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.     

Isolation and culture of rat seminal vesicle epithelial cells : The use of the secretory protein SVS IV as a functional probe

A method for the isolation and culture of seminal vesicle epithelial cells obtained from control and androgen-primed sexually-immature, uncastrated rats is described. This method allows the establishment of monolayer cultures from aggregates of seminal vesicle epithelial cells isolated after trypsin and collagenase digestion. Phase contrast and transmission electron microscopic methods demonstrate that cell aggregates, after attaching to the substrate, establish within 48 h a colony-like, epithelial-like growth pattern. Immunofluorescent localization studies of SVS IV, an androgen-dependent secretory protein purified from rat seminal vesicle secretion, show that cultured seminal vesicle epithelial cells are immunoreactive. An electrophoretic analysis of [35S]methionine-labeled secretory proteins immunoprecipitated with rabbit anti-SVS IV serum demonstrate that, whereas SVS IV is newly-synthesized and accumulated in the medium of cultured seminal vesicle cells established from androgen primed rats, cultured cells from control rats appear to synthesize and accumulate SVS IV in a precursor form. Results of this work show that seminal vesicle epithelial cells in culture not only retain several structural features representative of the tissue but also serve as a potential system for the study of androgen action.     

The relationship between the morphology of cell organelles and kinetics of the secretory process in male sex accessory glands of mice

Summary Two male sex accessory glands of the mouse, seminal vesicle and coagulating gland, were compared with the aim of relating differences in the morphology of organelles to the kinetics of the secretory process. The epithelial cells of the two glands were assessed by morphometric analysis, cytochemical staining, and electron-microscopic autoradiography after administration of a labeled amino acid. The rough endoplasmic reticulum of the seminal vesicle comprised narrow parallel cisternae, while that of the coagulating gland was greatly distended and occupied a much larger percentage of the cytoplasmic volume. Radioactively labeled products were secreted much more rapidly in the seminal vesicle than in the coagulating gland. The primary point of difference in kinetics of intracellular transport between the two glands was in exit of material from the rough endoplasmic reticulum. The more rapid drainage of the rough endoplasmic reticulum may be related to its relatively greater membrane surface density and lesser internal volume. In contrast, similarities in size and cytochemical staining in the Golgi apparatus of the two glands were accompanied by similar kinetics of intracellular transport of secretory protein through this organelle.     

Fine structure of male genital tracts of some Acrididae and Tettigoniidae (Insecta: Orthoptera)

A morphological, histological and ultrastructural study was carried out on the spermiducts and seminal vesicles of some species of Acrididae and Tettigoniidae. In all the species examined, the spermiducts and seminal vesicles have a monolayered secretory epithelium. Only the species of Acrididae have the sac with a flattened epithelium. Furthermore, in the most distal tubule region of the seminal vesicles of Eyprepocnemis plorans plorans, a rather characteristic secretory mechanism was found: the cytoplasm of the epithelial cells contained a large vesicle delimited by tightly packed microvilli. Numerous small vesicles open into this large vesicle which gradually dilates to merge with the apical plasma membrane releasing its contents into the lumen. Spermiophagic activity was found in all the species investigated. In the Tettigoniidae, this activity was found only in some epithelial cells of the seminal vesicle wall; in the species of the Acrididae the spermiophagic activity was carried out in the spermiduct lumen by an epithelial‐type cellular group. Spermiophagic activity is discussed as well as its role in the reproduction of these insects.     

Ultrastructure and innervation of water buffalo (Bubalus bubalis) seminal vesicle.

The lining epithelium of secretory end pieces and central glandular duct in the seminal vesicle of the water buffalo (Bubalus bubalis) consists of columnar principal and small polymorphous basal cells. A system of intercellular and even intracellular canaliculi enlarges the secretory surface. The most prominent organelle of the columnar principal cells is the granular endoplasmic reticulum, forming large aggregates of parallel lamellae. Using antibodies against the neural cell adhesion molecule L1 and the neural marker protein gene product 9.5 (PGP 9.5), the innervation pattern of the seminal vesicle becomes evident. The muscular layer surrounding the propria contains a dense network of unmyelinated fibers. Thicker bundles traverse the muscular layer to reach the propria. Around glandular secretory tubules and below the epithelial lining of the glandular duct a tightly woven subepithelial plexus is observed which sends short penetrating branches into the basal zone of the epithelium. These intraepithelial nerves are devoid of Schwann cells and basal lamina (naked axons) and are situated within the intercellular spaces between principal and basal cells. Acetylcholinesterase histochemistry with short (1-2 h) incubation times, dopamine-beta-hydroxylase immunohistochemistry and ultrastructural study of transmitter-containing vesicles was performed. The results suggest that muscular contraction in the seminal vesicle is predominantly under the influence of the sympathetic nervous system, whereas secretory epithelial function is regulated by both sympathetic and parasympathetic fibers.     

Secretory cell activity in the hamster seminal vesicle following castration. A morphometric ultrastructural study

The ultrastructure of hamster seminal vesicle epithelium was studied 7, 14, 21 and 28 days after castration using a stereological approach. The results show that castration promotes epithelial reorganization, mainly characterized by reduced epithelial cell size and number, decreased rough endoplasmic reticulum and Golgi complex, increased lysosomes and lipid droplets, increased apical secretory granule size and number, and increased intracellular secretory products per average epithelial cell. It is concluded that after testosterone withdrawal the secretory activity of hamster seminal vesicle epithelial cells, although reduced, is not abolished, and that exocytosis is relatively more reduced than secretory protein production. We suggest that an extracellular androgen source is responsible for secretory activity not being lost in the epithelial cells of castrated hamster seminal vesicle.     

Polarized epithelial cells of the hamster seminal vesicle in a serum-free bicameral culture system: evidence of secretory and endocytic activities

Primary cultures of epithelial cells from the hamster seminal vesicle were established in a chemically defined medium supplemented with hormones and growth factors. Epithelial cell clusters were prepared combining enzymatic dissociation and mechanical disaggregation and then seeded in bicameral systems equipped with collagen-membrane inserts. A growth curve was generated and the cells were characterized morphologically and morphometrically by light and electron microscopy. The immunocytochemical detection of cytokeratins and the measurement of transepithelial electrical resistance were also performed. The secretory activity was studied by fluorography using L-[³⁵S]methionine as a precursor, and endocytosis was approached using horseradish peroxidase as a tracer. Our results show that epithelial cells of the seminal vesicle can be grown as a monolayer of morphological and functionally polarized cells which retain secretory and endocytic activities. These cell cultures might therefore prove useful to investigate further the regulation of secretion and endocytosis in the seminal vesicle and are a promising model to approach, in a broader scope, cell polarity and protein sorting and targeting.     

Fine structure of the seminal vesicle epithelium of the mouse and golden hamster

The seminal vesicle epithelium of the mouse and golden hamster was examined by light microscopy and by transmission and scanning electron microscopy. By transmission electron microscopy, in the seminal vesicle epithelium of both animals secretory epithelial cells which consisted of mostly light and a few dark cells were observed. The epithelial cells possessed secretory granules which contained a densely stained core. The secretory granules in the mouse epithelium reacted weakly with periodic acid-Schiff (PAS) stain and were slightly stained with alcian blue (AB), and those in the golden hamster exhibited strongly positive reactions with PAS and AB. The nuclei in the mouse tissue were spherical or ovoid, and those in the golden hamster tissue had a few lobes. By scanning electron microscopy, the apical surfaces of most of the epithelial cells were commonly flat or domed, and those of some epithelial cells protruded into the lumen as apocrine-like processes, or possessed small and large orifices. Besides the epithelial cells, there were cells characterized by pseudopodium-like cytoplasmic projections, a few membranous structures, an irregular nucleus, and cytoplasm containing a few dense bodies, in the basal portions of the epithelial cells, or between the basal lamina and the epithelial cells. These cells of the two species were similar in their features.     

Fine structural studies of rat seminal vesicle in castrated and intact animals following estrogen treatment.

The effect of estradiol and/or testosterone upon secretion by seminal vesicle in castrated and intact rats was assessed in young adult Sprague-Dawley rats, using light microscopy (LM), transmission (TEM) and scanning (SEM)electron microscopy. Hormones were injected daily for ten days beginning ten days after castrations were performed. The normal rat seminal vesicle, as revealed by SEM, was characterized by a large saccular lumen with highly folded walls. Cell surfaces were covered with microvilli, or occasionally displayed a protruding, ruffled surface, sparsely covered with short microvilli. Cytology was normal in testosterone-treated animals. Estradiol treatment of castrated animals stimulated secretion by seminal vesicle epithelial cells as evidenced by the presence of normal secretory bodies, the presence of RER, and moderately hypertrophied Golgi complexes. These glands were not heavier than were glands from castrated, untreated animals, although the epithelial cells were significantly taller. Secretion was maintained in intact animals treated with estradiol, although glands were smaller and epithelial height was reduced. Estradiol and testosterone treatment in combination did not appear to have an additive effect on secretion, weight of the gland, or epithelial height. The following results support the hypothesis that estrogen-induced prolactin synthesis and release may be involved in the mechanism by which estradiol effected stimulation of seminal vesicle epithelium. Prolactin-treated, castrated animals exhibited focal areas of stimulated epithelium. In hypophysectomized animals (untreated controls), the seminal vesicle epithelium retained some secretory bodies and secretory fluid in the glandular lumen; epithelial height was taller than that in castrated controls. Estrogen treatment reduced the epithelial height to that of castrated controls; there was no evidence of secretion. This suggests that in the absence of anterior pituitary hormones, including prolactin, the stimulatory effect of estradiol on seminal vesicle epithelium was nullified. In adrenalectomized/castrated animals, estradiol treatment stimulated secretion in seminal vesicle epithelium just as in non-adrenalectomized/castrated animals. This indicates that the adrenal gland plays a non-essential role in the action of estrogen on seminal vesicle epithelium.     

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.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants

Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.     

Identification of low density lipoprotein receptor-related protein-2/megalin as an endocytic receptor for seminal vesicle secretory protein II

The low density lipoprotein receptor-related protein-2/megalin (LRP-2) is an endocytic receptor that is expressed on the apical surfaces of epithelial cells lining specific regions of the male and female reproductive tracts. In the present study, immunohistochemical staining revealed that LRP-2 is also expressed by epithelial cells lining the ductal region and the ampulla of the rat seminal vesicle. To identify LRP-2 ligands in the seminal vesicle, we probed seminal vesicle fluid with 125I-labeled LRP-2 in a gel-blot overlay assay. A 100-kDa protein (under non-reducing conditions) was found to bind the radiolabeled receptor. The protein was isolated and subjected to protease digestion, and the proteolytic fragments were subjected to mass spectroscopic sequence analysis. As a result, the 100-kDa protein was identified as the seminal vesicle secretory protein II (SVS-II), a major constituent of the seminal coagulum. Using purified preparations of SVS-II and LRP-2, solid-phase binding assays were used to show that the SVS-II bound to the receptor with high affinity (Kd = 5.6 nM). The binding of SVS-II to LRP-2 was inhibited using a known antagonist of LRP-2 function, the 39-kDa receptor-associated protein RAP. Using a series of recombinant subfragments of SVS-II, the LRP-2 binding site was mapped to a stretch of repeated 13-residue modules located in the central portion of the SVS-II polypeptide. To evaluate the ability of LRP-2 to mediate 125I-SVS-II endocytosis and lysosomal degradation, ligand clearance assays were performed using differentiated mouse F9 cells, which express high levels of LRP-2. Radiolabeled SVS-II was internalized and degraded by the cells, and both processes were inhibited by antibodies to LRP-2 or by RAP. The results indicate that LRP-2 binds SVS-II and can mediate its endocytosis leading to lysosomal degradation.     

Ultrastructural and histological study of degenerative changes in the antennal glands, hepatopancreas, and midgut of grass shrimp exposed to two dithiocarbamate biocides

The histological and ultrastructural alterations observed in the antennal glands, hepatopancreas, and midgut of grass shrimp exposed to either a 50% potassium dimethyldithiocarbamate biocide (Busan-85; 5–60 ppb) for 14 days, or to a different biocide, composed of 15% sodium dimethyldithiocarbamate and 15% disodium ethylene bisdithiocarbamate (Aquatreat DNM-30), for 3–4 days (60–140 ppb) and 28–35 days (40–120 ppb), were compared and contrasted with the normal morphological features in control shrimp. Only those experimental shrimp that exhibited various degrees of branchial abnormality were examined. Although the alterations in Busan-exposed shrimp were generally more pronounced, the antennal glands of 32 out of 36 experimental shrimp exhibited abnormalities that were manifested primarily as increased secretory activity by the labyrinth cells. In dithiocarbamate-exposed shrimp with “black gills”, the labyrinth epithelium exhibited moderate nuclear hypertrophy, apparent cell sloughing, intense secretory activity, and occasional melanized lesions; alterations in the antennal gland coelomosac included nuclear pyknosis, a general deterioration of podocyte organization, and an unusual increase in hemolymph density adjacent to affected tissues. Although there was an apparent increase in mitotic activity in the hepatopancreatic tubules of shrimp exposed to Aquatreat for 28–35 days, degenerative changes were most frequent and extensive in the hepatopancreas and midgut of dithiocarbamate-exposed shrimp with “black gills”. These observed changes included the diminution of the basal midgut and hepatopancreatic tubular system, moderate midgut hypertrophy, pronounced activity by the hepatopancreatic fixed phagocytes, development of mitochondrial inclusions and megamitochondria, loss of cytoplasmic density, hepatopancreatic nuclear pyknosis, and irreversible degeneration of hepatopancreatic tubule apices. This study suggests that some of the observed abnormal/pathological changes are the indirect consequence of branchial degeneration. A number of possible defensive reactions to dithiocarbamate poisoning, including heterostasis, phagocytosis, encapsulation, and the possible participation of reserve inclusion cells are proposed.     

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.     

Prostate androgen receptor: immunohistological localization and mRNA characterization

Four androgen receptor (AR) specific monoclonal antibodies were used for the immunohistochemical localization of AR in the human prostate tissue. The prostate tissue consisted of alveoli embedded in fibromuscular stroma and lined with a single layer of columnar secretory epithelial cells. The immunoreactive ARs were found predominantly in the nuclei of epithelial cell, suggesting ARs, like estrogen receptors and progesterone receptors, are mainly nuclear proteins. Northern blot hybridization showed that AR mRNA is about 9 kilobases (kb) and relative abundant in the androgen-sensitive organs, such as ventral prostate, dorsolateral prostate and seminal vesicle.     

Cadmium toxicity on cultured neonatal rat hepatocytes: biochemical and ultrastructural analyses.

The effects of cadmium exposure on the protein secretory functions of cultured neonatal rat hepatocytes were analyzed by both two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and electron microscopy. [35S]Methionine-labelled protein secretion was significantly depressed by cadmium exposure in a dose-dependent manner (1, 10 and 100 microM). Protein secretory patterns resolved by 2D-PAGE and analyzed by autoradiography showed that besides albumin and transferrin, three polypeptide spots decreased their radiolabelling intensities, whereas four spots appeared due to cadmium exposure. Ultrastructural alterations in cultured neonatal rat hepatocytes induced by cadmium exposure were characterized by condensation of the nuclear chromatin, appearance of intra-nuclear inclusions, decrease in number of microvilli, increase in number of intra-mitochondrial granules and transformation of rough endoplasmic reticulum to cytoplasmic vesicles in a dose-dependent manner. Both biochemical and ultrastructural findings indicate that cadmium adversely affects the protein secretory functions of cultured neonatal rat hepatocytes.     

Functional disturbance of rat sexual accessory glands in an early phase of lead intoxication

In order to investigate the effects of chronic lead intoxication on sexual accessory glands adult male Wistar rats were poisoned by ad libitum ingestion of lead acetate at concentrations of 0.5 g/l and 1.0 g/l for 90 d. Blood lead exhibited a significant increase in both treated groups. A decrease in hematocrit and hemoglobin, besides a rise in glycemic levels, confirmed lead intoxication. No sign of lesion was detected upon histologic examination of prostate, seminal vesicle, and coagulating gland. A morphometric technique revealed that, in this early phase of intoxication, no alteration occurred in the prostate's and seminal vesicle's epithelial height. However, a significant decrease in fructose content was observed in both lead treated groups. The results, revealing a precocious envolvement of male sexual accessory glands in lead intoxication, were related to disturbance in plasma testosterone level and also probably in androgen receptors.