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Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants
陆生植物游动雄配子中心粒、基体和鞭毛的透射电子显微镜观察   

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Plant Physiology
Jun 2017

Abstract

Motile male gametes (spermatozoids) of land plants are coiled and contain a modified and precisely organized complement of organelles that includes a locomotory apparatus with two to thousands of flagella. Each flagellum is generated from a basal body that originates de novo as a centriole in spermatogenous cell lineages. Much of what is known about the diversity of plant male gametes was derived from detailed transmission electron microscopic studies. Because the process of spermatogenesis results in complete transformation of the shape and organization of these cells, TEM studies have yielded a wealth of information on cellular differentiation. Because green algal progenitor groups contain centrioles and a variety of motile cells, land plant spermatozoids also provide a plethora of opportunities to examine the evolution and eventual loss of centrioles and locomotory apparatus during land colonization.

Here we provide a brief overview of the studies and methodologies we have conducted over the past 20 years that have elucidated not only the structural diversity of these cells but also the development of microtubule organizing centers, the de novo origin of centrioles and the ontogeny of structurally complex motile cells.

Keywords: Blepharoplast (生毛体), Basal bodies (基体), Centrioles (中心粒), Extracellular matrix (细胞外基质), Flagella (鞭毛), Transmission electron microscopy (透射电子显微镜观察)

Background

Motile gametes of land plants are strikingly diverse and develop through transformations that involve repositioning, and reshaping of cellular components, and the assembly of a complex locomotory apparatus (Renzaglia and Garbary, 2001; Lopez and Renzaglia, 2008). Because of constraints imposed by cell walls, elongation of the cell and flagella is around the periphery of a nearly spherical space, resulting in a coiled configuration of the mature gamete (Renzaglia and Garbary, 2001; Lopez and Renzaglia, 2014). The degree of coiling varies from just over one to as many as 10 revolutions per cell. The number of flagella per gamete is even more variable, ranging from two in bryophytes (mosses, hornworts, liverworts and most lycophytes) to an estimated 1,000-40,000 in Ginkgo and cycads, the earliest divergent seed plant lineages. Following the diversification of Ginkgo and cycads, all vestiges of basal bodies and flagella were lost in the remaining seed plants that utilize pollen tubes to deliver non-motile sperm to egg cells (Southworth and Cresti, 1997).

It is widely known that vegetative plant cells lack centrioles and the centrosome is elusive. A lesser-known fact is that in plants with motile sperm cells, centrioles arise de novo during the penultimate or ultimate mitotic divisions that produce the nascent spermatid in antheridia (Renzaglia and Carothers, 1986; Vaughn and Renzaglia, 1998; Vaughn and Harper, 1998; Renzaglia and Maden, 2000; Vaughn and Renzaglia, 2006). In these cell lineages, centriolar centrosomes serve as the nucleation site for spindle microtubules and thus bear striking parallels with centrioles of animal and protist cells. In the developing sperm cells, the centrioles reposition, anchor to form the distinctive basal bodies, and elongate to produce the 2-40,000 flagella in each gamete. These changes occur in synchrony with cell elongation, and the entire process of cytomorphogenesis is guided by the production of unique arrays of microtubules, and fibrillar and lamellar bands or strips. Because of the exclusive occurrence of basal bodies, flagella and associated complexes in developing male gametes, studies of spermatogenesis have revealed important information on the structure, composition, and developmental changes in microtubule arrays as they relate to the cell cycle, microtubule organizing centers (MTOCs), and cellular differentiation in plants. The purpose of this review is to describe the method used in transmission electron microscopic examination and to demonstrate how this approach has advanced understanding of basal bodies, flagella/cilia, and associated structures in land plants.

Materials and Reagents

  1. Transmission electron microscope (TEM)
    1. Scintillation vials with aluminum covered caps (Fisher Scientific, catalog number: 03-340-4B)
      Manufacturer: DWK Life Sciences, Kimble®, catalog number: 7450320 .
    2. BEEM embedding capsules size ‘00’ (Electron Microscopy Sciences, catalog number: 70000-B )
    3. Formfar Carbon Film 200 mesh Ni grids (Electron Microscopy Sciences, catalog number: FCF200-Ni )
    4. Copper 200 mesh grids (Electron Microscopy Sciences, catalog number: EMS200-Cu )
    5. Sperm cells
      1. Megaceros flagellaris
      2. Phaeoceros carolinianus
      3. Phylloglossum drummondii
      4. Ginkgo biloba
      5. Angiopteris evecta
      6. Conocephalum conicum
      7. Ceratopteris richardii
      8. Riccardia multifida
      9. Aulacomnium palustre
      10. Equisetum arvense
    6. Ethanol (Decon Labs, catalog number: 2705HC )
    7. Low viscosity resin
    8. Glutaraldehyde (Electron Microscopy Sciences , catalog number: 16120 )
    9. Sorensens phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
    10. Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
    11. Potassium ferrocyanide (Fisher Scientific, catalog number: P236-500 )
    12. Uranyl acetate (Polyscience, catalog number: 21447-25 )
    13. 100% methanol
    14. Lead nitrate (Electron Microscopy Sciences, catalog number: 17900 )
    15. Sodium citrate (Electron Microscopy Sciences, catalog number: 21140 )
    16. 1 N NaOH
    17. 100% propylene oxide (Electron Microscopy Sciences, catalog number: 20401 )
    18. 2.5% glutaraldehyde (see Recipes)
    19. 0.05 M phosphate buffer (pH 7.2) (see Recipes)
    20. 4% aqueous osmium tetroxide (see Recipes)

Equipment

  1. Transmission electron microscope (TEM)
    1. Diamond knife (Diatome, specs: Ultra, 45°, 4 mm, Wet)
    2. Transmission electron microscope (Hitachi, model: HF7100 )

Procedure

  1. Transmission electron microscope (TEM)
    TEM examination has revealed diagnostic developmental features and structural peculiarities of plant spermatozoids. Three examples that illustrate these plant specific features are superficially described herein: basal body origin (Figure 1), locomotory apparatus development (Figure 2) and basal body/flagella structure (Figure 3). The de novo appearance of basal bodies takes several unique forms, the most notable of which are bicentrioles and blepharoplasts. Sperm cells of bryophytes and most lycophytes are biflagellated and in these plants, basal bodies originate as two centrioles attached end to end (Figure 1A). Centrioles have the typical complement of nine overlapping triplet microtubules around a central core or hub (Figure 1B). Each nascent sperm cell inherits one bicentriole and during development, the two centrioles separate, reposition and become the basal bodies in the biflagellate gametes. In ferns, blepharoplasts form in spermatogenous cells. Each blepharoplast is spherical and contains the central hubs of all basal bodies in a dense matrix that will be assembled in the young spermatid (Figure 1C).
    An alternate mode of origin of centrioles occurs in Phylloglossum, one of two lycophytes with multiple flagella. In this case, the centrioles originate as a branched unit of central hubs on to which the triplets assemble directly (Figure 1D). The 20 or so basal bodies formed this way separate and align around the anterior of the developing spermatid and elongate into the flagella. In the seed plant, Ginkgo biloba, a pair of massive blepharoplasts in the sperm mother cell assembles approximately 1,000 centrioles around an electron dense unit that contains spherical electron lucent areas occasionally containing basal bodies (Figure 1E). The cycads produce a blepharoplast similar to that of Ginkgo, but much larger, producing an estimated 40,000-50,000 basal bodies (Gifford and Larson, 1980).


    Figure 1. Origin of basal bodies in spermatogenous cells. A. The bicentriole of bryophytes and most lycophytes is represented here in Megaceros flagellaris, a hornwort. It consists of two centrioles attached end-to-end by a central core (arrow). B. Cross section of a bicentriole of Phaeoceros carolinianus, a hornwort, showing the nine triplets in the centriole/basal body arranged around a central core or hub. C. The blepharoplast of ferns as illustrated in Ceratopteris richardii contains numerous basal body hubs embedded in an amorphous dense matrix. D. A pair of branched centriolar units forms in the spermatid mother cell of Phylloglossum drummondii, one of two lycophytes with approximately 20 flagella. Triplet microtubules are assembled directly on the hub with A microtubules produced first (arrow) followed by B and C microtubules. E. A massive blepharoplast in the sperm mother cell of Ginkgo biloba has approximately 1,000 centrioles around an electron dense unit that contains spherical electron lucent areas occasionally containing basal bodies (arrow). Bars = 0.2 µm (A, C, D), 0.1 µm (B), 1.0 µm (E).

    In the nascent spermatid, basal bodies separate and migrate as other elements of the locomotory apparatus develop. This repositioning is often in association with a dense microtubule-organizing center from which numerous microtubules emanate (Figure 2A). In ferns, basal bodies are assembled around the central hubs as A, B, and C microtubules of the triplets are added (Figure 2B). Then the short basal bodies migrate in concert with construction of the multilayered structure, composed of a lamellar strip and band of microtubules (Figure 2C), until they are positioned around the anterior coils of the cell in a precise arrangement (Figure 2D). While repositioning, basal bodies begin to elongate from both ends. At the proximal end, more triplets are added to elongate the basal body and in ferns the hub forms a highly elongated extension that is transient and disappears in the late stage spermatozoid (Figure 2E). A transition zone (Figure 2E) that contains the stellate configuration begins formation during basal body repositioning (Figure 2D).


    Figure 2. Microtubule-organizing centers and development of the locomotory apparatus in spermatids (developing sperm cells). A. Dense microtubule-organizing center (MTOC) with emanating microtubules (*) and three basal bodies in Phylloglossum drummondii. The MTOC guides basal body placement. B. In spermatids of Ceratopteris richardii, short basal bodies seen in cross section (left arrow) and longitudinal section (right arrow) are developing and reorganized in an electron-dense matrix. C. The reorganization of basal bodies in C. richardii occurs in synchrony with the development of a multilayered structure (mls) that is composed of a lamellar strip and band of microtubules. D. During the assembly of the anterior locomotory apparatus, the basal bodies of C. richardii grow from both ends resulting in neatly aligned elongated basal bodies; the posterior transition region with a stellate pattern (sp) is beginning to elongate in these basal bodies. E. Long hub extensions (h) in Angiopteris evecta, a eusporangiate fern, are visible after the flagella initiate elongation but prior to completion of sperm cell differentiation. The extensions are transient and thus are lacking in the mature gamete. Basal bodies (bb) and the stellate pattern (sp) in the transition region are located internal to emergence of flagella from the cell body (arrow). Bars = 0.2 µm (A-E).

    Proximal basal body elongation is critical in positioning basal bodies in mosses and liverworts spermatozoids that have two dimorphic, staggered basal bodies (Figures 3A-3C). Staggering is achieved by proximal elongation of usually three ventral triplets in one basal body that becomes very long (Figure 3A) and is eventually anchored some distance from the less elongated anterior basal body. Slight proximal elongation of dorsal triplets in the anterior basal body anchors it within the multilayered structure (Figure 3B). A peculiar feature of moss gamete development is the existence of a single microtubule that strays from the microtubular band and appears to guide the ventral triplets into position during elongation of the posterior basal body (Figure 3C).


    Figure 3. Structure of mature basal bodies and flagella. A. Posterior basal body of Riccardia multifida, a simple thalloid liverwort, consists of elongated ventral triplets (arrow) and hub (h) that extend well beyond the original basal body (bb). B. Dimorphic basal bodies of Conocephalum, a complex thalloid liverwort, in cross section. The multilayered structure (mls) subtends an anterior basal body on the right with hub and six triplet extensions and posterior basal body on the left that consists solely of three ventral triplet extensions. C. The posterior basal body of mosses, illustrated in Aulacomnium palustre, consists of a hub and three ventral triplet extensions that connect to and are guided to their final position by a single stray microtubule (arrow). Visible in this cross section is the anterior flagellum on the right just exterior to the band of microtubules of the multilayered structure. D. Longitudinal section of basal bodies and flagella in the lycophyte Phylloglossum, illustrating the basal body (F1), transition zone (F2) and flagellum (F3) that correspond to the cross sections from right to left in figure F in Equisetum. Notably, the stellate pattern of the transition zone is located anterior and posterior to the emergence of the flagellum from the cell body (arrow). E. Cross section of two basal bodies at the stellate pattern of the transition region and two flagella in Phylloglossum. The stellate pattern (left arrow) that is still contained in the cell body consists of nine overlapping triplets and the one that is in the flagellar shaft (right arrow) has nine overlapping doublets with a single central microtubule. F. Cross sections of basal body, stellate pattern and axoneme at the levels indicated as F1, F2 and F3 in figure D. G. Cross sections of flagella in Ceratopteris demonstrating the typical 9+2 configuration of land plant axonemes. Bars = 0.2 µm (A-F).

    Note: For each solution change below, completely saturate tissue by adding solution until it rises over the material in the vial/ tube (this could be anywhere from 1 ml to 5 ml (or more) of solution depending on how much tissue is in each tube or vial).
    1. Fix antheridial tissue (either excised or whole gametophytes if very small e.g., Ceratopteris fern male gametophytes) in 2.5% glut in 0.05 M sodium phosphate buffer (pH 7.2).
    2. Rinse three times (15 min each) in 0.05 M phosphate buffer (pH 7.2).
    3. Post-fix for 2 h at room temperature in aqueous 2% osmium tetroxide (optional with 1.5% potassium ferrocyanide).
    4. Rinse three times in double distilled autoclaved water (10 min each), dehydrated through 25%, 50%, 75% and 95% ethanol (20 min each), rinsed twice in 100% ethanol (20 min each), then rinse three times in 100% propylene oxide (15 min each).
    5. Infiltrate in 25%, 50% and 75% in low viscosity resin (diluted with propylene oxide) for 12 h each at 4 °C and soaked in three changes of pure resin for 8 h each.
    6. Place one or two antheridia (depending on size) per BEEM embedding capsule and polymerize in oven for 48 h at 60 °C.
    7. Cut gold/silver sections with a diamond knife, and collect on either formvar films using slot (1 x 2 mm) or on 200 mesh copper grids.
    8. Post-stain with 2% uranyl acetate (2 min) followed by basic lead citrate (5 min) prior to viewing in a Hitachi HF7100 .

Recipes

  1. 0.01 M phosphate buffer (pH 7.2)
    Add 5 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 95 parts double distilled autoclaved water
  2. 0.05 M phosphate buffer (pH 7.2)
    Add 25 parts 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 75 parts double distilled autoclaved water
  3. 2.5% glutaraldehyde
    1. Add 1 part 10% glutaraldehyde to 1 part double distilled autoclaved water
    2. Add 1 part 0.20 M Sorensen’s phosphate buffer (pH 7.2) to 1 part double distilled autoclaved water
    3. Add 1 part 5% glutaraldehyde to 1 part 0.10 M Sorensen’s phosphate buffer (pH 7.2)
  4. 0.05 M sodium cacodylate buffer (pH 7.2)
    Add 1 part 0.30 M sodium cacodylate buffer (pH 7.2) to 6 parts double distilled autoclaved water
  5. 2% aqueous osmium tetroxide
    Add 1 part 4% aqueous osmium tetroxide to 1 part double distilled autoclaved water
  6. 0.02 M phosphate buffer (pH 7.2)
    Add 1 part 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 10 parts double distilled autoclaved water
  7. 0.05 M PIPES buffer (pH 7.2)
    Add 1 part 0.30 M PIPES buffer (pH 7.2) to 6 parts double distilled autoclaved water
  8. 1.5% potassium ferrocyanide
    Add 1.5 g potassium ferrocyanide to 10 ml double distilled autoclaved water
  9. 2% uranyl acetate
    Add 2 g uranyl acetate to 10 ml double distilled autoclaved water or 100% methanol
  10. Reynold’s lead citrate
    1. Add 1.33 g lead nitrate and 1.76 g sodium citrate to 30 ml boiled double distilled autoclaved water that has cooled to room temperature and shake for 30 min
    2. Add 8 ml 1 N NaOH
    3. Add 12 ml double distilled autoclaved water

Acknowledgments

This research was supported by research grants (DEB-0322664, DEB-0423625, DEB0521177, and DEB-0228679) from the National Science Foundation as part of the Research Experience for Undergraduates and Assembling the Tree of Life Programs.

References

  1. Gifford, E. M. and Larson, S. (1980). Developmental features of the spermatogenous cell in Ginkgo biloba. Am J Bot 62: 119-124.
  2. Lopez, R. A. and Renzaglia, K. S. (2008). Sperm cell architecture, insemination, and fertilization in the model fern, Ceratopteris richardii. Sex Plant Reprod 21: 153-167.
  3. Lopez, R. A. and Renzaglia, K. S. (2014). Multiflagellated sperm cells of Ceratopteris richardii are bathed in arabinogalactan proteins throughout their development. Am J Bot 101: 2052-2061.
  4. Renzaglia, K. S. and Carothers, Z. B. (1986). Ultrastructural studies of spermatogenesis in the Anthocerotales. IV. The blepharoplast and mid-stage spermatid of Notothylas. J Hattori Bot Lab 60: 97-104.
  5. Renzaglia, K. S. and Garbary, D. J. (2001). Motile male gametes of land plants: Diversity, development, and evolution. Crit Rev Sci 20:107-213.
  6. Renzaglia, K. S. and Maden, A. R. (2000). Microtubule organizing centers and the origin of centrioles during spermatogenesis in the pteridophyte Phylloglossum. Micros Res Tech 49: 496-505.
  7. Southworth, D. and Cresti, M. (1997). Comparison of flagellated and nonflagellated sperm in plants. Am J Bot 84: 1301-1311.
  8. Vaughn, K. C. and Harper, J. D. (1998). Microtubule-organizing centers and nucleating sites in land plants. Int Rev Cytol 181: 75-149.
  9. Vaughn, K. C. and Renzaglia, K. S. (1998). Origin of bicentrioles in anthocerote spermatogenous cells. In Bates, J. W., Ashton, N., Duckett, J. G. (Eds.). Bryology for the Twenty-First Century.
  10. Vaughn, K. C. and Renzaglia, K. S. (2006). Structural and immunocytochemical characterization of the Ginkgo biloba L. sperm motility apparatus. Protoplasma 22: 165-173.

简介

土地植物的运动雄性配子(精子)卷曲,并含有修改和精确组织的细胞器的补体,其包括具有两至数千个鞭毛的运动器官。每个鞭毛都是从起源于新生细胞的基底体产生的,作为精子细胞谱系中的中心粒。关于植物雄性配子多样性的许多知识来自详细的透射电子显微镜研究。由于精子发生过程导致这些细胞的形状和组织的完全转化,TEM研究已经产生了关于细胞分化的大量信息。由于绿藻藻类祖细胞含有中心粒和各种运动细胞,所以土地植物精子也提供了大量的机会来检验土地殖民时期的中心粒和运动器具的演化和最终的损失。
在这里,我们简要概述了我们在过去20年进行的研究和方法,不仅阐明了这些细胞的结构多样性,而且阐明了微管组织中心的发展。 >中心粒的起源和结构复杂的运动细胞的个体发育。
【背景】土地植物的运动配子是非常多样化的,通过涉及重新定位和重塑细胞组分以及组装复杂运动器官的变革来发展(Renzaglia和Garbary,2001; Lopez和Renzaglia,2008)。由于细胞壁施加的限制,细胞和鞭毛的伸长在几乎球形的空间周围,导致成熟配子的盘绕构型(Renzaglia和Garbary,2001; Lopez和Renzaglia,2014)。卷取程度从每个单元只有一到多到十转。每个配子的鞭毛数量甚至更为可变,从苔藓植物(苔藓植物, 。随着银杏和苏铁的多样化,剩余的利用花粉管将非活动精子输送到卵细胞的种子植物中,基底体和鞭毛的所有残留都丢失(Southworth and Cresti,1997)。
众所周知,营养植物细胞缺少中心粒,中心体是难以捉摸的。一个鲜为人知的事实是,在具有活动精子细胞的植物中,在生产新生精子细胞在花药中的倒数第二或最终有丝分裂分裂期间,中心粒起始于新生(Renzaglia和Carothers,1986; Vaughn和Renzaglia, 1998; Vaughn和Harper,1998; Renzaglia和Maden,2000; Vaughn和Renzaglia,2006)。在这些细胞谱系中,中心粒子作为纺锤体微管的成核位点,因此与动物和原生生物细胞的中心粒子具有显着的平行。在发育中的精子细胞中,中心粒重新定位,锚定形成独特的基体,并在每个配子中伸长以产生2-40,000鞭毛。这些变化与细胞延伸同步发生,并且细胞形态发生的整个过程由独特的微管阵列以及纤维和片状带或条带的引导引导。由于发生雄性配子的基础体,鞭毛和相关复合体的独家发生,精子发生的研究揭示了微管阵列的结构,组成和发育变化的重要信息,因为它们与细胞周期,微管组织中心(MTOCs) )和植物细胞分化。这项审查的目的是描述透射电子显微镜检查中使用的方法,并说明这种方法如何对陆生植物中的基底,鞭毛/纤毛和相关结构有深入的了解。

关键字:生毛体, 基体, 中心粒, 细胞外基质, 鞭毛, 透射电子显微镜观察

材料和试剂

  1. 透射电子显微镜(TEM)
    1. 具有铝盖帽的闪烁瓶(Fisher Scientific,目录号:03-340-4B)
      制造商:DWK Life Sciences,Kimble ®,目录号:7450320.
    2. BEEM嵌入胶囊尺寸'00'(电子显微镜科学,目录号:70000-B)
    3. Formfar碳膜200目Ni网格(电子显微镜科学,目录号:FCF200-Ni)
    4. 铜200目网格(电子显微镜科学,目录号:EMS200-Cu)
    5. 精子细胞
      1. Megaceros flagellaris
      2. > us us us us>>>。。。。

      3. 银杏叶
      4. Angiopteris evecta
      5. Conocephalum conicum
      6. Ceratopteris richardii

      7. > re re re>>>。。。。。。

    6. 乙醇(Decon Labs,目录号:2705HC)
    7. 低粘度树脂
    8. 戊二醛(Electron Microscopy Sciences,目录号:16120)
    9. Sorensens磷酸盐缓冲液,0.2M,pH7.2(Electron Microscopy Sciences,目录号:11600-10)
    10. 四氧化锇(Electron Microscopy Sciences,目录号:19150)
    11. 亚铁氰化钾(Fisher Scientific,目录号:P236-500)
    12. 乙酸铀酯(Polyscience,目录号:21447-25)
    13. 100%甲醇
    14. 硝酸铅(电子显微镜科学,目录号:17900)
    15. 柠檬酸钠(电子显微镜科学,目录号:21140)
    16. 1 N NaOH
    17. 100%环氧丙烷(Electron Microscopy Sciences,目录号:20401)
    18. 2.5%戊二醛(见食谱)
    19. 0.05 M磷酸盐缓冲液(pH 7.2)(见配方)
    20. 4%四氧化锇水溶液(参见食谱)

设备

  1. 透射电子显微镜(TEM)
    1. 钻石刀(Diatome,规格:Ultra,45°,4 mm,湿)
    2. 透射电子显微镜(日立,型号:HF7100)

程序

  1. 透射电子显微镜(TEM)
    TEM检查显示了植物精子的诊断发育特征和结构特征。说明这些植物特异性特征的三个实例在本文中表面描述:基础体起源(图1),运动器官发育(图2)和基础体/鞭毛结构(图3)。基底体的新生外观采取几种独特的形式,其中最着名的是双核生物和睑状体。苔藓植物和大多数lycophytes的精子细胞双歧杆菌,而在这些植物中,基底体起始于两端并列连接的中心粒(图1A)。 Centrioles在中心核心或集线器周围有9个重叠三重态微管的典型补体(图1B)。每个新生精子细胞都继承了一种二百里酵母,在发育过程中,两个中心粒分离,重新定位并成为双鞭配子中的基础体。在蕨类植物中,睑缘状体在精细细胞中形成。每个睑缘质体是球形的,并且包含将被组装在年轻精子细胞中的致密基质中的所有基体的中心轮毂(图1C)。
    中心粒的另一种起源模式发生在具有多个鞭毛的两个lycophytes之一的Phylloglossum 中。在这种情况下,中心粒起源于中心轮毂的分支单元,三联体直接组装在该中心毂上(图1D)。 20个左右的基底体以这种方式形成分离,并且围绕着发育中的精子细胞的前部并排并进入鞭毛。在种子植物中,银杏叶中的一对大量眼睑毛细胞在电子致密单元周围组装大约1000厘沲,其中包含偶尔含有基体的球形电子透射区域(图1E)。苏铁生产类似于银杏的眼睑鳞状细胞,但要大得多,产生大约40,000-50,000个基底体(Gifford和Larson,1980)。


    图1.精子细胞中基础体的起源。 :一种。苔藓植物和大多数lycophytes的二百里香在这里代表了巨型鞭毛虫(Megaceros flagellaris),一种伏地草。它由两个由中心核心(箭头)端对端连接的中心线组成。 B. Ph inian a inian a a B. B. B. B.。。。。B. horn,,,,,。。。。。。。。。。。。。。。。。。。。。。 C.镰刀菌(Ceratopteris richardii)中所示的蕨类植物的睑缘质体包含嵌入非晶密集基质中的许多基体体心。 D.一种分枝的中心单位形成在具有大约20个鞭毛的两个淋巴细胞之一的Phylloglossum drummondii的精子细胞母细胞中。将三倍体微管直接装配在毂上,首先(箭头)和B和C微管产生一个微管。 E.银杏的精子母细胞中的大量眼睑细胞在电子致密单元周围具有约1,000个中心粒子,其中包含偶尔含有基体(箭头)的球形电子透射区域。棒=0.2μm(A,C,D),0.1μm(B),1.0μm(E)
    在新生精子细胞中,基体随着其他器官的发育而分离和迁移。这种重新定位通常与致密的微管组织中心相关联,许多微管从该中心发出(图2A)。在蕨类植物中,基本体被组装在中心轮毂周围,因为加入三联体的A,B和C微管(图2B)。然后,短基础体与由层状条和微管带组成的多层结构(图2C)一起迁移,直到它们以精确的布置定位在细胞的前线圈周围(图2D)。在重新定位时,基底体从两端开始伸长。在近端,添加更多的三联体以延长基底体,并且在蕨类植物中,轮毂形成高度伸长的延伸,其是短暂的并且在晚期精子中消失(图2E)。包含星状构型的过渡区(图2E)在基体重新定位期间开始形成(图2D)。


    图2.微管组织中心和精子细胞(发育精子细胞)中的运动器官的发展。 :一种。密集的微管组织中心(MTOC)具有发出的微管(*)和三个基础体,在"Phylloglossum drummondii"中。 MTOC指导基体放置。 B.在Ceratopteris richardii的精子细胞中,在横截面(左箭头)和纵向截面(右箭头)中看到的短基底体在电子致密基质中发展和重组。 C.基层体重组C. richardii 与由层状带和微管带组成的多层结构(mls)的发展同步发生。 D.在前运动器械的组装过程中,C的基体。 richardii 从两端生长,导致整齐排列的细长基底体;具有星状图案(sp)的后过渡区域在这些基体中开始延长。 E. fl is f ern fl after after after f f f f f f f f。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。延伸是短暂的,因此在成熟的配子中是缺乏的。过渡区域中的基底体(bb)和星状图案(sp)位于从细胞体(箭头)出现的鞭毛的内部。棒=0.2μm(A-E)。

    近端基体延长对于定位基础体在苔藓和具有两个二形交错基底体的肝脏精液中是至关重要的(图3A-3C)。通过在一个基底体中通常三个腹侧三联体的近端伸长来实现交错,其变得非常长(图3A),并且最终锚定在距离较不细长的前基底部一定距离处。前基底体中背侧三联体的轻微近端延伸将其固定在多层结构内(图3B)。苔藓配子发育的一个独特特征是存在从微管带流出的单个微管,并且似乎在后基底体伸长期间将腹侧三联体引导到位(图3C)。


    图3.成熟基底体和鞭毛的结构 A.多发性心脏的后基底体,简单的沙拉鱼肝,由细长的腹侧三联体(箭头)和轮毂h)远远超出原始基体(bb)。 B.双锥体基底体,一种复杂的沙门氏菌,横截面。多层结构(mls)对着右侧的前基底体,其中包括轮毂和六个三线制延伸部和左侧的后基底体,其仅由三个腹侧三联体延伸部分组成。 C.麝香的后基底体,如ium re re re,,,,,,,,,and and and and and and and and and。。。。。by((((((((((((((((((((((((((在这个横截面中可以看出,右侧的前鞭毛只在多层结构的微管带外面。 D.Leophophyte Phylloglossum中的基底体和鞭毛的纵剖面,示出了对应于从右到左的横截面的基体(F1),过渡区(F2)和鞭毛(F3) Equisetum 中的F图。值得注意的是,过渡区的星状图案位于细胞体的鞭毛出现之前和之后(箭头)。 E.过渡区域的星状图案的两个基底体的横断面以及fl叶中的两个鞭毛。仍然包含在细胞体中的星状图案(左箭头)由九个重叠的三联体组成,并且位于鞭毛轴(右箭头)中的星形图案(左箭头)具有与单个中心微管的九个重叠的双峰。 F.图D中F1,F2和F3表示的基础体,星状图和轴突的横截面。G.镰刀菌中的鞭毛的横截面证明了陆地植物轴突的典型的9 + 2构型。棒=0.2μm(A-F)。

    注意:对于下面的每个溶液变化,通过加入溶液完全饱和组织,直到其上升到小瓶/管中的材料上(这可以是1ml至5ml(或更多种)溶液的任何位置,这取决于多少组织在每个管或小瓶中)
    1. 如果在0.05M磷酸钠缓冲液(pH7.2)中的2.5%过氧化物中,固定植物组织(如果非常小的切割或全配子体,例如蕨类蕨类配子体)。
    2. 在0.05M磷酸缓冲液(pH 7.2)中冲洗三次(每次15分钟)。
    3. 在2%四氧化锇(1.5%的亚铁氰化钾)中,室温下固定2小时。
    4. 在双蒸蒸压灭菌水(每次10分钟)中冲洗三次,通过25%,50%,75%和95%乙醇脱水(每次20分钟),在100%乙醇(每次20分钟)中漂洗两次,然后冲洗三次在100%环氧丙烷中(每次15分钟)
    5. 在4℃下,在低粘度树脂(用环氧丙烷稀释)中浸泡25%,50%和75%12小时,浸泡3次纯树脂8小时。
    6. 每个BEEM包埋胶囊放置一个或两个花药(取决于大小),并在60℃下在烘箱中聚合48小时。
    7. 用金刚石切割金/银切片,并使用槽(1 x 2 mm)或200目铜网格收集在任何一种形状的胶片上。
    8. 用2%乙酸铀酰(2分钟)后染色,然后加入碱性柠檬酸铅(5分钟),然后在日立HF7100中观察。

食谱

  1. 0.01M磷酸盐缓冲液(pH7.2)
    加入5份0.20 M Sorenson磷酸盐缓冲液(pH 7.2)至95份双蒸高压灭菌水
  2. 0.05 M磷酸盐缓冲液(pH 7.2)
    加入25份0.20 M Sorenson磷酸盐缓冲液(pH 7.2)至75份双蒸压灭菌水
  3. 2.5%戊二醛
    1. 将1份10%戊二醛加入1份双蒸煮蒸压水中
    2. 将1份0.20 M Sorensen磷酸盐缓冲液(pH 7.2)加入1份双蒸压蒸煮水中
    3. 加入1份5%戊二醛至1份0.10M索伦森磷酸缓冲液(pH7.2)
  4. 0.05 M二甲胂酸钠缓冲液(pH 7.2)
    加入1份0.30 M二甲胂酸钠缓冲液(pH 7.2)至6份双蒸压蒸煮水
  5. 2%四氧化锇水溶液
    将1份4%四氧化锇水溶液加入1份双蒸精制高压灭菌水中
  6. 0.02 M磷酸盐缓冲液(pH 7.2)
    加入1份0.20 M Sorenson磷酸盐缓冲液(pH 7.2)至10份双蒸压灭菌水
  7. 0.05 M PIPES缓冲液(pH 7.2)
    将1份0.30 M PIPES缓冲液(pH 7.2)加入6份双蒸煮蒸压水中
  8. 1.5%亚铁氰化钾
    加入1.5克亚铁氰化钾至10ml双蒸高压灭菌水中
  9. 2%乙酸铀酰
    加入2g乙酸双氧铀至10ml双蒸高压灭菌水或100%甲醇
  10. 雷诺的柠檬酸铅
    1. 将1.33克硝酸铅和1.76克柠檬酸钠加入到已冷却至室温并摇动30分钟的30毫升煮沸的双蒸煮高压灭菌水中。
    2. 加入8 ml 1N NaOH
    3. 加入12毫升双蒸高压灭菌水

致谢

这项研究得到了国家科学基金资助的研究资助(DEB-0322664,DEB-0423625,DEB0521177和DEB-0228679)的支持,作为本科生研究经验和组装生命之树计划的一部分。

参考

  1. Gifford,EM和Larson,S。(1980)。< a class ="ke-insertfile"href ="https://www.jstor.org/stable/2442543https:/www.jstor.org/stable/2442543 ?seq = 1"target ="_ blank">银杏叶中精子细胞的发育特征 Am J Bot 62:119-124。 />
  2. Lopez,R.A。和Renzaglia,K.S。(2008)。 精液细胞结构,授精和受精在蕨类植物,Cer藜(Ceratopteris richardii)。 Sex Plant Reprod 21:153-167。
  3. Lopez,RA和Renzaglia,KS(2014)。  >>>>>>>>>>>>> Cer>> ii>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
  4. Renzaglia,KS和Carothers,ZB(1986)。 J Hattori Bot Lab 60:97-104。
  5. Renzaglia,KS和Garbary,DJ(2001)。  土地活动男性配子植物:多样性,发展和进化。 Crit Rev Sci 20:107-213。
  6. Renzaglia,KS和Maden,AR(2000)。< a class ="ke-insertfile"href ="http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0029(20000601)49: 5%3C496 :: AID-JEMT12%3E3.0.CO; 2-H /摘要"target ="_ blank">微管组织中心和蕨类植物精子发生过程中的中心粒起源于蕨类植物 / a> Micros Res Tech 49:496-505。
  7. Southworth,D。和Cresti,M.(1997)。植物中鞭毛和非鞭毛状精子的比较 Am J Bot 84:1301-1311。
  8. Vaughn,KC和Harper,JD(1998)。  微管组织中心和陆地植物中的成核地点。 Int Rev Cytol 181:75-149。
  9. Vaughn,KC和Renzaglia,KS(1998)。  anthocerote spermatogenous cells中的bicentrioles的起源。在Bates,JW,Ashton,N.,Duckett,JG(Eds。)。二十一世纪的苔藓学。
  10. Vaughn,KC和Renzaglia,KS(2006)。  结构和银杏的免疫细胞化学表征。精子活力装置。原生质体 22:165-173。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Renzaglia, K. S., Lopez, R. A., Henry, J. S., Flowers, N. D. and Vaughn, K. C. (2017). Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants. Bio-protocol 7(19): e2448. DOI: 10.21769/BioProtoc.2448.
  2. Renzaglia, K. S., Villarreal, J. C., Piatkowski, B. T., Lucas, J. R. and Merced, A. (2017). Hornwort Stomata: Architecture and Fate Shared with 400-Million-Year-Old Fossil Plants without Leaves. Plant Physiol 174(2): 788-797.
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