Disrupted riboflavin metabolism impairs circadian rhythm in ovarian granulosa cells: a novel mechanism driving DOR.

Abstract

Diminished ovarian reserve (DOR) is a major cause of female infertility and a significant indicator of reproductive aging, yet its underlying pathogenesis remains poorly understood. In this study, we initially assessed the expression of core circadian genes in ovarian granulosa cells from DOR patients and in ovaries from cyclophosphamide (Cy)-induced DOR model rats. We observed dysregulated expression of circadian genes in DOR ovaries. Proteomic analysis further revealed a significant decrease in riboflavin kinase (RFK) expression in DOR rat ovaries, concomitant with abnormal elevation of the clock proteins BMAL1. RFK deficiency impairs riboflavin metabolism, thereby inhibiting the synthesis of flavin adenine dinucleotide (FAD). FAD competes with CRY proteins for binding to the E3 ubiquitin ligase substrate receptor, thereby reducing CRY degradation, while the CRY inhibits the transcriptional activity of BMAL1. Subsequent molecular experiments demonstrated that RFK knockdown not only reduced FAD synthase (FADS) levels but also exacerbated CRY1/2 degradation, disrupted circadian regulation, impaired ovarian granulosa cell function, and accelerated ovarian aging. Our findings indicate that aberrant riboflavin metabolism may accelerate ovarian functional decline by compromising circadian regulation. Collectively, this study elucidates a pivotal role for RFK in DOR pathogenesis and provides a theoretical foundation for developing novel therapeutic strategies targeting circadian pathways.