Unregulated Expression of the Imprinted Genes H19 and Igf2r in Mouse Uniparental Fetuses (2002)

The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms. The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms. Imprinted genes are genes that are expressed solely from one parental allele and are reciprocally suppressed in the other parental allele. The parental specific expression of the genes is governed by epigenetic genome modification (genomic imprinting), which is thought to involve the methylation of cytosine in CpG dinucleotides (1.Sasaki H. Jones P.A. Chaillet J.R. Ferguson-Smith A.C. Barton S.C. Reik W. Surani M.A. Genes Dev. 1992; 6: 1843-1856Crossref PubMed Scopus (225) Google Scholar, 2.Ferguson-Smith A.C. Sasaki H. Cattanach B.M. Surani M.A. Nature. 1993; 362: 751-755Crossref PubMed Scopus (354) Google Scholar, 3.Bartolomei M.S. Webber A.L. Brunkow M.E. Tilghman S.M. Genes Dev. 1993; 7: 1663-1673Crossref PubMed Scopus (415) Google Scholar, 4.Stoger R. Kubiccka P. Liu C.G. Kafri T. Razin A. Cedar H. Barlow D.P. Cell. 1993; 73: 61-71Abstract Full Text PDF PubMed Scopus (501) Google Scholar, 5.Razin A. Cedar H. Cell. 1994; 77: 473-476Abstract Full Text PDF PubMed Scopus (319) Google Scholar), as well as by conformational change of chromatin structure (2.Ferguson-Smith A.C. Sasaki H. Cattanach B.M. Surani M.A. Nature. 1993; 362: 751-755Crossref PubMed Scopus (354) Google Scholar, 6.Feil R. Kelsey G. Am. J. Hum. Genet. 1997; 61: 1213-1219Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and a time lag in DNA replication (6.Feil R. Kelsey G. Am. J. Hum. Genet. 1997; 61: 1213-1219Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). However, the precise mechanism of the parental specific expression remains unknown.It is generally known that parental allele-specific expressions of maternally and paternally expressed imprinted genes are maintained in mouse parthenogenetic/gynogenetic and androgenetic embryos (7.Walsh C. Glaser A. Fundele R. Ferguson-Smith A. Barton S. Surani M.A. Ohlsson R. Mech. Dev. 1994; 46: 55-62Crossref PubMed Scopus (55) Google Scholar). Because these uniparental embryos are constructed from only the maternal or paternal genome, they are often used in the study of imprinting mechanisms as models of the parental expression of imprinted genes. Recently, an attempt was made to identify novel imprinted genes by estimating differences in imprinted gene expression between parthenogenetic and androgenetic embryos using the subtraction method (8.Kaneko-Ishino T. Kuroiwa Y. Miyoshi N. Kohda T. Suzuki R. Yokoyama M. Viville S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1995; 11: 52-59Crossref PubMed Scopus (253) Google Scholar, 9.Miyoshi N. Kuroiwa Y. Kohda T. Shitara H. Yonekawa H. Kawabe T. Hasegawa H. Barton S.C. Surani M.A. Kaneko-Ishino T. Ishino F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1102-1107Crossref PubMed Scopus (136) Google Scholar) and fluorescent differential display screening (10.Hatada I. Morita S. Obata Y. Sotomaru Y. Shimoda M. Kono T. J. Biochem. (Tokyo). 2001; 130: 187-190Crossref PubMed Scopus (61) Google Scholar). However, no study has examined in detail whether or not parental specific expressions of imprinted genes are always maintained in uniparental embryos.In the present study, to confirm the parental specific expression of imprinted genes in uniparental embryos, we screened the expression of the maternally expressed imprinted genes H19 (11.Bartolomei M.S. Zemel S. Tilghman S.M. Nature. 1991; 351: 153-155Crossref PubMed Scopus (954) Google Scholar), Igf2r (12.Barlow D.P. Stoger R. Herrmann B.G. Saito K. Schweifer N. Nature. 1991; 349: 84-87Crossref PubMed Scopus (709) Google Scholar), and p57KIP2 (13.Hatada I. Mukai T. Nat. Genet. 1995; 11: 204-206Crossref PubMed Scopus (262) Google Scholar) and the paternally expressed imprinted genes Snrpn (14.Cattanach B.M. Barr J.A. Evans E.P. Burtenshaw M. Beechey C.V. Leff S.E. Brannan C.I. Copeland N.G. Jenkins N.A. Jones J. Nat. Genet. 1992; 2: 270-274Crossref PubMed Scopus (156) Google Scholar, 15.Leff S.E. Brannan C.I. Reed M.L. Ozcelik T. Francke U. Copeland N.G. Jenkins N.A. Nat. Genet. 1992; 2: 259-264Crossref PubMed Scopus (219) Google Scholar), Ndn (16.Yang T. Adamson T.E. Resnick J.L. Leff S. Wevrick R. Francke U. Jenkins N.A. Copeland N.G. Brannan C.I. Nat. Genet. 1998; 19: 25-31Crossref PubMed Scopus (259) Google Scholar), Zfp127 (16.Yang T. Adamson T.E. Resnick J.L. Leff S. Wevrick R. Francke U. Jenkins N.A. Copeland N.G. Brannan C.I. Nat. Genet. 1998; 19: 25-31Crossref PubMed Scopus (259) Google Scholar), Peg1/Mest (8.Kaneko-Ishino T. Kuroiwa Y. Miyoshi N. Kohda T. Suzuki R. Yokoyama M. Viville S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1995; 11: 52-59Crossref PubMed Scopus (253) Google Scholar,17.Sado T. Nakajima N. Tada M. Takagi N. Dev. Growth Differ. 1993; 35: 551-560Crossref Scopus (44) Google Scholar), and Peg3 (18.Kuroiwa Y. Kaneko-Ishino T. Kagitani F. Kohda T. Li L.L. Tada M. Suzuki R. Yokoyama M. Shiroishi T. Wakana S. Barton S.C. Ishino F. Surani M.A. Nat. Genet. 1996; 12: 186-190Crossref PubMed Scopus (214) Google Scholar) by semi-quantitative RT-PCR. 1The abbreviations used are: RTreverse transcriptionIVFin vitro fertilizationRFLPrestriction enzyme (BglI) fragment length polymorphismLPfull-length polymorphismDMRdifferentially methylated region 1The abbreviations used are: RTreverse transcriptionIVFin vitro fertilizationRFLPrestriction enzyme (BglI) fragment length polymorphismLPfull-length polymorphismDMRdifferentially methylated region The results showed that the H19 and Igf2r genes were irregularly expressed in paternal alleles of androgenetic embryos at the respective levels of 12 and 40% of the control biparental fetuses, although androgenetic fetuses have only the paternal alleles of the H19 and Igf2r genes in which the alleles are naturally silenced in biparental embryos. In addition, we performed a quantitative analysis of the expressions of these genes in individual uniparental fetuses using real time quantitative RT-PCR (19.Gibson U. Heid C. Williams P. Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1769) Google Scholar, 20.Wittwer C. Ririe K. Andrew R. David D. Gundry R. Balis U. BioTechniques. 1997; 22: 176-181Crossref PubMed Scopus (820) Google Scholar) and attempted to clarify the gene regulation mechanism by methylation analysis of the gene regulatory regions.MATERIALS AND METHODSProduction of Fetuses at Day 9.5 of GestationB6CBF1 (C57BL/6N × CBA) mice were used as oocyte donors to produce embryos. They were superovulated with injections of 5 IU of equine chorionic gonadotropin (Peamex, Sankyo, Ltd., Tokyo, Japan) and 5 IU of human chorionic gonadotropin (Puberogen, Sankyo, Ltd.) given 48 h apart. Oocytes at metaphase II were released from the oviducts 13–16 h after the human chorionic gonadotropin injection, and the cumulus cells were removed by digestion with 300 units/ml hyaluronidase in M2 medium (21.Quinn P. Barros C. Whittingham D. J. Reprod. Fertil. 1982; 66: 161-168Crossref PubMed Google Scholar). To produce diploid parthenogenetic embryos by artificial activation, oocytes were cultured for 2 h in a drop of Ca2+-free M16 medium (22.Whittingham D. J. Reprod. Fertil. 1971; 14: 7-12Google Scholar) containing 10 mmSrCl2 (Sr2+) (23.Bos-Mikich A. Swann K. Whittingham D.G. Mol. Reprod. Dev. 1995; 41: 84-90Crossref PubMed Scopus (133) Google Scholar). The oocytes were then cultured in M16 medium supplemented with 0.4% bovine serum albumin for 4 days in an atmosphere of 5% CO2, 5% C2, and 90% N2 at 37 °C. During the culture in the Sr2+ medium and following the first 4 h of culture in the M16 medium, 5 μg/ml cytochalasin B was added to the medium to induce diploidy by inhibiting a second polar body extrusion. Androgenetic embryos were produced by in vitro fertilization (IVF) of enucleated oocytes according to the procedure previously reported (24.Kono T. Sotomaru Y. Sato Y. Nakahara T. Mol. Reprod. Dev. 1993; 34: 43-46Crossref PubMed Scopus (47) Google Scholar). When required, pronuclear transfer was performed to produce diploid androgenones. Control biparental embryos were produced by IVF.To identify the expressed alleles, biparental and androgenetic embryos were constructed using mice that had a polymorphism in their genomic DNA sequences. For the H19 gene, B6.Spretus-H19 congenic mice (established in our laboratory) having a restriction enzyme (BglI) fragment length polymorphism (RFLP) were used. For the Igf2r gene, JF1 mice having the full-length polymorphism (LP) were used.H19 gene knock-out (H19-KO) mice (25.Ripoche M.A. Kress C. Poirier F. Dandolo L. Genes Dev. 1997; 11: 1596-1604Crossref PubMed Scopus (205) Google Scholar), in which the 3-kb transcription unit of the H19 gene was deleted, were used to identify the expression leakage of the H19 gene from the paternally suppressed allele in the absence of the maternally expressed allele. Fertilized embryos were constructed from H19-KO mice oocytes and B6CBF1 mice sperm by IVF.The obtained blastocysts were transferred to the uterine horns of CD-1 females on day 3 of pseudopregnancy (2.5 days postcoitum), and the fetuses were recovered at day 9.5 of gestation. For the following procedures, fetuses that had more than 15 somites and possessed a heartbeat were selected and introduced to RNA extraction.Analysis of Gene Expression (RT-PCR)Total RNA was isolated from the parthenogenetic, androgenetic, and control biparental fetuses, one by one, at day 9.5 of gestation using an SV total RNA isolation kit (Promega Co., Ltd., Madison, WI). After quantitative measurement of the total RNA extracted from each embryo using a GeneQuant Pro spectrum analyzer (Amersham Biosciences, Inc.), the fetuses from which more than 1 μg of RNA was extracted were subjected to cDNA synthesis. First strand cDNA was synthesized from 1 μg of the total RNA from each embryo using a Superscript reverse transcriptase II kit (Invitrogen) according to the manufacturer's instructions. To determine the biological activity in each sample, the expression of the β-actin gene (26.Tokunaga K. Taniguchi H. Yoda K. Shimizu M. Sakiyama S. Nucleic Acids Res. 1986; 14: 2829Crossref PubMed Scopus (535) Google Scholar) was first analyzed before the analysis of the imprinted gene expression. For semi-quantitative analysis of imprinted gene expression, the cDNA was diluted to concentrations of 1/1, 1/10, 1/100, and 1/1000 before being subjected to PCR reaction. PCR was carried out in a 50-μl reaction buffer containing 1.25 units of Taq DNA polymerase (Takara Shuzo Co., Ltd., Kyoto, Japan) in a GeneAmp PCR system (PerkinElmer Life Sciences). The following primer sets were used: 5′-TGTAAACCTCTTTGGCAATGCTGCC-3′ and 5′-TATTGATGGACCCAGGACCTCTGGT-3′ for the H19 gene; 5′-GGGTACTTTGCTTTTGGGTA-3′ and 5′-TTCGACCTATAAGAAGCCTT-3′ for Igf2r; 5′-GCCGGGTGATGAGCTGGGAA-3′ and 5′-AGAGAGGCTGGTCCTTCAGC-3′ for p57KIP2; 5′-ATACTGGCATTGCTCGTGTG-3′ and 5′-TGGAGGAGGCATGCCTATAC-3′ for Snrpn; 5′-GTTCTTCCTTCTCTGATGAC-3′ and 5′-CACAAGTTAACAAGTGCAC-3′ for Znf127; 5′-ATTCGCAACAATGACGGC-3′ and 5′-TGAGGTGGACTATTGTGTCACC-3′ for Peg1/Mest; 5′-TAGTCCTGTGAAGGTGTGGG-3′ and 5′-GTAGGGAATGGGTTGATTTGG-3′ for Peg3; and 5′-AGCGGCCTCCTTACCCAACTTCAG-3′ and 5′-AAGGCCTGCTGAAGTAGAAGCCG-3′ for Igf2. For the RFLP analysis in H19, this specific primer set was used: 5′-TGCTCCAAGGTGAAGCTGAAAG-3′ and 5′-GTAGGGCATGTTGAACACTTTATG-3′.Quantitative analysis of gene expression was performed by means of real time quantitative PCR (19.Gibson U. Heid C. Williams P. Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1769) Google Scholar, 20.Wittwer C. Ririe K. Andrew R. David D. Gundry R. Balis U. BioTechniques. 1997; 22: 176-181Crossref PubMed Scopus (820) Google Scholar). After adding a fluorescent reagent, SYBER Green I, to the PCR reaction, the quantity of PCR products was detected by monitoring the luminous intensity with a LightCycler (Roche Diagnostics). From the resulting indexes, the initial quantity of the cDNA/RNA of interest was estimated. The primers used were 5′-CATGTCTGGGCCTTTGAA-3′ and 5′-TTGGCTCCAGGATGATGT-3′ for the H19 gene, 5′-CGGGCGTGTCCTACAAGTA-3′ and 5′-CGGCCTGAGTGAACTTTCAC-3′ for the Igf2r gene, and 5′-GTGGATTCAGGTTTCATG-3′ and 5′-GGCCCAGATATAGAATGT-3′ for the antisense Igf2r RNA (Air). As control samples, the fetuses from fertilized embryos were analyzed as well.Analysis of Methylation in the H19 and Igf2r GenesIn the CpG island sequences of the H19 and Igf2r (regions II) genes of androgenetic, parthenogenetic (only for the Igf2r gene), and normal fetuses, the methylation pattern was analyzed by Southern hybridization using genome DNAs that had been digested by methyl-sensitive restriction enzymes. To obtain DNA fragments containing the CpG islands, extracted genome DNAs were digested by BamHI for the promoter region of the H19 gene and by PvuII for region II of the Igf2r gene. The digested DNAs were then digested by HpaII and applied to electrophoresis (1.2% agarose). The DNAs were transferred to nylon membrane (Hybond-N+; Amersham Biosciences, Inc.) and then subjected to Southern hybridization. Hybridization was performed for 15 h at 56 °C using AlkPhos DIRECT labeling reagent (RPN3680; Amersham Biosciences, Inc.). The probes used were a 2.6-kilobase pair BamHI fragment for the H19 gene (27.Sasaki H. Allen N.D. Surani M.A. EXS. 1993; 64: 469-486PubMed Google Scholar) and PCR products obtained using the primer set 5′-AACCCTCGGAACCCTGCCCTT-3′ and 5′-GAGGGTAGGATTCCGTTGCAAGG-3′ for region II of the Igf2r gene; all probes were alkaline phosphatase-labeled by the reagent. The hybridized membrane was treated with CDP-Star (RPN3682; Amersham Biosciences, Inc.), and the intensity of the band was measured by an LAS1000 image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).RESULTSTo confirm that the parental expressions of imprinted genes are maintained in uniparental embryos, we first examined the expression of three maternally expressed genes, H19, Igf2r, and p57KIP2, and five paternally expressed genes, Snrpn, Zfp127, Peg1/Mest, Peg3, and Igf2, in each individual parthenogenetic and androgenetic fetus at day 9.5 of gestation by semi-quantitative RT-PCR (Fig. 1). The Igf2r and H19 genes were unexpectedly found to be expressed in androgenetic fetuses. The expression levels of these genes were roughly 10–50% of those in the control fetuses. The other genes maintained their parental expression levels in each of the uniparental fetuses.Quantities of the unregulated expression of the H19 and Igf2r genes in androgenetic fetuses were further analyzed by real time quantitative RT-PCR. The results showed that androgenetic embryos expressed the H19 gene at a mean of 12% (ranging from 0.2 to 55%, n = 12) and the Igf2r gene at a mean of 40% (ranging from 3 to 84%, n = 12) of the level in the control fetuses (n = 12) (Fig. 2, a and b). The expression alleles were accessed using androgenetic and biparental fetuses constructed from B6CBF1 and congenic (for the H19 gene)/JF1 (for the Igf2r gene) mice genomes, which contain polymorphic sequences in the exon region between the strains (Fig. 3, a and b). The results showed that both genes were expressed from both alleles in the androgenetic fetuses. It was also confirmed that both genes were not expressed from the maternal allele in the biparental fetuses. Moreover, to access the leakage of expression from the paternal allele in the biparental fetuses, the quantity of H19 gene expression in H19-KO × B6CBF1 fetuses was analyzed by real time quantitative PCR. The results showed that the paternal allele of the H19-KO × B6CBF1 fetuses (n = 11) greatly suppressed the H19 gene (0.1% of B6CBF1 ×H19-KO fetuses (n = 11), Fig. 3c). These observations suggest that the regulation mechanism for parental specific suppression of the H19 and Igf2r genes should be relaxed in androgenetic fetuses.Figure 2Quantitative analysis of the H19(a) and Igf2r (b) gene expressions in control biparental (C), androgenetic (A), and parthenogenetic (P) fetuses by real time RT-PCR. Twelve samples were examined for each of these three groups. The expression level of each fetus is indicated with a dot. The average of the expression levels in the control fetuses was taken as a standard (100%). Shaded boxes indicate the average expression level in each group (H19 and Igf2r genes: 12 and 40%, respectively, in androgenetic fetuses, and 482 and 1259%, respectively, in parthenogenetic fetuses).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Analysis of allele-specific expression of the H19 and Igf2r genes in the biparental and androgenetic fetuses at day 9.5 of gestation. a, RFLP analysis of the H19 gene expression using B6CBF1 and B6.Spretus-H19 congenic (Cong.) mice. In the B6CBF1 × congenic and congenic × B6CBF1 biparental fetuses, expressions were theoretically detected only in the maternal B6CBF1 (521 bp) and congenic (384 bp) mouse alleles, respectively. In the androgenetic fetuses (A: B6CBF1 +Cong.), gene expressions were detected in both alleles. b, LP analysis of the Igf2r gene expression using B6CBF1 and JF1 mice. In the B6CBF1 × JF1 and JF1 × B6CBF1 fetuses, expressions were theoretically detected only in the maternal B6CBF1 (240 bp) and JF1 (220 bp) mouse alleles, respectively. In the androgenetic fetuses (A: B6CBF1 + JF1), gene expressions were detected in both alleles. c, quantitative analysis of H19 gene expression by real time RT-PCR in biparental fetuses of which the male and female alleles were derived from B6CBF1 and H19-deficient mice (F1 × KO) and from H19-deficient and B6CBF1 mice (KO × F1). Eleven samples were examined for each of the two groups. The expression level of each fetus is indicated with a dot. The average of the expression levels in the F1 × KO fetuses was taken as a standard (100%). Shaded boxes indicate the average expression level in each group (KO × F1 fetuses: 0.1%).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The quantitative analysis in parthenogenetic fetuses also showed unregulated expression of the H19 and Igf2r genes. The quantity of expression of the maternally expressed imprinted genes in parthenogenetic fetuses was thought to be roughly twice that of biparental fetuses, because two maternal alleles exist in the parthenogenetic fetuses. However, the parthenogenetic fetuses expressed the H19 gene at a mean of 482% (ranging from 317 to 742%, n = 12) and the Igf2r gene at a mean of 1259% (ranging from 347 to 2692%, n = 12), of the level in control fetuses (n = 12) (Fig. 2, a and b). This suggests that the expression of the H19 and Igf2r genes was not controlled by the dose regulation mechanism.Generally, parental expression of imprinting genes is closely related to the methylation level of the CpG islands (1.Sasaki H. Jones P.A. Chaillet J.R. Ferguson-Smith A.C. Barton S.C. Reik W. Surani M.A. Genes Dev. 1992; 6: 1843-1856Crossref PubMed Scopus (225) Google Scholar, 2.Ferguson-Smith A.C. Sasaki H. Cattanach B.M. Surani M.A. Nature. 1993; 362: 751-755Crossref PubMed Scopus (354) Google Scholar, 3.Bartolomei M.S. Webber A.L. Brunkow M.E. Tilghman S.M. Genes Dev. 1993; 7: 1663-1673Crossref PubMed Scopus (415) Google Scholar, 4.Stoger R. Kubiccka P. Liu C.G. Kafri T. Razin A. Cedar H. Barlow D.P. Cell. 1993; 73: 61-71Abstract Full Text PDF PubMed Scopus (501) Google Scholar). The H19 gene on the paternal genome is silenced whereas the regulatory region of the paternal allele in the CpG island is hypermethylated (28.Sasaki H. Ferguson-Smith A.C. Shum A.S. Barton S.C. Surani M.A. Development. 1995; 121: 4195-4202PubMed Google Scholar). Conversely, the Igf2r gene on the maternal allele is activated when the region of the maternal allele is hypermethylated (4.Stoger R. Kubiccka P. Liu C.G. Kafri T. Razin A. Cedar H. Barlow D.P. Cell. 1993; 73: 61-71Abstract Full Text PDF PubMed Scopus (501) Google Scholar,29.Wutz A. Smrzka O. Schweifer N. Schellander K. Wagner E.F. Barlow D.P. Nature. 1997; 389: 745-749Crossref PubMed Scopus (478) Google Scholar). To investigate whether this phenomenon is seen in the gene expressions in androgenetic fetuses, the methylation levels in the promoter region of the H19 gene and the CpG island of the Igf2r gene were analyzed by Southern analysis (Fig. a and using a The results that the HpaII in the of the H19 gene were in the androgenetic fetuses, that the unregulated expression is by this methylation (Fig. the other all of the HpaII in the Igf2r gene were in the androgenetic fetuses expression of the gene (Fig. This suggests that other regulation be in the of the regulated expression of the Igf2r gene in androgenetic fetuses. The HpaII in the H19 and Igf2r genes were in in the biparental fetuses, and those in the Igf2r gene were hypermethylated in parthenogenetic fetuses, the of maternal analysis of the DNA methylation of the H19 and Igf2r genes in control biparental (C), parthenogenetic and androgenetic fetuses at day 9.5 of gestation. a, methylation of the H19 gene. In the control the sequences in the CpG island bp) were digested fragments by HpaII in with the gene expression. In the androgenetic fetuses, of the sequences were digested by HpaII which showed the methylation related to the gene expression. b, methylation of the Igf2r gene. After the by of the sequences in region 2 bp) were digested and fragments in the control fetuses, and no was digested in the parthenogenetic these to the gene expression In the sequences in the androgenetic fetuses were digested by that of the gene Large Image Figure ViewerDownload Hi-res image Download further the unregulated expression of the Igf2r gene, the expression of antisense Igf2r RNA (Air) A. Smrzka O. Schweifer N. Schellander K. Wagner E.F. Barlow D.P. Nature. 1997; 389: 745-749Crossref PubMed Scopus (478) Google Scholar), which the expression of the Igf2r gene, was The results showed that expression of the was also unregulated in the androgenetic and parthenogenetic fetuses. The mean expression of the was (ranging from to n = and (ranging from to n = of that in the control fetuses (n = in the androgenetic and parthenogenetic fetuses, respectively (Fig. This suggests that parental specific regulation in this region is in the uniparental fetuses.Figure analysis of the expressions in the control biparental (C), androgenetic (A), and parthenogenetic (P) fetuses by real time RT-PCR. samples were examined for each of these three groups. The expression level of each fetus is indicated with a dot. The average of the expression levels in the control fetuses was taken as a standard (100%). Shaded boxes indicate the average expression level in each group in androgenetic fetuses and in parthenogenetic fetuses).View Large Image Figure ViewerDownload Hi-res image Download that the parental specific expression of imprinted genes is maintained in uniparental fetuses, is from the of expressed alleles that the expression in parthenogenetic and androgenetic fetuses be twice that in the biparental In the present study, the maternally expressed genes H19 and Igf2r were unexpectedly expressed in androgenetic fetuses and in parthenogenetic fetuses. Recently, we also the unregulated expression of the paternally expressed gene in maternal alleles in parthenogenetic fetuses Y. Y. T. Obata Y. Suzuki H. I. I. Kono T. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). These indicated that imprinted genes are to their imprinted expression in mouse uniparental previously reported that the expression of the paternally expressed gene in androgenetic fetuses was to the Y. Y. I. Kono T. J. Reprod. Dev. 2001; Scopus Google Scholar). in the present study the expressions of the H19 and Igf2r genes in parthenogenetic fetuses were more than two and the respectively, and the expressions were from that of the gene in androgenetic fetuses. This shows that expression from maternal alleles of the H19 and Igf2r genes in parthenogenetic fetuses are not regulated those in biparental fetuses. The of gene regulation were to from the of the paternal genome in parthenogenetic fetuses. It has been reported that fetuses maternal for the H19 gene at twice the dose of normal fetuses, that the dose maternal allele is maintained in the absence of a paternal allele and of the DNA of the H19 gene (2.Ferguson-Smith A.C. Sasaki H. Cattanach B.M. Surani M.A. Nature. 1993; 362: 751-755Crossref PubMed Scopus (354) Google Scholar). This suggests that the expression of the H19 gene in parthenogenetic fetuses is by an in the regulation of a gene of the region of in or the other study confirmed that biparental fetuses maternally specific expressions of the H19 and Igf2r genes, whereas the androgenetic fetuses unexpectedly expressed the H19 and Igf2r genes at 12 and 40%, respectively, of the levels in biparental fetuses in which the unregulated expressions from both paternal alleles. a RT-PCR to Sasaki (1.Sasaki H. Jones P.A. Chaillet J.R. Ferguson-Smith A.C. Barton S.C. Reik W. Surani M.A. Genes Dev. 1992; 6: 1843-1856Crossref PubMed Scopus (225) Google Scholar) previously reported that the expression of the paternally expressed gene in parthenogenetic fetuses was of the level in They that this expression leakage from two silenced maternal alleles in diploid parthenogenetic fetuses and was twice that of the maternal allele in the biparental fetuses. The expression of the H19 and Igf2r genes in androgenetic fetuses at a level from the leakage expression seen in the gene. This suggests that be a regulatory which is with the