Figure 4.

Expression of cumulus cell genes critical for regulation of cumulus function and oocyte maturation in Type 1 compared with excessive cpFSH-induced Types 2, 3, and 4 ovulatory-size follicles. Small ovarian reserve Holstein heifers in our previous study (Clark et al., 2022b) were subjected to ovarian stimulation with IS-cpFSH (control) or Ex-cpFSH doses and cumulus cells removed from the different types ovulatory-size follicles and subjected to RNAseq and bioinformatic analyses (Table 1 footnote). Types 1, 2, 3, and 4 ovulatory-size follicles are depicted along the x axis. Data points within each panel reflect mean expression values for replicates of each gene based on FPKM (y axis) determined in our previous study (Clark et al., 2022b). Analysis of our previously published data identified 17 shared cumulus DEGs (FDR <0.05 to FDR <0.01) common to all the Type 2, 3, and 4 ovulatory-size follicles. These 17 Ex-cpFSH-induced cumulus cell DEGs all have well-established roles in modulation of FSH action and cumulus cell function including induction of cumulus cell expansion and regulation of oocyte maturation (Table 1). Gene symbols are depicted at the top of each panel (names and functions in Table 1). Within each panel, the large black circles denote mean FPKM values, small gray circles denote each sample, and vertical lines denote SEM. Based on statistical analysis results (using DSeq2 software) from our previous study (Clark et al., 2022b), the horizontal lines at the top of each panel represent comparisons between Type 1 versus 2, 3, or 4 ovulatory-size follicles. Asterisks above each horizontal line indicate if the FDR values for each mean comparison differed significantly (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). IS-cpFSH, industry-standard commercial FSH-enriched porcine pituitary preparation; Ex-cpFSH, excessive commercial FSH-enriched porcine pituitary preparation; FPKM, fragments per kilobase per million mapped reads; DEG, differentially expressed gene; FDR, false discovery rate.