Numb regulates meiotic spindle organisation in mouse oocytes
H. Lv A , J. C. Wang A , K. L. Wu A , X. Gao A , L. C. Wang B , L. You A and Z. J. Chen A CA Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Key Laboratory of Reproductive Medicine, Shandong Province, Jinan 250021, China.
B Department of Central Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China.
C Corresponding author. Email: chenzijiang@hotmail.com
Reproduction, Fertility and Development 22(4) 664-672 https://doi.org/10.1071/RD09236
Submitted: 14 September 2009 Accepted: 12 October 2009 Published: 11 March 2010
Abstract
Numb is an adaptor protein that controls the fate of cells in different species through asymmetrical inheritance by sibling cells during division. It has been investigated extensively in mitosis, mostly in neural progenitor cells, but its function in meiosis remains unknown. The present study was designed to investigate the expression, subcellular localisation and functional roles of Numb during mouse oocyte meiotic maturation. Using real-time polymerase chain reaction and western blotting, we found that the expression of Numb increased from the germinal vesicle (GV) to MII stages. Immunofluorescent staining revealed that Numb was mainly concentrated in the GV before meiosis resumption, aggregated in the vicinity of the chromosomes after GV breakdown and then localised to the spindle poles from prometaphase I to MII. Nocodazole treatment resulted in spindle destruction and Numb diffusion into the cytoplasm. However, Numb appeared at the spindle poles again once the spindles had formed when nocodazole-treated oocytes were washed and cultured for spindle recovery. Depletion of Numb by RNA interference resulted in chromosome misalignment, spindle deformation and even doubled spindle formation. Our results suggest that Numb is critical for spindle organisation during mouse oocytes meiosis. The present study provides evidence of a new function for Numb in addition to its action as a cell fate-determining factor.
Additional keywords: cell fate-determining factor, meiosis, mouse oocyte, Numb, spindle organisation.
Acknowledgements
This work was supported by the National Basic Research Program of China (973 Program, 2006CB944004), National Natural Science Foundation (China, Canada; 30611120524) and Natural Science Foundation of Shandong Province (Z2002C05).
Battaglia, D. E. , Goodwin, P. , Klein, N. A. , and Soules, M. R. (1996). Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum. Reprod. 11, 2217–2222.
| PubMed |
Bray, S. J. (2006). Notch signalling: a simple pathway becomes complex. Nat. Rev. Mol. Cell Biol. 7, 678–689.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Brunet, S. , and Maro, B. (2005). Cytoskeleton and cell cycle control during meiotic maturation of the mouse oocyte: integrating time and space. Reproduction 130, 801–811.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Buescher, M. , Yeo, S. L. , Udolph, G. , Zavortink, M. , Yang, X. , Tear, G. , and Chia, W. (1998). Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells. Genes Dev. 12, 1858–1870.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Dumont, J. , Million, K. , Sunderland, K. , Rassinier, P. , Lim, H. , Leader, B. , and Verlhac, M. H. (2007). Formin-2 is required for spindle migration and for the late steps of cytokinesis in mouse oocytes. Dev. Biol. 301, 254–265.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Horvitz, H. R. , and Herskowitz, I. (1992). Mechanisms of asymmetric cell division: two Bs or not two Bs, that is the question. Cell 68, 237–255.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Jacobs, C. , and Shapiro, L. (1998). Microbial asymmetric cell division: localization of cell fate determinants. Curr. Opin. Genet. Dev. 8, 386–391.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Jan, Y. N. , and Jan, L. Y. (1998). Asymmetric cell division. Nature 392, 775–778.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Knoblich, J. A. , Jan, L. Y. , and Jan, Y. N. (1995). Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Knoblich, J. A. , Jan, L. Y. , and Jan, Y. N. (1997). The N terminus of the Drosophila Numb protein directs membrane association and actindependent asymmetric localization. Proc. Natl Acad. Sci. USA 94, 13 005–13 010.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Livak, K. J. , and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCt method. Methods 25, 402–408.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Longo, F. J. , and Chen, D. Y. (1985). Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus. Dev. Biol. 107, 382–394.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Lu, B. , Jan, L. Y. , and Jan, Y. N. (1998). Asymmetric cell division: lessons from flies and worms. Curr. Opin. Genet. Dev. 8, 392–399.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Manandhar, G. , Schatten, H. , and Sutovsky, P. (2005). Centrosome reduction during gametogenesis and its significance. Biol. Reprod. 72, 2–13.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Maro, B. , and Verlhac, M. H. (2002). Polar body formation: New rules for asymmetric divisions. Nat. Cell Biol. 4, E281–E283.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Maro, B. , Johnson, M. H. , Pickering, S. J. , and Flach, G. (1984). Changes in the actin distribution during fertilisation of the mouse egg. J. Embryol. Exp. Morphol. 81, 211–237.
| PubMed |
Maro, B. , Howlett, S. K. , and Webb, M. (1985). Non-spindle microtubule organizing centers in metaphase II-arrested mouse oocytes. J. Cell Biol. 101, 1665–1672.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Messinger, S. M. , and Albertini, D. F. (1991). Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J. Cell Sci. 100, 289–298.
| PubMed |
Pece, S. , Serresi, M. , Santolini, E. , Capra, M. , Hulleman, E. , Galimberti, V. , Zurrida, S. , Maisonneuve, P. , Viale, G. , and Di Fiore, P. P. (2004). Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis. J. Cell Biol. 167, 215–221.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Roegiers, F. , and Jan, J. Y. (2004). Asymmetric cell division. Curr. Opin. Cell Biol. 16, 195–205.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Roncarati, R. , Sestan, N. , Scheinfeld, M. H. , Berechid, B. E. , Lopez, P. A. , Meucci, O. , McGlade, J. C. , Rakic, P. , and D’Adamio, L. (2002). The gamma-secretase-generated intracellular domain of beta-amyloid precursor protein binds Numb and inhibits Notch signaling. Proc. Natl Acad. Sci. USA 99, 7102–7107.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Schuh, M. , and Ellenberg, J. (2007). Self-organization of MTOCs replaces centrosome function during a centrosomal spindle assembly in live mouse oocytes. Cell 130, 484–498.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Shen, Q. , Zhong, W. , Jan, Y. N. , and Temple, S. (2002). Asymmetric Numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. Development 129, 4843–4853.
| PubMed |
Spana, E. P. , and Doe, C. Q. (1996). Numb antagonizes Notch signaling to specify sibling neuron cell fates. Neuron 17, 21–26.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Spana, E. P. , Kopczynski, C. , Goodman, C. S. , and Doe, C. Q. (1995). Asymmetric localization of Numb autonomously determines sibling neuron identity in the Drosophila CNS. Development 121, 3489–3494.
| PubMed |
Strome, S. (1993). Determination of cleavage planes. Cell 72, 3–6.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Tong, C. , Fan, H. Y. , Lian, L. , Li, S. W. , Chen, D. Y. , Schatten, H. , and Sun, Q. Y. (2002). Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation, fertilization, and early embryonic mitosis. Biol. Reprod. 67, 546–554.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Uemura, T. , Shepherd, S. , Ackerman, L. , Jan, L. Y. , and Jan, Y. N. (1989). Numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos. Cell 58, 349–360.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Verdi, J. M. , Schmandt, R. , Bashirullah, A. , Jacob, S. , Salvino, R. , Craig, C. G. , Program, A. E. , Lipshitz, H. D. , and McGlade, C. J. (1996). Mammalian NUMB is an evolutionarily conserved signaling adapter protein that specifies cell fate. Curr. Biol. 6, 1134–1145.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Verlhac, M. H. , Lefebvre, C. , Guillaud, P. , Rassinier, P. , and Maro, B. (2000). Asymmetric division in mouse oocytes: with or without Mos. Curr. Biol. 10, 1303–1306.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Vinot, S. , Le, T. , Maro, B. , and Louvet-Vallée, S. (2004). Two PAR6 proteins become asymmetrically localized during establishment of polarity in mouse oocytes. Curr. Biol. 14, 520–525.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Wirtz-Peitz, F. , Nishimura, T. , and Knoblich, J. A. (2008). Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization. Cell 135, 161–173.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Xiong, B. , Li, S. , Ai, J. S. , Yin, S. , Ouyang, Y. C. , Sun, S. C. , Chen, D. Y. , and Sun, Q. Y. (2008). BRCA1 is required for meiotic spindle assembly and spindle assembly checkpoint activation in mouse oocytes. Biol. Reprod. 79, 718–726.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yan, B. , Omar, F. M. , Das, K. , Ng, W. H. , Lim, C. , Shiuan, K. , Yap, C. T. , and Salto-Tellez, M. (2008). Characterization of Numb expression in astrocytomas. Neuropathology 28, 479–484.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yao, L. J. , Zhong, Z. S. , Zhang, L. S. , Chen, D. Y. , Schatten, H. , and Sun, Q. Y. (2004). Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos. Biol. Reprod. 70, 1392–1399.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yu, L. Z. , Xiong, B. , Gao, W. X. , Wang, C. M. , and Zhong, Z. S. , et al. (2007). MEK1/2 regulates microtubule organization, spindle pole tethering and asymmetric division during mouse oocyte meiotic maturation. Cell Cycle 6, 330–338.
| PubMed |
Zheng, Z. Y. , Li, Q. Z. , Chen, D. Y. , Schatten, H. , and Sun, Q. Y. (2005). Translocation of phospho-protein kinase Cs implies their roles in meiotic-spindle organization, polar-body emission and nuclear activity in mouse eggs. Reproduction 129, 229–234.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zhong, W. , Feder, J. N. , Jiang, M. M. , Jan, L. Y. , and Jan, Y. N. (1996). Asymmetric localization of a mammalian Numb homologue during mouse cortical neurogenesis. Neuron 17, 43–53.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
