Retinoic Acid Regulates Calcium Signaling to Promote Mouse Ovarian Granulosa Cell Proliferation

Normal development of ovarian follicles is critical for female reproduction and endocrine function. We have identified retinoic acid (RA) and the RA-degrading enzyme CYP26B1 as regulators of ovarian follicle development and showed that RA and a CYP26 inhibitor stimulated ovarian granulosa cell proliferation. The mechanism underpinning RA-dependent proliferation, however, is not known. The current study was designed to examine the role of intracellular calcium (Ca2+) signaling in mediating the effects of RA on primary mouse granulosa cell proliferation. In single-cell Ca2+ imaging experiments, treatment of cultured granulosa cells with RA increased the steady-state Ca2+ content of the endoplasmic reticulum (ER) stores. This correlated with increased store-operated Ca2+ entry (SOCE) and enhanced inositol 1,4,5-trisphosphate receptor (IP3R)-dependent Ca2+ release. In proliferation assays, RA treatment or Cyp26b1 knockdown stimulated proliferation, whereas Cyp26b1 overexpression inhibited proliferation. When RA was given together with 2-aminoethoxydiphenylborane (2-APB), a blocker of IP3R-dependent ER Ca2+ release and SOCE, with xestospongin C, a selective IP3R- receptor antagonist, or with 3,5-bis (trifluoromethyl)pyrazole (BTP-2), a specific SOCE blocker, the stimulatory effect of RA on cell proliferation was abolished. Further investigation showed that treatment with 2-APB or BTP-2 inhibited RA induction of RA response element (RARE) activation in granulosa cells, confirming an important role for Ca2+ signaling in mediating RA actions. Overall, these data support a model in which RA regulates ovarian follicle development by stimulating granulosa cell proliferation and that this stimulatory effect is at least in part driven by the modulation of Ca2+ signaling.