118 THE PEAK OF OXYGEN CONSUMPTION AT THE TIME OF SPERM PENETRATION IS ASSOCIATED WITH AN INCREASE IN REACTIVE OXYGEN SPECIES PRODUCTION IN BOVINE ZYGOTES

Abstract

Oxygen is essential for the generation of cellular energy (ATP) via oxidative phosphorylation, and thus oxygen consumption is a key indicator of metabolic activity within cells. Increased oxidative activity at the time of fertilization has been described for marine invertebrate oocytes. The objective of the present study was to determine if changes in oxygen consumption would occur at the time of fertilization and cell cleavage in bovine zygotes. The oxygen consumption of presumptive zygotes (n = 57) was measured individually and continuously from 6 until 30 h after IVF using the Embryoscope^™ (Unisense Fertilitech A/S). A control group of in vitro matured oocytes (n = 9) that had not been fertilized was also measured continuously from 6 h after transfer to IVF medium. Time-lapse images were acquired during the measurements at intervals of approximately 36 min and used for evaluation of the developmental progress. Oocytes/zygotes were subsequently stained with Hoechst 33342 for more accurate classification by fluorescence microscopy. Cohorts of oocytes/zygotes (n = 55) were individually stained with REDOX-Sensor Red CC-1 and Hoechst 33342 at 0, 7, 12, 17 and 24 h after IVF and imaged by confocal microscopy to determine the REDOX potential and the distribution pattern as well as nuclear stage. Data was analyzed statistically using Proc Mixed, SAS. The mean oxygen consumption of the developing zygotes peaked markedly at 9 h after fertilization (0.552 nL h^–1; value 14% over and above baseline oxygen consumption) and fell abruptly until 20 h after IVF (0.482 nL h^–1). Following this, an oxygen peak of lower magnitude (0.487 nL h^–1; value 1% over and above baseline oxygen consumption) was observed at the time of the first cleavage (detected between 21 to 32 h). The curve pattern for the control group was statistically different: the mean oxygen consumption was at its greatest from 8 to 12 h after IVF and then fell gradually until 30 h after IVF with no visible small increase in oxygen consumption at the supposed time of first cleavage. Mean respiration rates of the oocytes in the control group were significantly greater than those of the developing zygotes at all time points during the measurements. The mean pixel intensity of the REDOX-Sensor Red CC-1 staining was significantly greater at 7 h and at 24 h after IVF, when compared with the other group. The two distinct peaks of oxygen consumption were coincident with the REDOX pulses occurring at the time of sperm penetration and cell cleavage. However, due to the considerably higher magnitude of the oxygen peak occurring around the time of sperm penetration (signaling fertilization) we were led to speculate that only this peak was associated with an increased reactive oxygen species production. The REDOX pulse observed at the time of cleavage did not seem to be an oxygen-derived REDOX pulse because the oxygen consumption by the bovine zygotes was only changed marginally.

ASL is supported by FCT, Portugal. The authors thank Unisense-Fertilitech for their collaboration.