Calcium ion regulation by BAPTA-AM and ruthenium red improved the fertilisation capacity and developmental ability of vitrified bovine oocytes

Abstract

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca²⁺]i). However, little information about the mechanism by which vitrification increases [Ca²⁺]i levels or a procedure to regulate [Ca²⁺]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca²⁺]i, endoplasmic reticulum Ca²⁺ (ER Ca²⁺), and mitochondrial Ca²⁺ (mCa²⁺) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca²⁺ release into the cytoplasm, consequently increasing the [Ca²⁺]i and mCa²⁺ levels. Supplementing the cells with 10 μM 1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca²⁺]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 μM ruthenium red (RR) significantly inhibited the Ca²⁺ flux from the cytoplasm into mitochondria; maintained normal mCa²⁺ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.