Use of fetal bovine serum to protect against zona hardening during preparation of mouse oocytes for cryopreservation.

Abstract

Addition of 20% fetal bovine serum (FBS) to media used for cryopreservation does not reduce the premature release of cortical granules but does prevent their action on the zona pellucida and thereby prevents zona hardening. In this paper, it is shown that the washing period required for removal of FBS is less than 12 min for cumulus-free oocytes and between 150 and 170 min for cumulus-intact oocytes. When these washing periods are observed after exposure of oocytes to the cryoprotectant dimethylsulphoxide (DMSO; 1.5 M) in the presence of 20% FBS, a subsequent exposure to calcium ionophore A23187 or a second exposure to 1.5 M DMSO both lead to zona hardening. This result suggests that sufficient cortical granules remain to elicit a block to polyspermy at fertilization. Oocytes, which had been exposed to DMSO and FBS and then washed free of both, were fertilized in vitro; the incidence of polyspermy was not found to be elevated over the level found in controls.