Interrelationships between amphiregulin, kisspeptin, FSH and FSH receptor in promotion of human ovarian cell functions

Abstract

The aim of this study was to investigate: (1) the ability of granulosa cells to produce amphiregulin (AREG), kisspeptin (KISS) and FSH receptor (FSHR); (2) the role of AREG and KISS in the control of ovarian functions; (3) the effect of FSH and KISS on AREG; and (4) the ability of KISS to affect FSHR and to modify FSH action on AREG output by human ovarian granulosa cells. We examined: (1) time-dependent accumulation of AREG; (2) effects of AREG (0, 1, 10, 100 ng/mL) and KISS (0, 1, 10, 100 ng/mL) on granulosa cell functions; and (3) the effects of KISS (0, 1, 10, 100 ng/mL), FSH (0, 1, 10, 100 ng/mL), and their combinations on AREG release. Viability, markers of proliferation [accumulation of proliferating cell nuclear antigen (PCNA) cyclin B1 and sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy6-nitro)benzene sulfonic acid hydrate (XTT formazan)] and apoptosis (accumulation of bax, caspase 3 and terminal deoxynucleotidyl transferase dUTP nick-end labelling), accumulation of KISS, FSHR and steroid hormones, and AREG release were analysed by Trypan blue exclusion test, quantitative immunocytochemistry, XTT, terminal deoxynucleotidyl transferase dUTP nick-end labelling assays and enzyme-linked immunosorbent assay. AREG promoted cell viability, proliferation and steroid hormone output, and inhibited apoptosis. KISS (1 and 10 ng/mL) stimulated viability, proliferation, steroid hormone release and occurrence of FSHR and suppressed apoptosis and AREG output; KISS (100 ng/mL) had the opposite effect. FSH stimulated AREG release, whilst addition of KISS reversed this FSH effect. FSH mimicked and promoted the inhibitory effect of KISS on AREG release. These results suggest an intra-ovarian production and a functional interrelationship between AREG, KISS, FSH and FSHR in direct regulation of basic ovarian cell functions.