• 29
    Dec
  • Tight Junctions in Human Ovarian Surface Epithelium: MATERIAL AND METHODS

Patient Material

OSE cells and tissue biopsies from normal ovaries were obtained from women operated on for benign nonovarian diseases at the Gynecology Unit, Sahlgrenska University Hospital, Goteborg, Sweden. OSE cells for culture were obtained from a total of 32 women. Normal ovarian tissue biopsies were collected from a total of nine women. A description of age at time for operation, reproductive status, and parity are found in Table 1. The study was approved by the Ethical Committee of Goteborg University and the patients had given their informed consent.

Harvest of Cells and Culture Conditions

To obtain OSE, cytobrushes (Cytobrush Plus; Medscand AB, Malmo, Sweden) were used as the first step after entering the peritoneal cavity at laparotomy or laparoscopy. The brushes were simultaneously rotated and moved 2-3 times over the ovarian surface, exerting only a slight pressure to minimize the risk of damaging or disrupting the underlying basal membrane and tunica albuginea. The brushes were withdrawn and immediately placed in culture medium, taken to the laboratory, and gently rubbed against each other to release the cells.

The cells were then centrifuged for 5 min at 300 X g, diluted in fresh culture medium, and equally seeded into two 30-mm diameter Petri dishes (Falcon; Becton Dickinson, Meylan, France). OSE cells were cultured in a 1:1 mixture of M199:MCDBl05 medium (Sigma Chemicals, St Louis, MO) supplemented with 15% fetal bovine serum (FBS; Life Technologies Ltd., Paisley, UK) and penicillin-streptomycin (100 IU/ml-100 |xg/ml; Life Technologies Ltd.). Culture medium was exchanged every 2-3 days. When the cells reached confluence, cells were split and further cultured in Transwell inserts or Petri dishes according to each experiment.

An ovarian cancer cell line, NIH: OVCAR-3 (OVCAR), was purchased from American Type Culture Collection (ATCC; Rockville, MD). OVCAR cells were cultured in a 1:1 mixture of M199:MCDB105 medium (Sigma) supplemented with 10% FBS (Life Technologies Ltd.) and penicillin-strep-tomycin (100 IU/ml-100 |xg/ml; Life Technologies Ltd.). OVCAR cells were grown in culture inserts and trans epithelial resistance (TER) was measured. As positive control in the immunoblot experiments, Madin-Darby canine kidney (MDCK) cells (a kind gift from Dr. Nillson, Gote-borg, Sweden) were used. MDCK cells were cultured in Eagle minimal essential medium (Sigma Chemical Co.), supplemented with 10% fetal bovine serum (Life).

Immunofluorescence

Cells in first or second passage were grown on 18 X 18-mm glass cover slips (Histolab; Histolab Products AB, Goteborg, Sweden) until confluence. Cover slips were then removed from the culture dish, washed in PBS, fixed in cold acetone for 15 min, air dried at room temperature, and frozen at —20°C until analysis. Fresh-frozen tissue biopsies were cryosec-tioned and fixed as described for cultured cells. Cultured cells and tissue sections were incubated with 5% nonfat milk for 30 min, followed by incubation with primary antibodies against TJ proteins overnight: rabbit polyclonal antibody to ZO-1, occludin, and claudin-1 (1:500; Zymed Laboratories, San Francisco, CA). Mouse monoclonal antibodies against cy-tokeratin 8 (1:400; DAKO, Copenhagen, Denmark), cytokeratin AE1/AE3 (1:50; Boehringer Mannheim, Mannheim, Germany), E-cadherin (1:100; Transduction Laboratories, Nottingham, Aylesbury, UK), and N-cadherin (1:100; Zymed Laboratories) were used to further characterize epithelial features of the cells. Bound antibodies were visualized by biotinylated secondary horse anti-rabbit (1:200; Vector Laboratories, Burlingame, CA) followed by streptavidin-fluorescein isothiocyanate (1:200; Amersham, Buckinghamshire, UK) while the nuclei were counterstained with Hoechst 33342 (1 |xg/ml; Molecular Probes Inc., Eugene, OR). In the control sections, which showed only negligible signals, the first antibody was replaced by 5% nonfat milk in PBS. Sections and cells were mounted in 4-diazabicyclo-2, 2, 2-octane (Dabco; Fluka, Buchs, Switzerland) and photographed with a Nikon ECLIPSE E600 fluorescence microscope (Nikon Corporation, Tokyo, Japan) using a digital cytoversion program (Applied Imaging Corporation, Santa Clara, CA).

Immunoblot Analysis

Confluent secondary cultures were solubilized in lysis buffer consisting of 62.5 mM Tris, 20% glycerol, and 2% SDS. The protein concentration of the supernatant was determined with the Micro BCA protein assay kit according to the manufacturer’s instructions (Pierce, Rockford, IL). Samples were diluted in SDS-sample buffer and heated at 70°C for 10 min. Fifty micrograms of total protein from each sample were loaded into each lane of a SDS-polyacrylamide gel (NuPAGE 4-12% Bis-Tris Gel; Invi-trogen Ltd., Paisley, UK) and separated by electrophoresis. Proteins were transferred to polyvinyldifluoride membrane (Amersham) using a blotting system (Novex miniblot; Invitrogen Ltd.) and incubated with primary antibodies: occludin (1:500) and claudin-1 (1:500). MDCK cells were used as positive control for TJ-expressing epithelia. Prestained standards (see blue; Invitrogen Ltd.) were used as molecular weight markers. Immuno-reactive protein was visualized by chemiluminescence using alkaline phosphatase-conjugated secondary goat anti-rabbit (1:30 000) (Tropix, Bedford, MA) and CDP Star (Tropix) as substrate. The membrane was exposed to enhanced chemiluminescence film (Amersham).

Resistance Measurements

OSE cells were seeded with a density of 9.0 X 104/cm2 on 6.5-mm transparent Transwell filter inserts with a membrane pore size of 0.4 |xm (Transwell-Clear; Corning COSTAR, Costar Europe Ltd., Badhoevedorp, The Netherlands). Culture medium was added into both chambers and routinely exchanged every 2-3 days. TER was measured every 2-3 days by a combined Millicell Electrical Resistance System (ERS; Millipore Corp., Bedford, MA) according to the manufacturer’s instructions. TER was measured in OSE secondary cultures from seven different women. For each culture, cells were seeded in triplicate wells. TER (see Figs. 5 and 6) was determined by subtracting the background resistance of a blank filter and multiplying by the area of the monolayer (0.33 cm2 for the 6.5mm inserts).

To further evaluate TJ function, filter-cultured OSE were subjected to chelation of extracellular Ca2+ with 2 mM ethyleneglycol-fa’s-(a-aminoeth-yl ether)-A^A^-tetraacetic acid (EGTA; Sigma) to rapidly disrupt cell-cell contacts. Confluent OSE was treated with EGTA for 30 min and then transferred back to EGTA-free media. TER was measured every 5 min during the EGTA experiment, and after 45 min, 12 h, and 24 h during the recovery period. Confluent OSE from the same patient, continuously cultured with ordinary medium, was measured at the same time points as controls. For each culture, TER was measured in triplicate wells. This experiment was performed on cultures from three different patients.

Data and Statistics for Resistance Measurements

The results from TER measurements were shown as median, top, and lowest range of the data from three measured wells for each time point. The possible statistical difference between EGTA test group and control group within the same patient were examined by the Mann-Whitney test. Differences were assumed to be significant at P < 0.05.

Electron Microscopy

Filter-grown OSE primary and secondary cultures from four women (2 pre- and 2 postmenopausal; see Table 1) were fixed for 1 h in 2.5% glutaraldehyde in 0.05 M sodium cacodylate, pH 7.4, followed by postfixation for 1 h in 1% OsO4. After dehydration in ethanol series, the specimens were embedded in epoxy resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a Zeiss electron microscope.

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