MRNA transcript profiling of human oocytes at different maturational stages and after in vitro maturation

Abstract

To catalogue and compare genes expressed in human oocytes at germinal vesicle (GV) or metaphase II (MII) stage and after in vitro maturation (IVM). Descriptive research assessing unfertilized oocytes by microarray. GV, in vivo matured MII or IVM-MII oocytes were collected and processed rapidly. RNA was extracted and amplified using a 2-round in vitro transcription procedure, then labeled and hybridized to Applied Biosystems Human Genome Survey Microarrays. This array interrogates 29,098 genes. Up- or down-regulated genes were classified according to the molecular function, biological process, or pathway in which they participate. Statistical analysis was employed to compare each class of genes between samples. The in vitro transcription protocol generated an average of 12 μg of RNA per sample. GV, in vivo matured-MII and IVM-MII oocytes expressed 14,188, 16,402 and 18,299 genes, respectively. There was extensive overlap in the genes expressed by each group, but also some significant differences. Samples showing the least similarity in expression profile were GV and in vivo MII oocytes. IVM-MIIs also displayed significant differences in expression when compared to in vivo MII oocytes, indicating that the process of in vitro maturation does not perfectly mimic that achieved in vivo. A disproportionate number of genes for storage proteins displayed down-regulation in IVM-MII oocytes (relative to in vivo MII). A reduction in expression was also seen for genes involved in processes of homeostasis. Significant up-regulation of gene expression for the ubiquitin proteasome pathway was observed in both IVM-MII and GV samples. This study provided a detailed insight into gene expression of human oocytes, assessing virtually every gene in the genome. GV oocytes were highly active for processes related to transcription, RNA processing and early stages of protein synthesis, whereas MII oocytes were active for pathways involved in processing of mature proteins. The results suggest that although IVM-MII oocytes closely resemble in vivo-MII oocytes for pathways related to nuclear maturity, a number of those associated with cytoplasmic functions continue to be expressed in a GV-like manner. Additionally, IVM-MII oocytes have significant differences in the expression of genes related to cellular storage and homeostasis. The pathways abnormally expressed during IVM highlight specific deficiencies in culture media and point the way to further optimization of IVM protocols.