Expression analysis of genes implicated in meiotic resumption in vivo and developmental competence

Abstract

This thesis investigated the gene expression in bovine oocytes during meiotic resumption, at 6 h post LH surge, coinciding with germinal vesicle breakdown, which was supposed to give a picture of the major cell cycle regulation changes, cytoskeleton rearrangement and chromosome alignment. Presumptive competent oocytes were used to construct and hybridize the DNA microarray using Suppression Subtraction Hybridization (SSH), to unravel the potential transcripts involved in meiotic resumption and possibly developmental competence. The mRNA expression of several genes was further studied using quantitative real-time PCR. The roles of these genes during maturation have not been entirely characterized, but the results presented in this thesis will support the establishment of new hypothesis regarding the resumption of meiotic arrest in bovine oocytes and provide useful information for the development of efficient maturation media for in vitro embryo production. In this Thesis, we used also oocytes collected from cows stimulated with FSH classified as deviant and normal based on the steroid hormones profile, and oocytes collected from slaughterhouse ovaries and matured in vitro, to investigate whether the divergent culture conditions is accompanied by altered mRNA levels of the different selected genes playing important roles in organelles translocation and spindle integrity and formation using real-time PCR. The analysis of six mRNAs suggested that both the exogenous FSH induced intrafollicular environment and in vitro maturation and culture conditions may affect the developmental competence by affecting the expression of these genes. mRNAs representing the major metabolic pathways involved in lipid metabolism were also investigated in this thesis using normal in vivo matured oocytes.The results, provide new information regarding long chain fatty acid transport into the oocytes and regulation of energy requirement during maturation and blastocyst stage using lipid as a substrate. Furthermore, the aberrant expression of lipid metabolism mRNAs levels in the oocyte matured in vitro, as well as that in deviant oocyte from stimulated cows, in addition to that in the in vitro produced blastocysts suggests a defect in fatty acid metabolism. This thesis has revealed many of the new basic properties of the mammalian oocyte, and based on this information a plausible model for regulation of meiotic resumption and proper oocyte function is possible.