Ultrastructural and proliferative features of the ventral lobe of the prostate in non-obese diabetic mice (NOD) following androgen and estrogen replacement associated to insulin therapy

Diabetes causes harmful effects on prostatic function. Thus, the aims of this study were to characterize morphological and proliferative features of the prostate of diabetic mice after long-term glycemic control and testosterone and estrogen replacement. A total of 48 mice (Nod and BALBc) were used. After 20 days in a diabetic state, the mice were divided into six groups: the control group received a 5 mL/kg dose of peanut oil; the diabetic group received the same treatment as the control group; the diabetic-insulin group received 4 IU doses of insulin; the diabetic-testosterone group received a 5 mg/kg dose of testosterone cypionate; the diabetic-estrogen group received a 25 μg/kg dose of 17β-estradiol; the diabetic-insulin-testosterone-estrogen group received insulin, testosterone and estrogen at the same concentration as the other groups. After 20 days, the ventral lobe was processed for morphological and immunological analyses. The results showed structural disorganization, which was more intense in the diabetic group than in the other groups. The diabetic state showed a proliferation and apoptosis rate that was two times higher than that found in the control group. To conclude, diabetes disturbed the prostatic secretory activity and the association of insulin, testosterone and estrogen was crucial for glandular structural restoration, characterizing the complex activity of the prostate. The imbalance verified between the proliferation process and apoptosis in diabetic mice showed diabetes to be a triggering factor for prostatic pathogenesis.     

Structure and ultrastructure of the ventral prostate of isogenic mice (C57B1/6J) submitted to chronic alcohol ingestion

Morphological and functional alterations caused by chronic alcohol ingestion in testes and accessory sex organs have been studied both in man and in laboratory animals. The aim of the present study was to examine the possible occurrence of deleterious effects of chronic alcohol ingestion on the secretory epithelium of the ventral prostate of mice. Twenty-four adult male C57BL/6J mice were divided into three groups. The alcohol-treated group was allowed to drink only 6% (v/v) ethanol, the isocaloric group received a diet of water/sucrose with a calorie content equivalent to a 6% alcohol solution and the control group received water. Both groups were fed ad libitum with solid Purina rat chow. After 120 days, animals from each group were anesthetized with ethyl ether, weighed and processed for light and transmission electron microscopy. The results demonstrated reduction in the glandular epithelium cell height and disorganization of the Golgi complex. Moreover, abundant membrane-bound structures, most likely representing cytoplasmic material, were observed, as well as accumulation of dense bodies. Statistical analysis showed that bodyweight gain was similar for both groups. In conclusion, chronic alcohol ingestion has harmful effects on the secretory epithelium cells of the ventral lobe of the prostate of mice after 120 days of treatment.     

Expression signature of the mouse prostate

Genetically engineered mice are being used increasingly for delineating the molecular mechanisms of prostate cancer development. Epithelium-stroma interactions play a critical role in prostate development and tumorigenesis. To better understand gene expression patterns in the normal sexually mature mouse prostate, epithelium and stroma were laser-capture microdissected from ventral, dorsolateral, and anterior prostate lobes. Genome-wide expression was measured by DNA microarrays. Our analysis indicated that the gene expression pattern in the mouse dorsolateral lobe was closest to that of the human prostate peripheral zone, supporting the hypothesis that these prostate compartments are functionally equivalent. Stroma from a given lobe had closer gene expression patterns with stroma from other lobes than epithelium from the same lobe. Stroma appeared to have higher expression complexity than epithelium. Specifically, stromal cells had higher expression levels of genes implicated in cell adhesion, muscle development, and contraction, in structural constituents of cytoskeleton and actin binding, and in components such as sarcomere and extracellular matrix collagen. Among the genes that were enriched in the epithelium were secretory proteins, including seminal vesicle protein secretion 2 and 5. Surprisingly, prostate stroma expressed many osteogenic molecules, as confirmed by immunohistochemistry. A "bone-like" environment in the prostate may predispose prostate cells for survival in the bone. Chemokine Cxcl12 but not its receptor, Cxcr4, was expressed in normal prostate. In prostate tumors, interestingly, Cxcl12 was up-regulated in epithelial cells with a concomitant expression of Cxcr4. Expression of both the receptor and ligand may provide an autocrine mechanism for tumor cell migration and invasion.     

Hoxb13 is required for normal differentiation and secretory function of the ventral prostate

The murine prostate is a structure that is made up of four distinct lobes; the dorsal and lateral prostates (often grouped together as the dorsolateral prostate), the anterior (coagulating gland) and the ventral prostate. Previous work has implicated Hox genes in the development of these structures, but how each lobe acquires unique identities for specific functions has not been addressed. In this study, the ventral prostate-specific function of Hoxb13 is described. Mice lacking Hoxb13 function show normal numbers of duct tips, but mice mutant for both Hoxb13 and Hoxd13 exhibit severe hypoplasia of the duct tips, revealing a role for Hoxb13 in ventral prostate morphogenesis. Additionally, a ventral lobe-specific defect was identified in Hoxb13 mutants wherein the epithelium is composed of simple cuboidal cells rather than of tall columnar cells. Ventral prostate ducts appear devoid of contents and do not express the ventral prostate-specific secretory proteins p12, a kazal-type protease inhibitor and p25, a spermine binding protein. These defects are not due to reduction of Nkx3.1 expression or to a global effect on androgen receptor signaling. These results suggest a specific role for Hoxb13 in a differentiation pathway that gives the ventral prostate epithelium a unique identity, as well as a more general role in ventral prostate morphogenesis that is redundant with other Hox13 paralogs.     

Morphometric and morphological features of the ventral prostate in rats submitted to chronic nicotine and alcohol treatment

Clinical studies analyzing simultaneous nicotine-alcohol use by patients showed important alterations in various organic systems such as: respiratory, digestory, and genital. Also, the prostatic morphology and physiology have been analyzed, specially due to large occurrence of prostatic diseases. Then, this work aimed at determining the structure and ultrastructure of the prostatic stroma and epithelium, as well as the stroma epithelium interactions from rats submitted to simultaneous long-term alcohol-nicotine treatment. A total of 40 male rats were divided into four groups: control group (10 animals) received tap water; alcoholic group (10 animals) received diluted 10% Gay Lussac ethanol; nicotine group (10 animals) received a 0.125mg/100g of body weight dose of nicotine injected subcutaneosly on a daily basis; nicotine-alcohol group (10 animals) received simultaneous alcohol and nicotine treatment. After 90 days of treatment, the animals were sacrificed and samples from the ventral lobe of the prostate were collected and processed for transmission electron and light microscopies. The results showed atrophied epithelium; prostatic intra-epithelial neoplasia; dilated cisterns of the granular endoplasmic reticulum, large amounts of collagen fibers besides inflammatory cells, specially in the alcoholic and nicotine-alcohol groups. Therefore, it could be concluded that the association between alcohol and nicotine caused the impairment of the prostatic secretory process. Moreover, this association is related to prostatic pathogenesis, which could lead to late glandular malignancy.     

Prostatic stromal microenvironment and experimental diabetes

The diabetes causes alterations in various organ systems, including the male accessory sex glands. The prostate is very important in the reproductive process and it is a frequent target of malignant changes. The aim of this work was to demonstrate the histochemical and ultrastructural alterations in the prostate of diabetic animals. Two groups of animals were utilized: control and non-obese diabetic mice (NOD). Twelve days after the characterization of diabetic status the ventral prostate was collected, fixed in Karnovsky and paraformaldehyde, processed for histochemistry and TEM associated to stereology. The results showed reduction of the epithelial area and increasing of the stromal area with muscular and collagen hypertrophy in the prostatic gland. It was characterized the development of prostatic intraepithelial neoplasia, inflammatory processes and dilation of the organelles involved in the secretory process. It was concluded that diabetes besides damaging the reproductive process, affects the glandular homeostasis favoring the development of prostatic pathologies.     

Cellular changes in the prostatic stroma of glucocorticoid-treated rats

Glucocorticoid hormones (GCs) have been widely used for the treatment of prostate cancer because of their inhibitory property against tumour growth. However, their mechanism of action in the prostate has received little attention. Excess GCs can lead to peripheral insulin resistance resulting in hyperglycaemia and hyperinsulinaemia. Insulin plays an important role as a cellular stimulant and high levels are related to low levels of androgens. Our objective has been to describe the effects of insulin resistance induced by dexamethasone treatment on the morphology of rat ventral prostate. Male adult Wistar rats received daily intraperitoneal injections of dexamethasone or saline for five consecutive days after which the rats were killed and the ventral prostate was removed, weighed and prepared for conventional and transmission electron microscopy (TEM). Dexamethasone treatment resulted in atrophy and decreased proliferative activity of prostatic epithelial cells. TEM analysis revealed changes in the epithelium-stroma interface, with some interruptions in the basement membrane. Fibroblasts showed a secretory phenotype with dilated endoplasmic reticulum. Smooth muscle cells exhibited a contractile pattern with 50% atrophy, an irregular membrane and twisted nuclei. Mitochondrial alterations, such as enlarged size and high electron density in the mitochondrial matrix, were also detected in smooth muscle cells. Insulin resistance induced by dexamethasone is thus associated with epithelial atrophy similar to that described for diabetic rats. However, GCs are responsible for morphological changes in the stromal cell population suggesting the activation of fibroblasts and atrophy of the smooth muscle cells.     

Short-term effects of mating on the accessory sex glands of the male rat

Mating in the rat was associated with a significant reduction in the tissue concentrations of the presumptive secretory products of the male accessory sex glands: prostatein and the amines, putrescine, spermidine and spermine (ventral prostate lobe), zinc (lateral prostate lobe) and fructose (coagulating gland). The amount of secretory product discharged and the time taken to restore precopulatory levels differed for the different lobes. Within 12-24 h of the mating period, the activity of ornithine decarboxylase and cytosolic oestrogen binding in the ventral prostate lobe underwent a transient increase which lasted 2-3 days. No change was observed in prolactin binding. Circulating testosterone concentrations were significantly elevated above control values 12 h after the start of mating but were significantly lower than control values at 24 h. A gradual recovery to concentrations in controls occurred over the next 2-3 days. None of these changes could be explained by alterations in gonadotrophin or prolactin release.     

Expression of the ZNT (SLC30) family members in the epithelium of the mouse prostate during sexual maturation

A prostate contains ~10-fold higher zinc than other soft organs. The function of the prostate is to produce a zinc-enriched seminal fluid. To establish a protein expression profile for zinc transporters involved in zinc efflux and intracellular sequestration/storage in the mouse prostate during sexual maturation, ZNT expression were investigated by immunohistochemistry. Our study demonstrated that ZNT proteins were differentially expressed in the prostate during sexual maturation. ZNT1 was mainly detected on the lateral membrane of the epithelium. Other ZNTs examined resided intracellularly. Among differences were a staining of ZNT2/ZNT5 in the ER-rich area of the epithelium in the anterior lobe, a staining of ZNT2 along the lateral and apical membrane, a luminal border staining of ZNT4, a staining of ZNT5 in the Golgi area of the epithelium in the ventral lobe, a uniform expression of ZNT6 across the lobes and ages, and a staining of ZNT7 in all lobes across ages.     

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 markers and apoptosis in the prostate of diabetic rats and the influence of vitamin C treatment

Negative consequences of diabetes on the prostate such as involution are associated with diminished testosterone, insulin deficiency, and hyperglycemia. The contributions of oxidative damage, which usually increases with diabetes, are unknown for these alterations. This study evaluated the impact of streptozotocin-induced diabetes on the biomarkers of the antioxidant system of rat ventral prostate, the influence of vitamin C supplementation on these biomarkers, and on the balance between cell proliferation and death. Diabetes (D) was induced in Wistar male rats by streptozotocin (5 mg/100 g b.w., i.p.). Control animals (C) were injected with a vehicle. Vitamin C (150 mg/kg b.w./day) supplementation was introduced by gavage in diabetes (D + V) as well as control (C + V) groups. Thirty days after diabetes onset, the rats were killed and the ventral prostates were analyzed using light microscopy, immunocytochemistry, and biochemical assays for biomarkers of oxidative stress. In comparison to control groups, the levels of circulating testosterone, proliferating, and androgen receptor-positive cells decreased in diabetic groups regardless of vitamin C treatment whereas apoptosis was increased. The levels of superoxide dismutase and glutathione peroxidase did not change, but the levels of glutathione-S-transferase (GST) were increased in diabetic prostate. Vitamin C supplementation normalized GST activity and recovered the apoptotic rates in the prostate. In conclusion, GST is a good indicator of compensatory oxidant defense in the prostate at earlier stages of diabetes and vitamin C improves its activity and attenuates apoptosis in the gland.     

Experimental alcoholism and pathogenesis of prostatic diseases in UChB rats

Previous studies have shown that long-term alcohol treatment has negative effects on prostatic stromal-epithelial interaction. Thus, the aim of the present study was to analyze the histochemical, immunohistochemical and ultrastructural alterations that occur in the prostatic stroma and epithelium of rats submitted to chronic alcohol ingestion and alcohol abstinence, as well as to establish the relationship between these changes and prostatic diseases. Thirty male rats (10 Wistar and 20 UChB rats) were divided into three experimental groups: the control group received tap water, the alcoholic group received ethanol diluted to 10 degrees G.L. for 150 days, and the abstinent group received the same liquid diet as the alcoholic group up to 120 days of treatment and only tap water for 30 days thereafter. At the end of treatment, all animals were sacrificed and the ventral lobe of the prostate was removed and processed for histochemical, immunohistochemical and ultrastructural analyses. In addition, plasma testosterone levels were measured. The results showed prostatic intraepithelial neoplasia, infolding of the epithelium towards the stroma, stromal hypertrophy and the presence of inflammatory cells in alcoholic animals. In the abstinent group, alterations were noted mainly in the stromal area. In conclusion, ethanol triggers alterations in prostatic epithelial and stromal compartments, affecting the stromal microenvironment and predisposing the organ to pathological processes.     

Impact of experimental diabetes and insulin replacement on epididymal secretory products and sperm maturation in albino rats

The present study is aimed to explore the impact of experimental diabetes and insulin replacement on epididymal secretory products, sperm count, motility, and fertilizing ability in albino rats. Prepubertal and adult male Wistar strain rats were made diabetic with a single intraperitoneal injection of streptozotocin (STZ), at 120 and 65 mg/kg body weight for prepubertal and adult rats, respectively. After 3 days of STZ administration, insulin was given to a group of diabetic rats at a dose of 3 U/100 g body weight, subcutaneously and killed after 20 days of treatment. STZ‐diabetes significantly reduced the epididymal tissue concentrations of testosterone, androgen‐binding protein, sialic acid, glycerylphosphoryl choline, and carnitine, suggesting its adverse effects on the secretory activity and concentrating capacity of epididymal epithelium. Impaired cauda epididymidal sperm motility and fertility (in vivo) of STZ‐diabetic rats imply the defective sperm maturation. Insulin replacement prevented these changes either partially or completely. From the above findings, it is evident that STZ‐diabetes has an adverse effect on sperm maturation, which may be due to the decrease in the bioavailability of testosterone and epididymal secretory products. J. Cell. Biochem. 108: 1094–1101, 2009. © 2009 Wiley‐Liss, Inc.     

Histological,histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda,Cephalaspidea)

Due to its biological and systematic importance, the morphology and function of the male copulatory apparatus of Haminoea navicula, a Cephalaspidea gastropod mollusk, was investigated by light and electron microscopy. These systems are poorly understood in haminoids, but are often used in the taxonomy of the genus. In H. navicula, the male copulatory apparatus comprises the penis within a penial sheath, a seminal duct and the prostate with two lobes. The penis is a muscular structure with a tip covered by spikes formed by muscular cells. The penial sheath consists of muscular tissue folds lined by an epithelium. Below this epithelium, polysaccharide‐secreting cells and pigment cells were observed. A large number of vacuolar cells were found below the ciliated epithelium of the seminal duct. The proximal lobe of the prostate was formed by tubules that could be divided in basal, middle and apical zones, containing cells that secrete polysaccharides and proteins. The tubules of the prostate distal lobe contained a single type of secretory cells with vesicles that were stained by histochemical techniques for detection of polysaccharides and proteins. Ciliated cells were present along the tubules in both lobes of the prostate. This study revealed a complex prostate with five types of secretory cells, which secrete substances that should be involved in the maintenance and eventually also in the maturation of spermatozoa. A comparison with previous publications, shows that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non‐European species attributed to this genus.     

Development of some male characteristics supported by oestrone but not dehydroepiandrosterone in the boar

Prepubertally castrated boars received subcutaneous injections twice weekly, from 13 to 35 weeks of age, of dehydroepiandrosterone (2 mg/5 kg) or oestrone (1 mg/5 kg). Dehydroepiandrosterone did not support the growth and secretory activity of the accessory organs, or induce copulatory behaviour. However, oestrone caused hypertrophy of the prostate, seminal vesicles and bulbourethral glands which was due to an increase in fibrous stromal tissue and not to the secretory epithelium. Oestrone also induced some male mating behaviour patterns in the presence of an oestrous gilt, although penile extrusion and ejaculation did not occur. The morphological and behavioural effects of the steroid treatments were supported by steroid profiles in blood plasma as seen in comparison with androgen and oestrogen values of intact and untreated castrated boars. It is concluded that oestrogen in the intact boar might enhance the secretion of the accessory organs by affecting the neural control of the secretory processes rather than by increasing the amount of secretory epithelium in the glands.     

Senescence and steroid hormone receptor reactivities in accessory sex glands of elderly rats (Sprague-Dawley) following exogenous hormonal therapy

The aim of this study was to characterize the stromal and epithelial distribution of AR, ERα and ERβ reactivities in the different accessory sex glands of elderly rats and during strong hormonal changes. Ten month old male rats were divided into six senile groups and submitted to treatment: Senile/Control group (SC); Senile/Testosterone group (ST): Senile/Estrogen group (SE); Castrated group (CA); Castrated/Testosterone group (CT); Castrated/Estrogen group (CE). After a 30-day treatment, the prostatic ventral lobe (VL), dorsal lobe (DL) and coagulating gland (CG) samples were processed for immunohistochemistry and Western Blotting. The results showed that AR immunoreactivity was characterized in the epithelium of VL and DL in senile/control rats and senile rats submitted to exogenous hormonal therapy. AR reactivity in the coagulating gland was verified predominantly in the stromal cells in the different experimental groups. ERα reactivity occurred predominantly in the stromal compartment in all accessory sex glands. In the DL and CG, ERα immunoreactivities were intense in the groups which received testosterone (ST) and estrogen (SE). ERβ immunoreactivity in the CG was verified in the stromal compartment in the different experimental groups, showing a positive response to both increased testosterone and estrogen levels. ERβ reactivity, in the DL, was intensified in the stroma of senile rats with higher serum testosterone levels, and in senile rats with increased serum estrogen levels, especially in the glandular epithelium. Thus, the results revealed different distribution pattern of steroid hormone receptors in each one of the prostatic lobes in senescence, especially in the prostate dorsal lobe and coagulating gland, which is a fundamental factor due to the fact that major prostatic diseases occur in a later period of life.     

Lobe-specific proteome changes in the dorsal-lateral and ventral prostate of TRAMP mice versus wild-type mice

The transgenic adenocarcinoma of mouse prostate (TRAMP) is the most widely used transgenic model for prostate cancer chemoprevention studies. Although two lobe‐specific lineages of carcinogenesis have been described, the molecular mechanisms are still poorly defined. Here, we concurrently profiled the proteome of dorsal‐lateral (DLP) and ventral (VP) prostate lobes of both TRAMP and littermate WT C57BL/6 mice of 18 wk by 2‐D LC‐MALDI‐TOF/TOF with iTRAQ labeling. A total of 483 and 748 proteins were identified at critical false discovery rates of 1 and 5%. In WT mice, 84 proteins were found to have different expression levels between DLP and VP. In TRAMP mice, 118 proteins significantly changed in DLP and/or VP during TRAMP carcinogenesis. Among them, 55 and 36 proteins were uniquely changed in DLP or VP lobe, respectively, and 27 proteins in both DLP and LP lobe. Ingenuity Pathway Analysis was able to segregate proteins changed in two lobes into different pathway networks. In addition to serving as reference for prostate proteomic profiles, our data suggest that different sets of proteins are involved in the carcinogenesis in DLP versus VP in the TRAMP model.     

The EZC-prostate model: noninvasive prostate imaging in living mice

Recently, progress in the development of prostate-specific promoters and high resolution imaging techniques has made real-time monitoring of transgenic expression possible, opening a vista of potentially important in vivo models of prostate disease. Herein, we describe a novel prostate reporter model, called the EZC-prostate model that permits both ex vivo and in vivo imaging of the prostate using a sensitive charge-coupled device. Firefly luciferase and enhanced green fluorescent protein were targeted to the prostate epithelium using the composite human kallikrein 2 (hK2)-based promoter, hK2-E3/P. In EZC-prostate mice, the ventral and dorsal/lateral prostate lobes were brilliant green under fluorescence microscopy, with expression localized to the secretory epithelium. In contrast, enhanced green fluorescent protein was undetectable in the anterior lobes of prostate, seminal vesicles, testes, liver, lung, and brain. The kinetics of luciferase activity in intact and castrated living mice monitored with the IVIS charge-coupled device-based imaging system confirmed that firefly luciferase expression was largely prostate restricted, increased with age up to 24 wk, and was androgen dependent. Decreases in reporter expression after 24 wk may reflect well known, age-related decreases in androgen signaling with age in humans. Ex vivo imaging of microdissected animals further confirmed that the luminescence detected in living mice emanated predominately from the prostate, with minor signals originating from the testes and cecum. These data demonstrate that the hK2-E3/P promoter directs strong prostate-specific expression in a transgenic mouse model. Multigenic models, generated by crosses with various hyperplastic and neoplastic prostate disease models, could potentially provide powerful new tools in longitudinal monitoring of changes in prostate size, androgen signaling, metastases, or response to novel therapies without sacrificing large cohorts of animals.     

The response of female urogenital tract epithelia to mesenchymal inductors is restricted by the germ layer origin of the epithelium: prostatic inductions

The epithelium of the mammalian vagina arises from two distinct germ layers, endoderm from the urogenital sinus and mesoderm from the Müllerian ducts. While neonatal vaginal epithelium can be induced to form prostate which is normally an endodermal derivative, it has not been determined whether this ability to form prostate is shared by both mesoderm- and endoderm-derived vaginal epithelia. To test the competence of vaginal epithelia we have isolated sinus-derived and Müllerian-derived vaginal epithelia from newborn mice, combined them with rat urogenital sinus mesenchyme, and grown the tissue recombinants for 4 weeks in male athymic nude mice. Endoderm-derived sinus vaginal epithelium was induced to form prostatic tissue which expressed prostate-specific secretory proteins in 21 of 23 tissue recombinants. Müllerian-derived vaginal epithelium formed small ducts and cysts lined by a simple epithelium. These latter tissue recombinants lacked any evidence of prostatic secretory proteins. Similarly, endoderm-derived urethral epithelium was induced to form prostate (17 of 17 cases), while mesoderm-derived uterine epithelium was not (0 of 13 cases). Therefore, the ability to form prostatic epithelium was limited to endodermal derivatives of the urogenital tract.