母源蛋白RBBP7在小鼠卵母细胞发育成熟过程中的功能研究
摘要
视网膜母细胞瘤结合蛋白7(RBBP7)是一种具有肿瘤抑制作用的核蛋白,有研究表明RBBP7蛋白可以通过干扰正常的细胞周期和增加细胞凋亡进而抑制肿瘤细胞的转化。在本实验室的前期工作中,运用二维电泳和质谱鉴定等蛋白质组学技术建立了小鼠MII期(第二次减数分裂中期)卵细胞的蛋白表达谱。RBBP7就是在该谱系中鉴定得到的蛋白之一。且在激活后其含量下降,表明RBBP7可能在卵母细胞的发育成熟中发挥重要的作用。但RBBP7蛋白在小鼠卵细胞和早期胚胎中的功能研究尚属空白。
本研究首先用免疫组化、免疫荧光等方法研究RBBP7在小鼠卵巢组织中的定位。免疫组织化学的结果显示RBBP7蛋白主要定位于各级卵母细胞的细胞核。免疫荧光结果显示RBBP7定位于GV期卵母细胞的细胞核,GVBD期时则弥漫分布于胞浆,MI期定位于纺锤体上,AI期定位于纺锤体的中体上,MII期与纺锤体共定位。为研究RBBP7蛋白在小鼠卵母细胞发育成熟过程中的作用,应用显微注射的方式将RBBP7 siRNA显微注射至GVBD期卵母细胞胞浆中,活细胞工作站观察卵母细胞的发育过程。结果发现:RBBP7蛋白的减少最终不会导致第一极体排放数目的异常,但是在第一极体的排放过程中,RBBP7蛋白的减少会导致卵母细胞胞质分裂的比率增高,同源染色体不分离或不均等分离的比率明显增高,最终分裂沟回缩,产生一个双核细胞,第一极体排放障碍。
基于以上实验表型,接下来研究在小鼠卵母细胞中RBBP7影响第一次减数分裂过程中同源染色体分离的机制。应用小鼠卵巢全蛋白免疫共沉淀分析相互作用蛋白,结果显示RBBP7可以与VCP相互作用。VCP的作用是调节有丝分裂后期纺锤体的解聚。RBBP7特异定位于第一次减数分裂后期纺锤体的中体,因此推测RBBP7可能作为一种纺锤体组装因子,通过与VCP的相互作用,进而干扰第一次减数分裂后期纺锤体的解聚,从而影响同源染色体的精确分离,最终产生一个双核细胞。
Retinoblastoma binding protein 7 (RBBP7) is a nuclear protein with tumor inhibitory effect, previous studies showed that RBBP7 protein can interfere with normal cell cycle and increase the apoptosis of tumor cells to inhibit the conversion. Our laboratory studied the protein expression of mouse MII (metaphase of the second meiosis) oocytes and parthenogenetically activated oocytes by 2D gel electrophoresis, through mass spectrum identification, bioinformatical analysis. RBBP7 decreased in the 2D gel of parthenogenetically activated oocytes. This indicates that RBBP7 may play an important function in the maturation of mouse oocytes. But there is no report on the functional reasearch of RBBP7 in the oocyte maturation and embryo development.
We study the location of RBBP7 in mouse ovary with immunohistochemistry and immunofluorescence first. Immunohistochemistry of ovary sections indicate that RBBP7 distribute in the nucleus of oocytes. Immunofluorescence show that, RBBP7 locates in the nucleus of the germinal vesicle (GV) oocytes and when the germinal vesicle breakdown (GVBD), RBBP7 is in the cytoplasm. During MI (the metaphase of the first meiosis) RBBP7 is in the spindle. In AI(antaphase of the first meiosis), RBBP7 is in the midbody of the spindle. Specially RBBP7 has the same localtion with the spindle in the MII oocytes. To study the function of RBBP7 in mouse oocyte development and maturation, we microinject RBBP7 siRNA into the cytoplasm of mouse GVBD oocytes, continuous observation using time-lapse video miscroscopy finds that when RBBP7 is knock down,there is no difference in the number of oocytes which emission the first polar body, but many of the RBBP7 depleted oocytes has the increased cytokinesis of the first meiosis, the homologous chromosome can not segregate and at last, the cleavage furrow displays fusion with the oocyte, and finally the cells will be binucleate cells.
With above phenotypes, we then studied the mechanism that how RBBP7 influence the segregation of homologous chromosome. Immunoprecipitation analysis with mouse ovaries finds that RBBP7 can function with VCP. VCP can regulate the disassembly of the spindle in the anaphase of mitosis. RBBP7 specially locate in the midbody of the spindle in the anaphase of meiosis I. So we suggest that RBBP7 may be a spindle assembly factor and RBBP7 can function with VCP to regulate the spindle disassembly in female meiosis I to regulate the homologous chromosme segregation.
本研究首先用免疫组化、免疫荧光等方法研究RBBP7在小鼠卵巢组织中的定位。免疫组织化学的结果显示RBBP7蛋白主要定位于各级卵母细胞的细胞核。免疫荧光结果显示RBBP7定位于GV期卵母细胞的细胞核,GVBD期时则弥漫分布于胞浆,MI期定位于纺锤体上,AI期定位于纺锤体的中体上,MII期与纺锤体共定位。为研究RBBP7蛋白在小鼠卵母细胞发育成熟过程中的作用,应用显微注射的方式将RBBP7 siRNA显微注射至GVBD期卵母细胞胞浆中,活细胞工作站观察卵母细胞的发育过程。结果发现:RBBP7蛋白的减少最终不会导致第一极体排放数目的异常,但是在第一极体的排放过程中,RBBP7蛋白的减少会导致卵母细胞胞质分裂的比率增高,同源染色体不分离或不均等分离的比率明显增高,最终分裂沟回缩,产生一个双核细胞,第一极体排放障碍。
基于以上实验表型,接下来研究在小鼠卵母细胞中RBBP7影响第一次减数分裂过程中同源染色体分离的机制。应用小鼠卵巢全蛋白免疫共沉淀分析相互作用蛋白,结果显示RBBP7可以与VCP相互作用。VCP的作用是调节有丝分裂后期纺锤体的解聚。RBBP7特异定位于第一次减数分裂后期纺锤体的中体,因此推测RBBP7可能作为一种纺锤体组装因子,通过与VCP的相互作用,进而干扰第一次减数分裂后期纺锤体的解聚,从而影响同源染色体的精确分离,最终产生一个双核细胞。
Retinoblastoma binding protein 7 (RBBP7) is a nuclear protein with tumor inhibitory effect, previous studies showed that RBBP7 protein can interfere with normal cell cycle and increase the apoptosis of tumor cells to inhibit the conversion. Our laboratory studied the protein expression of mouse MII (metaphase of the second meiosis) oocytes and parthenogenetically activated oocytes by 2D gel electrophoresis, through mass spectrum identification, bioinformatical analysis. RBBP7 decreased in the 2D gel of parthenogenetically activated oocytes. This indicates that RBBP7 may play an important function in the maturation of mouse oocytes. But there is no report on the functional reasearch of RBBP7 in the oocyte maturation and embryo development.
We study the location of RBBP7 in mouse ovary with immunohistochemistry and immunofluorescence first. Immunohistochemistry of ovary sections indicate that RBBP7 distribute in the nucleus of oocytes. Immunofluorescence show that, RBBP7 locates in the nucleus of the germinal vesicle (GV) oocytes and when the germinal vesicle breakdown (GVBD), RBBP7 is in the cytoplasm. During MI (the metaphase of the first meiosis) RBBP7 is in the spindle. In AI(antaphase of the first meiosis), RBBP7 is in the midbody of the spindle. Specially RBBP7 has the same localtion with the spindle in the MII oocytes. To study the function of RBBP7 in mouse oocyte development and maturation, we microinject RBBP7 siRNA into the cytoplasm of mouse GVBD oocytes, continuous observation using time-lapse video miscroscopy finds that when RBBP7 is knock down,there is no difference in the number of oocytes which emission the first polar body, but many of the RBBP7 depleted oocytes has the increased cytokinesis of the first meiosis, the homologous chromosome can not segregate and at last, the cleavage furrow displays fusion with the oocyte, and finally the cells will be binucleate cells.
With above phenotypes, we then studied the mechanism that how RBBP7 influence the segregation of homologous chromosome. Immunoprecipitation analysis with mouse ovaries finds that RBBP7 can function with VCP. VCP can regulate the disassembly of the spindle in the anaphase of mitosis. RBBP7 specially locate in the midbody of the spindle in the anaphase of meiosis I. So we suggest that RBBP7 may be a spindle assembly factor and RBBP7 can function with VCP to regulate the spindle disassembly in female meiosis I to regulate the homologous chromosme segregation.
引文
1. X.G. Minyue Ma, Fuqiang Wang, Chun Zhao, Zichuan Liu, Zhonghua Shi, Yufeng Wang, Ping Zhang, Kemei Zhang, Ningling Wang, Min Lin, Zuomin Zhou, Jiayin Liu, Qingzhang Li, Liu Wang, Ran Huo, Jiahao Sha, and Qi Zhou. Protein Expression Profile of the Mouse Metaphase-II Oocyte. J Proteome Res ,2008,(7) :4821-4830
2. Ahringer, J. NuRD and SIN3: Histone deacetylase complexes in development. Cell, 2000,(16):351-356
3. Zhang, Q., Vo, N., and Goodman, R. H.. Histone binding protein RbAp48 interacts with a complex of CREB binding protein and phosphorylated CREB. Molecular and Cellular Biology, 2000,(20):4970–4978
4. Sif, S., Saurin, A. J., Imbalzano, A. N., and Kingston, R. E. Purification and characterization of mSin3A-containing Brg1 and Brm chromatin remodeling complexes,Genes Dev,2001,(15):603–618
5. Vermaak, D., Wade, P. A., Jones, P. L., Shi, Y.-B., and Wolffe, A. P. Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte. Molecular and Cellular Biology ,1999,(19):5847–5860
6. Parthun, M. R., Widom, J., and Gottschling, J. The major cytoplasmic histone acetlytransferase in yeast: links to chromatin replication and histone metabolism.Cell ,1996,(87):85–94
7. Verreault, A., Kaufman, P. D., Kobayashi, R., and Stillman, B. Nucleosomal DNA regulates the core histone binding subunit of the human HAT1 acteyltransferase. Curr.Biol,1998,(8):96–108
8. Riehman TJ,Sawyer MM, Johnson DI.Saceharomyces cerevisiae Cde42p localizes to cellular membranes and clusters atsites ofpolarized growth.Cell,2002, (3):458-468
9. David A. Guertin, Susanne Trautmann, and Dannel McCollum. Cytokinesis in Eukaryotes. Microbiology and molecular biology reviews,2002,(66):155–178
10. MAKOTO HIBI, AKIRA NAGASAKI, MASAYUKI TAKAHASHI, AKIHIKO YAMAGISHI and TARO Q.P. UYEDA. Dictyostelium discoideum talin A is crucial for myosin II-independent and adhesion-dependent cytokinesis.Journal of Muscle Research and Cell Motility,2004,(25):127–140
11. Jill Kuglin Schweitzer and Crislyn D’Souza-Schorey. Finishing the job: cytoskeletal and membrane events bring cytokinesis to an end. Experimental Cell Research,2004,(295):1– 8
12. Balasubramanian M K,Bi E, Glotzer M. Comparative analysis in budding yeast, fission yeast and animal cells. Curr Biol,2004,(14):806-818
13. Vernos I, Karent E. The Mitotic Spindle : a Self—Made Machine. Science,2001,(294):543—547
14. Jing Yang, Susan McLeskey Kiefer and Michael Rauchman. Characterization of the Gene Encoding Mouse Retinoblastoma Binding Protein-7, a Component of Chromatin-Remodeling Complexes. GENOMICS,2002,(80):4
15. Vermaak, D., Wade, P. A., Jones, P. L., Shi, Y.-B., and Wolffe, A. P. Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte. Mol. Cell Biol,1999,(19):5847–5860
16. Parthun, M. R., Widom, J., and Gottschling J. The major cytoplasmic histone acetlytransferase in yeast: links to chromatin replication and histone metabolism. Cell, 1996,(87):85–94
17. Verreault, A., Kaufman, P. D., Kobayashi, R., and Stillman, B. Nucleosomal DNA regulates the core histone binding subunit of the human HAT1 acteyltransferase. Curr.Biol,1998,(8):96–108
18. D. Moir, D. Botstein. Determination of the order of gene function in theyeast nuclear division pathway using cs and ts mutants, Genetics,1982,(100):565–577
19. Yohei Sasagawa, Kunitoshi Yamanaka, Shingo Nishikori, Teru Ogura. Caenorhabditis elegans p97/CDC-48 is crucial for progression of meiosis I.Biochemical and Biophysical Research Communications,2007,(358):920–924
20. Kan Cao, Reiko Nakajima, Hemmo H. Meyer, and Yixian Zheng. The AAA-ATPase Cdc48/p97 Regulates Spindle Disassembly at the End of Mitosis. Cell ,2003,(115):355–367
21. Brian H. Lee and Angelika Amon. Role of Polo-like Kinase Meiosis I Chromosome Segregation. Science,2003,(300):482
22. Anton Khmelinskii and Elmar Schiebel. Assembling the spindle midzone in the right place at the right time. Cell Cycle,2008,(7):1-4
23. Oliver J. Gruss and Isabelle Vernos. The mechanism of spindle assembly: functions of Ran and its target TPX2. The Journal of Cell Biology,2004,(949):166
24.AaronC.Groen. ThomasJ. Maresca.JesseC. Gatlin.EdwardD. Salmon.and TimothyJ. Mitchison. Functional Overlap of Microtubule Assembly Factorsin Chromatin-Promoted Spindle Assembly. Molecular Biology of the Cell,2009, (20):2766-2773
25. Iva Kronja.Anamarija Kruljac-Letunic.Ma?¨wen Caudron-Herger.Peter Bieling and Eric Karsenti. XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract. Molecular Biology of the Cell,2009,(20):2684–2696
26. Kuliev A.Cieslak J.Ilkevitch Y. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod Biomed ,2003,(1):54-59
27. Verlinsky Y.Cieslak J.Ivakhnenko V. Prepregnancy genetic testing for age-related aneuploidies by polar body analysis. Genet Test,1997-1998,(4):231-235
28. Verlinsky Y.Cieslak J.Ivakhnenko V. Prevention of age-related aneuploidies by polar body testing of oocytes. J Assist Reprod Genet,1999,(4):165-169
29. Kuliev A.Verlinsky Y. Meiotic and mitotic nondisjunction:lessons from preimplantation genetic diagnosis. Hum Reprod Updte,2004,(5):401-407
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5.Pacchierotti F.Adler I D.Eichenlaub-Ritter U. Gender effects on the incidence of aneuploidy in mammalian germ cells.Environ Res. 2007,(1):46-69
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2. Ahringer, J. NuRD and SIN3: Histone deacetylase complexes in development. Cell, 2000,(16):351-356
3. Zhang, Q., Vo, N., and Goodman, R. H.. Histone binding protein RbAp48 interacts with a complex of CREB binding protein and phosphorylated CREB. Molecular and Cellular Biology, 2000,(20):4970–4978
4. Sif, S., Saurin, A. J., Imbalzano, A. N., and Kingston, R. E. Purification and characterization of mSin3A-containing Brg1 and Brm chromatin remodeling complexes,Genes Dev,2001,(15):603–618
5. Vermaak, D., Wade, P. A., Jones, P. L., Shi, Y.-B., and Wolffe, A. P. Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte. Molecular and Cellular Biology ,1999,(19):5847–5860
6. Parthun, M. R., Widom, J., and Gottschling, J. The major cytoplasmic histone acetlytransferase in yeast: links to chromatin replication and histone metabolism.Cell ,1996,(87):85–94
7. Verreault, A., Kaufman, P. D., Kobayashi, R., and Stillman, B. Nucleosomal DNA regulates the core histone binding subunit of the human HAT1 acteyltransferase. Curr.Biol,1998,(8):96–108
8. Riehman TJ,Sawyer MM, Johnson DI.Saceharomyces cerevisiae Cde42p localizes to cellular membranes and clusters atsites ofpolarized growth.Cell,2002, (3):458-468
9. David A. Guertin, Susanne Trautmann, and Dannel McCollum. Cytokinesis in Eukaryotes. Microbiology and molecular biology reviews,2002,(66):155–178
10. MAKOTO HIBI, AKIRA NAGASAKI, MASAYUKI TAKAHASHI, AKIHIKO YAMAGISHI and TARO Q.P. UYEDA. Dictyostelium discoideum talin A is crucial for myosin II-independent and adhesion-dependent cytokinesis.Journal of Muscle Research and Cell Motility,2004,(25):127–140
11. Jill Kuglin Schweitzer and Crislyn D’Souza-Schorey. Finishing the job: cytoskeletal and membrane events bring cytokinesis to an end. Experimental Cell Research,2004,(295):1– 8
12. Balasubramanian M K,Bi E, Glotzer M. Comparative analysis in budding yeast, fission yeast and animal cells. Curr Biol,2004,(14):806-818
13. Vernos I, Karent E. The Mitotic Spindle : a Self—Made Machine. Science,2001,(294):543—547
14. Jing Yang, Susan McLeskey Kiefer and Michael Rauchman. Characterization of the Gene Encoding Mouse Retinoblastoma Binding Protein-7, a Component of Chromatin-Remodeling Complexes. GENOMICS,2002,(80):4
15. Vermaak, D., Wade, P. A., Jones, P. L., Shi, Y.-B., and Wolffe, A. P. Functional analysis of the SIN3-histone deacetylase RPD3-RbAp48-histone H4 connection in the Xenopus oocyte. Mol. Cell Biol,1999,(19):5847–5860
16. Parthun, M. R., Widom, J., and Gottschling J. The major cytoplasmic histone acetlytransferase in yeast: links to chromatin replication and histone metabolism. Cell, 1996,(87):85–94
17. Verreault, A., Kaufman, P. D., Kobayashi, R., and Stillman, B. Nucleosomal DNA regulates the core histone binding subunit of the human HAT1 acteyltransferase. Curr.Biol,1998,(8):96–108
18. D. Moir, D. Botstein. Determination of the order of gene function in theyeast nuclear division pathway using cs and ts mutants, Genetics,1982,(100):565–577
19. Yohei Sasagawa, Kunitoshi Yamanaka, Shingo Nishikori, Teru Ogura. Caenorhabditis elegans p97/CDC-48 is crucial for progression of meiosis I.Biochemical and Biophysical Research Communications,2007,(358):920–924
20. Kan Cao, Reiko Nakajima, Hemmo H. Meyer, and Yixian Zheng. The AAA-ATPase Cdc48/p97 Regulates Spindle Disassembly at the End of Mitosis. Cell ,2003,(115):355–367
21. Brian H. Lee and Angelika Amon. Role of Polo-like Kinase Meiosis I Chromosome Segregation. Science,2003,(300):482
22. Anton Khmelinskii and Elmar Schiebel. Assembling the spindle midzone in the right place at the right time. Cell Cycle,2008,(7):1-4
23. Oliver J. Gruss and Isabelle Vernos. The mechanism of spindle assembly: functions of Ran and its target TPX2. The Journal of Cell Biology,2004,(949):166
24.AaronC.Groen. ThomasJ. Maresca.JesseC. Gatlin.EdwardD. Salmon.and TimothyJ. Mitchison. Functional Overlap of Microtubule Assembly Factorsin Chromatin-Promoted Spindle Assembly. Molecular Biology of the Cell,2009, (20):2766-2773
25. Iva Kronja.Anamarija Kruljac-Letunic.Ma?¨wen Caudron-Herger.Peter Bieling and Eric Karsenti. XMAP215–EB1 Interaction Is Required for Proper Spindle Assembly and Chromosome Segregation in Xenopus Egg Extract. Molecular Biology of the Cell,2009,(20):2684–2696
26. Kuliev A.Cieslak J.Ilkevitch Y. Chromosomal abnormalities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod Biomed ,2003,(1):54-59
27. Verlinsky Y.Cieslak J.Ivakhnenko V. Prepregnancy genetic testing for age-related aneuploidies by polar body analysis. Genet Test,1997-1998,(4):231-235
28. Verlinsky Y.Cieslak J.Ivakhnenko V. Prevention of age-related aneuploidies by polar body testing of oocytes. J Assist Reprod Genet,1999,(4):165-169
29. Kuliev A.Verlinsky Y. Meiotic and mitotic nondisjunction:lessons from preimplantation genetic diagnosis. Hum Reprod Updte,2004,(5):401-407
1.Heffner L J. Advanced Maternal Age:How Old Is Too Old ? N Engl J Med. 2004,(19):1927-9
2.Shi Q.Martin R H. Aneuploidy in human sperm:a review of the frequency and distribution of aneuploidy,effects of donor age and lifestyle factors.Cytogenet Cell Genet. 2000,(3-4):219-26
3.Templado C.Bosch M.Benet J. Frequency and distribution of chromosome abnormalities in human spermatozoa. Cytogenet Genome Res. 2005,(3-4):199-205
4.Shi Q.Martin R H. Spontaneous frequencies of aneuploid and diploid sperm in 10 normal Chinese men:assessed by multicolor fluorescence in situ hybridization.Cytogenet Cell Genet. 2000,(1-2):79-83
5.Pacchierotti F.Adler I D.Eichenlaub-Ritter U. Gender effects on the incidence of aneuploidy in mammalian germ cells.Environ Res. 2007,(1):46-69
6.Kuliev A.Verlinsky Y. Meiotic and mitotic nondisjunction:lessons from preimplantation genetic diagnosis.Hum Reprod Update. 2004,(5):401-7
7.Wells D.Escudero T.Levy B. First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril. 2002,(3):543-9
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