L26/O-216 O-GlcNAcylation-mediated FOXL2 destabilization underlies reduced estrogen synthesis via CYP19A1 in vitrified ovarian tissue

Le résumé fourni par la source

Abstract Study question Does vitrification impaire the estrogen-producing capacity of ovarian tissue by altering O-linked N-acetylglucosaminylation (O-GlcNAcylation), and what is the underlying mechanism? Summary answer Vitrification-induced hyper-O-GlcNAcylation of FOXL2 attenuated its transcriptional regulation of CYP19A1 and consequently reduced estrogen biosynthesis. What is known already Advances in ovarian tissue cryopreservation have expanded fertility preservation to diverse clinical settings. Vitrification prevents ice-crystal damage through rapid glass-state transition and offers advantages in speed and efficiency. However, the high concentrations of cryoprotectants required may exert cytotoxic effects, potentially impairing not only follicular reserve but also ovarian steroidogenic function, including estrogen synthesis. O-GlcNAcylation is a dynamic post-translational modification that modulates protein stability and transcriptional activity in hormonal regulation and metabolism. Whether vitrification alters ovarian estrogen synthesis via O-GlcNAcylation remains unknown. Study design, size, duration Ovarian cortical tissues were obtained from five patients undergoing laparoscopic ovarian cystectomy. Fresh and vitrified tissues were compared for estrogen secretion and O-GlcNAcylation levels. The mechanism by which vitrification influences estrogen production was further investigated using primarily cultured ovarian granulosa cells. Participants/materials, setting, methods Ovarian cortical strips (1.0 × 1.0 cm) from each patient were equally divided into fresh and vitrified groups for histological analysis and 3-day culture to measure estradiol secretion. CYP19A1 expression and FOXL2 O-GlcNAcylation were assessed by western blotting and immunoprecipitation. O-GlcNAcylation sites on FOXL2 were identified by LC–MS/MS analysis. In human granulosa cells, the impact of O-GlcNAcylation on FOXL2 protein stability and its transcriptional regulation of CYP19A1 were examined using chromatin immunoprecipitation (ChIP). Main results and the role of chance Estrogen secretion was significantly reduced in vitrified-thawed ovarian tissue compared with fresh ovarian tissue (0.13±0.05 vs. 0.69±0.3, P<0.05), concomitant with increased O-GlcNAcylation of the transcription factor FOXL2 (P< 0.05). In human ovarian granulosa cells, enhancement of O-GlcNAcylation using Thiamet G (TMG), an inhibitor of O- GlcNAcase, significantly decreased FOXL2 protein stability in a time-dependent manner (P< 0.05). ChIP experiments revealed that impaired FOXL2 stability attenuated its binding to CYP19A1 gene promoter, resulting in a reduced CYP19A1 expression and estrogen biosynthesis (P< 0.05). LC-MS/MS analysis revealed identified Ser255 as a putative O-GlcNAcylation site on FOXL2. Human granulosa cells transfected with the Ser255-G-FOXL2 mutant exhibited significantly higher CYP19A1 expression and estrogen biosynthesis than cells transfected with wild-type-FOXL2 under conditions of enhanced O-GlcNAcylation with Thiamet G (P< 0.05). These results indicate that vitrification-induced hyper-O-GlcNAcylation of FOXL2 is a key mechanism underlying impaired estrogen synthesis in ovarian tissue. Limitations, reasons for caution This study is primarily based on in vitro models. Whether pharmacological modulation of O-GlcNAcylation during vitrification can improve estrogen production in ovarian tissue requires further validation at the tissue level. Wider implications of the findings These findings identify O-GlcNAcylation-mediated FOXL2 dysfunction as a novel mechanism underlying impaired estrogen synthesis after vitrification and may guide the optimization of ovarian tissue cryopreservation strategies. Trial registration number No

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.