O-154: The effect of chelating cytosolic calcium on the viability of vitrified mouse oocytes

Abstract

Vitrification is an alternative to cryopreserve MII oocytes, however, the embryonic potential of oocytes vitrified by choline chloride DMSO-EG-sucrose vitrification system is still low. Recently, it has been reported that DMSO and EG may induce the increase of intracellular calcium. This increase in calcium may have detrimental effects: premature cortical granule release, zona pellucida hardening, metaphase-to-anaphase transition, and/or egg activation. Preventing the increase of intracellular calcium by calcium-free media combined with a cell membrane permeable calcium chelator was carried out to assess the viability of mouse oocytes exposed to a choline chloride DMSO-EG-sucrose based vitrification system. Prospective study using mouse oocytes at a University-based research laboratory. MII oocytes were collected from 6-8 weeks old CB6F1 mice by hyperstimulation of 5IU PMSG and 5IU hCG. MII oocytes were denuded by brief exposure to 100iu/ml hyaluronidase, and then cultured in vitro less than 1hour prior to exposure of choline chloride DMSO-EG-sucrose vitrification system. Oocytes were loaded in pulled straw and heat-sealed just before plunging directly into liquid nitrogen. The experimental groups include presence or absence of calcium in the vitrification system, and concentration of BAPTA AM, a cell permeant calcium chelator. Group 1, calcium-based PBS; Group 2: calcium-free PBS; Groups 3 - 7: calcium-free PBS + varying concentrations (0.1μM, 0.5μM, 5μM, 50μM and 500μM) of BAPTA AM. After thawing, eggs were allowed to recover for one hour. Surviving oocytes were inseminated by capacitated sperm. Embryos were cultured in sequential Quinn’s Media (Sage) supplemented with 6% plasmanate, and monitored at day 1 and day 5(Table 1). Day 5 blastocysts were graded and corroborated by double staining of Oct 3/4 (detect functional inner cell mass) and of Hoechst 33342 (detect total embryonic cell number). Mouse oocytes, in response to 0.1μM BAPTA based vitrification system, display high rate of survival (97%) and cleavage (71%), comparable to that response to calcium based and calcium-free based vitrification system. However, raising the concentration of BAPTA to 0.5μM and above, the rate of survival (<82%) and cleavage (<55%) declined dramatically. Furthermore, more embryos in group of 0.1μM BAPTA develop into good (45% ≥3BB) and excellent (32% ≥4AA) blastocyst than in other groups. The group of 0.5μM BAPTA yielded less blastocysts, but still generated a number of good and excellent blastocysts comparable to the group of 0.1μM BAPTA. BAPTA influences the viability of mouse oocytes in a concentration dependant manner. BAPTA, at a concentration of 0.1μM (equivalent to cytosolic Ca2+ concentration -10-7 M = 0.1μM), exhibits the maximum benefit to improve the viability of vitrified oocytes. The effect of BAPTA on post-implantation embryos and full-term offspring needs to be addressed in further study.