Transcriptome variance in single oocytes within, and between, genotypes.

The zygote of sexually reproducing organisms contains a combination of parental genomes, and all subsequent cells of the embryo are derived from this original genotype. Although clonal, it is not known howmuch genetic variation exists in progeny of this original cell, or between cells of the same lineage resulting from this zygote. Oocytes in mammals, especially humans, have prolonged developmental histories and each may be quite different in terms of gene expression. It is clear that oocyte quality can differ significantly within a cohort, and the variation in early developmental success from each oocyte can be dramatic. Oocyte quality is ultimately best measured by the success of the embryo, but other features, such as normalcy of the mRNApopulation,may be important criteria to identify such potential. Here, we test the variation in steady-state levels of mRNAs in mouse oocytes to establish a baseline of ‘‘normal’’ variation, and compare it mRNA levels of individual oocytes of poor quality. We sequenced to saturation the mRNA from five wild-type oocyte samples (three individual oocytes, and two pools of five oocytes each from two wild-type mice) and 16 Taf4b-deficient oocyte samples (12 individual oocytes and four pools of 5 or 10 oocytes each from two Taf4b-deficient mice). The Taf4b-deficient mice are known to have oocytes that appear morphologically normal (Fig. 1A,B), but are of poor quality with regard to successful embryogenesis. This genotype was selected as a model for human premature ovarian insufficiency (POI; Lovasco et al., 2010). Taf4b-null animals are viable as adults, but the oocytes they make die prematurely in adults, leading to a POI phenotype, and any oocytes that mature and are fertilized do not develop past the twoto four-cell stage (Falender et al., 2005; Lovasco et al., 2010). The hypothesis tested here is that the transcriptome of the Taf4b-deficient oocyte differs significantly from that of the wild-type oocyte. To properly assess this, we also needed to determine the variance between individual oocytes to ascribe significance to the comparison. This dataset was generated by high-throughput DNA sequencing following transcriptome amplification (Reich et al., 2011) and compared within and between genotypes to determine the variance. To test the fidelity of the amplification process for this protocol, prior to and independent of high-throughput DNA sequencing, oocytes from awild-type mouse were isolated and pooled before lysing. Following DNase treatment, one oocyte-equivalent was isolated and the cDNA library was synthesized. The resulting library was diluted 100 times, the approximate volume of a single polar body, which is important if a polar body were to be used to determine the oocyte quality without harming theoocyte (Reich et al., 2011). Threesamples from this pool were independently amplified, and each technical replicate was tested by quantitative RT-PCRT (qPCR) as ameasure of the fidelity of the amplification procedure (Reich et al., 2011). Overall, low technical variation was detected, providing confidence in the protocol (Fig. 1C). We do not know what kinds of bias the amplification procedure may have, but based on these results, the amplification appears to be consistent. The starting material for a polar body is * Corresponding author: 185 Meeting Street Box G, Brown University Providence, RI 02912. E-mail: [email protected]