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We report the first demonstration of oocyte mRNA detected in its sibling polar body. Oogenesis is a conserved process that requires gene transcription and storage of RNA for development until the embryonic genome is activated. Analysis of oocyte mRNA profiles detected in polar bodies may allow evaluation of gene expression in single oocytes without destroying the cell.
We biopsied the polar bodies from eggs of the starfish, Asterina miniata, without disrupting either cell. The estimated volume of an A. miniata oocyte is 3.05×10−3pl, nearly 2,000 times greater than that of its polar body, which has an estimated volume of 1.77×10−6pl. 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 supplemental material). We first tested whether the polar body had detectable ribosomal RNA using 1/30th of the RT-reaction from each cell and we consistently amplified the appropriate product from each cell (supplemental figure 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 ratio=0.40; p=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 figure 3).
We compared the level of mRNA for six genes in seven different sibling pairs. The ΔCt 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 25 to 16,000 fold relative differences in mRNA abundance between oocytes and polar bodies. Since the variance within sample replicates is less than 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 body with its sibling oocyte. It will be important to examine cell-to-cell variability among developmentally critical gene transcripts in polar bodies and to test whether variability between individuals may reflect biological differences between oocytes. Variation between different oocytes may reflect dynamic changes in mRNA abundance during oocyte development or the maturation process. Translating this work into humans is promising; although the human oocyte is smaller (120μm diameter compared to 180μm), the polar body is comparable or larger than the starfish polar body (Veeks, 1990). Technological advances including incorporation of non-biased amplification of mRNA may permit clinical analysis of human oocyte gene expression from a single polar body. Such a development may aid with the assessment of oocyte quality and developmental competence in patients using assisted reproductive technologies.
We are greatly appreciative of the contributions from Linda Sousa, who assisted with microdissection and polar body biopsies, Drs. Mamiko Yajima, Julian Wong and S. Zak Schwartz for their assistance with imaging, as well as Iris Chen for statistical analysis. This research was supported by grants from the NIH, the NSF, and the Provost of Brown University and lab and financial support from the Center for Reproduction & Infertility at Women & Infants Hospital of Rhode Island.