Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Calcium signalling in cattle cumulus-oocyte complexes during in vitro fertilisation (#479)

Hanna McLennan 1 , Melanie Sutton-McDowall 1 , Jeremy Thompson 1 , Sabrina Heng 2
  1. Discipline of Obstetrics and Gynaecology, School of Medicine, The University of Adelaide, Adelaide, SA, Australia
  2. Department of Chemistry, School of Physical Sciences, The University of Adelaide, Adelaide, SA, Australia

Oocyte activation, or oocyte-embryo transition, following fertilisation is driven by repetitive calcium oscillations that regulate mitochondrial activity, zona hardening, pronuclear formation and syngamy. Calcium oscillations have been extensively studied in mouse oocytes but minimal studies have been conducted in larger mammals such as cows. Furthermore, it is unknown if cumulus cells play a role in oocyte activation signalling despite the fact they remain present for in vivo fertilisation. Therefore, the aim of this study was to investigate calcium changes in the cattle cumulus-oocyte complex (COC) and its surrounding media.


After 23 hours of in vitro maturation, COCs were inseminated in vitro with frozen-thawed spermatozoa, with a no sperm negative control. At approximately 3 hours post-insemination, COCs to be imaged were cultured in 5 μM of the calcium fluorophore Fluo-4AM for 30 minutes, washed and transferred into 2 μl drops covered with mineral oil in glass-bottomed confocal dishes. COCs were imaged using a confocal microscope every 2 minutes for 8 hours. Changes in the fluorescence intensity were measured in defined regions; namely the oocyte, cumulus vestment and surrounding media.


Fluorescence intensity peaked within the oocyte and cumulus cells, with timing corresponding to expected sperm entry time in cattle (399-405 minutes), followed by 2-4 fold intensity increase in the surrounding culture media. In the no sperm control group, fluorescence remains relatively constant and any fluorescence loss is random and not synchronised like their fertilised counterparts. Waves of calcium fluorescence within the oocyte originated from a single point, expected to be the point of sperm fusion as suggested in the literature. Furthermore, increases in fluorescence in the surrounding media following fertilisation may be indicative of successful fertilisation.


This study demonstrates that cumulus cells are involved in calcium homeostasis during fertilisation, though their exact role remains to be elucidated.