Abstract

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.

INTRODUCTION

Imprinted genes are expressed monoallelically in a parent-of-origin specific manner. To date, 96 imprinted sequences have been identified in the mammalian genome (reviewed in 1). These genes have been shown to play important roles in embryonic growth and development, placental function and postnatal behavior (reviewed in 2–4). The aberrant expression of imprinted genes has been implicated in the development of several human disorders, including Beckwith–Weidemann syndrome, Prader-Willi and Angelman syndromes, as well as cancer (reviewed in 5).

Genomic imprinting is controlled by epigenetic mechanisms, as DNA sequence alone cannot distinguish between parental alleles or control the allele-specific expression of genes. Among known epigenetic modifications, the best characterized of these is the methylation of cytosine residues in DNA. Many imprinted genes have a differentially methylated region (DMR), with sex-specific methylation patterns inherited from the gametes (6–8). In addition, DNA methylation is a both reversible and heritable modification that can be stably maintained after cell division.

Methylation patterns required for genomic imprinting undergo dynamic regulation during mammalian development. Patterns present in the mature gametes must be maintained throughout the life of the organism following fertilization, with the exception of cells in the germline. In the mouse, it has been shown that methylation patterns are almost completely erased in the germline by 13.5dpc (days post-coitum), around the time the germ cells enter the genital ridge (9–13). These patterns must then be re-established in the developing embryo in a sex-specific manner. The establishment of methylation at imprinted genes occurs at different times in the male and female germlines. In the female germline, methylation acquisition at imprinted loci begins postnatally, during the oocyte growth phase (9,14–17). Acquisition of methylation patterns begins early in the oocyte growth phase, and occurs throughout oocyte growth. Methylation is acquired in a locus-specific manner, with some genes achieving their full methylation status only very late in oocyte growth (15–17). Since imprint acquisition occurs over a relatively long period of time, concerns have been raised that the establishment of maternal imprints may be susceptible to perturbation.

The use of exogenous gonadotrophins to synchronize and/or induce ovulation has been used in both research models and as a part of fertility treatments in women. However, these hormonal treatments may force oocytes to undergo the growth and maturation phases too rapidly. As such, the acquisition of methylation imprints may be perturbed in oocytes undergoing superovulation. Previous studies have shown that superovulation results in delayed embryo development, decreased implantation rates and increased postimplantation loss (18–21). A few studies have examined the effect of superovulation on DNA methylation and imprinting in the oocyte or the embryo. An initial study showed an increase in aberrant 5-methyl-cytosine staining in two-cell embryos (22). Recently, an examination of the methylation of the DMRs of three maternally imprinted genes (Peg1, Kcnq1ot1 and Zac) in oocytes found no difference in the acquisition of imprints at these loci following superovulation. In contrast, an increase in methylation at the DMR of the paternally methylated gene H19, which is normally unmethylated in oocytes, was observed (8).

In this study, we investigated the impact of superovulation followed by in vivo development on the acquisition and/or maintenance of imprinting in the embryos and placentas collected at 9.5 days of pregnancy in the mouse. In addition, to understand the contribution of the superovulated uterus to changes in imprinted gene expression, we performed embryo transfer experiments. We provide evidence that events occurring prior to fertilization or during the preimplantation period, such as hormonal stimulation or its subsequent effects on the uterine environment, can affect imprinted genes much later in development.

RESULTS

Effects of superovulation/in vivo development on the growth or development of 9.5dpc embryos

Following superovulation, female mice were mated and embryos were allowed to develop in vivo to midgestation (9.5dpc). There was no difference in the percentage of viable embryos between control and superovulated matings; however, there was an increase in the number of embryos undergoing resorption following superovulation (Kruskal–Wallis test, control versus superovulated, P < 0.05; Table 1). There was also an increase in the percentage of delayed embryos following superovulation (Fisher’s exact test, control versus superovulated, P < 0.001; Table 1), but there was no difference in the percentage of embryos having gross abnormalities (Table 1). The average crown-rump length of control embryos was 2.73 ± 0.09 mm, which was not significantly different from that of embryos from superovulated mothers (3.37 ± 0.07 mm). Similarly, the overall development of the embryos, as assessed by somite number, was not affected (20.86 ± 1.03 for control embryos versus 22.16 ± 1.40 for superovulated embryos).

Table 1.

Litter characteristics of control and superovulated litters following in vivo development

  No. of implantation sites No. of resorption sites (%) Numbers collecteda Numbers viable (%)b Numbers delayed (%) No. with morphological abnormality (%)c 
Control Litter 1 1 (11.1) 8 (100) 1 (12.5) 0 (0) 
 Litter 2 14 1 (7.1) 12 12 (100) 0 (0) 0 (0) 
 Litter 3 12 0 (0) 11 11 (100) 1d 0 (0) 
 Total 35 2 (5.7) 31 31 (100) 1 (3.2) 0 (0) 
Superovulated Litter 1 31 n.d. 12 11 (91.7) 12 (100) 1 (8.3) 
 Litter 2 10 2 (20.0) 8 (80.0) 0 (0) 0 (0) 
 Litter 3 25 0 (0) 16 12 (75.0) 0 (0) 3 (18.8) 
 Litter 4 1 (14.3) 4 (100) 3 (75.0) 1 (25.0) 
 Litter 5f n.d. 1 (100) 0 (0) 0 (0) 
 Litter 6f 30 18 (60.0) 12 7 (58.3) 11 (91.7) n.d.e 
 Total 104 21 (44.7)* 53 43 (81.1) 26 (49.1)** 5 (12.2) 
  No. of implantation sites No. of resorption sites (%) Numbers collecteda Numbers viable (%)b Numbers delayed (%) No. with morphological abnormality (%)c 
Control Litter 1 1 (11.1) 8 (100) 1 (12.5) 0 (0) 
 Litter 2 14 1 (7.1) 12 12 (100) 0 (0) 0 (0) 
 Litter 3 12 0 (0) 11 11 (100) 1d 0 (0) 
 Total 35 2 (5.7) 31 31 (100) 1 (3.2) 0 (0) 
Superovulated Litter 1 31 n.d. 12 11 (91.7) 12 (100) 1 (8.3) 
 Litter 2 10 2 (20.0) 8 (80.0) 0 (0) 0 (0) 
 Litter 3 25 0 (0) 16 12 (75.0) 0 (0) 3 (18.8) 
 Litter 4 1 (14.3) 4 (100) 3 (75.0) 1 (25.0) 
 Litter 5f n.d. 1 (100) 0 (0) 0 (0) 
 Litter 6f 30 18 (60.0) 12 7 (58.3) 11 (91.7) n.d.e 
 Total 104 21 (44.7)* 53 43 (81.1) 26 (49.1)** 5 (12.2) 

n.d. = not done.

aImplantation sites containing embryo tissue were collected. In the superovulated group, for Litter 1, data are available for the 12 embryos collected; for Litter 5, data are available for the one embryo collected.

bViable embryos were defined as embryos having a heart beat, regardless of stage of development.

cDetailed information regarding morphological abnormalities can be found in Supplementary Material, Tables S1A and B.

dThis embryo was not collected.

eAll embryos in this litter were too delayed to assess gross morphology.

fThese litters were not included in allele-specific expression analysis, litter 5 due to small numbers and litter 6 due to the abnormality of the entire litter.

*P < 0.05, Kruskal–Wallis test, control versus superovulated.

**P < 0.001, Fisher’s exact test, control versus superovulated.

Superovulation/in vivo development causes biallelic expression of Snrpn in postimplantation placentas

In order to examine the effect of superovulation on the expression of imprinted genes, RNA was extracted from embryo and placenta tissues and cDNA was generated, followed by allele-specific expression analysis. Four genes were assayed, the maternally methylated genes Snrpn and Kcnq1ot1(or Lit1), and the paternally methylated gene H19 as well as Igf2, which shares a DMR with H19. These genes have well-defined DMRs which have been shown to have gamete-specific differences in methylation (6,23).

Snrpn is normally expressed only from the paternal allele, owing to methylation of the maternal allele during oogenesis (24). We defined samples having 10% or less expression from the normally silent maternal allele as being monoallelic. Using this criterion, the proportion of embryo samples with biallelic expression of Snrpn following superovulation was 13.8% (Fig. 1B and Supplementary Material, Table S1B), which is not significantly different from control embryos (6.5%; Fig. 1A and Supplementary Material, Table S1A). In the placenta, biallelic expression of Snrpn was observed in 3.2% of the control placentas (Fig. 1C and Supplementary Material, Table S1A), but there was a significantly higher proportion of samples with biallelic expression in the placentas from superovulated females (37.8%, Fisher’s exact test, control versus superovulated, P < 0.001; Fig. 1D and Supplementary Material, Table S1B). These results show that the imprinted paternal expression of Snrpn in the placenta is susceptible to disruption by superovulation.

Figure 1.

Allele-specific expression analysis of Snrpn in 9.5dpc embryos (A and B) and placentas (C and D) collected from control (A and C) or superovulated (B and D) females following development in vivo. Gray bars represent maternal expression, black bars represent paternal expression.

Figure 1.

Allele-specific expression analysis of Snrpn in 9.5dpc embryos (A and B) and placentas (C and D) collected from control (A and C) or superovulated (B and D) females following development in vivo. Gray bars represent maternal expression, black bars represent paternal expression.

Superovulation/in vivo development does not affect the expression of Kcnq1ot1

Kcnq1ot1 is an intronic paternally expressed antisense RNA within a large imprinting cluster on mouse chromosome 7. This region is syntenic to human chromosome 11p15.5, which contains the Beckwith–Weidemann causative region. Expression of Kcnq1ot1 is regulated by maternal methylation of a 2 kb region termed the KvDMR within intron 10 of the Kcnq1 locus (25). Methylation of the KvDMR is also important for the imprinted expression of several other genes in the cluster, including Tssc3, Cdkn1c, Kcnq1 and Ascl2 (26,27). We determined whether samples exhibited monoallelic or biallelic expression of Kcnq1ot1 using a qualitative assay. Following superovulation, the expression of Kcnq1ot1 was unchanged in both embryos and placentas (Fig. 2 and Supplementary Material, Tables S1A and B). These results suggest that the establishment and maintenance of imprinted expression of Kcnq1ot1 in the placenta is more resistant to disruption by superovulation than is Snrpn.

Figure 2.

Allele-specific expression analysis of Kcnq1ot1 in 9.5dpc embryos and placentas collected from control or superovulated females following development in vivo. Black bars represent control samples (N = 31 embryos, 29 placentas), gray bars represent superovulated samples (N = 33 embryos, 39 placentas).

Figure 2.

Allele-specific expression analysis of Kcnq1ot1 in 9.5dpc embryos and placentas collected from control or superovulated females following development in vivo. Black bars represent control samples (N = 31 embryos, 29 placentas), gray bars represent superovulated samples (N = 33 embryos, 39 placentas).

Superovulation/in vivo development results in biallelic expression of H19 in postimplantation placentas

The paternally methylated gene H19 is normally expressed strictly from the maternal allele. The imprinted expression of H19 is the result of paternal methylation marks that are established during spermatogenesis and maintained through early embryogenesis (28). The expression of H19 was biallelic in 19.4% of control (Fig. 3A and Supplementary Material, Table S1A), and 13.5% of superovulated embryos (Fig. 3B and Supplementary Material, Table S1B). However, in the placenta, none of the control samples exhibited biallelic expression (Fig. 3C and Supplementary Material, Table S1A), whereas 64.1% of the placentas from superovulated females expressed H19 from both alleles (Fig. 3D and Supplementary Material, Table S1B). This represents a highly significant increase in the proportion of placentas with biallelic expression of H19 following superovulation (Fisher’s exact test, control versus superovulated, P < 0.001). These results indicate that the paternal expression of H19 in the placenta is susceptible to perturbation by superovulation.

Figure 3.

Allele-specific expression analysis of H19 in 9.5dpc embryos (A and B) and placentas (C and D) collected from control (A and C) or superovulated (B and D) females following development in vivo. Details as described in Figure 1.

Figure 3.

Allele-specific expression analysis of H19 in 9.5dpc embryos (A and B) and placentas (C and D) collected from control (A and C) or superovulated (B and D) females following development in vivo. Details as described in Figure 1.

Superovulation/in vivo development does not affect allelic expression of Igf2

As we observed an increase in the proportion of placenta samples with biallelic expression of H19 following superovulation/in vivo development, we examined the allelic expression of Insulin-like growth factor-2 (Igf2). Igf2 and H19 share a DMR, which acts as a methylation-sensitive boundary element. In the absence of methylation on the maternal allele, CTCF binding at the DMR prevents the Igf2 promoter from accessing enhancer elements located downstream of H19, resulting in strictly paternal expression of Igf2. We did not observe a difference in the proportion of samples having biallelic expression of Igf2, as strictly monoallelic expression was observed in almost all the samples (Supplementary Material, Tables S1A and B).

Superovulation/in vivo development does not affect the methylation of the Snrpn and H19 DMRs

To determine if the biallelic expression of Snrpn or H19 was due to a loss of methylation at their respective DMRs, bisulfite conversion and sequencing was undertaken using selected embryos and placentas from the previous experiments. Superovulated samples were selected to represent different combinations of biallelic and monoallelic expression in the embryo and placenta for both genes. Given the many different permutations of these criteria, and the technical difficulty of bisulfite sequencing, four superovulated samples were selected for bisulfite sequencing of both embryo and placenta. The results of the bisulfite sequencing are shown in Figures 4 and 5, and the samples are numbered with reference to the numbering in Figures 1 and 3.

Figure 4.

Allele-specific methylation status of Snrpn in 9.5dpc embryos and placentas collected from control (A) and superovulated (B) CD1 female mice. Numbers beside each sample correspond to the number of each sample in Figures 1 and 3. Each line represents an individual clone; open circles denote unmethylated CpG sites and closed circles denote methylated CpG sites.

Figure 4.

Allele-specific methylation status of Snrpn in 9.5dpc embryos and placentas collected from control (A) and superovulated (B) CD1 female mice. Numbers beside each sample correspond to the number of each sample in Figures 1 and 3. Each line represents an individual clone; open circles denote unmethylated CpG sites and closed circles denote methylated CpG sites.

Figure 5.

Allele-specific methylation status of H19 in 9.5dpc embryos and placentas collected from control (A) and superovulated (B) CD1 female mice. Numbers beside each sample correspond to the number of each sample in Figures 1 and 3. Each line represents an individual clone; open circles denote unmethylated CpG sites and closed circles denote methylated CpG sites.

Figure 5.

Allele-specific methylation status of H19 in 9.5dpc embryos and placentas collected from control (A) and superovulated (B) CD1 female mice. Numbers beside each sample correspond to the number of each sample in Figures 1 and 3. Each line represents an individual clone; open circles denote unmethylated CpG sites and closed circles denote methylated CpG sites.

Bisulfite sequencing analysis of the Snrpn DMR of control (Fig. 4A) and superovulated (Fig. 4B) embryos and placentas revealed that methylation was not affected following superovulation. Similarly, for the H19 DMR, bisulfite sequencing analysis of control (Fig. 5A) and superovulated (Fig. 5B) embryos and placentas showed that methylation was not affected by the administration of exogenous hormones. Although subtle effects would not be detected using the bisulfite sequencing assay, the results suggest that the loss of imprinted expression of these two genes was unlikely to be the result of a notable or significant loss of methylation in the regions examined.

The overall methylation at the Snrpn and H19 DMRs is lower in the placenta than in the embryo

From the DNA methylation data described above, it was noted that the methylation of DMRs in the placenta appeared to be less complete than in the embryo. The maternal allele of Snrpn was highly methylated (94% of CpGs methylated) in all six embryos examined, while in the placenta, the methylation levels were somewhat lower (85% of CpGs methylated). Similarly, for the paternal allele of H19, the levels of methylation appeared to be higher in the six embryos (90% of CpGs methylated) than in the placentas (78% of CpGs methylated). There was no observable difference in the levels of methylation on the unmethylated alleles for either gene. These data suggest that methylation in the placenta may not be subject to the same tight regulation as in the embryo.

Effects of embryo transfer of superovulated blastocysts on overall growth or development of the embryos

Since the embryos in the previous experiment developed in vivo, we undertook embryo transfer experiments to alleviate possible effects of the environment and/or litter size (Table 1). Ten or fewer blastocysts were transferred to each uterine horn, in an attempt to more closely model the normal litter size for CD1 mice. Each recipient received embryos from a single donor female.

The blastocysts transferred to pseudopregnant CD1 females from superovulated females exhibited a significant decrease in implantation rate when compared to control donors (t-test, control versus superovulated, P < 0.05; Table 2), as well as an increase in the number of embryos with delayed development (Fisher’s exact test, control versus superovulated, P < 0.05; Table 2). There was no difference in the percentage of embryos that were viable, or in the percentage of implantation sites that were undergoing resorption. There was also no difference in the number of embryos with gross abnormalities; although this number was quite high in both groups (Table 2). The average crown-rump length of control embryos was 4.21 ± 0.31 mm, which was not significantly different from that of embryos from superovulated mothers (3.77 ± 0.23 mm). However, the overall development of the embryos, as assessed by somite number, was affected by superovulation and embryo transfer (30.92 ± 1.94 for control embryos versus 26.17 ± 1.43 for superovulated embryos, Kruskal–Wallis test, P < 0.01).

Table 2.

Litter characteristics of control and superovulated litters following embryo transfer

  No. of blastocysts transferred No. of implantation sites (%) No. of resorption sites (%) Numbers collected (%)a Numbers viable (%)b Numbers delayed No. with morphological abnormality (%)c 
Control Litter 1 10 8 (80.0) 2 (25.0) 6 (75.0) 6 (100) 0 (0) 0 (0) 
 Litter 2 10 9 (90.0) 1 (11.1) 8 (88.9) 7 (87.5) 1 (12.5) 3 (37.5) 
 Litter 3 10 9 (90.0) 6 (66.7) 3 (33.3) 2 (66.6) 0 (0) 1 (33.3) 
 Litter 4 10 9 (90.0) 1 (11.1) 7 (77.8) 5 (62.5) 0 (0) 1 (14.3) 
 Litter 5 13 11 (84.6) 2 (18.2) 8 (72.7) 6 (54.5) 1 (12.5) 1 (12.5) 
 Total 53 46 (86.8) 12 (22.6) 32 (69.6) 26 (81.3) 2 (6.3) 6 (18.8) 
Superovulated Litter 1 20 8 (40.0) 2 (25.0) 6 (75.0) 5 (83.3) 6 (100) 2 (33.3) 
 Litter 2 12 4 (33.3) 0 (0) 4 (100) 3 (75.0) 0 (0) 1 (25.0) 
 Litter 3 13 11 (84.6) 6 (54.5) 5 (45.5) 3 (60.0) 0 (0) 3 (60.0) 
 Litter 4 19 17 (89.5) 4 (23.5) 12 (70.5) 9 (75.0) 1 (8.3) 1 (8.3) 
 Litter 5 13 7 (53.8) 2 (28.6) 5 (71.4) 5 (100) 1 (20.0) 2 (40.0) 
 Total 77 47 (61.0)* 14 (29.8) 32 (68.1) 25 (78.1) 8 (17.0)** 9 (19.1) 
  No. of blastocysts transferred No. of implantation sites (%) No. of resorption sites (%) Numbers collected (%)a Numbers viable (%)b Numbers delayed No. with morphological abnormality (%)c 
Control Litter 1 10 8 (80.0) 2 (25.0) 6 (75.0) 6 (100) 0 (0) 0 (0) 
 Litter 2 10 9 (90.0) 1 (11.1) 8 (88.9) 7 (87.5) 1 (12.5) 3 (37.5) 
 Litter 3 10 9 (90.0) 6 (66.7) 3 (33.3) 2 (66.6) 0 (0) 1 (33.3) 
 Litter 4 10 9 (90.0) 1 (11.1) 7 (77.8) 5 (62.5) 0 (0) 1 (14.3) 
 Litter 5 13 11 (84.6) 2 (18.2) 8 (72.7) 6 (54.5) 1 (12.5) 1 (12.5) 
 Total 53 46 (86.8) 12 (22.6) 32 (69.6) 26 (81.3) 2 (6.3) 6 (18.8) 
Superovulated Litter 1 20 8 (40.0) 2 (25.0) 6 (75.0) 5 (83.3) 6 (100) 2 (33.3) 
 Litter 2 12 4 (33.3) 0 (0) 4 (100) 3 (75.0) 0 (0) 1 (25.0) 
 Litter 3 13 11 (84.6) 6 (54.5) 5 (45.5) 3 (60.0) 0 (0) 3 (60.0) 
 Litter 4 19 17 (89.5) 4 (23.5) 12 (70.5) 9 (75.0) 1 (8.3) 1 (8.3) 
 Litter 5 13 7 (53.8) 2 (28.6) 5 (71.4) 5 (100) 1 (20.0) 2 (40.0) 
 Total 77 47 (61.0)* 14 (29.8) 32 (68.1) 25 (78.1) 8 (17.0)** 9 (19.1) 

aImplantation sites containing embryo tissue were collected.

bViable embryos were defined as embryos having a heart beat, regardless of stage of development.

cDetailed information regarding morphological abnormalities can be found in Supplementary Material, Tables S1A and B.

*P < 0.05, t-test, control versus superovulated.

**P < 0.05, Fisher’s exact test, control versus superovulated.

Monoallelic expression of Snrpn is not affected by superovulation and embryo transfer

Following embryo transfer, allele-specific expression analysis revealed that Snrpn was expressed biallelically in 8.7% of naturally cycling control embryos (Fig. 6A and Supplementary Material, Table S2A) and in 18.8% of superovulated embryos (Fig. 6B and Supplementary Material, Table S2B). This apparent increase of biallelic expression following superovulation and embryo transfer was not statistically significant. Similarly, in the placenta, biallelic expression of Snrpn was observed in 26.1% of control placentas (Fig. 6C and Supplementary Material, Table S2A) and in 37.5% of superovulated placentas (Fig. 6D and Supplementary Material, Table S2B); this difference was not statistically significant.

Figure 6.

Allele-specific expression analysis of Snrpn in 9.5dpc embryos (A and B) and placentas (C and D). Donor CD1 female mice were either naturally cycling (control) (A and C) or superovulated (B and D). Gray bars represent maternal expression, black bars represent paternal expression.

Figure 6.

Allele-specific expression analysis of Snrpn in 9.5dpc embryos (A and B) and placentas (C and D). Donor CD1 female mice were either naturally cycling (control) (A and C) or superovulated (B and D). Gray bars represent maternal expression, black bars represent paternal expression.

Superovulation and embryo transfer result in biallelic expression of H19 in the placenta

Following embryo transfer, biallelic expression of H19 was observed in 29.2% of embryos following transfer of control blastocysts (Fig. 7A and Supplementary Material, Table S2A) and of 39.4% of embryos following transfer of blastocysts collected from superovulated donor females (Fig. 7B and Supplementary Material, Table S2B). There was no statistically significant difference between the groups. However, biallelic expression of H19 was observed in 26.1% of placentas following transfer of control blastocysts (Fig. 7C and Supplementary Material, Table S2A) and in a significantly higher proportion of placentas (58.1%) following transfer of blastocysts from superovulated donors (Fig. 7D and Supplementary Material, Table S2B, Fisher’s exact test, control versus superovulated, P < 0.03). These results suggest that the imprinted expression of H19 in the placenta is particularly susceptible to perturbation.

Figure 7.

Allele-specific expression analysis of H19 in 9.5dpc embryos (A and B) and placentas (C and D). Donor CD1 female mice were either naturally cycling (control) (A and C) or superovulated (B and D). Details as for Figure 6.

Figure 7.

Allele-specific expression analysis of H19 in 9.5dpc embryos (A and B) and placentas (C and D). Donor CD1 female mice were either naturally cycling (control) (A and C) or superovulated (B and D). Details as for Figure 6.

Superovulation results in increased levels of Igf2 expression in the placenta

We examined the effect of superovulation on the total expression of an important growth factor, the Insulin-like growth factor-II (Igf2) gene in the placenta. Igf2 expression in the placenta is critical for both placental and fetal growth (29), as it regulates the development of the diffusional permeability capacity of the placenta (30). While we did not observe any differences in the total levels of Igf2 expression in the embryos (data not shown), expression of Igf2 in placentas was increased following superovulation after both in vivo development and embryo transfer (n = 14 placentas for the in vivo control group, n = 15 placentas for all other groups; Fig. 8). This result was particularly interesting since the placenta retained strictly monoallelic expression as shown for the superovulation/in vivo development group. This result shows that superovulation can affect the placental expression of Igf2, a gene that is closely linked to H19.

Figure 8.

Analysis of Igf2 expression in 9.5dpc placentas. *P > 0.03 **P > 0.008. (N = 15 placentas for in vivo control group, 16 placentas for all other groups).

Figure 8.

Analysis of Igf2 expression in 9.5dpc placentas. *P > 0.03 **P > 0.008. (N = 15 placentas for in vivo control group, 16 placentas for all other groups).

DISCUSSION

Previous studies examining the effects of superovulation have largely concentrated on the growth and development of the embryo. Despite the strong association of imprinted genes with embryo growth (reviewed in 2), little has been done to investigate the possible epigenetic effects of superovulation. An investigation of 5-methylcytosine staining in two-cell mouse embryos suggested that superovulation might lead to epigenetic abnormalities in the resultant embryos (22). Subsequently, bisulfite sequencing of DMRs of several imprinted genes in both mouse and human oocytes demonstrated both gain and loss of methylation (8). Our approach in the current paper differs from earlier studies in extending the examination of epigenetic outcomes in the offspring of superovulated mothers to the postimplantation period and allowing imprinted genes in both the embryo and the placenta to be examined.

We have shown that superovulation followed by in vivo development results in biallelic expression of Snrpn and H19 in 9.5dpc placenta, while expression of Kcnq1ot1 was unaffected. Although several studies have shown an effect of preimplantation culture on imprinted gene expression (31–34), the expression of Snrpn has not always been examined; thus, it is possible that the imprinted expression of Snrpn is also affected by these manipulations. As the methylation pattern that confers genomic imprinting at H19 is established in the male germline (35–38), the biallelic expression of H19 is somewhat surprising. The administration of the exogenous hormones takes place prior to ovulation and fertilization, and was therefore expected to only affect genes that are imprinted in the female germline. Our result suggests that it is not the establishment of imprinting that is affected, but rather its maintenance. We envision two possible causes of disruption to paternal imprints. First, superovulation may result in the ovulation of abnormal oocytes, either by forcing the oocytes to develop too quickly or by rescuing oocytes that have already been selected for atresia. As a result, these oocytes may be defective in their ability to maintain imprints during the preimplantation period. The second possibility is that administration of exogenous hormones may alter the milieu of the oviduct and/or uterus, which in turn may have a negative effect on the maintenance of genomic imprinting in the conceptus. In fact, the imprinting of Snrpn was unaffected in superovulated embryos and placentas that had been transferred to an unstimulated uterus, which provides further support for an effect of hormonal milieu on the maintenance of imprinted gene expression. We cannot, however, rule out that imprint acquisition is affected by superovulation because we have only examined two of the many genes known to be methylated during oogenesis. Our group and others have previously shown that methylation imprints are acquired asynchronously during the oocyte growth phase (24,39). As such, genes that acquire their methylation imprints later during oocyte growth, such as Peg1, may be more susceptible to disruption by ovarian stimulation. This is also supported by the relative protection of Kcnq1ot1 to the effects of superovulation, as imprint acquisition at the KvDMR occurs relatively early during oocyte growth (39).

As no change was seen in the expression of Kcnq1ot1 following superovulation, this suggests that superovulation may not be a major contributor to the increased incidence of Beckwith–Weidemann observed following assisted reproductive technologies (ARTs) (40–43). A small number of samples did exhibit biallelic expression of Kcnq1ot1 in the placenta following superovulation, but the matched embryos exhibited strictly monoallelic expression. However, a recent study of a small number of human oocytes collected following ovarian stimulation showed a complete loss of methylation at the KvDMR in one of the 16 oocytes analyzed (44). Further studies will be required to elucidate the effect of superovulation on the Kcnq1ot1 locus at different stages during the reproductive cycle.

While only the monoallelic expression of H19 in the placenta was affected following superovulation and embryo transfer, these results may be misleading. There was an increase in the proportion of samples that were biallelic for Snrpn in the placenta, and in the proportion of samples that were biallelic for H19 in the embryo and placenta following embryo transfer alone (embryo transfer control group). A direct comparison of the in vivo and embryo transfer groups may not be appropriate, due to the difference in approach taken. However, these results suggest that the embryo transfer procedure can perturb imprinted gene expression, and that further study is required. There are many aspects of the embryo transfer procedure that could cause disruption of imprinted gene expression, such as the flushing of blastocysts from the uterus and/or embryo culture. A recent study has shown that the embryo transfer procedure alone results in loss of imprinting at several loci in both the placenta and the yolk sac at 9.5dpc, with more severe effects when embryo culture preceded the embryo transfer (45). Additionally, preimplantation culture has been shown to have an effect on the expression of several imprinted genes (31–34). Although these studies examined the consequence of longer exposures to culture media, an effect due to shorter culture times cannot be ruled out. Furthermore, the blastocysts are transferred to a uterus that is delayed by 1 day relative to the donors. Although the recipients are naturally cycling females, the uterine milieu will not be synchronized with the donor embryos, and this may affect the embryo. All of these features of embryo transfer may contribute to the biallelic expression of imprinted genes.

The monoallelic expression of imprinted genes in the placenta appears to be particularly susceptible to perturbation. Loss of imprinting at H19 was observed specifically in the placenta following preimplantation culture in Whitten’s media (34). In a subset of placentas from embryos cultured in Whitten’s media, biallelic expression of Snrpn was also observed (34). Here, we have shown loss of imprinting at both Snrpn and H19 in the placenta following superovulation. These results suggest that maintenance of imprinting is regulated differentially in the embryo and in the placenta. As previously discussed by Mann et al. (34), there are several possible reasons why trophectoderm-derived tissues might be more susceptible to epigenetic disruption. One potential reason for the increased effect seen in placentas is that the trophectoderm cells are the outer cells of the blastocyst and are therefore in direct contact with the environment. We have observed that the disruption of Snrpn imprinting following superovulation is slightly more pronounced in the placenta, which may be a result of these cells being in direct contact with an altered uterine milieu. A second possibility is that there is less redundancy in epigenetic modifications in the placenta. Some groups have suggested that imprinting in the placenta is regulated primarily by histone modifications, and that methylation does not play an important role in imprinted gene expression in this tissue (46,47). This may be supported by our observation that the overall levels of methylation at the DMRs of both Snrpn and H19 appear to be lower in placentas than in embryos, without an effect on the monoallelic expression of these genes. However, as the normal patterns of methylation of Snrpn and H19 have not been well characterized in the placenta, further studies are required.

We did not observe changes in the methylation at either of the DMRs examined following superovulation, despite the appearance of biallelic expression. The regions we have examined are known to acquire methylation during gametogenesis (24) and therefore are important for the initial establishment of imprinted expression. In the mouse, the DMR of H19 is approximately 2.0 kb long, and methylation is also observed in the promoter region located upstream of the DMR (48–50). We have only examined a region of approximately 420 bp within the DMR, which includes two of the four CTCF binding sites (6). As such, it is possible that methylation in a different region of the DMR is of greater importance for the maintenance of imprinting during postimplantation development. Alternatively, it may not be overall levels of methylation that are important for maintaining imprinted expression. Rather the methylation of specific CpG dinucleotides might be of key importance, such as binding sites for proteins like methyl CpG-binding protein which may then recruit other proteins involved in chromatin modifications (reviewed in 51). Similarly, the DMR1 of Snrpn is 6.6 kb in length. We have examined a region of approximately 430 bp within DMR1 (6), and therefore cannot conclude that methylation is not affected at all, or if the observed biallelic expression has occurred in the absence of any methylation changes. A recent study showed that for several imprinted genes, including Snrpn, tandem repeats outside the DMR1 region are important for the maintenance of imprinted expression. These regions also become methylated when part of an imprinted transgene and this methylation is maintained throughout preimplantation development (52). This supports the idea that regions other than those known to be important for imprint establishment are required for maintaining imprinting. Finally, we are proposing that maintenance of DNA methylation is affected by superovulation. Thus, failure to maintain imprints due to compromised oocyte quality could occur at any time during preimplantation development, affect only a subset of blastomeres and be difficult to detect using bisulfite sequencing.

Previous studies which focused on the growth and development of embryos showed that superovulation resulted in smaller embryos by weight (20,21). Developmental delay following superovulation and embryo transfer have also been reported (20,21). However, in our current study, we did not observe an effect on embryo size or somite number following superovulation/in vivo development. In the previous studies, embryo size was assessed at 18dpc or at 14dpc, respectively, whereas we have assessed size at 9.5dpc. Although we have not seen any effects on the growth of the embryo, this may be due to the early time point examined. Prior to 9.5dpc, it is thought that the placenta does not play a critical role in supporting the embryo (53–56). As we are examining embryos prior to this critical period, it may be too early to observe defects of growth.

As the primary effect of superovulation on imprinted genes appears to be in the placenta, we have also examined the expression of Igf2, a critical factor in placental growth and nutrient transfer to the placenta. Following superovulation, the expression of Igf2 was increased in both the in vivo and embryo transfer groups despite the fact that Igf2 expression was monoallelic in the placenta following in vivo development. Elevated levels of Igf2 in the placenta have been correlated with fetal growth restriction in humans (57) and sheep (58) and with early embryonic lethality of somatic cell nuclear transfer derived cows (59). Little is known about mechanisms regulating the total levels of Igf2 expression in the placenta. It is known that the expression of Igf2 in the labyrinthine trophoblast occurs exclusively from a placenta-specific promoter (P0) (29,60). In the other placental cell types, Igf2 is expressed from the fetal promoters, P1, P2 and P3 (29,61). All of these promoters result in an identical protein; however, the presence of multiple promoters with differential usage dependent on cell type suggests that regulation of Igf2 expression in the placenta is complex.

Even in the absence of methylation changes, the biallelic expression of imprinted genes is the cause for concern. Several studies have shown postnatal phenotypes as a result of disruption of imprinted genes during early development. Ecker et al. (62) demonstrated that preimplantation culture in either KSOM or Whitten’s media resulted in behavioral changes, increased anxiety and deficits in spatial memory in adult mice. Embryo culture in the presence of fetal calf serum was shown to affect the expression of several imprinted genes, but also to result in increased weight, locomotor deficits, organomegaly and increased anxiety in adult mice (63). In addition to mouse models, patients with Beckwith–Weidemann Syndrome, which is often associated with alterations in imprinted gene expression, are more susceptible to neoplasia (64). Several groups have raised concerns about the association of rare imprinting diseases with ARTs, including ovarian hyperstimulation (16,65). Analyses of affected children have consistently shown methylation defects at the DMRs of SNRPN (Angelman Syndrome), KCNQ1OT1 (Beckwith–Weidemann Syndrome) and most recently PEG1/MEST (Silver–Russell Syndrome) (40–43,66,67). While we did not observe changes in methylation at the DMRs examined in this study, we must also consider the possibility of long-term effects as a result of biallelic expression of imprinted genes.

In this study, we have shown that superovulation results in biallelic expression of two imprinted genes, Snrpn and H19, and the increased expression of an important growth factor, Igf2, in the postimplantation period. Our results, and those of Mann et al. (34), demonstrate that trophectoderm-derived tissues are more susceptible to disruption of imprinted genes than the embryo proper. These results lead us to suggest that placental tissue from babies born following ARTs may be used to screen for epigenetic disruption. Early screening using this tissue may lead to earlier intervention or better surveillance of children who may be at risk later in life.

MATERIALS AND METHODS

Embryo collection and mice

Embryos and placentas were obtained from crosses of CD1 female mice (Charles River Canada, St Constant, QC, Canada) to C57Bl/6J (CAST7) male mice (68), to allow for the identification of alleles. Eight-week-old CD1 female mice were superovulated by injection of 5 IU of pregnant mares’ serum gonadotropin (Sigma) and 5 IU human chorionic gonadotropin (Sigma). The presence of a vaginal plug was designated at 0.5dpc. Embryos and placentas were collected at 9.5dpc. For embryo transfer experiments, females were superovulated and mated to C57Bl/6J (CAST7) males, as above. Embryos were collected at 3.5dpc into HEPES-buffered KSOM, and were then immediately transferred to 2.5dpc pseudopregnant CD1 females as described (69). Embryos and placentas were collected at 9.5dpc relative to the recipient female. Embryos and placentas were bisected transversely to allow for DNA and RNA extraction from the same embryo. Experiments were performed in compliance with guidelines established by the Canadian Council for Animal Care.

RNA isolation and allele-specific expression assays

RNA was isolated from one half of bisected embryos or placentas using the HighPure RNA Tissue Kit (Roche Molecular Biochemicals), with minor modifications to the manufacturer’s protocol. cDNA synthesis was performed using SuperScriptII (Invitrogen), random hexamers (Invitrogen) and 80 ng of total RNA. Allele-specific H19 and Snrpn LightCycler (LC) expression assays were carried out on cDNA using a LC Real-Time PCR System (Roche Biochemicals). The H19 LC assay was performed as described previously (68,70), except that 1 µl of cDNA was used. The Snrpn LC assay was performed as described (68), without the addition of DMSO and with 1 µl of cDNA. After background subtraction, the relative contribution of each allele was calculated as the peak area of the melting curve generated at the allele-specific temperature.

Kcnq1ot1 expression assay

The Kcnq1ot1 expression LightCycler assays were performed on cDNA prepared as above. The Kcnq1ot1 primers, LitLC-F (5′-GAGGCTCAGGTCTCTGTTGG-3′) and Lit1LC-R (5′-TTACAGCGGAAAGCACTCCT-3′) were used to amplify a 311 bp region of the Kcnq1ot1 region (AF119385; 71). Fluorescent resonance energy transfer hybridization probes were designed to the CD1 amplicon. The Kcnq1ot1 sensor probe (5′-AGAGTTGATTTAAAGGACCAAGGCCA-FL-3′; Idaho Technologies) spans a single nucleotide polymorphism at nucleotide 3939 between CD1 (T) and CAST (G) and was labeled with fluoroscein at the 3′ end. The Kcnq1ot1 anchor probe (5′-RED640-GGCCCAAACCTTAGTCCTCCATTTTG-P-3′) was labeled with LC-Red640 at the 5′ end and phosporylated at the 3′ end. To a Ready-to-Go PCR bead (GE Healthcare), 8.12 μl of H2O and 0.38 µl of TaqStart Antibody (BD Biosciences Clontech) were added, and the reaction was incubated at room temperature for 5 min. After incubation, 1.5 µm MgCl2, 0.2 µm of each primer and 0.3 µm of each probe was added to the mix and the volume brought to 12.5 µl. From this reaction mix, 10 µl was removed and placed in a LC glass capillary (Roche Molecular Biochemicals), and 1 µl of cDNA plus 9 µl of H20 were added for a final volume of 20 µl. After an initial denaturation step at 95°C for 30 s, amplification was performed for 35 cycles at 95°C for 0 s, 60°C for 15 s and 72°C for 13 s. A single fluorescence acquisition occurred at the end of each annealing step. After amplification, a final denaturation and annealing step was conducted (95°C for 0 s, 50°C for 2 min) followed by a melting curve analysis with fluorescence acquisition occurring continuously as the temperature increased from 50 to 85°C at a rate of 0.05°C/s. After background subtraction, the presence or absence of each allele was determined based on the presence of peaks at the allele-specific temperature, approximately 65.5°C for CAST7 and 68.5°C for CD1 (LC Data Analysis Software, Roche Molecular Biochemicals).

Igf2 expression assay

Igf2 expression was performed on cDNA as above. The Igf2 primers, Igf2-F (5′-ATCTGTGACCTCCTCTTGAGCAGG-3′) and Igf2-R (5′-GGGTTGTTTAGAGCCAATCAA-3′) were used to amplify a 202 bp region of Igf2 (NM 010514). To a Ready-to-Go PCR bead, 0.3 µm of each primer and H2O were added. After incubating for 5 min at room temperature, 1 µl of cDNA was added. After an initial denaturation step at 94°C for 5 min, amplification was performed for 40 cycles at 95°C for 15 s, 56°C for 30 s and 72°C for 30 s. One microliter of Tsp509I restriction enzyme was added to 10–15 µl of PCR product and digestion was allowed to proceed at 65°C for 2 h. The digested samples were run on a 7% acrylamide gel. The digested CD1 allele produces fragments of 178 and 24 bp, while the digested CAST7 allele produces fragments of 163, 24 and 15 bp.

DNA isolation and bisulfite sequencing

DNA was isolated from one half of bisected embryos or placentas and bisulfite treatment was carried out as previously described (6). Nested PCR amplification of H19 and Snrpn was conducted with the same primers as previously described (6). We examined a total of 15 CpG sites in the imprint control region (ICR) upstream of the H19 transcriptional start site (Genbank acc. no. U19619, nucleotides 1304-1726). This region includes the two 5′ CTCF binding sites (R1 and R2), but does not include the two 3′ CTCF binding sites (R3 and R4) (70). We examined 16 CpG sites in the exon 1 region of Snrpn (Genbank acc. no. AF081460, nucleotides 2151-2570). The presence of both alleles in the PCR products was confirmed using restriction fragment length polymorphisms generated by SNPs between the CD1 and C57Bl/6J (CAST7) strains. Digestion of 5 µl of the purified PCR products [Snrpn with SwaI (NEB) and H19 with DpnII (NEB) restriction enzymes] was followed by gel electrophoresis in 1.5% agarose. For each sample, a minimum of two independent PCR products was subcloned using the TOPO TA Cloning Kit (Invitrogen), and plasmid DNA was purified using the QIAprep Spin Miniprep Kit (Qiagen). Ten to twenty clones were analyzed per PCR product, and clones containing the appropriate insert were sequenced using an ABI 3100 sequencer.

Quantification of Igf2 gene expression

Real-time quantitative RT–PCR was conducted using a two-step approach; the cDNA prepared for the allele-specific expression assays was used as the template for SybrGreen (Qiagen). The primers for total Igf2 expression were the same as those used for allele-specific expression analysis (above). Samples were analyzed in duplicate and the threshold cycle (CT) was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (primers Gapdh-F: 5′-TGTTTGTGATGGGTGTGAA-3′ and Gapdh-R: 5′-TCCTCAGTGTAGCCCAAGAT-3′) using an MX4000 (Stratagene). The mRNA level for each sample relative to one control sample was calculated using the relative CT method.

Statistical analysis

Comparisons of litter characteristics between control and superovulated groups were first subjected to Levene’s test for homogeneity of variance. For groups with equal variances, comparisons were further analyzed by one-way ANOVA, while for groups with unequal variances the comparisons were analyzed by the Kruskal–Wallis test for non-parametric data. Some groups were analyzed using Fisher’s exact test, these are indicated in the text. Comparisons of allele-specific expression assay groups were analyzed using Fisher’s exact test. Bisulfite sequencing groups were compared using a t-test for unpaired samples and Levene’s test for equality of variances was applied using SPSS 14.0 for Windows. Comparisons of Igf2 expression levels were analyzed using the least square ANOVA and the linear mixed effect model using SAS (Cary, NC, USA).

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG Online

FUNDING

This work is supported by a grant from the Canadian Institutes of Health Research (CIHR) to J.M.T. A.L.F. is the recipient of a CIHR Graduate Studentship. J.M.T. is a James McGill Professor and a Scholar of the Fonds de la Recherche en santé du Québec (FRSQ). This work was part of the Program on Oocyte Health funded under the Healthy Gametes and Great Embryos Strategic Initiative of the CIHR Institute of Human Development, Child and Youth Health.

ACKNOWLEDGEMENTS

We thank Drs Marisa Bartolomei, Raluca I. Verona and Rocio M. Rivera from the University of Pennsylvania for assistance in setting up the allele-specific H19 and Snrpn assays as well as communication of data prior to publication. Dr Bartolomei also kindly provided the original CAST7 mice for our colony. We also thank Drs D. Arnold, G. Hooker and X. Zhang for assistance with statistical analysis, and K. Christensen, A. Lawrance, K. Niles and L. Pickell for thoughtful discussion.

Conflict of Interest statement. None declared.

REFERENCES

1
Wood
A.J.
Oakey
R.J.
Genomic imprinting in mammals: emerging themes and established theories
PLoS Genet.
 , 
2006
, vol. 
2
 pg. 
e147
 
2
Smith
F.M.
Garfield
A.S.
Ward
A.
Regulation of growth and metabolism by imprinted genes
Cytogenet. Genome Res.
 , 
2006
, vol. 
113
 (pg. 
279
-
291
)
3
Fowden
A.L.
Sibley
C.
Reik
W.
Constancia
M.
Imprinted genes, placental development and fetal growth
Horm. Res.
 , 
2006
, vol. 
65
 
Suppl. 3
(pg. 
50
-
58
)
4
Isles
A.R.
Holland
A.J.
Imprinted genes and mother-offspring interactions
Early Hum. Dev.
 , 
2005
, vol. 
81
 (pg. 
73
-
77
)
5
Murrell
A.
Genomic imprinting and cancer: from primordial germ cells to somatic cells
Sci. World J.
 , 
2006
, vol. 
6
 (pg. 
1888
-
1910
)
6
Lucifero
D.
Mertineit
C.
Clarke
H.J.
Bestor
T.H.
Trasler
J.M.
Methylation dynamics of imprinted genes in mouse germ cells
Genomics
 , 
2002
, vol. 
79
 (pg. 
530
-
538
)
7
Geuns
E.
De Rycke
M.
Van Steirteghem
A.
Liebaers
I.
Methylation imprints of the imprint control region of the SNRPN-gene in human gametes and preimplantation embryos
Hum. Mol. Genet.
 , 
2003
, vol. 
12
 (pg. 
2873
-
2879
)
8
Sato
A.
Otsu
E.
Negishi
H.
Utsunomiya
T.
Arima
T.
Aberrant DNA methylation of imprinted loci in superovulated oocytes
Hum. Reprod.
 , 
2007
, vol. 
22
 (pg. 
26
-
35
)
9
Szabo
P.E.
Mann
J.R.
Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting
Genes Dev.
 , 
1995
, vol. 
9
 (pg. 
1857
-
1868
)
10
Kato
Y.
Rideout
W.M.
3rd
Hilton
K.
Barton
S.C.
Tsunoda
Y.
Surani
M.A.
Developmental potential of mouse primordial germ cells
Development
 , 
1999
, vol. 
126
 (pg. 
1823
-
1832
)
11
Hajkova
P.
Erhardt
S.
Lane
N.
Haaf
T.
El-Maarri
O.
Reik
W.
Walter
J.
Surani
M.A.
Epigenetic reprogramming in mouse primordial germ cells
Mech. Dev.
 , 
2002
, vol. 
117
 (pg. 
15
-
23
)
12
Lee
J.
Inoue
K.
Ono
R.
Ogonuki
N.
Kohda
T.
Kaneko-Ishino
T.
Ogura
A.
Ishino
F.
Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells
Development
 , 
2002
, vol. 
129
 (pg. 
1807
-
1817
)
13
Szabo
P.E.
Hubner
K.
Scholer
H.
Mann
J.R.
Allele-specific expression of imprinted genes in mouse migratory primordial germ cells
Mech. Dev.
 , 
2002
, vol. 
115
 (pg. 
157
-
160
)
14
Bao
S.
Obata
Y.
Carroll
J.
Domeki
I.
Kono
T.
Epigenetic modifications necessary for normal development are established during oocyte growth in mice
Biol. Reprod.
 , 
2000
, vol. 
62
 (pg. 
616
-
621
)
15
Obata
Y.
Kono
T.
Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth
J. Biol. Chem.
 , 
2002
, vol. 
277
 (pg. 
5285
-
5289
)
16
Lucifero
D.
Chaillet
J.R.
Trasler
J.M.
Potential significance of genomic imprinting defects for reproduction and assisted reproductive technology
Hum. Reprod. Update
 , 
2004
, vol. 
10
 (pg. 
3
-
18
)
17
Hiura
H.
Komiyama
J.
Shirai
M.
Obata
Y.
Ogawa
H.
Kono
T.
DNA methylation imprints on the IG-DMR of the Dlk1-Gtl2 domain in mouse male germline
FEBS Lett.
 , 
2007
, vol. 
581
 (pg. 
1255
-
1260
)
18
Fossum
G.T.
Davidson
A.
Paulson
R.J.
Ovarian hyperstimulation inhibits embryo implantation in the mouse
J. In Vitro Fert. Embryo Transf.
 , 
1989
, vol. 
6
 (pg. 
7
-
10
)
19
Ertzeid
G.
Storeng
R.
Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice
J. Reprod. Fertil.
 , 
1992
, vol. 
96
 (pg. 
649
-
655
)
20
Ertzeid
G.
Storeng
R.
The impact of ovarian stimulation on implantation and fetal development in mice
Hum. Reprod.
 , 
2001
, vol. 
16
 (pg. 
221
-
225
)
21
Van der Auwera
I.
D’Hooghe
T.
Superovulation of female mice delays embryonic and fetal development
Hum. Reprod.
 , 
2001
, vol. 
16
 (pg. 
1237
-
1243
)
22
Shi
W.
Haaf
T.
Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure
Mol. Reprod. Dev.
 , 
2002
, vol. 
63
 (pg. 
329
-
334
)
23
Yatsuki
H.
Joh
K.
Higashimoto
K.
Soejima
H.
Arai
Y.
Wang
Y.
Hatada
I.
Obata
Y.
Morisaki
H.
Zhang
Z.
, et al.  . 
Domain regulation of imprinting cluster in Kip2/Lit1 subdomain on mouse chromosome 7F4/F5: large-scale DNA methylation analysis reveals that DMR-Lit1 is a putative imprinting control region
Genome Res.
 , 
2002
, vol. 
12
 (pg. 
1860
-
1870
)
24
Lucifero
D.
Mann
M.R.
Bartolomei
M.S.
Trasler
J.M.
Gene-specific timing and epigenetic memory in oocyte imprinting
Hum. Mol. Genet.
 , 
2004
, vol. 
13
 (pg. 
839
-
849
)
25
Mancini-DiNardo
D.
Steele
S.J.
Ingram
R.S.
Tilghman
S.M.
A differentially methylated region within the gene Kcnq1 functions as an imprinted promoter and silencer
Hum. Mol. Genet.
 , 
2003
, vol. 
12
 (pg. 
283
-
294
)
26
Horike
S.
Mitsuya
K.
Meguro
M.
Kotobuki
N.
Kashiwagi
A.
Notsu
T.
Schulz
T.C.
Shirayoshi
Y.
Oshimura
M.
Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith–Wiedemann syndrome
Hum. Mol. Genet.
 , 
2000
, vol. 
9
 (pg. 
2075
-
2083
)
27
Fitzpatrick
G.V.
Soloway
P.D.
Higgins
M.J.
Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1
Nat. Genet.
 , 
2002
, vol. 
32
 (pg. 
426
-
431
)
28
Tremblay
K.D.
Saam
J.R.
Ingram
R.S.
Tilghman
S.M.
Bartolomei
M.S.
A paternal-specific methylation imprint marks the alleles of the mouse H19 gene
Nat. Genet.
 , 
1995
, vol. 
9
 (pg. 
407
-
413
)
29
Constancia
M.
Hemberger
M.
Hughes
J.
Dean
W.
Ferguson-Smith
A.
Fundele
R.
Stewart
F.
Kelsey
G.
Fowden
A.
Sibley
C.
, et al.  . 
Placental-specific IGF-II is a major modulator of placental and fetal growth
Nature
 , 
2002
, vol. 
417
 (pg. 
945
-
948
)
30
Sibley
C.P.
Coan
P.M.
Ferguson-Smith
A.C.
Dean
W.
Hughes
J.
Smith
P.
Reik
W.
Burton
G.J.
Fowden
A.L.
Constancia
M.
Placental-specific insulin-like growth factor 2 (Igf2) regulates the diffusional exchange characteristics of the mouse placenta
Proc. Natl Acad. Sci. USA
 , 
2004
, vol. 
101
 (pg. 
8204
-
8208
)
31
Sasaki
H.
Ferguson-Smith
A.C.
Shum
A.S.
Barton
S.C.
Surani
M.A.
Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos
Development
 , 
1995
, vol. 
121
 (pg. 
4195
-
4202
)
32
Doherty
A.S.
Mann
M.R.
Tremblay
K.D.
Bartolomei
M.S.
Schultz
R.M.
Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo
Biol. Reprod.
 , 
2000
, vol. 
62
 (pg. 
1526
-
1535
)
33
Khosla
S.
Dean
W.
Brown
D.
Reik
W.
Feil
R.
Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes
Biol. Reprod.
 , 
2001
, vol. 
64
 (pg. 
918
-
926
)
34
Mann
M.R.
Lee
S.S.
Doherty
A.S.
Verona
R.I.
Nolen
L.D.
Schultz
R.M.
Bartolomei
M.S.
Selective loss of imprinting in the placenta following preimplantation development in culture
Development
 , 
2004
, vol. 
131
 (pg. 
3727
-
3735
)
35
Davis
T.L.
Trasler
J.M.
Moss
S.B.
Yang
G.J.
Bartolomei
M.S.
Acquisition of the H19 methylation imprint occurs differentially on the parental alleles during spermatogenesis
Genomics
 , 
1999
, vol. 
58
 (pg. 
18
-
28
)
36
Davis
T.L.
Yang
G.J.
McCarrey
J.R.
Bartolomei
M.S.
The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development
Hum. Mol. Genet.
 , 
2000
, vol. 
9
 (pg. 
2885
-
2894
)
37
Ueda
T.
Abe
K.
Miura
A.
Yuzuriha
M.
Zubair
M.
Noguchi
M.
Niwa
K.
Kawase
Y.
Kono
T.
Matsuda
Y.
, et al.  . 
The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development
Genes Cell
 , 
2000
, vol. 
5
 (pg. 
649
-
659
)
38
Shamanski
F.L.
Kimura
Y.
Lavoir
M.C.
Pedersen
R.A.
Yanagimachi
R.
Status of genomic imprinting in mouse spermatids
Hum. Reprod.
 , 
1999
, vol. 
14
 (pg. 
1050
-
1056
)
39
Hiura
H.
Obata
Y.
Komiyama
J.
Shirai
M.
Kono
T.
Oocyte growth-dependent progression of maternal imprinting in mice
Genes Cell
 , 
2006
, vol. 
11
 (pg. 
353
-
361
)
40
DeBaun
M.R.
Niemitz
E.L.
Feinberg
A.P.
Association of in vitro fertilization with Beckwith–Wiedemann syndrome and epigenetic alterations of LIT1 and H19
Am. J. Hum. Genet.
 , 
2003
, vol. 
72
 (pg. 
156
-
160
)
41
Maher
E.R.
Brueton
L.A.
Bowdin
S.C.
Luharia
A.
Cooper
W.
Cole
T.R.
Macdonald
F.
Sampson
J.R.
Barratt
C.L.
Reik
W.
, et al.  . 
Beckwith–Wiedemann syndrome and assisted reproduction technology (ART)
J. Med. Genet.
 , 
2003
, vol. 
40
 (pg. 
62
-
64
)
42
Gicquel
C.
Gaston
V.
Mandelbaum
J.
Siffroi
J.P.
Flahault
A.
Le Bouc
Y.
In vitro fertilization may increase the risk of Beckwith–Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene
Am. J. Hum. Genet.
 , 
2003
, vol. 
72
 (pg. 
1338
-
1341
)
43
Cox
G.F.
Burger
J.
Lip
V.
Mau
U.A.
Sperling
K.
Wu
B.L.
Horsthemke
B.
Intracytoplasmic sperm injection may increase the risk of imprinting defects
Am. J. Hum. Genet.
 , 
2002
, vol. 
71
 (pg. 
162
-
164
)
44
Geuns
E.
Hilven
P.
Van Steirteghem
A.
Liebaers
I.
De Rycke
M.
Methylation analysis of KvDMR1 in human oocytes
J. Med. Genet.
 , 
2007
, vol. 
44
 (pg. 
144
-
147
)
45
Rivera
R.M.
Stein
P.
Weaver
J.R.
Mager
J.
Schultz
R.M.
Bartolomei
M.S.
Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development
Hum. Mol. Genet.
 , 
2007
, vol. 
17
 (pg. 
1
-
14
)
46
Lewis
A.
Mitsuya
K.
Umlauf
D.
Smith
P.
Dean
W.
Walter
J.
Higgins
M.
Feil
R.
Reik
W.
Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation
Nat. Genet.
 , 
2004
, vol. 
36
 (pg. 
1291
-
1295
)
47
Umlauf
D.
Goto
Y.
Cao
R.
Cerqueira
F.
Wagschal
A.
Zhang
Y.
Feil
R.
Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes
Nat. Genet.
 , 
2004
, vol. 
36
 (pg. 
1296
-
1300
)
48
Olek
A.
Walter
J.
The pre-implantation ontogeny of the H19 methylation imprint
Nat. Genet.
 , 
1997
, vol. 
17
 (pg. 
275
-
276
)
49
Thorvaldsen
J.L.
Duran
K.L.
Bartolomei
M.S.
Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2
Genes Dev.
 , 
1998
, vol. 
12
 (pg. 
3693
-
3702
)
50
Tremblay
K.D.
Duran
K.L.
Bartolomei
M.S.
A 5′ 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development
Mol. Cell Biol.
 , 
1997
, vol. 
17
 (pg. 
4322
-
4329
)
51
Li
E.
Chromatin modification and epigenetic reprogramming in mammalian development
Nat. Rev. Genet.
 , 
2002
, vol. 
3
 (pg. 
662
-
673
)
52
Reinhart
B.
Paoloni-Giacobino
A.
Chaillet
J.R.
Specific differentially methylated domain sequences direct the maintenance of methylation at imprinted genes
Mol. Cell Biol.
 , 
2006
, vol. 
26
 (pg. 
8347
-
8356
)
53
Guillemot
F.
Caspary
T.
Tilghman
S.M.
Copeland
N.G.
Gilbert
D.J.
Jenkins
N.A.
Anderson
D.J.
Joyner
A.L.
Rossant
J.
Nagy
A.
Genomic imprinting of Mash2, a mouse gene required for trophoblast development
Nat. Genet.
 , 
1995
, vol. 
9
 (pg. 
235
-
242
)
54
Anson-Cartwright
L.
Dawson
K.
Holmyard
D.
Fisher
S.J.
Lazzarini
R.A.
Cross
J.C.
The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta
Nat. Genet.
 , 
2000
, vol. 
25
 (pg. 
311
-
314
)
55
Voss
A.K.
Thomas
T.
Gruss
P.
Mice lacking HSP90beta fail to develop a placental labyrinth
Development
 , 
2000
, vol. 
127
 (pg. 
1
-
11
)
56
Ono
R.
Nakamura
K.
Inoue
K.
Naruse
M.
Usami
T.
Wakisaka-Saito
N.
Hino
T.
Suzuki-Migishima
R.
Ogonuki
N.
Miki
H.
, et al.  . 
Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality
Nat. Genet.
 , 
2006
, vol. 
38
 (pg. 
101
-
106
)
57
Street
M.E.
Seghini
P.
Fieni
S.
Ziveri
M.A.
Volta
C.
Martorana
D.
Viani
I.
Gramellini
D.
Bernasconi
S.
Changes in interleukin-6 and IGF system and their relationships in placenta and cord blood in newborns with fetal growth restriction compared with controls
Eur. J. Endocrinol.
 , 
2006
, vol. 
155
 (pg. 
567
-
574
)
58
de Vrijer
B.
Davidsen
M.L.
Wilkening
R.B.
Anthony
R.V.
Regnault
T.R.
Altered placental and fetal expression of IGFs and IGF-binding proteins associated with intrauterine growth restriction in fetal sheep during early and mid-pregnancy
Pediatr. Res.
 , 
2006
, vol. 
60
 (pg. 
507
-
512
)
59
Oishi
M.
Gohma
H.
Hashizume
K.
Taniguchi
Y.
Yasue
H.
Takahashi
S.
Yamada
T.
Sasaki
Y.
Early embryonic death-associated changes in genome-wide gene expression profiles in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo
Mol. Reprod. Dev.
 , 
2006
, vol. 
73
 (pg. 
404
-
409
)
60
Moore
T.
Constancia
M.
Zubair
M.
Bailleul
B.
Feil
R.
Sasaki
H.
Reik
W.
Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2
Proc. Natl Acad. Sci. USA
 , 
1997
, vol. 
94
 (pg. 
12509
-
12514
)
61
Constancia
M.
Dean
W.
Lopes
S.
Moore
T.
Kelsey
G.
Reik
W.
Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19
Nat. Genet.
 , 
2000
, vol. 
26
 (pg. 
203
-
206
)
62
Ecker
D.J.
Stein
P.
Xu
Z.
Williams
C.J.
Kopf
G.S.
Bilker
W.B.
Abel
T.
Schultz
R.M.
Long-term effects of culture of preimplantation mouse embryos on behavior
Proc. Natl Acad. Sci. USA
 , 
2004
, vol. 
101
 (pg. 
1595
-
1600
)
63
Fernandez-Gonzalez
R.
Moreira
P.
Bilbao
A.
Jimenez
A.
Perez-Crespo
M.
Ramirez
M.A.
Rodriguez De Fonseca
F.
Pintado
B.
Gutierrez-Adan
A.
Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior
Proc. Natl Acad. Sci. USA
 , 
2004
, vol. 
101
 (pg. 
5880
-
5885
)
64
Elliott
M.
Maher
E.R.
Beckwith–Wiedemann syndrome
J. Med. Genet.
 , 
1994
, vol. 
31
 (pg. 
560
-
564
)
65
Gosden
R.
Trasler
J.
Lucifero
D.
Faddy
M.
Rare congenital disorders, imprinted genes, and assisted reproductive technology
Lancet
 , 
2003
, vol. 
361
 (pg. 
1975
-
1977
)
66
Halliday
J.
Oke
K.
Breheny
S.
Algar
E.D.J.A.
Beckwith–Wiedemann syndrome and IVF: a case-control study
Am. J. Hum. Genet.
 , 
2004
, vol. 
75
 (pg. 
526
-
528
)
67
Kagami
M.
Nagai
T.
Fukami
M.
Yamazawa
K.
Ogata
T.
Silver–Russell syndrome in a girl born after in vitro fertilization: partial hypermethylation at the differentially methylated region of PEG1/MEST
J. Assist. Reprod. Genet.
 , 
2007
, vol. 
24
 (pg. 
131
-
136
)
68
Mann
M.R.
Chung
Y.G.
Nolen
L.D.
Verona
R.I.
Latham
K.E.
Bartolomei
M.S.
Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos
Biol. Reprod.
 , 
2003
, vol. 
69
 (pg. 
902
-
914
)
69
Nagy
A.
Gertsenstein
M.
Vintersten
K.
Behringer
R.
Manipulating the Mouse Embryo: A Laboratory Manual
 , 
2002
3rd edn.
Cold Spring Harbor Laboratory Press
70
Thorvaldsen
J.L.
Mann
M.R.
Nwoko
O.
Duran
K.L.
Bartolomei
M.S.
Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting
Mol. Cell Biol.
 , 
2002
, vol. 
22
 (pg. 
2450
-
2462
)
71
Smilinich
N.J.
Day
C.D.
Fitzpatrick
G.V.
Caldwell
G.M.
Lossie
A.C.
Cooper
P.R.
Smallwood
A.C.
Joyce
J.A.
Schofield
P.N.
Reik
W.
, et al.  . 
A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith–Wiedemann syndrome
Proc. Natl Acad. Sci. USA
 , 
1999
, vol. 
96
 (pg. 
8064
-
8069
)