Effects of low methyl donor levels in culture medium during mouse follicle culture on oocyte imprinting establishment.

Imprinted genes are differentially methylated during gametogenesis to allow parent-of-origin specific monoallelic expression. We previously demonstrated normal imprinting establishment at four key imprinted genes in mouse MII oocytes after in vitro follicle culture. Commercially available culture media feature a wide range of methyl donor levels. The aim was to study the effect of low methyl donor (methionine, vitamin B12, folic acid, choline and vitamin B6) levels during follicle culture on DNA methylation acquisition at imprinted genes in mouse oocytes. Follicle culture performed under low methyl donor levels led to decreased antral follicle development: mean (SD) antral follicle rate was 87.5 (12.6)% versus 97.7 (4.3)% in control conditions, P <0.05; and to a dramatic decrease in polar body (PB) oocyte rate: mean (SD) PB oocyte rate was 38.7 (25.5)% versus 96.1 (7.1)%; P <0.001. The methylation status of differentially methylated regions (DMRs) of 4 key imprinted genes was studied (by bisulphite sequencing) in normal-looking PB and GVBD-arrested oocytes obtained from follicle culture under low methyl donor levels. DMRs of Snrpn, Igf2r, and H19 showed no alteration in DNA methylation, but at Mest DMR in PB oocytes we found a significant reduction in DNA methylation compared to control follicle culture (% DNA methylation was respectively 89.9% and 98.2%, P = 0.0014). In conclusion, restriction of methyl donors during follicle culture led to a dramatic decrease in PB oocyte rate, but induced no or only minor DNA methylation alterations at the studied regulatory sequences of key imprinted genes in oocytes.

Jaar van publicatie:2010

Trefwoorden:methyl, follicle culture, mouse, oocyte, GVBD, DMR