Oral Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2013

Haemoglobin: A gas transport molecule lost during oocyte in vitro maturation (IVM). (#152)

Hannah M Brown 1 , Karen L Kind 1 , Darryl L Russell 1 , Rebecca L Robker 1 , Robert B Gilchrist 1 , Jeremy G Thompson 1
  1. The Robinson Institute, Research Centre for Reproductive Health, School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide, SA, Australia

Haemoglobin is the iron-containing oxygen-transport metalloprotein in erythrocytes of all vertebrates.  In addition to oxygen, haemoglobin also binds carbon dioxide, nitric oxide and carbon monoxide.  Decades of research have intricately characterised the role of haemoglobin in erythrocytes, however more recently, haemoglobin has been described in neurons, macrophages, alveolar cells, mesangial cells in the kidney and in endothelial cells.  Micro-array analysis comparing in vivo and in vitro matured cumulus oocyte complexes (COCs) revealed a drastic reduction of haemoglobin A (Hba) expression in COCs matured in vitro, which was confirmed by PCR.  Temporal gene expression analysis was then undertaken from isolated cumulus and granulosa cells and expression patterns described for the haemoglobin subunits, and members of the heme biosynthesis pathway.  Both haemoglobin alpha (Hba) and beta (Hbb) were significantly regulated in cumulus cells by hCG administration with a 9.9 and 5.2-fold increase respectively, following 12 h hCG administration.  Hba and Hbb were also regulated in granulosa cells at 12 h following hCG (4.7 and 4.6-fold respectively). Additionally, haptoglobin, a protein capable of binding free haemoglobin was also increased more than 5-fold following hCG treatment in granulosa cells. Interestingly, bisphosphoglycerate mutase (Bpgm), responsible for the synthesis of 2,3-bisphosphoglycerate (2,3-BPG), a glycolysis intermediate which mediates oxygen binding to Hb was dramatically down regulated in both cumulus and granulosa cells following hCG administration.  Protein analysis revealed a unique spatial distribution of HBA in the COC.  In vivo, in immature COCs (eCG 48 h), HBA was widely and uniformly distributed within the cytoplasm of the oocyte, whilst in mature COCs (hCG 12 h), HBA was found in clumps within the cytoplasm, and in the perivitelline space.  Following ovulation (hCG 16 h), HBA was again more uniformly distributed within the oocyte.  Following in vitro maturation (IVM), the oocyte was completely devoid of HBA, but interestingly, HBA was able to accumulate within the oocyte following the addition of either purified HB or ferrous-HB during maturation.  While the role for haemoglobin within the oocyte remains elusive, we propose it is important for oxygen transfer and/or nitric oxide signalling in the oocyte, both of which are critical for reproductive success.