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Biology of Reproduction, Vol 21, 773-786, Copyright © 1979 by Society for the Study of Reproduction
1 Reproductive Endocrinology Program,
Department of Pathology,
The University of Michigan
Ann Arbor, Michigan 48109 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- Similar numbers of granulosa cells (7-8 x 106 per ovary) were obtained either by direct physical
expression or by pretreatment of the ovaries followed by physical expression. However, the ability
of the granulosa cells to exclude trypan blue dye was consistently improved 2-3-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 2-6-fold, 5-10-fold and 10-20-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.
-aminoethyl ether)-N,N’-tetracetic 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.
Note:
ACKNOWLEDGMENTS
Sincere thanks are extended to Dr. A. Rees Midgley, Jr. for frequent support and suggestions during
the entire course of this study, to Dr. George E.
Palade for making the suggestions which led to the
initial experiments on ovarian perfusion, to Dr. Leo E.
Reichert of Emory University for the generous provision of purified FSH and to Ms. Janet Poppe without
whose technical assistance much of the work could
not have been done.
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