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Regulation of Rat Granulosa Cell Differentiation by Extracellular Matrix Produced by Bovine Corneal Endothelial Cells^*

ANCHEL FURMAN, SIEGFRIED ROTMENSCH, FORTUNE KOHEN, SHLOMO MASHIACH and ABRAHAM AMSTERDAM

Department of Hormone Research, The Weizmann Institute of Science Rehouot 76100, Israel
Department of Obstetrics and Gynecology, The Chaim Sheba Medical Center Tel-Hashomer, Israel

Address requests for reprints and all correspondence to: Abraham Amsterdam, Ph.D., Department of Hormone Research, The Weizmann Institute of Science, Rehovot 76100 Israel.

Abstract

The effect of bovine corneal extracellular matrix (ECM) on gonadotropin-primed rat granulosa cells in vitro was studied by examining the following parameters: 1) rate of cell attachment to culture dishes; 2) modulation of cell morphology; 3) specific binding of [¹²⁵I]human(h)CG to LH/hCG receptors; 4) cAMP response to hCG stimulation; and 5) basal and hCG stimulated progesterone production. Attachment of cells to culture dishes occurred significantly earlier on ECM, as compared with uncoated dishes (6 h vs. 24 h). Cells grown on ECM were epitheloid and organized in multilayer aggregates, closely resembling their organization in the intact wall of the ovarian follicle. In contrast, cultures on uncoated dishes grew as a monolayer of markedly flattened cells. A 2-fold increase in number of LH/hCG receptors occurred on ECM within 48 h, probably due to de novo synthesis. Scatchard analysis revealed no change in hormone affinity to the receptor during the culture period [association constant (Ka) = 2.5 x 10¹⁰M^–1 for hCG]. Cells grown on ECM had a parallel increase in cAMP responsiveness to hCG stimulation. Cells grown in serum-free medium on ECM-coated dishes preserved only 50% of LH/hCG receptors and cAMP responsiveness after 48 h. Cells cultured on ECM showed a marked elevation in progesterone production even in the absence of gonadotropin stimulation, whereas cells grown on uncoated dishes almost completely lost their ability to produce progesterone both in the presence and absence of hCG. These results indicate that ECM plays a substantial role in the maintenance and further propagation of granulosa cell differentiation in vitro. (Endocrinology 118: 1878–1885, 1986)

Footnotes

^* This work was supported by a grant from the Rockefeller Foundation.

Fanny and Samuel Kay Research Fellow at the Weizmann Institute of Science.

Incumbent of the Joyce and Ben B. Eisenberg Professorial Chair in Molecular Endocrinology and Cancer Research.

Received August 9, 1985.

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