230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation

Abstract

The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge. In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation. Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs. These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.