Patterns of intracellular Ca2+ concentrations in fertilized bovine eggs

Abstract

Sperm-induced calcium (Ca2+) changes were examined in zona pellucida- intact, mature bovine eggs injected with the fluorescent Ca2+ indicator fura-2 dextran (fura-2 D). Fifty four percent (37/68) of the dye-injected, inseminated bovine eggs were fertilized and 43% (16/37) of the fertilized eggs exhibited Ca2+ elevations during the time of measurement. All (16/16) of the eggs with Ca2+ elevations were fertilized but none of the unfertilized eggs (0/31) showed intracellular Ca2+ elevations. Six of 13 eggs that were later examined and found to be fertilized at the time of the Ca2+ recordings did not show sperm-induced Ca2+ elevations. Fifty percent (8/16) of the eggs with Ca2+ elevations exhibited a single Ca2+ rise as a response to sperm penetration during the 60-min period in which these eggs were monitored. Twelve percent (2/16) of the eggs responded with two Ca2+ elevations spaced by 50- and 51-min intervals and 38% (6/16) of the eggs exhibited multiple elevations with intervals of 15-29 min. In the latter group, one egg was polyspermic. The mean amplitude of the sperm-induced Ca2+ elevations was 564 ± 58 nM. Eggs with single elevations reached higher peak concentrations than eggs with multiple elevations (p < 0.05). The mean duration of the Ca2+ elevations was 166 ± 13 sec and was similar among eggs with different Ca2+ patterns. The first elevations detected occurred at a mean of 6.6 ± 0.5 h after insemination. Fertilization in this study was confirmed by looking at pronuclear formation 16 h post-insemination or by DNA staining immediately after the fluorescence readings. Eggs exhibiting Ca2+ elevations ranged in stage of fertilization from just penetrated to pronuclear. Injection of inositol 1,4,5 trisphosphate (5 μM in the injection pipette) into 6 bovine eggs induced an immediate Ca2+ elevation with a mean peak Ca2+ value of 700 ± 60 nM and a mean duration of 103 ± 21 sec. Incubation of bovine eggs with 200 μM thimerosal induced periodic Ca2+ rises. The mean number of Ca2+ elevations observed in 35 min of recordings was 3.0 ± 0.5 (n = 9, range 1-5). The mean peak Ca2+ value of the first thimerosal-induced Ca2+ elevation was 990 ± 210 nM. The results of this study indicate that fertilization can evoke intracellular Ca2+ elevations in bovine eggs and that the periodicity of these Ca2+ elevations is different among eggs. Furthermore, both inositol 1,4,5 trisphosphate and thimerosal were able to induce intracellular Ca2+ release in bovine eggs.