Profiling Methylenetetrahydrofolate Reductase Throughout Mouse Oocyte and Preimplantation Embryo Development

Abstract

The global DNA methylation pattern is erased and re-established during oogenesis and again in preimplantation (PI) embryo development. Understanding where these methyl groups come from and how the process of methylation is regulated is important, as disruptions could result in detrimental effects. The methionine cycle that produces the cellular methyl pool is linked to the folate cycle. The key enzyme linking theses cycles is Methylenetetrahydrofolate Reductase (MTHFR) which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Mthfr RNA and protein are present throughout mouse oocyte and PI embryo development, including the germinal vesicle, MII egg, 1-cell embryo, 2-cell embryo, morula and blastocysts. In MII eggs the protein appears to be heavier than in any other stage. This was reversed by treatment with Lambda Protein Phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. MTHFR was progressively phosphorylated beginning shortly after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing after egg activation using strontium chloride. Potential kinases responsible for the phosphorylation of MTHFR have been identified however not in oocytes or PI embryos. DYRK1A/1 and GSK3A/B have both been suggested to mediate the phosphorylation, however when inhibited showed no effect on the oocyte sample. An LC-MS/MS assay was attempted to measure the activity of MTHFR in wildtype and knockout mouse liver samples, however unsuccessful in the amounts needed to be used for comparison to oocytes. Overall, MTHFR is present in the developing stages of interest and is mediated in some capacity by phosphorylation modifications around the MII stage of development.