147 EFFECT OF OVULATORY FOLLICLE DIAMETER ON THE OOCYTE TRANSCRIPTOME IN BEEF COWS

Abstract

Inadequate oocyte competence is a potential explanation for reduced pregnancy rates and(or) embryonic/fetal mortality when small dominant follicles are induced to ovulate prematurely with gonadotropin releasing hormone (GnRH). Our hypothesis was that the physiological status of an ovulatory follicle has a direct effect on competence of the oocyte and resulting embryo. The objective was to determine if the transcriptome of oocytes differ depending on whether they are collected from small or large dominant follicles following a GnRH-induced gonadotropin surge. Suckled beef cows (n = 350) were pre-synchronized with a 5-day CIDR protocol. Following pre-synchronization, GnRH1 was administered on Day –9, prostaglandin F on Day –2, GnRH2 (to initiate the ovulatory process) on Day 0, and dominant follicles were transvaginally aspirated on Day 1 before follicular rupture. On Day 0, cows were divided into small (<11.7 mm; no oestrus expression), or large (>12.5 mm; no oestrus expression) groups based on dominant follicle diameter. Oocytes were individually collected after aspiration, and RNA was later extracted from pools of 4 oocytes (n = 6 oocyte pools from both small and large follicles) and sequenced on an Illumina HiSEqn 2000 (single reads, 100 bases; Illumina Inc., San Diego, CA, USA). The sequences were tiled against a ~23 500 member bovine transcript reference obtained from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Tiling to target transcripts required a minimum match of 50 bases with at least 96% identity. Tiling counts were displayed as reads per kilobase of transcript per million mapped reads (RPKM) values, which were obtained by correcting for the length of each target (in kb) and the number of total tiled reads (in millions). Differences between groups were defined by two-tailed t-test and gene lists were selected based on P-values <0.02. Numerous differences in transcript abundance were characterised between oocytes from small and large dominant follicles. Follistatin-like 5 expression was increased (P < 0.02) in oocytes collected from large dominant follicles compared with oocytes collected from small dominant follicles. Several genes were associated with the ubiquitin pathway (e.g. ubiquitin conjugating enzyme, ubiquitin like protein-7) and were up-regulated (P < 0.02) in oocytes from large versus small dominant follicles. In addition, 4 members of the zinc finger protein family were up-regulated (P < 0.02) and 4 members were down-regulated (P < 0.02) in oocytes from large compared with small dominant follicles. In summary, some of the genes that were highly differentially regulated in bovine oocytes between small and large dominant follicles included members of the zinc finger and ubiquitin pathways, which may reflect differences in transcriptional regulation and protein turnover, respectively, between oocytes collected from large and small follicles.

Study was supported by AFRI Grant no. 2013–67015–21076 from the USDA National Institute of Food and Agriculture (Washington, DC).