The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes

The 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 (MeSO) 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 MeSO 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 MeSO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to MeSO 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 MeSO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions.