Vitrification uses cryoprotectants and liquid nitrogen, which may cause osmotic stress and cryodamage to oocytes. Autophagy is widely considered as a survival or responsive mechanism to various environmental and cellular stresses. However, the status of autophagy in vitrified-warmed oocytes has not been studied. In this work, we investigated if vitrification-warming process induces autophagy in mouse oocytes. Four-week-old female ICR mice and GFP-LC3 transgenic mice were used. The mice were superovulated with 5IU PMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts. Oocytes obtained from several mice were pooled and divided into three groups. Group1: fresh oocytes. Group2: oocytes treated with vitification solutions (1.3 M EG+1.1 M DMSO and 2.7 M EG+2.1 M DMSO+0.5 M sucrose for 2.5 min) and warming solutions (0.5 M, 0.25, 0,125, and 0 M sucrose at intervals 2.5 min). Group3: vitrified-warmed oocytes (loaded onto an EM copper grid, and were stored in LN2 for 2 weeks). RT-PCR and confocal live imaging of GFP-LC3 were performed to examine the effects of vitrification-warming process on autophagy in oocytes. In RT-PCR analyses, expression of autophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, and Beclin1 was examined. Expression of Atg7 and Atg12 was slightly reduced in Group 3 (vitrified-warmed oocytes). The expression levels of other Atg genes did not change. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that some vitrified-warmed oocytes showed green puncta which indicate autophagic activation. All oocytes of Group 1 and Group 2 show no puncta formation. Our results suggest that induction of autophagy may serve as an indicator of conditions of vitrification-warming process. Moreover, it offers the possibility that development of methods to modulate autophagic response during cryopreservation could improve efficacy of oocyte cryopreservation.

• Soyoung Bang(Department of Biomedical Science & Technology, Konkuk University)
• Hyejin Shin(Department of Biomedical Science & Technology, Konkuk University)
• Hyunjung Jade Lim(Department of Biomedical Science & Technology, Konkuk University)