精子细胞核的显微观察、三维重建和体积测量

Yukinosuke Ohnishi; Takashi Okamoto

doi:10.21769/BioProtoc.1437

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Peer-reviewed

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

精子细胞核的显微观察、三维重建和体积测量

YO Yukinosuke Ohnishi

Takashi Okamoto email

发布: 2015年04月05日第5卷第7期 DOI: 10.21769/BioProtoc.1437 浏览次数: 8731

评审: Tie LiuAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Aug 2014

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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 (卵细胞)

Materials and Reagents

Transgenic rice plants (Oryza sativa L. cv. Nipponbare) expressing SUN2-GFP or H2B-RFP fusion proteins that are targeted to nuclear membrane or nuclear chromatin, respectively (Ohnishi et al., 2014) (see Note 1)
Mannitol (Wako Chemicals USA, catalog number: 133-00845 )
370 mosmol/kg H₂O (330 mM) mannitol solution (autoclaved)
450 mosmol/kg H₂O (385 mM) mannitol solution (autoclaved)
Mineral oil (Sigma-Aldrich, catalog number: M8410-100ML )

Equipment

Inverted fluorescent microscope (OLYMPUS, model: BX-71 )
Confocal laser scanning (CLS) microscope (ZEISS, model: LMS 710 )
Non-treated plastic dishes with diameter of 3.5 cm (Iwaki, catalog number: 1000-035 )
Glass base dishes with diameter of 3.5 cm (Iwaki, catalog number: 3971-035 ) (see Note 2)
Coverslips (24 x 40 mm) (Thermo Fisher Scientific, catalog number: 125485J ) (siliconized at the edges with 5% dichloromethylsilane in 1,1,1-trichloroethane) (see Note 3)
Glass capillaries made from 50 μl aspirator tubes (Drummond Scientific Company, catalog number: 2-000-050 ) (Figure 1A-B) (see Note 4)
Manual handling injector (ST Science, type UJB)
Environmental chamber (Koito Industries Ltd., catalog number: K30-7248 )

Software

IMARIS software (Bitplane)
Note: A core scientific software module that delivers the necessary functionality for data management, visualization, analysis, segmentation and interpretation of 3D and 4D microscopy datasets.

Procedure

English

中文翻译

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Ohnishi, Y. and Okamoto, T. (2015). Microscopic Observation, Three-dimensional Reconstruction, and Volume Measurements of Sperm Nuclei. Bio-protocol 5(7): e1437. DOI: 10.21769/BioProtoc.1437.
Ohnishi, Y., Hoshino, R. and Okamoto, T. (2014). Dynamics of male and female chromatin during karyogamy in rice zygotes. Plant Physiol 165(4): 1533-1543.