The utilization of in vitro oocyte maturation protocols for assisted reproduction in humans has potential advantages for lowering treatment costs, increasing patient safety, reducing exogenous hormonal treatments, reducing the number of clinical consultations required, and treating polycystic ovarian disease. However, the efficiency of embryo development following in vitro oocyte maturation procedures remains low. Identification of mechanisms for the resumption of meiosis in oocytes will facilitate the development of strategies to improve the effectiveness of assisted reproduction techniques by providing potential opportunities to increase the developmental competence of oocytes through the arrest of nuclear maturation while allowing cytoplasmic maturation to proceed during continued culture. Oocytes that have been exposed to a gonadotropin stimulus during in vitro maturation are significantly more competent to support embryonic development following fertilization. In mammals, the gonadotropin signal for the initiation of meiosis requires the expression of newly transcribed mRNAs within the cumulus cells that surround the oocyte. The identities of these key transcripts have not been obtained for any species to date. The long-term goal is to understand the regulation of mammalian oocyte maturation in an effort to improve methodologies for the production of embryos in vitro. Defects in oocyte competency adversely affect embryo development following in vitro fertilization and are influenced by exposure to gonadotropins during oocyte maturation. The specific objective of the current proposal is to identify and characterize transcriptionally regulated mRNAs associated with gonadotropin-mediated resumption of meiosis in routine cumulus-oocyte complexes (COC) by serial analysis of gene expression (SAGE). Using cultured cumulus-oocyte complexes, we have established a model useful for identification of new transcripts required for the resumption of meiosis in the mouse. In this model, cumulus-oocyte complexes exposed to FSH undergo germinal vesicle breakdown by 3 h after the initiation of in vitro culture; however, if a transcriptional inhibitor is included at the start of culture or up to 20 min after the start of culture, the majority of COC will remain arrested at the germinal vesicle stage. Thus, key transcripts required for the resumption of meiosis are being synthesized within the first 20-30 min of COC culture. Serial Analysis of Gene Expression (SAGE) libraries will be created from two groups of COC cultured in either presence or absence of a transcriptional inhibitor (DRB). COC will be harvested 25 min after the start of culture and used to generate SAGE libraries. Comparisons of SAGE-tag sequences generated from these two libraries will allow for the identification of newly transcribed mRNAs whose expression occurs coincident with the period of transcriptional commitment required for gonadotropin-mediated resumption of meiosis. Differential expression of the candidate gene sequences will be verified using reverse transcription-polymerase chain reaction (RT-PCR).
| Farin, C E; Rodriguez, K F; Alexander, J E et al. (2007) The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro. Anim Reprod Sci 98:97-112 |
| Rodriguez, K F; Blomberg, L A; Zuelke, K A et al. (2006) Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression. Physiol Genomics 27:318-27 |
