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Granulosa Cell Differentiation in Vitro: Induction and Maintenance of Follicle-Stimulating Hormone Receptors by Adenosine 3'5'-Monophosphate^*

Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, Maryland 20205

Address all correspondence and requests for reprints to: Michael Knecht, Ph.D., Building 10, Room 8C407, National Institutes Health, Bethesda, Maryland 20205.

Abstract

Granulosa cell differentiation is stimulated in vitro by FSH and other agents that increase cAMP production. To determine if alterations in FSH receptors are associated with cAMP-mediated granulosa cell maturation, FSH-binding sites were measured during culture of undifferentiated granulosa cells from hypophysectomized diethylstilbestrol-implanted rats. FSH receptors decreased rapidly in the absence of stimulatory ligands, with loss of 75% and 90% of the FSH-binding activity from freshly prepared cells after 24 and 48 h, respectively. The decline in FSH receptors during culture was accompanied by a corresponding decrease in cAMP responses to added FSH. In cells cultured with FSH, the available FSH receptor content fell to 15% after 8 h, then rose to 25% of the initial receptor levels from 24–48 h of culture. Cells treated with 8-bromo-cAMP or choleragen for 48 h retained about 40% and 90%, respectively, of their initial FSH-binding activity. Although choleragen did not prevent the 60–70% fall in FSH binding during the first 6 h of culture, it increased receptors 2.5-fold from 12–48 h. Also, the addition of choleragen after 3, 6, or 12 h of culture elevated FSH receptors at 48 h in proportion to the amount of cAMP produced. The FSH receptors induced by choleragent treatment were functionally active, as shown by their ability to mediate FSH-stimulated cAMP production. A GnRH agonist prevented the choleragen- induced rise in FSH receptors and cAMP accumulation from 24–48 h of culture. Scatchard analysis revealed a single population of FSH receptors after all treatments, with association constant of 5 x 10⁹ M^–1. Freshly prepared cells contained approximately 1600 FSH binding sites/cell, while treatment with choleragen or FSH for 48 h restored receptor levels to 1150 and 500 sites/cell, respectively. These results indicate that the expression of functional FSH receptors during granulosa cell maturation is mediated by cAMP. (Endocrinology 113: 949, 1983)

Footnotes

^* This work was supported in part by Public Health Service International Research Fellowship 1-F05-TW03116-01 (to T.R.).

Received November 17, 1982.

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