Abstract
Simple SummaryThe permeability of the plasma membrane to water and cryoprotectants is a critical factor in the effective vitrification of oocytes. The goal of this study is to better understand the pathways used to transport water and other cryoprotectants through the plasma membrane of bovine in vitro matured oocytes, with a focus on the role of aquaporin 7 (AQP7). We demonstrated that cryoprotectants stimulated AQP3 and AQP7 but not AQP9 expression in mature bovine oocytes. Dimethyl sulfoxide upregulates AQP3 expression, while ethylene glycol upregulates AQP7 expression in oocytes in a CPA-dependent fashion. We also demonstrated that exogenous expression of aquaglyceroporins such as AQP7 is possible in in vitro matured oocytes. When permeability values for membrane transport of dimethyl sulfoxide, ethylene glycol and sucrose were assessed, we observed that AQP7 overexpressed oocytes are more permeable to water in the presence of dimethyl sulfoxide solution. These biophysical characteristics, together with the use of membrane transport modeling, will allow re-evaluation and possibly improvement of previously described protocols for bovine oocyte cryopreservation.Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic Me2SO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of Me2SO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to Me2SO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to Me2SO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions.
Highlights
Recent developments in vitrification technology have enhanced the efficiency of bovine oocyte cryopreservation in terms of oocyte survival and embryo development rates after warming
Immunofluorescence revealed that AQP7 protein expression increased (p < 0.05) when bovine oocytes were exposed to ethylene glycol (EG) while no changes in AQP7 fluorescence intensity was observed when oocytes were exposed to Me2 SO or sucrose when compared to the control group
Water and cryoprotectant agents (CPAs) transport rates across the plasma membrane are low in simple diffusion, but they are significantly higher in facilitated diffusion where
Summary
Recent developments in vitrification technology have enhanced the efficiency of bovine oocyte cryopreservation in terms of oocyte survival and embryo development rates after warming. One of the key issues in cryobiology is the potentially lethal mechanical stresses that cells experience when exposed to high-osmolarity solutions that enable exchange of intracellular water with cryoprotectant agents (CPAs). This concern about such osmotic stress is especially acute for the ultra-fast-cooling technique known as vitrification, which uses high concentrations of CPAs to prevent the formation of lethal intracellular ice crystals [3]. The movement of water and solutes across the cell membrane plays a crucial role in cell viability because it influences the major forms of cell injury caused by cryopreservation, including damage from intracellular ice crystal formation, CPA toxicity, and osmotic stress during the addition and removal of the CPA [6,7]
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