- Email: [email protected]
Age-related changes in the localization of DNA methyltransferases during meiotic maturation in mouse oocytes
Age-related changes in the localization of DNA methyltransferases during meiotic maturation in mouse oocytes 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. (Fertil Steril 2011;95:1531–4. 2011 by American Society for Reproductive Medicine.) Key Words: DNA methyltransferase, aging, oocytes, in vitro maturation
Correlations between progressive decline in female fertility and age have been observed for many years (1). 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 (2). In recent years, several reports have demonstrated that DNA methylation is affected by maternal age (3–5). 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 (6, 7). The mammalian Dnmt family has five members: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L (6, 7). It has been shown that Dnmt enzymes other than Dnmt2 contribute to the methylation pattern acquisition during gametogenesis and embryogenesis (7–10). 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 (11, 12), and the transcription profile of Dnmt3 is expressed in growing oocytes (13, 14). Previous studies Lu Zhang, M.Sc.a Dan-Yu Lu, M.Sc.a Wan-Yun Ma, Ph.D.b Ying Li, M.D.a a Department of Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, People’s Republic of China b Key Laboratory for Atomic and Molecular Nanosciences of the Ministry of Education, Department of Physics, Tsinghua University, Beijing, People’s Republic of China Received February 25, 2010; revised June 9, 2010; accepted June 16, 2010; published online August 2, 2010. L.Z. has nothing to disclose. D-Y.L. has nothing to disclose. W-Y.M. has nothing to disclose. Y.L. has nothing to disclose. Supported by the Major Research Program of National Natural Science Foundation of China (No. 90919012) and the Chinese National Basic Research Program (973 Program; no. 2007CB5119004). Reprint requests: Ying Li, M.D., Department of Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xueyuan Rd, Haidian District, Beijing, 100191, People’s Republic of China (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.06.050
of Dnmt focused primarily on the gene expression that takes place during oogenesis (3, 9, 10, 15, 16). 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-ml drop of a rabbit polyclonal anti-Dnmt1, anti-Dnmt3a, antiDnmt3b, 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
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1 Immunofluorescent localization of Dnmts in pubertal and aging mouse oocytes at various stages of meiotic progression. Representative micrographs of IVM oocytes from (A, C, E, G) aging and (B, D, F, H) pubertal mice show the distribution of (A, B) Dnmt1, (C, D) Dnmt3a, (E, F) Dnmt3b and (G, H) Dnmt3L. GV-intact oocytes were matured in vitro for 0 (GV), 3, 5, 7, 9, or 16 hours and then immunostained with specific antibodies. Each antibody was visualized using Quantum dot 585 (yellow), and the DNA was counterstained using Hoechst33342 (blue). Three independent experiments were performed, which used 30 oocytes per group. Arrowheads indicate the misalignment of chromosomes; arrows show the chromatin of the first polar body. Scale bars ¼ 20 mm.
Zhang. Dnmts change in oocytes during mouse aging. Fertil Steril 2011.
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Dnmts change in oocytes during mouse aging
Vol. 95, No. 4, March 15, 2011
585-streptavidin conjugate (diluted 1:50 in PBS; Invitrogen, Carlsbad, CA; cat. no. Q10111MP) for 1 hour. Nuclei were counterstained with 5 mg/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 (17). 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), PMIstage (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 GVoocytes, 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 (17–20). Our study illustrates that the age of the female can perturb oocyte maturation, which is corroborated by the Jones study (20). 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
Fertility and Sterility
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 (15). Lees-Murdock et al. (15) 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 (7, 21, 22). 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 (6, 14, 23). 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. (14) 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 (24–26). 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 (3). 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) (23, 27–29) involved in meiosis-specific chromosomal segregation and chromatin structure (30). Changes in DNA methylation may be associated with altered chromosome architecture (24, 31). 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.
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REFERENCES 1. Klein J, Sauer MV. Assessing fertility in women of advanced reproductive age. Am J Obstet Gynecol 2001;185:758–70. 2. Plachot 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;3:627–35. 3. Hamatani T, Falco G, Carter MG, Akutsu H, Stagg CA, Sharov AA, et al. Age-associated alteration of gene expression patterns in mouse oocytes. Hum Mol Genet 2004;13: 2263–78. 4. Pan H, Ma P, Zhu W, Schultz RM. Age-associated increase in aneuploidy and changes in gene expression in mouse eggs. Dev Biol 2008;316: 397–407. 5. Lopes FL, Fortier AL, Darricarrere N, Chan D, Arnold DR, Trasler JM. Reproductive and epigenetic outcomes associated with aging mouse oocytes. Hum Mol Genet 2009;18: 2032–44. 6. Margot JB, Ehrenhofer-Murray AE, Leonhardt H. Interactions within the mammalian DNA methyltransferase family. J Mol Biol 2003;4:7. 7. Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet 2000;9:2395–402. 8. Okano M, Bell DW, Haber DA, Li En. DNA Methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999;99:247–57. 9. Lucifero D, La Salle S, Bourc’his D, Martel J, Bestor TH, Trasler JM. Coordinate regulation of DNA methyltransferase expression during oogenesis. BMC Dev Biol 2007;7:36. 10. Ratnam S, Mertineit C, Ding F, Howell CY, Clarke HJ, Bestor TH, et al. Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development. Dev Biol 2002;245:304–14.
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11. Howell CY, Bestor TH, Ding F, Latham KE, Mertineit C, Trasler JM, et al. Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 2001;104:829–38. 12. Cardoso MC, Leonhardt H. DNA methyltransferase is actively retained in the cytoplasm during early development. J Cell Biol 1999;147:25–32. 13. Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the establishment of maternal genomic imprints. Science 2001;294:2536–9. 14. Hata 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;129:1983–93. 15. Lees-Murdock DJ, Shovlin TC, Gardiner T, De Felici M, Walsh CP. DNA methyltransferase expression in the mouse germ line during periods of de novo methylation. Dev Dyn 2005;232:992–1002. 16. La Salle S, Mertineit C, Taketo T, Moens PB, Bestor TH, Traslera JM. Windows for sex-specific methylation marked by DNA methyltransferase expression profiles in mouse germ cells. Dev Biol 2004;268:403–15. 17. Nichols SM, Gierbolini L, Gonzalez-Martinez JA, Bavister BD. Effects of in vitro maturation and age on oocyte quality in the rhesus macaque Macaca mulatta. Fertil Steril 2010;93:1597–600. 18. Zuccotti M, Boiani M, Garagna S, Redi CA. Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging. Mol Reprod Dev 1998;50:305–12. 19. Liu L, Keefe DL. Ageing-associated aberration in meiosis of oocytes from senescence-accelerated mice. Hum Reprod 2002;17:2678–85. 20. Jones KT. Meiosis in oocytes: predisposition to aneuploidy and its increased incidence with age. Hum Reprod Update 2008;14:143–58. 21. Turek-Plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett 2005;10:631–47.
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22. Pradhan S, Esteve PO. Mammalian DNA (cytosine5) methyltransferases and their expression. Clin Immunol 2003;109:6–16. 23. Deplus R, Brenner C, Burgers WA, Putmans P, Kouzarides T, Launoit Y, et al. Dnmt3L is a transcriptional repressor that recruits histone deacetylase. Nucleic Acids Res 2002;30:3831–8. 24. Bernardino-Sgherri J, Flagiello D, Dutrillaux B. Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes. Cytogenet Genome Res 2002;99:85–91. 25. Chambeyron S, Bickmore WA. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription. Genes Dev 2004;18:1119–30. 26. Morey C, Da Silva NR, Perry P, Bickmore WA. Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation. Development 2007;134:909–19. 27. Rountree MR, Bachman KE, Baylin SB. DNMT1 binds HDAC2 and a new corepressor, DMAP1, to form a complex at replication foci. Nat Genet 2000;25:269–77. 28. Fuks F, Burgers WA, Godin N, Kasai M, Kouzarides T. Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription. EMBO J 2001;20: 2536–44. 29. Bachman KE, Rountree MR, Baylin SB. Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin. J Biol Chem 2001;276:32282–7. 30. Kim JM, Liu H, Tazaki M, Nagata M, Aoki F. Changes in histone acetylation during mouse oocyte meiosis. J Cell Biol 2003;162:37–46. 31. Matarazzo MR, Boyle S, D’Esposito M, Bickmore WA. Chromosome territory reorganization in a human disease with altered DNA methylation. Proc Natl Acad Sci U S A 2007;104:16546–51.
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SUPPLEMENTAL TABLE 1 Assessment of nuclear maturation of cultured oocytes based on morphology.
Aging mouse oocytes GVBD (% SEM) PB1 (% SEM) n Pubertal mouse oocytes GVBD (% SEM) PB1 (% SEM) n
0h
3h
5h
7h
9h
16 h
00 00 195
44.75 5.83a 00 185
55.82 3.40a 00 258
56.28 2.76a 1.4 1.40 198
66.68 5.50a 3 1.37 201
68.85 4.23a 50.73 3.18a 151
00 00 285
70.24 4.45a 00 259
74.83 3.53a 00 292
77.18 4.06a 2.52 0.82 338
81.91 2.79a 5.35 1.77 332
83.41 2.82a 62.55 3.37a 392
Note: GVBD% ¼ the percentage of germinal vesicle breakdown; PB1% ¼ the percentage of the first polar body extrusion; n ¼ 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. Zhang. Dnmts change in oocytes during mouse aging. Fertil Steril 2011.
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Correlations between progressive decline in female fertility and age have been observed for many years (1). 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 (2). In recent years, several reports have demonstrated that DNA methylation is affected by maternal age (3–5). 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 (6, 7). The mammalian Dnmt family has five members: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L (6, 7). It has been shown that Dnmt enzymes other than Dnmt2 contribute to the methylation pattern acquisition during gametogenesis and embryogenesis (7–10). 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 (11, 12), and the transcription profile of Dnmt3 is expressed in growing oocytes (13, 14). Previous studies Lu Zhang, M.Sc.a Dan-Yu Lu, M.Sc.a Wan-Yun Ma, Ph.D.b Ying Li, M.D.a a Department of Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, People’s Republic of China b Key Laboratory for Atomic and Molecular Nanosciences of the Ministry of Education, Department of Physics, Tsinghua University, Beijing, People’s Republic of China Received February 25, 2010; revised June 9, 2010; accepted June 16, 2010; published online August 2, 2010. L.Z. has nothing to disclose. D-Y.L. has nothing to disclose. W-Y.M. has nothing to disclose. Y.L. has nothing to disclose. Supported by the Major Research Program of National Natural Science Foundation of China (No. 90919012) and the Chinese National Basic Research Program (973 Program; no. 2007CB5119004). Reprint requests: Ying Li, M.D., Department of Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, 38 Xueyuan Rd, Haidian District, Beijing, 100191, People’s Republic of China (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2010.06.050
of Dnmt focused primarily on the gene expression that takes place during oogenesis (3, 9, 10, 15, 16). 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-ml drop of a rabbit polyclonal anti-Dnmt1, anti-Dnmt3a, antiDnmt3b, 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
Fertility and Sterility Vol. 95, No. 4, March 15, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
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FIGURE 1 Immunofluorescent localization of Dnmts in pubertal and aging mouse oocytes at various stages of meiotic progression. Representative micrographs of IVM oocytes from (A, C, E, G) aging and (B, D, F, H) pubertal mice show the distribution of (A, B) Dnmt1, (C, D) Dnmt3a, (E, F) Dnmt3b and (G, H) Dnmt3L. GV-intact oocytes were matured in vitro for 0 (GV), 3, 5, 7, 9, or 16 hours and then immunostained with specific antibodies. Each antibody was visualized using Quantum dot 585 (yellow), and the DNA was counterstained using Hoechst33342 (blue). Three independent experiments were performed, which used 30 oocytes per group. Arrowheads indicate the misalignment of chromosomes; arrows show the chromatin of the first polar body. Scale bars ¼ 20 mm.
Zhang. Dnmts change in oocytes during mouse aging. Fertil Steril 2011.
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Dnmts change in oocytes during mouse aging
Vol. 95, No. 4, March 15, 2011
585-streptavidin conjugate (diluted 1:50 in PBS; Invitrogen, Carlsbad, CA; cat. no. Q10111MP) for 1 hour. Nuclei were counterstained with 5 mg/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 (17). 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), PMIstage (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 GVoocytes, 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 (17–20). Our study illustrates that the age of the female can perturb oocyte maturation, which is corroborated by the Jones study (20). 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
Fertility and Sterility
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 (15). Lees-Murdock et al. (15) 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 (7, 21, 22). 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 (6, 14, 23). 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. (14) 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 (24–26). 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 (3). 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) (23, 27–29) involved in meiosis-specific chromosomal segregation and chromatin structure (30). Changes in DNA methylation may be associated with altered chromosome architecture (24, 31). 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.
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SUPPLEMENTAL TABLE 1 Assessment of nuclear maturation of cultured oocytes based on morphology.
Aging mouse oocytes GVBD (% SEM) PB1 (% SEM) n Pubertal mouse oocytes GVBD (% SEM) PB1 (% SEM) n
0h
3h
5h
7h
9h
16 h
00 00 195
44.75 5.83a 00 185
55.82 3.40a 00 258
56.28 2.76a 1.4 1.40 198
66.68 5.50a 3 1.37 201
68.85 4.23a 50.73 3.18a 151
00 00 285
70.24 4.45a 00 259
74.83 3.53a 00 292
77.18 4.06a 2.52 0.82 338
81.91 2.79a 5.35 1.77 332
83.41 2.82a 62.55 3.37a 392
Note: GVBD% ¼ the percentage of germinal vesicle breakdown; PB1% ¼ the percentage of the first polar body extrusion; n ¼ 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. Zhang. Dnmts change in oocytes during mouse aging. Fertil Steril 2011.
Fertility and Sterility
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