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Xanthine Oxidase and Oxidoreductase Corneal Epithelium

Xanthine oxidoreductase exists in two functionally distinct forms, xanthine dehydrogenase (XDH, D-form) and xanthine oxidase (XOX, O-form). Under normal conditions, the larger part of the enzyme occurs in an NAD⁺-dependent dehydrogenase form that produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase which produces reactive oxygen species (oxygen radicals and/or hydrogen peroxide) and urate.

The physiological role of the enzymes is still not completely understood. Some authors have ascribed a bactericidal function to xanthine oxidoreductase, whereas others have proposed an anti-oxidant function based on the production of urate. Moreover, the physiological role of the O-form of xanthine oxidoreductase (xanthine oxidase) has been discussed. Van den Munckhof described the possible function of this enzyme in the oesophagus and tongue, in the skin and ejková et al. (2001) in the cornea. These authors suggested that under normal conditions, xanthine oxidase is secreted by the epithelial cells and may play a role in anti-bacterial defence by generating superoxide anions and/or hydrogen peroxide.

Xanthine oxidoreductase and xanthine oxidase have been demonstrated in various tissues, including liver, skin, heart, kidney, and intestine. ejková and Lojda (1996) and ejková et al. (2002) demonstrated xanthine oxidase activity in rabbit and human cornea, and in human and bovine retina. ejková and Lojda (1996) and ejková et al. (2001) described that reactive oxygen species generated by xanthine oxidase may contribute to the damage of rabbit corneas irradiated with UV-B light. ejková et al. (1998) hypothesized that xanthine oxidase is present in the tear fluid and is involved in additional damage of superficial layers of corneal epithelium after prolonged wearing of contact lenses, long-lasting hypoxia, and rapid reoxygenation of the cornea after contact lens removal. discussed the involvement of xanthine oxidase in acute uveitis, whereas suggested that xanthine oxidase is involved in corneal damage after excimer laser therapy. Cekic et al (1999) hypothesized that xanthine oxidase has damaging effects on the lens in alloxan-induced diabetic rats and Kuriyama et al (2001) suggested that toxic oxygen species generated by xanthine oxidase are involved in retinal ischemia–reperfusion injury.

To examine the possible participation of xanthine oxidase-generated reactive oxygen species in oxidative eye damage, we decided to localize enzyme histochemically and immunohistochemically xanthine oxidoreductase and xanthine oxidase in the epithelium of bovine, pig, guinea-pig, and rat corneas, where these enzymes have not yet been demonstrated.

Bovine, pig, guinea-pig, and rat corneas of normal eyes were employed (6 corneas in every group). Immediately after the death of the animals (ages of the animals were as follows: cows, 12 years; pigs, six months; guinea-pigs, 2 months; rats, 3 months), the anterior eye segments were cut out, quenched in light petroleum and chilled with an acetone dry-ice mixture. Cryostat sections of 10 ?m thickness were cut on a cryostat (Leica CM 1900; Leica Instruments GmbH, Heidelberg, Germany) and transferred to glass slides.

Xanthine oxidase activity was detected by capturing hydrogen peroxide by cerium ions as described by. Shortly; sections were fixed in 2% glutaraldehyde at 4°C for 1 min, rinsed in distilled water and incubated at 37°C for 3 h. The incubation medium contained 100 mM Tris–maleate buffer (pH 8.0), 10 mM cerium chloride, 100 mM sodium azide, and 0.5 mM hypoxanthine. The visualization step was performed by incubating sections for 25 min at 37°C in 100 mM sodium acetate buffer (pH 5.3), containing 42 mM cobalt chloride, 100 mM sodium azide, 1.4 mM diaminobenzidine, and 0.6 mM hydrogen peroxide. After visualization, sections were mounted in glycerol jelly. Controls were performed by incubating in the medium without substrate or in the presence of substrate and 1 mM allopurinol, an inhibitor of xanthine oxidase.

Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt method as modified by Frederiks and Bosch (1995). Shortly, unfixed sections were incubated at 37°C for 30 min in a medium consisting of 100 mM phosphate buffer (pH 8.0) containing 18% PVA, 0.5 mM hypoxanthine, 1 mM NAD, 5 mM tetranitro BT and 0.45 mM phenazine methosulphate (PMS). After incubation, sections were washed in 100 mM phosphate buffer (pH 5.3, 60°C), rinsed in distilled water and embedded in glycerol jelly. Hypoxanthine and/or NAD were omitted from the incubation medium in control incubations.

For the immunohistochemical localization of xanthine oxidoreductase and xanthine oxidase, the following primary antibodies were used: polyclonal rabbit anti-bovine xanthine oxidase antibody (Chemicon International, Temecula, CA, USA), polyclonal rabbit anti-human xanthine oxidase antibody (Biogenesis, Dorset, UK) and monoclonal mouse anti-human xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody (Lab Vision, Fremont, CA, USA). Unfixed cryostat sections were postfixed in acetone at 4°C for 4 min. Subsequently, anti-mouse/rabbit-HRP/DAB Ultravision Detection System (Lab Vision) was employed as recommended by the manufacturer: hydrogen peroxide block (12 min), ultra V block (5 min), primary antibody incubation (60 min), biotinylated goat anti-mouse/rabbit incubation (10 min), and streptavidin peroxidase incubation (12 min). The visualization was performed using freshly prepared DAB substrate/chromogen solution. The optimal dilution of the primary antibodies was 4 ?g/ml, and the incubation in a humidity chamber lasted for 60 min at room temp. After visualization, sections were mounted in Aquatex (Merck, Darmstadt, Germany). In controls, sections were incubated in the absence of primary antibody.

Xanthine oxidoreductase and xanthine oxidase (both the proteins and their enzymatic activities) were present in the corneal epithelium of bovine (1–4), pig (5–8), guinea-pig (9–12), and rat (13–16) eyes. As compared with the histochemical demonstration of xanthine oxidoreductase activity in bovine, pig, and rat corneal epithelium, the immunohistochemical demonstration of xanthine oxidoreductase in corneal epithelium of the same animals revealed more intense staining (compare 1 with 3, 5 with 7, and 13 with 15). Only in guinea-pig corneal epithelium are the enzyme histochemical and immunohistochemical staining patterns nearly identical (compare 9 with 11). It should be mentioned that the antibody used for the demonstration of xanthine oxidoreductase localized not only xanthine oxidoreductase, but also aldehyde oxidase in corneal epithelium. In immunohistochemical studies, polyclonal rabbit anti-human xanthine oxidase antibody and polyclonal rabbit anti-bovine xanthine oxidase antibody were used for the detection of xanthine oxidase in the corneal epithelium of all animals investigated. We detected xanthine oxidase protein in the epithelium of bovine, pig, guinea-pig, and rat corneas with both antibodies. We did not obtain significant differences in staining pattern using these two antibodies. The xanthine oxidase protein was demonstrated in 2, 6, 10 and 14 with the bovine xanthine oxidase antibody.

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Nuclei of C-maf Mrna was Detected in Skin

Maf is a family of oncogenes originally identified in an avian oncogenic retrovirus, AS42, encoding bZIP transcription factors. Recently, two maf-related clones, c-maf (maf-2) and mafB (maf-1), were isolated from genes of the rat liver complementary DNA library. It has been reported that genes of the maf family have essential roles in embryonic development and cellular differentiation. Both genes are expressed in a wide variety of tissues including spleen, kidney, lens, brain and liver; but so far, maf gene expression has never been investigated in skin development. The present study was performed to analyze the expression of c-maf-1 and mafB genes in rat skin in embryonic stages from 15 days onwards using in situ hybridization.

Fischer rats were used in the present study and were purchased from Nippon Clea (Shizuoka, Japan). The embryos were obtained from pregnant rats of embryonic days (ED) 15, 16 and 19 and were rinsed briefly with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and fixed in the same solution at 4°C overnight. The skin of rats on postnatal days (PD) 3 and 5 were also used to investigate maf gene expression. Fixed tissues were embedded in paraffin. Sections, 5 ?m thick, were cut. Digoxigenin (DIG)-labeled single-strand RNA probes were prepared using a DIG RNA labeling kit (Boehringer Mannheim, Germany) according to the manufacturer’s protocol. To prepare the rat maf probe, the full-length fragment of rat c-maf and mafB cDNA were subcloned into a pBluescript SK (?) plasmid (Stratagene, La Jolla, CA, USA). This plasmid was either linearized with EcoRI and transcribed with T7 RNA polymerase to prepare an anti-sense probe or linearized with HindIII and transcribed with T3 RNA polymerase to prepare the sense probe. Hybridization was carried out as described previously.

The expression of c-maf mRNA was first detected on ED 16 in the nuclei of cells in the basal layer of developing epidermis. On day 19, high expression was found in the nuclei of basal keratinocytes and developing hair germs. On PD 3, c-maf mRNA could not be demonstrated in the epidermis and hair follicles. Nonspecific staining was found at the edge of the corneal layer with both DIG-labeled anti-sense and sense RNA probes. On the other hand, high expression of c-maf mRNA was detected in the nuclei of cells in developing cerebral cortex on ED 19 which has been used as positive control. MafB has similar expression patterns as c-maf (data not shown). Table 1 summarizes expression patterns of both mafs.

Figure 1. Expression of c-maf mRNA in the skin of rat embryos and neonatal rats. On ED 19, high c-maf mRNA levels were detected in the nuclei of developing epidermal keratinocytes of the basal layer (open arrows) (A), and in the developing hair germs (B). On PD 3, c-maf mRNA could not be detected in basal epidermal keratinocytes (arrow heads) (C), and in hair follicles (open arrows) (D). A negative control section incubated with the DIG-labeled sense c-maf RNA probe did not contain any positive staining in the skin at ED 16 (E). High expression of c-maf mRNA was detected in the nuclei of cells in the developing cerebral cortex on ED 19 as positive control (F). Bars, 25 ?m.

As already mentioned, the maf oncogene (v-maf) was initially identified in an avian oncogenic retrovirus, AS42, which induces musculoaponeurotic fibrosarcoma in vivo and transforms chicken embryo fibroblasts in vitro. Several maf-related genes have been identified, and the maf gene family includes the large maf genes (c-maf, mafB and Nrl) and the small maf genes (mafK, mafG and mafF). Recently, it was found that both c-maf and mafB genes participate in transcriptional regulation during the development of the lens in rats. In the present study, high expression of both maf mRNAs was detected in the nuclei of basal keratinocytes and in the hair germs in developing rat epidermis on ED 19. Maf mRNA was not detected in the upper layer of differentiated epidermis on ED 19. Furthermore, maf mRNA expression was lost in postnatal rat skin. On the basis of data already available including the present results, it is concluded that c-maf and mafB may be involved in the embryonic development of the epidermis and hair follicles, but not in cellular differentiation of the skin.

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B10.BR Leydig Cells Y Chromosome Spermatozoa

Androgens are essential in males for the development and maintenance of specific reproductive organs such as testes, prostate, and epididymis. The biochemical aspects of androgen metabolism are now of special interest since recent data imply that estrogens play an important role in modulating the function of the male gonad, especially in germ cell development. The formation of estrogens from androgens depends on functionally active aromatase, a microsomal enzyme (also known as estrogen synthase, P450arom, EC 1.14.14.1), that has been demonstrated in both Leydig and Sertoli cells, and recently in germ cells of various animal species.

The mammalian Y chromosome plays a crucial role in sex determination and fertility of males. Over the last decade, various Y-chromosome microdeletions have been identified in human infertile patients with azoospermia or oligozoospermia. In mice, the Y-del mutation is caused by partial deletion in the long arm of the Y chromosome. This mutation was found during routine chromosomal analysis of B10.BR males in The National Institute of Genetics (Mashima, Japan). Since 1987, these mutants have been bred at the Department of Genetics and Evolution of Jagiellonian University (Kraków, Poland). They have been extensively studied with respect to sperm quality and their fertilization capacity. In comparison with the B10.BR strain, its congenic mutant strain is characterized by increased numbers of abnormal spermatozoa and low fertilization efficiency as demonstrated in vitro. In mutant males with the deletion in the long arm of the Y chromosome, a high percentage of sperms with abnormal heads has been observed, and among them class 1b, which is characterized by flattening of the acrosomal cap, is predominant. Moreover, formation of the acrosome during spermiogenesis is altered in mutant mice. Other important testicular parameters have also been analyzed such as testis weight, proportion of abnormal tubules, number of Sertoli cells, number of pachytene spermatocytes, and number of step-9 spermatids. Because spermatozoa of control and mutant mice are qualitatively and quantitatively different, we decided to compare expression of aromatase as well as steroidogenic activity in testes of both strains, especially because aromatase is a key enzyme in the modulation of balance between sex-steroid hormones.

Bishop et al (1985) identified a mouse Y-derived sequence, Y353/B, which detects multiple copies of related sequences on the mouse Y long arm. The exact function of Y353/B-related sequences is yet unknown, but it has been demonstrated that it is part of an expressed region, and a strong candidate to contain the Smy gene. Later, tested the presence of Y353/B-related sequences in mutant males with Y-chromosome deletions.

Southern-blot analysis was performed on DNA from B10.BR-Y^del mice and the congenic B10.BR strain to test copy numbers of the Y353/B-related sequence. Furthermore, testes of these two strains of mice, B10.BR and B10.BR-Y^del, respectively, were examined using immunohistochemistry, Western-blot analysis, and radioimmunological assays, to localize aromatase, and to measure steroid hormone levels, respectively.

Animals were bred at the Department of Genetics and Evolution of Jagiellonian University. All mice were given commercial pelleted diet, water ad libitum, and maintained under a 12 h light–dark cycle. Thirty-five mature males (2–3 months old) were used for the experiments. They were killed by cervical dislocation. Testes were used for either isolation of Leydig cells and in vitro cultures, or routine histology. Furthermore, immunocytochemistry on cultured cells or immunohistochemistry on testicular sections was applied.

Experiments were performed in accordance with Polish legal requirements, under the licence provided by the Commission of Bioethics at Jagiellonian University.

Genomic DNA was isolated from the tail. DNA (20 ?g of each sample) was restricted with EcoRI, electrophoresed on a 1% agarose gel, and transferred onto nitrocellulose membranes (Hybond C; Amersham, Buckinghamshire, UK). Membranes were hybridized with ³²P-labeled Y353/B probe (Rediprime II Kit, [-³²P] dCTP, Amersham;) at 65 °C, overnight. After hybridization, membranes were washed at high stringency (0.2×SSC at room temperature for 30 min, 0.1×SSC, 0.1% SDS at 65 °C for 2×1 h) and exposed overnight at ?70 °C. To check the integrity of DNA and its quantity, the membranes were stripped and re-hybridized with Zfy-derived probe. These experiments were repeated 4 times.

Eight decapsulated testes from each mouse strain were used for the preparation of Leydig cell suspensions according to the procedure previously described in detail. Briefly, crude suspensions of Leydig cells were obtained by short digestion (10 min, at 37°C) using trypsin in PBS (0.25%), and then they were serially sieved through steel meshes with pore sizes of 156 and 74 ?m, respectively (US Standard Sieve series with ASTME 11 Specifications; Dual MFG, Chicago, IL, USA). Supernatants were collected and centrifuged at 180g for 5 min. The resulting cell pellets were washed twice and subjected to purification by centrifugation on a continuous 10–90% (v/v, 50 ml) Percoll gradient (Pharmacia, Uppsala, Sweden) using a method described by with some modifications. After centrifugation at 800g for 25 min at 4°C, because the low temperature significantly prevented cell aggregation, the cell bands containing Leydig cells were collected, washed, and centrifuged once or twice at room temperature using low-speed centrifugation (90g for 10 min). Leydig cells were cultured for 48 h in 24-well culture dishes (Nunc, Kalmstrup, Denmark) in Eagle’s medium supplemented with 2% calf serum and -glutamine (Laboratory of Sera and Vaccines, Lublin, Poland). Penicillin (120 iu/ml) was also added. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ in air. The purity of the cells was approx. 87% and was checked by a histochemical assay of ?⁵, ?-hydroxysteroid dehydrogenase (?⁵, ?-HSD) activity. Viability of the cells as assessed by the trypan blue exclusion test was over 95%. Purity and viability of the cells were determined in triplicate. For immuncytochemical purposes, each well was closed with a round coverslip with an appropriate diameter. Culture media were collected and stored at ?20° for hormone measurements.

It has been previously reported that spermatozoa of B10.BR-Y^del mice possess a cytoplasmic droplet attached to their flagella. Therefore, only spermatozoa of this strain were isolated to demonstrate the presence of aromatase in cytoplasmic droplets. Sperm cell isolation has been described in detail. Small drops of suspensions of sperm cells were transferred to slides precoated with poly-lysine and smears were carefully made. Immunocytochemistry was applied to detect aromatase in cytoplasmic droplets (see below).

Testes of five animals of both strains were fixed in 4% paraformaldehyde and embedded in paraplast (Monoject Scientific Division of Scherwood Medical, St. Louis, MO, USA). Sections (6 ?m thick) were mounted on slides coated with 3-aminopropyl-triethoxysilane (APES; Sigma, St. Louis MO, USA), deparaffinized, and rehydrated. To optimize immunohistochemical staining, sections were immersed in 10 mM citrate buffer (pH 6.0) and treated in a microwave oven (2×5 min, 600 W) for high-temperature antigen unmasking. Testicular sections were stained 4 times and staining was scored for the presence and intensity of immunostaining. Aromatase staining was designated as absent, weak, moderate, or strong on the basis of visual examination of cytoplasmic localization of the antigen.

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Mucous Granules of Positive Goblet Cells

Methylxanthines induce bronchospasmolytic effects in asthmatic patients due to their nonspecific inhibition of phosphodiesterases and a subsequent increase in intracellular levels of the second messenger cAMP. Methylxanthines also increase ciliary beat frequency and stimulate mucociliary transport. Previous studies have demonstrated overstimulation and damage to tracheal goblet cells as well as a substantial decrease in the proportion of goblet cells containing sialylated glycoconjugates after intravenous administration of aminophylline.

Fucosylated glycoconjugates have been identified by in both secretory granules and plasma membranes of cells in the respiratory tract. Glycoconjugates are components of secreted mucus and contribute substantially to viscoelastic properties of mucus. Both secreted glycoconjugates and those bound to cellular surfaces also serve as adhesion sites for antigens. An increased proportion of fucosylated glycoconjugates in the mucus of the respiratory tract has been described in sinusitis, chronic bronchitis, asthma, cystic fibrosis, and acute bronchiolitis in rats. In healthy adult rats, the (1-2) linkage of fucose to galactose is the most common linkage. Fucosylation in other linkages, (1-3), (1-4), and (1-6), respectively, to N-acetyl glucosamine increases under pathological conditions such as inflammation or cystic fibrosis at the expense of fucosylation in the (1-2) position and of sialylation.

Since the administration of aminophylline significantly lowers the proportion of sialylated glycoconjugate-containing tracheal goblet cells, we have evaluated the proportion of fucosylated glycoconjugate-containing goblet cells in the present study.

Sixteen SPF New Zealand White male rabbits (Charles River, Sulzfeld, Germany; mean body weight 2.31±0.38 kg) were used in the study. Six rabbits were given intravenous injections of the methylxanthine bronchodilator drug aminophylline (Syntophyllin inj.; Hoechst-Biotika, Martin, Slovakia; 5 mg/kg/body weight), and six rabbits were administered intravenously the methylxanthine vasodilator drug containing 4 mg etophylline and 1 mg theophylline per kg/body weight (Oxantil inj.; Hoechst-Biotika). The dose of Syntophyllin was chosen because it is the recommended dose for intravenous treatment of acute asthmatic attacks and this was the rationale for the selected dose of Oxantil as well. The material was collected at 15 and 30 min after treatment. Four untreated healthy rabbits served as controls. The specimens were collected immediately after the induction of anaesthesia.

The middle portions of tracheae between the 15th and 20th tracheal rings were formalin-fixed, paraffin-embedded, and sections of 5–7 ?m thickness were cut. Combined staining with alcian blue at pH 2.5 and PAS was used to demonstrate total acidic and neutral glycoconjugates and thus the total number of goblet cells. Lectin histochemical methods were used to detect fucosylated glycoconjugates. The legume lectin Ulex europaeus agglutinin I (ULE-I), detecting terminal or branched fucose (1-2) linked to an oligosaccharide, and the ascomycete orange-peel mushroom Aleuria aurantia lectin (AAL), detecting (1-6)-, (1-3)-, and (1-4)-linked fucose residues, were employed (Vector Laboratories, Burlingame, CA, USA). After dewaxing and rehydrating, endogenous peroxidase was blocked and the sections were incubated with either biotinylated ULE-I or biotinylated AAL alone or in combination with concentrations of 30 ?g/ml for 60 min at room temperature. The sections were then incubated with a solution of streptavidin–horseradish peroxidase conjugate (Vector Laboratories) in a concentration of 2 ?g/ml for 45 min, followed by a visualisation step using FAST™ DAB Peroxidase Substrate Tablets (Sigma-Aldrich Chemie, Deisenhofen, Germany) in combination with CuSO₄ to enhance staining. The blocking of endogenous peroxidase was verified by omitting the first step of the method. Specific lectin binding was verified by a 15-min preincubation of lectins with control substrate 0.2 M -fucose, preceding the incubation of sections.

Only goblet cells that were stained and contained well-developed granules were evaluated. The granules had to occupy at least 2/3 of a cell. Simultaneous application of both lectins enabled identification of goblet cells containing glycoconjugates that were positive for both lectins; therefore, the overlap of AAL- and ULE-I-positive goblet cells could be calculated. Venn’s diagrams enabled the calculation of goblet cells containing ULE-I-positive granules only and goblet cells containing AAL-positive granules only.

For statistical evaluation, numbers of goblet cells in five categories (all goblet cells, ULE-I-positive goblet cells, AAL-positive goblet cells, all lectin-positive goblet cells, and ULE-I- and AAL-positive goblet cells) were evaluated by the chi-square test of homogeneity in frequency tables, using Yates’ correction for low frequencies and the McNemar test of symmetry, respectively, when appropriate. These data were also evaluated by one-way ANOVA with the Kruskal–Wallis post hoc test and Levene’s test for equal variances followed by the Aspin-Welch test, when appropriate (Statistica v.6.0 software; StatSoft, Tulsa, OK, USA). The significance of differences between fucosylated glycoconjugate-detecting methods within individual groups was tested with the matched t-test, Spearman rank correlation, the matched sign test, and the Wilcoxon’s paired test (BMDP New System software; Statistical Solutions, Saugus, MA, USA).

The experimental procedures were performed under general anaesthesia (ketamine 35 mg/kg and xylazine 5 mg/kg, intramuscularly) and after the local subcutaneous infiltration of the ventral cervical field with procaine. The experimental procedure was approved by the Animals Protection Expert Commission of the Faculty.

The tracheae of both control and treated rabbits were lined with pseudostratified columnar ciliated epithelium largely composed of ciliated cells, goblet cells, and basal cells. The height of the epithelium was approximately 25–30 ?m. The distribution pattern of secretory elements was irregular. By using conventional histochemical methods, the secretory elements revealed typical staining patterns according to the type of glycoconjugates they contained. PAS-positive mucous granules were stained magenta; alcian blue-stained mucous granules were stained blue. Some goblet cells were stained in various shades of violet; these cells were considered to contain mixtures of acidic and neutral glycoconjugates. The appearance of goblet cells stained with lectins was identical in control and treated rabbits. Staining with ULE-1 resulted in stained mucous granules in goblet cells, either as a homogeneous content of granules, or as darkly-stained rings; staining of the zone of cilia occurred only in the close vicinity of apical surfaces of goblet cells. AAL staining resulted in positive individual mucous granules as dark rings. The zone of the cilia was always densely stained.

Figure 1. ULE-I staining of mucous granules in a goblet cell in tracheal epithelium of a rabbit at 15 min after intravenous administration of Oxantil. Note that a goblet cell contains unstained mucous granules (open arrow) in close vicinity of a goblet cell containing stained granules. Bar, 20 ?m.

Figure 2. AAL staining of mucous granules in a goblet cell (arrow) in tracheal epithelium of a rabbit at 15 min after intravenous administration of Oxantil. Bar, 20 ?m.

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Sildenafil Citrate Corpus Cavernosum of Blood

Sildenafil citrate has been shown to be highly effective in the treatment of erectile dysfunction and it is used to treat impotence of various etiologies with good tolerance.

The penis is composed of three columns of erectile tissue each enclosed by its own dense fibrous connective tissue. Corpora cavernosa, two of the three columns of erectile tissue, are positioned dorsally, and the third column, the corpus spongiosum, is positioned ventrally. The corpus spongiosum surrounds the urethra. The three corpora are surrounded by loose connective tissue and smooth muscle cells. The vascular spaces of the corpora cavernosa are larger centrally and smaller peripherally. All corpora include vascular spaces that receive blood from branches of the deep dorsal and cavernosal arteries of the penis.

Figure 1. Schematic drawing of a transverse section of the rat penis showing the corpus cavernosum and corpus spongiosum surrounding the urethra. Dorsal nn, dorsal nerves; and dorsal aa, dorsal arteries.

Sildenafil citrate, a pyrazolo-pyrimidinyl-methylpiperazine compound, is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5. It enhances the relaxant effect of nitric oxide (NO) on the corpus cavernosum by inhibiting PDE5, which is responsible for degradation of cGMP in the tissue. Inhibition of PDE5 by sildenafil citrate increases cGMP levels in the corpora, causing smooth muscle relaxation and blood flow into the penis during the local release of NO due to sexual stimulation. This is the main action of sildenafil citrate on the physiologic mechanisms of erection. However, we do not know the long-term effects of the use of sildenafil citrate on the corpora of the penis. In addition, histopathological and ultrastructural effects of the use of sildenafil citrate on corpus cavernosum components are not known. Therefore, we decided to investigate the histopathological and ultrastructural effects of sildenafil citrate in the rat corpus cavernosum using light and electron microscopical techniques.

Twenty male 10–12-week-old Wistar rats with an average weight of 250 g were used. Rats were fed standard diet and were divided into two groups of 10 rats each. The first group was used as control and the second group was treated with 2 mg/kg body weight/day sildenafil citrate orally via gavage on alternate days (3 days in a week) for 4 weeks. All male rats were coupled with female rats overnight for sexual activity only on the days when sildenafil was given. The impact of sildenafil citrate on sexual function was evaluated.

The Ethics Committee at Celal Bayar University, School of Medicine, approved our study protocol.

After 4 weeks, all rats were anesthetized with methoxyflurane and sacrificed by cervical dislocation. Penile tissue from the middle part of the penis was collected from each rat. Tissue was fixed using 10% formalin during 24 h for light microscopy, and 2.5% glutaraldehyde during 4 h for electron microscopy.

All specimens were fixed in a solution of 10% formalin during 24 h. Specimens were washed and soaked in a graded series of ethanol. Then they were embedded in paraffin. Sections (5 ?m thick) were cut and prepared for both histochemical and immunohistochemical staining. Periodic acid Schiff (PAS) staining was used for histological diagnosis.

Specimens were processed for light microscopy and sections were incubated at 60°C overnight and then dewaxed in xylene for 30 min. After soaking in a decreasing series of ethanol, sections were washed with distilled water and phosphate-buffered saline (PBS) for 10 min. Sections were then treated with 2% trypsin in 50 mM Tris buffer (pH 7.5) at 37°C for 15 min and washed with PBS. Sections were delineated with a Dako pen (Dako, Glostrup, Denmark) and incubated in a solution of 3% H₂O₂ for 15 min to inhibit endogenous peroxidase activity. Then, sections were washed with PBS and incubated for 18 h at 4°C with primary antibody, a polyclonal anti-inducible NOS (iNOS) in a 1:100 dilution (Zymed, San Francisco CA, USA). Afterwards, sections were washed 3 times for 5 min each with PBS, followed by incubation with biotinylated IgG (Dako) and then with streptavidin-peroxidase conjugate (Dako). All incubation steps were separated by 3 washing steps. After washing 3 times for 5 min with PBS, sections were incubated with a solution containing 3-amino-9-ethylcarbazole (Dako) for 5 min to visualize immunolabelling and then with Mayer’s hematoxylin. Sections were covered with mounting medium and were analyzed light microscopically with a BX 40 microscope (Olympus, Tokyo, Japan). Control samples were processed in an identical manner, but the primary antibody was omitted. Two observers blinded to clinical information evaluated the staining scores independently.

1 mm³ of specimens were fixed with 2.5% glutaraldehyde for 4 h. Then, postfixation was performed using osmium tetroxide for 1.5 h. Specimens were processed by standard dehydration in a graded series of ethanol before infiltration and embedding in Epon 812 (Fluka, Buchs, Switzerland). Ultrathin sections were cut on an ultracut R ultramicrotom (Leica, Nussloch, Germany) and stained with uranyl acetate-lead citrate before viewing with a 9S electron microscope (Zeiss, Oberkochen, Germany).

Measurements of the surface area of blood vessels were performed to assess vascular density in all samples. Three micrographs from each sample were randomly selected and for the estimation of surface areas of blood vessels, the unbiased stereology method was used. A cycloid test system, with its minor axis parallel to the vertical direction, was randomly translated in x and y directions on the micrograph. The test system has a known length of cycloid per point (l/p). In order to estimate surface density, we performed two measurements on each image: first, the number of intersections between the cycloid lines and the boundary of interest (I); and second, the number of points that were found within the reference space (P). Surface areas of blood vessels were then estimated with the formula: surface area of blood vessel 2?I/((l/p)?P).

The thickness of the basal lamina was also measured using micrographs. All measurements were performed by three investigators who were blinded to the micrographs. Each investigator reported his/her results separately. The mean of the results of the three investigators was used in the statistical analysis. All data were expressed as the mean±standard error of the mean. Data comparisons were performed using an ANOVA test, with P<0.05 for the level of significance.

The histological analysis revealed that the corpus cavernosum, which is the main anatomic structure used during erection and consists of numerous vascular spaces with wide and irregular shaped lumina that are lined by endothelial cells, was elongated and the number of vascular spaces was increased in sildenafil-treated rats. The amount of connective tissue in the corpus cavernosum was clearly increased and dense collagen and smooth muscle fibers were observed in treated rats. The lamina basalis of blood vessels of treated rats in the corpus cavernosum appeared to be thicker than in control rats after staining with PAS.

Figure 2. Light micrographs of penile tissue of control rats (A,C) and rats treated with sildenafil (B,D) after PAS staining. The dorsal vein (V) and corpus cavernosum (CC) show increased density of collagen bundles (asterisk) in the stroma of the corpus cavernosum and a thicker basement membrane (arrows) around arteries and vessels is present in treated rats. Magnifications: (A,B) ×100; and (C,D) ×200.

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Pinealectomized Rats were Treated with Melatonin

The pineal secretory product melatonin was recently shown to have free radical scavenging properties. Melatonin also activates several antioxidative enzymes including glutathione (GSH) peroxidase, modulates gene expression for several protective enzymes and reduces lipid peroxidation. Additionally, the antioxidant effects of melatonin are probably based on its stimulatory effect on the expression of superoxide dismutase, GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase and its inhibitory effect on nitric oxide synthase expression. On the other hand, the role of physiological levels of melatonin, which are known to decrease with age, in the prevention of this damage is not yet known. It regulates a wide variety of biological rhythms, including cardiac rhythms. Physiological and/or pharmacological doses of melatonin affect a variety of heart functions. These effects on the heart may be mediated directly and/or indirectly.

Pinealectomy-induced biochemical and behavioral changes have been the subject of many studies, but morphological changes and particularly cardiovascular changes have been less well studied. Since it was reported that pinealectomy may cause hypertension up to 60 days, we have used rats that were pinealectomized at 2 months before treatment with melatonin to eliminate any possible effect of pinealectomy-induced hypertension.

We have explored whether reduced melatonin levels in the circulation play a critical role in damage to the heart as a result of pinealectomy. Although the retina has a high capacity to synthesize melatonin, it does not seem to contribute significantly to plasma melatonin levels probably because of rapid catabolism in the retina. Night-time blood melatonin peaks are eliminated after pinealectomy. We also investigated the effects of melatonin on pinealectomy-induced changes of malondialdehyde (MDA), a major lipid peroxidation product, and GSH, which is a major protectant against lipid peroxidation. It has been proposed that antioxidants that maintain levels of GSH may restore cellular defense mechanisms against lipid peroxidation. Additionally, serum cholesterol levels and heart weight were determined. The present study was designed to investigate the effects of physiological and pharmacological levels of melatonin on rat hearts using pinealectomy and exogenous melatonin.

Female Wistar rats, weighing 150–200 g, were kept at a constant temperature (21±2°C) and humidity (60%) with a 12/12 h light/dark cycle. Rats were pinealectomized or not at 2 months before treatment with melatonin.

Pinealectomy was performed as described by. Rats were anesthetized with ketamine hydrochloride (75 mg/kg) and xylazine (8 mg/kg) which were administered intraperitoneally (i.p.) before operation. The entire procedure was completed within 15 min.Pinealectomy was confirmed by histological evaluation of the gland of each animal. Rats were divided into two groups: pinealectomized (n=12) and non-pinealectomized (n=6). Subsequently, pinealectomized rats were distributed over two groups: one group of rats were treated ip with vehicle, and one group with 4 mg/kg melatonin (Sigma, St. Louis, MO, USA) for 3 days. Non-pinealectomized rats were injected with vehicle only. At 24 h after the last treatment, rats were sacrificed, hearts and aortas were quickly removed and hearts were divided into two equal longitudinal parts. One of the parts was placed in a solution of 10% formaldehyde for routine histopathological examination using light microscopy. The other half of the hearts was placed in liquid nitrogen and stored at ?70°C until assayed for levels of MDA and GSH. Trunk blood was collected to determine the serum levels of cholesterol. Additionally, the weight of the hearts was determined. For these studies, melatonin was dissolved in ethanol and diluted in saline to give a final concentration of 5% ethanol.

All experiments were performed in accordance with the guidelines for animal research of the National Institute of Health and were approved by the Committee for Animal Research at Inonu University, Malatya, Turkey.

Heart tissue (200 mg) was homogenized in an ice-cold solution of KCl (150 mM) for the determination of MDA levels. MDA levels in homogenates were determined spectrophotometrically by measuring thiobarbituric acid-reactive substances. Levels of GSH were determined by spectrophotometry based on the use of Elman’s reagent. Results are expressed as nmol/g tissue. Serum levels of cholesterol were determined using an Olympus Autoanalyser (Olympus, Tokyo, Japan). Heart MDA and GSH levels were measured to compare our data with those in the literature and to assess the status of lipid peroxidation and antioxidant capacity in the animals in the three groups. Serum cholesterol levels were also found to be related to pinealectomy in animal experiments.

Samples of heart and aorta that had been fixed in 10% formaldehyde were routinely processed and paraffin embedded. Subsequently, 5 ?m-thick sections were stained by the following methods: hematoxylin and eosin (H&E), Van Gieson, Masson’s trichrome and PAS/Alcian blue at pH 2.5 (PAB).

Levels of MDA and GSH in the heart and serum cholesterol levels were analyzed by one-way ANOVA. Post hoc comparisons were performed using Tukey tests. The weight of hearts was analyzed by a Student’s t-test. Differences were considered significant when p<0.05. All results are expressed as mean±SEM.

Biochemical data with respect to tissue levels of MDA and GSH, and serum cholesterol levels are shown in Table 1. Heart MDA levels were significantly higher in the pinealectomized group than in the sham-operated group and the group of pinealectomized rats treated with melatonin; however, GSH levels were within the normal range, irrespective of the treatment. Serum cholesterol levels were higher in the pinealectomized group than in the sham-operated group and the group of pinealectomized rats treated with melatonin.

Hearts of pinealectomized rats were heavier than those of sham-operated rats, whereas hearts of pinealectomized rats that had been treated with melatonin had similar weights as the pinealectomized group.

Microscopical examination of the hearts and aortas revealed pathological effects of pinealectomy. First, we noticed myocardial fibrosis in both pinealectomized rats and pinealectomized rats treated with melatonin but not in the hearts of sham-operated rats. Fibrosis was detected optimally with Masson’s trichrome staining method. Second, myxomatous degeneration of heart valves was observed in pinealectomized rats and pinealectomized rats treated with melatonin, which showed similarities with the floppy mitral valve syndrome in humans, and this degeneration was best demonstrated with PAB staining. These changes may have occurred as follows: attenuation of the fibrosa layer of the valve was accompanied by focal thickening of the spongiosa layer and concomitant deposition of mucoid (myxomatous) material. Thickening of the mural endocardium of the left atrium was also observed. Coronary arteries were difficult to find, whereas aortas did not exhibit calcification or degeneration of elastic tissue. These morphological alterations were similar in the pinealectomized rats and pinealectomized rats treated with melatonin.

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Caveolin-1 in PBS Goblet Cells and Anti Caveolin

Caveolin-1 is an integral membrane protein, with both the N- and C-terminal domains being cytoplasmic, and is the principal component of caveolar membranes. Caveolae are specialised vesicular microdomains of the plasma membrane that are formed by localised accumulation of cholesterol, glycosphingolipids, and caveolin. Caveolae can be found individually or in clusters and are found in most cell types, but they are most abundant in adipocytes, endothelial cells, fibroblasts, and muscle cells. Functionally, caveolae have been implicated in endothelial transcytosis, potocytosis, and signal transduction. Caveolin-1 can play a role in intracellular vesicular and cholesterol trafficking.

Recently, caveolin-1 has been found unexpectedly in the cytoplasm, mitochondria and elements of the secretory pathways of exocrine secretory cells. These data suggest that caveolin-1 may also exist in a soluble form as a cytosolic protein or associated with secretory products. However, the function of secreted caveolin-1 is still unknown, but a role in extracellular lipid transport has been hypothesised.

To better understand this role of caveolin-1, we have immunohistochemically localised caveolin-1 in secretory cells of the gastro-intestinal tract of an amphibian, the red-legged frog Rana aurora aurora. We have previously described the gastro-oesophageal mucosa of this anuran. In the red-legged frog, as in other Ranidae, such as Rana esculenta, pepsinogen in the gastric juice is for the larger part synthesised by oesophageal glandular cells, whereas hydrochloric acid is produced by oxyntic cells in fundic glands. Thus, there is a gradient in the production of proteolytic enzymes and hydrochloric acid along the oral–aboral axis of the gastro-oesophageal tract, as has been reported for other non-mammalian vertebrates. Since peptic glands are particularly voluminous and secrete abundant amounts of pepsinogen, the oesophageal mucosa of this frog seems to represent an appropriate model to investigate the presence and functions of caveolin-1 in the secretory pathway.

Specimens of R. a. aurora from western North America were obtained from Minervini (Bari, Italy).

Four adult specimens of R. a. aurora were sacrificed after ether anaesthesia and their digestive tracts were quickly removed and processed for embedding in Technovit 8100 (Heraeus-Kulzer, Wehrheim, Germany). Small samples were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4, for 4 h at 4°C. After several rinses in PBS, the samples were incubated overnight at 4°C in PBS containing 6.8% sucrose. They were then dehydrated at 4°C in a graded series of acetone. Specimens were incubated in the Technovit 8100 monomer solution for 6 h at 4°C under gentle stirring. Polymerisation was carried out on ice for 3 h. Semithin sections (3 ?m thick) were cut with glass knives using an Ultratome (LKB, Uppsala, Sweden). Before histochemical and immunohistochemical staining, sections were mounted on microscope slides coated with polylysin and were incubated for 5 min at 37°C in 0.01% trypsin and 0.1% CaCl₂, in PBS, pH 7.8, for antigen retrieval.

Zymogen granules were identified with a modified Bowie’s staining method according to, or with a combined PAS-Bowie staining method to demonstrate both mucus and zymogen granules simultaneously. All sections were counterstained with haemallum.

Some sections were stained using the Masson–Fontana silver method to detect argentaffin endocrine cells.

Caveolin-1 and pepsinogen were localised using the peroxidase–antiperoxidase (PAP) method. Endogenous peroxidase was blocked by incubating sections in PBS containing 1% H₂O₂ for 10 min at room temperature. After several rinses in PBS, sections were incubated for 2 h at 37°C in a humid Petri dish with the primary antibodies (anti-caveolin-1: ABR, Golden CO, USA; anti-pepsinogen-1: DPC Biermann, Bad Nauheim, Germany) diluted 1:100 in PBS containing 1% normal goat serum (blocking buffer). Sections were washed in blocking buffer and incubated for 1 h at 37°C with goat anti-rabbit IgG (Sigma, St. Louis MO, USA) diluted 1:100 in blocking buffer. After several washes in blocking buffer, sections were incubated with horseradish PAP complex (Sigma) at a dilution of 1:100 for 1 h at 37°C. Finally, immunolabelling was visualised by incubation with 3-3?-diaminobenzidine–H₂O₂ medium for 10 min at room temperature.

Controls were performed by using antibodies pre-adsorbed with the respective antigens or by omitting the primary antibodies.

Images were captured using an E600 photomicroscope equipped with a digital camera DMX1200 (Nikon, Kawasaki, Japan).

Small samples of gastro-oesophageal mucosa obtained from four adult specimens of R. a. aurora were fixed in a mixture of 3% paraformaldehyde and 1% glutaraldehyde in 0.1 M PBS, pH 7.4, for 4 h at 4°C, and then postfixed in 1% OsO₄ in PBS for 30 min at 4°C. Fixed specimens were washed in several changes of PBS and dehydrated in a graded series of ethanol. Finally, samples were embedded in Epon (Taab, Redding, UK). Sections were mounted on formvar-coated nickel grids and stained routinely with uranyl acetate and lead citrate. Finally, grids were observed with an EM 109 electron microscope (Zeiss, Oberkochen, Germany).

For immunoelectron microscopy, ultrathin non-osmicated sections, mounted on formvar-coated gold grids, were treated with 0.05 M glycine in PBS buffer for 15 min at room temperature. Grids were incubated for 30 min at room temperature with 1% BSA in PBS containing 0.2% gelatin (PBG) and then placed on a drop of anti-caveolin-1, diluted 1:100 in PBG overnight at 4°C. The grids were rinsed with PBG, and then incubated in a dilution of 1:10 of 10-nm gold-conjugated anti-rabbit IgG (Sigma) in PBG for 1 h at room temperature. After several rinses in PBG and distilled water, the grids were lightly stained with uranyl acetate and lead citrate.

Immunolabelling controls were performed by using antibodies pre-adsorbed with antigens or by omitting the primary antibodies.

The oesophagus of the frog R. a. aurora was found to be lined with a pseudostratified ciliated epithelium containing abundant amounts of mucous goblet cells. Two different types of goblet cells were found in the epithelium. Type-I goblet cells were characterised by supranuclear cytoplasm containing large PAS-positive secretory vesicles that were often confluent. Type-II goblet cells were thinner with ovoid basal nuclei and numerous small secretory granules in the supranuclear cytoplasm. The granules, which were PAS positive, often showed weak immunostaining of pepsinogen.

Figure 1. Gastro-oesophageal mucosa of the frog R. a. aurora stained with different histochemical methods. (A–C) Oesophageal mucosa. (A) The ciliated epithelium contains two types of goblet cells (g1, and g2). Oesophageal glands consist of serous (peptic) cells (s) and mucous cells (m). PAS-Bowie-haemallum staining. (B) Numerous pepsinogen-positive granules (arrows) are present in serous cells (s) in oesophageal glands. Anti-pepsinogen staining and haemallum. (C) Secretory granules (arrows) are stained for caveolin-1. (s) Serous cells. Anti-caveolin-1 and haemallum staining. (D–F) Gastric mucosa. (D) The mucosa is lined with a single layer of mucus-secreting cells (sc). Gastric glands mainly consist of mucous neck cells (nc) and oxyntic cells (ox). PAS-Bowie-haemallum staining. (E) Oxyntic cells (ox) are not positive after anti-pepsinogen staining. Anti-pepsinogen and haemallum staining. (F) Immunostaining is not present in oxyntic cells (ox) after staining with anti-caveolin-1. Anti-caveolin-1 and haemallum staining. Bars: (A,D–F) 30 ?m; (B,C) 25 ?m.

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PPAR was Performed in Pbst Samples

Number and volume of peroxisomes are increased and peroxisomal ?-oxidation enzymes such as acyl-CoA oxidase (AOX) are induced when responsive animals are exposed to peroxisome proliferators. This phenomenon is known as peroxisome proliferation and it has been observed in several fish species after treatment with fibrate hypolipidemic drugs and a diverse array of environmental organic pollutants. In zebrafish Danio rerio, exposure to the organochlorine pesticide methoxychlor, the phthalate dibutylphthalate, the alkylphenol 4-tert-octylphenol and estrogens causes an increase in liver peroxisomal volume, surface and numerical densities together with a significant induction of AOX activity. Because of this significant response to pollutant exposure, peroxisome proliferation has been proposed in recent years as a novel early warning biomarker that may be useful in marine pollution monitoring.

Peroxisome proliferation is mediated by peroxisome proliferator-activated receptors (PPARs), which belong to the nuclear receptor superfamily. These receptors are ligand-activated transcription factors. Hitherto, three PPAR subtypes have been identified in several mammals and some amphibians: PPAR, PPAR? and PPAR?. In addition, PPARs have been found in several fish species such as salmon (Salmo salar), turbot (Scophthalmus maximus) and plaice (Pleuronectes platessa). In zebrafish (Danio rerio), 100 bp of the DNA-binding domain of each of the three subtypes, and two different isotypes of PPAR? have been cloned. In spite of our increased knowledge on PPAR genes in fish, tissue and cell distribution patterns of PPAR proteins and their specific functions are not well understood.

In a previous study, we have detected expression of the three subtypes of PPARs in various tissues of adult zebrafish by immunohistochemistry using commercially available antibodies against PPAR, PPAR? and PPAR?. PPAR was predominantly expressed in tissues that catabolize large amounts of fatty acids, such as liver parenchymal cells, proximal tubules of kidney, enterocytes and pancreas. PPAR? showed a widespread distribution and was expressed in liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes and male and female gonads. PPAR? expression was weak and was only detected in pancreatic cells, intestine and gonads. Although zebrafish is a very convenient model species for laboratory studies, this tropical freshwater species cannot be used as sentinel for marine pollution monitoring studies. Therefore, these investigations were extended in the present study to other fish species such as the gray mullet Mugil cephalus, which is very abundant in estuaries and coastal areas in Europe.

Recently, liver peroxisomes of gray mullet have been characterized ultrastructurally, immunocytochemically and morphometrically. Furthermore, mullet liver peroxisomes exhibit variations depending on physiological and environmental conditions, thus constituting an appropriate model to study regulation of PPAR expression. The aim of the present study was to investigate immunohistochemically expression and distribution patterns of PPAR, PPAR? and PPAR? in the various cell types of gray mullet liver.

Three-month-old BALB/c mice were used as positive controls and were obtained from the central animal facility of the University of Heidelberg (for immunohistochemistry) and of the University of the Basque Country (for Western blotting).

Adult mullets of 27–38.5 cm length (3–8 g liver weight) were sampled from the Plentzia estuary (43°24?N, 2°56?W). For all studies, fish were anesthetized by immersion in a saturated solution of 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO, USA) before being sacrificed.

Rabbit anti-mouse PPAR, PPAR? and PPAR? were raised by the Genosys Division of Sigma (St. Louis, MO, USA) against the 1–18, 1–14 and 1–16 amino acids, respectively, of the A domains of the respective proteins.

Livers from gray mullet and mouse were dissected and immediately homogenized in RIPA buffer (PBS buffer, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) using a Potter S homogenizer (Braun, Melsungen, Germany) in an ice-water cooled bath. The samples were ultracentrifuged in a Centrikon T-1065 with a TFT 65.13 rotor (Kontron Instruments, Munich, Germany) at 100,000g for 1 h at 4°C. The resulting pellet was removed and the supernatant was frozen at ?80°C. Total protein content was determined using a DC protein assay kit (BioRad, Hercules, CA, USA) which is based on the Lowry assay using ?-globulin as standard.

SDS-PAGE was performed using minigels consisting of a 4% stacking gel and 12% running gel in a Mini-Protean II electrophoresis cell (BioRad). Samples were prepared with a final protein concentration of 5 ?g/?l and 50 ?g of protein as well as molecular weight standards were loaded on the gels. After electrophoresis, separated proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK). Membranes were washed in phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST) and were blocked for 1 h at room temperature with 5% milk powder dissolved in PBST. Incubations with the diluted specific antibodies (1:200 in PBST containing 0.1% milk powder) were performed overnight at 4°C. Membranes were washed for 4×10 min in PBST and incubated with an HRP-coupled anti-rabbit secondary antibody (Sigma; diluted 1:10,000 in PBST containing 0.1% milk powder) for 1 h at room temperature. After 2×5 min and 3×10 min washes with PBST, HRP activity was visualized by chemiluminescence using an ECL kit (Amersham Biosciences).

Mullet liver was immersion fixed in Carnoy fixative for 1 h. The tissue was dehydrated through a graded series of ethanol, and embedded in paraffin. Sections (1.5 ?m thick) were cut using a 2000R microtome (Leica Microsystems, Wetzlar, Germany) and picked up onto SuperFrost Plus glass slides (Menzel-Glaser, Braunschweig, Germany). Mouse liver samples were fixed in paraformaldehyde and processed similarly for paraffin embedding.

Sections were deparaffinized in xylol and rehydrated through a graded series of ethanol ending in water. Sections were stained with or without antigen retrieval. For antigen retrieval, sections were treated using a microwave or with trypsin. Microwaving was performed three times for 5 min in a conventional household microwave (740 W; Sanyo, Chatsworth, CA, USA), using citrate buffer (0.01 M, pH 6.0), followed by 20 min cooling without changing the buffer. Digestion was performed with 0.001% (w/v) trypsin for 10 min or 0.01% (w/v) trypsin for 5 min (lyophilized porcine pancreas trypsin; Sigma) at 37°C. Only microwave pretreatment was applied to mouse liver. After antigen retrieval, sections were washed 2×5 min in TBS (0.05 M Tris–HCl, 0.15 M NaCl) and incubated in 3% hydrogen peroxide dissolved in methanol for blocking endogenous peroxidase. Sections were washed 3×10 min in TBS and then blocked with TNB blocking buffer, supplied in the Tyramide Signal Amplification TSA™-Indirect kit (NEN, Boston, MA, USA) for 1 h at room temperature. After 2×5 min washing steps using TBS, sections were treated overnight with the primary antibodies diluted in TNB solution containing 0.05% Tween. Dilutions of the primary antibodies were 1:200 for mouse and 1:500 for mullet. Samples were then washed 2×5 min with TBS and incubated with the anti-rabbit biotinylated secondary antibody for 1 h and additionally with peroxidase-coupled extravidin (Sigma), according to the manufacturer’s instructions. Staining reaction was performed using a NovaRed kit (Vector, Burlingame, CA, USA).

Controls consisted of sections that were incubated in blocking solution instead of the primary antibody solution.

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Addition of Rinsed Out Egg Activation

Many molecules and biochemical cascades that mediate sperm–egg interactions and egg activation have been discovered, but much still remains to be understood. Studies in the past were often focused on the identification of specific molecules such as egg and sperm receptors and egg activation and transduction pathways, but relatively few studies dealt with more general factors such as charge and pH that may affect these processes.

Histochemical studies on cells and tissues usually involve the use of dyes and fixed non-living material, but we have developed other histochemical approaches that identify cellular properties using derivatized agarose beads and various compounds to probe the biochemical components of fertilization and egg activation processes in living systems. The present study is the third presented in this journal that examines fertilization and egg activation in a model living system using free sugars and amino acids as probes instead of derivatized beads or dyes.

In previous studies, we used derivatized agarose beads as a model non-living system to show that charge–charge bonding may be involved in controlling cellular adhesive interactions. We followed up these studies examining sperm–egg interactions in the sea urchin model to show that negatively charged phosphorylated amino acids and sugars block sea urchin fertilization, whereas uncharged molecules had no inhibitory effect. These results were intriguing, suggesting that charge–charge interactions may be involved in sperm–egg binding.

Here we expand these studies by examining the effects of positively charged, negatively charged and uncharged molecules on fertilization and egg activation. In the early experiments on negatively charged molecules, no attempt was made to determine whether charged molecules affect sperm, eggs or both, whether their effect was due to a change in sea water pH, or whether the effects are due to chelation of positive ions such as Ca²⁺ or Zn²⁺.

A resolution of these questions should help to determine how charged molecules are involved. Answers to these questions may also lead to a better understanding of sperm–egg interactions and egg activation. In addition, this information may lead to a new approach in the development of contraceptives. In the present studies, we explore these questions by:

(1)examining the effects of positively charged, negatively charged and uncharged molecules on fertilization and egg activation;

(2)performing rinse-out experiments that remove charged molecules from eggs before the addition of sperm or addition of an artificial egg activator;

(3)determine whether the pH of sea water is affected by the added molecules;

(4)addition of Ca²⁺ or Zn²⁺ to examine the chelation issue, that is also examined in (2) above.

The results show that sperm and eggs are exquisitely sensitive to charged molecules. In at least one case, it is shown that a charged molecule with the same structural formula as another, but that is a ketone instead of an aldehyde, may reveal new information on species-specific components involved in the fertilization process.

Lytechinus pictus sea urchins were purchased from Marinus Inc. (Long Beach, CA, USA). Gametes were obtained and treated as previously described. The fertilization was performed in some experiments after the addition of specific sugars or amino acids as described below as. All reagents were obtained from Sigma (St. Louis, MO, USA). Experiments were run in triplicate by at least two investigators and the results were recorded as percentages of eggs displaying fertilization membranes 10 min after the addition of sperm or ionophores with standard error of the mean or standard deviation (pH readings were recorded as means±standard deviation). In some cases, fertilization and/or activation was monitored by establishing whether eggs were cleaved at 1.5–2 h after fertilization.

For pH-adjustment experiments, the pH of each solution consisting of artificial sea water (ASW) was adjusted with Trizma base (Sigma) or HCl to be as close to pH 8.0 as possible. The pH was checked at the start and end of all experiments. Suspensions of eggs in 100 ?l of pH-unadjusted solutions, pH-adjusted solutions or ASW controls were placed in three separate droplets on a microscope slide. Approx 1 ?l of undiluted sperm was added to each drop with the blunt end of a flat toothpick.

Calcium ionophore A23187 and other reagents were obtained from Sigma. The calcium ionophore (5 mg) was dissolved in 9.55 ml dimethylsulfoxide (DMSO). This stock solution of 10 mM was stored in 100 ?l aliquots in small foil-wrapped tubes and these aliquots were further diluted with 50 ml DMSO and stored in 2 ml aliquots (2 ?M ionophore concentration) in foil-wrapped tubes at ?20°C. Final stock solutions (40 ?l) were used in experiments and this concentration of DMSO alone had no effect on egg activation. For the non-rinse-out experiments, 465 ?l of egg suspension and 500 ?l of either ASW or test compound in ASW and 40 ?l of ionophore were mixed. For the rinse-out experiments, 495 ?l of egg suspension and 500 ?l of either ASW or test compound in ASW were mixed. Eggs were allowed to settle and rinsed 3 times with ASW, pH 8, followed by addition of 40 ?l ionophore.

For the rinse-out fertilization experiments, eggs were washed 3 times in ASW, pH 8.0, and then incubated in 1 ml aliquots of solutions to be tested for 10 min. Eggs were allowed to settle and the supernatant was removed and replaced with ASW, pH 8.0. Eggs were allowed to settle and the supernatant was removed and replaced with ASW once again. Diluted sperm (5 ?l of 10-times diluted sperm in ASW) was added to each sample. Sperm motility was observed and fertilization was evaluated by the presence of fertilization membranes and egg cleavage at 1.5–2.0 h afterwards. The non-rinse-out samples were handled as above except that the original solutions were never rinsed out.

Fig. 1 shows that the positively charged amino acid -arginine (50 mM) inhibited fertilization whether or not it was rinsed out from eggs prior to the addition of sperm. Similar results were obtained with negatively charged o-phospho- -serine, o-phospho- -threonine, -glucosamine-6-phosphate, and -ribose-1-phosphate, whereas other negatively charged molecules ( -mannose-1-phosphate, -glucose-6-phosphate, -glucose-1-phosphate, -glucose-6-sulfate and -maltose-1-phosphate) were only inhibitory when not rinsed out before the addition of sperm. Fructose-1-phosphate, however, was never inhibitory even in the non-rinsed-out samples. Uncharged molecules ( -threonine, -serine, -valine, -mannose, -glucose, -maltose, -fructose, and -ribose) had no effect. -glucosamine and -glucosamine-6-sulfate inhibition was partially restored when rinsed out.

Most of the molecules that were inhibitory at 50 mM were not inhibitory at 5 mM, except for o-phospho- -threonine and o-phospho- -serine that were still inhibitory at 5 mM whether or not these negatively charged molecules were rinsed out prior to the addition of sperm. -arginine and -ribose-1-phosphate inhibition at 5 mM was partially restored when rinsed out prior to the addition of sperm.

Table 1 shows that sperm cells were motile in sugar-phosphate samples whether or not the solutions were rinsed out prior to the addition of sperm, except for -glucosamine-6-phosphate. In the presence of 50 mM o-phospho- -serine and o-phospho- -threonine, sperm cells were not motile in the rinsed-out or non-rinsed-out samples, whereas in the presence of 5 mM, sperm cells were motile in the rinsed-out samples but not in the non-rinsed-out samples. -arginine affected sperm motility only occasionally.

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Positive Cells Gastrointestinal Tract were Spindle Shaped

The grass lizard, Takydromus wolteri Fischer, belonging to the Laceridae in the order of Lacertinae, lives in eastern part of Russia, China and Korea and is considered to be a primitive palearctic lacertid, most closely related to Lacerta vivipara. They have a distinct white ventrolateral band running from the tip of the snot to the groin. In Korea, numbers and habitats of this grass lizard have dramatically decreased because of pollution and immigration of foreign species of amphibians and reptiles having similar feeding habits.

Gastrointestinal endocrine cells are dispersed in epithelia and glands of the gastrointestinal tract and synthesize various types of hormones and play an important role in physiological functions of the gastrointestinal tract. The study of gastrointestinal endocrine cells is considered to be important for phylogenetic purpose. In addition, distribution patterns and the relative frequency of these endocrine cells vary with animal species and feeding habits. Although many studies have elucidated distribution patterns and the relative frequency of different types of endocrine cells in the gastrointestinal tract of various vertebrates, immunohistochemical studies on reptilia have received little attention until now. Studies have been performed on reptile species because their phylogenetic tree is found between amphibians and mammals and reports show characteristic distribution patterns and a relative frequency of gastrointestinal endocrine cells and have been compared with those of other vertebrates. Many studies have dealt with the identification of regulatory peptides in the gastrointestinal tract in reptile species using silver techniques, radioimmunochemical or immunohistochemical methods. Distribution patterns and relative frequency of these endocrine cells in reptile species varied with species and feeding habits. For example, different from amphibians and mammals, cells positive for insulin have been demonstrated in intestinal parts in reptiles. However, gastrointestinal endocrine cells have not been studied yet in gastrointestinal tract of the grass lizard, Takydromus wolteri Fischer. Therefore, we monitored these cells in the grass lizard using specific immunohistochemical methods and different antibodies raised against bovine Sp-1/chromogranin (BCG), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon, insulin, human pancreatic polypeptide (HPP) and secretin, and the results were compared with those obtained in other reptile species.

Five adult grass lizards (45–50 mm in length) of the Laceridae, Takydromus wolteri Fischer, were captured around Kyungpook, Korea. Both males and females were used in our study. After phlebotomy from the head, samples of six parts of the gastrointestinal tract (from proximal to distal: esophagus, fundus, pylorus, duodenum, small and large intestine) were fixed in Bouin’s solution, according to. After paraffin embedding, serial sections (3–4 ?m thick) were prepared. Sections were deparaffinized, rehydrated and stained with hematoxylin and eosin for light microscopic examination of the normal alimentary architecture. Other sections were used for immunostaining using the peroxidase anti-peroxidase (PAP) method. Blocking of nonspecific peroxidase reactions was performed with normal goat serum prior to incubation with the specific antibodies. After rinsing in phosphate buffered saline (PBS; 0.01 M, pH 7.4), sections were incubated with secondary antibodies (goat anti-rabbit IgG or goat anti-guinea pig IgG, dilution, 1:200; Sigma, St. Louis, MO, USA). Sections were then washed in PBS buffer and finally incubated with PAP complex (dilution, 1:200; Sigma). The peroxidase reaction was carried out using a solution 3,3?-diaminobenzidine tetrahydrochloride containing 0.01% H₂O₂ in Tris-HCl buffer (0.05 M, pH 7.6). After immunostaining, sections were analysed with the use of a light microscope.

Specificity of the immunohistochemical staining methods was determined as recommended by, including preincubation of the antibodies with their corresponding antigens. The relative frequency of each type of endocrine cell was scored using 5 categories: (?), not present; (±), rare occurrence; (+), only a few cells present; (++), moderate amounts of cells present; (+++), numerous amounts of cells present.

Nine types of endocrine cells were detected with the antibodies against BCG, serotonin, somatostatin, gastrin, CCK-8, glucagon, insulin, HPP and secretin. The distribution patterns and relative frequencies of these endocrine cells in the gastrointestinal tract of the grass lizard are shown in Table 2. In addition, most endocrine cells were spindle shaped (open cell type), whereas cells that were spherical in shape (closed cell type) were occasionally found in stomach and large intestine.

BCG-positive cells were detected throughout the whole gastrointestinal tract including esophagus and most predominant frequencies were detected in the stomach. Cells stained for BCG showing a spherical to spindle shape were dispersed throughout the esophageal mucosa and especially in esophageal glands. In addition, polymorphic cells were demonstrated in epithelia and gastric glands of the stomach. Spindle-shaped BCG-positive cells having relatively short cytoplasmic processes were detected in epithelia of the small intestine. Spherical-shaped BCG-positive cells were observed in the large intestinal mucosa and appeared to be crowded in some regions.

Serotonin-positive cells were found in various relative frequencies throughout the entire gastrointestinal tract including the esophagus and showed the highest frequency in the pylorus. They were the most predominant cell types found in this study. Spindle-shaped cells having relatively short cytoplasmic process were dispersed throughout esophageal mucosa but they were not detected in other gastrointestinal epithelia. Cells showing a polymorphic shape were demonstrated in regions of gastric glands. Spindle-shaped cells having relatively long cytoplasmic processes ending in the lumen were detected in epithelia of small intestine but spherical-shaped cells were found in the large intestinal mucosa.

Cells stained for somatostatin were demonstrated throughout the entire gastrointestinal tract except for esophagus and large intestine and were most predominant in pylorus and duodenum. Spherical-shaped cells were located in regions of gastric glands but spindle-shaped somatostatin-positive cells having relatively long cytoplasmic processes were detected in epithelia of small intestine.

Gastrin-positive cells were restricted to pylorus and duodenum and they showed spherical to spindle-shaped morphology in regions of gastric glands in the pylorus, but they were not found in epithelia of the pylorus. Spindle-shaped gastrin-positive cells having relatively long cytoplasmic process were detected in epithelia of the duodenum.

Figure 4. Gastrin- and CCK-8-positive cells in the gastrointestinal tract of the grass lizard, Takydromus wolteri. Note that gastrin-positive cells are restricted to pylorus (a) and duodenum (b), and CCK-8-positive cells to pylorus (c, d), duodenum (e) and small intestine (f; arrowhead). Scale bar, 33 ?m.

CCK-8-positive cells were observed from pylorus to small intestine and showed the highest frequency in pylorus. Spherical to spindle-shaped cells were located in regions of gastric glands but they were not found in other epithelia. Spindle-shaped cells with cytoplasmic processes were detected in epithelia of small intestine.

Glucagon-positive cells were restricted to duodenum and small intestine. Spindle-shaped cells were detected in the epithelia of small intestine. These cells were crowded in some parts of the duodenum.

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