侧金盏花双受精进程研究

应用荧光显微镜和常规石蜡切片观察侧金盏花(Adonis amurensis)花粉管生长和受精作用的全过程。结果表明,侧金盏花为湿型柱头,授粉后1–2小时,花粉粒与柱头识别;授粉后2–4小时,花粉粒萌发;授粉后4–6小时,花粉管进入柱头。侧金盏花的受精模式为珠孔受精,授粉后10小时,精子被释放;授粉后30小时,精核与卵核融合;授粉后7天合子形成;授粉后15天合子进入分裂期,合子休眠期为8天。2个极核在受精前不融合,授粉后14–16小时,精核与1个极核融合;授粉后20–22小时,受精极核与另1个极核融合形成初生胚乳核。双受精作用属于有丝分裂前配子融合型。通过实验确定了侧金盏花受精过程的雌雄性细胞融合形态变化与相应经历的时间及其合子休眠期。研究结果丰富了侧金盏花胚胎学资料,对其今后的育种及转基因研究具有重要意义。     

被子植物受精机制的研究进展

被子植物的受精是一个复杂而精巧的过程。花粉管到达子房,通过退化助细胞进入胚囊,释放出两个精细胞。原来在花粉管中相互联结的两个精细胞在退化助细胞中分开,一个与卵细胞融合,另一个与中央细胞融合,完成双受精。目前对双受精过程中有关雌、雄配子识别的机制还知之甚少。本文介绍了目前被子植物精、卵细胞融合前后的细胞周期变化、退化助细胞的功能、精细胞在退化助细胞中迁移的研究动态、精细胞的倾向受精和卵细胞的激活等被子植物受精生物学领域中的一些新的研究成果和发展趋势。     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

Gamete attachment process revealed in flowering plant fertilization

Sex-possessing organisms perform sexual reproduction, in which gametes from different sexes fuse to produce offspring. In most eukaryotes, one or both sex gametes are motile, and gametes actively approach each other to fuse. However, in flowering plants, the gametes of both sexes lack motility. Two sperm cells (male gametes) that are contained in a pollen grain are recessively delivered via pollen tube elongation. After the pollen tube bursts, sperm cells are released toward the egg and central cells (female gametes) within an ovule (Fig. 1). The precise mechanism of sperm cell movement after the pollen tube bursts remains unknown. Ultimately, one sperm cell fuses with the egg cell and the other one fuses with the central cell, producing an embryo and an endosperm, respectively. Fertilization in which 2 sets of gamete fusion events occur, called double fertilization, has been known for over 100 y. The fact that each morphologically identical sperm cell precisely recognizes its fusion partner strongly suggests that an accurate gamete interaction system(s) exists in flowering plants.Open in a separate windowFigure 1.Illustration of the fertilization process in flowering plants. First, each pollen tube accesses an ovule containing egg and central cells. Next, the 2 sperm cells face the female gametes in the ovule after the pollen tube bursts. Finally, each sperm cell simultaneously fuses with either egg or central cell.     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚.要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料.以前的研究表明,烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显.这种异型性的精细胞可能与倾向受精有关.烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞.用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞.用微量酶液可使两个精细胞分开.然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体.这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础.本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件.     

Recent progress on sperm characterization in flowering plants

The organization, isolation and physiology of the angiosperm male gamete has recently emerged as an area of special interest. Detailed in vivo studies have revealed: (1) that the functional unit of male reproduction is a'male germ unit,'composed of two sperm cells and a physically associated vegetative nucleus; (2) that the two sperm cells are often cytoplasmically dimorphic, and (3) that some sperm cells appear to undergo preferential fertilization, preferentially fusing with either the egg or the proendospermaric central cell. Male gamete isolation has provided enrichments of up to 10⁷ cells ml^–1, with sperm integrity and viability demonstrated by the flunrochromatic reaction and ATP levels. Although results from biochemical and hybridoma antibody characterization are still preliminary, they indicate that sperm cells possess polypeptide expression patterns that are different from those of surrounding cells. These findings suggest the existence of an independent developmental program in the angiosperm male gamete.     

黑节草双受精过程及胚胎发育的研究

黑节草从传粉到受精约需１３０ｄ,精子在花粉管中形成,胚囊发育属蓼型胚囊,因反足细胞较早退化,故受精前胚囊多只由卵器和中央细胞组成。精卵核融合时,精核染色质进入卵核后凝集成颗粒状,并在原位与卵核的染色质融合,雌、雄性核仁一直维持至合子的第一次分裂期前。双受精作用正常,属于有丝分裂前配子融合类型,初生胚乳核发生２－３次分裂后逐渐退化消失,胚的发育局限于球形胚阶段。     

The egg cell is preferentially fertilized in Arabidopsis double fertilization

Each cyclin-dependent kinase a;1 mutant pollen grain contains a single sperm-like cell that can fertilize egg cells, similar to sperm cells. Pollination assays with mutant pollen demonstrated that the egg cell is preferentially fertilized in Arabidopsis.     

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.     

Isolation of two populations of sperm cells from the pollen tube of Torenia fournieri

The two sperm cells of Torenia fournieri are dimorphic. The dimorphic character suggests that they might be preferentially involved in fertilization during in vivo fusion with the egg cell and central cell. To probe the mechanism of preferential fertilization, it is necessary to use the most current molecular techniques. For this purpose, populations of >1000 individuals of the two dimorphic sperm cells, Sua (unassociated with the vegetative nucleus) and Svn (associated with the vegetative nucleus) were isolated from pollen tubes that had grown out of the cut ends of the styles. The two sperm cells released from pollen tubes remained attached to one another. When the two attached sperm cells were transferred into a solution containing 0.01% cellulose, 0.01% pectinase, and 5% mannitol, the connection between the two cells disappeared, and they were easily separated using a micromanipulator. The collection of these two individual populations containing over a thousand cells will permit research on gametic recognition at the molecular level.     

SEXUAL REPRODUCTION IN EPHEDRA NEVADENSIS (EPHEDRACEAE): FURTHER EVIDENCE OF DOUBLE FERTILIZATION IN A NONFLOWERING SEED PLANT

Since the initial discovery of double fertilization in angiosperms in 1898, a number of reports of double fertilization-like events in the genus Ephedra have appeared. Until recently, convincing documentation of double fertilization in Ephedra had not been presented. In Ephedra nevadensis, following entry of a single binucleate sperm cell into the egg cell, one sperm nucleus migrates in a chalazal direction to fuse with the egg nucleus. Contemporaneous with this first fertilization event, the ventral canal nucleus regularly migrates from its initially apical position within the egg cell to a more central position within the egg cytoplasm, where it fuses with a second sperm nucleus. Based on quantitative microspectrofluorometric analysis, occasional supernumerary nuclei within the egg cell (derived by migration through pores in the cell walls between jacket cells and the central cell or egg cell) can be ruled out as participating in the second fertilization event. The evolutionary establishment of double fertilization in Ephedra (or its ancestors) was dependent on a number of specific developmental preconditions: 1) persistence of the ventral canal nucleus (which is degenerate in many groups of nonflowering seed plants) through the time of normal fertilization; 2) regular displacement of the ventral canal nucleus from its initially apical position within the egg cell to a position within the egg cytoplasm where fusion of the egg nucleus with the first sperm nucleus earlier occurred; 3) acquisition of egg-like features by the ventral canal nucleus that allow it to attract and fuse with a sperm nucleus; and 4) consistent entry of a second sperm nucleus into the archegonial cavity to participate in a second fertilization event. Although it cannot be determined definitively whether double fertilization in Ephedra is evolutionarily homologous with double fertilization in flowering plants, comparative evidence is consistent with the hypothesis that double fertilization arose in a common ancestor of the Gnetales and angiosperms.     

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes . The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10⁸/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO₂浓度升高可以诱导植物叶片气孔关闭, 提高植物对高浓度CO₂的适应性。但植物如何感知CO₂浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚。利用高通量、非侵入的远红外成像技术, 建立了拟南芥(Arabidopsis thaliana)气孔对CO₂浓度变化反应相关的突变体筛选技术, 筛选出对环境CO₂浓度敏感的拟南芥突变体ecs1。遗传学分析表明, ecs1为单基因隐性突变体, 突变基因ECS1编码一个跨膜钙离子转运蛋白。与野生型拟南芥相比, 360 μL·L^–1CO₂可引起ecs1突变体叶片温度上升和气孔关闭, ecs1突变体对900 μL·L^–1CO₂长时间处理具有较强的适应性。进一步的实验表明, 360μL·L^–1CO₂即可诱导ecs1突变体叶片积累较高浓度的H₂O₂, 而900 μL·L^–1CO₂才能够诱导野生型拟南芥叶片积累H₂O₂。因此, ECS1可能参与调节高浓度CO₂诱导的拟南芥气孔关闭和H₂O₂产生, H₂O₂可能作为第二信号分子介导CO₂诱导拟南芥气孔关闭的反应。     

Double fertilization on the move

Double fertilization is a flowering plant mechanism whereby two immotile sperm cells fertilize two different female gametes. One of the two sperm cells fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce the endosperm. Despite the biological and agricultural significance of double fertilization, the mechanism remains largely unknown owing to difficulties associated with the embedded structure of female gametes in the maternal tissue. However, molecular genetic approaches combined with novel live-cell imaging techniques have begun to clarify the actual behavior of the sperm cells, which is different from that described by previous hypotheses. In this review article, we discuss the mechanism of double fertilization based on the dynamics of the two sperm cells in Arabidopsis.     

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca²⁺-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca²⁺ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca²⁺ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 μM Fluo-3 for 30 min at 30  °C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca²⁺]_cyt of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.     

Ovule,Female and Male Gametophyte and Fertilization of Kingdonia uniflora Balfour F. et W. W. Smith

The structure of ovule, female and male gametophyte, double fertilization and the distrubution of starch grains during the fertilization have been studied. The main results are as follows: ( 1 ) Ovule The ovule is anatropous, unitegmic and tenuinucellate. The nucetlus appears cylindric, since megaspores and embryo sac development, its internal cells of nucellus become disorganized, so that only a single layer of epidermal cells remains toward the side of the micropyle, On the other hand, the integument is not as long as nucellus, as a result micropyle is not formed. And no vascular bundle is found in the integument. (2) Female gametophyte The mature embryo sac is slender and is composed of an egg cell, two synergids, a central cell and three antipodal cells. The egg cell is situated slightly away from the tip of embryo sac. Some of them contain starch grains. Synergids occupy the tip of embryo sac. Its wall at micropylar region appears irregular in thickenes and irregular in ingrowths to form the filiform apparatus. The centrateell is very large, and strongly vacuolated Two polar nuclei come to contact closely with each other, but not fuse, or to fuse into a large secondary nucleus before fertilization. The polar nuclei or the secondary nucleus are usually situated at the middle-lower position of the central cell or nearer to the chalazal end above the antipodal cell. It is different from egg cell, no starch grains are found here. In most embryo sacs three antipodal cells are found. They are not as large as those in other plants of Ranunculaceae. But six antipodal cells or the antipodal cell with two nuclei may rarely be found. Like synergid, the wall of them appears not only irregularly thickened, but clearly with irregular ingrowths. In a few antipodal cells the starch garins are usually found near the nucleus. By the end of fertilization, antipodal cells become disintegrated. (3) Male gametophyte Most pollen grains are two-celled when shedding, and rich in starch grains. A few of them contain single nucleus or three-celled. (4) The double fertilization The fertilization of Kingdonia unifiora Balfour f. et W, W. Smith is wholly similar to some plants of Ranunculaceae studied. First, the pollen tube penetrates a degenerating synergid. And the pollen tube discharges its contents with two sperm nuclei into the degenerating synergid cell. One of the two sperms fuses with the nucleus of the egg, and the other fuses with two polar nuclei or the secondary nucleus of the central cell. If one sperm nucleus at first fuses with one of the polar nuclei, and then the fertilized polar nuclei again fuses with other polar nucleus. Secondly, the fertilization of the polar nuclei or the secondary nuclei completes earlier than that of the egg. The primary endosperm nucleus begins to divide earlier than the zygote. It seems that one of the sperm nuclei come to contact with egg nucleus, the other has already fused with polar nuclei or the secondary nucleus. The zygote with a single nucleolus appears until the endosperm with 16–20 cell. Thirdly, before and after fertilization there are one to some small nucleoli in egg nucleus and polar nuclei or secondary nucleus. However they increase in quantity from the beginning of the fusion of male nucleis. These nucleoli quite differ from male nucleoli by their small size, and most of them disappear at the end of fertilization. It may be concluded that the small nucleoli increase in quantity is related to the fusion of male and female nuclei. In the duration of fertilization, in ovule starch distribution is in the basal region of integument. But in embryo sac, onlysome egg cells, or zygotes contain starch grains, a part of which was brought in by pollen tube. Sometimes the starch grains are found in some synergids and antipodal cells. No starch grains are found in the central cell.     

Sperm Identification in Maize by Fluorescence in Situ Hybridization

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. Despite the obvious advantages of being able to recognize and then track, separate, and analyze one sperm type from the other, it has not been possible because of the lack of sufficient detectable differences between the two types of sperms. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Our results are consistent with conclusions from previous genetic studies related to B chromosome behavior during pollen formation. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random.     

A novel signalling mechanism for generating ca2+ oscillations at fertilization in mammals

At fertilization in mammals the sperm activates the egg by triggering a series of oscillations in the intracellular free Ca²⁺ concentration. The precise sequence of events that occur between sperm-egg contact and the increases in intracellular Ca²⁺ remains unknown. Here, we discuss recent evidence supporting the hypothesis that a cytosolic sperm protein enters the egg after gamete membrane fusion and triggers Ca²⁺ oscillations from within the egg cytoplasm. Biochemical studies suggest that there exists a novel sperm protein, named oscillin, that specifically comigrates with Ca²⁺ oscillation-inducing activity. Oscillin has been immunolocalised to the region of the sperm that first fuses with the egg. The concept of a specific protein that triggers Ca²⁺ oscillations may have wider physiological significance since sperm oscillin can induce Ca²⁺ oscillations in somatic cells, such as neurons and hepatocytes. Unravelling the novel signalling system involved in mammalian fertilization may help reveal some fundamental molecular mechanisms responsible for triggering cytoplasmic Ca²⁺ oscillations.     

烟草精细胞两个群体的分离

高等植物的倾向受精是一个非常吸引人的研究课题,目前对其机理还不清楚。要想探索高等植物倾向受精现象,前提之一是要分离出一定数量的两个精细胞群体作为分子生物学研究方法的材料。以前的研究表明, 烟草(Nicotiana tabacum L.)花粉管中的两个精细胞体积差异明显。这种异型性的精细胞可能与倾向受精有关。烟草是二胞型花粉,生殖细胞只在体内生长的花粉管中才分裂形成两个精细胞。用体内/体外技术培养出花粉管后,爆破花粉管即可释放出花粉管内含物,其中包括两个精细胞。用微量酶液可使两个精细胞分开。然后用显微操作器可挑选出两个大小不同、数量上千的精细胞群体。这种单一纯化的精细胞群体为用分子生物学方法区分两个精细胞的DNA和蛋白质差异打下基础。本研究是高等植物的第二例、二胞花粉植物中的第一例分离两个特定精细胞群体的尝试,为构建烟草两个精细胞的cDNA文库创造了条件。