153 Fertilisation of Cattle Oocytes is Linked to Novel Waves of a Ca²⁺ Fluorophore in Cumulus Cells

Abstract

During fertilization, multiple intracellular calcium (Ca²⁺) oscillations are initiated after sperm binding to the oocyte vitelline membrane. This Ca²⁺ signalling has been extensively studied in denuded mouse and Xenopus oocytes but minimally studied in larger mammals. Cows in particular are unusual, as the few studies on oocyte activation have observed fewer Ca²⁺ oscillations during fertilisation compared with mice. Furthermore, cattle intracytoplasmic sperm injection (ICSI) is inefficient, despite parthenogenetic activation occurring readily. We hypothesise that cumulus cells are important for cattle oocyte activation at fertilisation. Here, we assessed the behaviour of Ca²⁺oscillations in fertilising intact cattle cumulus–oocyte complexes (COC). Abattoir-derived cattle COC were matured and fertilised in vitro using Bovine Vitro Media Suite (IVF Vet Solutions). The COC were stained 3.5 h after insemination with the Ca²⁺ fluorescent probe Fluo4AM (5 μM, Molecular Probes Inc., Eugene, OR, USA) for 30 min, washed, and imaged every 5 min for 6 h in a Fluoview FV10i incubating time-lapse confocal microscope (Olympus) before being returned to culture. Embryo development was assessed at Day 8 to confirm fertilisation. Fluo4AM fluorescence intensity was assessed using FIJI ImageJ. Mean relative intensity over time was graphed for specific regions of interest and the area under graphs was calculated to quantify differences for comparison using a Mann-Whitney Test (mean ± SEM). Experiment 1 (4 reps of 10 COC) compared confirmed fertilised v. uninseminated; experiment 2 (2 reps of 10 COC) compared inseminated COC ± 10 μM BAPTA-AM (Ca²⁺ chelator, Sigma-Aldrich, St. Louis, MO, USA). There were distinct coordinated waves of differing Fluo4AM intensity in both the oocyte and the cumulus cells surrounding the confirmed fertilised oocytes. This contrasted to the random uncoordinated flashes of Fluo4AM fluorescence in the cumulus cells of the uninseminated oocytes. The fluorescence pattern in +BAPTA-AM COC matched the random flashes observed in the uninseminated group of experiment 1. The fluorescence in the media surrounding the COC immediately following the Fluo4AM waves spiked and then plateaued at a higher level of fluorescence. This was quantified by assessing the area under the graph for 1 h of the plateau following the fluorescence spike. There were no differences between confirmed fertilised (346.4 ± 41.62) and uninseminated groups (239.8 ± 32.08; P > 0.05), but this was affected by differences in cumulus dispersal due to the presence or absence of sperm. Experiment 2 used BAPTA-AM to block oocyte activation with sperm present in both groups and showed a significant difference between the fluorescence increase in the media of the 2 groups (–BAPTA-AM: 311.2 ± 31.57, +BAPTA-AM: 201.4 ± 26.59; P < 0.03). Although the physiological significance has yet to be determined, we have observed a novel Ca²⁺ wave in the cumulus cells that could be linked to oocyte activation in cattle. There was a significant increase in Fluo4AM fluorescence in the media surrounding the COC, which may indicate cumulus cells are releasing Ca²⁺ at the time of oocyte activation.