213 Using mRNA from cytoplasmic biopsies to assess molecular maturation and developmental potential of bovine oocytes

Embryos resulting from assisted reproductive technologies, such as in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI), develop with lower efficiencies than embryos resulting from their in vivo counterparts. The reasons behind the developmental discrepancies remain largely unknown. Because the oocyte is the primary determinant of embryo developmental success, it is reasonable to consider inherent oocyte quality as a possible cause. The hypothesis for this project is that there are distinct mRNA transcript patterns, or molecular “fingerprints,” that distinguish high- versus low-quality oocytes developing within the same environment. In this study, small cytoplasmic biopsies were removed from metaphase II (MII) bovine oocytes via micromanipulation and utilised for relative transcript abundance profiling. Preliminary experiments showed that oocyte developmental potential was unchanged after biopsy (development to blastocyst after parthenogenetic activation was 26.7% for biopsied oocytes versus 27.9% for control oocytes [P > 0.05]) and that biopsy mRNA content was highly representative of the transcript levels in the oocytes those biopsies were derived from (average R² = 0.8339 for quantitative PCR Ct values across 17 oocyte/biopsy couplets). In order to investigate the relationship between oocyte developmental potential and cytoplasmic mRNA content, 40 MII oocytes were biopsied and then parthenogenetically activated to stimulate development and cultured in vitro in a well of the well (WoW) format. Following an eight-day development period, embryos that reached the blastocyst stage (success) and embryos that failed to develop were identified. This was repeated five times, for a total of 240 biopsied oocytes across six experimental replicates. Across all replicates, 48/240 (20.0%) of the biopsied oocytes developed into blastocysts. The BioMark single cell qPCR system from Fluidigm was used to evaluate the relative transcript levels of 48 genes in the biopsies from 45 of the oocytes that developed successfully and 45 that failed to develop properly. The functional categories of the 48 genes include apoptosis, oocyte-specific, epigenetic, metabolism, housekeeping, pluripotency, and RNA processing. After applying false discovery rate corrections to raw Ct and delta Ct data analysed by ANOVA, none of the 48 genes were significantly differentially expressed in biopsies from successful versus failed oocytes. In conclusion, the evidence gathered to this point does not support the hypothesis of differential transcript patterns between the cytoplasmic biopsies from oocytes of divergent developmental success. However, only a small subset of genes was targeted for this preliminary analysis, and an expanded scope would increase the likelihood of finding transcripts associated with developmental competence. Furthermore, this study provides valuable technical innovation and establishes a functional framework for future research into the relationship between cytoplasmic biomolecules and oocyte quality.