Fertility potential of mouse oocytes revealed by cytosolic calcium after cryopreservation and in vitro maturation

Abstract

OBJECTIVE: To test whether cryopreservation of mouse oocytes at an intermediate stage of maturation in vitro is optimal for developmental competence. DESIGN: Germinal vesicle (GV) oocytes were matured in vitro to metaphase II (MII) with or without cryopreservation at different stages. At MII they were activated by intracytoplasmic sperm injection (ICSI) and intracellular calcium oscillations were monitored to predict developmental competence. MATERIALS AND METHODS: 6 week-old B6D2F1 mice were primed with PMSG and oocytes harvested for cryopreservation in 1.5M 1,2-propanediol at GV, pro-metaphase I (GVBD) or MII stages, or used as fresh controls. Oocytes were matured to the MII stage after thawing, and ICSI was performed with B6D2F1 sperm using a Piezo drill. Calcium oscillations were subsequently measured in oocytes loaded with Fura-2/AM using a fluorescence microscope and InCyt 2 software to measure fluorescence ratios at 340/380 nm and determine intracellular calcium. The data were analyzed by non-parametric statistical tests and ANOVA. RESULTS: There were no significant differences in survival between the three stages at cryopreservation (84-94%). Fresh oocytes exhibited normal calcium spikes every 15-20 min post-fertilization lasting 3-4 hr. After cryopreservation at GV or GVBD stages, maturation to MII and ICSI, persistent calcium oscillations were induced similar to fresh oocytes, but they were truncated or absent in oocytes cryopreserved at the MII stage. CONCLUSIONS: Cryopreservation of oocytes prior to the MII stage appeared to improve developmental potential as indicated by their ability to support normal intracellular calcium oscillations. There was no reduction of survival after thawing. Since calcium signaling is essential for successful fertilization and an excellent indicator of competence, there may be an unexpected advantage of banking oocytes at GV or GVBD stages instead of MII, perhaps from a longer cellular recovery time after the stress of cryopreservation.