214 OOSNCR1, a novel oocyte-specific long non-coding RNA in cattle, is cytoplasmic and maternally derived

In mammals, proper development during early embryogenesis relies heavily on the regulation of maternal transcripts. Transcripts must undergo a gradual degradation accompanied by a concomitant activation of the embryonic genome. Long noncoding RNAs (lncRNAs) have been documented to regulate various cellular processes, including modulation of gene expression and epigenetic status. Recently, lncRNAs have been functionally characterised as key regulators of embryonic genome activation in humans and mice. Using RNA-sequencing, our laboratory identified 1,535 lncRNAs in bovine oocytes. The objectives of this study were to determine whether OOSNCR1, a novel lncRNA, is maternal in origin, and to localise the transcript in germinal vesicle (GV) oocytes using fluorescence in situ hybridisation (FISH). Ovaries were collected from a commercial abattoir and aspirated oocytes were subjected to in vitro embryo production, where presumptive zygotes were treated with an RNA polymerase inhibitor, a-amanitin, or fixed for FISH. For experiment one, presumptive zygotes were randomly assigned at 12 hpi to either control or a-amanitin (25 mg/mL) treated culture medium. All embryos were cultured in pools of 10 (n = 6) until either the fourth embryonic division at 52 hpi or Day 8 blastocysts. All embryos collected at 52 hpi for analysis were at the 16-cell stage. All samples were snap-frozen in liquid nitrogen and stored at −80°C until isolated. RNA isolation was performed followed by cDNA synthesis. RT-qPCR analysis was performed, and data were analysed via the standard curve method using GFP as an exogenous control. All data are expressed as relative lncRNA abundance. Embryo expression data were log-transformed, and a Student’s t-test was performed to compare treated and control embryos. For localisation studies, GV oocytes were fixed, permeabilised, and hybridised to a custom probe specific to OOSNCR1. Following hybridisation, oocytes were whole mounted to slides and imaged using confocal microscopy. Embryo expression data revealed no significant difference between transcript amount in treated (15.46 ± 0.0074) versus control embryos (15.60 + 0.0049; P = 0.34), signifying a maternal origin. Embryos cultured in a-amanitin versus control revealed blastocyst rates of 0% and 50%, respectively. FISH detected OOSNCR1 throughout the cytoplasm of the GV oocyte. Together, these data indicate OOSNCR1 is maternally derived and localised to the cytoplasm of GV oocytes. Future studies aim to localise OOSNCR1 throughout early embryonic development and elucidate its functional role during early embryonic development.