Microscopic Observation, Three-dimensional Reconstruction, and Volume Measurements of Sperm Nuclei

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.