Human Molecular Genetics, 2001, Vol. 10, No. 10 1093-1100
© 2001 Oxford University Press
Paternal monoallelic expression of PEG3 in the human placenta
1Research Group in Human Reproductive Immunobiology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK, 2Sub-department of Animal Behaviour, University of Cambridge, Madingley CB3 8AA, UK and 3Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge CB2 1QR, UK
Received 31 January 2001; Revised and Accepted 13 March 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting is the phenomenon whereby mono-allelic expression of certain genes occurs depending on their parental origin. The observation that imprinting only occurs in placental mammals has led to the suggestion that it may play a role in this form of reproduction. In the present study we have investigated the pattern of expression of the human PEG3 gene in the early to term placenta, as well as the uterus and ovary, using RT–PCR, northern blot and in situ hybridization. A comparison is made with the expression of Peg3 in the mouse by histochemical staining in ßgeo knock out mice. We have demonstrated high levels of PEG3 in the human placenta and have localized the signal to the layer of villous cytotrophoblast cells. In contrast, the pattern of expression of Peg3 in the mouse placenta is less restricted, the message being present in all trophoblast populations. Thus, expression of PEG3/Peg3 in the human and mouse placenta is not directly comparable. We have also detected PEG3 message in the ovarian stroma. We have sequenced the human PEG3 gene from exon 3 to exon 9. By utilizing a polymorphism detected in exon 9, we have established that only the paternal allele is expressed in human placenta. Human PEG3 is therefore maternally imprinted as in mouse.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The phenomenon whereby mono-allelic expression of certain genes occurs depending on their parental origin is termed genomic imprinting. The different roles the paternal and maternal genomes might play is illustrated in the original murine experiment that led to the idea of imprinting (1). Parthenogenotes were seen to give rise to embryos that, despite being otherwise normal, lacked proper development of the extra-embryonic membranes required for placental formation. Conversely, androgenotes had normal extra-embryonic tissues but failure of embryonic development. A human equivalent of the murine experiments is the androgenetic complete hydatidiform mole, a condition where there is abundant placental tissue but poorly developed or absent embryo. Paternally expressed genes would, therefore, appear to have a crucial influence on extra-embryonic and, ultimately, placental development.
At present there is not much information available on paternally expressed genes in the placenta. There was a recent report on such a gene MEST/Mest (also known as Peg1) which was observed to be expressed in murine extra-embryonic mesoderm derivatives and in human trophoblast (2). Another gene that appears to be a logical candidate for further investigation is PEG3/Peg3 (paternally expressed gene 3). Peg3 was identified in a systemic screen using subtraction hybridization with cDNA from normal and parthenogenetic mouse embryos and located to proximal chromosome 7 (3). Highest expression was seen in the adult mouse brain but there was also strong expression in the extra-embryonic tissues of the day 8 mouse embryo (4). Perhaps the most exciting insight into Peg3 has followed the study of the phenotype of knock out mice created by insertion of a ßgeo selection cassette into exon 5 (5). Peg3-deficient mice were viable and fertile but it was noted that the first litters of mutant females often failed to survive to weaning due to impaired maternal behaviour, such as nest building, gathering the pups or keeping them warm. The placentae of mutant embryos were also found to be smaller and there was fetal growth retardation. The human homologue, PEG3, has now been identified and located to the homologous chromosome 19q13.4 with a high degree of conservation seen between the human and mouse amino acid sequences of exon 9, although the human PEG3 appears to lack the first proline-rich repeat seen in the mouse (6). Highest expression was found in the ovary, testis and placenta by northern blot analysis but the exact cell type within these tissues where the gene is expressed has not yet been identified. The imprinted status of human PEG3 is not known.
In the present study, we have investigated the pattern of expression of human PEG3 in the early and term placentae, as well as the uterus and ovary, using RT–PCR, northern blot and in situ hybridization (ISH). A comparison is made with the expression of Peg3 in the mouse placenta at varying gestations by histochemical staining for ß-galactosidase in ßgeo knock out mice. We have also sequenced the human PEG3 gene from the ATG in exon 3 to exon 9 and have determined its imprinted status by utilizing a polymorphism detected in exon 9.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression of human PEG3 detected by RT–PCR and northern blotting.
To provide some insight into the expression patterns of PEG3 in human tissues associated with pregnancy and in cell lines that might be of use in further experiments, we used RT–PCR to detect mRNA transcripts of the gene. PCR primers were devised by deducing the probable exons from the human genomic sequence in GenBank (accession no. AC006115) and the murine amino acid sequence (GenBank accession no. AF038939). Although PEG3 shares exons 3–7 with the gene ZIM2 (7), the 3' primers were PEG3-specific, thus ensuring the specificity of the PCR reaction. This was confirmed on sequencing the PCR products. PEG3 mRNA message levels were semi-quantitatively estimated using RT–PCR of the housekeeping gene ß actin as reference. After one round of PCR amplification, a positive signal was detected in placenta (first trimester), villous trophoblast, choriocarcinoma cell lines, JEG-3 and JAR, and embryonal carcinoma lines, Tera-1 and Tera-2, as well as some endometrial samples from throughout the menstrual cycle. An amplified band of 751 bp was observed on gel electrophoresis. Second round amplification was required to detect low levels of message in extravillous trophoblast, term placenta, the choriocarcinoma cell line BeWo, decidual tissue and decidual NK cells, most of the endometrial samples (7 out of 11), and peripheral blood lymphocytes. An amplified fragment of 303 bp was obtained. Of the three NK cell lines tested (NK92, NKL and YT), only NKL gave a positive result and the other leucocyte lines tested (U937, macrophage line and JURKAT, T cell line) had no detectable message. Decidual macrophages and the putative trophoblast lines, TCL-1 and HT-1, were also negative (Fig. 1).
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Northern blotting was employed to give a more reliable estimate of the relative levels of PEG3 message in two samples of maternal decidua and three samples of placental tissue (data not shown). The RT–PCR first and second round amplified fragments were used in the form of 32P-labelled probes, both of which detected a single band of
9 kb. The considerably higher levels of message in first trimester placenta compared with decidual tissue were striking. The blots were then stripped and hybridized with a ß actin probe as a control. The hybrid bands were quantified as described in Materials and Methods and placental samples were found to give readings 20- to 60-fold higher than for the decidual samples.
Expression of PEG3 revealed by ISH
The second round RT–PCR product was used to derive the riboprobes for ISH and this had 72 of the 303 bp in common with ZIM2 but, as was seen from the results, specific expression was detected in placenta, which has been reported to have no ZIM2 expression (7).
Pregnancy tissues used for ISH included placental and the corresponding decidual tissue from first trimester samples (6–11 weeks) and a pregnant uterus (10–12 weeks gestation) showing both free floating and anchoring villi attached to and invading the decidua, an ectopic tubal pregnancy with villi and invasion of the Fallopian tube wall and a term placental sample. All first trimester samples showed the same clear and specific pattern of expression; a strong PEG3 hybridization signal was detected in the villous cytotrophoblast layer but was not detectable in any other cell type [Fig. 2A–C and D(iii)]. Immunohistochemical staining for cytokeratin was performed on sections cut from the same blocks to reveal the different trophoblast populations [Fig. 2A–E(i)]. It was striking that expression was down-regulated as villous trophoblast differentiated to the various extravillous trophoblast populations. This was true in the floating villi, where the extravillous cytotrophoblast columns form at the tips of the villi [Fig. 2D(iii)], and in the anchoring villi, where the fetal cells start to invade into the maternal tissue. This occurred whether trophoblast infiltration was into normal decidua or into the wall of the Fallopian tube in the ectopic pregnancy [Fig. 2A and B(iii)]. The second pathway of differentiation is from villous to the overlying syncitiotrophoblast layer of the placental villi and here also PEG3 was sharply down-regulated [Fig. 2C(iii)]. No signal was detected in the mesenchymal stromal cells or in the endothelial cells of the villous core and there was no detectable hybridization of the probe within the maternal decidua. [Fig. 2A(iii)]. Villous cytotrophoblast cells are sparse at term and difficult to identify using light microscopy of haemotoxylin and eosin (H&E)-stained sections. Interestingly, a strong signal was still detectable in these remaining scattered focal villous cytotrophoblast but again, no other cells showed detectable hybridization with the PEG3 probe [Fig. 2E(iii)]. Endometrial samples from throughout the menstrual cycle also gave no detectable signal on hybridizing with the PEG3 antisense probe. There was no specific staining of any sections with the sense probe.
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Previously it had been reported that on northern blotting human ovarian tissue as well as placenta exhibited high levels of PEG3 message (6). ISH on ovarian tissue and the Fallopian tube was therefore carried out to identify the cell type(s) expressing the message. A strong hybridization signal was seen in the cells of the ovarian stroma, including the thecal layers around the follicles. The surface ovarian epithelium, follicular cells, fimbriae and all layers of the tubal wall were negative [Fig. 2F(iii)].
Imprinting of human PEG3
The identity of the first and second round RT–PCR products was confirmed on sequencing the excized gel bands and the sequence of exons 3 to the start of exon 9 was thus ascertained. The deduced amino acid sequence of exons 3–8 showed 73% homology with the mouse protein over this region (Fig. 3). Overall similarity between the human and mouse protein sequence, including exon 9, was 58%.
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To determine whether PEG3 is imprinted we looked for polymorphisms in the PEG3 sequence. No polymorphisms were found in the 5' portion of the PEG3 message when sequencing the 750 bp PCR product from placental cDNA samples from four individuals and the 300 bp fragment from an additional 10 individuals. However, on devising new primers to the 3' end of the PEG3 message a polymorphism was discovered 41 bp from the end of the open reading frame (codon 1452). The most frequent allele, PEG3*01, had a guanine residue (cgc) and the less frequent allele, PEG3*02, had an adenine (cac) nucleotide. This is a non-synonymous substitution that would lead to a change from arginine to histidine in the amino acid sequence (R1452H). However, no change to restriction enzyme sites in the DNA occurs with this polymorphism.
This polymorphism was then used to type genomic DNA from placentae of 32 unrelated pregnancies by PCR and sequencing. Gene frequencies of 87.5% for the PEG3*01 allele and 12.5% for the PEG3*02 allele were found. Six heterozygotes (PEG3*01/*02) were detected and in all these placentae only one of the alleles was found in cDNA samples, three expressing the PEG3*01 allele and three expressing the PEG3*02 allele. This demonstrates that the human PEG3 gene is monoallelically expressed in human placenta.
To determine whether the paternal or maternal allele is expressed we then typed the maternal PEG3 gene using decidual genomic DNA from the six women whose placentae showed heterozygosity of genomic DNA. Four mothers were found to be heterozygotes (Fig. 4A). Only two were homozygous for the PEG3*01 (PEG3*01/*01) allele and in these cases the corresponding placentae only transcribed the PEG3*02 allele, hence showing that the expressed gene is paternally derived (Fig. 4B). The sequencing data showing genomic DNA of the mother (Fig. 4C) of the placenta (Fig. 4D) and cDNA of the placenta (Fig. 4E) is shown.
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placenta, father’s genotype unknown). (A) PEG3 is imprinted in the placental samples. Heterozygous placentae (genomic DNA) are found to express only one of the PEG3 alleles (cDNA). (B) PEG3 is seen to be imprinted and expressed solely from the paternal allele. The allele expressed by these heterozygous placentae is not inherited from the mother. (C–E) Show sequence tracings for one of the informative (B) pairings. Genomic DNA from the mother has a single G peak (PEG3*01) whereas the heterozygous placenta shows a double G/A peak in the genomic DNA sample (D) but a single A peak (PEG3*02) in the cDNA sample (E).In cDNA samples from the heterozygous decidua again only one of the alleles was detected, showing that the imprint is retained in this adult tissue as has been reported for Mest/Peg1 expression in mouse decidua (2). To summarize the findings, human PEG3, as for mouse Peg3, is paternally expressed.
Expression of Peg3 in mouse placenta
Localization of Peg3 expression in the mouse placenta and uterus was investigated using mice in which the Peg3 gene had been silenced by insertion of a ß-galactosidase neomycin fusion gene into exon 5 (5). This disruption of Peg3 transcription leaves the upstream tissue-specific promoter sequences intact and functional and therefore wherever Peg3 would normally be transcribed, ß-galactosidase activity will be substituted. At 9 days post-coitum (d.p.c.), embryos in utero derived from a cross of mutant male with wild-type female, stained for ß-galactosidase activity, showed activity in the embryo, in the yolk sac surrounding the embryo and in the trophoblast of the developing placenta (Fig. 5A–C). Neither the primary fetal-derived giant cells nor the maternal decidual tissue stained blue with the Xgal substrate. At 15 d.p.c. the well-developed placenta showed positive staining of most of the trophoblast populations (Fig. 5D and E). Two different patterns of staining were observed; strong uniform staining of cells in the spongiotrophoblast layer and secondary giant cells with weaker punctate staining in the labyrinth (Fig. 5E and F). Expression in the yolk sac was maintained at day 15 within the mesodermal component (Fig. 5G).
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Expression of Peg3 in mouse maternal tissues
Mutant female mice were mated with normal males to allow investigation of Peg3 expression in the maternal tissues during pregnancy. At 9 d.p.c. faint X gal staining was observed at the border of the antimesometrial decidua and the myometrium and seemed to be present in more than one cell type (Fig. 5J). It was noticeable that the implanted embryos appeared to be abnormally closely spaced. No staining of other areas of the decidua was seen, including the abundant NK cells around the implantation site (data not shown). To investigate Peg3 expression at an earlier stage the uterine horn at 5 d.p.c. was Xgal-stained and macroscopically showed weak patches of staining along the uterus with stronger punctate staining of the attached ovary, fimbriae and oviduct (Fig. 5H). In section the blue stain in the uterus was seen in an area of adipose tissue attached to the wall, in the media of several blood vessels and in other scattered cells in the myometrium (Fig. 5I).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Peg3 is a maternally imprinted gene originally described in the mouse (3). In this study, we have shown that the human homologue PEG3 is imprinted also with expression from the paternal allele. This, therefore, follows the general rule that genes imprinted in the mouse are similarly imprinted in humans (8). In the context of reproduction, the ‘tug-of-war’ hypothesis proposes that paternally expressed genes including PEG3/Peg3 have a growth-promoting influence on the fetus and placenta in the battle for nutrients during pregnancy whereas maternally expressed genes provide the necessary restraint to ensure survival of the mother (9).
We have demonstrated the expression of PEG3 in the human placenta. The levels of message are considerably higher in the placenta than in the corresponding decidua. ISH has localized the signal to the layer of villous cytotrophoblast cells in both first trimester and term placenta. No other placental cells express PEG3. This pattern of distribution is, therefore, different to that reported for MEST (PEG1), where message was detected in the stromal and endothelial cells of the villous mesenchyme as well as in trophoblast (2). Villous cytotrophoblast is the population with proliferative potential, as they frequently display mitotic figures and express proliferation markers such as Ki67 (10). This finding would accord with the hypothesis that PEG3 has a growth-promoting influence on the placenta. Indeed, Peg3-deficient mice are observed to have a smaller placenta than wild-type controls. Interestingly, HASH2, a maternally expressed gene, is observed in extravillous trophoblast (11). This trophoblast population invades into decidua at the implantation site but is generally no longer proliferative. Thus, paternally expressed PEG3 may be involved in placental growth whereas maternally expressed HASH2 controls the extent of placental invasion.
The function of PEG3 is not known. Its zinc finger motif suggests that it may act as a transcription factor. HASH2 is a member of the basic helix–loop–helix gene family which function as lineage-specific transcription factors. Both of these imprinted genes, therefore, could have important downstream effects on the expression of other trophoblast genes. For example, EGF-R and c-erbB2 have reciprocal expressions in villous and extravillous trophoblast, respectively (12), and the non-classical class I, HLA-G, is expressed by extravillous but not by villous trophoblast (13). Work on mouse Peg3 has suggested that it is implicated in p53-mediated apoptosis and has a role in the TNF-NF
B signal pathway, although this has not been confirmed by others (14–16). Such pathways could provide a mechanism for regulating cell numbers in the placenta.
The expression of Peg3 in the mouse placenta appears to be less restrictive than in the human placenta in that all trophoblast populations, including spongio- and labyrinthine trophoblast as well as secondary giant cells, express message for this gene in the mouse. Restricted expression to the proliferating trophoblast population is not found. Thus, the expression of PEG3/Peg3 in humans and mice is not directly comparable. This is not surprising in view of the fact that the placentae in these two species are structurally very different, even though they are both defined as haemochorial. There is now increasing evidence that the evolutionary conservation of the coding sequence of developmental genes does not necessarily mean that their temporal or spatial expression patterns are equally conserved (17).
Besides the placenta, high levels of PEG3 message had also been reported in human ovarian tissue (6). By ISH, we have now confirmed that the signals originate from cells of the ovarian stroma, including the thecal layers around the follicles. A paternally expressed gene in the ovary could also impact on reproductive success since the ovarian stroma is the source of many important hormones that prime the uterus in preparation for pregnancy. In the mouse also there appears to be expression in the adult decidua and also in the ovary and oviduct but how this may compromise reproductive success is not known. The observation of impaired maternal behaviour towards her offspring in Peg3-deficient mice suggests that activity in the brain is yet another way in which this gene influences reproduction. In this situation, the paternal gene appears to affect the behaviour of his female offspring to benefit his grandchildren. A similar phenomenon was seen also in the Mest-deficient mouse (18). It would be particularly interesting in this context to see if human PEG3 is also imprinted in the brain and whether the impairment of maternal behaviour in the mouse model has a counterpart in the human population.
Of the many hypotheses proposed to explain the evolutionary rationale of genomic imprinting, the ‘tug-of-war’ model in relation to reproduction appears to be the most successful in accommodating the available data for individual imprinted genes. In support of this, imprinting is only observed in eutherian mammals and marsupials but not in egg-laying monotremes. Much of the research to date has focused on the role of imprinted genes in the growth and developmental pathways of the fetus and placenta, but it is now clear that these genes can act in other ways to affect reproductive performance. For example, imprinting in the brain may affect maternal behaviour and nurturing of her young. Thus, imprinted genes can act at many levels to influence the fetal–maternal relationship.
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MATERIALS AND METHODS |
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Tissues and cells
First trimester normal placental and decidual tissue was obtained from patients undergoing therapeutic termination of pregnancy at Addenbrooke’s hospital (Cambridge, UK). Ethical approval had been granted. Pieces of tissue (
100 mg) were frozen immediately in liquid nitrogen and stored at –80°C until required for RNA or DNA extraction. Tissues for ISH were immediately fixed in neutral-buffered formalin (16 h) and processed the next day for paraffin wax sections. Isolation of highly purified trophoblast cell populations has been previously described (12). Briefly, this involved a short trypsin digestion of the placental tissue to give single cell suspensions which were either harvested directly or labelled with mAb to EGFR and cerbB2, which react with villous or extravillous trophoblast cells, respectively. The labelled cells were then flow-sorted to obtain >99.5% purity. Decidual NK cells were purified to >97% purity after disaggregation of the decidual tissue with collagenase and selection on a CD56 MACS bead column as described by Verma et al. (19). Term placenta, ovary, pregnant uterus, endometrial and ectopic pregnancy samples were obtained from archival material. The choriocarcinoma cell lines, JEG-3, BeWo and JAR, the monocyte-like line U937, the T cell line Jurkat and the embryonal carcinoma cell lines, Tera-1 and Tera-2, were purchased from the American Tissue Culture collection and maintained in culture according to the conditions recommended. The NK-derived cell lines NK-92, NKL and YT were obtained from Immune Medicine, Dr M.J. Robertson (Harvard University, MA) and Dr G. Griffiths (Oxford University, UK), respectively. Culture conditions have been published elsewhere (19). Placental derived lines TCL-1 and HT-1 were kindly made available by Dr M.H.F. Sullivan (Hammersmith Hospital, London, UK) and Dr C.-K. Ho (Veterans General Hospital, Taipei, Taiwan) and maintained in culture in RPMI with 10% fetal bovine serum.
PCR analysis and sequencing of human PEG3
Total RNA was isolated from tissue or cell samples using the RNeasy Mini kit according to the manufacturer’s instructions (Qiagen) and first-strand cDNA synthesized using MMLV-RT and oligo(dT) primer from the ProSTAR First-Strand RT–PCR kit following the instruction manual (Stratagene). Genomic DNA was isolated from placental and decidual samples using the QIAamp Tissue kit (Qiagen). Genomic DNA was subjected to a single round of amplification by PCR and cDNA to one or two rounds in a nested PCR to improve detection levels. The primers and programs used were: (i) 5' primers, first round, cDNA, exon 3, 5'-AAGCTCGTCACTCTGCTGGA-3' and exon 9, 5'-GCTGCTGGATCACTGACTCC-3'. The following cycling conditions were used: initial denaturation at 94°C for 3 min followed by 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 1 min for 30 cycles with a final extension at 72°C for 8 min. (ii) 5' primers, second round, cDNA, exon 7, 5'-TCGCTGAGGACAGGAAACCT-3' and exon 9, 5'-ACTCCCTTGCTCTTCCCGAT-3'. One microlitre from the first round reaction volume of 30 µl was subjected to PCR for 22 cycles with steps of 30 s duration at 94, 57 and 72°C and a final extension at 72°C for 8 min. (iii) 3' end primers, cDNA and genomic DNA, exon 9, 5'-CAGGGCTTAAACGTAGAGGC-3' with 5'-CTCAGCCAGTGTGGGTATTC-3'. The PCR program was as for the 5' primers in the first round. (iv) The second pair of 3' end primers were 5'-GTCTGTGGGCAGCTCTTCAA-3' and 5'-CCAGGTAAGGTACCTCTGCA-3'. The PCR annealing temperature and cycle number were as for the other 3' end pair.
As a measure for varying mRNA levels between samples, PCR of the housekeeping gene ß actin was carried out as described previously (20). The amplified PCR products were gel-purified and sequenced using dye terminator automated sequencing (Applied Biosystems). The amplified products from the first and second round PCR using the 5' primers were also cloned into the vector, pCR11 (Invitrogen), sequenced to ascertain that there were no Taq mistakes and used as probes for northerns and ISH.
Northern blot hybridization
To analyse relative expression levels of PEG3 in fetal placenta and maternal decidua, total RNA aliquots were electrophoresed, blotted onto Hybond N (Amersham Pharmacia) and probed with the amplified PCR products, which were 32P-labelled using the random priming method. Probe hybridization was carried out overnight at 42°C and washing was done at 65°C in 2x SSC, 0.1% SDS twice, 15 min before exposing to film overnight at –70°C or for 1 h at room temperature. A ß actin probe was used as a control to allow comparison of PEG3 message levels. The hybrid bands were imaged and quantified using a Cyclone PhosphoImager (Packard).
ISH
ISH was carried out on paraffin-embedded sections of tissues cut onto slides coated with 3-aminopropyl-triethoxy-silane. The protocol used in this study was based on that described previously (21). The RNA probes were derived from the 303 bp PEG3 fragment, which encompassed sequence from exon 7 to 9, and had been ligated into pCR11. Single stranded sense and antisense probes were radiolabelled with (33P)UTP using a T7/SP6 MAXIscript in vitro transcription kit (Ambion). The sections were dewaxed, washed and pretreated with proteinase K before hybridizing with the RNA probes at 55°C overnight. After washing and treatment with RNase A the slides were coated in Hypercoat LM-1 emulsion (Amersham Pharmacia) and left for 4 weeks exposure before being developed and counterstained with Mayer’s haemalum.
Histochemical staining
An immunohistochemical stain for cytokeratin using the MNF116 mouse monoclonal antibody (Dako) was used to identify trophoblast in the serial sections from tissues used for ISH, employing methods described previously (22). Other sections were stained with H&E to visualize cell morphology.
Peg3-deficient mice
Peg3ßgeo mutant mice were generated as described (5). Briefly the Peg3 gene was mutated by insertion of a ßgeo selection cassette into the coding exon 5 using gene targeting. Paternal inheritance of the mutant gene effectively silenced all Peg3 expression in the offspring of heterozygous males and wild-type females and ß-galactosidase expression was substituted in these individuals. Nine and 15 d.p.c. embryos in utero from matings of normal females and mutant males plus 5 and 9 d.p.c. uterus samples from mutant females were collected. Samples were frozen in isopentane in liquid nitrogen and stored at –70°C until required for frozen sections. Ten-micrometre cryosections were air-dried and fixed for 10 min in 4% paraformaldehyde in PBS at 4°C, washed for 30 min in rinse buffer (0.1 M sodium phosphate pH 7.3, 2 mM magnesium chloride, 0.01% Triton X) before incubation in the dark for 6–16 h in freshly prepared Xgal solution comprising rinse buffer plus 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide and 1 mg/ml Xgal (Roche Diagnostics). Sections were then rinsed in PBS and counterstained with haematoxylin. For whole mounts, fresh tissue was fixed for 1 h in neutral buffered formalin before staining with Xgal as for the sections.
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ACKNOWLEDGEMENTS |
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We thank our obstetric colleagues and the staff at Addenbrooke’s hospital for collecting the placental samples, Anna Wilson and Rafia Al-Lamki for tissue sectioning and Di Licence, Sharzia Malik, Rob Sherwin, Lucy Gardner and Francesca Stewart for their invaluable advice and technical help.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1223 333727; Fax: +44 1223 765065; Email: akk27@cam.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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