27 - 30 May 2005

SETTING AND UPSETTING IMPRINTS: IMPLICATIONS FOR ART Jacquetta M Trasler McGill University-Montreal Children’s Hospital Research Institute and the Departments of Pediatrics Human Genetics and Pharmacology & Therapeutics McGill University Montreal Canada

A number of studies published over that last few years have suggested that there is an increased incidence of rare genomic imprinting disorders associated with alterations in the methylation of imprinted genes in children conceived by human assisted reproductive technologies (ART).

Genomic imprinting is initiated in the germline, persists through preimplantation development and involves the formation of an epigenetic ‘mark’ at specific loci in a parent-of-origin-specific manner, such that a subset of genes is expressed monoallelically in the resulting offspring. ART includes hormonal stimulation protocols as well as the isolation, handling and culture of gametes and early embryos at times when imprinted genes are likely to be particularly vulnerable to external influences.

The most well studied epigenetic ‘mark’ that distinguishes the maternal and paternal alleles of imprinted genes is DNA methylation. To better understand how genomic imprinting defects could be related to ART we have used the mouse model to:1) examine the methylation status of several imprinted genes during male and female germ cell development, 2) determine the expression and roles of the DNA methyltransferase (Dnmt) enzymes in establishing and maintaining gametic imprints, and 3) examined effects of superovulation and culture conditions on imprinted gene methylation.

Bisulfite sequencing was used to determine the methylation status of multiple CpG sites in imprinted genes as well as the timing of paternal and maternal methylation imprint establishment in the two germ lines. In the male, the H19 methylation imprint is initiated prenatally during embryonic germ cell development and is complete by the pachytene phase of postnatal spermatogenesis.

In contrast, examination of the methylation status of Igf2r, Snrpn, Peg1 and Peg3 during oocyte development identified the postnatal growth phase (post-pachytene) of oogenesis as the crucial period during which maternal imprints are set down in the female. Thus methylation imprints are established at different developmental times in the parental germ lines, suggesting that male and female germ cells may be susceptible to conditions that perturb imprints at sex-specific stages of prenatal and postnatal gametogenesis.

Expression studies and gene-targeting experiments have been used to determine the roles of the Dnmt enzymes in imprinting. The recently discovered Dnmt3 enzymes, Dnmt3a, Dnmt3b, and Dnmt3l are developmentally expressed during postnatal oocyte growth, with Dnmt3l transcripts being particularly abundant. Dnmt1 and the three Dnmt3 enzymes are differentially expressed in a stage-dependent manner in prenatal and postnatal male germ cells, coinciding with the timing of methylation establishment and maintenance in the male germ line. Gene-targeting experiments by others indicate essential roles for the Dnmt3 enzymes in establishing methylation imprints.

Results to date suggest that the different Dnmt enzymes perform distinct functions during germ cell development and early embryogenesis. In preliminary studies we have compared the methylation of Snrpn and H19 in embryos derived from naturally cycling versus superovulated females.

The results suggest that some imprinted genes such as H19, which are known to be sensitive to alterations in culture conditions, may be affected by superovulation, while others, such as Snrpn, may be more resistant. Similar results were seen in oocyte culture experiments.

Our studies suggest the need for more research to model techniques used in ARTs and test for epigenetic effects in the offspring.

List of abstracts from the 3rd International Conference on the Female Reproductive Tract