Rat Ventral Prostate Research Articles

Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats

The Developmental Origins of Health and Disease (DOHaD) concept has provided the framework to assess how early life experiences can shape health and disease throughout the life course. While maternal malnutrition has been proposed as a risk factor for the developmental programming of prostate cancer (PCa), the molecular mechanisms remain poorly understood. Using RNA-seq data, we demonstrated deregulation of miR-206-Plasminogen (PLG) network in the ventral prostate (VP) of young maternally malnourished offspring. RT-qPCR confirmed the deregulation of the miR-206-PLG network in the VP of young and old offspring rats. Considering the key role of estrogenic signaling pathways in prostate carcinogenesis, in vitro miRNA mimic studies also revealed a negative correlation between miR-206 and estrogen receptor α (ESR1) expression in PNT2 cells. Together, we demonstrate that early life estrogenization associated with the deregulation of miR-206 networks can contribute to the developmental origins of PCa in maternally malnourished offspring. Understanding the molecular mechanisms by which early life malnutrition affects offspring health can encourage the adoption of a governmental policy for the prevention of non-communicable chronic diseases related to the DOHaD concept.

Long exposure to a mixture of endocrine disruptors prediposes the ventral prostate of rats to preneoplastic lesions.

Endocrine disruptors (ED) are compounds dispersed in the environment that modify hormone biosynthesis, affecting hormone-dependent organs such as the prostate. Studies have only focused on evaluating the effects of ED alone or in small groups and short intervals and have not adequately portrayed human exposure. Therefore, we characterized the prostate histoarchitecture of rats exposed to an ED mixture (ED Mix) mimicking human exposure. Pregnant females of the Sprague-Dawley strain were randomly distributed into two experimental groups: Control group (vehicle: corn oil, by gavage) and ED Mix group: received 32.11mg/kg/day of the ED mixture diluted in corn oil (2ml/kg), by gavage, from gestational day 7 (DG7) to post-natal day 21 (DPN21). After weaning at DPN22, the male pups continued to receive the complete DE mixture until they were 220days old when they were euthanized. The ED Mix decreased the epithelial compartment, increased the fractal dimension, and decreased glandular dilation. In addition, low-grade prostatic intraepithelial neoplasia was observed in addition to regions of epithelial atrophy in the group exposed to the ED Mix. Exposure to the mixture decreased both types I and III collagen area in the stroma. We concluded that the ED Mix was able to cause alterations in the prostatic histoarchitecture and induce the appearance of preneoplastic lesions.

Exposure to Dichlorvos pesticide alters the morphology of and lipid metabolism in the ventral prostate of rats.

Organophosphate pesticides are widely used in agriculture, leading to soil, water, and food contamination. Among these compounds is Dichlorvos [O,O-dimethyl O-(2,2-dichlorovinyl)phosphate, DDVP], which is listed as a highly toxic compound by the Environmental Protection Agency and World Health Organization. Exposure to DDVP can result in nervous, respiratory, hepatic, and reproductive abnormalities, in addition to endocrine disrupting, mutagenic, and carcinogenic effects. Little is known about the impacts of DDVP on the reprogramming of lipid metabolism, which is also associated with the development and progression of cancer, since the tumor cells need to recruit, capture, and use fatty acids to compose their building membranes. This study aimed to evaluate the influence of the pesticide DDVP on lipid metabolism in the prostate, after chemical induction by the carcinogen N-methyl-N-nitrosourea (MNU). For this, 32 Fischer rats aged 90days were randomly divided into four experimental groups: Control, DDVP, MNU, and MNU + DDVP. The MNU and MNU + DDVP groups underwent chemical induction with MNU (15mg/kg) and the DDVP and MNU + DDVP groups received a diet supplemented with DDVP (10mg/kg). Histopathological analyses of the rat ventral prostate showed 100% incidence of epithelial hyperplasia in the MNU and MNU + DDVP groups. This finding was accompanied by an increase of the epithelial compartment in the MNU + DDVP group. Immunolocalization of important proteins linked to lipid metabolism has been established. In the MNU + DDVP group, Western blotting analyses pointed out an increased expression of the protein LIMP II (Lysosomal Integral Membrane Protein-II), which is correlated with the capture and distribution of lipids in tumor cells. Together, these results indicate that the association of a low dose of DDVP with MNU was able to promote alterations in the morphology and lipid metabolism of the rat ventral prostate, which may be related to tumor progression in this organ.

Comparative analysis of male reproductive accessory glands in bats Histiotus velatus (Vespertilionidae), Molossus rufus (Molossidae), and Peropteryx leucoptera (Emballonuridae).

Despite the high number of species and wide geographic dispersion, reproductive accessory glands (RAGs) of bats have traditionally received little attention in the literature, with some species not even having a basic description of their composition and structure. Thus, this study aimed to analyze and compare the composition, anatomy, and histology of male RAGs of bat species belonging to three of the largest (cosmopolitan) bat families: Vespertilionidae (Histiotus velatus), Molossidae (Molossus rufus), and Emballonuridae (Peropteryx leucoptera), in order to understand the variations in the bat RAGs. The results showed that the RAGs of H. velatus, M. rufus, and P. leucoptera are composed of an intra-abdominal prostatic complex, associated with the urethra, urethral glands, and a pair of inguinal bulbourethral glands; without ampullary glands or seminal vesicles. The prostatic complex can be composed of two (M. rufus and P. leucoptera) or three (H. velatus) prostatic regions, and can be compact (P. leucoptera), semi-lobed (M. rufus), or multilobed (H. velatus). Each prostatic region has unique and distinct characteristics, with the ventral region presenting a holocrine nature, exclusive to bats; while the dorsal and/or dorsolateral regions have similar characteristics to the ventral prostate of rats and to the human peripheral zone.

Changes in the Expression and Functional Activities of C-X-C Motif Chemokine Ligand 13 (CXCL13) in Hyperplastic Prostate

C-X-C motif chemokine ligand 13 (CXCL13), a member of the CXC subtype in chemokine superfamily, affects numerous biological processes of various types of cells and the progress of a great number of clinical diseases. The purpose of the current study was to reveal the internal mechanism between CXCL13 and benign prostatic hyperplasia (BPH). Human serum, prostate tissues and human prostate cell lines (BPH-1, WPMY-1) were utilized. The effect of recombinant human CXCL13 (rHuCXCL13) protein and the influences of the knockdown/overexpression of CXCL13 on two cell lines were studied. Rescue experiments by anti-CXCR5 were also conducted. In vivo, rHuCXCL13 was injected into the ventral prostate of rats. Additionally, a tissue microarray of hyperplastic prostate tissues was constructed to analyze the correlations between CXCL13 and clinical parameters. CXCL13 was highly expressed in the prostate tissues and upregulated in the BPH group. It was observed that CXCL13 modulated cell proliferation, apoptosis, and the epithelial-mesenchymal transition (EMT) through CXCR5 via AKT and the ERK1/2 pathway in BPH-1, while it contributed to inflammation and fibrosis through CXCR5 via the STAT3 pathway in WPMY-1. In vivo, rHuCXCL13 induced the development of rat BPH. Additionally, CXCL13 was positively correlated with the prostate volume and total prostate specific antigen. Our novel data demonstrated that CXCL13 modulated cell proliferation, cell cycle, the EMT of epithelial cells, and induced the fibrosis of prostatic stromal cells via a variety of inflammatory factors, suggesting that CXCL13 might be rediscovered as a potential therapeutic target for the treatment of BPH.

CHANGES IN LIPID PEROXIDATION IN THE REPRODUCTIVE ORGANS OF MALE RATS DUE TO THE TRANSPLACENTAL ACTION OF CHEMICAL ENDOCRINE DISRUPTORS

Abstract. In everyday life, people come into contact with chemical products that have hormone-like properties, for example, antiandrogenic (dibutyl phthalate, DBP) or estrogenic (bisphenol A, BPA). For a number of years, the issue of the potential harm of low doses of these endocrine disruptors (ED) for a developing fetus after entering the body of a pregnant mother has been discussed. Taking into consideration the ED ability to overcome the placental barrier, and the fact that one of the general mechanisms of the pathogenic effect of ED on humans and animals is oxidative stress, it is reasonable to study the state of lipid peroxidation (LPO) in the reproductive organs of adult male offspring, which was subjected to prenatal effects of low doses of DBP and BPA. The aim is to elucidate the prenatal effect of low doses of DBP and BPA on the content of LPO products in the ventral prostate (VP) and gonads of adult male rats. Material and methods. Wistar rats received orally an oil solution of DBP at 100 mg / kg bw. or BPA in Dorfman gel at 25 μg / kg bw per day from 15 to 21 days of pregnancy, control animals – carriers of drugs. In male offspring of 6 and 18 months of age, the contents of LPO products – malonic dialdehyde (MDA) and diene conjugates (DC) – were determined per mg of protein in the VP and testes. Results. In the testes of young rats prenatally exposed to DBP, the contents of both studied LPO products were significantly increased. The content of MDA in the VP of aging experimental rats exceeded 6 times, and DC – 1.5 times the values of control animals. The expression levels of MDA and DC in the testes of experimental animals increased by 134 % and 37 %, respectively. In the testes of 6-month-old rats exposed to BPA in utero, the contents of LPO products significantly increased in comparison with the control group: MDA by 155 %, DC – by 16 %. In the VP, the content of MDA significantly increased, DC did not differ from the control group. Conclusions. The result of oral administration of DBP to rats at a threshold dose relative to reproductive fetotoxicity during the last week of pregnancy is the activation of LPO in the testes of young and in the VP and testes of aging offspring. Oral administration of a subthreshold BPA dose to rats in the last week of pregnancy leads to the activation of LPO in the testes and the VP of young offspring. Key words: dibutyl phthalate, bisphenol A, prenatal action, lipid peroxidation, male rats, ventral prostate, testes.

Pesticide Dichlorvos Promotes Morphological Alternations in the Rat Prostate

The complex process of carcinogenesis can be modulated by the action of environmental endocrine disruptors, such as the organophosphate pesticide Dichlorvos (DDVP), capable of interfering in homeostasis of different organs and leading to the development of neoplastic lesions. Considering that prostate cancer is the second most common neoplasm in men, worldwide, it is emphasized the importance of studying morphological alterations using animal models. In rodents, tumor initiation can be induced by the carcinogen N-methyl-N-nitrosourea (MNU) and tumor promotion can be stimulated by the administration of testosterone, combinated or not with the use of pesticides. The present study aimed to evaluate the influence of DDVP on the development of morphological alterations in the ventral prostate of rats, after chemical induction by MNU. Thereunto, 32 Fischer 344 rats, aged 90 days, were randomly separated into four experimental groups: Control, DDVP, MNU, MNU+DDVP. For chemical induction, the MNU and MNU+DDVP groups were inoculated with MNU (15 mg/kg), followed by daily subcutaneous injections of testosterone cypionate (2.5 mg/kg), for 20 days. Animals of the DDVP and MNU+DDVP groups, from 120 to 240 days old, received the basal diet supplemented with 10 mg/kg of DDVP. Subsequent to the experimental period, the prostatic ventral lobe of each animal was collected and subjected to morphometric-stereological and histopathological analysis, for the classification of the lesions found. The incidence of alterations in epithelium and stroma of all experimental groups was evaluated, such as epithelial hyperplasia (increased cell population with stratification, without atypia) (Figure A and B); atrophy (retraction of the epithelium with reduction of the secretory cytoplasm) (Figure C and D); periacinary inflammatory foci (presence of inflammatory cell infiltrate) (Figure E and F); cell atypia with presence of cytoplasmic inclusions (Figure G and H) and metaplasia with stromal hyperplasia (change in the pattern of the original epithelium without proliferative change, with an increase in the adjacent stroma) (Figure I to L). The results showed a significant increase of epithelial hyperplasia areas in MNU and MNU+DDVP groups, with 100% of the animals showing this lesion (Table 1), which demonstrates a relation between chemical induction and the proliferative process. The morphometric-stereological analysis confirmed this finding, revealing a relatively greater volume of the epithelial compartment in MNU+DDVP, compared to the Control group (Table 2). Thus, it can be concluded that DDVP, even in low concentration, associated with chemical induction by MNU, was able to disturb the morphology of the ventral prostate, promoting proliferative epithelial lesions, strongly associated with the process of carcinogenesis.

The Effects of Long-Term Immunosuppressive Therapies on the Structure of the Rat Prostate

Background: Little is known about the overall impact of immunosuppressive drugs on the prostate. The study aimed to determine the impact of different protocols of immunosuppressive treatment on the structure of the rat ventral prostate. Methods: For 6 months, 48 male Wistar rats received immunosuppressive drugs: cyclosporin A, tacrolimus, mycophenolate mofetil, rapamycin, and prednisone, according to three-drug protocols. Light and transmission electron microscopic studies, and quantitative evaluation of immunohistochemical expression of selected intermediate filaments, CD117+ mast cells, and CD138+ plasma cells were performed in the rat ventral prostate. Results: In all experimental groups, acini focal hyperplasia, changes to the ultrastructure of the glandular epithelium, changes in the expression of cytokeratins and desmin, and numerous mast and plasma cells in the prostate stroma were found. In cyclosporine-A-based groups, atrophy and numerous intracellular vacuoles were observed. In groups where a three-drug treatment was replaced with rapamycin, morphological alterations were less severe compared to those without conversion. Conclusions: In the rat ventral prostate, (1) immunosuppressive protocols affect the morphology and immunohistochemical expression of intermediate filaments, (2) morphological alterations, expression, and localization of selected proteins are not connected with adenocarcinoma development, and (3) conversion of the treatment to rapamycin may prevent hyperplastic abnormalities.

Effects of adjuvant-induced arthritis on the ventral prostate of rats treated with angiotensin AT1 receptor blocker

AimsTo analyze the prostatic compartments, extracellular matrix, microvascularization, transforming growth factor-beta (TGF-β) and angiotensin II receptors type 1 (AT1) levels, and histopathology of the ventral prostate in a rat model for rheumatoid arthritis, and to evaluate the effect of angiotensin II AT1 receptor blocker (ARB) in the disease. Main methodsFifteen male rats (90 days old) were divided into three groups (n = 5/group): control, adjuvant-induced arthritis without (AIA) or with AT1 receptor blocker (AIA + ARB). Animals were euthanized 60 days after immunization. The ventral prostate was collected, weighed, and processed for histological and immunohistochemical analysis. Key findingsOur results show that AIA increases production of the prostatic epithelium and extracellular matrix, accompanied by a reduction in the number of tissue capillaries. ARB treatment promotes decreased production of extracellular matrix and increased TGF-β and AT1 receptor immunostaining. SignificanceAIA may activate specific mechanisms that modify the prostatic environment; the use of ARB attenuates some altered prostate parameters in a rat model for arthritis.

The role of WNT10B in normal prostate gland development and prostate cancer

WNT signaling is implicated in embryonic development, and in adult tissue homeostasis, while its deregulation is evident in disease. This study investigates the unique roles of canonical WNT10B in both normal prostate development and prostate cancer (PCa) progression. Organ culture and rat ventral prostates (VPs) were used to study Wnt10b ontogeny and growth effect of WNT10B protein. PB-SV40 LTag rat VPs were utilized for Wnt expression polymerase chain reaction (PCR) array and immunohistochemistry. Human localized PCa tissue microarrays (TMAs) were investigated for differential WNT10B expression. Human RNA-seq data sets were queried for differential expression of WNT10B in metastatic and localized PCa. Knockdown of WNT10B in PC3 cells was utilized to study its effects on proliferation, stemness, epithelial to mesenchymal transition (EMT), and xenograft propagation. Wnt10b expression was highest at birth and rapidly declined in the postnatal rat VP. Exogenous WNT10B addition to culture developing VPs decreased growth suggesting an antiproliferative role. VPs from PB-SV40 LTag rats with localized PCa showed a 25-fold reduction in Wnt10b messenger RNA (mRNA) expession, confirmed at the protein level. Human PCa TMAs revealed elevated WNT10B protein in prostate intraepithelial neoplasia compared with normal prostates but reduced levels in localized PCa specimens. In contrast, RNA-seq data set of annotated human PCa metastasis found a significant increase in WNT10B mRNA expression compared with localized tumors suggesting stage-specific functions of WNT10B. Similarly, WNT10B mRNA levels were increased in metastatic cell lines PC3, PC3M, as well as in HuSLC, a PCa stem-like cell line, as compared with disease-free primary prostate epithelial cells. WNT10B knockdown in PC3 cells reduced expression of EMT genes, MMP9 and stemness genes NANOG and SOX2 and markedly reduced the stem cell-like side population. Furthermore, loss of WNT10B abrogated the ability of PC3 cells to propagate tumors via serial transplantation. Taken together, these results suggest a dual role for WNT10B in normal development and in PCa progression with opposing functions depending on disease stage. We propose that decreased WNT10B levels in localized cancer allow for a hyperproliferative state, whereas increased levels in advanced disease confer a stemness and malignant propensity which is mitigated by knocking down WNT10B levels. This raises the potential for WNT10B as a novel target for therapeutic intervention in metastatic PCa.

An improved acridine orange staining of DNA/RNA

New tools are desirable to examine the metabolic state of individual cells within tissues. We proposed a fluorescence-based procedure consisting of acridine orange staining and fast green counterstaining (AO-FG) to improve the selectivity of the former for nucleic acids (acridine orange stains both DNA and RNA with different fluorescence colors), with no interference from proteins. We compared this test with the biochemical quantification of the relative amounts of RNA and DNA in selected rat ventral prostate samples and PC3 cells. The epithelium of the prostate gland is highly active metabolically for the production of secretions. Differences in AO-RNA staining were revealed and correlated with the metabolic state of the epithelium. Specificity was confirmed by RNase A. To assess how AO-FG staining correlates with the metabolic state of the cell, we cultured PC3 cells in different concentrations of glucose and measured the ratios between the amounts of RNA and DNA. In parallel, similar cultures were subjected to AO-FG, and the staining pattern correlated closely (r2=0.886) with the obtained biochemical results. The results confirmed that the combined use of AO and FG is useful for detecting DNA and RNA simultaneously, as well as for assessing quantitatively the transcriptional activity of individual cells and their changes in response to experimental manipulation.

PESTICIDE DICHLORVOS ALTERS THE EXPRESSION OF HORMONAL RECEPTORS IN RAT VENTRAL PROSTATE

Organophosphorus pesticides are very effective, widely used, and pose a great risk to the environment and the health of various organisms, including humans. Dichlorvos (DDVP) is one of these pesticides and it has a mutagenic and carcinogenic potential for humans. Many pesticides, such as DDVP, act as endocrine disrupters and can alter endocrine homeostasis. These compounds act via androgen receptors (AR) or via estrogen receptors (ERs), promoting endocrine and reproductive changes, such as prostate cancer. Both androgens and estrogens are closely linked to the control of proliferation and cell death mechanisms that control normal prostate growth. Changes in the expression and activity of ARs and ERs are essential for understanding the process of carcinogenesis in the prostate.The present project aims to evaluate the expression of the AR, ERα and ERβ receptors in the ventral prostate of rats after exposure to the pesticide Dichlorvos, associated or not to chemical induction by MNU.A total of 40 rats of the Fischer 344 strain, at the age of 90 days, were used. Rats were randomly assigned to four experimental groups: Sham (G1), Sham + DDVP (G2), MNU (G3), MNU + DDVP (G4). For chemical induction, G3 and G4 were inoculated with MNU at 15 mg/kg, followed by daily subcutaneous injections of 2.5 mg/kg of testosterone cypionate for 20 days. Animals of the G2 and G4 groups, from 120 to 240 days old, received the basal diet supplemented with 10 mg/kg of DDVP. Western Blotting analysis of the ventral prostate was performed for AR (fig. 1), ERα (fig. 2) and ERβ (fig. 3) with β‐actin (fig. 4) loading control.In the densitometric analysis, it was verified that the expression of AR is higher in the G2 group than in the G1. When comparing the G3 group to the G1 group, it is noted that the expression of AR is higher in the G3 group. Thus, it is possible to infer that the expression of AR increases, both after exposure to DDVP and after chemical induction by MNU (fig. 5).In the densitometric analysis, it was also verified that ERα expression is higher in the G2 group than in the G1 group. When comparing the G3 group to the G1 group, it is noted that the ERα expression is higher in the G3 group. It is also observed that the ERα expression is higher in the G4 group than in the G2 group. The G4 group, on the other hand, presents ERα expression equal to that of G3. Thus, it is possible to infer that ERα expression increases, both after exposure to DDVP and after chemical induction by MNU (fig. 6).It has also been shown that ERβ expression is lower in the G2 group than in the G1 group. When comparing the G3 group to the G1 group, it is noted that the ERβ expression is lower in the G3 group. It is also observed that the ERβ expression is lower in the G4 group than in the G2 group. The G4 group, on the other hand, has lower ERβ expression than G3. Thus, it is possible to infer that ERβ expression decreases, both after exposure to DDVP and after chemical induction by MNU (fig. 7).Thus, we can conclude that the exposure to the pesticide DDVP alters the expression of hormone receptors in the ventral prostate of rats and can act as an endocrine disruptor.Support or Funding InformationFapesp 2014/07329‐0This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PESTICIDE DICHLORVOS PROMOTES CHANGES IN IMMUNOLOCALIZATION OF INFLAMMATORY MEDIATORS IN VENTRAL PROSTATE OF RATS AFTER CHEMICAL INDUCTION BY MNU

Organophosphorus pesticides, such as dichlorvos (DDVP), are widely used in agricultural, commercial, industrial and domestic areas, even though there are many studies that prove their action as endocrine disruptors. They are able to interfere with homeostasis maintained by hormones, disrupting the hypothalamic‐pituitary‐testicular axis and inducing inflammation.Inflammation stimulates carcinogenesis, causing damage to cells and the genome, promoting cell replacement and creating a tissue microenvironment rich in cytokines and growth factors that may increase cell replication, angiogenesis, and repair tissue. The inflammatory process predisposes individuals to various types of cancer, as it induces the production of cytokines such as Tumor necrosis factor alpha (TNFα) which is involved in the development of prostate cancer. Inflammation can be triggered by Toll‐Like receptors (TLRs) which may result in the activation of, for example, Nuclear factor kappa B (NFκB), which plays an important role in cancer responses, inflammation, stress and cell differentiation.Thus, the objective of this work was to evaluate the immunolocalization of inflammatory mediators such as TNFα, TLR4 and NFκB in the ventral prostate of rats after exposure to DDVP, associated or not with chemical induction by N‐methyl‐N‐nitrosourea (MNU).A total of 40 rats of the Fischer 344 strain were used, at the age of 90 days. Rats were randomly assigned to four experimental groups: Sham (G1), Sham + DDVP (G2), MNU (G3), MNU + DDVP (G4). For chemical induction, G3 and G4 were inoculated with MNU at 15 mg / kg, followed by daily subcutaneous injections of 2.5 mg / kg of testosterone cypionate for 20 days. Animals of the G2 and G4 groups, from 120 to 240 days old, received the basal diet supplemented with 10 mg / kg of DDVP. Immunohistochemical analysis of the ventral prostate were performed.TNFα was detected in the whole cytoplasm of luminal epithelial cells in the ventral prostate of all experimental groups, with intense immunoreactivity in the apical region and moderate immunoreactivity in the region of the Golgi apparatus of these cells. The G1 group showed TNFα nuclear localization (fig. 1).The presence of TLR4 in luminal epithelial cells was detected. In groups G1, G2 and G3 there was immunolabeling in the whole cytoplasm of the cells, being the most intense expression in the Golgi apparatus region. In the G4 group, there was immunolabeling in the whole cytoplasm as well, but with moderate intensity in the Golgi apparatus and intense in the apical membrane of the cells (fig. 2).In all experimental groups, NFkB was immunolocalized in the whole cytoplasm of luminal epithelial cells in the ventral prostate. In addition, the G1 and G2 groups showed intense immunoreactivity in the apical region and only the G3 and G4 groups showed immunoreactivity in the perinuclear region and in the nuclear envelope (fig. 3).Thus, we can conclude that exposure to DDVP may alter the immunolocalization pattern of inflammatory mediators, such as NFκB, TNFα and TLR4. And, when associated DDVP with chemical induction by MNU, these changes are accentuated.Support or Funding InformationFAPESP 2017/08505‐5This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heparanase-1 activity and the early postnatal prostate development.

Ventral prostate (VP) morphogenesis starts during embryonic development and continues for the first three postnatal weeks. Heparan sulfate (HS) affects paracrine signaling. Heparanase-1 (HPSE) is the only enzyme capable of cleaving HS. HPSE releases the HS bioactive fragment and mobilizes growth factors. Little is known, however, about HS turnover and HPSE function during VP morphogenesis. In this study, we measured HSPG expression and analyzed the expression and distribution of HPSE in the rat VP. HPSE was predominantly expressed by the VP epithelium. The VP was treated with heparin in ex vivo cultures to interfere with HS and resulted in delayed epithelial growth. Hpse knockdown using siRNA delayed epithelial growth in the first postnatal week ex vivo, which was similar to treating with the lower concentration of heparin. Hpse silencing was related to changes in HS chain length (as determined by size-exclusion chromatography, up-regulation of Mmp9, and down-regulation of Mmp2 expression). It also down-modulated ERK1/2 phosphorylation, suggesting a reduction in signaling, likely due to decreased HS cleavage and growth factor bioavailability. Our results showed that HPSE played a role in early epithelial growth during the first week of VP postnatal development. Developmental Dynamics 248:211-220, 2019. © 2019 Wiley Periodicals, Inc.

Hyaluronan content and distribution in the rat ventral prostate after castration.

Hyaluronan (HA) has been implicated in tissue remodeling, healing, and tumor growth. This study investigated the variation in hyaluronan content, distribution, and metabolism in the rat ventral prostate (VP) in response to androgen deprivation after castration. The mRNA abundance of hyaluronan synthases (Has1-3) and hyaluronidases (Hyal 1-3) were assessed by reverse transcription (RT)-PCR and immunohistochemistry, respectively. The results demonstrated an increased concentration, but an overall reduction in HA content. HA was located in both epithelium and stroma of the prostate of both the noncastrated and castrated animals. Quantitative RT-PCR (qRT-PCR) showed that Has1 and Has2 are major synthases, and that Hyal 1 was the predominant hydrolase expressed in the VP. qRT-PCR also showed that Has1 and Has2 mRNA increased transiently after castration, whereas Has3 mRNA declined markedly. While Hyal 1 mRNA increased slowly up to day 21 after castration, Hyal 2 and Hyal 3 mRNA dropped significantly. CD44 was found in the epithelial cells and in some stromal cells in both hormonal conditions. In conclusion, castration results in increased abundance of Has1 and Has2 mRNA, but is associated with a decrease in the total content of HA, with an increased concentration, and a predominance of short-chain HA molecules.

Pathological lesions and global DNA methylation in rat prostate under streptozotocin-induced diabetes and melatonin supplementation.

Chronic hyperglycemia increases production of reactive oxygen species, which favors carcinogenesis. The association between diabetes and prostate cancer is controversial. Melatonin has antioxidant, anti-inflammatory, and antiproliferative properties. We investigated whether low doses of melatonin prevent the tissue alterations caused by diabetes and alter prostate histology of healthy rats. We also investigated whether experimental diabetes promoted the development of pathological lesions in the ventral prostate of rats. Melatonin was provided in drinking water (10 μg/kg/day) from age 5 weeks until the end of experiment. Diabetes was induced at 13 weeks by administration of streptozotocin (40 mg/kg, ip). Rats were euthanized at 14 or 21 weeks. Histological and stereological analyses were carried out and the incidence and density of malignant and pre-malignant lesions were assessed. Immunohistochemical assays of α-actin, cell proliferation (PCNA), Bcl-2, glutathione S-transferase (GSTPI), and DNA methylation (5-methylcytidine) were performed. Melatonin did not elicit conspicuous changes in the prostate of healthy animals; in diabetic animals there was a higher incidence of atrophy (93%), microinvasive carcinoma (10%), proliferative inflammatory atrophy, PIA (13%), prostatitis (26%), and prostate intraepithelial neoplasia, PIN (20%) associated with an increase of 40% in global DNA methylation. Melatonin attenuated epithelial and smooth muscle cell (smc) atrophy, especially at short-term diabetes-and normalized incidence of PIN (11%), inflammatory cells infiltrates, prostatitis (0%) and PIA (0%) at long-term diabetes. MLT was effective in preventing inflammatory disorders and PIN under diabetic condition. Although MLT has antioxidant action, it did not influence DNA methylation and not avoid carcinogenesis at low doses.

Early detection of prostate carcinogens by immunohistochemistry of HMGB2.

Screening prostatic carcinogens is time-consuming due to the time needed to induce preneoplastic and neoplastic lesions. To overcome this, we investigated alternative molecular markers for detection of prostatic carcinogens in a short period in rats. After treatment with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), expression of high-mobility group protein B2 (HMGB2) was up-regulated in rat ventral prostate. To evaluate the applicability of HMGB2 in the early detection of carcinogenicity of chemicals using animal models, we examined HMGB2 expression in prostate of rats. Six-week-old male F344 rats were gavaged for four weeks with a total of eight individual chemicals, divided into two categories based on prostate carcinogenicity. Animals were sacrificed at the end of the study and HMGB2 immunohistochemistry was performed. HMGB2 expression in least one prostate lobe was significantly increased by all four prostate carcinogens compared with the controls. In contrast, the four chemicals that were not carcinogenic in the prostate did not cause HMGB2 up-regulation. Additionally, high HMGB2 expression in neoplastic lesions in both rat and human was detected. Therefore HMGB2 expression may be a good screening tool for the identification of potential of prostate carcinogens.

The effect of diabetes mellitus on the rat ventral prostate and the possible protective role of Ginkgo biloba extracts

Context Diabetes mellitus (DM) is a worldwide common disease that causes many complications in the male genital system including the prostate gland. Many studies have shown that extracts of Ginkgo biloba have antioxidant and free radical eliminating effects. Aim The present study aims to explore the histological effects of DM on the ventral prostate gland in rats and the possible protective role of Ginkgo extracts. Materials and methods Forty-five adult albino male rats were chosen for this study and they were classified into three equal groups. The control group was divided into two subgroups: the subgroup 1 rats received distilled water for 6 weeks; the subgroup 2 rats were injected intraperitoneally by a single dose of 0.15 ml 0.1 mol/l citrate buffer. To induce diabetes rats in the diabetic and protective groups were administered 60 mg/kg streptozotocin intraperitoneally, a week later rats in the protective group received Ginkgo extract orally in a dose of 100 mg/kg/day for 6 weeks. Six weeks after diabetes induction, samples from the ventral prostate from all groups were obtained for histological examination. Results No structural differences between the two control subgroups were observed. The diabetic group showed focal epithelial stratification with pyknotic nuclei resting on the disrupted basement membrane. By electron microscope (EM) the cells had irregular nuclei, few mitochondria, many cytoplasmic vacuoles, dilated rough endoplasmic reticulum (rER) cisternae, and few apical microvilli. In other areas, the cells were destructed with dissolved cytoplasm; many fragmented rounded bodies were observed in the lumen. The Ginkgo was found to improve most of the prostatic changes associated with DM. Conclusion Ginkgo extracts improve the prostatic changes produced by DM.

Influence of postnatal prolactin modulation on the development and maturation of ventral prostate in young rats.

Besides androgenic dependence, other hormones also influence the prostate biology. Prolactin has been described as an important hormone associated with maintenance of prostatic morphophysiology; however, there is a lack of information on the involvement of prolactin during prostate development and growth. This study aimed to evaluate whether perinatal prolactin modulation interferes with rat ventral prostate (VP) development and maturation. Therefore, prolactin or bromocriptine (an inhibitor of prolactin release from the pituitary) were administered to Sprague Dawley rats from postnatal Day (PND) 12 to PND 21 or 35. Animals were then killed and serum hormonal quantification, VP morphological-stereological and immunohistochemical analyses and western blotting reactions were employed. Our results demonstrate that prolactin blockage increased serum testosterone on PND 21, which reflected an increase in anogenital distance. Although prolactin modulation did not interfere with VP weight, it modified VP morphology by dilating the acinar lumen and reducing epithelial cell height. Prolactin activated the signal transducer and activator of transcription (STAT) downstream pathway, increased androgen receptor expression and epithelial proliferation. In addition, prolactin and bromocriptine also increased expression of cytokeratin 18, a marker of luminal-differentiated cells. In conclusion, the VP responds to prolactin modulation through a mechanism of increasing the epithelial proliferative response and dynamics of cell differentiation, especially in animals treated for a more prolonged period.

Macrophage roles in the clearance of apoptotic cells and control of inflammation in the prostate gland after castration.

Androgen deprivation results in massive apoptosis in the prostate gland. Macrophages are actively engaged in phagocytosing epithelial cell corpses. However, it is unknown whether microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis (LAP) is involved and contribute to prevent inflammation. Flow cytometry, RT-PCR and immunohistochemistry were used to characterize the macrophage subpopulation residing in the epithelial layer of the rat ventral prostate (VP) after castration. Stereology was employed to determine variations in the number of ED1 and ED2. Mice were treated with either chloroquine or L-asparagine to block autophagy. M1 (iNOS-positive) and M2 macrophages (MRC1+ and ARG1+) were not found in the epithelium at day 5 after castration. The percentage of CD68+ (ED1) and CD163+ (ED2) phenotypes increased after castration but only CD68+ cells were present in the epithelium. RT-PCR showed increased content of the autophagy markers Bcl1 and LC3 after castration. In addition, immunohistochemistry showed the presence of LC3+ and ATG5+ cells in the epithelium. Double immunohistochemistry showed these cells to be CD68+ /LC3+ , compatible with the LAP phenotype. LC3+ cells accumulate significantly after castration. Chloroquine and L-asparagine administration caused inflammation of the glands at day 5 after castration. CD68+ macrophages phagocytose apoptotic cell corpses and activate the LAP pathway, thereby contributing to the preservation of a non-inflammed microenvironment. Marked inflammation was detected when autophagy blockers were administered to castrated animals.