Oocyte-secreted growth differentiation factor-9 (GDF9) plays critical roles in oocyte development by directing differentiation of the cumulus cell lineage from the granulosa cells to provide essential metabolic support for the oocyte. How oocyte-secreted GDF9 effects are restricted to cumulus cells is unknown. Recent reports show that GDF9 effects on cumulus cells can be disrupted with exogenous heparin. Heparan sulfate proteoglycans (HSPGs) are cell-surface glycosaminoglycans with a similar structure to heparin. We hypothesised that endogenous HSPGs sequester GDF9 at cumulus cell surfaces thereby restricting signalling during oocyte maturation. To explore this, we first determined the temporal expression of heparan-sulfate synthesising enzymes during maturation of cumulus oocyte complexes (COCs). We found that Ext1 was significantly induced by 3- and 6-fold at 4- and 16-h of in vivo maturation (IVV), respectively, when compared to immature COCs (P<0.05). However, Ext1 was dysregulated during in vitro maturation (IVM) with significantly less Ext1 transcript at 4- and 16-h of maturation compared to IVV. Similarly, Ext2 was significantly reduced in IVM at 8- and 16-h of maturation compared to IVV (P<0.05). This was supported by a significant increase in sulphated glycosaminoglycan (including heparan sulfate) during IVV with no increase observed during IVM. To confirm the role of endogenous heparan sulfate, we conducted a CRISPR-Cas9 knock-down of Ext1 in murine COCs using lentiviral delivery during pre-maturation. Murine COCs were collected 44 h post-eCG and transduced with lentivirus containing Ext1-specific guide RNA and Cas9 protein. COCs were maintained in meiotic arrest in maintenance culture media containing 1 x 10-8 M estradiol and 1 uM milrinone. Thirty hours post-transduction, GFP fluorescence was observed to confirm viral transduction. Successful gene knockdown was verified by cumulus expansion and qPCR for cumulus- and granulosa-cell specific mRNA transcripts. Collectively, the data suggest that heparan sulfates play an essential role in GDF9-signalling during oocyte maturation.