Polycystic ovary syndrome (PCOS), characterized by a high androgen-to-estrogen ratio, follicular arrest and anovulation, is a reproductive and endocrine disorder affecting 10% of women of reproductive age worldwide ¹. Abnormalities in ovarian granulosa cells contribute to PCOS pathogenesis, although the underlying molecular mechanisms remain unclear. Previously, we demonstrated multi-drug resistance protein 4 (MRP4), a membrane protein of the ATP-binding cassette transporter subfamily C encoded by the human gene ABCC4, to be essential to uterine signaling pathways (i.e., prostaglandin E2 and Wnt/β-catenin) required for embryo implantation and female fertility². In the present study, we hypothesized that MRP4 might play a role in granulosa cells for ovarian functions.

We first examined MRP4 expression in mouse ovarian tissues and found its expression mainly in granulosa cells initiating at primary follicles and increasing gradually as follicles grow and peaking at late antral follicles. We next generated transgenic mouse models with granulosa-specific knockout of MRP4 by crossing MPR4-floxed (Abcc4^fl/fl) mice with Cyp19a1–Cre- or Amh-Cre-inserted one. Such conditional (granulosa-specific) knockout mice (cKO) were compared with Cre negative controls (Abcc4^fl/fl). Although these cKO mice were viable and grossly normal, they exhibited clear reproductive abnormalities in adulthood. Estrous cycle analysis showed a significantly (p <0.05) shortened estrus phase and prolonged diestrus/metestrus in cKO (n = 7 of Amh-Cre and n = 9 of Cyp19a1-Cre) in comparison to the controls (n = 12). Histological examination and follicle counting cross the entire ovary revealed a marked (p <0.05) accumulation of small follicles (primary and secondary) accompanied by a significant (p <0.05) reduction in large antral follicles in the cKO mice (n = 5 of Amh-Cre and n = 9 of Cyp19a1-Cre) versus those in controls (n = 12). In addition, cKO ovaries contained thin-walled oocyte-lacking follicular cysts, which was very rare (1 cyst in 12 ovaries) in the controls. Fertility testing over consecutive pregnancies demonstrated reduced reproductive capacity of the cKO (n = 5). Moreover, granulosa expression level of anti-Mullerian hormone (AMH) was significantly elevated in cKO ovaries, particularly in large follicles, which together suggested a PCOS-like follicular arrest phenotype in cKO mice. Consistently, spatial transcriptomic analysis of ovaries from the mice revealed a pathological granulosa cell transition in the cKO. The cluster of cells associated with steroidogenic function reduced in number, whereas a distinct population enriched in adhesion- and scaffold-related genes became predominant in cKO ovaries. Quantitative PCR analysis confirmed that primary granulosa cultures from MRP4 knockout mice exhibited downregulation of key steroidogenic markers, including Cyp11a1, Cyp19a1, and Fshr with impaired gonadotropin responsiveness. Mechanistically, intracellular cAMP- and Ca²⁺-overload was found in primary granulosa cells of MRP4 knockout, linking transporter loss to altered signaling homeostasis and granulosa dysfunction.

Taken together, these findings have revealed previously undefined role of MRP4 in regulating granulosa cell function and folliculogenesis. Deficiency of MPR4 triggers in a pathological transition of granulosa cells resulting in PCOS-like follicular arrest in mice, providing a novel mechanism for granulosa abnormalities in PCOS.