Dehydration-based preservation of cat cumulus-oocyte complexes is not improved by membrane-permeable trehalose.

Abstract

INTRODUCTION: Dry-preservation of gametes offers a promising alternative to cryobanking by potentially storing cells at ambient temperatures; however, successful preservation of whole oocytes remains limited by dehydration-induced cellular damages. This study evaluated whether intracellular delivery of trehalose via a membrane-permeable derivative, trehalose hexaacetate (6-O-Ac-Tre), could enhance dehydration tolerance of domestic cat cumulus-oocyte complexes (COCs), a relevant model for mammalian fertility preservation. METHODS: Cat COCs were incubated with 0, 3, 10, or 30&#x202f;mM of 6-O-Ac-Tre to assess intracellular trehalose incorporation and cytotoxicity. Non-cytotoxic doses (3 and 10&#x202f;mM) were then tested for their ability to mitigate cellular damage following 10 or 15&#x202f;min of microwave-assisted drying and rehydration, with outcomes including DNA integrity, mitochondrial membrane potential, cell membrane integrity, and meiotic maturation competence. RESULTS: Overnight exposure to 6-O-Ac-Tre enabled dose-dependent intracellular accumulation of trehalose. However, cytotoxicity examination revealed that prolonged exposure to 30&#x202f;mM 6-O-Ac-Tre reduced survival and oocytes' meiotic and developmental competence (&#x202f;<&#x202f;0.05). Although DNA integrity was largely preserved after drying (&#x202f;>&#x202f;0.05), meiotic maturation of oocytes was severely compromised (&#x202f;<&#x202f;0.05). Pre-incubation with 3 or 10&#x202f;mM 6-O-Ac-Tre did not mitigate (&#x202f;>&#x202f;0.05) a reduction in mitochondrial membrane potential, cell membrane integrity, and meiotic competence. DISCUSSION: These findings demonstrated that, while 6-O-Ac-Tre effectively delivered trehalose into COCs, this approach did not improve dehydration tolerance of whole oocytes under the conditions tested. Collective results reflected limitations of the 6-O-Ac-Tre delivery strategy owing to its potential cytotoxicity at higher concentrations. Alternative trehalose delivery conditions or approaches need to be explored to facilitate the development of effective dry-preservation strategies for oocytes.