13-cis-retinoic acid modulates porcine ovarian granulosa cell differentiation via retinoic acid signaling-FoxA1 axis: Implications for ovarian follicular development and luteinization.

Abstract

13-cis-Retinoic acid (13cRA), a vitamin A derivative clinically employed in cancer therapy, demonstrates novel regulatory effects on ovarian follicular development and luteinization through granulosa cell proliferation and differentiation in this study. Our murine model revealed that 13cRA administration significantly increased the number of primary follicle and corpus luteum by ovarian sectioning and follicle counting, indicating accelerated follicular maturation and ovulation competence. In porcine ovarian granulosa cell cultures, 13cRA treatment induced cell cycle progression (G1-S phase transition), and promoted cell proliferation while up-regulating differentiation markers luteinizing hormone receptor (LHR) and progesterone receptor (PGR). Cellular retinoic acid-binding protein CRABP2-mediated mechanisms were identified as pivotal through gain/loss-of-function experiments. Overexpression of CRABP2 amplified 13cRA-induced LHR and PGR expression, while knockdown of CRABP2 by specific siRNA resulted in the opposite effect. The retinoid acid signaling axis was further delineated through receptor-specific modulation. 13cRA promoted RARβ/RXRβ receptor heterodimer formation, with pharmacological activation (Adapalene) potentiating LHR and PGR expression and receptor knockdown diminishing their expression. RA metabolic regulation studies revealed that CRABP1 and CYP26 isoforms (CYP26A1 and CYP26B1) as functional antagonists of 13cRA activity were demonstrated through siRNA silencing or CYP26 inhibitor R115866 treatment experiments. Notably, we identified that FoxA1, as a negative regulator of granulosa cell proliferation and differentiation, mediated the effect of 13cRA on granulosa cell differentiation through siRNA transfection and FoxA1 recombinant protein treatment experiments. 13cRA-mediated FoxA1 expression suppression occurred through CRABP2-dependent nuclear shuttling and RARβ/RXRβ receptor heterodimer activating, with combinatorial modulation of CRABP1/CYP26 system components (CYP26A1 and CYP26B1) significantly altering FoxA1 regulatory dynamics. Finally, we conclude that 13cRA can inhibit FoxA1 expression through RA signaling molecules (CRABP2, RARβ/RXRβ heterodimer, CRABP1, CYP26A1 and CYP26B1) and thus promote the differentiation of porcine ovarian granulosa cells.