Age-related changes in the localization of DNA methyltransferases during meiotic maturation in mouse oocytes
2010; Elsevier BV; Volume: 95; Issue: 4 Linguagem: Inglês
10.1016/j.fertnstert.2010.06.050
ISSN1556-5653
AutoresLu Zhang, Danyu Lu, Wanyun Ma, Ying Li,
Tópico(s)Genetic Syndromes and Imprinting
ResumoThe effects of maternal aging on the localization of DNA methyltransferases were evaluated during mouse oocyte maturation using fluorescence staining. And we conclude that maternal aging affects the cytoplasmic-to-nuclear trafficking of DNA methyltransferases in mouse oocytes during the time from germinal vesicle breakdown to metaphase I. The effects of maternal aging on the localization of DNA methyltransferases were evaluated during mouse oocyte maturation using fluorescence staining. And we conclude that maternal aging affects the cytoplasmic-to-nuclear trafficking of DNA methyltransferases in mouse oocytes during the time from germinal vesicle breakdown to metaphase I. Correlations between progressive decline in female fertility and age have been observed for many years (1Klein J. Sauer M.V. Assessing fertility in women of advanced reproductive age.Am J Obstet Gynecol. 2001; 185: 758-770Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). It has been well recognized that poor oocyte quality is responsible for an overall reduction in fertility and the age-related decline in female fertility (2Plachot M. Veiga A. Montagut J. de Grouchy J. Calderon G. Lepretre S. et al.Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multicentric study.Hum Reprod. 1988; 3: 627-635Crossref PubMed Scopus (197) Google Scholar). In recent years, several reports have demonstrated that DNA methylation is affected by maternal age (3Hamatani T. Falco G. Carter M.G. Akutsu H. Stagg C.A. Sharov A.A. et al.Age-associated alteration of gene expression patterns in mouse oocytes.Hum Mol Genet. 2004; 13: 2263-2278Crossref PubMed Scopus (397) Google Scholar, 4Pan H. Ma P. Zhu W. Schultz R.M. Age-associated increase in aneuploidy and changes in gene expression in mouse eggs.Dev Biol. 2008; 316: 397-407Crossref PubMed Scopus (210) Google Scholar, 5Lopes F.L. Fortier A.L. Darricarrère N. Chan D. Arnold D.R. Trasler J.M. Reproductive and epigenetic outcomes associated with aging mouse oocytes.Hum Mol Genet. 2009; 18: 2032-2044Crossref PubMed Scopus (55) Google Scholar). DNA methylation, catalyzed by DNA methyltransferases (Dnmts) that can be divided into de novo and maintenance methyltransferases, is essential for chromatin remodeling, genomic imprinting, and X chromosome inactivation (6Margot J.B. Ehrenhofer-Murray A.E. Leonhardt H. Interactions within the mammalian DNA methyltransferase family.J Mol Biol. 2003; 4: 7Google Scholar, 7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar).The mammalian Dnmt family has five members: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L (6Margot J.B. Ehrenhofer-Murray A.E. Leonhardt H. Interactions within the mammalian DNA methyltransferase family.J Mol Biol. 2003; 4: 7Google Scholar, 7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar). It has been shown that Dnmt enzymes other than Dnmt2 contribute to the methylation pattern acquisition during gametogenesis and embryogenesis (7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar, 8Okano M. Bell D.W. Haber D.A. Li E.n. DNA Methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4413) Google Scholar, 9Lucifero D. La Salle S. Bourc'his D. Martel J. Bestor T.H. Trasler J.M. Coordinate regulation of DNA methyltransferase expression during oogenesis.BMC Dev Biol. 2007; 7: 36Crossref PubMed Scopus (92) Google Scholar, 10Ratnam S. Mertineit C. Ding F. Howell C.Y. Clarke H.J. Bestor T.H. et al.Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development.Dev Biol. 2002; 245: 304-314Crossref PubMed Scopus (166) Google Scholar). Dnmt1 is considered the major maintenance enzyme during replication. Dnmt3a, Dnmt3b, and Dnmt3L are required for de novo methylation. Several reports have provided that a variant Dnmt1 protein called Dnmt1o is found in the cytoplasm of metaphase II oocytes (11Howell C.Y. Bestor T.H. Ding F. Latham K.E. Mertineit C. Trasler J.M. et al.Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene.Cell. 2001; 104: 829-838Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 12Cardoso M.C. Leonhardt H. DNA methyltransferase is actively retained in the cytoplasm during early development.J Cell Biol. 1999; 147: 25-32Crossref PubMed Scopus (131) Google Scholar), and the transcription profile of Dnmt3 is expressed in growing oocytes (13Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Dnmt3L and the establishment of maternal genomic imprints.Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar, 14Hata K. Okano M. Lei H. Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar). Previous studies of Dnmt focused primarily on the gene expression that takes place during oogenesis (3Hamatani T. Falco G. Carter M.G. Akutsu H. Stagg C.A. Sharov A.A. et al.Age-associated alteration of gene expression patterns in mouse oocytes.Hum Mol Genet. 2004; 13: 2263-2278Crossref PubMed Scopus (397) Google Scholar, 9Lucifero D. La Salle S. Bourc'his D. Martel J. Bestor T.H. Trasler J.M. Coordinate regulation of DNA methyltransferase expression during oogenesis.BMC Dev Biol. 2007; 7: 36Crossref PubMed Scopus (92) Google Scholar, 10Ratnam S. Mertineit C. Ding F. Howell C.Y. Clarke H.J. Bestor T.H. et al.Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development.Dev Biol. 2002; 245: 304-314Crossref PubMed Scopus (166) Google Scholar, 15Lees-Murdock D.J. Shovlin T.C. Gardiner T. De Felici M. Walsh C.P. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.Dev Dyn. 2005; 232: 992-1002Crossref PubMed Scopus (69) Google Scholar, 16La Salle S. Mertineit C. Taketo T. Moens P.B. Bestor T.H. Traslera J.M. Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells.Dev Biol. 2004; 268: 403-415Crossref PubMed Scopus (175) Google Scholar). However, the effects of maternal age on the expression of Dnmt proteins during meiosis have not been fully elucidated. The aim of the present study was to observe the age-related changes in localization of Dnmt proteins during mouse oocyte maturation.Female Kun-Ming mice (CAMS, Beijing, China) were housed and bred under standard conditions (temperature 22 ± 2°C, relative humidity 55% ± 5%, 12-hour light-dark cycle) with food and water available ad libitum according to the Chinese National Standard (GB14925-2001). All the animal experiments were approved by the Institutional Animal Welfare and Ethics Committee of Peking University (No. IRBLA2009-020).Fifty 7–8-week-old (pubertal) and fifty 40–47-week-old (aging) female mice were sacrificed by cervical dislocation, and their oocytes were isolated by puncturing the ovarian follicles with a sterile needle in human tubular fluid (Millipore, Bedford, MA). The female reproductive lifespan in Kun-Ming mice extends to 45 weeks, so this time point is a functional approximation of human perimenopause. Only the denuded oocytes displaying an intact germinal vesicle (GV) were collected for further culture. Immature oocytes were incubated in human tubular fluid medium supplemented with 10% FBS under liquid paraffin oil at 37°C in a humidified atmosphere of 5% CO2 in air. Oocytes were matured as one of six groups over the entire course of maturation: 0, 3, 5, 7, 9, and 16 hours. Oocytes after in vitro maturation (IVM) were immediately fixed in 4% paraformaldehyde for 30 min.Fixed oocytes in each experimental group were permeabilized with 0.5% Triton-X-100 for 30 minutes, followed by three washes in phosphate-buffered saline (PBS). Afterward, oocytes were washed again and blocked in 2% normal goat or rabbit serum blocking solution for 30 minutes and then incubated in a 10-μl drop of a rabbit polyclonal anti-Dnmt1, anti-Dnmt3a, anti-Dnmt3b, or a goat polyclonal anti-Dnmt3L antibody (diluted 1:50 in PBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; cat. no. sc-20701, sc-20703, sc-20704, sc-10239) overnight at 4°C, respectively. After washing, samples were immersed in a biotinylated goat anti-rabbit or rabbit anti-goat IgG (diluted 1:100 in PBS; Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes. Finally, rinsed oocytes were reacted with Quantum dot 585-streptavidin conjugate (diluted 1:50 in PBS; Invitrogen, Carlsbad, CA; cat. no. Q10111MP) for 1 hour. Nuclei were counterstained with 5 μg/ml Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) for 15 minutes. For negative controls, primary antibody was omitted.Quantum dot 585–labeled Dnmts were observed with a Bio-Rad MRC 1024MP two-photon laser scanning microscope at 780-nm excitation and 585-nm emission with 100× oil objective. Nuclei stained with Hoechst33342 were simultaneously imaged via the other channel.Differences among groups were analyzed with GLM using SAS9.2. Results were expressed as mean ± SEM. P < 0.05 was considered statistically significant. For all results, the examples shown are representative of three replications.To study the effect of maternal aging on oocyte maturation, we examined two key components of morphologic nuclear maturation: the percentage undergoing germinal vesicle breakdown (GVBD) and the first polar body (PB1) extrusion, because it is much more difficult and complicated to examine cytoplasmic maturation (17Nichols S.M. Gierbolini L. Gonzalez-Martinez J.A. Bavister B.D. Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.Fertil Steril. 2010; 93: 1597-1600Abstract Full Text Full Text PDF Scopus (17) Google Scholar). At 3 hours after maturation, the percentage of GVBD was only 45% in the aging oocytes, far lower than the 70% seen in the pubertal oocytes. After 16 hours in culture, the GVBD rate of 69% and the PB1 rate of 51% in the aging oocytes were significantly less than the 83% and the 63% seen in the pubertal oocytes, respectively (Supplemental Table 1, available online).To better depict the process of maturation, oocytes were classified as: GV-stage (0 hours), GVBD-stage (3 hours after IVM), PMI-stage (prometaphase of meiosis I, 5–7 hours after IVM), metaphase I stage (metaphase of meiosis I, 9 hours after IVM), or metaphase II stage (metaphase of meiosis II, 16 hours after IVM). (Anaphase and telophase of meiosis I are not easily discerned because of their short duration.)As shown in Figure 1, Dnmt1 was localized to the cytoplasm of aging (Fig. 1A) and pubertal (Fig. 1B) mouse oocytes during meiotic maturation. Dnmt3a, Dnmt3b, and Dnmt3L were shown surrounding the chromosomes of pubertal mouse oocytes at the GVBD and PMI stages, and were localized to the cytoplasm at other stages (Fig. 1D, 1F, 1H). However, in the aging groups, Dnmt3a and Dnmt3b were detected only in the cytoplasm (Fig. 1C, 1E). Dnmt3L was seen around the nucleus of GV oocytes, and localized to the cytoplasm at other stages (Fig. 1G).This study verified that aging influences the morphology of nuclear maturation, because nuclear maturation was significantly slower in the aging mouse oocytes (Supplemental Table 1, available online). Aging is also correlated with an increase in the aneuploidy rate of oocytes (17Nichols S.M. Gierbolini L. Gonzalez-Martinez J.A. Bavister B.D. Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.Fertil Steril. 2010; 93: 1597-1600Abstract Full Text Full Text PDF Scopus (17) Google Scholar, 18Zuccotti M. Boiani M. Garagna S. Redi C.A. Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging.Mol Reprod Dev. 1998; 50: 305-312Crossref PubMed Scopus (32) Google Scholar, 19Liu L. Keefe D.L. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice.Hum Reprod. 2002; 17: 2678-2685Crossref PubMed Google Scholar, 20Jones K.T. Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age.Hum Reprod Update. 2008; 14: 143-158Crossref PubMed Scopus (166) Google Scholar). Our study illustrates that the age of the female can perturb oocyte maturation, which is corroborated by the Jones study (20Jones K.T. Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age.Hum Reprod Update. 2008; 14: 143-158Crossref PubMed Scopus (166) Google Scholar).Based on this observation, we compared the localization of Dnmts in aging and pubertal mouse oocytes matured in vitro. First, these data provide direct evidence that aging does not alter the localization of Dnmt1 in oocytes, because Dnmt1 is cytoplasmic both in the pubertal and aging groups. Second, we suggest that the effects of aging on the localization of Dnmt3a and Dnmt3b in oocytes are similar. There is a lack of cytoplasm around chromosome-cytoplasm trafficking in the aging group, which is a notable event for Dnmt3a and Dnmt3b to perform in de novo methylation (15Lees-Murdock D.J. Shovlin T.C. Gardiner T. De Felici M. Walsh C.P. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.Dev Dyn. 2005; 232: 992-1002Crossref PubMed Scopus (69) Google Scholar). Lees-Murdock et al. (15Lees-Murdock D.J. Shovlin T.C. Gardiner T. De Felici M. Walsh C.P. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.Dev Dyn. 2005; 232: 992-1002Crossref PubMed Scopus (69) Google Scholar) proposed that Dnmts are all synthesized and stored in the cytoplasm and move to the nucleus to perform their enzymatic function, moving back out when they are no longer required. Therefore, we speculate that Dnmt3a and Dnmt3b may share common features in aging: the regulatory mechanism of nuclear trafficking is disrupted in aging mouse oocytes, which prevents Dnmt3a and Dnmt3b to come in contact with chromosomes during meiosis. This defect in regulating protein accumulation could induce multiple maternal abnormalities, such as aberrant methylation, dysregulated gene transcription and chromosome instability (7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar, 21Turek-Plewa J. Jagodzinski P.P. The role of mammalian DNA methyltransferases in the regulation of gene expression.Cell Mol Biol Lett. 2005; 10: 631-647PubMed Google Scholar, 22Pradhan S. Esteve P.O. Mammalian DNA (cytosine-5) methyltransferases and their expression.Clin Immunol. 2003; 109: 6-16Crossref PubMed Scopus (81) Google Scholar). Third, we confirm that the effect of aging on the localization of Dnmt3L in oocytes is unique, because it is the only enzyme appearing around the chromatin in aging mouse oocytes. Thus, Dnmt3L is an indispensable protein for establishing maternal genomic imprinting in aging mouse oocytes. However, the cooperative relationship to Dnmt3a and Dnmt3b has been changed in aging mouse oocytes, which implies that the regulatory mechanism of Dnmt3 family collaboration may be associated with aging in maternal imprint establishment (6Margot J.B. Ehrenhofer-Murray A.E. Leonhardt H. Interactions within the mammalian DNA methyltransferase family.J Mol Biol. 2003; 4: 7Google Scholar, 14Hata K. Okano M. Lei H. Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 23Deplus R. Brenner C. Burgers W.A. Putmans P. Kouzarides T. Launoit Y. et al.Dnmt3L is a transcriptional repressor that recruits histone deacetylase.Nucleic Acids Res. 2002; 30: 3831-3838Crossref PubMed Scopus (144) Google Scholar).Last, we hypothesize that localization of Dnmts in aging mouse oocytes during meiosis may be correlated with chromosome structure. Interestingly, all of the Dnmts interacting with chromosomes are on the exterior surface of the chromosome, rather than the interior. Unlike our study in mouse oocytes, Hata et al. (14Hata K. Okano M. Lei H. Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar) reported that Dnmt3L binds and co-localizes with Dnmt3a and Dnmt3b in the nuclei of NIH3T3 cells. One possible explanation for this finding is that Dnmts perform a specific function to interact with the exterior surface of chromosomes in mouse oocytes without being internalized, because most of the regions that have active genes modified by the Dnmts localize to the edge or to the outside of the chromosome territory (24Bernardino-Sgherri J. Flagiello D. Dutrillaux B. Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes.Cytogenet Genome Res. 2002; 99: 85-91Crossref PubMed Scopus (7) Google Scholar, 25Chambeyron S. Bickmore W.A. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.Genes Dev. 2004; 18: 1119-1130Crossref PubMed Scopus (509) Google Scholar, 26Morey C. Da Silva N.R. Perry P. Bickmore W.A. Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation.Development. 2007; 134: 909-919Crossref PubMed Scopus (152) Google Scholar). This precise function guarantees the establishment of the correct maternal imprint. Regarding the aging group, transcripts for both maintenance DNA methyltransferase and de novo methyltransferase, as well as histone deacetylase 2 were changed in aging oocytes (3Hamatani T. Falco G. Carter M.G. Akutsu H. Stagg C.A. Sharov A.A. et al.Age-associated alteration of gene expression patterns in mouse oocytes.Hum Mol Genet. 2004; 13: 2263-2278Crossref PubMed Scopus (397) Google Scholar). In addition, there is a great deal of evidence showing that Dnmt1, Dnmt3a, Dnmt3b, and Dnmt3L are transcriptional repressors through their ability to associate with the histone deacetylase (HDAC) (23Deplus R. Brenner C. Burgers W.A. Putmans P. Kouzarides T. Launoit Y. et al.Dnmt3L is a transcriptional repressor that recruits histone deacetylase.Nucleic Acids Res. 2002; 30: 3831-3838Crossref PubMed Scopus (144) Google Scholar, 27Rountree M.R. Bachman K.E. Baylin S.B. DNMT1 binds HDAC2 and a new corepressor, DMAP1, to form a complex at replication foci.Nat Genet. 2000; 25: 269-277Crossref PubMed Scopus (854) Google Scholar, 28Fuks F. Burgers W.A. Godin N. Kasai M. Kouzarides T. Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription.EMBO J. 2001; 20: 2536-2544Crossref PubMed Scopus (463) Google Scholar, 29Bachman K.E. Rountree M.R. Baylin S.B. Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin.J Biol Chem. 2001; 276: 32282-32287Crossref PubMed Scopus (373) Google Scholar) involved in meiosis-specific chromosomal segregation and chromatin structure (30Kim J.M. Liu H. Tazaki M. Nagata M. Aoki F. Changes in histone acetylation during mouse oocyte meiosis.J Cell Biol. 2003; 162: 37-46Crossref PubMed Scopus (233) Google Scholar). Changes in DNA methylation may be associated with altered chromosome architecture (24Bernardino-Sgherri J. Flagiello D. Dutrillaux B. Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes.Cytogenet Genome Res. 2002; 99: 85-91Crossref PubMed Scopus (7) Google Scholar, 31Matarazzo M.R. Boyle S. D'Esposito M. Bickmore W.A. Chromosome territory reorganization in a human disease with altered DNA methylation.Proc Natl Acad Sci U S A. 2007; 104: 16546-16551Crossref PubMed Scopus (56) Google Scholar). Alternatively, abnormal DNA methylation associated with aging may be responsible for the alteration of chromosome territory.Maternal aging affects the cytoplasmic-to-nuclear trafficking of DNA methyltransferases in mouse oocytes during the time from germinal vesicle breakdown to metaphase I. Correlations between progressive decline in female fertility and age have been observed for many years (1Klein J. Sauer M.V. Assessing fertility in women of advanced reproductive age.Am J Obstet Gynecol. 2001; 185: 758-770Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). It has been well recognized that poor oocyte quality is responsible for an overall reduction in fertility and the age-related decline in female fertility (2Plachot M. Veiga A. Montagut J. de Grouchy J. Calderon G. Lepretre S. et al.Are clinical and biological IVF parameters correlated with chromosomal disorders in early life: a multicentric study.Hum Reprod. 1988; 3: 627-635Crossref PubMed Scopus (197) Google Scholar). In recent years, several reports have demonstrated that DNA methylation is affected by maternal age (3Hamatani T. Falco G. Carter M.G. Akutsu H. Stagg C.A. Sharov A.A. et al.Age-associated alteration of gene expression patterns in mouse oocytes.Hum Mol Genet. 2004; 13: 2263-2278Crossref PubMed Scopus (397) Google Scholar, 4Pan H. Ma P. Zhu W. Schultz R.M. Age-associated increase in aneuploidy and changes in gene expression in mouse eggs.Dev Biol. 2008; 316: 397-407Crossref PubMed Scopus (210) Google Scholar, 5Lopes F.L. Fortier A.L. Darricarrère N. Chan D. Arnold D.R. Trasler J.M. Reproductive and epigenetic outcomes associated with aging mouse oocytes.Hum Mol Genet. 2009; 18: 2032-2044Crossref PubMed Scopus (55) Google Scholar). DNA methylation, catalyzed by DNA methyltransferases (Dnmts) that can be divided into de novo and maintenance methyltransferases, is essential for chromatin remodeling, genomic imprinting, and X chromosome inactivation (6Margot J.B. Ehrenhofer-Murray A.E. Leonhardt H. Interactions within the mammalian DNA methyltransferase family.J Mol Biol. 2003; 4: 7Google Scholar, 7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar). The mammalian Dnmt family has five members: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L (6Margot J.B. Ehrenhofer-Murray A.E. Leonhardt H. Interactions within the mammalian DNA methyltransferase family.J Mol Biol. 2003; 4: 7Google Scholar, 7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar). It has been shown that Dnmt enzymes other than Dnmt2 contribute to the methylation pattern acquisition during gametogenesis and embryogenesis (7Bestor T.H. The DNA methyltransferases of mammals.Hum Mol Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1571) Google Scholar, 8Okano M. Bell D.W. Haber D.A. Li E.n. DNA Methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.Cell. 1999; 99: 247-257Abstract Full Text Full Text PDF PubMed Scopus (4413) Google Scholar, 9Lucifero D. La Salle S. Bourc'his D. Martel J. Bestor T.H. Trasler J.M. Coordinate regulation of DNA methyltransferase expression during oogenesis.BMC Dev Biol. 2007; 7: 36Crossref PubMed Scopus (92) Google Scholar, 10Ratnam S. Mertineit C. Ding F. Howell C.Y. Clarke H.J. Bestor T.H. et al.Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development.Dev Biol. 2002; 245: 304-314Crossref PubMed Scopus (166) Google Scholar). Dnmt1 is considered the major maintenance enzyme during replication. Dnmt3a, Dnmt3b, and Dnmt3L are required for de novo methylation. Several reports have provided that a variant Dnmt1 protein called Dnmt1o is found in the cytoplasm of metaphase II oocytes (11Howell C.Y. Bestor T.H. Ding F. Latham K.E. Mertineit C. Trasler J.M. et al.Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene.Cell. 2001; 104: 829-838Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 12Cardoso M.C. Leonhardt H. DNA methyltransferase is actively retained in the cytoplasm during early development.J Cell Biol. 1999; 147: 25-32Crossref PubMed Scopus (131) Google Scholar), and the transcription profile of Dnmt3 is expressed in growing oocytes (13Bourc'his D. Xu G.L. Lin C.S. Bollman B. Bestor T.H. Dnmt3L and the establishment of maternal genomic imprints.Science. 2001; 294: 2536-2539Crossref PubMed Scopus (1052) Google Scholar, 14Hata K. Okano M. Lei H. Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar). Previous studies of Dnmt focused primarily on the gene expression that takes place during oogenesis (3Hamatani T. Falco G. Carter M.G. Akutsu H. Stagg C.A. Sharov A.A. et al.Age-associated alteration of gene expression patterns in mouse oocytes.Hum Mol Genet. 2004; 13: 2263-2278Crossref PubMed Scopus (397) Google Scholar, 9Lucifero D. La Salle S. Bourc'his D. Martel J. Bestor T.H. Trasler J.M. Coordinate regulation of DNA methyltransferase expression during oogenesis.BMC Dev Biol. 2007; 7: 36Crossref PubMed Scopus (92) Google Scholar, 10Ratnam S. Mertineit C. Ding F. Howell C.Y. Clarke H.J. Bestor T.H. et al.Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development.Dev Biol. 2002; 245: 304-314Crossref PubMed Scopus (166) Google Scholar, 15Lees-Murdock D.J. Shovlin T.C. Gardiner T. De Felici M. Walsh C.P. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.Dev Dyn. 2005; 232: 992-1002Crossref PubMed Scopus (69) Google Scholar, 16La Salle S. Mertineit C. Taketo T. Moens P.B. Bestor T.H. Traslera J.M. Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells.Dev Biol. 2004; 268: 403-415Crossref PubMed Scopus (175) Google Scholar). However, the effects of maternal age on the expression of Dnmt proteins during meiosis have not been fully elucidated. The aim of the present study was to observe the age-related changes in localization of Dnmt proteins during mouse oocyte maturation. Female Kun-Ming mice (CAMS, Beijing, China) were housed and bred under standard conditions (temperature 22 ± 2°C, relative humidity 55% ± 5%, 12-hour light-dark cycle) with food and water available ad libitum according to the Chinese National Standard (GB14925-2001). All the animal experiments were approved by the Institutional Animal Welfare and Ethics Committee of Peking University (No. IRBLA2009-020). Fifty 7–8-week-old (pubertal) and fifty 40–47-week-old (aging) female mice were sacrificed by cervical dislocation, and their oocytes were isolated by puncturing the ovarian follicles with a sterile needle in human tubular fluid (Millipore, Bedford, MA). The female reproductive lifespan in Kun-Ming mice extends to 45 weeks, so this time point is a functional approximation of human perimenopause. Only the denuded oocytes displaying an intact germinal vesicle (GV) were collected for further culture. Immature oocytes were incubated in human tubular fluid medium supplemented with 10% FBS under liquid paraffin oil at 37°C in a humidified atmosphere of 5% CO2 in air. Oocytes were matured as one of six groups over the entire course of maturation: 0, 3, 5, 7, 9, and 16 hours. Oocytes after in vitro maturation (IVM) were immediately fixed in 4% paraformaldehyde for 30 min. Fixed oocytes in each experimental group were permeabilized with 0.5% Triton-X-100 for 30 minutes, followed by three washes in phosphate-buffered saline (PBS). Afterward, oocytes were washed again and blocked in 2% normal goat or rabbit serum blocking solution for 30 minutes and then incubated in a 10-μl drop of a rabbit polyclonal anti-Dnmt1, anti-Dnmt3a, anti-Dnmt3b, or a goat polyclonal anti-Dnmt3L antibody (diluted 1:50 in PBS; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; cat. no. sc-20701, sc-20703, sc-20704, sc-10239) overnight at 4°C, respectively. After washing, samples were immersed in a biotinylated goat anti-rabbit or rabbit anti-goat IgG (diluted 1:100 in PBS; Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes. Finally, rinsed oocytes were reacted with Quantum dot 585-streptavidin conjugate (diluted 1:50 in PBS; Invitrogen, Carlsbad, CA; cat. no. Q10111MP) for 1 hour. Nuclei were counterstained with 5 μg/ml Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) for 15 minutes. For negative controls, primary antibody was omitted. Quantum dot 585–labeled Dnmts were observed with a Bio-Rad MRC 1024MP two-photon laser scanning microscope at 780-nm excitation and 585-nm emission with 100× oil objective. Nuclei stained with Hoechst33342 were simultaneously imaged via the other channel. Differences among groups were analyzed with GLM using SAS9.2. Results were expressed as mean ± SEM. P < 0.05 was considered statistically significant. For all results, the examples shown are representative of three replications. To study the effect of maternal aging on oocyte maturation, we examined two key components of morphologic nuclear maturation: the percentage undergoing germinal vesicle breakdown (GVBD) and the first polar body (PB1) extrusion, because it is much more difficult and complicated to examine cytoplasmic maturation (17Nichols S.M. Gierbolini L. Gonzalez-Martinez J.A. Bavister B.D. Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.Fertil Steril. 2010; 93: 1597-1600Abstract Full Text Full Text PDF Scopus (17) Google Scholar). At 3 hours after maturation, the percentage of GVBD was only 45% in the aging oocytes, far lower than the 70% seen in the pubertal oocytes. After 16 hours in culture, the GVBD rate of 69% and the PB1 rate of 51% in the aging oocytes were significantly less than the 83% and the 63% seen in the pubertal oocytes, respectively (Supplemental Table 1, available online). To better depict the process of maturation, oocytes were classified as: GV-stage (0 hours), GVBD-stage (3 hours after IVM), PMI-stage (prometaphase of meiosis I, 5–7 hours after IVM), metaphase I stage (metaphase of meiosis I, 9 hours after IVM), or metaphase II stage (metaphase of meiosis II, 16 hours after IVM). (Anaphase and telophase of meiosis I are not easily discerned because of their short duration.) As shown in Figure 1, Dnmt1 was localized to the cytoplasm of aging (Fig. 1A) and pubertal (Fig. 1B) mouse oocytes during meiotic maturation. Dnmt3a, Dnmt3b, and Dnmt3L were shown surrounding the chromosomes of pubertal mouse oocytes at the GVBD and PMI stages, and were localized to the cytoplasm at other stages (Fig. 1D, 1F, 1H). However, in the aging groups, Dnmt3a and Dnmt3b were detected only in the cytoplasm (Fig. 1C, 1E). Dnmt3L was seen around the nucleus of GV oocytes, and localized to the cytoplasm at other stages (Fig. 1G). This study verified that aging influences the morphology of nuclear maturation, because nuclear maturation was significantly slower in the aging mouse oocytes (Supplemental Table 1, available online). Aging is also correlated with an increase in the aneuploidy rate of oocytes (17Nichols S.M. Gierbolini L. Gonzalez-Martinez J.A. Bavister B.D. Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta.Fertil Steril. 2010; 93: 1597-1600Abstract Full Text Full Text PDF Scopus (17) Google Scholar, 18Zuccotti M. Boiani M. Garagna S. Redi C.A. Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging.Mol Reprod Dev. 1998; 50: 305-312Crossref PubMed Scopus (32) Google Scholar, 19Liu L. Keefe D.L. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice.Hum Reprod. 2002; 17: 2678-2685Crossref PubMed Google Scholar, 20Jones K.T. Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age.Hum Reprod Update. 2008; 14: 143-158Crossref PubMed Scopus (166) Google Scholar). Our study illustrates that the age of the female can perturb oocyte maturation, which is corroborated by the Jones study (20Jones K.T. Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age.Hum Reprod Update. 2008; 14: 143-158Crossref PubMed Scopus (166) Google Scholar). Based on this observation, we compared the localization of Dnmts in aging and pubertal mouse oocytes matured in vitro. First, these data provide direct evidence that aging does not alter the localization of Dnmt1 in oocytes, because Dnmt1 is cytoplasmic both in the pubertal and aging groups. Second, we suggest that the effects of aging on the localization of Dnmt3a and Dnmt3b in oocytes are similar. There is a lack of cytoplasm around chromosome-cytoplasm trafficking in the aging group, which is a notable event for Dnmt3a and Dnmt3b to perform in de novo methylation (15Lees-Murdock D.J. Shovlin T.C. Gardiner T. De Felici M. Walsh C.P. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.Dev Dyn. 2005; 232: 992-1002Crossref PubMed Scopus (69) Google Scholar). Lees-Murdock et al. (15Lees-Murdock D.J. Shovlin T.C. Gardiner T. De Felici M. Walsh C.P. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation.Dev Dyn. 2005; 232: 992-1002Crossref PubMed Scopus (69) Google Scholar) proposed that Dnmts are all synthesized and stored in the cytoplasm and move to the nucleus to perform their enzymatic function, moving back out when they are no longer required. 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Third, we confirm that the effect of aging on the localization of Dnmt3L in oocytes is unique, because it is the only enzyme appearing around the chromatin in aging mouse oocytes. Thus, Dnmt3L is an indispensable protein for establishing maternal genomic imprinting in aging mouse oocytes. However, the cooperative relationship to Dnmt3a and Dnmt3b has been changed in aging mouse oocytes, which implies that the regulatory mechanism of Dnmt3 family collaboration may be associated with aging in maternal imprint establishment (6Margot J.B. Ehrenhofer-Murray A.E. Leonhardt H. Interactions within the mammalian DNA methyltransferase family.J Mol Biol. 2003; 4: 7Google Scholar, 14Hata K. Okano M. Lei H. Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar, 23Deplus R. Brenner C. Burgers W.A. Putmans P. Kouzarides T. Launoit Y. et al.Dnmt3L is a transcriptional repressor that recruits histone deacetylase.Nucleic Acids Res. 2002; 30: 3831-3838Crossref PubMed Scopus (144) Google Scholar). Last, we hypothesize that localization of Dnmts in aging mouse oocytes during meiosis may be correlated with chromosome structure. Interestingly, all of the Dnmts interacting with chromosomes are on the exterior surface of the chromosome, rather than the interior. Unlike our study in mouse oocytes, Hata et al. (14Hata K. Okano M. Lei H. Li E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice.Development. 2002; 129: 1983-1993Crossref PubMed Google Scholar) reported that Dnmt3L binds and co-localizes with Dnmt3a and Dnmt3b in the nuclei of NIH3T3 cells. One possible explanation for this finding is that Dnmts perform a specific function to interact with the exterior surface of chromosomes in mouse oocytes without being internalized, because most of the regions that have active genes modified by the Dnmts localize to the edge or to the outside of the chromosome territory (24Bernardino-Sgherri J. Flagiello D. Dutrillaux B. Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes.Cytogenet Genome Res. 2002; 99: 85-91Crossref PubMed Scopus (7) Google Scholar, 25Chambeyron S. Bickmore W.A. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.Genes Dev. 2004; 18: 1119-1130Crossref PubMed Scopus (509) Google Scholar, 26Morey C. Da Silva N.R. Perry P. Bickmore W.A. Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation.Development. 2007; 134: 909-919Crossref PubMed Scopus (152) Google Scholar). 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Dutrillaux B. Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes.Cytogenet Genome Res. 2002; 99: 85-91Crossref PubMed Scopus (7) Google Scholar, 31Matarazzo M.R. Boyle S. D'Esposito M. Bickmore W.A. Chromosome territory reorganization in a human disease with altered DNA methylation.Proc Natl Acad Sci U S A. 2007; 104: 16546-16551Crossref PubMed Scopus (56) Google Scholar). Alternatively, abnormal DNA methylation associated with aging may be responsible for the alteration of chromosome territory. Maternal aging affects the cytoplasmic-to-nuclear trafficking of DNA methyltransferases in mouse oocytes during the time from germinal vesicle breakdown to metaphase I. Table 1. Tabled 1Assessment of nuclear maturation of cultured oocytes based on morphology.0 h3 h5 h7 h9 h16 hAging mouse oocytes GVBD (% ± SEM)0 ± 044.75 ± 5.83aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.55.82 ± 3.40aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.56.28 ± 2.76aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.66.68 ± 5.50aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.68.85 ± 4.23aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point. PB1 (% ± SEM)0 ± 00 ± 00 ± 01.4 ± 1.403 ± 1.3750.73 ± 3.18aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point. n195185258198201151Pubertal mouse oocytes GVBD (% ± SEM)0 ± 070.24 ± 4.45aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.74.83 ± 3.53aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.77.18 ± 4.06aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.81.91 ± 2.79aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point.83.41 ± 2.82aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point. PB1 (% ± SEM)0 ± 00 ± 00 ± 02.52 ± 0.825.35 ± 1.7762.55 ± 3.37aStatistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point. n285259292338332392Note: GVBD% = the percentage of germinal vesicle breakdown; PB1% = the percentage of the first polar body extrusion; n = number of cultured oocytes per time point.a Statistically significant difference (P < 0.05) between the aging and pubertal groups at the specified time point. Open table in a new tab Note: GVBD% = the percentage of germinal vesicle breakdown; PB1% = the percentage of the first polar body extrusion; n = number of cultured oocytes per time point.
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