65 THE EFFECTS OF CUMULUS CELLS ON DEVELOPMENTAL RATES OF VITRIFIED MOUSE OOCYTES IN C57BL/6J STRAIN

Abstract

Unfertilized oocytes are one of the most desired germ-cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including IVF and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. We have recently reported that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence (Kohaya et al. 2011 J. Reprod. Dev. 57, 675–680). Since the previous study was carried out using ICR mice (closed colony), we examined whether our protocol can be applied for C57BL/6J mice (inbred strain), which are commonly used for production of transgenic and knockout mice. The effect of cumulus cells on the ability of C57BL/6J mouse oocytes to be fertilized and develop in vitro was examined. Cumulus oocyte complexes (COC) derived from female mice with super ovulation were collected by flushing. Cumulus cells were removed for a portion of the oocytes (DO) using hyarulonidase. Oocytes from both treatment groups (COC and DO) were then vitrified according to the protocol we previously reported (Kohaya et al. 2011). After warming, vitrified COC and DO were used for IVF. All percentage data were subjected to arcsine transformation before statistical analysis. Data were analyzed by one-way ANOVA and Tukey’s test. Significance was considered at P < 0.05. The pronuclear formation rate of vitrified DO after IVF (20/58, 33.3%) was reduced compared with vitrified COC (55/90, 62.1%). Vitrified COC showed significantly (P < 0.05) higher developmental ability to develop into the 2-cell (50/90, 57.0%) and blastocyst stages (42/90, 45.9%) compared with vitrified DO [24.8% (16/58) and 18.4% (11/58), respectively]. The vitrified COC developed to term at a high success rate (51/90, 56.7%) being equivalent to the rate obtained with IVF using fresh COC (52/90, 57.8%). Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence. These findings will contribute to improve oocytes vitrification in not only experimental animals but also in clinical application in human infertility.