270 IN VITRO MATURATION ALTERS THE EXPRESSION OF SPECIFIC PUTATIVELY IMPRINTED GENES IN BOVINE MII OOCYTES

Abstract

Genomic imprinting is an epigenetic form of gene regulation resulting in only one parental allele being expressed. Imprinted genes have diverse functions including the regulation of growth and development in mammals. Errors in genomic imprinting have been associated with human disease (e.g. Beckwith-Wiedemann Syndrome) and large offspring syndrome of in vitro-produced ruminant fetuses. The aim of this study was to investigate the effect of in vitro maturation (IVM) on the expression of specific putatively imprinted genes in the bovine oocyte. Cumulus-enclosed immature oocytes were collected from cattle after 6 injections of 50 mg FSH with transvaginal aspiration (TVA) performed 48 h post-final FSH injection. The oocytes were cultured in groups of 10 for 23 h in a defined maturation medium (G-Mat) with 5 mg of HSA and 100 ng mL^-1 of epidermal growth factor (EGF; group A) or with 20% serum and 100 ng mL^-1 of EGF (group B) at 38.5°C in 6% CO₂ in air. In vivo-matured oocytes (group C) were also collected via TVA after the administration of 6 FSH injections (50 mg), prostaglandin (PG) with last FSH injection, and GnRH 37 h post-PG. Total RNA was extracted from individual MII oocytes in all 3 groups, and expression profiles of putatively imprinted genes (Igf2r, Peg3, and Snrpn) were assayed by real-time PCR (Roche Applied Biosciences, Indianapolis, IN, USA), relative to the housekeeping gene GAPDH. Statistical analysis of expression profiles was performed using REST software. Expression of the Igf2r and Peg3 putatively imprinted genes was significantly up-regulated in individual in vitro-matured MII oocytes (groups A and B, n = 5 replicates per group) when compared with control, individual in vivo-matured MII oocytes (group C, n = 5 replicates; P < 0.05). Gene expression did not differ between in vitro- and in vivo-matured MII oocytes for the putatively imprinted gene, Snrpn. In conclusion, following in vitro maturation of bovine oocytes, the putatively imprinted genes Igf2r and Peg3 were aberrantly expressed in individual oocytes relative to in vivo controls. Both of these putatively imprinted genes have been implicated in the regulation of growth and apoptotic pathways during mammalian development. Analysis of such putatively imprinted genes will facilitate the development of more suitable oocyte maturation conditions.

This research was supported by the Serono Research Institute.