Transcriptome profile of oocytes expressing a catalytically inactive knock-in allele of Ago2

The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because miRNA function is suppressed in mouse oocytes, it has been proposed that endo-siRNAs may have a role during female meiosis. By utilizing a catalytically inactive knock-in allele of AGO2 specifically in oocytes, we disrupt the function of siRNAs and analyze the transcriptome of these oocytes in comparison with Ago2 null, and Dicer null oocytes. Germinal vesicle-intact, full-grown oocytes were collected from eCG-primed females expressing a catalytically inactive knock-in allele of Ago2 (Ago2 ADH) specifically in oocytes. Oocytes were also collected from Ago2 null and Ago2 fl/fl oocytes, as well as Dicer wild-type and null mice. Oocytes were freed of attached cumulus cells by pipetting. Twenty oocytes collected from 3 different mice were used for high-throughput RNA sequencing. Three replicates for all genotypes except Ago2 null (two). The mouse background strain is "mostly" C57BL6, i.e., it is a cross between 3 different transgenic mice of different strains.核糖核酸酶III（RNase III）家族酶DICER可同时合成微小RNA（microRNAs，miRNAs）与内源性短干扰RNA（endogenous short interfering RNAs，endo-siRNAs）。小鼠卵母细胞中Dicer基因敲除会引发雌性不育，其原因是减数分裂I期出现发育缺陷。鉴于小鼠卵母细胞内微小RNA的功能受到抑制，有研究推测内源性短干扰RNA可能在雌性减数分裂过程中发挥调控作用。本研究通过在卵母细胞中特异性表达催化失活型AGO2敲入等位基因，阻断siRNA的功能，并分别与AGO2敲除纯合子、Dicer敲除纯合子卵母细胞的转录组进行对比分析。本研究从经孕马血清促性腺激素（equine chorionic gonadotropin，eCG）预处理、且卵母细胞特异性表达催化失活型AGO2敲入等位基因（Ago2 ADH）的雌性小鼠中，收集生发泡（germinal vesicle）完整的成熟卵母细胞；同时还收集了AGO2敲除纯合子、AGO2 flox/flox（Ago2 fl/fl）卵母细胞，以及Dicer野生型与敲除纯合子小鼠的卵母细胞。通过吹打操作去除卵母细胞表面附着的卵丘细胞（cumulus cells）。从3只不同小鼠体内收集的20枚卵母细胞作为一组，用于高通量RNA测序（high-throughput RNA sequencing）。除AGO2敲除纯合子组设置2次生物学重复外，其余所有基因型组均设置3次生物学重复。本实验所用小鼠的背景品系"整体以C57BL/6为主"，即由3种不同品系的转基因小鼠杂交获得。