Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep Robert Feil, 1 Sanjeev Khosla, 1 Pietro Cappai, 2 Pasqualino Loi 2 1 Programme in Developmental Genetics, The Babraham Institute, Cambridge CB2 4AT, United Kingdom 2 Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo (Sassari), Italy Received: 13 May 1998 / Accepted: 16 July 1998 Abstract. Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthe- nogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theo- ries of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have inves- tigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic devel- opment in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnor- malities are most likely caused by imprinted genes. We demon- strate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These obser- vations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants. Key words: Genomic imprinting—Epigenetic— Evolution—Sheep—IGF2—PEG1/MEST Introduction In humans and mice, the maternal and paternal genomes are func- tionally different and are therefore both required for normal em- bryonic development. The distinct contributions from male and female gametes are mediated by genomic imprinting, an epigenetic mechanism that gives rise to differential expression of the mater- nally and paternally inherited alleles of certain genes (Hall 1990; John and Surani 1996). Dissection of the parental contributions, by analysis of mice that are uniparentally disomic for the entire ge- nome or for individual chromosomes, has established that mater- nally and paternally imprinted genes play different and rather op- posite developmental roles (Surani et al. 1984; McGrath and Solter 1984; Cattanach and Kirk 1985; Hall 1990). An extreme conse- quence of imprinting is that parthenogenetic mouse embryos (which have two maternal and no paternal genome) are small and die at early postimplantation stages because of the lack of pater- nally expressed genes (Surani et al. 1984; McGrath and Solter 1984). Indeed, it has been demonstrated for the Igf2, Peg1/Mest, Peg3, and Snrpn imprinted genes, which are paternally expressed, that they are fully repressed in parthenogenetic mouse embryos (Sasaki et al. 1992; Walsh et al. 1994; Barr et al. 1995; Kaneko- Ishino et al. 1995; Szabo ´ and Mann 1996). In the mouse, 27 parental allele-specifically expressed genes have been identified to date (Beechey and Cattanach 1998) and for two of these it has been shown that they are imprinted in rats as well (Pedone et al. 1994; Overall et al. 1997). Although compara- tive studies have established that most of the imprinted rodent genes are parental allele-specifically expressed in humans as well (John and Surani 1996), and vice versa, it is not known whether genomic imprinting is conserved among other mammalian groups. Many theories have been developed that consider the evolution of genomic imprinting (Hurst 1997), possibly the most inclusive of these says that imprinting evolved because of the conflicting in- terests of maternal and paternal genes in relation to transfer of nutrients from the mother to her offspring during pre- and post- natal development (the ‘‘conflict hypothesis’’: Haig and Graham 1991; Moore and Haig 1991). It would be important to know to which extent genomic imprinting is conserved among eutherian mammals in order to evaluate this and other evolutionary theories of imprinting. We set out to investigate its conservation in a bovid ruminant, the domestic sheep (Ovis aries). First, because rumi- nants are phylogenetically quite distinct from the rodent and pri- mate lineages from which (based on comparative studies on mi- tochondrial proteins) they diverged about 110 and 100 million years ago, respectively (Penny and Hasegawa 1997, and references therein; Janke et al. 1997). Secondly, in contrast to rodents and primates, which have a placenta that invades all uterine layers and acquires nutrients directly from maternal blood vessels, ruminants have a nonaggressive form of placentation, with a chorion that does not invade the uterine layers (Steven 1975). Comparatively, implantation is also delayed in ruminants. In sheep, for example, the total gestation length is about 150 days, with gastrulation tak- ing place at day 9.5 and implantation (indicated by placentome organization) occurring between days 23 and 25 of gestation (Steven 1975). The pronounced differences in placentation be- tween ruminants and other groups of placental mammals should allow evaluation of other evolutionary theories which say that imprinting prevents ovarian trophoblast disease and restrains ag- gressive placentas from harming the pregnant mother (Hall 1990; Varmuza and Mann 1994). We recently demonstrated that parthenogenetic sheep concep- tuses can be produced efficiently by chemical activation of meta- phase II oocytes with a combination of ionomycin and 6- dimethylaminopurine. This preliminary study (Loi et al. 1998) showed that, when cultured in vivo to the blastocysts stage and transferred into recipient ewes, thus-derived parthenogenetic em- bryos can develop to day 21 of gestation, but apparently not to later fetal stages of development. For our analysis of imprinting we derived a larger number of day 21 parthenogenetic fetuses and compared these with appropriate biparental control fetuses. Devel- Correspondence to: R. Feil Mammalian Genome 9, 831–834 (1998). © Springer-Verlag New York Inc. 1998 Incorporating Mouse Genome