| The gametes of maize and tobacco were isolated and fused by in vitro fertilization system to investigated post-fertilization events. We focused on several key events during fertilization from membrane fusion to karyogamy of sperm cells and egg cells. The purpose of the study is to seek detailed cytologic events triggered by fertilization and to understand the mechanism underlying the events. The main results are as follows:1. Various systems by using electric pulse, calcium, or polyethylene glycol have been developed in the past decade for the in vitro fusion of angiosperm gametes. These in vitro systems provide a new way to study the fertilization mechanisms of plants. However, it is still important to develop new in vitro fusion system to improve efficiency and understand the natural condition of fertilization. In this study, we developed a novel bovine serum albumin (BSA)-mediated fusion system for the in vitro fusion of maize gametes. The in vitro fusion of the isolated single egg cell andsperm cell of maize was observed microscopically in the BSA solution and the fertilized egg cell showed normal cell wall regeneration and nuclear division. The result indicated that the new fusion system had little negative effect on the activity of gametes. The effects of the BSA concentration, pH value and calcium level on the efficiency of the maize gamete fusion were also assessed. BSA concentration and pH value did significantly affect the efficiency of the gamete fusion. Calcium was not necessary for the gamete fusion when BSA was present, and 2mM calcium even inhibited the fusion of gametes. The optimal solution for the gamete fusion contained 0.1% BSA, pH 6.0. The fusion frequency was as high as 96.7% in that optimal solution. The two sperm cells came from one pollen both could fuse with egg cells by using the BSA fusion system. This new in vitro fertilization system offers an alternative tool for the in vitro study of fertilization mechanisms with much simpler manipulating procedure than PEG fusion system. For its ability of inducing gamete fusion with or without calcium, the BSA fusion system will be especially useful for the in vitro study of the calcium dynamics and the resource of calcium involving in egg cytosolic calcium increase during angiosperm fertilization.2. Double fertilization is a key process of sexual plant reproduction in higher plants. The role of calcium in the activation of female cells through fertilization has recently received a great deal of attention. The establishment of a Ca2+-imaging technique for living, single, female sexual cells is a difficult but necessary prerequisite for evaluating the role of Ca2+ in the transduction of external stimuli, including the fusion with sperm, to internal cellular processes. This study describes the use of Fluo-3 for reporting the Ca2+ signal in isolated, single, female sexual cells, the egg cell, and central cells of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 pM Fluo-3 for 30 min at 30°C. We found that probenecid, an inhibitor of anion transporters, was essential for successful loading. Under this condition, the dynamic range of Fluo-3 in the female sexual cells was 11.46, which was sufficient to detect the transient change in Ca2+ concentration ([Ca2+]c) in single female cells. Several key factors dealing with in vitro fertilization were also investigated to test their possible effects on the [Ca2+]c of the female sexualcells. Gentle mechanical stimulation was found to have no evident effect on [Ca2+]c. After incubation in bovine serum albumin (BSA)-fusion medium for several hours, the female cell showed no evident change in [Ca2+]c. In contrast, the female cell exhibited a clear change in [Ca2+]c after being incubated in polyethylene glycol (PEG)-fusion medium for minutes. These results indicated that the BSA-fusion system was superior to the PEG-fusion system for detecting calcium fluctuations in female sexual cells during fertilization.3. Egg cells have the ability to avoid polyspermy. In the present study, we investigated the mechanism underlying the blocks to polyspermy in maize. The PEG fusion system and BSA fusion system were used. In both systems, maize egg cells contract in 2 min after fusion and refresh to round after 7-40 min. In the period of during and after contraction, the second fusion events could not occur. However, tobacco female gametes did not contract after fusion with maize sperm cells. Tobacco female gametes could fuse with several maize sperm cells. These results suggested that the egg contraction involved in blocks to polyspermy. In the solution containing calcium, after fertilization maize egg cells contract acutelier than that of without calcium. Maize sperm cells could not trigger the change of cytosolic calcium of tobacco female gametes after their fusion. However, maize sperm cells could trigger calcium influx into maize egg cells and calcium increase in maize egg cells. These results suggested that the calcium increase in egg is the early signal of blocks to polyspermy.4. A very important event during fertilization is that the male nucleus must integrate into female gamete to fulfill karyogamy. However, the cellular mechanisms underlying the migration of male nucleus and female nucleus are not yet understood in angiosperms. We investigated the mechanism underlying the migration of male nucleus and female nucleus in maize and tobacco. In animals, male pronucleus and female pronucleus migrate to each other after fertilization. However, the situation was different in angiosperms. Our results showed that male nucleus migrated to unmoved female nuclear after fertilization. In maize, the velocity of sperm nucleus migration to egg nucleus was 0.26+0.09 ^m/s. In tobacco, the velocity of sperm nuclear migrationto egg nuclear was 0.026+0.001 ^m/s; the velocity of sperm nuclear migration to polar nuclei was 0.045+0.004 /*m/s. Cytoskeleton inhibitors were used to study the molecular mechanism of sperm nuclear migration. Several actin filament-disrupting drugs including cytochalasin B, latrunculin A and jasplakinolide inhibited sperm nuclear migration. However, microtubule inhibitor colchicine and oryzalin both had no effect on sperm nuclear migration. These results indicated that sperm nuclear migration to female nuclear was dependent on actin filament but not microtubule. In an attempt to identify the driving force behind sperm nuclear migration, the involvement of an actin-dependent motors, myosin, was investigated. A common myosin inhibitors 2, 3-butanedione monoxime (BDM) was used for further investigation. Sperm nuclear migration could not be inhibited by 20 mM BDM, and the velocity of sperm nuclear migration was not affected. On the contrary, mitochondrial migration was inhibited by 20 mM BDM. These results indicated that myosin did not involve in sperm nuclear migration and we suggested another actin-dependent force, Actin-related protein complex (Arp2/3), mighe be involved in sperm nuclear migration.5. In vitro fertilization technique enable us to get fertilized zygotes by the fusion of gametes from two incompatible species for the barriers before the fusion of membrane of sperm cells and egg cells are avoided. We isolated tobacco egg cells or central cells as female gametes, and tobacco sperm cells or generative cells and maize sperm cells as male gametes. We induce the fusion of different pair of female gametes and male gametes by in vitro fertilization system and study the development of fusion products. From membrane fusion to karyogamy, the cytologic events are similar in different fusion pairs. Male nucleus migrated to female nucleus and reached it in 20-25 min. Using the central cells as female cells, male nucleus began to fuse with one of the polar nuclei 7 h after fusion. Ten hours after fusion, the male nucleus completed the fusion with one of the polar nuclei. The three nuclei fuse to a large nucleus 17 h after cell fusion. Using the egg cells as female cells, male nucleus began to fuse with egg nucleus 4 hours after fusion. Seven hours after fusion, the male nucleus fulfilled the fusion with egg nucleus. The two nuclei fuse to a large nucleus17 h after fusion. Using the central cells as female cells, the fusion products of it and maize sperm cells did not divide, while the fusion products of it and tobacco generative cells developed into coenocyte. Maize sperm cells, tobacco generative cells and tobacco sperm cells carried different development information, thus led to different downstream development events. Maize sperm cells could not activate tobacco central cells. Generative cells partly substituted the function of sperm during fertilization. Although the barrier of distant hybridization could be overcome by in vitro fertilization, the fusion products could not develop beyond karyogamy. These results indicated that interspecific barrier is still present at gamete level in later events.
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