Dual Effects of Superovulation: Loss of Maternal and Paternal Imprinted Methylation in a Dose-dependent Manner

Superovulation or ovarian stimulation is currently an indispensable assisted reproductive technology (ART) for human subfertility/infertility treatment. Recently, increased frequencies of imprinting disorders have been correlated with ARTs. Significantly, for Angelman and Beckwith-Wiedemann Syndromes, patients have been identified where ovarian stimulation was the only procedure used by the couple undergoing ART. In many cases, increased risk of genomic imprinting disorders has been attributed to superovulation in combination with inherent subfertility. To distinguish between these contributing factors, carefully controlled experiments are required on spontaneously ovulated, in vivo-fertilized oocytes and their induced-ovulated counterparts, thereby minimizing effects of in vitro manipulations. To this end, effects of superovulation on genomic imprinting were evaluated in a mouse model, where subfertility is not a confounding issue. This work represents the first comprehensive examination of the overall effects of superovulation on imprinted DNA methylation for four imprinted genes in individual blastocyst stage embryos. We demonstrate that superovulation perturbed genomic imprinting of both maternally and paternally expressed genes; loss of Snrpn, Peg3 and Kcnq1ot1 and gain of H19 imprinted methylation were observed. This perturbation was dose-dependent, with aberrant imprinted methylation more frequent at the high hormone dosage. Superovulation is thought to primarily affect oocyte development; thus, effects were expected to be limited to maternal alleles. Our study revealed that maternal as well as paternal H19 methylation was perturbed by superovulation. We postulate that superovulation has dual effects during oogenesis, disrupting acquisition of imprints in growing oocytes, as well as maternal-effect gene products subsequently required for imprint maintenance during pre-implantation development

ATRX Partners with Cohesin and MeCP2 and Contributes to Developmental Silencing of Imprinted Genes in the Brain

Human developmental disorders caused by chromatin dysfunction often display overlapping clinical manifestations, such as cognitive deficits, but the underlying molecular links are poorly defined. Here, we show that ATRX, MeCP2, and cohesin, chromatin regulators implicated in ATR-X, RTT, and CdLS syndromes, respectively, interact in the brain and colocalize at the H19 imprinting control region (ICR) with preferential binding on the maternal allele. Importantly, we show that ATRX loss of function alters enrichment of cohesin, CTCF, and histone modifications at the H19 ICR, without affecting DNA methylation on the paternal allele. ATRX also affects cohesin, CTCF, and MeCP2 occupancy within the Gtl2/Dlk1 imprinted domain. Finally, we show that loss of ATRX interferes with the postnatal silencing of the maternal H19 gene along with a larger network of imprinted genes. We propose that ATRX, cohesin, and MeCP2 cooperate to silence a subset of imprinted genes in the postnatal mouse brain