Granulosa Cell and Follicle Incubations
Immature (25-day-old) female Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were housed in environmentally-controlled rooms with food and water ad libitum. Upon arrival, rats were given a single s.c. injection of 10 IU of eCG to promote growth of a cohort of healthy antral follicles over a subsequent 46-h period. For analysis of potassium efflux in individual granulosa cells, cells were collected by needle puncture of the largest follicles present within the gonadotropin-stimulated ovaries, washed, and cultured for 24 h at 1 X 106 cells/ml in McCoy-5a medium containing 100 U/ml penicillin and 75 U/ml streptomycin sulfate.
The cells were cultured in chambers constructed by placing double-stick tape across poly-L-lysine-coated slides and suspending a cover-slip between the pieces of tape. Infusions of cells, treatments, and washes were carried out by placing the solution on one side and allowing capillary action to draw the liquid in. One hour before analysis, PBFI was added to a final concentration of 5 ^M. After a 1-h incubation, the cells were washed in PBS containing trypan blue to allow discrimination between cells undergoing apoptosis versus those that had undergone necrosis. Slides were then analyzed by fluorescence microscopy (excitation at 340 nm; emission at 505 nm).