173 Size Matters: a Follicle-of-Origin Effect on the Preovulatory Epigenetic Constitution of Germinal Vesicle-Stage Porcine Oocytes

Abstract

The goal of this study was to evaluate global levels of a variety of histone modifications at different lysine (K) residues on Histone 3 (H3) within the chromatin of porcine germinal vesicle (GV)-stage oocytes that were aspirated from follicles of different sizes. We hypothesised that we would see evidence of a transition from open, transcriptionally active chromatin (in oocytes from smaller, growing follicles) to more closed, transcriptionally silent chromatin associated with fully grown oocytes (aspirated from large, preovulatory follicles). Cumulus-enclosed oocytes were aspirated from small (<3 mm) or large (>7 mm) follicles from abattoir-derived pig ovaries. Oocytes were denuded immediately after aspiration and then immunoprobed with antibodies specific for trimethylated (me3) H3K4, H3K9me3, and H3K27me3. Background-corrected nuclear fluorescence levels for each histone mark were collected from multiple oocytes from each of at least three experimental replicates (aspiration days). Data were subjected to one-way ANOVA with a Bonferroni multiple testing correction to determine whether there were differences in fluorescence intensities in the nuclei (germinal vesicles) of oocytes from small v. large follicles. Oocytes from large follicles displayed more intense nuclear staining for all 3 histone marks: average nuclear H3K4me3 intensity was 31.4% higher (P = 0.0004), H3K9me3 was 70.3% higher (P = 0.0218), and H3K27me3 was 32.0% higher (P = 0.0231) in oocytes from large follicles. An ancillary analysis of the data revealed no effect (P > 0.1) of pubertal status (i.e. whether small and large follicles were aspirated from pre- v. post-pubertal ovaries) on the intensity of nuclear fluorescence for any of the marks evaluated. In continuation, 3 oocytes from both follicle types were collected on each of 6 aspiration days (i.e. 18 individual oocytes from each follicle type), and the mRNA from these were used for an RT-qPCR experiment to detect the relative abundance of transcripts from 21 different genes coding for histone methyltransferase or demethylase enzymes in oocytes from large v. small follicles. Of the 21 genes tested, 5 genes (KDM4C, KDM4D, KDM5B, KDM5C, and SETD7) were not detectable in our individual oocyte samples, but transcripts from 6 of the 16 remaining genes (KDM6A, KMT2B, MLL3, SETD1B, SETDB1, and SUV39H2) were shown to be significantly more abundant in oocytes from large follicles (at least 2-fold greater abundance and P < 0.05). Although our expectation-that histone marks (and related transcripts) would consistently reflect a globally “repressive” chromatin configuration in oocytes from large follicles and a more “open” configuration in oocytes from small follicles-turned out to be untrue, the evidence suggests that the epigenetic constitution of oocytes from small follicles may indeed vary from that of oocytes from large, preovulatory follicles, and this phenomenon warrants further investigation.