Modulation of imprinted gene expression following superovulation.

Although assisted reproductive technologies increase the risk of low birth weight and genomic imprinting disorders, the precise underlying causes remain unclear. Using a mouse model, we previously showed that superovulation alters the expression of imprinted genes in the placenta at 9.5days (E9.5) of gestation. Here, we investigate whether effects of superovulation on genomic imprinting persisted at later stages of development and assess the surviving fetuses for growth and morphological abnormalities. Superovulation, followed by embryo transfer at E3.5, as compared to spontaneous ovulation (controls), resulted in embryos of normal size and weight at 14.5 and 18.5days of gestation. The normal monoallelic expression of the imprinted genes H19, Snrpn and Kcnq1ot1 was unaffected in either the placentae or the embryos from the superovulated females at E14.5 or E18.5. However, for the paternally expressed imprinted gene Igf2, superovulation generated placentae with reduced production of the mature protein at E9.5 and significantly more variable mRNA levels at E14.5. We propose that superovulation results in the ovulation of abnormal oocytes with altered expression of imprinted genes, but that the coregulated genes of the imprinted gene network result in modulated expression.

Betaine homocysteine methyltransferase is active in the mouse blastocyst and promotes inner cell mass development.

Methyltransferases are an important group of enzymes with diverse roles that include epigenetic gene regulation. The universal donor of methyl groups for methyltransferases is S-adenosylmethionine (AdoMet), which in most cells is synthesized using methyl groups carried by a derivative of folic acid. Another mechanism for AdoMet synthesis uses betaine as the methyl donor via the enzyme betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5), but it has been considered to be significant only in liver. Here, we show that mouse preimplantation embryos contain endogenous betaine; Bhmt mRNA is first expressed at the morula stage; BHMT is abundant at the blastocyst stage but not other preimplantation stages, and BHMT activity is similarly detectable in blastocyst homogenates but not those of two-cell or morula stage embryos. Knockdown of BHMT protein levels and reduction of enzyme activity using Bhmt-specific antisense morpholinos or a selective BHMT inhibitor resulted in decreased development of embryos to the blastocyst stage in vitro and a reduction in inner cell mass cell number in blastocysts. The detrimental effects of BHMT knockdown were fully rescued by the immediate methyl-carrying product of BHMT, methionine. A physiological role for betaine and BHMT in blastocyst viability was further indicated by increased fetal resorption following embryo transfer of BHMT knockdown blastocysts versus control. Thus, mouse blastocysts are unusual in being able to generate AdoMet not only by the ubiquitous folate-dependent mechanism but also from betaine metabolized by BHMT, likely a significant pool of methyl groups in blastocysts.

Reproductive and epigenetic outcomes associated with aging mouse oocytes.

Female aging entails a decline in fertility in mammals, manifested by reduced oocyte reserves and poor oocyte quality accompanied by chromosomal anomalies and reduced litter size. In addition to compromised genetic integrity, recent studies suggest that epigenetic mechanisms may be altered in aging oocytes, with age affecting the expression of DNA methyltransferases, which catalyze the important epigenetic modification, DNA methylation. Loss of DNA methylation patterns, most notably for imprinted genes, is lethal to mouse embryos. To investigate how maternal age affects embryonic development and underlying DNA methylation patterns, young and aged C57BL/6 females were mated with C57BL/6 or C57BL/6(CAST7) males to allow for the identification of parental alleles; resulting blastocysts and mid-gestation embryos and placentas were evaluated. Although pregnancy, ovulation and implantation rates were similar between age groups, an age-related increase in resorption sites, morphological abnormalities and delayed development was found. Interestingly, placental morphology was also perturbed by aging, with elevated numbers of trophoblast giant cells in aged pregnancies. Normal monoallelic expression of the imprinted genes H19 and Snrpn was unaltered in blastocysts from aged females. We failed to observe any age-related changes in methylation of the differentially methylated regions of imprinted genes Snrpn, Kcnq1ot1, U2af1-rs1, Peg1, Igf2r and H19. Restriction Landmark Genome Scanning showed no significant differences in genome-wide DNA methylation in embryos and placentas, regardless of maternal age. Our findings demonstrate that maternal age affects post-implantation embryo and placental development; however embryos capable of developing to mid-gestation appear to undergo normal acquisition and maintenance of DNA methylation patterning.

Superovulation alters the expression of imprinted genes in the midgestation mouse placenta.

Imprinted genes play important roles in embryonic growth and development as well as in placental function. Many imprinted genes acquire their epigenetic marks during oocyte growth, and this period may be susceptible to epigenetic disruption following hormonal stimulation. Superovulation has been shown to affect growth and development of the embryo, but an effect on imprinted genes has not been shown in postimplantation embryos. In the present study, we examined the effect of superovulation/in vivo development or superovulation/3.5dpc (days post-coitum) embryo transfer on the allelic expression of Snrpn, Kcnq1ot1 and H19 in embryos and placentas at 9.5 days of gestation. Superovulation followed by in vivo development resulted in biallelic expression of Snrpn and H19 in 9.5dpc placentas while Kcnq1ot1 was not affected; in the embryos, there was normal monoallelic expression of the three imprinted genes. We did not observe significant DNA methylation perturbations in the differentially methylated regions of Snrpn or H19. Superovulation followed by embryo transfer at 3.5dpc resulted in biallelic expression of H19 in the placenta. The expression of an important growth factor closely linked to H19, Insulin-like growth factor-II, was increased in the placenta following superovulation with or without embryo transfer. These results show that both maternally and paternally methylated imprinted genes were affected, suggesting that superovulation compromises oocyte quality and interferes with the maintenance of imprinting during preimplantation development. Our findings contribute to the evidence that mechanisms for maintaining imprinting are less robust in trophectoderm-derived tissues, and have clinical implications for the screening of patients following assisted reproduction.

Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta.

Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition to mediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine and immune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have been identified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGC subtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCs that surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes: spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of the placenta. All four TGC subtypes could be identified based on the expression patterns of four genes: Pl1, Pl2, Plf (encoded by genes of the prolactin/prolactin-like protein/placental lactogen gene locus), and Ctsq (from a placental-specific cathepsin gene locus). Each of these subtypes was detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces Pl1/Plf+ TGCs preferentially. Furthermore, cell lineage tracing studies indicated unique origins for different TGC subtypes, in contrast with previous suggestions that secondary TGCs all arise from Tpbpa+ ectoplacental cone precursors.