ABSTRACT: The proprotein convertases (PCSKs) are serine proteases with a wide range of biological substrates. Pcsk3, 5, and 6 expression is upregulated in the mouse follicle during ovulation, but their functions in the ovary are underexplored. Using an ex vivo ovulation assay, we observed that a PCSK inhibitor (PCI) impairs follicular rupture and reduces cumulus cell adherence to the (COC) matrix. PCI treatment of COCs matured in vitro recapitulated loss of cumulus cell adherence late in the maturation period (12h) and revealed that PCI does not affect COC viability or the morphokinetics of oocyte maturation. Therefore, we hypothesized that the PCSKs have a role in maintaining structural integrity of the COC matrix. We first sought to determine which PCSKs may regulate COC matrix integrity. Using multiple approaches to assess PCSK3, 5, and 6 RNA and/or protein in the COC, we identified PCSK5A as the most likely regulator of COC matrix organization. To determine which pathways are dysregulated by PCI treatment, we conducted bulk RNA sequencing comparing control and PCI-treated COCs collected 0, 4, 8, or 12h after onset of in vitro maturation. 8h PCI-treated COCs clustered with 12h control COCs, indicating that PCI accelerates the normal temporal expression profile of COCs. Pathway analysis revealed that PCI treatment upregulated pathways related to metabolism and cell motility, and downregulated pathways corresponding to ECM organization, cell adhesion, and TGF-beta signaling. We then conducted proteomics of CTL vs. PCI-treated COCs at 10h to determine potential PCSK substrates that are affected by PCI treatment. Proteomics detected 79 proteins altered by PCI treatment, with functions related to ECM remodeling, cell adhesion/migration, cell signaling pathways, and metabolism. Notably, the oocyte proteome was stable with PCI treatment, consistent with earlier findings that the effect of PCI appears to be specific to cumulus cells. To validate PCI-induced ECM changes, we evaluated the PCI-treated COC matrix with H&E and a hyaluronic acid binding protein assay, which confirmed disruption of COC matrix integrity with PCSK inhibition. We then validated the effect of PCI on cumulus cell migration first with scanning electron microscopy, which revealed increased plasma membrane blebbing on the surface of PCI-treated cumulus cells. Live tracking of COC expansion showed that compared to control cumulus cells, PCI-treated cumulus cells have an increased mean square displacement and dysregulated anomalous diffusion, consistent with changes to cumulus cell migration. Based on the dysregulation of ECM organization and TGF-beta signaling pathways observed we PCI-treatment, we identified GDF9 as a substrate of interest, as it is a TGF-beta ligand with known PCSK consensus sequences and roles in COC expansion. Supplementation of recombinant GDF9 fully rescued COC matrix integrity, suggesting that GDF9 likely acts downstream of the PCSKs to regulate matrix organization. In conclusion, this study utilized a multi-omics approach to identify novel roles and molecular targets of the PCSKs in the COC during ovulation, with implications on development of fertility therapeutics and non-hormonal contraceptives.
