Abstract
Purpose: We demonstrated that briefly treating ovaries with collagenase before exposure to a hyperosmotic solution preserved secondary follicles. However, preserving antral follicles remained difficult. We investigated whether the absence of lipids in warming (thawing) solution is a limiting factor. Methods: Frozen ovary was thawed in a lipid-containing solution and investigated for the effect on the structure, function, and viability of granulosa cells, which are important for antral follicle function. Results: Lipids incorporated into the granulosa cell membrane protected the distribution of actin structures lining the cell membrane and maintained the expression of paxillin, actin-related adapter proteins, and YAP, which induces genes critical for granulosa cell function, to support the survivability of granulosa cells in antral follicles. It also preserved the function of frozen ovaries for 3 months after transplantation into the ovarian bursa of recipient hosts, significantly increasing the total number of viable offspring. Conclusions: Lipids-key components of serum-protected the granulosa cell membrane during warming by preventing the efflux of fatty acids for maintenance of proper localization of the actin cytoskeleton and membrane proteins, supporting normal antral follicle function. The novel method combining collagenase pretreatment with a lipid-containing warming solution offers a promising approach to maintaining the function of cryopreserved ovaries.