During fertilisation the oocyte undergoes a dramatic increase in metabolic activity as it transitions into an embryo in a process called oocyte activation. Our pilot study shows a loss of Fluo4AM-labelled calcium fluorescence in the cumulus cells during time-lapse imaging of oocyte activation, but it is unknown how the cumulus cells are changing. This study hypothesises that this fluorescence loss is due to the cumulus cells losing their membrane integrity.
Abattoir-derived ovaries were aspirated to collect COCs, which were matured for 22 hours and fertilised with frozen-thawed spermatozoa including a no sperm control. Approximately 3 hours later, COCs were stained with 5μM Fluo4AM to label calcium, washed and put into individual 2μl drops of 1X DeadRed stock solution in a glass-bottomed confocal dish to assess cumulus cell membrane integrity. The COCs were imaged every 5 minutes for 6 hours before being returned to culture to assess development at Day 8. FIJI image analysis software was used to measure the fluorescence intensity changes in the cumulus vestment.
The mean fluorescence intensity of DeadRed staining in the cumulus cells significantly increases (Paired t-test, p<0.001) following a significant decrease in Fluo4AM fluorescence (Paired t-test, p<0.0001) at the time of oocyte activation in cattle. This loss was co-ordinated in sperm exposed COCs compared to random loss in the no sperm control but there was no relationship to embryo development. A 30% DMSO concentration with DeadRed showed the oocyte fluoresce red with no cumulus cell change, indicating the loss of membrane integrity is not due to oocyte death. However, the DeadRed fluorescence increase is only seen when both Fluo4AM and laser exposure are present.
It is concluded the change in cumulus cells at the time of oocyte activation is a result of membrane integrity loss, but this effect is induced by the imaging methodology.