Detection of oocyte mRNA in starfish polar bodies.

Oogenesis is a conserved process that requires gene transcription and storage of RNA for development until the embryonic genome is activated (Evsikov andEviskov, 2009). Analysis of oocyte mRNA profiles detected in polar bodies may allow evaluationof geneexpression in single oocyteswithout destroying the cell. Here we demonstrate the detection of such oocyte mRNA in its sibling polar body. We biopsied polar bodies from eggs of the starfish, Asterina miniata, without disrupting either cell. The estimated volume of an A. miniata oocyte is 3.05 10 3 pl, nearly 2,000 times greater than that of its polar body, which has an estimated volume of 1.77 10 6 pl. Individual polar bodies and oocytes were isolated and transferred to a reaction buffer, heat lysed, DNAse treated, and reverse transcribed without isolation of the RNA (Protocol described in the Supplemental Material). We first tested if the polar bodyhaddetectable ribosomalRNAusing 1/30th of the RT-reaction from each cell, and we consistently amplified the appropriate product from each cell (Supplemental Fig. 2). We then tested for specific mRNAs; these too were detectable with greater success for transcripts that were more abundant (had lower Ct values) in the sibling oocyte (Table I). Detection of a specific mRNA transcript in the polar body decreased 60% for every unit increase in corresponding Ct value for that transcript in its sibling oocyte (odds ratio1⁄4 0.40; P1⁄4 0.01). This result provides a baseline from which to predict transcripts that may be detectable in polar bodies if levels in the oocyte are known. Confocal imaging supports the hypothesis that representative ooplasm containing mitochondria is extruded with the first polar body (Supplemental Fig. 3). We compared the level of mRNA for six genes in seven different sibling pairs. The ~Ct value between oocytes and polar bodies for a given gene ranged from 4.6 for eukaryotic initiation factor 2 (eif2) transcripts to 14.0 for histone2A (h2a) transcripts. These values correspond to 25to 16,000-fold relative differences in mRNA abundance between oocytes and polar bodies. Since the variance within sample replicates is <2%, variability between transcripts may reflect a difference in retention or localization between these sibling cells. Further research is needed to correlate variation of mRNA levels in a polar bodywith its sibling oocyte. It will be important to examine cell-to-cell variability among developmentally critical gene transcripts in polar bodies and to test if variability between individuals may reflect biological differences between oocytes as these may reflect dynamic changes in mRNA abundance during oocyte maturation. Translating this technique to the evaluation of human oocytes is promising; although the human oocyte is smaller (120mm diameter compared to 180mm), the polar body is comparable or larger than the starfish polar body (Veeck, 1990). Technological advances including incorporation of non-biased amplification of mRNAmay permit clinical analysis of human oocyte gene expression from a single polar body (Noutsias et al., 2008). Such a method may aid with the assessment of oocyte quality and developmental competence in patients using assisted reproductive technologies.