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Developmental Coordination of Luteinizing Hormone/Human Chorionic Gonadotropin (hCG) Receptors and Acute hCG Responsiveness in Cultured and Freshly Harvested Porcine Granulosa Cells^*

Departments of Obstetrics and Gynecology and Physiology, Duke University Medical Center Durham, North Carolina 27710

Address requests for reprints to: Dr. Jeffrey V. May, Box 3323, Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710.

Abstract

A fundamental characteristic of ovarian antral follicle development is the progressive differentiation of the granulosa cells, a process marked by an increase in their complement of LH receptors. In this study we investigated the coordinated expression of [¹²⁵I]iodo-hCG binding sites and hCGsensitive cAMP production in intact cells from normally differentiating antral follicles of increasing size and in cells maintained in monolayer culture under conditions known to facilitate differentiation. In addition to hCG responsiveness, basal, FSHstimulated, and cholera toxin-stimulated cAMP production were compared.

Granulosa cell [¹²⁵I]iodo-hCG binding capacity was directly related to follicle size; thus, binding provided a convenient marker of cell maturation, which, in turn, reflected the state of follicle development. Basal and hCG-stimulated cAMP production increased as a function of cellular LH/hCG receptor binding. Whereas basal activity increased linearly, and proportionately with LH/hCG receptor binding, hCG-stimulated cAMP production was not linearly proportional. FSH responsiveness in terms of cAMP production declined as a function of LH/hCG receptor binding, exhibiting first order decay. While the patterns of FSH- and hCG-stimulated cAMP production were inversely related throughout much of follicle development, cholera toxin (CTX) responsiveness of cells remained constant until the very late preovulatory stages. Granulosa cells from large follicles (rhean diameter, >8 mm), having been exposed to the endogenous LH surge, exhibited high [¹²⁵I]iodo-hCG binding but severely impaired hCG- and CTX-stimulated cAMP production, suggesting desensitization of adenylyl cyclase.

In cultured granulosa cells, increased [¹²⁵I]iodo-hCG binding induced by insulin and FSH was paralleled by an increased ability to generate cAMP in response to hCG. This coordinated expression of receptor and responsiveness was similar to that observed with progressively higher states of differentiation in freshly harvested cells. CTX-stimulated cAMP production in cells maintained in vitro was elevated and independent of LH receptor levels and, thus, was similar to that exhibited by freshly harvested cells. Granulosa cell cAMP production in response to acute FSH stimulation after chronic FSH treatment during culture was consistently lower than that of freshly harvested cells, a phenomenon that appeared to be related to the chronic dose of FSH used in vitro.

Cells cultured with insulin alone, having markedly reduced levels of LH/hCG receptor binding relative to those of cells cultured with insulin plus FSH, responded to hCG with 3- to 5-fold more cAMP per unit [¹²⁵I]iodo-hCG binding than did cells treated with FSH plus insulin. The mechanisms underlying this difference are not known, but may reflect heterologous desensitization due to chronic FSH treatment and/or the intracellular conditions that affect the rate of enzymatic synthesis of cAMP.

Our results suggest that during granulosa cell differentiation in vivo and in vitro, the LH/hCG receptors induced are functionally linked to adenylyl cyclase. The developmental patterns of expression of LH/hCG-binding activity and LH/hCG-, CTX-, and FSH-stimulated adenylyl cyclase activities are very similar in vivo and in vitro. The individual parameters, however, appear to be modulated independently under a variety of physiological circumstances in vivo and in vitro. (Endocrinology 114: 153, 1984)

Footnotes

^* This work was presented in part at the 63rd Annual Meeting of The Endocrine Society, Cincinnati, OH, June 17-19, 1981 (Abstract 790). It was supported in part by Grant HD-11827 from the NICHHD, USPHS.

Received January 4, 1983.

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