Development of Polyspermic Rice Zygotes

Abstract

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In most animals and fucoid algae, polyspermy block occurs at the plasmogamy step. Because the polyspermy barrier in animals and in fucoid algae is incomplete, polyspermic zygotes are generated by multiple fertilization events. However, these polyspermic zygotes with extra centrioles from multiple sperms show aberrant nuclear and cell division. In angiosperms, polyspermy block functions in the egg cell and the central cell to promote faithful double fertilization, although the mechanism of polyspermy block remains unclear. In contrast to the case in animals and fucoid algae, polyspermic zygotes formed in angiosperms are not expected to die because angiosperms lack centrosomes. However, there have been no reports on the developmental profiles of polyspermic zygotes at cellular level in angiosperms. In this study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells, and monitored their developmental profiles. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle, as observed during mitosis of a diploid zygote. The two-celled proembryos further developed and regenerated into triploid plants. These findings suggest that polyspermic plant zygotes have the potential to form triploid embryos. Polyspermy in angiosperms might be a pathway for the formation of triploid plants, which can contribute significantly to the formation of autopolyploids.

Figure 1.

Production and development of polyspermic rice zygotes. A, Schematic illustration of procedure to produce polyspermic rice zygotes. An egg cell was fused with a sperm cell to produce a monospermic zygote (first fusion). Within 10 min after first fusion, the monospermic zygote was fused with a second sperm cell to produce a polyspermic zygote (second fusion). B, C, D, E, F, G, H, I, J, K, and L, Development of polyspermic rice zygotes produced by in vitro fusion. The polyspermic zygote (B, bright-field image; C, fluorescent image) developed into a globular embryo-like structure (D, bright-field image; E, fluorescent image), cell mass (F), and white callus (G) during culture in liquid N6D medium. When the white callus was subsequently cultured on solid regeneration medium, it formed multiple shoots (H) and then plantlets were obtained (I). Plantlets were grown in soil and formed flowers (J). The flowers (3x in K) were larger than those of diploid plants (2x in K), and awns were well developed in possible triploid flowers (3x in L). M, N, Ploidy level of rice plant regenerated from polyspermic zygote. After nuclei were extracted from leaves of wild-type rice plants (M) or from leaves of wild-type rice plants and plants regenerated from polyspermic zygotes (N), DNA content per nucleus was measured by flow cytometry. Light-green and pink colored circles in (A) indicate sperm and egg nuclei, respectively. Arrowheads in (L) indicate awns. Scale bars = 20 μm in (B–E), 100 μm in F, 1 mm in (G and H), and 1 cm in (I, K, and L).

Figure 2.

Two karyogamy pathways in polyspermic triploid zygotes. A, Karyogamy in a monospermic diploid zygote. An egg cell was fused with a sperm cell expressing H2B-GFP, and the resulting triploid zygote was serially observed. The sperm nucleus fluorescently labeled with H2B-GFP migrated adjacent to the egg nucleus after gamete fusion (a, b). Then, when the sperm nucleus fused with the egg nucleus, the sperm chromatin began to decondense (c, d). Decondensation of sperm chromatin further progressed (e, f) and karyogamy was completed (g–j). B, One karyogamy pathway in polyspermic zygote. An egg cell was fused with a sperm cell expressing H2B-GFP, and then the fused gamete further fused with another sperm cell. The resulting triploid zygote was serially observed. Two sperm nuclei were clearly visible in the egg cell after its fusion with sperm cells (a, b). One of the two sperm nuclei first fused with the egg nucleus (c, d), and then the second sperm nucleus fused with the egg nucleus (e, f), resulting in a triploid zygotic nucleus (g–j). C, Another karyogamy pathway in polyspermic zygote. Polyspermic zygote was prepared as in (B) and the triploid zygote was serially observed. Two sperm nuclei were observed in the egg cell after its fusion with sperm cells (a, b), and then these two sperm nuclei fused together (c–f). Then the united sperm nuclei further fused with the egg nucleus (g, h), resulting in a triploid zygotic nucleus (i, j). Top panels are fluorescent images, and bottom panels are merged bright-field and fluorescent images. Arrowhead in (Bc) indicates sperm chromatin, which is decondensing in fused nucleus. Scale bars = 20 μm.

Figure 3.

Organization of actin filaments and migration of sperm nuclei in a polyspermic zygote during karyogamy. An egg cell expressing Lifeact-tagRFP was sequentially fused with two sperm cells expressing H2B-GFP, and the resultant zygote was serially observed under a fluorescent microscope. Because two sperm nuclei were observed at different focal planes, images for two nuclei were separately captured and presented in (A–C) and (D–F). The first sperm nucleus migrated toward the egg nucleus (A, B) and fused with the egg nucleus, resulting in decondensation of sperm chromatin in the fused nucleus (C). The second sperm nucleus migrated toward the egg nucleus (D–F) and fused with the diploid zygote nucleus, resulting in decondensation of sperm chromatin in the triploid nucleus (G). Insets in (D) and (E) show enlarged views of the sperm nuclei enclosed within the squares. Actin filaments in these insets were represented by white color. The asterisk in (A) indicates the egg nucleus surrounded by actin filaments. Arrows in (C) and (G) indicate decondensing chromatin derived from the first sperm nucleus. Arrowhead in (G) indicates decondensing chromatin derived from the second sperm nucleus. Scale bars = 20 μm in (A–G), 5 μm in the insets.

Figure 4.

Mitotic division of polyspermic zygote. After in vitro fusion of an egg cell with a sperm cell expressing H2B-GFP, the fused gamete was further fused with another sperm cell. The polyspermic zygote was then cultured and mitotic division was serially monitored. Chromatin and chromosomes were labeled with H2B-GFP in polyspermic zygote (A–D). Chromosomes originated from the triploid zygotic nucleus were arranged at the equator (E–H), and then separated to form two daughter nuclei (I–N). Top and bottom panels are fluorescent and bright-field images, respectively. Scale bar = 20 μm.

Figure 5.

Microtubule organization during mitotic division of polyspermic zygotes. Polyspermic zygotes were fixed at each mitotic phase, and microtubule structure and chromosome organization were visualized by immuno-fluorescent staining with anti-α-tubulin antibody and DAPI staining, respectively. Representative images are presented. Images in top and center panels show immuno-fluorescent staining and DAPI staining, respectively. Bottom panels show merged images. Scale bars = 10 μm in (A–L), 20 μm in (M–O).