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Embryo |
Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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form) and late (ß form) stage embryos. The paternally imprinted genes Gnas, Grb10, and Xist were expressed in both in vitro-fertilized (IVF) and parthenogenetically activated (PA) blastocysts as expected. Of the four maternally imprinted genes expressed in the blastocyst (Mest, Ndn, Nnat, and Sgce), Nnat alone showed differential mRNA expression between IVF and PA blastocysts, suggesting imprinting by this stage of development. In conclusion, seven of the eight genes investigated showed mRNA expression during preimplantation development, indicating a potential role during early development. Also significant is the observation that Nnat is imprinted by the blastocyst stage of development although the other genes are not, indicating a temporal imprinting program.
early development, embryo, developmental biology, gene regulation, in vitro fertilization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During development, DNA imprints are erased in the primordial germ cells (PGCs) and reestablished during gametogenesis. This is most easily seen by changes in methylation patterns, with demethylation seen at differentially methylated regions (DMRs) in imprinted genes [2]. This cycle is then repeated during early embryonic development. Following fertilization, there is a global demethylation of the genome by active and passive mechanisms, followed by genome-wide remethylation beginning at the 8–16-cell stage in the cow [2].
Many imprinted genes are essential for fetal development, in both growth regulation and brain development [3]. It is also known that improper regulation of imprinted genes can cause developmental abnormalities. Because imprints are reestablished during preimplantation development, it was our aim to determine the mRNA expression patterns of several imprinted genes in cattle. Both maternally (Dlk1, Mest, Ndn, Nnat, Sgce) and paternally (Grb10, Gnas, and Xist) imprinted genes were selected.
Of the maternally imprinted genes, Dlk1 mRNA is expressed in mouse embryos during postimplantation development, beginning between embryonic Day 7 (e7) and e11, as well as in the adult pancreas, bone marrow, adrenal, and pituitary glands [4]. Mest has recently been shown to have two isoforms: isoform 1 expressed from the paternal allele; and isoform 2, with the promoter in Exon 2, expressed biallelically [5]. This complicates the findings in the human embryo, in which a small but detectable amount of Mest transcript, considered to be leaky transcription, was reported to be expressed from the maternal allele [6]. Ndn mRNA is paternally expressed in newborn mouse brain, and is found in many human tissues, with highest expression in the brain and placenta [7]. Nnat mRNA is also paternally expressed in the human and mouse, with human Nnat expression in the central nervous system from midgestation through early postnatal development, and in the adult anterior pituitary [8]. Nnat also has alternate splice forms ( and ß), either with or without exon 2 [9]. Sgce mRNA is predominantly expressed from the paternal allele in all mouse adult tissues, except for weak maternal expression in the brain. Sgce mRNA is also expressed in the mouse embryo, with initial expression in the allantoic region around Day 9.5 [10].
Paternally imprinted genes included Gnas, Grb10, and Xist. Gnas encodes the G protein -subunit G(S) and several other gene products. It is paternally imprinted in the mouse, showing maternal expression in renal proximal tubules and adipose tissue, but biallelic expression in most other tissues, including the embryo [11]. Growth factor receptor-bound protein 10 (Grb10) is maternally expressed in most tissues and is known to bind the insulin and insulin-like growth factor I (IGF-I) receptors. Xist transcripts have previously been detected in bovine embryos, with single-round polymerase chain reaction (PCR) showing detectable levels from the 8-cell stage onwards. Transcripts can be found in both male and female morulae and in female blastocysts [12].
To test for the presence of several gene transcripts in a single oocyte or embryo, amplification of single-cell cDNA was performed primarily as described [13]. Amplified cDNA was tested for several control genes. Eight putatively imprinted genes (Dlk1, Gnas, Grb10, Mest, Ndn, Nnat, Sgce, and Xist) were then analyzed in the bovine oocyte and embryo samples, with several genes being described in embryos for the first time. Putative imprinted genes were also tested for differential mRNA expression patterns between in vitro-fertilized (IVF) and parthenogenetically activated (PA) blastocysts. It was the aim of the present study to describe the mRNA expression of several putative imprinted genes in the mammalian embryo and to determine the status (imprinted or not) of several genes during bovine preimplantation development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental procedures were approved by the Monash University Animal Ethics Committee for animal experimentation and were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals.
Oocyte and Embryo Collection
Bovine intact cumulus oocytes complexes (COCs) were matured in TCM199 medium supplemented with 10 µg/ml FSH (Ovagen, Pacific Vet, Christchurch, New Zealand), 0.1 IU/ml LH (Chorulon, Intervet Pty. Ltd., Bendigo East, VIC, Australia), 1 µg/ml ß-estradiol, 100 µM cysteamine, 1 ng/ml IGF-I, and 10% fetal calf serum, FCS (CSL Ltd., Parkville, VIC, Australia) for 20 h at 39°C in a humidified atmosphere of 5% CO2 in air. Following maturation (20–22 h), COCs were fertilized in Fert-Talp medium supplemented with 0.5 mg/ml gentamycin sulphate, 30 µg/ml heparin, 1.65 µg/ml hypotaurine, 0.27 µg/ml epinephrine, and 4.5 µg/ml penicillamine [14], and cultured in synthetic oviductal fluid (SOF) medium [15] supplemented with 30 µl/ml Basal Medium Eagle (BME) essential amino acid solution, 10 µl/ml Minimum Essential Medium (MEM) nonessential amino acid solution, 200 µM L-glutamine, 50 µg/ml gentamycin sulfate, 100 µg/ml sodium tricitrate (Selby Scientific, Clayton, Victoria, Australia), 500 µg/ml myo-inositol and 8 mg/ml BSA (ICPbio, Henderson, Aukland, New Zealand) until the desired stage of development. Embryos were cultured in 4-well plates in gassed foil bags (West Packaging Services, Carrum Downs, Melbourne, NSW, Australia) containing 5% O2, 5% CO2, and 90% N2, and incubated at 39°C in a humidified atmosphere of 5% CO2.When culturing to the blastocyst stage, 5% fibroblast growth factor 4 (FGF4)-conditioned medium (mouse embryonal carcinoma cells) [16] was added on Day 5, along with 10% charcoal-treated FCS and 25 ng/ml heparin.
For mRNA isolation, in vitro-matured (IVM) oocytes were chosen following the removal of cumulus cells by vortexing for 3.5 min in the presence of hyaluronidase. Embryos were collected at the following hours postfertilization (hpf), 2-cell (31 hpf), 4-cell (42 hpf), 8-cell (49 hpf), 16–32-cell (92 hpf), compacting morulae (139 hpf), blastocysts (168 hpf), and hatched blastocysts (187 hpf).
Parthenogenetic Activation
IVM oocytes were denuded of cumulus cells and checked for the presence of a polar body at 20–22 h postmaturation. Oocytes with a polar body were deemed mature and were used for parthenogenetic activation. Briefly, oocytes were placed into 5 µM calcium ionophore A23187 in Hepes-buffered TCM199 for 4 min, washed three times, and placed into TCM199 with 8 mg/ml BSA and 2 mM 6-dimethylaminopurine (6-DMAP) for 5 h in 5% CO2 in air [17].
Messenger RNA Extraction, Reverse Transcription, and PCR
Single oocytes or embryos were taken at various stages of development and placed fresh into 3–5 µl lysis buffer consisting of 0.8% Igepal, 5 mM DTT, and 1 IU/µl RNAsin, and snap frozen. The Dynabeads mRNA purification kit (Dynal, Inc., Carlton South, VIC, Australia) was used to isolate mRNA from oocytes and single embryos within 1 h of being snap frozen. Magnetic beads (Dynabeads) with mRNA attached were resuspended in 3 µl of deionized H2O/ sample. Reverse transcription (RT) reactions were then carried out as described in Clontech's (Becton Dickinson, North Ryde, NSW, Australia) SMART cDNA synthesis kit in the presence of the Dynabeads. Half of the RT reaction (5 µl) was then used for each cDNA amplification step according to SMART cDNA synthesis kit protocols. Reactions were loaded onto a preheated thermocycler (95°C), and then underwent 29 cycles at the following PCR parameters: 95°C for 25 sec, 65°C for 30 sec and 68°C for 7 min, followed by incubation at 10°C until tested. Samples (5 µl) were electrophoresed on a 1.2% agarose gel for confirmation of amplification and then stored at -80°C until use.
Gene-Specific PCR
Amplified cDNA was diluted 1:10 and then used to test for the control genes poly(A) polymerase and Oct4, as well as GDF9 (oocyte expression) and interferon 
(IFN-) (blastocyst expression). PCR amplification was performed in 25 µl reactions containing 200 µmol/L of each dNTP, 1.9 mmol/L MgCl2, 10 pmol each primer and 1.2 Units of Taq DNA polymerase in the supplied buffer (Fisher Biotech Int. Ltd, Subiaco, WA, Australia). The cycling parameters for these genes were: 95°C for 45 sec, 56°C for 45 sec and 72°C for 1 min, followed by an extension at 72°C for 5 min. Bovine cDNA sequence data were obtained for each of the putatively imprinted genes with the use of primers designed from available human, mouse and bovine EST sequence data where available. All primers used are shown in Table 1. Xist and IFN- bovine primers were ordered as previously published [18, 19]. All primers designed within imprinted genes encompassed at least one intron, with the exception of Ndn and Xist whose genes do not contain introns within the coding region. These primers were initially tested on bovine fibroblast or trophectoderm cell cultures. Amplified products were subcloned into pGEM T Easy Vectors (Promega, Annandale, NSW, Australia) and sequenced for confirmation of primer specificity. All bovine sequences showed significant homology to human and mouse sequences (>80% homology; data not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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transcripts were detected in one of the three morula-staged embryos, and then in three of three blastocysts and three of three hatched blastocysts (Fig. 1).
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Dilutions of the amplified cDNA were then tested for the eight genes of interest, both the paternally imprinted (maternally expressed) genes and the maternally imprinted (paternally expressed) genes. The paternally imprinted genes included Gnas, Grb10, and Xist. The maternally imprinted genes included Dlk1, Mest, Ndn, Nnat, and Sgce. Of the eight putatively imprinted genes examined, Dlk1 and Mest (isoform 1) were not detected following 35 cycles of PCR amplification in the MII oocytes or any of the preimplantation embryo stages (Fig. 2). The remaining genes showed a variety of mRNA expression patterns (Fig. 2). Gnas transcripts were found in 3 of the 15 samples between the oocyte and 16–32-cell stage, and then in all samples starting from the morula stage. Grb10 transcripts were expressed in most samples from the 2-cell stage onwards. Mest (isoform 2) transcripts were detected in all but one blastocyst sample. Ndn mRNA was expressed in two of three 8-cell embryos and in all samples starting from the 16–32-cell stage. Nnat transcripts were present in the oocyte and off by the 8-cell stage, then reappeared at the blastocyst stage, with stronger expression in Day 8 than in Day 7 blastocysts. Interestingly, the ß isoform of Nnat was the predominant form in early embryos, and the isoform was stronger in the blastocysts. Sgce transcripts were present in most samples from the oocyte to the 16–32-cell stage of development. Finally, Xist transcripts were detected in two of three 16–32-cell stage embryos, all morulae, and two of six blastocysts.
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To determine whether the genes studied were imprinted at the bovine blastocyst stage of development, parthenogenetic blastocyst samples were also created. In this experiment, Day 7 IVF embryos (n = 3) were compared to Day 7 PA embryos (n = 3; Fig. 3). Dlk1, Mest (isoform 1), and Sgce were not detected in either IVF or PA blastocyst samples. Of the remaining genes tested, Gnas and Xist are maternally expressed and should be found in both IVF and PA blastocysts. Xist is found in only one of the blastocysts, suggesting that only one blastocyst is female. Mest, Nnat, and Ndn, if imprinted at the blastocyst stage, should be expressed only in IVF blastocysts. Nnat mRNA was expressed as predicted in three of three IVF blastocysts and zero of three PA blastocysts. Mest (isoform 2) and Ndn mRNA were expressed in all but one sample of the IVF and PA blastocysts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Three paternally imprinted genes, Gnas, Grb10, and Xist, were investigated. Gnas and Grb10 transcripts were found in most samples, as would be expected for fairly ubiquitously expressed genes. There was also mRNA expression in both the IVF and PA blastocysts as expected for maternally expressed genes, although only one PA blastocyst contained Grb10 transcripts. Xist transcripts were first detected at the 16–32-cell stage. Transcripts were then detected in all morulae and in two of six blastocysts. Our results therefore confirm previous reports in which Xist mRNA was expressed in bovine male and female morulae and in female blastocysts [12, 25].
The remaining five genes were maternally imprinted. Of these, Dlk1 mRNA expression was not detected in any of the oocyte or embryo samples. Mest has two isoforms, isoform 1 being imprinted and isoform 2 not imprinted. Human embryos have been tested, with expression being seen at various stages of development, but isoform 1 is not distinguished from isoform 2 [13]. In our bovine samples, the imprinted isoform 1 was not expressed in any of the stages tested and isoform 2 was found in all but one of the samples analyzed, showing a strikingly different mRNA expression pattern between the two isoforms. Ndn encodes the protein necdin, which in the mouse is expressed only from differentiated neurons in the brain, and in the human is expressed in many tissues with highest levels in the brain and placenta [8]. Expression was also found in one of four morulae and one of four human blastocysts tested [13]. We report that Ndn mRNA is expressed following the maternal-to-zygotic transition (MZT) during bovine preimplantation development (8–16-cell onwards). The methylation status of Ndn has also been reported in mouse oocytes and embryos, with maternal methylation persisting until the morula stage but being lost in the blastocyst stage [26]. This methylation data may help explain the biallelic expression pattern seen in our bovine blastocysts. If the methylation mark is also present until the morula stage in the bovine embryo before being erased, then maternal transcripts may still be present at the blastocyst stage of development.
Nnat mRNA is expressed in the oocyte, degraded, and then reappears at the blastocyst stage. This later expression in the blastocyst is seen only in IVF and not in PA blastocysts, suggesting that Nnat is imprinted by this stage of development. This is the first report of the imprinted behavior of bovine Nnat. It is also interesting to note that there appears to be differential expression of the and ß isoforms at different stages of preimplantation development, with the form primarily expressed in the oocyte and the ß form being predominant in the blastocysts. The functional significance of this switch in isoform abundance will require further investigation.
Sgce, the final maternally imprinted gene, was expressed in the oocyte, with transcripts detected until the 16–32-cell stage (MZT). Sgce was not found in either IVF or PA blastocysts, and therefore imprinting status of this gene could not be inferred for this gene. Sgce is widely expressed in the mouse, including expression in the embryonic Day 14 (E14) mouse embryo. It is unclear whether Sgce, which encodes the epsilon-sarcoglycan protein, has a role in oocyte development, fertilization, or the early cleavage stages of development. Mutations in this gene are known to cause myoclonus-dystonia syndrome, causing myoclonic jerking of the arms and axial muscles as well as psychiatric abnormalities in many patients [27, 28].
Of the eight putatively imprinted genes investigated during bovine preimplantation development, seven showed mRNA expression, confirming the belief that imprinted genes are often crucial for fetal and placental growth. Of the seven genes expressed, Sgce appeared to be expressed early and then shut off, indicating a potential role in the oocyte, fertilization, or initial cleavage events. Ndn and Xist transcripts were detected following the MZT, and Gnas, Grb10, and Mest (isoform 2) appear to be on at all stages. Nnat, the only gene that showed allelic expression differences, appears to be turned on at the blastocyst stage, but was also present in some oocyte and embryo samples up to the 8-cell stage. We can now report that Nnat is imprinted in cattle as well as in humans and mice. The fact that Nnat is imprinted at the blastocyst stage and Ndn is not shows that imprinting is not fully established by the blastocyst stage of development in cattle. Further studies are needed to definitively determine the imprinting status of the other genes and to determine the timing of imprint establishment during early development. Also of interest is the importance of these genes in the growth and development of the fetus and placenta following nuclear transfer. Nuclear transfer is known to produce embryos that display gene expression patterns unlike those of IVF embryos, which give over a 60–70% survival to term [29, 30]. Future studies will analyze the differences in imprinted gene expression patterns between IVF and NT embryos during embryonic development in order to understand the reasons for the high embryo/fetus mortality and abnormality rates following nuclear transfer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Nancy T. Ruddock, Centre for Early Human Development, Level 3, Monash Institute of Reproduction and Development, 27-31 Wright Street, Clayton, VIC, 3168, Australia. FAX: 61 3 9594 7311; nancy.ruddock{at}med.monash.edu.au
Received: 12 August 2003.
First decision: 27 August 2003.
Accepted: 1 December 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-sarcoglycan are maternally imprinted genes: identification by a subtractive screen of novel uniparental fibroblast lines. Mol Cell Biol 2000 20:93308-3315-Sarcoglycan, a broadly expressed homologue of the gene mutated in limb-girdle muscular dystrophy 2D. J Biol Chem 1997 272:5132534-32538-sarcoglycan cause myoclonus-dystonia syndrome. Nat Genetics 2001 29:66-69[CrossRef][Medline]This article has been cited by other articles:
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  A. Thurston, J. Taylor, J. Gardner, K. D Sinclair, and L. E Young Monoallelic expression of nine imprinted genes in the sheep embryo occurs after the blastocyst stage Reproduction, January 1, 2008; 135(1): 29 - 40. [Abstract] [Full Text] [PDF]
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 I-M. Siu, R. Bai, G. L. Gallia, J. B. Edwards, B. M. Tyler, C. G. Eberhart, and G. J. Riggins Coexpression of neuronatin splice forms promotes medulloblastoma growth Neuro-oncol, January 1, 2008; 10(5): 716 - 724. [Abstract] [Full Text] [PDF]
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