Luteinizing hormone receptor induction in dispersed granulosa cells requires estrogen

Studies on granulosa cell responses in vitro have routinely utilized cell preparations in which intercellular gap-junctions are maintained. The present study was conducted to determine if disruption of these junctions, prior to culture, would affect subsequent follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction on these cells. Granulosa cells were expressed from ovaries of diethylstilbestrol (DES)-primed immature rats after a short incubation of the excised ovaries in culture medium alone or medium containing 6.8 mM EGTA. The latter procedure disrupts gap-junctions between granulosa cells thus providing a predominantly mono-dispersed cell suspension. The two cell preparations were cultured, separately, for 72 h in sterile polypropylene tubes in media containing either FSH or FSH plus various steroids (estradiol, testosterone, or 5 alpha-dihydrotestosterone (DHT)). LH receptor content of cells was determined at 72 h of culture. Both the cell yield and the proportion of viable cells obtained were enhanced by the EGTA pretreatment. LH receptors were induced in all FSH-containing cultures of non-dispersed granulosa cells. In the dispersed cell cultures, FSH alone failed to induce LH receptors. The inclusion of either estradiol or testosterone but not DHT with FSH, however, restored LH receptor induction to levels comparable to non-dispersed cultures. LH receptors were not induced in cultures of either cell preparation with steroids alone. Aromatase activity, however, was stimulated in both cell preparations by FSH alone. These results suggest that cell-cell communication may be necessary for LH receptor induction in granulosa cells and that estradiol (or an aromatizable androgen) can promote intercellular interactions if this communication has been disrupted.