Ovarian Granulosa Cells Isolated with EGTA and Hypertonic Sucrose: Cellular Integrity and Function

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Abstract
Chemical treatments previously shown to disrupt gap junctions were applied to rat ovaries with antral follicles prior to expressing the granulosa cells with a blunt spatula. Exposure of the ovaries to 6.8 mM EGTA [ethyleneglycol-bis-(.beta.-aminoethyl ether)-N,N''-tetraacetic acid] and 0.5 M sucrose either by perfusion in situ or incubation in vitro generated monodisperse suspensions of granulosa cells with improved integrity as evaluated by several criteria. Similar numbers of granulosa cells (7-8 .times. 106 per ovary) were obtained either by direct physical expression or by pretreatment of the ovaries followed by physical expression. The ability of the granulosa cells to exclude trypan blue dye was consistently improved 2-to 3-fold by pretreatment with EGTA and hypertonic sucrose (from 25% to 40-80%). Likewise, in vitro synthetic capacities for protein, RNA and DNA were enhanced 2- to 6-fold, 5-to 10-fold and 10-to 20-fold, respectively. A qualitative one-to-one correspondence between protein synthesis and vital dye exclusion was demonstrable but the quantitative relationship between dye exclusion and macromolecular precursor incorporation appeared to be nonlinear. Cells obtained using the chemical pretreatments also demonstrated better survival in minimal medium for at least the first 12 h of culture. The enhancements observed could not be obtained by reversing the order of the chemical treatments or by treating granulosa cells after physical removal from ovarian follicles. Pretreatment of ovaries with EGTA and hypertonic sucrose appears to be a reliable procedure for improving the yield of monodisperse, viable, biochemically intact granulosa cells for use in in vitro examinations of follicular physiology and function.