Evaluating the Impact of Membrane-Permeable Trehalose on the Survival and Dehydration Tolerance of Cumulus-Oocyte Complexes

Pei-Chih Lee¹ and Pierre Comizzoli¹

1. Smithsonian’s National Zoo and Conservation Biology Institute, Washington, D.C., USA

Abstract Text: The increasing global demand for fertility preservation is fueling interest in simpler and more economical dry-preservation methods for long-term storage at non-freezing temperatures, as an alternative to cryopreservation. While successful drying and storage at non-cryogenic temperatures of gamete nuclei have been reported, dehydration of whole germ cells remains a challenge. A key obstacle is effectively introducing into mammalian cells enough trehalose, a non-permeable disaccharide known to protect organisms against dehydration damage. This step is crucial before stably maintaining viable gametes in a dried state. An engineered trehalose derivative, trehalose hexaacetate (6-OAc-Tre), possesses high membrane permeability and can be converted to regular trehalose by intracellular esterases. The objective of the study was to evaluate the impact of the membrane-permeable trehalose on the survival and dehydration tolerance of cumulus-oocyte complexes (COCs) in the domestic cat model. COCs (N = 1200 in 6 replicates) collected from adult cats were co-incubated with 0, 3, 10, or 30 mM of 6-OAc-Tre at 38.5 °C for 4 hours or overnight in the presence of milrinone to prevent spontaneous meiotic resumption. Significant increase (P < 0.05) of intracellular trehalose (Megazyme trehalose assay) was only detectable after overnight incubation with 10 or 30 mM of 6-OAc-Tre. However, cytotoxicity examination revealed that prolonged exposure to 30 mM 6-OAc-Tre led to a lower percentage of survival (P < 0.05; Propidium Iodide exclusion assay; N = 214 in 4 replicates) and reduced oocyte competence for maturation, cleavage, and blastocyst formation (P < 0.05; in vitro maturation and fertilization; N = 313 in 4 replicates). We then evaluated the effect the two least detrimental concentrations (3 and 10 mM) of 6-OAc-Tre on dehydration tolerance of COCs. After overnight trehalose loading, COCs were dehydrated in 0.3 M external trehalose via microwave-assisted drying for 10 or 15 min, and then rehydrated immediately to assess their survival (N = 234 in 4 replicates) and DNA integrity (TUNEL assay; N = 112 in 4 replicates) of the oocytes. Without intracellular trehalose (0 mM control groups), only 33 ± 1% and 14 ± 8% of COCs survived after 10 and 15 min of dehydration, respectively. COCs pre-incubated with 3 mM 6-OAc-Tre had higher percentage of survival (54 ± 14%; P < 0.05) than the 10 mM counterparts (18 ± 11%) after 10 min of dehydration. No significant difference (P > 0.05) in percentages of survival was observed among all treatment groups after 15 min of dehydration (Range, 3 to 14%). DNA integrity in the oocytes was maintained (P > 0.05) after dehydration, regardless of 6-OAc-Tre concentration and drying time (Range, 62 to 86%). In sum, we confirmed the effectiveness of utilizing 6-OAc-Tre for intracellular trehalose loading. However, collective results also highlight the delicate balance between the protective property of trehalose and the potential cytotoxicity of its engineered derivative. Exploration of safer and more reliable trehalose delivery approaches are required to facilitate the development of effective dry-preservation strategies for oocytes.