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a University of Notre Dame, Department of Biological Sciences, Notre Dame, Indiana 46556-0369
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TGF-), serine proteases, corticosteroids, bacterial lipopolysaccharide, and the nitric oxide generator SNAP ([±]-S-nitroso-N-acetylpenicillamine). Overall, while several compounds caused significant effects, none were able to reproduce the increase in TOP RNA and protein that occurs in vivo, suggesting that the natural mediator of TOPs may still be untested, or that a combination of mediators may be involved. Finally, coelomic fluid inhibited the growth of the Gram negative bacterium, P. aeruginosa, and this inhibition was lost following immunoprecipitation of TOPs. This suggests that one function of TOPs may be to protect ovulated eggs from bacterial infection.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The amino acid sequence of TOPs is homologous to that of mammalian secretory leukocyte proteinase inhibitor (SLPI) [2], a serine protease inhibitor [3, 4]. Previous studies have demonstrated that brook trout coelomic fluid inhibits the activity of three serine proteases (trypsin, chymotrypsin, and pancreatic elastase) and that a significant portion of this inhibitory capacity is a result of the TOPs present in the coelomic fluid [5]. Along with its actions as a protease inhibitor, SLPI exhibits antibacterial activity [6,7]. In order to learn more about the possible biological function of TOPs, the ability of coelomic fluid to inhibit bacterial growth and the participation of TOPs in this inhibition were investigated.
Finally, while TOPs increase dramatically at the time of ovulation, factors affecting the regulation of their mRNA and protein are unknown. Considering that TOPs may be similar in structure and function to SLPI, the effects of several compounds that have proven to regulate SLPI were investigated for their ability to regulate TOP RNA and protein levels. Like TOPs, SLPI is induced during specific stages of development or under certain physiological conditions. For example, in the porcine uterus, SLPI increases during middle to late pregnancy [8], and inflammatory conditions increase SLPI expression in the lungs [9]. The up-regulation of SLPI during lung inflammation may be initiated by the release of elastase from infiltrating neutrophils [10, 11]. Significant up-regulation of SLPI mRNA also occurs in the mouse lung within 10 h of nasal inoculation with Streptococcus pneumoniae [12]. Thus, SLPI is up-regulated by mediators that are present during inflammatory events or bacterial infections. Given that SLPI is up-regulated during inflammation and in response to bacteria, several inflammatory mediators and bacterial compounds were tested for their ability to regulate TOP RNA and protein. In addition, since TOPs are highly up-regulated during ovulation, the effects of several relevant ovarian hormones and ovulatory mediators were investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Mature brook trout (300–400 g) were purchased during the reproductive season from a commercial hatchery (Homestead Trout Farm; Grand Haven, MI) and held under natural photoperiods in 1100-L tanks supplied with flow-through well water at 12°C.
Analysis of TOPs in Relation to Reproductive Stage
Collection of ovarian tissue and coelomic fluid The reproductive stage of individual trout was determined as previously described [13]. Ovarian tissue was collected from females at four reproductive stages: 1) 3 wk prior to GVBD; 2) 12 h after GVBD; 3) 24 h after GVBD, but prior to ovulation; and 4) 12 h postovulation. Prior to ovulation, there is a small amount of fluid in the peritoneal cavity. However, after ovulation a significant quantity of fluid accumulates around the ovulated eggs in the peritoneal cavity, and this fluid is referred to as coelomic fluid. Coelomic fluid was collected at five stages: 1) prior to GVBD; 2) 12 h after GVBD; 3) 24 h after GVBD, but prior to ovulation; 4) at ovulation; and 5) 12 h postovulation.
To collect ovarian tissue, trout were overanesthetized in 2-phenoxyethanol and decapitated. The ovaries were quickly removed and deyolked (pre-GVBD and ovulating ovaries) by pressing them between two stainless steel screens and continuously applying ice-cold Cortland medium [14] with a squirt bottle. An aliquot of the tissue was immediately processed for RNA isolation (see below), and the remaining tissue was stored in liquid nitrogen for subsequent Western analysis. In addition, ovaries from three fish at 12 h postovulation were processed for in situ hybridization, immunocytochemistry, and histological staining (see below).
Coelomic fluid is produced in abundance only after ovulation. Therefore, to collect coelomic fluid from preovulatory fish, strips of filter paper were briefly laid against the inner peritoneal cavity wall prior to ovarian tissue collection. The fluid-soaked filter paper was placed in a 0.6-ml microcentrifuge tube that had been previously punctured with a hypodermic needle to create a small hole at the bottom. The 0.6-ml tube was then placed in a 2-ml microcentrifuge tube and centrifuged at 12 000 x g for 10 min to collect the coelomic fluid from the filter paper. Coelomic fluid from postovulatory fish was collected from lightly anesthetized trout by stripping the eggs and fluid onto a stainless steel screen placed over a large beaker. Fluid was collected from the beaker and spun at 2000 x g for 15 min to remove red blood cells and other debris.
Western analysis of ovarian tissue and coelomic fluid A slice of partially thawed ovarian tissue was weighed and placed in 5 µl cold protein extraction buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 10 mM benzamidine) per milligram of tissue. To release protein, tissues were homogenized (Tissue Tearor; Biospec, Bartlesville, OK) and sonicated (Heat Systems-Ultrasonics, Farmingdale, NY) on ice 3 times with 10-sec, 50-watt bursts. Insoluble material was removed by centrifugation at 12 000 x g for 10 min. The supernatant was removed and assayed for protein concentration using the BCA protein assay (Pierce, Rockford, IL). The protein content of coelomic fluid was also determined using this assay.
For Western analysis, 10 µg of total protein from ovarian tissue or coelomic fluid was run on a 10% reducing SDS-PAGE gel. After electrophoresis, proteins were transferred to Westran-polyvinylidene fluoride (PVDF) membranes (Schleicher and Schuell, Keene, NH) using a Trans-Blot SD Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA) for 30 min at 15 V. Membranes were subsequently placed in blocking buffer (5% nonfat dried milk in TBS-T; 20 mM Tris base, pH 7.6, 137 mM NaCl, 0.1% Tween 20) overnight at 4°C. Western analysis was performed using the ECL+ Western Blotting Detection System (Amersham Pharmacia Biotech, Piscataway, NJ), and all subsequent steps were performed at room temperature. Briefly, membranes were incubated with a primary MBP-TOP-2 antibody (1:1000) [2] for 1 h. Membranes were rinsed for 15 min in TBS-T, followed by three 5-min washes. Membranes were incubated for 1 h in anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Pierce) at a 1:10 000 dilution, and the blots were again rinsed with wash buffer for 15 min, followed by three 5-min washes. The ECL+ detection solution mixture consists of a Lumigen PS-3 acridan substrate that, in the presence of HRP and peroxide, generates acridinium ester intermediates that can be detected by means of chemiluminescence. This detection solution was incubated with the membrane for 5 min, and the membrane was wrapped in plastic wrap and placed directly on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) for visualization and quantification of immunogenic bands.
Because of the large number of samples assayed, numerous Western blots were performed. This required a standard protein preparation that could be immunologically detected on each blot and used to normalize densitometric measurements between blots. This normalization was performed using a smaller recombinant TOP protein (TOP-1) that had been previously cloned as a fusion protein containing maltose-binding protein (MBP) [2]. A 1.5-µg aliquot of a Factor Xa-digested MBP-TOP-1 fusion protein preparation was run on each Western for normalization. The result of the proteolysis was a 21-kDa immunogenic band corresponding to TOP-1 that could be completely resolved from MBP and was used for normalization on all Western blots as previously described [2].
Densitometry and analysis of immunogenic bands Densitometry of immunogenic bands was performed using ImageQuant software (Molecular Dynamics). At each reproductive stage, protein samples were taken from 4–8 individual trout and analyzed by Western blotting (described above). Sample band intensities were standardized to the 21-kDa TOP-1 band by dividing the optical density (OD) of the sample band by the OD of the TOP-1 band. Means of relative intensity were calculated by reproductive stage for each immunogenic band. Differences for each individual band across reproductive stages was determined by examining the relative intensities for each band by ANOVA followed by Tukey's HSD (honestly significant difference). Significant differences among bands in a single stage were analyzed by t-test (ovarian tissue) or ANOVA followed by Tukey's HSD (coelomic fluid). Statistical significance was defined as P < 0.05.
Localization of TOPs in the Ovary
Tissue preparation Ovarian tissues taken at 12 h postovulation were fixed for 4 h in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7), then rinsed in tap water overnight and held in 50% ethanol. Dehydration (increasing ethanol), clearing (toluene), and paraffin infiltration were performed in a Citadel 2000 Tissue Processor (Shandon, Pittsburgh, PA). Tissues were embedded in plastic molds in paraffin using a Histoembedder (Leica, Wetzlar, Germany). Tissue sections (7 µm) were cut and placed on Fisherbrand Superfrost microscope slides (Fisher, Pittsburgh, PA).
In situ hybridization The TOP-1 cDNA was labeled as a probe using the BioPrime DNA Labeling System (GibcoBRL, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, 100 ng of TOP-1 cDNA was boiled for 5 min with 20 µl of a 2.5-strength random primer solution and then chilled on ice. A 5-µl aliquot of 10-strength dNTP (1 mM biotin dCTP, 1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP) was added to this mixture, and the total volume was brought to 49 µl with distilled (d)H2O. The mixture was incubated at 37°C for 1 h with 1 µl of Klenow fragment; then 5 µl of stop buffer was added. The mixture was precipitated with ethanol and 3 M sodium acetate (pH 5.5) at -70°C for 15 min and pelleted by centrifugation at 12 000 x g for 10 min at 4°C. The pellet was dried 5 min and resuspended in 50 µl dH2O. The precipitation and resuspension steps were repeated once.
In situ hybridization was performed using an in situ hybridization detection system (Dako, Carpinteria, CA). Unless indicated, all steps were performed at room temperature. Slides were first treated sequentially as follows: 2 times, 5 min each, in toluene; 2 times, 5 min each, in 100% ethanol; 3 min in 95% ethanol, 3 min in 80% ethanol, 3 min in DEPC-treated dH2O, and 3 min in PBS (140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4 in DEPC-treated dH2O, pH 7.4) + 0.01% DEPC. Slides were incubated in proteinase K (40 mg/100 ml) for 7 min, rinsed in PBS, incubated in 4% paraformaldehyde (in PBS) for 5 min, and then rinsed three times in PBS. Using the biotin blocking system (Dako) to block endogenous avidin and biotin activity, the sections were incubated for 10 min in avidin, rinsed twice in PBS, incubated 10 min in biotin, and again rinsed twice in PBS. Sections were prehybridized for 2 h at 43°C in a prehybridization buffer containing 86 µl double-strength hybridization buffer (4-strength SSC [single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate], 0.2 M sodium phosphate, pH 6.5, double-strength Denhardt's solution, 0.1 mg/ml sodium azide), 4.6 µl calf thymus DNA, and 106 µl 20% dextran sulfate in formamide previously boiled for 10 min and held on ice. Sections were hybridized overnight at 43°C in hybridization buffer (prehybridization buffer) containing the TOP-1 probe and tRNA.
The second day, slides were washed in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) for 2 min and again in TBS for 5 min. Two additional washes were performed in 0.2-strength SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.0) at 43°C for 15 min each. To detect TOP RNA, sections were incubated for 30 min in streptavidin, washed for 5 min in TBS, incubated for 30 min in biotinylated alkaline phosphatase, and washed in levamisole (10 mg/50 ml TBS). Diluted substrate solution was added to each section and allowed to develop overnight. Slides were then washed in dH2O for 5 min and dehydrated. Coverslips were mounted using Permount (Fisher).
Immunocytochemistry Immunocytochemistry was performed using the ABC Staining System (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's instructions. All steps were carried out at room temperature. Slides were first placed sequentially in each of the following treatments: 3 times, 5 min each, toluene; 2 times, 10 min each, 100% ethanol; and 2 times, 10 min each, 95% ethanol. Sections were then washed in dH2O for 5 min. Sections were incubated for 5 min in 0.1% hydrogen peroxide to quench endogenous peroxidase activity, then washed in PBS two times, 5 min each. Sections were blocked for 1 h in blocking serum (1.5% in PBS). Primary antibody incubation was performed for 30 min using the MBP-TOP-2 antibody diluted 1:200 in blocking serum, then washed 3 times, 5 min each, in PBS. Sections were then incubated for 30 min in biotinylated, anti-rabbit secondary antibody diluted at 1.0 µg/ml in blocking serum; they were then washed 3 times, 5 min each, in PBS. TOP protein was visualized by adding 3 drops of peroxidase substrate to the sections for 10 min, then washing 5 min in dH2O. Slides were then dehydrated, and coverslips were mounted using Permount.
Hematoxylin and eosin staining Adjacent sections were cleared in toluene, hydrated in decreasing ethanoic solutions, and stained in hematoxylin (Gill's formulation stain #2; Fisher) and counterstained in eosin (Fisher). Sections were rapidly dehydrated in ethanol, cleared in toluene, and mounted using Permount.
Effects of Coelomic Fluid on Bacterial Growth
Preparation of coelomic fluid Coelomic fluid from 4 individual trout was concentrated 10-fold using Centriprep-10 centrifugal concentrators (Amicon, Beverly, MA). A 200-µl aliquot of the 10-strength coelomic fluid from each fish was boiled for 10 min, held on ice for 1 min, then centrifuged 12 000 x g for 10 min. The supernatant was removed and saved on ice. Immunoprecipitation of TOPs from coelomic fluid was performed as previously described [5]. Aliquots of coelomic fluid from 4 individual female brook trout were mixed separately in a 1:4 ratio (total protein) with a TOP antibody overnight at 4°C. Protein A-coated agarose beads (Pierce) were added to the fluid-antibody mixture at a ratio of 2 ml beads:17.81 mg antibody and mixed for 2 h at room temperature. The mixture was centrifuged at 12 000 x g for 10 min at 4°C. The supernatant was removed and analyzed by Western blotting for TOPs. The immunoprecipitation procedure was repeated until TOPs were no longer detectable on Western blots. Prior to use, immunoprecipitated fluid from the 4 trout was concentrated to a volume of 20 µl using Centricon-10 concentrators (Amicon).
Antibacterial assay Bacterial cultures (Baccilus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphlococcus aureus, S. faecalis) were maintained on NZY agar plates (5 g NaCl, 2 g MgSO4·7H2O, 5 g yeast extract, 10 g casein enzymatic hydrolysate, 20 g bacto-agar in 1.0 L dH2O, pH 7). To produce a stock culture, 20 ml of LB medium (10 g NaCl, 10 g bactotryptone, 5 g yeast extract in 1.0 L dH2O, pH 7) was inoculated with each bacterium and allowed to grow at 37°C and 300 rpm for 6 h. The culture was centrifuged for 10 min at 5000 rpm. The supernatant was discarded, and the cells were resuspended in 5 ml of 10 mM MgSO4. An aliquot of each stock culture was diluted to OD600 = 1.0 in MgSO4, and these diluted cultures were used for subsequent experiments.
Brook trout coelomic fluid was first tested for its specificity in inhibiting the growth of the five bacteria listed above. Top agar (5 g NaCl, 2 g MgSO4 x 7H2O, 5 g yeast extract, 10 g casein enzymatic hydrolysate, 7 g bacto-agar in 1.0 L dH2O, pH 7) was prepared by boiling in a microwave, cooling for 10 min at room temperature, then holding in a 50°C water bath. The diluted bacterial cultures described above were added to the top agar in a ratio of 5 µl diluted culture/1 ml top agar, mixed by inversion, and poured evenly over an NZY agar plate. The top agar was allowed to set for 10 min. Treatments were applied to the plate by placing 1/4 inch blank paper disks (BBL; Becton Dickinson, Cockeysville, MD) onto the top agar, then pipetting 20 µl of each treatment onto a disk. Disk treatments were as follows: erythromycin (15 µg; Sensi-Disc; Becton Dickinson) for B. cereus, P. aeruginosa, S. aureus, and S. faecalis; kanamycin (30 µg) for E. coli, 10-strength coelomic fluid from each of the 4 trout, boiled 10-strength coelomic fluid from each of the 4 trout, 10% BSA in PBS (pH 8.4; pH of trout coelomic fluid), and a blank treatment disk.
After a general survey of the five bacteria listed above, the antibacterial capacity of various coelomic fluid preparations against the growth of P. aeruginosa was further assessed on NZY agar plates. For this experiment, top agar containing P. aeruginosa was added to four plates (one plate per trout) as described above. For a dose-dependent study, 10-strength coelomic fluid from 4 individual brook trout (see above) was diluted to 5-strength, 2.5-strength, and single-strength using PBS (pH 8.4). Disk treatments were as follows: erythromycin (15 µg) Sensi-Disc, 20 µl each of 10-strength, 5-strength, 2.5-strength, single-strength coelomic fluid; 20 µl of boiled 10-strength coelomic fluid; 20 µl of concentrated TOP immunoprecipitated fluid; 20 µl of 10% BSA in PBS, 20 µl of PBS; and a blank disk. Plates were placed lid side up in a 37°C incubator and grown for 18 h. The plates were then scanned (ScanMaker III; Microtek, Redondo Beach, CA), and antibiotic activity was assessed by measuring the clear area surrounding each disk using ImageQuant (Molecular Dynamics).
Analysis of antibacterial activity To analyze the dose-dependent antibacterial data, the inhibited area surrounding each dilution treatment was standardized by dividing by the area surrounding the blank disk ("percent of negative control"), and means were calculated. To determine significant differences among the treatments, means were examined by ANOVA followed by Tukey's HSD.
To further analyze the effect of removing TOPs by immunoprecipitation, the degree of concentration of each of the 4 immunoprecipitated samples was calculated. First, a linear curve fit was made from a graph of the total amount of coelomic fluid protein versus the degree of concentration of coelomic fluid from each fish. Second, the degree of concentration was calculated for each immunoprecipitated sample using the equation for the coelomic fluid of each fish and the protein concentration of the corresponding immunoprecipitated sample. Confidence intervals of 95% were calculated for the dose-response curve generated by plotting the mean percent of negative control versus coelomic fluid dilution. The 95% confidence intervals were then used to assess the significance of the mean of the 4 immunoprecipitated samples relative to untreated coelomic fluid.
Regulation of TOP mRNA and Protein
In vitro incubations To collect ovarian tissue from females prior to GVBD, the fish were overanesthetized in 2-phenoxyethanol and then decapitated. Ovaries were quickly removed and placed in ice-cold Cortland medium [14]. The ovaries were dissected into groups of 5 intact follicles connected by extrafollicular tissue. A total of 10 follicles was placed in each well of a 12-well culture plate containing 3 ml Cortland medium supplemented with the appropriate treatment. Replicate treatments were made for RNA and protein isolation. The follicles were incubated at 12°C with periodic shaking. At time points between 1 and 24 h, follicle walls were dissected from the extrafollicular tissue and the oocytes. One replicate of follicles from each treatment was placed in 1 ml of Tri-reagent (Molecular Research Center, Inc., Cincinnati, OH), homogenized with a Tissue Tearor (Biospec) for 1 min and stored at -80°C until RNA isolation and analysis were performed as described below. The other replicate from each treatment was placed in a screw-cap microcentrifuge tube and stored at -80°C for analysis of TOP protein levels. Dissected follicle walls were also collected at 0 h for each fish.
Treatments were incubated in Cortland medium, and controls consisted of follicles incubated in Cortland medium supplemented with the treatment vehicle alone. The treatments performed in Cortland medium alone were LPS (lipopolysaccharide) from E. coli (10 µg/ml), P. aeruginosa (10 µg/ml), and S. minnesota (10 µg/ml), L- and D-NNA (NG-nitro-L-arginine; 4 µg/ml), L- and D-NAME (NG-L-arginine methyl ester, hydrochloride; 4 µg/ml), L- and D-NMMA (NG-monomethyl-L-arginine, monoacetate; 300 nM), L-arginine (4 µg/ml), 1,3-PBITU (S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea, 2HBr; 500 nM), conconavalin A (10 µg/ml), orthovanadate (100 nM), mammalian kallikrein (100 µM), cod trypsin (100 µM), salmon gonadotropin II (GTH; 200 ng/ml), IL (interleukin)-1ß (50 ng/ml), IL-6 (200 ng/ml), arachidonic acid (100 µg/ml), PGE2 (2 µg/ml), and PGF2 (2 µg/ml). The treatment performed in Cortland medium with dimethyl sulfoxide (1 µl/ml) was a combination of PMA (phorbol 12-myristate 13-acetate) and A23187 (0.05 µg/ml each). The treatment performed in Cortland medium with 10 mM acetic acid + 0.1% BSA (1 µl/ml) was TGF- (transforming growth factor-; 100 ng/ml). The treatments performed in Cortland medium with ethanol (1 µl/ml) were forskolin (10 mM), E2 (estradiol; 1 µg/ml), SNAP ([±]-S-nitroso-N-acetylpenicillamine; 100 µM), dexamethasone (2 µg/ml), and cortisol (0.5 µg/ml). All test compounds were obtained from either Sigma (St. Louis, MO) or Calbiochem (La Jolla, CA). Salmon GTH II was a gift from Dr. P. Swanson (National Marine Fisheries Service, Seattle, WA). Test concentrations were based on the results of past experiments performed in the lab with certain agents, or on the literature involving the effects of certain compounds on SLPI levels.
RNA analysis The RNA in the frozen Tri-reagent homogenate was isolated as previously described [15, 16]. Briefly, the homogenate was thawed and incubated at room temperature for 5 min prior to addition of 0.2 ml chloroform. Samples were vigorously shaken for 15 sec, incubated at room temperature for 10 min, and centrifuged at 12 000 x g for 15 min at 4°C. The aqueous phase was transferred to a new tube, and the RNA was precipitated by adding 0.5 ml isopropanol, incubating at room temperature for 10 min, and centrifuging at 12 000 x g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed with 75% ethanol in DEPC-treated water. The pellet was air dried for 10 min and resuspended in 10 µl of Formazol (Molecular Research Center). The RNA concentration was determined by spectrophotometric analysis at 260 nm.
TOP cDNA probes hybridize with 2–4 bands on Northern blots of RNA from brook trout ovarian tissue [1, 2], and initial studies indicated that the two largest bands were readily quantifiable on Northern blots of RNA isolated from in vitro follicle incubations. Although the absolute levels of the two bands may differ, preliminary results indicated that changes measured in the expression levels of each individual band across time and within a treatment were similar. Therefore, dot-blot analysis was chosen as a method to more efficiently quantify changes in total TOP RNA levels from many samples. Expression of TOP mRNA was analyzed using dot-blot hybridization as described by Simmen et al. [17]. Briefly, 1.0 µg of RNA in Formazol was resuspended in 250 µl loading buffer (6% Formazol, 50% formamide in 50 mM Tris, pH 7.0, using DEPC-treated water). This was heated to 65°C for 5 min, and 250 µl of 20-strength SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.2) was added. Magnacharge nylon membranes (MSI, Westborough, MA) were wetted in DEPC water and incubated in 10-strength SSC for 5 min. The 96-well dot-blot apparatus (Schleicher and Schuell, Keene, NH) was assembled according to the manufacturer's instructions using one piece of pre-wetted GB002 blotting paper (Schleicher and Schuell) under the nylon membrane. Each well was filtered sequentially with the following: 500 µl 10-strength SSC, 500 µl RNA sample, 500 µl 20-strength SSC. Filters were UV crosslinked (UV Stratalinker 1800; Stratagene, La Jolla, CA), air dried, and stored under vacuum until hybridization.
Membranes were prehybridized in 10 ml of hybridization buffer containing 5-strength SSPE (0.75 M NaCl, 0.05 M sodium phosphate monobasic, 5 mM EDTA, pH 7.4), 0.1% SDS, 5-strength Denhardt's solution, 50% formamide, and 150 µg/ml sonicated calf thymus DNA for 2–4 h at 42°C in roller tubes. Membranes were hybridized overnight with a 32P-labeled DNA probe generated using the full-length brook trout TOP-2 cDNA, [-32P]dATP (3000 Ci/mmol; ICN, Irvine, CA) and the Prime-it-II kit (Stratagene). Labeled probes were separated from free label using Centri-Sep spin columns (Princeton Separations, Adelphia, NJ). Membranes were washed twice in medium-stringency buffer (single-strength SSPE, 0.1% SDS) at 45°C for 15 min with shaking and twice in high-stringency buffer (0.1-strength SSPE, 0.5% SDS) at 65°C with shaking. Membranes were exposed to phosphorimaging screens, and hybridization signals were detected and quantified using a Storm 840 PhosphorImager (Molecular Dynamics).
Protein Analysis
Ten partially thawed follicle walls were placed in 400 µl cold protein extraction buffer (50 mM Tris-HCl, 5 mM EDTA, 10 mM EGTA, 10 mM benzamidine, pH 7.5). Tissue was homogenized and sonicated on ice to release protein, and insoluble material was removed by centrifugation at 12 000 x g for 10 min. The supernatant was removed and assayed for protein content using the BCA protein assay (Pierce).
The MBP-TOP-2 polyclonal antibody detects 2–3 bands on Western blots of total protein from brook trout ovarian tissue [2], and initial studies indicated that the two largest bands were readily quantifiable on Western blots of total protein isolated from in vitro follicle incubations. Although absolute levels of the two bands may differ, the changes measured in the expression levels of each individual band across time and within a treatment were similar. Therefore dot-blot analysis was chosen as a method to more efficiently quantify changes in total TOP protein levels from many samples. Briefly, 1.0 µg of protein was diluted in 500 µl TBS (20 mM Tris base, pH 7.6, 137 mM NaCl). Westran-PVDF membranes (Schleicher and Schuell) were pre-wetted in methanol and incubated in TBS for 5 min. The 96-well dot-blot apparatus (Schleicher and Schuell) was assembled according to the manufacturer's instructions using one piece of GB002 blotting paper (Schleicher and Schuell), pre-wetted in TBS, under the membrane. Each well was filtered sequentially with the following: 500 µl TBS, 500-µl protein sample, 500 µl TBS. Membranes were subsequently placed in blocking buffer (5% nonfat dried milk in TBS with 0.1% Tween 20) overnight at 4°C. Western analysis was performed using the ECL+ Western Blotting Detection System as described above (Western Analysis).
Statistical analysis of treated samples The RNA and protein from follicles incubated in Cortland medium alone were used to assess the level of TOP transcription and translation in vitro in the absence of any treatments. Individual values were normalized at each incubation time by dividing by the 0-h control. Student's t-test was performed to determine statistical significance in relation to the 0-h control at P < 0.05.
For each treatment replicate, hybridization levels of RNA or protein from treated follicles (E) were normalized to corresponding (e.g., with/without vehicle) control levels (C) by the following calculation: "percent of control" = (E/C) x 100%. After means were calculated for experiments on 3–6 fish per treatment, percentages of control values for each treatment were analyzed by Student's one-sample t-test with statistical significance defined as P < 0.05. Means discussed in the Results section are presented as the "percent difference from control" (percent of control - 100) ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blots of coelomic fluid consistently contained three immunoreactive proteins of 53, 39, and 29 kDa. A 24-kDa protein was also observed in some individuals. Figure 1A shows a Western blot of coelomic fluid taken from a representative fish at each of the nine stages (lanes 1–5: the present study; lanes 6–9: [2]). Levels of all immunogenic proteins significantly increased at ovulation (Fig. 1B). However, prior to ovulation, they were barely detectable (Fig. 1B). Levels of each protein were significantly elevated at one or more postovulatory stages. For example, levels of the 53-kDa protein significantly increased at 48 h and 4 days postovulation. Levels of the 39-kDa protein significantly increased at 24 and 48 h postovulation. Levels of the 29-kDa protein significantly increased at 48 h postovulation. By 8 days postovulation, levels of all immunogenic proteins had decreased and were not significantly different from ovulatory levels. Because of low levels, statistical analysis of the 24-kDa TOP in the coelomic fluid was not performed.
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ANOVA was performed to determine significant differences among the 53-, 39-, and 29-kDa protein at each stage. Levels of the 29-kDa protein were significantly lower than levels of both the 53- and 39-kDa protein at 12 and 24 h postovulation, significantly lower than levels of the 39-kDa protein at 48 h postovulation, and significantly lower than levels of the 53-kDa protein at 4 days postovulation.
Expression of TOP-2 Immunogenic Proteins in Ovarian Tissue
The results obtained in the present study on ovarian TOP immunogenic proteins were evaluated with those obtained previously from studies using ovaries obtained at other reproductive stages [2]. Thus, the data in Figure 2 represent a compilation of the two studies. Western blots of ovarian tissue consistently contained two immunoreactive proteins with molecular masses of approximately 53 and 39 kDa, while samples from some individuals also contained a 29-kDa protein band (data not shown). Figure 2 represents the mean relative OD of the 53- and 39-kDa protein from 4–8 trout at each of the 10 reproductive stages (3 wk preGVBD, 12 and 24 h post-GVBD, 12 h postov: the present study; the remaining stages: [2]). In all stages prior to 24 h postovulation, the 53- and 39-kDa proteins displayed a similar pattern of expression, and the levels of each protein were not significantly different from their levels at ovulation (Fig. 2). In contrast, at 24 and 48 h postovulation, levels of the 53-kDa protein significantly increased and then returned to ovulatory levels at 4 and 8 days postovulation. Levels of the 39-kDa protein were not significantly different from ovulatory levels until 4 and 8 days postovulation, when they significantly decreased. Because of low levels, statistical analysis of the 29-kDa protein in the ovarian tissue was not performed.
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t-Tests were performed to determine significant differences between the 53- and 39-kDa protein at each stage. At 24 h postGVBD, levels of the 39-kDa protein were significantly greater than levels of the 53-kDa protein (Fig. 1B). In contrast, at 24 and 48 h postovulation, levels of the 53-kDa protein were significantly greater than levels of the 39-kDa protein.
Cellular Localization of TOPs
From the results of Northern [2] and Western (present study) analysis, TOP RNA and protein expression was greatest in the postovulatory ovary; therefore ovarian tissue obtained at 12 h postovulation was used to localize TOP RNA and protein to specific cell layers in the ovary. Adjacent sections stained with hematoxylin and eosin clearly showed distinct granulosa and theca layers in postovulatory follicles (Fig. 3F). Prior to ovulation, the theca and granulosa layers were stretched to accommodate the large (~4.0 mm) oocyte. However, after ovulation the follicle shrank dramatically; and the layers, particularly the granulosa, appeared dense, compacted, and multilayered (Fig. 3F).
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In situ hybridization using TOP-1 as a probe showed dense staining in the granulosa of postovulatory follicles (Fig. 3A). Staining also appeared intermittently in the ovarian surface epithelium (Fig. 3E). No staining was observed when the TOP-1 probe was not added during the hybridization step (Fig. 3B).
Immunocytochemistry using the MBP-TOP-2 antibody also localized TOP protein to the granulosa cells of postovulatory follicles (Fig. 3C). Positive staining was also observed within the open center of the follicle and presumably indicates the presence of TOPs in a fluid secreted by the postovulatory follicle. No staining was observed in sections incubated without the MBP-TOP-2 antibody (Fig. 3D).
Antibacterial Activity of Coelomic Fluid and TOPs
In the initial survey experiment, brook trout coelomic fluid inhibited the growth of P. aeruginosa but had no effect on the growth of B. cereus, E. coli, S. aureus, or S. faecalis (results not shown). Therefore, further tests were conducted only with P. aeruginosa.
Coelomic fluid dose-dependently inhibited growth of P. aeruginosa as assessed by measuring the affected area surrounding each treatment disk (Fig. 4). Affected areas produced by 10-strength, 5-strength, and 2.5-strength coelomic fluid treatments were all significantly greater than for the blank disk control (Fig. 4). When expressed on the basis of the degree of concentration, the mean for TOP immunoprecipitated coelomic fluid samples fell outside the 95% confidence interval for the curve generated by the means of the concentrated unprecipitated coelomic fluid samples (Fig. 5). This indicated that TOP immunoprecipitated coelomic fluid possessed significantly less antibacterial activity than untreated coelomic fluid from the identical fish when the actual concentration of the fluid was taken into account.
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In Vitro Expression and Regulation of TOP RNA and Protein
In the absence of any treatment, TOP RNA increased significantly after 1 h of in vitro incubation and remained significantly elevated from the 0-h control level through 12 h (Fig. 6). TOP protein increased significantly after 3 h of incubation and remained significantly elevated from the 0-h control level through 12 h (Fig. 6). While the effects of many different agonists or inhibitors were investigated on TOP RNA and protein using the in vitro incubation system, very few had any significant effects. In addition, these effects were usually observed only at isolated incubation times.
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Several treatments, including signal transduction mediators and TGF-, increased TOP RNA and/or protein levels at specific time points. Of the second messenger agonists tested, orthovanadate significantly increased TOP RNA above control levels at 3 h (78 ± 21%) and 6 h (88 ± 28%), while the combination of PMA and A23187 significantly increased TOP protein above control levels at 6 h (18 ± 65%) and 24 h (316 ± 16%). TGF- significantly increased TOP RNA above control levels at 6 h (95 ± 38%), and above control protein levels at 3 h (32 ± 13%).
Several treatments decreased the expression of TOP RNA and/or protein at specific incubation periods. The corticosteroid, dexamethasone, significantly decreased TOP protein below control levels at 12 h (-30 ± 14%); and the serine proteases, cod trypsin and mammalian kallikrein, significantly decreased TOP RNA below control values at 12 h (-32 ± 5%) and 24 h (-45 ± 15%), respectively. TOPs may possess antibacterial activity; therefore the effects of bacterial cell wall components, such as LPS, were examined. LPS from P. aeruginosa significantly decreased TOP protein at 3 h (-18 ± 5%), while LPS from E. coli or S. minnesota had no significant effects on either TOP RNA or protein levels. Finally, the nitric oxide donor, SNAP, significantly increased TOP RNA levels above control levels at 12 h (26 ± 7%) but significantly decreased TOP protein (-45 ± 14%) at the same time period. Either the nitric oxide synthase inhibitors tested had no significant effect, or the effects of the active L-enantiomer were mirrored by that of the inactive D-enantiomer.
Treatments that failed to produce significant (P < 0.05) effects on either TOP protein or RNA levels included forskolin, concanavalin A, 17,20ß-P, E2, salmon GTH II, arachidonic acid, PGE2, PGF2, IL-1ß, IL-6, and L-arginine.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In situ hybridization and immunocytochemistry revealed dense staining for TOP RNA and protein, respectively, in the granulosa. In the postovulatory ovary, the granulosa layer appears compacted and encircles an open center that once held the oocyte. This area would be confluent with the peritoneal cavity through a hole in the follicle wall through which the oocyte was released at ovulation. Therefore, the granulosa cells are strategically placed to secrete TOPs into the coelomic fluid during and after ovulation. In fact, some sections that were stained for immunocytochemistry revealed a fluid in this space that was positive for TOPs. The protein expression pattern reveals that TOPs are not secreted in abundance prior to ovulation, and that at ovulation TOP secretion into the coelomic fluid dramatically increases. Therefore, although TOPs may play a role in the postovulatory ovary, their presence in the coelomic fluid may be of greater importance and strongly suggests a postovulatory role. TOPs are homologous to mammalian SLPI [18], a serine protease inhibitor [19] with antiprotease activity against leukocyte proteases such as elastase. TOPs can also act as serine protease inhibitors [5] and therefore may inhibit nonspecific degradation of the postovulatory ovary or ovulated eggs by proteases released at the time of ovulation.
In this study, we show that TOP RNA and protein levels increase in vitro without exogenous treatment. This is also the case for uterine and bronchial SLPI [20, 21] and SKALP (skin-derived antileukoproteinase), a structurally related antiprotease that is induced upon wounding of human skin [22]. Therefore it is possible that simply the dissection of the ovarian tissue was enough to induce expression of TOPs. Nonetheless, the spontaneous increase measured in vitro allowed for the determination of stimulatory or inhibitory influences on TOP RNA and protein levels by comparing treated follicles to control follicles.
Using an in vitro incubation system, the effects of a number of agents on TOP RNA and protein were investigated. While some compounds had significant effects in these assays, the effects were generally not large, and they occurred at varying incubation times. The precise interpretation at this point of increases and decreases in mRNA and protein levels is obviously dependent on the synthesis, degradation, and secretion of TOP mRNA and protein during the treatments. Given that TOPs appear to be produced by the granulosa, and since whole follicles (oocytes surrounded by an intact follicle wall) were used for in vitro assays, it would not be possible to determine whether secretion was occurring. However, whole follicles were used in these studies since the extensive dissection necessary to obtain follicle walls might have further increased the spontaneous production of TOPs, masking the effects of some treatments.
TOP RNA and protein levels increase dramatically up to the time of ovulation and peak within 24 h postovulation [2]. The levels of several gonadal hormones including 17,20ß-P, estradiol, gonadotropins, and eicosanoids also change at the time of ovulation in brook trout [23, 24]. The window of expression for TOPs suggests that they may be tightly regulated by one or more of these factors, and therefore their effects on TOPs were studied. Surprisingly, however, none of these factors had significant effects on TOP RNA or protein.
Second messenger agonists such as forskolin that increase cAMP levels had no significant effects on TOP RNA or protein. However, agonists affecting other signal transduction pathways had strong stimulatory effects on TOPs. In the present study, orthovanadate significantly increased TOP RNA at time points up to 6 h. While orthovanadate had no significant effect on TOP protein, the combination of PMA and A23187 very strongly stimulated TOP proteins by 24 h. Similarly, SLPI mRNA and protein are elevated in bronchial epithelial cells after a 24-h incubation with PMA [21]. PMA classically stimulates protein kinase C [25], which is involved in the stimulation of ovulation in fish [26, 27], while orthovanadate is a general G-protein activator that stimulates ovulation and the production of inositol phosphates in goldfish follicles [28]. At the very least, the combined results with signal transduction mediators suggest that TOPs may be regulated through a G protein-mediated pathway that does not involve cAMP but may involve the activation of protein kinase C.
SLPI inhibits several serine proteases that are released during an inflammatory response, including neutrophil elastase [18, 29], cathepsin G [18], and chymase [30]. In turn, SLPI can also be regulated by the proteases that it inhibits. For example, treatment of airway epithelial cells with neutrophil elastase or cathepsin G increases SLPI mRNA but decreases SLPI protein levels [10]. Recently, a kallikrein/complement factor D homologue that increases at the time of ovulation was characterized from the brook trout ovary [31]. Kallikrein activity increases in the rat ovary up to the time of follicle rupture [32, 33], and kallikrein is a member of the chymotrypsin family of serine proteases [34]. Further, the TOPs present in the coelomic fluid inhibit trypsin activity [5], and trypsin is also a serine protease. Thus, since SLPI appears to be regulated by proteases, mammalian kallikrein and cod trypsin were tested for their effects on TOP RNA and protein levels. Addition of cod trypsin and mammalian kallikrein significantly decreased TOP RNA at 12 or 24 h but had no significant effect on protein levels.
SLPI mRNA increases in uterine endometrium cell culture after 6 h of incubation with TGF- [20]; therefore this growth factor was tested for its effect on TOPs. TGF- significantly increased TOP RNA at 6 h and TOP protein at 3 h. TGF- has been characterized as a paracrine and autocrine mediator in the ovary (for review, see [35]). In humans [36] and in the hen [37], TGF- immunostaining increases in the growing follicle, and TGF-/epidermal growth factor receptor binding has been characterized in the goldfish ovary [38]. TGF- is synthesized by several cell types involved in inflammation, including activated macrophages [39]. The number of macrophages significantly increases at the time of ovulation in the ovine ovary and remains elevated for at least 12 h [40]. Therefore, TGF-, produced by ovarian cells and/or macrophages, may stimulate TOPs in the postovulatory ovary to control protease activity during tissue reconstruction.
Salmonids ovulate their eggs into the peritoneal cavity, where they may be held for an extended period of time prior to spawning. During this time, the eggs are bathed in the coelomic fluid. The peritoneal cavity of brook trout is effectively in contact with the external environment by way of the ovipore. Bacteria, viruses, and fungi could gain access to the coelomic fluid through the ovipore and pose a threat to egg viability. In the present study, coelomic fluid specifically inhibited growth of P. aeruginosa, while fluid from which TOPs had been immunoprecipitated did not. These results strongly suggest that TOPs possess antibacterial activity against P. aeruginosa, a Gram negative bacterium. Since TOPs did not inhibit the growth of other bacteria, it is clear that they are not generally antibacterial but that the effects may be related to a specific action of TOPs on P. aeruginosa. P. aeruginosa is an opportunistic bacterium that secretes two proteases, LasA and LasB, which in combination possess elastase activity [41]. LasA is a serine protease, while Las B is a zinc metalloprotease. Given the antitrypsin activity of TOPs, the action of TOPs on this particular bacterium may be related to the inhibition of the LasA serine protease. The inhibition of bacterial growth by TOPs is not unexpected, since SLPI has also been reported to block E. coli and S. aureus [6, 7].
In conclusion, TOPs are up-regulated in the ovulatory and postovulatory brook trout ovary [2], and they function as serine protease inhibitors [5]. They are synthesized by the granulosa cells and released into the coelomic fluid that bathes the eggs after ovulation. One action of TOPs in this fluid may be to block the nonspecific degradation of the ovary or other tissues that could arise from proteases released as hundreds of eggs are being ovulated. The results of the present study also suggest that TOPs can inhibit the growth of certain bacteria, namely P. aeruginosa. Therefore, TOPs may also protect eggs from invading microbes. The regulation of TOP mRNA and protein at ovulation is still unclear. The treatments chosen in this study to investigate regulation reflected mediators and agonists that are present during ovulation, proteolysis, and bacterial infection. Although several treatments produced significant effects on TOP RNA and protein levels, none appeared to increase TOPs to the levels measured in vivo. Therefore, it may be that other mediators or even a combination of mediators would be necessary to produce the dramatic increase in TOPs that occurs during ovulation. In addition, the fact that TOP RNA and protein increase during in vitro incubations in the absence of any treatments suggests that TOPs may naturally have inhibitory regulators as well as stimulators.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by USDA grant #95-37203-1962; Enhancing Animal Reproductive Efficiency Program. 
2 Correspondence: Frederick William Goetz, University of Notre Dame, Department of Biological Sciences, P.O. Box 369, Notre Dame, IN 46556-0369. FAX: 219 631 7413; goetz.1{at}nd.edu
Accepted: November 12, 1999.
Received: September 20, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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