Abstract：
In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.
摘要：
在哺乳动物中,早期胚胎发育在很大程度上依赖于母体转录本的调节,包括储存在卵母细胞中的编码蛋白和非编码RNA.在这项研究中,三个牛卵母细胞的表达表达长链非编码RNA（lncRNAs），OOSNCR1，OOSNCR2和OOSNCR3在体细胞组织中表征，卵巢卵泡,以及整个早期胚胎发育。此外，使用siRNA介导的敲减方法研究了卵母细胞成熟和早期胚胎发育过程中每种转录物的功能需求.组织分布分析表明，OOSNCR1，OOSNCR2和OOSNCR3主要在胎儿卵巢中表达。卵泡细胞表达分析显示这些lncRNAs在卵母细胞中高表达,在卵丘细胞（CC）和壁颗粒细胞（mGC）中检测到少量表达。这三个基因的表达在卵母细胞成熟期间最高。受精时减少，并在16细胞阶段完全停止。未成熟卵母细胞中OOSNCR1，OOSNCR2和OOSNCR3的敲低是通过用靶向这些lncRNA的siRNA显微注射卵丘封闭的胚泡（GV）卵母细胞来实现的。击倒OOSNCR1，OOSNCR2和OOSNCR3不影响积云扩张，但授精后12小时的卵母细胞存活率显著降低。此外,未成熟卵母细胞中OOSNCR1，OOSNCR2和OOSNCR3的敲低导致胚泡发育率降低，与卵母细胞能力相关的基因表达减少，如核酶2（NPM2），生长分化因子9（GDF9），骨形态发生蛋白15（BMP15），和JY-1在MII卵母细胞中。本文的数据表明在牛卵母细胞成熟和早期胚胎发生期间OOSNCR1、OOSNCR2和OOSNCR3的功能需求。
