Transcriptional profile associated with human oocyte meiotic maturation. Transcriptional profile associated with human oocyte meiotic maturation

The transition from a transcriptionally active state (GV) to a transcriptionally inactive state (mature MII oocytes) is one of the requirements for the acquisition of oocyte developmental competence. After maturation, oocytes are mostly transcriptionally quiescent, and developmental competence prior to embryonic genome activation (EGA) relies on maternal RNA and proteins. The landscape of expressed genes at the MII stage could be mostly driven by post-transcriptional mechanisms, such as alternative splicing (AS). With the development of single cell transcriptome analysis, genome wide AS analysis becomes technically feasible and available to fully characterize the AS patterns in human oocytes. Profiling spliced mRNA isoforms might provide novel information on the molecular mechanisms driving early development, and might be a source of potential biomarkers of oocyte quality. The goal of the present study is to perform a transcriptomic analysis in oocytes at different stages of maturation, to identify the profiles of alternative spliced isoforms produced in both oocyte’ stages. Overall design: A total of 12 in vivo matured (MII) and 4 non-matured (GV stage) oocytes from 16 women undergoing oocyte donation were included in this study, processed and analyzed individually. Average women age was 28 ± 4.7 years (range 21-34), with a mean antral follicle count (AFC) of 21 ± 10.7 follicles (range 5-43). Oocytes were divided in 4 experimental groups (4 oocytes in each group) according to their maturation stage, and the age and AFC of the women (mean ± SD): GV oocytes from women up to 30 years old with high AFC (GV group; 26±4.1 years old and 27±13 follicles), MII oocytes from women up to 30 years old with high AFC (H-MII group; 26±4.6 years old and 24±3 follicles), MII oocytes from women with low AFC (L-MII group; 27±5.4 years old and 7±1 follicles) and MII oocytes from women above 31 years old with high AFC (O-MII group; 32.8±1.5 years old and 27±6 follicles). Total RNA from each oocyte independently was isolated, amplified, labeled, and hybridized on HTA 2.0 arrays (Affymetrix).

转录活跃状态（GV，生发泡期，germinal vesicle）向转录非活跃状态（成熟MII（减数分裂II期）卵母细胞）的转变，是卵母细胞获得发育潜能的必要条件之一。成熟后的卵母细胞大多处于转录静止状态，而胚胎基因组激活（EGA，embryonic genome activation）前的发育潜能依赖于母源RNA与蛋白质。MII阶段的表达基因谱，其调控大多由可变剪接（AS，alternative splicing）这类转录后机制所驱动。随着单细胞转录组分析技术的发展，全基因组可变剪接分析已在技术上具备可行性，得以全面解析人类卵母细胞中的可变剪接模式。对剪接mRNA异构体的分析，或可为解析早期发育的分子机制提供全新见解，同时也可能成为评估卵母细胞质量的潜在生物标志物来源。本研究的目标是对不同成熟阶段的卵母细胞开展转录组学分析，以鉴定两种卵母细胞成熟阶段所产生的可变剪接异构体谱。总体实验设计：本研究共纳入16名接受卵母细胞捐赠的女性的12枚体内成熟MII卵母细胞与4枚未成熟GV期卵母细胞，所有样本均单独处理与分析。受试者平均年龄为28±4.7岁（年龄范围21~34岁），平均窦卵泡计数（AFC，antral follicle count）为21±10.7个（计数范围5~43个）。根据卵母细胞成熟阶段，以及供体女性的年龄与窦卵泡计数，将样本分为4个实验组（每组包含4枚卵母细胞）：1. GV组：来自年龄≤30岁、高窦卵泡计数供体的GV期卵母细胞（供体年龄26±4.1岁，窦卵泡计数27±13个）；2. H-MII组：来自年龄≤30岁、高窦卵泡计数供体的MII卵母细胞（供体年龄26±4.6岁，窦卵泡计数24±3个）；3. L-MII组：来自低窦卵泡计数供体的MII卵母细胞（供体年龄27±5.4岁，窦卵泡计数7±1个）；4. O-MII组：来自年龄≥31岁、高窦卵泡计数供体的MII卵母细胞（供体年龄32.8±1.5岁，窦卵泡计数27±6个）。每枚卵母细胞的总RNA均独立提取、扩增并标记后，在Affymetrix HTA 2.0基因芯片上完成杂交。