123 CRYOPROTECTANT EXPOSURE AFFECTS THE PROTEOME OF THE MOUSE METAPHASE II OOCYTE

Abstract

The ability to routinely cryopreserve oocytes will have a significant impact on human- and domestic animal-assisted reproduction, and on wildlife conservation. However, success with oocyte cryopreservation has been relatively limited. Previous studies have shown that slow freezing alters mouse oocyte physiology, including the proteome. To improve cryopreservation techniques, it is vital to investigate the effects of the cryopreservation procedure on oocyte physiology. It was therefore the aim of this study to determine at which stage of the slow freezing procedure the proteome was affected, or whether exposure to the cryoprotectant (1,2-propanediol; PrOH) alone was responsible for cellular perturbations. Metaphase II (MII) oocytes were collected from superovulated F1 (C57BL/6 x CBA) mice at 13 h post-hCG. Denuded MII oocytes were randomly allocated to treatments that mimic parts of the slow freezing protocol: (A) 37�C for 20 min (control), (B) room temperature for 20 min (temperature control), (C) 1.5 M PrOH exposure for 20 min (i.e. the equilibration and dehydration step), and (D) 1.5 M PrOH exposure and seeding (i.e. the equilibration, dehydration, and seeding step). Oocytes were then collected in groups of 5 (n = 12 replicates per group), extracted, processed, and analyzed by time-of-flight mass spectrometry. Statistical analysis was performed with the Mann-Whitney non-parametric test. Oocytes maintained at room temperature for 20 min displayed a protein profile similar to that of oocytes incubated at 37�C. The protein expression profile was altered when oocytes were exposed to PrOH for twenty min, with several proteins showing at least a two-fold change of expression level. A similar effect on protein expression was also observed with 20 min of PrOH exposure plus seeding. On comparison with control oocytes, those exposed to PrOH (with or without seeding) had a significantly (P < 0.05) lower abundance of 6 proteins and up-regulation of 4 proteins. Analysis of the mouse MII oocyte at different stages of the slow freezing protocol revealed that there is negligible impact on protein expression when oocytes are maintained at room temperature for 20 min. However, PrOH exposure at room temperature for the same amount of time induces significant perturbation of the oocytes' proteome. These data demonstrate that temperature changes per se (during cooling to room temperature and seeding) impact negligibly on the oocyte proteome, whereas exposure of oocytes to 1.5 M PrOH alone, which mimics the equilibration phase of slow freezing, significantly alters the proteome. The detrimental effect on the oocyte proteome suggests that chronic exposure to PrOH during slow freezing negatively impacts oocyte cryopreservation. To conclude, analyzing the effects of cryopreservation on cell physiology is pivotal for allowing the selection of appropriate techniques and implementing appropriate modifications. This study was supported by a grant from Vitrolife.