[Frontiers in Bioscience E2, 1254-1264, June 1, 2010]

PAK1 regulates spindle microtubule organization during oocyte meiotic maturation

Sheng-Li Lin1,2, Shu-Tao Qi1,2, Shao-Chen Sun1, Ya-Peng Wang1,2, Heide Schatten3, Qing-Yuan Sun1

1State Key Laboratory of Reproductive Biology, Institute of Zoology, Beijing, China, 2Graduate School, Chinese Academy of Sciences, Beijing, China, 3Department of Veterinary Pathobiology, University of Missouri-Columbia, MO

TABLE OF CONENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Antibodies
3.2. Oocyte collection and culture
3.3. Taxol and nocodazole treatment of oocytes
3.4. IPA-3 treatment of oocytes
3.5. Morpholino oligonucleotide injection
3.6. Immunofluorescence and Confocal Microscopy
3.7. Immunoblotting Analysis
3.8. Statistics
4. Results
4.1. Expression of PAK1 during meiosis in mouse oocytes
4.2. Subcellular localization of PAK1 during mouse oocyte meiotic maturation
4.3. Localization of PAK1 in mouse oocytes treated with taxol and nocodazole
4.4. Spindle organization defects and decreased polar body extrusion after treatment with IPA-3
4.5.Spindle organization defects and decreased polar body extrusion after injection of PAK1 morpholino oligonucleotide
4.6. Effect of PAK1 inhibition on proper p-MEK1/2 localization
5. Discussion
6. Acknowledgement
7. References

1. ABSTRACT

P21-activated kinase 1 (PAK1), an effector of Rho GTPase Rac 1 and Cdc42, is required for mitotic progression. However, its functions in meiosis are unclear. In the present study, we examined the expression, localization and function of PAK1 during mouse oocyte meiotic maturation and found that PAK1 was mainly associated with the meiotic spindle microtubules. Taxol treatment resulted in localization of PAK1 on spindle and aster microtubules, while nocodazole treatment induced the dispersion of PAK1 protein into the cytoplasm. Loss-of-function of PAK1 by both inhibitor treatment and morpholino oligonuclotide injection caused disorganized spindles, decreased polar body extrusion and misaligned chromosomes. In addition, inhibition of PAK1 resulted in abnormal localization of mitogen-activated protein kinase kinase (MEK). Taken together, our results suggest that PAK1 plays an important role in spindle assembly and chromosome alignment during mouse oocyte meiotic maturation.