Figure 2: PCSK5A is the primary PCSK regulating cumulus cell function during ovulation.

A) Heatmap depicting expression of Pcsk3, 5, and 6 in early (4 h post-hCG) and late (12 h post-hCG) cumulus cells from a published single cell dataset of the mouse ovary throughout an in vivo time course of ovulation⁴¹. B) Representative brightfield images from RNA in situ hybridization depicting expression of Pcsk3 and Pcsk5 in COCs at 0 h, 4 h, 8 h, or 11 h post-hCG injection. C) Quantification of Pcsk3, 5, and 6 expressions throughout ovulation. Data are represented as mean ± SEM. p-values as follows: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001 by mixed-effects analysis. n=3-5 biological replicates per Pcsk. D) Representative brightfield images from immunohistochemistry depicting abundance of Pcsk3 and Pcsk5 in COCs at 0 h, 4 h, 8 h, or 11 h post-hCG injection. E) Quantification of PCSK3 and PCSK5 protein throughout ovulation. Data are represented as mean ± SEM. p>0.05 for all timepoint comparisons by one-way ANOVA. n=2 biological replicates per PCSK. F) Representative brightfield images of COCs treated with proprotein convertase inhibitor (PCI), Furin inhibitor (FII), or DMSO control (CTL) following culture and transfer out of the EmbryoScope+^™ dish. G) Graph showing COC area of CTL, FII-, or PCI-treated COCs following transfer out of the EmbryoScope+^™ dish. Data are represented as mean ± SEM. CTL and PCI data replotted from Fig 1E (experiments conducted in parallel). **** p < 0.0001 by Welch’s t-test. n=11-26 biological replicates.