Dynamics of Male and Female Chromatin during Karyogamy in Rice Zygotes

Abstract

In angiosperms, the conversion of an egg cell into a zygote involves two sequential gametic processes: plasmogamy, the fusion of the plasma membranes of male and female gametes, and karyogamy, the fusion of the gametic nuclei. In this study, the nuclei and nuclear membranes of rice (Oryza sativa) gametes were fluorescently labeled using histones 2B-green fluorescent protein/red fluorescent protein and Sad1/UNC-84-domain protein2-green fluorescent protein, respectively, which were heterologously expressed. These gametes were fused in vitro to produce zygotes, and the nuclei and nuclear membranes in the zygotes were observed during karyogamy. The results indicated that the sperm nucleus migrates adjacent to the egg nucleus 5 to 10 min after plasmogamy via an actin cytoskelton, and the egg chromatin then appears to move unidirectionally into the sperm nucleus through a possible nuclear connection. The enlargement of the sperm nucleus accompanies this possible chromatin remodeling. Then, 30 to 70 min after fusion, the sperm chromatin begins to decondense with the completion of karyogamy. Based on these observations, the development of early rice zygotes from plasmogamy to karyogamy was divided into eight stages, and using reverse transcription PCR analyses, paternal and de novo synthesized transcripts were separately detected in zygotes at early and late karyogamy stages, respectively.

Figure 1.

Rice gametes expressing the H2B-GFP, H2B-RFP, or SUN2-GFP fusion protein. A and B, An egg cell isolated from transgenic rice expressing H2B-GFP under control of the ubiquitin promoter visualized with bright-field (A) and fluorescence (B) microscopy. The arrowheads indicate nucleoli. C and D, An egg cell isolated from transgenic rice expressing H2B-RFP under control of the DD45 promoter visualized with bright-field (C) and fluorescence (D) microscopy. The arrowheads indicate nucleoli. E and F, A pollen grain expressing H2B-GFP under control of the ubiquitin promoter visualized with bright-field (E) and fluorescence (F) microscopy. Arrowheads and the arrow indicate sperm and vegetative nuclei, respectively. G and H, A pollen grain expressing H2B-GFP releasing its content in mannitol solution visualized with bright-field (G) and fluorescence (H) microscopy. Arrowheads indicate sperm cells, and the arrow indicates a possible vegetative nucleus. I to K, An egg cell expressing SUN2-GFP under control of the ubiquitin promoter visualized with fluorescence (I) and bright-field (J) microscopy. K contains the merged images of I and J. L to N, A pollen grain expressing SUN2-GFP releasing its content in mannitol solution (L). Two released sperm cells enclosed within the square in L were visualized with fluorescence microscopy (M). N contains the merged bright-field and fluorescent images. Bars = 20 μm. [See online article for color version of this figure.]

Figure 2.

Karyogamy in rice zygotes. A and B, Alignment of a sperm cell expressing H2B-GFP with a wild-type egg cell on one of the electrodes under an alternating current field in a fusion droplet. C to R, Gametes in B were electrically fused, and the resulting fused cell (zygote) was observed at the time points indicated. Top shows fluorescent images, and bottom shows merged bright-field and fluorescent images. Black and white arrowheads indicate sperm cells expressing H2B-GFP and nucleoli, respectively. Bar = 20 μm. [See online article for color version of this figure.]

Figure 3.

Timetable of sperm chromatin decondensation in the fused nuclei of 50 independent rice zygotes. Zygotes were produced by in vitro fusion of a wild-type egg cell with a sperm cell expressing a H2B-GFP fusion protein, and the fluorescence signal was observed every 15 to 20 min after fusion. The y axis indicates the number of zygotes in which initial decondensation of sperm chromatin was observed.

Figure 4.

Effects of latrunculin B and oryzalin on karyogamy in fused rice gametes. Egg cells were treated with inhibitors and used for electrofusion with sperm cells expressing H2B-GFP. A to F, Egg cells and fused cells that were treated with 200 nm latrunculin B. G to L, Egg cells and fused cells that were treated with 2 μm latrunculin B. M to R, Egg cells and fused cells that were treated with 100 μm oryzalin. After in vitro fertilization, the fused cells were observed at 10 to 15 min (A, B, G, H, M, and N), 80 to 120 min (C, D, I, J, O, and P), and 10 to 12 h (E, F, K, L, Q, and R) after fusion. In zygotes treated with 2 μm latrunculin B, the decondensation of sperm chromatin was not observed for 2 h after fusion (I and J), and the signal from H2B-GFP was detected in both sperm and egg nuclei at 12 h after fusion. Bar = 20 μm. [See online article for color version of this figure.]

Figure 5.

Actin organization and sperm nucleus migration. Egg cells expressing Lifeact-tagRFP without (A–F) or treated with (G–K) 2 μm latrunculin B were fused with sperm cells expressing H2B-GFP, and resultant zygotes were observed at the time point indicated. An arrow indicates the nucleus surrounded by actin filaments. Bars = 20 μm.

Figure 6.

Chromatin state during or after nuclear fusion in rice zygotes. Zygotes produced by electrofusion of a sperm cell expressing H2B-GFP and an egg cell expressing H2B-RFP were observed using a CLS microscope. The fluorescent signals from H2B-GFP and H2B-RFP are presented in top (A, D, G, J, M, P, and S) and middle (B, E, H, K, N, Q, and T), respectively, whereas bottom (C, F, I, L, O, R, and U) shows merged images. Bar = 10 μm.

Figure 7.

Changes over time in the volume of a rice sperm nucleus during contact with an egg nucleus. Zygotes produced by electrofusion of sperm cells expressing SUN2-GFP and an egg cell expressing H2B-RFP were observed using a CLS microscope. A, C, E, G, and I are CLS images, and B, D, F, H, and J are three-dimensional structure images. An arrowhead indicates the possible fusion point between the nuclear membranes. Arrows indicate the fusing area between sperm and egg nuclear membranes. Numbers in B, D, F, H, and J represent the volume of the sperm nucleus. Bars = 10 μm. K, Changes in the volume of the sperm nucleus before (A and B) or after (G and H) egg chromatin incursion. The data are means ± sds of three sperm nuclei.

Figure 8.

A schematic diagram of the progression of karyogamy in rice zygotes. After gamete fusion, the sperm nucleus migrates inside of the fused cell to appose the egg nucleus through possible actin filament-dependent machinery (stages I–III). Through a possible internuclear connection, female chromatin moves into the sperm nucleus, but sperm chromatin remains tightly packed (stage IV). Male chromatin begins decondensing (stage V), and male chromatin further uniformly distributes in accordance with the progression of nuclear fusion (stages VI and VII). The karyogamic event is finally completed at stage VIII, and thereafter, the zygote develops into an embryo. Light-blue and pink circles indicate sperm and egg nuclei/chromatin, respectively. Yellow indicates the merged sperm and egg chromatin.

Figure 9.

Expression patterns of five genes putatively induced after fertilization in rice zygotes. Semiquantitative RT-PCR was performed on total RNAs isolated from 10 to 12 zygotes at the appropriate karyogamic stages. Ubiquitin mRNA was used as an internal control. Supplemental Table S1 shows primer sequences.