General Features of Certain RNA Populations from Gametes and Cumulus Cells

Abstract

Abstract.Results described herein provide insight regarding certain features of gamete RNA and how they compare to cumulus cell RNA. In particular, 28S/18S rRNA ratio and size distribution of RNA molecules differed in total RNA from oocytes versus surrounding cumulus cells. Specifically, oocyte total RNA had a lower rRNA ratio and an increased abundance of smaller RNA sizes compared to RNA from surrounding cumulus. Extensive efforts demonstrated that observed differences were repeatable whether oocyte maturation occurred in vitro or in vivo, and were similar between the nuclear stages examined. Features of oocyte RNA were conserved across six mammalian species, yet differed from surrounding cumulus. Profiles of sperm RNA were also examined but had no discernible ribosomal RNA peaks and were conserved across four mammalian species. Because the oocyte and spermatozoon are highly specialized cells representing unique molecular entities required for proper embryo development, dissimilarities described herein likely represent real gamete versus cumulus RNA differences. Key words: Bioanalyzer, Cumulus, Oocyte, RNA, Sperm (J. Reprod. Dev. 56: [583][584][585][586][587][588][589][590][591][592] 2010) pon resumption of meiosis and germinal vesicle breakdown, oocytes become transcriptionally quiescent and must rely upon pools of RNA accumulated during the growth phase for protein synthesis [1]. Transcriptional inactivity and reliance upon maternal RNA stores persist until after fertilization and, in some species, until the third cleavage division [2]. In our laboratory, stress-induced perturbations in the total amount of polyadenylated RNA in oocytes during maturation were coincident with reductions in developmental competence [3]. Before performing microarray analyses to identify the impact on individual transcripts, assessment of RNA integrity was an important first step. To this end, initial efforts using microcapillary electrophoresis suggested differences between oocyte and cumulus RNA [4]. The first notable difference was that the rRNA ratio of total RNA from oocytes was significantly less than that observed in total RNA from their surrounding cumulus cells. A second notable feature was that oocyte RNA appeared to have an increased abundance of smaller RNA sizes.This serendipitous finding led us to conduct a series of investigations to address the extent to which certain features of oocyte RNA differ from surrounding cumulus, which are more somatic cell-like. In this regard, extensive effort was taken to determine whether RNA features were oocyte-specific or an artifact related to procedures or origin (i.e., from oocytes of antral follicles not otherwise destined to ovulate). Specifically, bovine oocyte RNA from different stages of in vitro maturation was evaluated in retrospect using data originating from different studies. When available, cumulus RNA characteristics were also included for comparison to RNA from oocytes. Additional effort was then put forth to examine 1) conservation of certain oocyte RNA feature...

Impact of Heat Stress Exposure During Meiotic Maturation on Oocyte, Surrounding Cumulus Cell, and Embryo RNA Populations

“…To this end, total RNA did not change during meiotic maturation which is consistent with previous findings for bovine oocytes [44,67]. The amount of RNA obtained for GVstage and matured oocytes was in agreement with others [~2 ng/ oocyte; 67, 68].…”

“…Microcapillary-electrophoretic separation and detection via laser-induced fluorescence using RNA 6000 Pico Lab Chips allowed for assessing size distribution of RNA [43] in oocytes, surrounding cumulus and resultant embryos as previously described by Payton et al [44]. For each sample (1 μl of eluted RNA), the Bioanalyzer software generated a rRNA ratio (area under 28S peak divided by area under 18S peak) and an RNA integrity number (RIN) which is an algorithm-based value taking into account all the regions of the electropherogram [43].…”

“…Total RNA was quantified using Quant-iT RiboGreen RNA Reagent and kit (Invitrogen, Carlsbad, CA, USA) by analyzing 1 μl of eluted RNA (diluted 200×) in triplicate using a TBS-380 fluorometer (Turner BioSystems, Sunnyvale, CA, USA). Amount of total RNA per oocyte was calculated from simple linear regression of a standard curve (1 to 400 ng/ml; interassay CV= 9.1% and 2.5% for 2000-and 200-fold dilution assays, respectively).Microcapillary-electrophoretic separation and detection via laser-induced fluorescence using RNA 6000 Pico Lab Chips allowed for assessing size distribution of RNA [43] in oocytes, surrounding cumulus and resultant embryos as previously described by Payton et al [44]. For each sample (1 μl of eluted RNA), the Bioanalyzer software generated a rRNA ratio (area under 28S peak divided by area under 18S peak) and an RNA integrity number (RIN) which is an algorithm-based value taking into account all the regions of the electropherogram [43].…”

Developmental competence of mammalian oocytes-insights into molecular research and the promise of microfluidic technology: a review

“…However, equine blastocysts obtained from heat-stressed oocytes had increased levels of HSP70 mRNA in comparison to non-heatstressed oocytes. Additionally, the overall mRNA population did not change during meiotic maturation, which is in agreement with results reported by Payton et al (2010). During meiotic maturation, a significant 30% to 50% decrease was observed in the poly (A) mRNA subpopulation (Lequarre et al 2004).…”

Effect of DNase treatment on RNA extraction from preimplantation murine embryos

“…Evaluation of RNA quality and purity are important steps prior to microarray or quantitative real-time polymerase chain reaction (RT-qPCR) analysis, especially when using preimplantation embryos, which are very limited samples of doubtful RNA quality (Payton et al, 2010). Bustin and Nolan (2004a) and Pfaffl (2005) have reported that there are some criteria that should be followed during RNA extraction procedures to obtain protein-and gene-free RNA that is not degraded, and does not contain enzymatic inhibitors or nucleases.…”