Acute high-dose irradiation disrupts cell adhesion and Silk-Ovarioid formation in human primary ovarian cells

Methods

Ovarian tissue was obtained from eight patients (aged 23–36 years) undergoing gender-affirming surgery at Karolinska University Hospital in Huddinge, Sweden. The tissue was separated into cortex and medulla and dissociated into cPOCs and mPOCs. Monolayer cultures of cPOCs and mPOCs were exposed to 10 Gy X-rays upon reaching confluency, or left unexposed as paired controls.

Following irradiation, cells were assessed for ATP content and mitochondrial dehydrogenase activity, followed by immunofluorescence staining, bulk RNA sequencing (Illumina Stranded mRNA Prep Ligation protocol; sequencing on the Illumina NovaSeq 6000 platform), bulk proteomic analysis (liquid chromatography–tandem mass spectrometry), and a functional assay for assessing their ability to form 3D Silk-Ovarioids. Results While irradiation did not significantly affect cell viability, immunofluorescence analyses revealed alterations in DNA damage response, apoptosis, and cell cycle regulation.

Transcriptomic analysis showed minimal changes at 1 h post-irradiation in both cPOCs and mPOCs. However, marked shifts in transcriptomic profiles were observed at 4 h (2,810 and 2,540 DEGs in cPOCs and mPOCs, respectively) and at 24 h (2,462 and 2,802 DEGs, respectively), including upregulation of the p53 pathway and downregulation of MYC targets, E2F targets, the G2/M checkpoint, and the mTORC1 pathway. At the proteomic level, differentially expressed proteins associated with cell adhesion, focal adhesion, and cadherin binding were detected at 24 h post-irradiation.

Functionally, irradiated cells demonstrated an impaired capacity to self-organize into 3D Silk-Ovarioids, indicating compromised cell–cell adhesion. Conclusion These findings reveal a novel mechanism by which radiotherapy may damage ovarian tissue independently of follicular loss, underscoring the need for targeted strategies to preserve somatic cell function in fertility preservation protocols.