Cellular and Molecular Life Sciences

, Volume 72, Issue 18, pp 3575–3586 | Cite as

Maternal PCBP1 determines the normal timing of pronucleus formation in mouse eggs

  • Zhonghua Shi
  • Chun Zhao
  • Ye Yang
  • Hui Teng
  • Ying Guo
  • Minyue Ma
  • Xuejiang Guo
  • Zuomin Zhou
  • Ran HuoEmail author
  • Qi ZhouEmail author
Research Article


In mammals, pronucleus formation, a landmark event for egg activation and fertilization, is critical for embryonic development. However, the mechanisms underlying pronucleus formation remain unclear. Increasing evidence has shown that the transition from a mature egg to a developing embryo and the early steps of development are driven by the control of maternal cytoplasmic factors. Herein, a two-dimensional-electrophoresis-based proteomic approach was used in metaphase II and parthenogenetically activated mouse eggs to search for maternal proteins involved in egg activation, one of which was poly(rC)-binding protein 1 (PCBP1). Phosphoprotein staining indicated that PCBP1 displayed dephosphorylation in parthenogenetically activated egg, which possibly boosts its ability to bind to mRNAs. We identified 75 mRNAs expressed in mouse eggs that contained the characteristic PCBP1-binding CU-rich sequence in the 3′-UTR. Among them, we focused on H2a.x mRNA, as it was closely related to pronucleus formation in Xenopus oocytes. Further studies suggested that PCBP1 could bind to H2a.x mRNA and enhance its stability, thus promoting mouse pronucleus formation during parthenogenetic activation of murine eggs, while the inhibition of PCBP1 evidently retarded pronucleus formation. In summary, these data propose that PCBP1 may serve as a novel maternal factor that is required for determining the normal timing of pronucleus formation.


hnRNP E1 mRNA stabilization Pronuclear development Microinjection 



We are grateful to Prof. Qiang Wang (Nanjing Medical University) for providing anti-myc antibody. We gratefully acknowledge Liwen Bianji for editing the article. This work is supported by the China 973 Program (2012CB944704) and the National Science Foundation of China (30700275).

Supplementary material

18_2015_1905_MOESM1_ESM.xlsx (13 kb)
Supplemental Table 1. The identification information of PCBP1 protein. (XLSX 12 kb)
18_2015_1905_MOESM2_ESM.xlsx (19 kb)
Supplemental Table 2. Maternal mRNAs suggested to be potential binding targets for PCBP1. (XLSX 19 kb)
18_2015_1905_MOESM3_ESM.tif (2.1 mb)
Supplemental Fig. 1. Only one band with predicted molecular weight was present on western blots of ovary protein extracts using anti-PCBP1 (a) or anti-H2A.X (b) antibody confirmed the specificity of these antibodies. (TIFF 2153 kb)
18_2015_1905_MOESM4_ESM.tif (10.4 mb)
Supplemental Fig. 2. RNase-free PBS (control group) or exogenous myc-Pcbp1 mRNA (overexpression group) was microinjected into MII eggs and then immunofluorescence detecting with anti-myc Tag antibody indicated that myc-PCBP1 protein was efficiently overexpressed. (TIFF 10699 kb)

Supplemental movie 1. The process of pronucleus formation in non-injected group. (MOV 1011 kb)

Supplemental movie 2. The process of pronucleus formation in IgG-injected group. (MOV 859 kb)

Supplemental movie 3. The process of pronucleus formation in anti-PCBP1 antibody-injected group. (MOV 1222 kb)


  1. 1.
    Horner VL, Wolfner MF (2008) Transitioning from egg to embryo: triggers and mechanisms of egg activation. Dev Dyn 237:527–544. doi: 10.1002/dvdy.21454 CrossRefPubMedGoogle Scholar
  2. 2.
    Krauchunas AR, Wolfner MF (2013) Molecular changes during egg activation. Curr Top Dev Biol 102:267–292. doi: 10.1016/B978-0-12-416024-8.00010-6 PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Malcuit C, Kurokawa M, Fissore RA (2006) Calcium oscillations and mammalian egg activation. J Cell Physiol 206:565–573. doi: 10.1002/jcp.20471 CrossRefPubMedGoogle Scholar
  4. 4.
    Stitzel ML, Seydoux G (2007) Regulation of the oocyte-to-zygote transition. Science 316:407–408. doi: 10.1126/science.1138236 CrossRefPubMedGoogle Scholar
  5. 5.
    Collas P (1998) Cytoplasmic control of nuclear assembly. Reprod Fertil Dev 10:581–592CrossRefPubMedGoogle Scholar
  6. 6.
    Poccia D, Collas P (1997) Nuclear envelope dynamics during male pronuclear development. Dev Growth Differ 39:541–550CrossRefPubMedGoogle Scholar
  7. 7.
    Perreault SD, Naish SJ, Zirkin BR (1987) The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disulfide bond content. Biol Reprod 36:239–244CrossRefPubMedGoogle Scholar
  8. 8.
    Clift D, Schuh M (2013) Restarting life: fertilization and the transition from meiosis to mitosis. Nat Rev Mol Cell Biol 14:549–562. doi: 10.1038/nrm3643 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Li L, Zheng P, Dean J (2010) Maternal control of early mouse development. Development 137:859–870. doi: 10.1242/dev.039487 PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Nakamura H, Wu C, Kuang J, Larabell C, Etkin LD (2000) XCS-1, a maternally expressed gene product involved in regulating mitosis in Xenopus. J Cell Sci 113(Pt 13):2497–2505PubMedGoogle Scholar
  11. 11.
    Ma M, Guo X, Wang F, Zhao C, Liu Z, Shi Z, Wang Y, Zhang P, Zhang K, Wang N, Lin M, Zhou Z, Liu J, Li Q, Wang L, Huo R, Sha J, Zhou Q (2008) Protein expression profile of the mouse metaphase-II oocyte. J Proteome Res 7:4821–4830. doi: 10.1021/pr800392s CrossRefPubMedGoogle Scholar
  12. 12.
    Wang S, Kou Z, Jing Z, Zhang Y, Guo X, Dong M, Wilmut I, Gao S (2010) Proteome of mouse oocytes at different developmental stages. Proc Natl Acad Sci USA 107:17639–17644. doi: 10.1073/pnas.1013185107 PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Ko JL and Loh HH (2005) Poly C binding protein, a single-stranded DNA binding protein, regulates mouse mu-opioid receptor gene expression. J Neurochem 93:749–761. doi: 10.1111/j.1471-4159.2005.03089.x CrossRefPubMedGoogle Scholar
  14. 14.
    Kim JH, Hahm B, Kim YK, Choi M, Jang SK (2000) Protein-protein interaction among hnRNPs shuttling between nucleus and cytoplasm. J Mol Biol 298:395–405. doi: 10.1006/jmbi.2000.3687 CrossRefPubMedGoogle Scholar
  15. 15.
    Weiss IM, Liebhaber SA (1994) Erythroid cell-specific determinants of alpha-globin mRNA stability. Mol Cell Biol 14:8123–8132PubMedCentralPubMedGoogle Scholar
  16. 16.
    Weiss IM, Liebhaber SA (1995) Erythroid cell-specific mRNA stability elements in the alpha 2-globin 3′ nontranslated region. Mol Cell Biol 15:2457–2465PubMedCentralPubMedGoogle Scholar
  17. 17.
    Thiele BJ, Doller A, Kahne T, Pregla R, Hetzer R and Regitz-Zagrosek V (2004) RNA-binding proteins heterogeneous nuclear ribonucleoprotein A1, E1, and K are involved in post-transcriptional control of collagen I and III synthesis. Circ Res 95:1058–1066. doi: 10.1161/01.RES.0000149166.33833.08 CrossRefPubMedGoogle Scholar
  18. 18.
    Paulding WR, Czyzyk-Krzeska MF (1999) Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3′-untranslated region. J Biol Chem 274:2532–2538CrossRefPubMedGoogle Scholar
  19. 19.
    Czyzyk-Krzeska MF, Bendixen AC (1999) Identification of the poly(C) binding protein in the complex associated with the 3′ untranslated region of erythropoietin messenger RNA. Blood 93:2111–2120PubMedGoogle Scholar
  20. 20.
    Evans JR, Mitchell SA, Spriggs KA, Ostrowski J, Bomsztyk K, Ostarek D, Willis AE (2003) Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo. Oncogene 22:8012–8020. doi: 10.1038/sj.onc.1206645 CrossRefPubMedGoogle Scholar
  21. 21.
    Pickering BM, Mitchell SA, Spriggs KA, Stoneley M, Willis AE (2004) Bag-1 internal ribosome entry segment activity is promoted by structural changes mediated by poly(rC) binding protein 1 and recruitment of polypyrimidine tract binding protein 1. Mol Cell Biol 24:5595–5605. doi: 10.1128/MCB.24.12.5595-5605.2004 PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Pickering BM, Mitchell SA, Evans JR, Willis AE (2003) Polypyrimidine tract binding protein and poly r(C) binding protein 1 interact with the BAG-1 IRES and stimulate its activity in vitro and in vivo. Nucleic Acids Res 31:639–646PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Zhong N, Radu G, Ju W and Brown WT (2005) Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C. Biochem Biophys Res Commun 338:855–861. doi: 10.1016/j.bbrc.2005.10.020 CrossRefPubMedGoogle Scholar
  24. 24.
    Lim J, Hao T, Shaw C, Patel AJ, Szabo G, Rual JF, Fisk CJ, Li N, Smolyar A, Hill DE, Barabasi AL, Vidal M and Zoghbi HY (2006) A protein–protein interaction network for human inherited ataxias and disorders of Purkinje cell degeneration. Cell 125:801–814. doi: 10.1016/j.cell.2006.03.032 CrossRefPubMedGoogle Scholar
  25. 25.
    Xia M, He H, Wang Y, Liu M, Zhou T, Lin M, Zhou Z, Huo R, Zhou Q, Sha J (2012) PCBP1 is required for maintenance of the transcriptionally silent state in fully grown mouse oocytes. Cell Cycle 11:2833–2842. doi: 10.4161/cc.21169 CrossRefPubMedGoogle Scholar
  26. 26.
    Huang XY, Guo XJ, Shen J, Wang YF, Chen L, Xie J, Wang NL, Wang FQ, Zhao C, Huo R, Lin M, Wang X, Zhou ZM, Sha JH (2008) Construction of a proteome profile and functional analysis of the proteins involved in the initiation of mouse spermatogenesis. J Proteome Res 7:3435–3446. doi: 10.1021/pr800179h CrossRefPubMedGoogle Scholar
  27. 27.
    Holcik M, Liebhaber SA (1997) Four highly stable eukaryotic mRNAs assemble 3′ untranslated region RNA-protein complexes sharing cis and trans components. Proc Natl Acad Sci USA 94:2410–2414PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Mignone F, Grillo G, Licciulli F, Iacono M, Liuni S, Kersey PJ, Duarte J, Saccone C and Pesole G (2005) UTRdb and UTRsite: a collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs. Nucleic Acids Res 33:D141–D146. doi: 10.1093/nar/gki021 PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Evsikov AV, Graber JH, Brockman JM, Hampl A, Holbrook AE, Singh P, Eppig JJ, Solter D, Knowles BB (2006) Cracking the egg: molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo. Genes Dev 20:2713–2727. doi: 10.1101/gad.1471006 CrossRefPubMedGoogle Scholar
  30. 30.
    Yeap BB, Voon DC, Vivian JP, McCulloch RK, Thomson AM, Giles KM, Czyzyk-Krzeska MF, Furneaux H, Wilce MC, Wilce JA, Leedman PJ (2002) Novel binding of HuR and poly(C)-binding protein to a conserved UC-rich motif within the 3′-untranslated region of the androgen receptor messenger RNA. J Biol Chem 277:27183–27192. doi: 10.1074/jbc.M202883200 CrossRefPubMedGoogle Scholar
  31. 31.
    Dai Y, Lee C, Hutchings A, Sun Y, Moor R (2000) Selective requirement for Cdc25C protein synthesis during meiotic progression in porcine oocytes. Biol Reprod 62:519–532CrossRefPubMedGoogle Scholar
  32. 32.
    Agrawal GK, Thelen JJ (2005) Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins. Proteomics 5:4684–4688. doi: 10.1002/pmic.200500021 CrossRefPubMedGoogle Scholar
  33. 33.
    Meng Q, Rayala SK, Gururaj AE, Talukder AH, O’Malley BW, Kumar R (2007) Signaling-dependent and coordinated regulation of transcription, splicing, and translation resides in a single coregulator, PCBP1. Proc Natl Acad Sci USA 104:5866–5871. doi: 10.1073/pnas.0701065104 PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Leffers H, Dejgaard K, Celis JE (1995) Characterisation of two major cellular poly(rC)-binding human proteins, each containing three K-homologous (KH) domains. Eur J Biochem 230:447–453CrossRefPubMedGoogle Scholar
  35. 35.
    Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W (2002) Histone H2A variants H2AX and H2AZ. Curr Opin Genet Dev 12:162–169CrossRefPubMedGoogle Scholar
  36. 36.
    Kleinschmidt JA, Steinbeisser H (1991) DNA-dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing. EMBO J 10:3043–3050PubMedCentralPubMedGoogle Scholar
  37. 37.
    Zuccotti M, Garagna S, Merico V, Monti M, Alberto Redi C (2005) Chromatin organisation and nuclear architecture in growing mouse oocytes. Mol Cell Endocrinol 234:11–17. doi: 10.1016/j.mce.2004.08.014 CrossRefPubMedGoogle Scholar
  38. 38.
    Tuteja N, Singh MB, Misra MK, Bhalla PL, Tuteja R (2001) Molecular mechanisms of DNA damage and repair: progress in plants. Crit Rev Biochem Mol Biol 36:337–397. doi: 10.1080/20014091074219 CrossRefPubMedGoogle Scholar
  39. 39.
    Dimitrov S, Dasso MC, Wolffe AP (1994) Remodeling sperm chromatin in Xenopus laevis egg extracts: the role of core histone phosphorylation and linker histone B4 in chromatin assembly. J Cell Biol 126:591–601CrossRefPubMedGoogle Scholar
  40. 40.
    Racki WJ, Richter JD (2006) CPEB controls oocyte growth and follicle development in the mouse. Development 133:4527–4537. doi: 10.1242/dev.02651 CrossRefPubMedGoogle Scholar
  41. 41.
    Inoue A, Zhang Y (2014) Nucleosome assembly is required for nuclear pore complex assembly in mouse zygotes. Nat Struct Mol Biol 21:609–616. doi: 10.1038/nsmb.2839 CrossRefPubMedGoogle Scholar
  42. 42.
    Wu BJ, Dong FL, Ma XS, Wang XG, Lin F, Liu HL (2014) Localization and expression of histone H2A variants during mouse oogenesis and preimplantation embryo development. Genet Mol Res 13:5929–5939. doi: 10.4238/2014.August.7.8 CrossRefPubMedGoogle Scholar
  43. 43.
    McLaren A, Bowman P (1973) Genetic effects on the timing of early development in the mouse. J Embryol Exp Morphol 30:491–498PubMedGoogle Scholar
  44. 44.
    Lonergan P, Khatir H, Piumi F, Rieger D, Humblot P, Boland MP (1999) Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos. J Reprod Fertil 117:159–167CrossRefPubMedGoogle Scholar
  45. 45.
    Fenwick J, Platteau P, Murdoch AP, Herbert M (2002) Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro. Hum Reprod 17:407–412CrossRefPubMedGoogle Scholar
  46. 46.
    Koyama K, Kang SS, Huang W, Yanagawa Y, Takahashi Y, Nagano M (2014) Aging-related changes in in vitro-matured bovine oocytes: oxidative stress, mitochondrial activity and ATP content after nuclear maturation. J Reprod Dev 60:136–142PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel 2015

Authors and Affiliations

  • Zhonghua Shi
    • 1
    • 3
  • Chun Zhao
    • 1
    • 3
  • Ye Yang
    • 1
    • 3
  • Hui Teng
    • 1
  • Ying Guo
    • 1
  • Minyue Ma
    • 1
    • 2
  • Xuejiang Guo
    • 1
  • Zuomin Zhou
    • 1
  • Ran Huo
    • 1
    Email author
  • Qi Zhou
    • 2
    Email author
  1. 1.State Key Laboratory of Reproductive Medicine, Department of Histology and EmbryologyNanjing Medical UniversityNanjingPeople’s Republic of China
  2. 2.State Key Laboratory of Reproductive Biology, Institute of ZoologyChinese Academy of SciencesBeijingPeople’s Republic of China
  3. 3.State Key Laboratory of Reproductive Medicine, Nanjing Maternity and Child Health HospitalNanjing Medical UniversityNanjingPeople’s Republic of China

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