Effects of microwave-assisted dehydration on the structure and function of ovarian cortex in the domestic cat model - New insights for future storage of living tissues at ambient temperatures†.

Developing protocols for ovarian cortex preservation paves the way for collection of valuable early germ cells from all female individuals, regardless of age and reproductive status. This study aimed to further optimize the microwave-assisted dehydration protocol by characterizing ovarian tissue stress response to drying in the domestic cat model. Ovaries from prepubertal cats were dissected using different techniques, exposed to different treatments, and analyzed for ovarian cortex integrity and function after dehydration and rehydration protocols. Using RNA-seq, we highlighted functions and pathways most affected by dehydration and selected marker genes for qPCR to test different protocol conditions. Switching to a biopsy puncher over our previously used dissection technique introduced the first major advancement in the protocol, improving follicular integrity and transcriptional activity after 10 min of drying compared to our previous reports. This change also improved expression of genes related to signaling, membrane transport, and transcription regulation after 10 min of drying (loss of 87.3% of water), while almost all genes were significantly downregulated after 15 min of drying (loss of 89.7% of water). Using lucifer yellow as a proxy for trehalose, we indirectly estimated trehalose uptake into follicles after membrane permeabilization with digitonin. Although increased digitonin incubation resulted in potentially higher uptake of trehalose, the associated membrane damage led to compromised follicular integrity and decreased transcriptional activity after dehydration. These results expand our knowledge of the ovarian tissue stress response to the dehydration challenge and bring us closer to optimizing the tissue preservation protocol at ambient temperatures.