Abstract
Karyogamy, a migration of the sperm nucleus toward the egg nucleus and their subsequent nuclear fusion, is an important biological event for initiating zygote formation toward early embryogenesis in angiosperms. However, how the male nucleus approaches and fuses with the female nucleus still remains unclear. Recently, time-lapse measurement of nuclear volume during karyogamic events revealed that the sperm nucleus enlarges during contact with the egg nucleus via possible one-directional migration of egg chromatin into sperm nucleus (Ohnishi et al., 2014). Here, we describe the protocol for microscopic observation, three-dimensional reconstruction, and volume measurements of sperm nuclei in rice zygotes/fused gametes, which are produced by an in vitro fertilization system (Uchiumi et al., 2006; Uchiumi et al., 2007). The present protocol will be applied for monitoring nuclear dynamics in cells during cell division, differentiation, de-differentiation and polarity formation as well as karyogamy progression.
Keywords: Egg cell, Karyogamy, Nuclear fusion, Sperm cell, Zygote
Materials and Reagents
Equipment
Software
Procedure
Figure 1. Glass capillary (A, B) and mannitol droplets on coverslip (C, D). A. A glass capillary drawn by hand. Tip region was boxed. B. Tip opening of the glass capillary in panel A. C and D. A photo (C) and an image (D) of mannitol droplets inside the mineral oil over the siliconized coverslip. Bars = 1 cm in A and C, 200 μm in B.
Notes
Acknowledgments
This work was supported, in part, by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nos. 21112007 and 26113715 to T.O.) and from the Japan Society for the Promotion of Science (No. 25650083 to T.O.). We thank Ms. Hiroki Maeda and Ms. Tomoko Mochizuki (Tokyo Metropolitan University) for technical assistance, and Dr. Min Yvonne Kim (University of California, Berkeley) for critical reading of the protocol.
References
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