Germline-specific epigenetic reprogramming in ICSI and cloned mice
Recent studies have demonstrated that the use of assisted reproductive technology (ART) and somatic cell nuclear transfer (SCNT) can induce primary epimuations at imprinted loci during early stages of development (Tamada and Kikyo, 2004; Laprise, 2009). Given the heritability of epigenetic modifications in somatic tissues, it is likely that any epimutation induced during embryogenesis will be maintained throughout the lifetime of the organism and could manifest as a disease phenotype. However, during gametogenesis inherited epigenetic marks are typically erased and new sex-specific epigenetic profiles are established in the mature gametes (Ariel et al., 1994; Yamazaki et al., 2003). While many studies have shown that ex vivo manipulations can induce imprinting errors in somatic cells (Doherty et al., 2000; Mann et al., 2003; Rivera et al., 2008), the likelihood of propagating such epimutations to subsequent generations has not been thoroughly investigated. The focus of this dissertation is to determine if epimutations induced by intracytoplasmic sperm injection (ICSI), a type of ART, or SCNT, the standard method used for reproductive cloning, are corrected by germline-specific epigenetic reprogramming. To this end, we analyzed allele-specific DNA methylation and expression of multiple imprinted genes in somatic and germ cells of mice produced by natural conception, ICSI, and SCNT. We found that epimutations are generated during the ICSI procedure more frequently than SCNT. In addition, we found that these epigenetic defects are corrected in the germ line and not transmitted to subsequent generations. Finally, we provide evidence that the use of superovulation may play a role in the induction of somatic epimutations.