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

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Abstract

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.

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Acknowledgments

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).

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    Authors and Affiliations

    1. State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing, 210029, People’s Republic of China

      Zhonghua Shi, Chun Zhao, Ye Yang, Hui Teng, Ying Guo, Minyue Ma, Xuejiang Guo, Zuomin Zhou & Ran Huo

    2. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China

      Minyue Ma & Qi Zhou

    3. State Key Laboratory of Reproductive Medicine, Nanjing Maternity and Child Health Hospital, Nanjing Medical University, Nanjing, 210011, People’s Republic of China

      Zhonghua Shi, Chun Zhao & Ye Yang

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    1. Zhonghua Shi
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    Correspondence to Ran Huo or Qi Zhou.

    Additional information

    Z. Shi, C. Zhao, and Y. Yang contributed equally to this work.

    Electronic supplementary material

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    Supplemental Table 1. The identification information of PCBP1 protein. (XLSX 12 kb)

    Supplemental Table 2. Maternal mRNAs suggested to be potential binding targets for PCBP1. (XLSX 19 kb)

    18_2015_1905_MOESM3_ESM.tif

    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

    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)

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    Shi, Z., Zhao, C., Yang, Y. et al. Maternal PCBP1 determines the normal timing of pronucleus formation in mouse eggs. Cell. Mol. Life Sci. 72, 3575–3586 (2015). https://doi.org/10.1007/s00018-015-1905-3

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