Chemicals and Reagents
Human pituitary FSH (EC-232-662-3, lot 092K1105, 7000 IU IRP 68/ 140 per mg), ionomycin, ATP, and DDE were purchased from Sigma-Aldrich Chemicals (Oakville, ON, Canada). Human chorionic gonadotropin (lot B18856, 3050 IU/mg, WHO 1st IRP 75/551) was purchased from Calbiochem (La Jolla, CA). All tissue culture supplies were purchased from Gibco Life Technologies (Burlington, ON, Canada). Fura-2 acetox-ymethyl ester was obtained from Molecular Probes (Eugene, OR). The gonadotropins were dissolved in normal saline before use, and DDE was dissolved in dimethyl sulfoxide (DMSO).
Approval for this work was obtained from the institutional research ethics board. Informed consent was obtained from each patient at the time of oocyte retrieval for IVF. Human granulosa cells were obtained at the time of oocyte retrieval from patients having IVF treatment at Hamilton Health Sciences Centre for Reproductive Care. Patients were treated with a long luteal protocol of GnRH agonist (Lupron; Abbott Laboratories, Montreal, QC, Canada; 0.5 mg/day for 10-14 days) and recombinant FSH (12-85 ampules, 75 IU/ampule Gonal F; Serono Canada, Ltd., Oakville,
ON, Canada) followed by hCG (Profasi; Serono). After removal of oocyte-cumulus complexes, the remaining follicular aspirates were transported to the research laboratory in polypropylene tubes. The cells and fluid were centrifuged for 4 min at 175 X g, the supernatant was removed, and 9 ml sterile distilled water was added to the granulosa cell pellet to lyse the red blood cells. After 20 sec, 1 ml 10X concentrated PBS was added to stop the reaction. The cells were pelleted by centrifugation for 4 min at 175 X g and resuspended in 3 ml of plating medium, which consisted of minimum essential medium containing 10% calf serum (Gibco Life Technologies), 100 units/ml penicillin, 0.1 mg/ml streptomycin, 200 mM L-glu-tamine, 10 mM nonessential amino acids, 50 |xl gentamicin, and 2.5 |xl amphotericin B (Sigma-Aldrich, St. Louis, MO). The granulosa cells were then plated at a concentration of 100-200 000 cells/dish on coverslips glued to the bottom of 35-mm Falcon culture dishes. After 2 days in culture, the granulosa-lutein cells (hereinafter referred to as granulosa cells) were labeled with 8 |xM Fura-2 acetoxymethyl ester for about 30 min. The medium used for analyzing Ca2+ changes was 10 mM Hepes (pH 7.4) containing 126 mM NaCl, 6 mM KCl, 10 mM glucose, 1.5 mM CaCl2, and 0.3 mM MgCl2. One run of each culture dish typically took about 20-30 min, and each experiment was performed in quadruplicate. At least 12 cells from a total of three separate patients on different days were monitored for each test substance.
Digital Fluorescence Ratio Calcium Imaging
Changes in Ca2+ concentration in the cytosol ([Ca2+]cyt) were measured using a dynamic digital Ca2+ imaging system (Image-I/FL; Universal Imaging Corporation, Downington, PA) with a lamp (HBO 100 W/DC; Zeiss, Thornwood, NY) coupled to an inverted microscope (IM 35; Zeiss) with a 100X oil immersion lens and a numerical aperture of 1.25, as previously described. A filter wheel held filters at 340 and 380 nm, which alternated, and images were captured on the first and second quadrant of the monitor screen. The ratio between these two wavelengths (340:380 nm) was displayed on the third quadrant, and the time event of the ratio changes at selected regions of the cells was displayed on the fourth quadrant. Emitted fluorescence was detected with a 540-nm filter. Images were integrated and collected with a camera (TM-720; Pulnix, Alexandria, VA; maximal at 3 sec/frame) initially at a speed of 15 sec/frame. When there were no [Ca2+]cyt oscillations or when a rapid transient elevation in [Ca2+]cyt occurred, the speed was slowed to 30 sec/frame to conserve digital memory space for data collection in experiments running longer than 30 min. The optical part ofthe microfluorometric system was custom-modified to reduce photobleaching, and each measurement was made in the dark. Exposure to light was minimized to further reduce photo-bleaching, and background values were preset by the defocusing technique. Prior to each set of experiments, the pseudocolor grading was calibrated against the in situ Fura-2 fluorescence ratio for maximum in the presence of 10 |xM ionomycin (a calcium ionophore) and for minimum in the presence of 5 mM EGTA (2.5 times more concentrated than the Ca2+ concentration in the cell medium). None of the drugs used in this work caused quenching of the fluorescence spectrum of Fura-2. Calibration of the flourescence signal at the end of each experiment with excess EGTA and ionomycin confirmed the reciprocal change of the intensities at 340 and 380 nm with the change of [Ca2+]cyt. Because the relative [Ca2+]cyt as reflected by the 340:380 nm ratio is not dependent on changes of absolute intensity at each wavelength, a noticeable loss of Fura-2, which sometimes occurred when [Ca2+]cyt rose too high, did not affect the ratio image of the cells. The software for the imaging processing converted the fluorescence data obtained at 340 and 380 nm to 340:380 nm ratios pixel by pixel, and the ratios were expressed by the corresponding pseudocolors. Further conversion from the ratio values to the Ca2+ concentration values was not attempted because of intrinsic problems in the estimation of absolute [Ca2+]cyt.
In all cases, granulosa cells were always exposed to 1-2 mM Ca2+ except for the experiments requiring Ca2+-free conditions, where the medium was replaced with Ca2+-free isotonic physiological medium containing 0.1 mM EGTA immediately prior to measurement. Although distilled and deionized water were used for the preparation of solutions, contaminating Ca2+ from containers and other chemicals may contribute up to 10 |xM Ca2+. Therefore, 0.1 mM EGTA was always included in the Ca2+-free medium. No changes in Ca2+ uptake could be elicited with up to 0.5% DMSO in the medium, the maximum concentration used in all these studies.