Molecular Human Reproduction, Vol. 7, No. 9, 839-844,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos
1 Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH and 2 Assisted Conception Unit, Department of Obstetrics and Gynaecology, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK
Abstract
Imprinted genes are characterized by expression of only one of the two alleles according to its inheritance from the mother or the father. This mono-allelic expression must arise from primary differential epigenetic modification of the parental alleles of the imprinted gene in the spermatozoon and the oocyte. Most of the information on the onset of imprinted gene expression, and on the molecular mechanisms regulating mono-allelic expression, have been derived from studies in the mouse. In this paper, we investigate the expression of seven imprinted genes in human preimplantation development. Due to limitations imposed by the rarity of human embryos available for research, our approach has been to screen amplified cDNA preparations prepared from human unfertilized oocytes and individual embryos at each of the 4-cell, 8-cell and blastocyst stages. Gene-specific primers were used to investigate expression of the imprinted genes by polymerase chain reaction (PCR) analysis of these amplified cDNA. We found that expression is inherently variable in the amplified cDNA from embryo to embryo but the use of several samples at each stage showed that the SNRPN, UBE3A and PEG1 genes are expressed throughout human preimplantation development. This was confirmed by direct analysis by gene-specific reverse transcription–PCR on a limited number of lysed embryos (one gene analysed per embryo). Thus, the amplified cDNA may be used to rapidly identify those imprinted genes expressed in preimplantation development and, hence, those genes amenable to investigation of the epigenetic mechanisms regulating mono-allelic expression. Confirmation of preimplantation expression also identifies those imprinted diseases amenable to preimplantation diagnosis, and the imprinted genes which may be used in assessment of possible perturbations of imprinting following new procedures in assisted reproduction. Our series of single embryo amplified cDNA are established as a valuable resource for comparative studies of gene expression within one embryo and between embryos throughout early human development. The amplified cDNA thus circumvent the need for a continuous supply of human embryos for studies on embryonic gene expression.
embryonic cDNA/genomic imprinting/human embryonic development/imprinted gene expression
Introduction
To date, over 20 genes which show imprinted mono-allelic expression, i.e. expression occurs predominantly, or exclusively, from one of the parental alleles, have been identified. [For a catalogue of imprinted genes, see http://cancer.otago.ac.nz/IGC/Web/home.html (Morison and Reeve, 1998
)]. Inherited diseases, and tumours involving aberrant expression of imprinted genes, show unusual patterns of inheritance, depending on the sex of the transmitting parent. These include neurological disorders mapping to chromosome 15q11–q13, such as Prader–Willi syndrome (PWS) and Angelman syndrome (AS) (Glenn et al., 1997), and conditions such as Beckwith–Wiedemann syndrome and Wilms' tumour, which are characterized by prenatal overgrowth and cancer and involve imprinted genes in the chromosomal region 11p15 (Ward, 1997). The early embryonic stages of human development are critical with respect to the regulation of imprinted gene expression as it is during this period that the epigenetic information marking the inherited maternal and paternal alleles from the oocyte and spermatozoon is interpreted and translated into the functional differences.
Clinical investigations relevant to assisted reproduction and preimplantation diagnosis and prevention of imprinted genetic disease require a clearer picture of the expression of these genes in the early embryo. There are concerns about the genetic risks of the possible disruption of imprinting by freezing of gametes, intracytoplasmic sperm injection (ICSI) of immature spermatids (Meschede et al., 1995), and the use of immature oocytes matured in vitro (Trounson et al., 1998) for IVF. It is also important to know the status of imprinting in human embryonic stem cells considering their proposed use in tissue transplantation therapy (Thomson et al., 1998). Before we can evaluate these concerns, we need to know which imprinted genes are already expressed in the human preimplantation embryo.
However, there are major limitations to studies on gene expression in the human due to the scarcity of embryos available for research and the associated ethical restrictions. In addition, the very few cells of the embryo limit the amount of information which may be obtained to only one, or very few, genes. In an attempt to overcome these limitations we have prepared amplified cDNA derived from human oocytes and individual preimplantation embryos. In this report, we investigate the use of these amplified cDNA to determine whether expression of seven imprinted genes can be detected in human preimplantation development.
Four of the imprinted genes analysed map to human chromosome 15q11–q13 region and are associated with Prader–Willi Syndrome [SNPRN (Reed and Leff, 1994); NDN (Jay et al., 1997; MacDonald and Wevrick, 1997); and IPW (Wevrick et al., 1994)] and Angelman syndrome [UBE3A (Kishino et al., 1997)]. The SNRPN gene codes for an RNA splicing factor (Reed and Leff, 1994) and is paternally expressed in a range of somatic tissues. The necdin gene (NDN) is expressed from the paternal allele in human fibroblasts and brain (Jay et al., 1997; MacDonald and Wevrick, 1997) and may play a role in the arrest of cell growth in murine post-mitotic neurones (Hayashi et al., 1995). The IPW gene codes for an untranslated transcript (Wevrick et al., 1994). The UBE3A gene, in the same chromosome region, is expressed only from the maternal allele in the brain (Rougeulle et al., 1997).
The other three imprinted genes investigated are PEG1/MEST, H19 and KVLQT1. The human PEG1/MEST gene (Paternally Expressed Gene 1/Mesoderm Specific Transcript), encoding a putative enzyme of the alpha hydrolase fold family, maps to human chromosome 7q32 (Kobayashi et al., 1997). H19 and KVLQT1 are both maternally expressed in somatic tissues and map to human chromosome 11p15.5. The H19 gene, like IPW, codes for an untranslated RNA (Brannan et al., 1990; Zhang and Tycko, 1992) and is proposed to be an oncofetal RNA (Ariel et al., 1997). The KVLQT1 gene is maternally expressed in most fetal tissues except the heart, where expression is bi-allelic (Lee et al., 1997). This gene is associated with the familial cardiac defect long-QT syndrome, Jervell and Lange–Nielsen syndrome and Beckwith–Wiedemann syndrome (Wang et al., 1996).
Our results using the amplified cDNA showed that the SNRPN, PEG1 and UBE3A genes are expressed in human preimplantation development. Although there was inherent embryo-to-embryo variability in expression observed in the amplified cDNA, even for embryos at the same morphological stage, the use of a sufficient number of individual embryos gave clear evidence as to whether a specific gene is expressed. The expression of these imprinted genes was confirmed by direct reverse transcription–polymerase chain reaction (RT–PCR) analysis of lysed individual embryos. Thus, our series of single embryo amplified cDNA preparations are invaluable for repetitive and comprehensive comparative assessments of gene transcription within single human embryos. These amplified cDNA, and libraries prepared from them, bypass the continual need for additional human embryos for research in embryonic gene expression.
Materials and methods
mRNA purification and preparation of amplified embryonic cDNA
Preparation of the amplified cDNA from human oocytes and 4-cell, 8-cell and blastocyst stage embryos has been previously described (Holding et al., 2000). Briefly, the oocytes and embryos were lysed in 3 µl of ice-cold lysis buffer [0.8% Igepal, Sigma, Poole, UK; 1 U/µl RNase inhibitor, Gibco BRL, Paisley, UK; and 5 mmol/l dithiothreitol (DTT), Gibco BRL] and the released mRNA molecules bound to oligo-(dT)-linked magnetic beads (Dynabeads, Dynal, UK). Preparations of mRNA from human 10 week fetal brain, muscle and gut tissue were treated in the same way to serve as somatic controls. Synthesis of cDNA on the mRNA bound to the beads, and PCR amplification of the cDNA, were carried out using the SMARTTM cDNA library construction kit (Clontech, Palo Alto, CA, USA).
PCR amplification of specific gene sequences in amplified cDNA preparations
Expression of the imprinted genes, SNRPN, NDN, IPW, PEG1, UBE3A, KVLQT1 and H19 in the amplified cDNA preparations derived from human oocytes and single preimplantation embryos, was detected by nested PCR with the primers given in Table I. Except for NDN, which has no introns, the primers span introns to eliminate misinterpretation due to possible genomic contamination. PCR amplification was conducted in 25 µl of reaction mixture, containing 200 µmol/l of each dNTP, 1 µmol/l of each primer and 2.5 U of Amplitaq DNA polymerase (Perkin Elmer, New Jersey, USA) in supplied buffer 1 (10 mmol/l Tris–HCI pH 8.3, 50 mmol/l KCI, 1.5 mmol/l MgCl2, 0.001% gelatin). With the exception of H19, the parameters for the PCR were two cycles of 93°C for 5 min, 60°C for 1 min and 73°C for 2 min, followed by 30 cycles of 93°C for 1 min, 60°C for 1 min and 73°C for 2 min. For the detection of expression of the H19 gene, the PCR cycling parameters were denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 1.5 min, 56°C for 1 min and 73°C for 1.5 min. One µl of the first round product was then transferred to 24 µl of reaction mixture for second round amplification, using the same conditions except for the substitution of the appropriate second round primers (see Table I).
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Expression of the housekeeping gene, ß-actin, was detected by 35 cycles of PCR with the primers given in Table I
and cycling parameters of 95°C for 1 min, 62°C for 1 min and 72°C for 1 min (469 bp PCR product between bp positions 441 and 910) (Ponte et al., 1984). Expression of the housekeeping gene, HPRT, was detected by nested PCR (see above) with the primers given in Table I and PCR cycling parameters of 95°C for 1 min, 60°C for 1 min and 72°C for 1 min (386 bp PCR product between bp positions 208 and 594) (Gibbs et al., 1989). For each gene-specific PCR, we used equivalent concentrations of amplified cDNA, determined by direct visualization of the cDNA by EtBr staining after electrophoresis. Despite equal loading of amplified cDNA, individual embryos, even at the same stage, show variability in the detection of expressed sequences. The interpretation of the data therefore relies on the use of amplified cDNA from several preimplantation embryos to give an overall picture of an expression profile.
RT–PCR to detect expression of imprinted genes directly in lysed embryos
The method of RT–PCR to detect expression of specific imprinted genes directly on lysates of individual embryos has been described (Huntriss et al., 1998). Briefly, human individual oocytes and 2-cell, 4-cell, 8-cell and blastocyst stage embryos were lysed in 1.5 µl RT lysis buffer [5 mmol/l dithiothreitol (DTT; Sigma), 1 U RNAsin (BCL, Roche Diagnostics Ltd, Lewes, UK), 0.8% Igepal (Sigma)] and RT was performed in a total volume of 5 µl of reaction mixture comprising the embryo lysates, 40 IU Superscript II reverse transcriptase (BRL, Life Technology, Paisley, Scotland), 0.9 mmol/l of each deoxyribonucleotide (Amersham Pharmacia Biotech, Amersham, UK), 2.25 µg random hexamer (BRL Life Technology), 5 mmol/l DDT (BRL) and 2 U RNAsin (BCL). PCR amplification of the imprinted gene sequences was carried out directly on these first strand cDNA preparations with the specific primers, as described above for the amplified cDNA.
Gel electrophoresis
Ten µl of PCR product was mixed with 2 µl of loading buffer and electrophoresed for 90 min at 120 V on a 1 or 2% agarose gel and visualized under short-wavelength UV light.
Results
Figure 1 shows the expression of the seven imprinted genes and the two control housekeeping genes in the series of single embryo amplified cDNA. Expression was variable from embryo to embryo, but overall the SNRPN, PEG1 and UBE3A genes were expressed, the NDN and IPW genes were only rarely expressed and the H19 and KVLQT1 genes were not detectably expressed during human preimplantation development. The low and sporadic expression of NDN and IPW was not due to contamination as many blanks were included. It is probable that these two genes are expressed as low abundance mRNA in the preimplantation embryos.
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This variability in detectable expression of specific genes from embryo to embryo, also seen with the control housekeeping genes ß-actin and HPRT, is partly due to the extreme sensitivity of procedures at the level of so few cells. It is also likely to be an inherent property of human preimplantation embryos consistent with the dynamic changes in mRNA levels during a period of degradation of maternal transcripts and onset of embryo-encoded transcripts. A decrease in actin transcripts at the 2–4-cell stage also has been shown in the mouse (Temeles et al., 1994
). Variable expression of specific genes from embryo to embryo may be expected to be even more prominent in the human embryos due to delayed, arrested or degenerate blastomeres [despite the fact that the embryos used here were all morphologically grade I and correct stage for age (Bolton et al., 1989)]. Despite the variability in expression from embryo to embryo, the inclusion of so many oocyte and embryo samples clearly discriminates the genes that are expressed at these preimplantation stages.
We confirmed expression of the three imprinted genes detected in the amplified cDNA (SNRPN, PEG1 and UBE3A) by direct RT–PCR on lysates from a limited number of human preimplantation embryos available (Figure 2). Each PCR reaction in this type of direct analysis requires a new embryo. We also used a limited number of individual embryos for direct analysis of expression of the H19 gene. As for the amplified cDNA, H19 was not shown to be expressed in the cleavage stage embryos. However, expression of H19 was detected in the single blastocyst analysed directly.
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Discussion
We have used amplified cDNA preparations from human oocytes and individual preimplantation embryos to investigate the expression of seven imprinted genes. We showed that the SNRPN, PEG1 and UBE3A genes are expressed in the human preimplantation embryo amplified cDNA and we confirmed this by direct analysis of lysed human preimplantation embryos. We also showed that expression of NDN and IPW was detected very rarely in the amplified cDNA (in two of the 16 samples, and in one of the 16 samples, respectively) and we suggest that mRNA corresponding to these genes is present in low abundance in human preimplantation development. Assuming that NDN and IPW are expressed at a low level, our results suggest that all four genes analysed in the Prader–Willi/Angelman region are expressed in human preimplantation development.
Although H19 expression was not detected in the amplified cDNA shown in Figure 1, in some repeat experiments we did detect H19 expression in one of the four blastocyst amplified cDNA (data not shown). H19 expression was also observed in the single blastocyst analysed directly by RT–PCR. In the mouse, there are conflicting reports about the time of onset of transcription of H19 either at the blastocyst stage (Tremblay et al., 1995) or after implantation (Szabo and Mann, 1995). KVLQT1 expression was not detectable at any stage during preimplantation development using the amplified cDNA.
The value of the resource of amplified cDNA preparations is that extensive comparative studies can be made on the expression of many genes from just one human embryo. Although the amplified cDNA may show inherent variability in expression of specific genes from embryo to embryo (due to the high sensitivity of procedures, the poor quality of human embryos, and the transition from maternal mRNA to embryonic mRNA), the use of amplified cDNA from several individual embryos throughout preimplantation development can give an overall picture with a clear indication of whether a specific gene is expressed. It should be emphasized that, due to the factors (outlined above) causing variability in detection of expression of specific genes, there is a considerable risk of spurious interpretation if investigation of expression of a specific gene is carried out in only one, or few, human embryos.
Our results indicate that SNRPN and PEG1 are candidates for further studies on mono-allelic expression at these early stages and on the possible molecular mechanisms involved. If mono-allelic expression of these genes occurs in preimplantation development, it would be expected to be from the paternal allele since it is the paternal allele that is expressed in somatic tissues. The UBE3A gene would be difficult to analyse since mono-allelic expression in somatic tissues is from the maternal allele and, if it occurs in the preimplantation embryos, it would be indistinguishable from maternal mRNA carried over from the oocyte.
We have previously shown mono-allelic expression from the paternal allele of the SNRPN gene at the 4-cell stage human embryo (Huntriss et al., 1998). This was done by direct analysis of lysates of human embryos shown to be polymorphic at the SNRPN alleles by prior analysis of parental samples of cumulus and spermatozoa. In the case of PEG1, informative polymorphic embryos for the analysis of the expression of parental alleles during preimplantation development have been very few and our results using lysed embryos remain equivocal at this time. In three 4-cell embryos investigated, one showed mono-allelic expression of PEG1 from the paternal allele, another showed bi-allelic expression (maternal transcripts carried over from the oocyte may still have been present in this embryo) and one was uninformative (J.Huntriss, V.Bolton and M.Monk, unpublished data). Some evidence has been presented by others that expression of another paternally expressed imprinted gene, IGFII, is mono-allelic from the paternal allele in human preimplantation embryos (Lighten et al., 1997).
Imprinted genes are believed to play a role in the parent–offspring conflict (Moore and Haig, 1991), whereby the parental alleles of these loci are believed to have different interests with respect to regulation of fetal, placental and neonatal growth. The mono-allelic expression of certain imprinted genes during preimplantation development thus indicates that these genes have the potential to affect human development according to this model. The expression of SNRPN, PEG1 and UBE3A in these early developmental stages must be noted in this respect, particularly the PEG1 gene, since the mouse homologue has been shown recently to play a role in embryonic growth (Lefebvre et al., 1998).
The amplified cDNA preparations may be used to examine the expression of other imprinted genes in preimplantation development. This will identify genes which may be investigated for the nature of the differential modification in spermatozoa and oocytes regulating mono-allelic expression. It will also indicate the imprinted diseases amenable to preimplantation genetic diagnosis by analysis of mRNA. The use of RT–PCR may be more reliable than genomic PCR in that it provides multiple copies of a cDNA template for PCR, the use of intron-spanning eliminates problems of genomic contamination, e.g. from spermatozoa, and RT–PCR may be the only way to diagnose imprinted gene defects where the defect is epigenetic. However, it must be borne in mind that, due to the embryo-to-embryo variability in specific gene expression seen with the amplified cDNA, only non-affected embryos can be identified.
Knowing which imprinted genes are expressed in human preimplantation development is important to provide criteria for monitoring the quality and normal development of the manipulated embryos in assisted reproduction. For example, following ICSI, the use of immature gametes, or oocyte maturation in vitro, we need to be able to test whether the mechanisms of imprinting have been disturbed in any way. So far, correct imprinting of six imprinted genes in postimplantation mouse embryos developed from oocytes fertilized by spermatid and sperm injections has been shown (Shamanski et al., 1999). However, this does not necessarily mean that this will be the case in humans. The knowledge of which imprinted genes are expressed in the human embryo, and whether the expression is already mono-allelic, provides us with important quality control measures to ensure the safety of these reproductive technologies.
Acknowledgements
We wish to thank the Birth Defects Foundation and Biotechnology and Biological Sciences Research Council for funding this work, and Cathy Holding and Tetsuya Goto for valuable discussions.
Notes
3 Present address: Division of Obstetrics and Gynaecology, D Floor, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, UK 
4 To whom correspondence should be addressed. E-mail: mmonk{at}ich.ucl.ac.uk
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Submitted on November 27, 2000; accepted on June 14, 2001.
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