CONTACT-DEPENDENT CELL-INTERACTIONS DETERMINE HORMONE RESPONSIVENESS AND DESENSITIZATION IN RAT GRANULOSA-CELLS

The maintenance of associations between granulosa cells (GCs) is neces sary for FSH-stimulated induction of LH receptors. In cultures in whic h these associations have been disrupted, FSH fails to induce LH recep tors. As FSH exerts its action in GCs via cAMP, me have examined if th e aggregation state of GCs plays a role in modulating FSH-stimulated c AMP production. GCs were obtained from the ovaries of diethylstilbestr ol-primed immature rats. Cells were prepared as aggregate or dispersed populations by isolating GCs in either the presence or absence of Ca2 +. Nonviable cells mere removed by a brief exposure to trypsin. We hav e shown previously that trypsin treatment in the absence of Ca2+ remov es a class of cell adhesion molecules, termed cadherins, from the plas ma membranes of GCs. Hence, the dispersed GCs are incapable of reaggre gating. Dispersed or aggregate GC preparations were incubated with dif ferent doses of human FSH (0-1 mu g) for 0-60 min in the presence of i sobutylmethylxanthine, a phosphodiesterase inhibitor. Incubations were terminated, and the cAMP accumulated was measured using a specific RI A As desensitization to hormonal stimuli is a characteristic property of many G protein-coupled response systems, cAMP production of cell ag gregates and dispersed cells in response to a repeated stimulation wit h FSH was assessed. Our results indicate that aggregate GCs have a sig nificantly attenuated cAMP response to all doses of FSH compared with dispersed GC preparations. Changing cell densities did not alter the n ature of these responses, indicating that nonspecific cell interaction s were not responsible for this difference. The number of FSH receptor s and their affinity were unaltered in the two cell preparations. Chol era toxin- and forskolin-stimulated cAMP production were similar in th e two preparations, demonstrating that the changes in responsiveness d id not arise from alterations in G protein activation or adenylate cyc lase activity. Only the aggregate GCs could be desensitized. The dispe rsed cells displayed undiminished cAMP responsiveness to a second FSH stimulation. Finally, culture of the GC preparations with cholera toxi n induced LH receptors in GC aggregates only. LH receptor induction in dispersed cell cultures required the addition of estradiol. These res ults indicate that contact-dependent cell interactions can modulate GC cAMP production in response to FSH, cAMP responses, however, were not the sole determinant of cell differentiation, as assessed by LH recep tor induction. We speculate that cell-cell interactions within the fol licular epithelium are important determinants for cell differentiation leading to follicle selection for ovulation or atresia.