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Biology of Reproduction 64, 918-926 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Culture of Preimplantation Mouse Embryos Affects Fetal Development and the Expression of Imprinted Genes1

Sanjeev Khosla3,a, Wendy Deana, David Brown4,b, Wolf Reika, and Robert Feil2,a

a Laboratory of Developmental Genetics and Imprinting and b Laboratory of Computational Neuroscience, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom

ABSTRACT

Culture of preimplantation mammalian embryos and cells can influence their subsequent growth and differentiation. Previously, we reported that culture of mouse embryonic stem cells is associated with deregulation of genomic imprinting and affects the potential for these cells to develop into normal fetuses. The purpose of our current study was to determine whether culture of preimplantation mouse embryos in a chemically defined medium (M16) with or without fetal calf serum (FCS) can affect their subsequent development and imprinted gene expression. Only one third of the blastocysts that had been cultured from two-cell embryos in M16 medium complemented with FCS developed into viable Day 14 fetuses after transfer into recipients. These M16 + FCS fetuses were reduced in weight as compared with controls and M16 fetuses and had decreased expression of the imprinted H19 and insulin-like growth factor 2 genes associated with a gain of DNA methylation at an imprinting control region upstream of H19. They also displayed increased expression of the imprinted gene Grb10. The growth factor receptor binding gene Grb7, in contrast, was strongly reduced in its expression in most of the M16 + FCS fetuses. No alterations were detected for the imprinted gene Mest. Preimplantation culture in the presence of serum can influence the regulation of multiple growth-related imprinted genes, thus leading to aberrant fetal growth and development.

developmental biology, gene regulation, IGF receptor, implantation/early development

INTRODUCTION

The oviduct provides the preimplantation mammalian embryo with a unique environment that nurtures the early embryo and thereby influences its further development. The use of specific media for in vitro culture of preimplantation embryos, mimicking the environment of the oviduct, has allowed examination of early development and manipulation of the mammalian genome [1]. However, there is increasing evidence that in vitro culture of preimplantation embryos can be associated with aberrant growth and specific phenotypic abnormalities during fetal and postnatal development [2–5]. In sheep and cattle, these aberrant phenotypes are collectively referred to as the large offspring syndrome. The main characteristic of this syndrome is increased fetal growth, and on average affected newborn animals are significantly heavier than controls [6]. The culture systems with which the large offspring syndrome has been associated are varied, but most involve the use of serum [5–7]. Little information has been obtained on growth effects in rodent species. However, mouse fetuses derived from in vitro cultured preimplantation embryos (from eight-cell stage to blastocysts) were lighter than those derived from control uterine blastocysts [8]. Pronuclear transfer in the mouse also has negative long-term implications for growth and development [9, 10].

Although the mechanisms responsible for the deregulation of development as a consequence of in vitro culture remain elusive, several hypotheses have been put forward to explain the diversity of abnormalities observed. Fragmentation of cytoplasm, documented for cultured bovine embryos [11], has been suggested as a possible reason for the deregulation, because interaction of cytoplasmic factors with the nucleus is essential for normal development [12–14]. Another proposal alleges that embryos do not receive the appropriate cues during in vitro culture and that this lack of signaling molecules could result in the deregulation of genes under their influence [5]. A third hypothesis is that in vitro culture of preimplantation embryos leads to aberrant epigenetic modifications in the genome. Such alterations would be maintained somatically and might affect gene expression at later stages of development [9, 15–17]. One heritable epigenetic feature that regulates gene expression in mammals is DNA methylation. During early embryonic development, extensive changes in genome-wide methylation take place [18], and therefore any perturbation caused in the methylation process could result in deregulation of development at later stages.

Imprinted genes are exceptional in that they are expressed in a parent-of-origin-specific manner. Genetic studies have shown that imprinting is evolutionarily conserved among eutherian mammals (e.g., mice, humans, sheep) and plays important roles in fetal growth and development [19–21]. In most imprinted gene loci, key regulatory regions have been identified that are methylated on one of the two parental chromosomes. For several of these imprinting control regions, the differential methylation originates from one of the germ lines and is maintained throughout pre- and postimplantation development [21–23]. This situation contrasts with that of nonimprinted genes, which acquire their methylation patterns after implantation [18, 24, 25]. Thus, imprinted genes may be particularly susceptible to methylation changes that occur during preimplantation development. Fetal abnormalities observed as a consequence of preimplantation in vitro culture have been proposed to result from aberrant changes in the methylation states of imprinted genes [9, 15, 16]. In addition, for several imprinted genes deregulation has significant effects on growth and development [19]. For instance, alterations in the expression of the insulin-like growth factor 2 gene (Igf2) have been shown to severely affect growth in mice [26–28]. In humans, deregulation of IGF2 has been implicated in cases of the congenital overgrowth syndrome, Beckwith-Weidemann syndrome (BWS) [29, 30], and several of the phenotypes observed in BWS are similar to the ones observed in the large offspring syndrome [6]. Our recent observations on embryonic stem (ES) cells support the hypothesis that imprinted genes may become deregulated as a consequence of culture. Upon derivation and subsequent culture of ES cells in medium complemented with serum, we observed aberrant methylation changes in a number of growth-related imprinted genes. Significantly, these epigenetic alterations were not corrected during postimplantation development and were associated with aberrant imprinted gene expression and phenotypic abnormalities [15]. In the current study, we investigated whether in vitro culture of preimplantation mouse embryos can affect their subsequent development and can lead to a deregulation of genomic imprinting. Hence, we determined the effects of embryo culture media with or without serum on pre- and postimplantation viability and examined the regulation of different growth-related imprinted genes.

MATERIALS AND METHODS

Embryo Culture and Blastocyst Transfer

To obtain one- or two-cell embryos, superovulation was induced in (C57BL/6J x CBA/Ca) F1 females by i.p. injection with 7.5 IU of eCG (Intervet UK Ltd., Cambridge, UK) followed 44–48 h later by an injection of 7.5 IU of hCG (Intervet UK Ltd.). Females were mated with males of the same genotype. The one-cell embryos were recovered from oviducts on Day 1, 21–23 h post-hCG. The two-cell embryos were recovered by flushing oviducts 32–34 h post-hCG injection. M16 medium (Sigma Ltd., Poole, Dorset, UK; containing 4 mg/ml BSA) was used in our studies. To test the effect of serum, 5% or 10% fetal calf serum (FCS; a single batch from Sigma) was added to the M16 medium (after heat treatment of the serum at 55°C for 30 min). Culture dishes with embryo-containing microdrops were incubated under oil at 37°C in an incubator equilibrated with 5% CO2 in air. Control blastocysts were obtained from females of the same genotype (by flushing the uterus on Day 4 postcoitum) and were transferred immediately into uteri of mature recipient (C57BL/6 x CBA) F1 females. To avoid artifacts due to fluctuations in the maternal contribution, the control (10 blastocysts/horn) and experimental (11 blastocysts/horn) blastocysts that were compared were always transferred contralaterally to recipient females. For this study, we chose to transfer experimental blastocysts to the left uterine horn and control blastocysts to the right uterine horn. From each group, only blastocysts that had a morphology typical of embryonic Day 4 were transferred (expanded blastocysts).

Analysis of Gene Expression and DNA Methylation

Simultaneous isolation of total RNA and genomic DNA from E14 fetuses was carried out using an RNA/DNA isolation kit following the manufacturer's (Qiagen Ltd., Crawley, West Sussex, UK) instructions. For expression analysis, 20 µg of total RNA from each sample was electrophoresed through 1.5% formaldehyde/3-N-morpholino-propanesulfonic acid (MOPS) agarose gels in 1x MOPS (0.2 M MOPS, pH 7.0; 0.5 M NaAc; 0.01 M EDTA). RNA was then blotted onto Hybond N+ membranes (Amersham-Pharmacia-Biotech Ltd., Little Chalfont, Buckinghamshire, UK), which were hybridized with radioactively labeled probes as previously described [20, 31]. The following probes were used: an intragenic 1-kilobase (kb) BamHI fragment from the H19 gene (H19-1) [32], a 0.9-kb KpnI-BamHI fragment from exon 6 of the Igf2 gene [31], a 2.5-kb EcoRI fragment from the Mest gene [33], a 499-base pair (bp) fragment from the 3' end of the Grb10 gene (nucleotides 1532–2030 in GenBank sequence U18996), a 372-bp fragment amplified by the polymerase chain reaction technique from the 5' untranslated region (UTR) of the Grb7 gene (nucleotides 112–464 in GenBank sequence M94450), and a 250-bp HindIII-PstI fragment from the 5' end of the Gapdh gene. For analysis of methylation, 10 µg of restriction enzyme-digested DNA was electrophoresed through 1% agarose gels in 0.5x TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA). DNA was transferred onto Hybond N+ membranes in 0.4 N NaOH, and hybridization was carried out with a 1.1-kb SacI-BstXI fragment from the H19 upstream region (H19-4) [32]. Following Northern and Southern hybridization, relative band intensities were determined by using the Quantity-One image software (BioRad Ltd., Hemel Hempstead, Herts, UK).

Statistical Analysis

The comparison between control and cultured groups was made using randomized block analysis of variance on the untransformed and logarithmically transformed data, where blocks were mouse litters. The results of this parametric analysis were confirmed using randomization tests. The logarithmic transformation was made to correct for any possible lack of homogeneity of variance within litter/treatment combinations. Applying the transformation before the analysis of variance did not affect the results. Correlation between fetal weight and gene expression was tested by performing multiple regression analyses on fetal weight and the Igf2, H19, Grb10, and Grb7 expression levels for fetuses in the control and serum groups. These tests were performed using the Genstat software [34].

RESULTS

Serum Affects Preimplantation Development

To analyze the effect of serum on preimplantation viability, one- and two-cell embryos were cultured to the blastocyst stage in M16 medium in the absence or presence of 5% or 10% FCS. Of all the one-cell embryos (n = 112) that were cultured in M16 medium, 92% (n = 103) had developed into cavitating or hatching blastocysts after 4 days. In M16 + 5% FCS, in contrast, only 50% (56 of 111) of the embryos became blastocysts. An even greater reduction in viability was observed in M16 + 10% FCS, in which only 35% (36 of 103) of the embryos developed into blastocysts. Most of the embryos that did not develop to the blastocyst stage in the M16 + FCS media (both the 5% and 10% FCS groups) were between the one- and the eight-cell stage after the 4 days of culture. Therefore, the presence of serum inhibits preimplantation development from one-cell embryos to blastocysts. Development from two-cell embryos, in contrast, was not inhibited by the presence of serum in the culture medium. In M16 medium, 99% (108 of 109) of the two-cell embryos had developed into blastocysts after 3 days of culture. Of the two-cell embryos placed in M16 + 5% FCS, 85% (245 of 290) developed to the blastocyst stage. Similarly, 85% (68 out of 80) of the two-cell embryos that were transferred into M16 + 10% FCS became blastocysts. The cavitated and hatching blastocysts cultured in the presence of serum appeared morphologically identical to those cultured in the absence of serum and to control uterine blastocysts (Fig. 1). In two additional culture experiments, embryos cultured in M16 + 5% FCS traversed the second cell cycle more rapidly and remained for an extended period in the third cell cycle as compared with those cultured in M16 medium alone (data not shown). However, both groups formed cavitated blastocysts after 4 days of culture (Fig. 1).



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  		FIG. 1. Comparison of blastocyst morphology. Blastocysts were cultured from two-cell embryos (M16 and serum group) or obtained from donor females at Day 4 postcoitum (control group). Cavitating and hatching blastocysts had a similar size and morphology in all three groups. Original magnification x100

Postimplantation Developmental Effects

To analyze whether serum during preimplantation culture affects postimplantation development, cultured blastocysts were transferred to the left uterine horn of recipient females. Control blastocysts flushed from a donor mother's uterus were transferred to the contralateral (right) horn of the same recipients. For these transfers, only fully expanded but not yet hatching blastocysts were selected. Because the percentage of blastocysts derived from one-cell embryos was greatly reduced in the presence of serum, all studies were carried out on blastocysts that had been grown in vitro from two-cell embryos. Fetuses derived from flushed control blastocysts (control group) were compared with fetuses derived from blastocysts that were cultured in vitro in the absence (M16 group) or presence (serum group) of 5% FCS. Fetuses were studied at embryonic Day 14 (E14) because rapid growth and organogenesis occurs at this stage of development and effects due to culture conditions would therefore be best distinguishable.

For computation of the viability of the groups of fetuses, each uterine horn was taken as a unit, and for each unit the ratio of viable fetuses to blastocysts transferred was calculated. Comparison of the averages of these ratios for each group showed that at E14 the percentage of viable fetuses derived from the serum group (33%, 66 of 202) was significantly lower (P < 0.001) than that derived from the control group (79%, 107 of 135). The percentage of viable fetuses developing from the M16 group of blastocysts (60%, 78 of 130), in contrast, did not differ significantly from that of the control group (P = 0.184). Therefore, the presence of serum during preimplantation development significantly decreased postimplantation viability. The finding that at E14 the dead conceptuses were partially resorbed cell masses (data not shown) suggested that loss occurred at an early postimplantation stage.

The average weights of the E14 fetuses were calculated for each recipient female. Comparisons were always made between fetuses of the two different groups dissected from the same recipient female. Also, to avoid minor effects that could have arisen from having reduced numbers of fetuses, comparison was made only when there were four or more fetuses present in each of the uterine horns. The results from the recipient females were then pooled, taking into account the differences that could have arisen between different recipients (see Materials and Methods). We found no difference in the average (±SEM) weights of fetuses from the control (147 ± 8 mg) and the M16 (153 ± 8 mg) groups (Fig. 2). However, fetuses of the serum group (139 ± 10 mg) were on average 20% lighter than the fetuses of the control group (175 ± 3 mg), and this difference was significant (P < 0.001, Fig. 2). In the comparison of M16 and serum group fetuses, a significant effect of serum was observed; serum group fetuses were on 12% lighter than M16 fetuses (183 ± 3 mg versus 160 ± 3 mg; P < 0.001, Fig. 2).



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  		FIG. 2. The effect of serum on fetal weight. Wet weights (in milligrams) of different groups of E14 fetuses were compared. The comparison between control and M16 media shows data for 2 recipients (11 and 17 fetuses, respectively); data from 3 recipients were pooled for the comparison between control and serum group fetuses (25 and 20 fetuses, respectively). The difference in average weight between the two independent control groups reflects maternal effects and differences in the timing of blastocyst transfer and dissection of fetuses. For comparison between M16 and serum groups, data from 3 recipients were pooled (22 and 14 fetuses, respectively). Error bars represent SEM. **P < 0.001

To determine whether preimplantation culture in the presence of serum can be compatible at a low frequency with viable development until birth, we transferred 39 blastocysts of the M16 + 5% FCS group into three recipient females. This transfer gave rise to three (8%) viable newborn animals (two and one pup each in two of the recipients) that had no apparent developmental abnormalities and were killed at 2 wk of age. A similar experiment on 30 M16 group blastocysts yielded nine (30%) apparently normal newborn animals.

Aberrant Expression of Growth-Related Imprinted Genes

We studied gene expression in the M16 and the serum group of E14 fetuses in comparison with that in the control E14 fetuses to determine the extent to which preimplantation culture in the M16 and M16 + 5% FCS media had altered the expression of imprinted genes. For the evaluation of fetal weights, comparison of expression levels was always between control and cultured groups within the same recipient, and results were pooled after taking maternal effects into consideration. Several growth-related imprinted genes were analyzed, and relative expression levels were calculated as the ratio of the imprinted gene's expression to the Gapdh signal. To allow statistical analysis, average ratios were adjusted to 1 for the groups of control fetuses (Fig. 3). We first analyzed the expression of the neighboring imprinted Igf2 and H19 genes on mouse distal chromosome 7. In the M16 group of fetuses, the levels of expression of the Igf2 (0.93 ± 0.07 relative to the control fetuses; P = 0.24) and H19 (0.83 ± 0.11 relative to controls; P = 0.07) genes were not significantly different from those in the control fetuses (Fig. 3A). However, the comparison of the control and serum group fetuses showed significantly lower (-31%) average levels of H19 expression in the serum group than in the control group (0.69 ± 0.05 relative to the controls; P < 0.001, Fig. 3B). The average level of Igf2 expression was also significantly reduced (-15%) in the serum group as compared with the control fetuses (0.85 ± 0.05 relative to controls fetuses; P < 0.001, Fig. 3B).



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  		FIG. 3. Reduced expression of Igf2, H19, and Grb7 and increased expression of Grb10 in the serum group fetuses. Mean expression levels were compared between the control and the M16 group (A) and between the control and the serum group (B). Relative expression levels were calculated as the ratio between the specific-transcript's signal and the Gapdh signal. For Igf2, measurements were performed on its 1.8-kb transcript (see Fig. 4A). The expression ratios for the various genes are represented relative to the control group's mean expression ratios (which were adjusted to 1). Open bars represent control group fetuses, solid bars represent M16 group fetuses, and shaded bars represent serum group fetuses. Error bars represent SEM. *P < 0.01; **P < 0.001

The growth factor receptor binding 10 gene (Grb10) is another imprinted gene whose product affects the IGF/INS (insulin) axis [35]. It is located on proximal mouse chromosome 11 and expressed from the maternal chromosome exclusively [36]. Analysis of Grb10 expression levels did not reveal differences between the M16 group of fetuses and the control fetuses (0.84 ± 0.1; P = 0.09). However, the serum group fetuses had a 27% higher Grb10 expression than the control fetuses, and this difference was significant (1.27 ± 0.11 relative to control; P < 0.01). The Grb10 probe used for Northern analysis hybridized with the 5.4-kb Grb10 transcript and cross-hybridized with a related transcript of 2.3 kb (indicated by an arrow in Fig. 4A), and we generated a specific probe for this transcript. Two additional members of the GRB protein family, GRB7 and GRB14, have been reported in mice and are encoded by genes that are homologous to Grb10 [37]. Using a Grb7-specific probe from the 5'-UTR of the gene we established that the 2.3-kb transcript corresponded to Grb7 (Fig. 4A). Hybridization with the Grb7 probe showed that in the serum group, the average Grb7 expression was substantially reduced (-76%) as compared with the control group (0.24 ± 0.06 relative to control; P < 0.001). In 17 of the 20 serum group fetuses, Grb7 expression was lower than in the control fetuses. The M16 group of fetuses, in contrast, did not show a significant difference in Grb7 expression as compared with control fetuses (0.75 ± 0.17 relative to controls; P = 0.17).



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  		FIG. 4. Serum-induced stochastic deregulation of Igf2, H19, Grb10, and Grb7. A) Northern hybridization analysis of control and serum group fetuses. H19, Igf2, Grb10, Grb7, Mest, and Gapdh transcripts were analyzed using gene-specific probes (see Materials and Methods). Transcript sizes are indicated in kilobases. All fetuses shown (1–12) originated from a single recipient female. B) For the same recipient female, wet weights and relative levels of H19, Igf2, Grb10, and Grb7 expression are shown for all of its fetuses. Crosses indicate data points for control fetuses, and dots are data points for serum group fetuses. Regression analysis between expression and weight was performed (lines)

Because the imprinting status of this gene had not been reported, we carried out an allele-specific analysis of Grb7 expression. Like Grb10, Grb7 is located on mouse chromosome 11, and we therefore analyzed mice that were either maternally (Matdi11) or paternally (Patdi11) disomic for chromosome 11 to ascertain its imprinting status. We found Grb7 expressed at equally high levels in kidneys dissected from adult Matdi11 and Patdi11 mice (Fig. 5), but the disomy-11 system did not allow us to ascertain its imprinting status in the embryo. We also analyzed the expression status of the growth-related imprinted gene Mest [33,38] but did not detect significant differences between control and M16 or serum group fetuses (Figs. 3 and 4A).



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  		FIG. 5. Analysis of the imprinting status of Grb7. Mice with maternal (Matdi11) and paternal (Patdi11) uniparental disomy for chromosome 11 were generated by intercrossing heterozygotes for the Robersonian translocation Rb(11;13)Bnr [67]. Total RNA for the northern hybridization was extracted from kidney because it is one of the few tissues in which Grb7 is highly expressed [48]. Transcript sizes are in kilobases

To test the correlation between fetal weight and gene expression, we performed regression analyses on fetal weight and the Igf2, H19, Grb10, and Grb7 expression levels for fetuses in the control and serum groups (as depicted in Fig. 4B for one recipient). These calculations showed that the effect of serum on weight could not be accounted for by the combined action of the four genes (P = 0.04) or by the combined effects of specific combinations of two or three of the genes. Thus, these genes are noncoordinate in their deregulated expression in individual fetuses. When we considered Grb7 on its own, however, its expression was correlated significantly (P < 0.001) with fetal weight, and the same was observed for H19 (P < 0.01) and Grb10 (P < 0.01) when considered on their own. Igf2, when considered on its own, yielded a nonsignificant association with fetal weight (P = 0.012). Even though most of the weight-reduced fetuses from the serum group had reduced expression of Igf2, H19, and Grb7, not all fetuses showed this pattern. Similarly, only a proportion of the serum group fetuses had increased levels of Grb10 expression. For the recipient shown in Figure 4B, for instance, fetus 10 was reduced in weight but showed normal levels of Igf2 and H19 expression. Although in this recipient Grb7 expression was reduced in all fetuses of the serum group, there were a few serum fetuses (three) from other recipients that had normal levels of Grb7 expression (data not shown). In addition, fetuses with lower Grb7 levels did not necessarily show low levels of H19 or Igf2. For example, weight-reduced fetus 10 showed low Grb7 but normal H19 and Igf2 expression. Conversely, although fetus 12 had reduced levels of Grb7, Igf2, and H19 expression, it was only slightly reduced in weight. Hence, in the serum group of fetuses, the Grb7, Grb10, Igf2, and H19 genes are deregulated in their expression in a stochastic and noncoordinate fashion.

Because at E14 the fetuses of the serum group were on average 20% reduced in weight, there was a possibility that they were developmentally behind relative to the control fetuses. Hence, the reduction in Igf2, H19, and Grb7 expression and the increase in Grb10 expression could reflect their developmental stage rather than these genes being involved in the growth reduction. However, on external and internal examination of developmental stage-specific characteristics we did not find evidence that the growth-reduced fetuses were developmentally delayed (data not shown). Furthermore, we examined the levels of Igf2, H19, Grb10, and Grb7 expression in control E13.5, E14, and E14.5 fetuses (four for each stage) that were of the same genotype as the experimental fetuses. This developmental analysis did not reveal detectable changes in expression levels during this 24-h period of fetal development (data not shown).

Gain of DNA Methylation at an Imprinting Control Element Upstream of H19

The allelic methylation status of a differentially methylated region (DMR) upstream of the H19 gene [39] is essential for the imprinted expression of both H19 and Igf2 [40]. To test whether methylation levels in this DMR were correlated with the differences in H19 expression levels observed between the control and the serum groups of fetuses, we assayed a methylation-sensitive AatII restriction site located 3939 bp upstream from the H19 transcription initiation site (Fig. 6A). In embryos, fetuses, and adult tissues, this AatII site is fully methylated on the paternal allele and is unmethylated on the maternal allele [39, 41]. In our assay, methylation was determined as the ratio of the undigested (SacI) fragment (methylated) to the AatII-digested (SacI) fragment (unmethylated). To ascertain obtained methylation ratios, Southern hybridization experiments (enzymatic digestion of DNA, gel electrophoresis, transfer to filter, and hybridization) and the subsequent measurement of band intensities were performed in duplicate. No significant difference was detected in the levels of H19 methylation between the control and M16 groups (Fig. 6B). However, in the serum group of fetuses, the methylation level was on average 19% higher than that in the control group of fetuses (P < 0.001). In some of the individual fetuses, the level of methylation was 25% higher than the control average value. Because only the paternal chromosome is normally methylated [39, 41], this finding suggests that the normally unmethylated maternal chromosome had become methylated at the AatII restriction site in 50% of the cells in these fetuses.



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  		FIG. 6. A) Methylation at an AatII site located upstream of H19. Genomic DNAs (1–12, corresponding to fetuses 1–12 in Fig. 4A) were digested with SacI and AatII and analyzed by Southern hybridization with probe H19-4. The upper band corresponds to the undigested (3.8-kb SacI) fragment, and the lower band corresponds to the (3.1 kb) AatII digestion product. The ratios between the intensities of these two bands (3.8-kb band:3.1-kb band) are indicated for each of the fetuses. B) Comparison of average methylation ratios (3.8-kb band:3.1-kb band) between control and the cultured groups of fetuses. Error bars represent SEM. **P < 0.001

DISCUSSION

Our results establish that serum in culture medium inhibits the in vitro development of preimplantation mouse embryos and affects their postimplantation developmental potential. The serum group embryos that were viable to E14 were reduced in weight and showed decreased expression of the imprinted Igf2 and H19 genes and increased expression of the imprinted gene Grb10. Another growth factor receptor binding gene, Grb7, was expressed at substantially reduced levels in almost all fetuses of the serum group.

Serum inhibited the in vitro growth of one-cell embryos; most of the embryos that had been cultured in M16 + FCS medium died or had development arrested between the one- and eight-cell stages. In agreement with reports on other defined media [42, 43], we did not observe a significant effect of M16 + FCS on development from two-cell stage embryos to blastocysts. The blastocysts that had been cultured from two-cell embryos in the presence of FCS appeared morphologically normal. However, 67% of these blastocysts did not survive to E14 after transfer into recipient females, and this result confirms findings by others on media complemented with serum [43, 44]. Thus, serum appears to have a direct or indirect adverse effect on gene(s) that are responsible for postimplantation development. FCS had a negative effect on the growth of many of the fetuses that survived to E14. However, at a strongly reduced frequency, preimplantation culture in the presence of serum can give rise to apparently normal development to term. M16 medium on its own seemed not to affect development to E14 in our study. This finding does not, however, exclude the possibility that serum-free M16 medium would not have effects at later fetal stages. For instance, a small reduction in fetal size at E17 of development has been noted in embryos that had been cultured from eight cells to blastocysts in medium without serum [8].

In the serum group of fetuses, the expression of the paternally expressed Igf2 gene was lower than that in control fetuses, whereas the maternally expressed Grb10 gene was increased in its expression. Given the growth-promoting roles of IGF2 via its activation of the IGF1 receptor, the observed reduction in Igf2 expression is likely to be a determinant in the reduced weight of the serum group fetuses. GRB10, however, is thought to have a negative effect on cellular proliferation because it binds the IGF1 receptor and the insulin receptor via its src homology 2 (SH2) domain and could thereby inhibit their intracellular signalling [35, 45]. The increased Grb10 expressed in the serum group fetuses, therefore, is also likely to contribute to the fetal growth reduction. Unexpectedly, expression of the Grb10-related gene Grb7 was strongly reduced in the serum group of fetuses. Like GRB10, GRB7 belongs to a family of growth receptor binding adapter proteins that link specific receptor tyrosine kinases and other tyrosine phosphorylated proteins to downstream effectors [37, 46, 47]. Although its precise role is unknown, GRB7 has been shown to interact with different receptor molecules, including HER2 (erbB2), the epidermal growth factor receptor, and the insulin receptor [46, 47]. Its receptor specificity and its tissue-specific pattern of expression [48] suggest that GRB7 plays a different role in development than does GRB10. GRB7 is overexpressed in a variety of human cancers and cancer cell lines, which suggests that it is involved in cellular proliferation [49, 50]. Although Grb7 is not imprinted in the adult mouse, this does not exclude the possibility of imprinted expression at embryonic or fetal stages. It also remains to be determined whether the observed reduction in Grb7 expression was caused by a direct or an indirect and possibly imprinted gene-mediated mechanism.

Statistical analysis indicated that the aberrant expression of the Igf2, Grb10, and Grb7 genes could not account entirely for the observed reduction in weight in the serum group of fetuses. For the aberrant Igf2, Grb10, and Grb7 expression to be the sole determinants of the growth phenotype, the association with fetal weight should have been apparent in all individual fetuses. Although we did observe a substantial (-76%) reduction of Grb7 expression, the association between reduced fetal weight and aberrant levels of Igf2, Grb10, and Grb7 expression was not apparent in all the serum group fetuses. Gene deregulation by culture in the presence of FCS may be a stochastic mechanism that affects the expression of multiple growth-related genes, including Igf2, Grb10, and Grb7. Such a stochastic effect would agree with our previous work on mouse ES cells, in which we demonstrated changes in imprinted gene expression as a consequence of in vitro culture (in the presence of FCS). In this ES cell study, alterations arose independently at different imprinted loci, and the alterations that were observed differed among cell lines [15]. Because ES cells are representative of the inner cell mass of blastocysts, culture can influence the epigenetic regulation of gene expression at this preimplantation stage.

Given the aberrant H19 and Igf2 expression in the serum cultured group, we analyzed a differentially methylated region upstream of H19 [39] that has been shown to be essential for the imprinting of H19 and Igf2 [40]. We found that the serum group of fetuses had significantly higher levels of methylation than the control fetuses. This increase in methylation was inversely correlated with decreased levels of H19 expression. In some of the fetuses, the levels of H19 methylation clearly indicated that in a considerable portion of cells they had acquired methylation on the normally unmethylated maternal chromosome, and this gain of maternal methylation is likely to be causally involved in the reduced H19 expression. Our data on fetuses extend the finding by Sasaki and coworkers [51] that culture of preimplantation mouse embryos can lead to deregulation of H19 imprinting in extraembryonic tissues. In these tissues, however, culture led to derepression of the normally silent H19 allele, and it is therefore unclear whether a common causal mechanism is involved. However, it may be that the directionality of the methylation changes at H19 is influenced by the culture medium used and by the presence of serum. In a recent study on the preimplantation stage of mouse development, Doherty and coworkers reported that culture of two-cell embryos to blastocysts in Whitten's medium (to which no serum was added) can lead to a loss of (paternal) methylation (and gain of expression) at the H19 gene [52]. In agreement with our studies on the serum group fetuses, we previously detected gain (rather than loss) of maternal H19 methylation upon culture of ES cells in FCS-supplemented medium [15, 32]. Using different ES cell lines, we established that methylation changes in H19 (and other imprinted genes) are not corrected during postimplantation development and are associated with aberrant imprinted gene expression [15]. Relative to the developmental maintenance of altered imprints, it should be interesting to determine whether the increased H19 methylation in the serum group of fetuses arose during the culture of preimplantation embryos, as has been shown for loss of H19 methylation in Whitten's medium [52]. The serum-induced gain of methylation at the H19 upstream region was not associated with a gain in expression of the flanking Igf2 gene, as might have been expected from the chromatin boundary function of this imprinting control center [40]. Rather, decreased levels of Igf2 expression were observed in the serum group fetuses. Whether Igf2 had become deregulated via an independent epigenetic mechanism is unclear, but apparently independent deregulation of IGF2 and H19 has also been observed in sporadic cases of BWS in humans [29, 30].

Our study raises the question as to how embryo and cell culture can lead to improper maintenance of allelic methylation at imprinted gene loci, in particular where serum complements the medium. There was a difference in the kinetics of growth in the embryos grown in M16 + 5% FCS as compared with embryos grown in M16 medium. In particular, the embryos cultured in the presence of serum seemed to traverse the second cell cycle more rapidly and remained for an extended period in the third cell cycle; such a biphasic effect of serum also has been reported for bovine embryos [53]. Relative to the cell cycle, epigenetic regulation at imprinting control regions is highly complex. It involves DNA methylation on one of the two parental chromosomes, specialized features of chromatin, and for some regions nonhistone protein binding to the unmethylated allele. Recent data suggest that an intricate interplay between DNA methylation and such chromatin features is involved in the somatic maintenance of imprinting during the cell cycle [32, 54]. Given this considerable complexity, (serum-induced) alterations in the first cell cycles of mammalian development can result in the improper maintenance of methylation and chromatin imprints. Although the precise causal mechanisms have not been determined, methyl-transferases and several chromatin regulatory complexes are cell cycle regulated and show dramatic alterations in their expression during preimplantation development [18, 55–57].

The findings in this study may be pertinent to other early embryonic interventions in the mouse that involve the use of culture media and to similar situations in other mammalian species. For instance, freezing of mouse embryos may affect fetal and postnatal development [58], and pronuclear transfer can lead to a decrease in growth of about 15% [10]. Different types of stress during early mouse development seem to have certain factors in common that can lead to a reduction in fetal growth. Such a directed acquisition of a phenotypic character as a consequence of environmental stress would agree with proposed theories on canalization and genetic assimilation [59, 60]. These theories apply in particular in instances where the newly arisen character is transmitted to the next generation(s), as for pronuclear transfer-induced growth retardation in mice [10]. In our current study on culture of mouse embryos, we could not address the heritability of the serum-induced growth reduction, but we did detect major directed shifts in the expression levels of Igf2, Grb10, and Grb7, genes that are involved in developmental signaling pathways. Support for a certain specificity in the deregulated gene expression is provided by our observation that Mest, another imprinted gene with a major impact on embryonic and fetal growth [38], was not altered in its expression by culture in M16 or M16 + FCS medium. In humans and ruminants, culture and manipulation-induced growth effects have also been reported. Human in vitro fertilization technologies, for instance, involve embryo culture and may give rise to reduced weight at birth [61, 62]. In cattle and sheep, intervention by manipulation and culture tends to lead to enhanced fetal growth [5, 6, 63]. In these ruminant species, other pathways may become activated or destabilized as a consequence of serum-induced environmental stress. However, in ruminant species the physiology of embryonic and extraembryonic development is dissimilar to that in multioffspring rodent species, in which the relative size of individual offspring is regulated to a greater extent by maternal effects [64–66]. In addition, the sera used in studies of different species are frequently of diverse origin and are sometimes treated differently before use [2]. Therefore, in spite of apparent differences in the long-term phenotypic effects of serum during preimplantation culture, comparable molecular mechanisms could be involved in different mammalian species, and it would be relevant to test this hypothesis in nonrodent model systems.

ACKNOWLEDGMENTS

We thank G. Konfortova for excellent technical assistance and Dr. G. Kelsey for helpful discussion and for generating the disomy-11 mice from Rb(11;13)Bnr translocation and PCE strains (which were a kind gift from Dr. B. Cattanach).

FOOTNOTES

First decision: 25 August 2000.

1 This work was supported by the Ministry of Agriculture, Fisheries and Food, the Biotechnology and Biological Sciences Research Council, and the Royal Society. Back

2 Correspondence and current address: Robert Feil, Institute of Molecular Genetics, CNRS, 1919 Route du Mende, 34293 Montpellier cedex 5, France. FAX: 33 4 67040231; feil{at}jones.igm.cnrs-mop.fr Back

3 Current address: Wellcome/CRC Institute of Cancer and Developmental Biology, Tennis Court Road, Cambridge CB2 1QR, UK. Back

4 Current address: Office for National Statistics, Ebury Gate, London SW1, UK. Back

Accepted: November 2, 2000.

Received: July 21, 2000.

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