were affected by the two androgens. Testosterone propionate was most effective in elevating zinc in the lateral lobe, particularly within the secretory components. In the ventral lobe both androgens caused an increase in subcellular zinc concentrations above control levels. The increase of nuclear and nucleolar zinc was related to the increase in nuclear activity and cellular response to the androgen administration.

The subcellular distribution of zinc in dog prostate studied by x-ray microanalysis.

X-ray microanalysis of zinc in ultrathin sections of dog prostate was performed by electron microscope microanalysis using the potassium pyroantimonate method of preparation. Prostates of both mature and immature dogs were examined and the metal was found to be localised primarily in the nucleolus, nuclear chromatin and secretory granules of epithelial cells. Differences in zinc concentrations were observed between mature and immature tissues, particularly in the nuclear chromatin. The metal was also incorporated into epithelial secretions, lysosomes and fibromuscular stroma. Variable binding of zinc to tissue components was revealed by a combination of histochemical precipitation and subcellular analysis.

The effect of testosterone and cadmium on the rat lateral prostate in organ culture.

Organ culture of rat lateral prostate was performed in the presence of testosterone and cadmium. Maintenance of epithelial cells did not occur even in the presence of the androgen, but basal cells were stimulated and replaced original epithelium. Testosterone alone caused a partial differentiation of these basal cells. Cadmium alone was found to enter the epithelial and basal cells and subsequently cause necrosis. The metal was subcellularly located in the nucleus and within cytoplasmic organelles. Cadmium appears to compete with zinc in cultured lateral prostate and affects the differentiation and maintenance of the epithelial growth.