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Scanning Electron Microscopy of Motile Male Gametes of Land Plants
陆生植物游动雄配子的扫描电子显微镜观察   

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Abstract

The only motile cells produced in land plants are male gametes (spermatozoids), which are reduced to non-flagellated cells in flowering plants and most gymnosperms. Although a coiled architecture is universal, the complexity of land plant flagellated cells varies from biflagellated in bryophytes to thousands of flagella per gametes in the seed plants Ginkgo and cycads. This wide diversity in number of flagella is associated with vast differences in cell size and shape. Scanning electron microscopy (SEM) has played an important role in characterizing the external form, including cell shape and arrangement of flagella, across the varied motile gametes of land plants. Because of the size and scarcity of released swimming sperm, it is difficult to concentrate them and prepare them for observation in the SEM. Here we detail an SEM preparation technique that yields good preservation of sperms cells across plant groups.

Keywords: Flagella(鞭毛), Male gamete(雄配子), Scanning electron microscopy(扫描电子显微镜观察), Spermatozoid(游动精子)

Background

Motile gametes of land plants are strikingly diverse and develop through transformations that involve repositioning cellular components and the assembly of a complex locomotory apparatus (Renzaglia and Garbary, 2001). 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. 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, and liverworts) to an estimated 1,000-40,000 in Ginkgo and cycads. 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). Male gametes provide a wealth of biological information, including biodiversity and cell differentiation and evolution (Garbary et al., 1993; Renzaglia et al., 1995; Renzaglia and Garbary, 2001; Renzaglia et al., 2000; Lopez-Smith and Renzaglia, 2008; Lopez and Renzaglia, 2014). Of the range of microscopic techniques utilized to characterize plant spermatozoids, SEM provides the most direct means of elucidating cell shape, and flagella number, length and arrangement. Together with TEM observations, SEM studies lead to comparative descriptions of gamete architecture, and organellar content and arrangement across plant lineages (Renzaglia et al., 2001 and 2002; Lopez-Smith and Renzaglia, 2008).

Materials and Reagents

Scanning Electron Microscopy (SEM) and the motile sperm cell architecture

  1. Pipette tips 1-200 µl (Carolina Biological Supply, catalog number: 215055 )
  2. Pasteur pipettes (Fisher Scientific, catalog number: 13-678-4)
    Manufacturer: Corning, catalog number: C7095B5X .
  3. 1.5-ml centrifuge tubes (USA Scientific, catalog number: 1615-5500 )
  4. Glass coverslips 22 x 22 mm (Fisher Scientific , catalog number: 12-542B )
  5. Male plants with mature sperm cells
    1. Pellia epiphylla
    2. Conocephalum conicum
    3. Equisetum arvense
    4. Ceratopteris richardii
  6. Sorensens phosphate buffer, 0.2 M, pH 7.2 (Electron Microscopy Sciences , catalog number: 11600-10 )
  7. Glutaraldehyde (Electron Microscopy Sciences , catalog number: 16120 )
  8. Sodium cacodylate buffer, 0.2 M, pH 7.4 (Electron Microscopy Sciences, catalog number: 11652 )
  9. Osmium tetroxide (Electron Microscopy Sciences, catalog number: 19150 )
  10. Ethanol (Decan Laboritories, catalog number: 2705HC )
  11. Hexamethyl disilizane (HMDS) (Electron Microscopy Sciences, catalog number: 16700 )
  12. 0.01 M phosphate buffer (pH 7.2) (see Recipes)
  13. 0.05 M phosphate buffer (pH 7.2) (see Recipes)
  14. 0.02 M phosphate buffer (pH 7.2) (see Recipes)
  15. 2.5% glutaraldehyde (see Recipes)
  16. 0.05 M sodium cacodylate buffer (pH 7.2) (see Recipes)
  17. 2% aqueous osmium tetroxide (see Recipes)

Equipment

  1. Scanning electron microscope (SEM) (FEI, model: QuantaTM 450 FEG )
  2. Shaker (Thermolyne, model: M-16715 )
  3. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: mySPINTM 6 , catalog number: 75004061)
  4. Oven (General Signal, model: Gravity convection )
  5. SEM Specimen Mount Stubs, Aluminum Slotted Head (Electron Microscopy Sciences, catalog number: 75220 )
  6. Samdri 790 Critical Point Dryer (Tousumis)
  7. Sputter coater (Denton Vacuum, model: Desk V )

Procedure

Scanning Electron Microscopy (SEM) and the motile sperm cell architecture:
SEM examination of mature sperm cells has enabled rapid visualization and comparisons of whole gamete architecture compared with laborious TEM observations and cell reconstructions. The diversity of gamete architecture and organization of flagella in mature cells is readily seen in the SEM (Figure 1).


Figure 1. Scanning electron micrographs of plant sperm cells. A. Pellia epiphylla, a simple thalloid liverwort. This is one of the longest sperm cells produced by bryophytes. The two flagella (f) are staggered in their insertion near the cell anterior (a) and the cell body consists of a long cylindrical nucleus (n) with a plastid and mitochondrion at the posterior (arrow). B. Conocephalum conicum, a complex thalloid liverwort. This sperm cell is much smaller than that of Pellia. The two flagella (f) are inserted anteriorly (a) and are much longer than the cell body, which consists of a cylindrical nucleus (n) and posterior cytoplasm that in this case is not fully extended. C. Equisetum arvense, a eusporangiate fern. The approximately 36 basal bodies (bb) attach flagella (f) along the anterior coils of the cell. The nucleus (n) extends to the cell posterior (not visible). D. Ceratopteris richardii, a leptosporangiate fern. This cell coils four times and has about 80 flagella (f) inserted anteriorly (a). The cylindrical nucleus (n) extends over three coils and tapers at the cell posterior. Bars = 5.0 µm (A), 1.0 µm (B, C, D).


  1. Using excised male tissue (antheridia of male gametophytes), place several antheridia in each droplet (ca. 100 µl) of 0.01 M phosphate buffer, pH 7.2 (see Recipes), on a dental wax plate, cover to retard evaporation, and leave overnight at room temperature to facilitate release of sperm. Alternatively, excised antheridia can be placed in 1.5 ml Eppendorf centrifuge tubes adding 1 ml of 0.01 M phosphate buffer, pH 7.2 to completely cover tissue and left overnight. Antheridial tissue can be removed and discarded the next day. Swimming sperm will be in the buffer.
  2. Remove tissue and place the droplet in a 1.5 ml Eppendorf centrifuge tube containing 1 ml 2.5% glutaraldehyde (see Recipes) in 0.05 M sodium cacodylate buffer, pH 7.2 (see Recipes).
  3. Shake tube gently and fix the spermatozoid suspension for 1 h at room temperature.
    Note: From this point on, spermatozoids are concentrated by centrifugation at 100 x g for 5 min between each solution change. Because the sperm are colorless, you will most likely not see a pellet after centrifugation.
  4. Remove the glutaraldehyde supernatant down to 0.1 ml leaving the pellet at bottom of tube. Rinse fixed sperm cells three times (10 min each) in 1 ml 0.05 M sodium cacodylate buffer.
  5. Post-fix tissue for 20 min in 1 ml 2% aqueous osmium tetroxide (see Recipes).
  6. Rinse specimens twice in 1 ml double distilled autoclaved water (10 min), once each in 25%, 50% and in 75% ethanol (10 min), twice in 100% ethanol (10 min), and then twice in 100% hexamethyl disilizane (HMDS; 10 min).
  7. Pipet sperm cells in 1 ml HMDS on to a glass coverslip affixed to a standard aluminum pin-type mounting stub and dry for 20 min in a 60 °C oven.
  8. Specimens on mounting stubs are sputter-coated with an ultra-thin layer (ca. 390 nm) of a conducting metal (palladium–gold) that prevents charging from non-conducting biological specimens, increases the number of secondary electrons from the specimen surface that improves topographical contrast, and minimizes damage to the specimen.
    Basic steps:
    1. Vacuum down sputter coater
    2. Out-gas with Argon gas
    3. Apply voltage
    4. Open gas to the specific emissions
    5. Coat with ca. 390 nm palladium–gold
  9. View on a high-resolution SEM.

Note: Often cells aggregate around the edges of the coverslip, so it is better to put a very small drop of sperm cell suspension in HMDS in the middle of the coverslip to dry.

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. 0.02 M phosphate buffer (pH 7.2)
    Add 1 part 0.20 M Sorenson’s phosphate buffer (pH 7.2) to 9 parts double distilled autoclaved water
  4. 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)
  5. 0.05 M sodium cacodylate buffer (pH 7.2)
    Add 1 part 0.30 M sodium cacodylate buffer (pH 7.2) to 5 parts double distilled autoclaved water
  6. 2% aqueous osmium tetroxide
    Add 1 part 4% aqueous osmium tetroxide to 1 part 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. Garbary, D. J., Renzaglia, K. S. and Duckett, J. G. (1993). The phylogeny of land plants: a cladistic analysis based on male gametogenesis. Plant Syst Evol 188: 237-269.
  2. Lopez, R. A. and Renzaglia, K. S. (2014). Multiflagellated sperm cells of Ceratopteris richardii are bathed in arabinogalactan proteins throughout development. Am J Bot 101(12): 2052-2061.
  3. Lopez-Smith, R. and Renzaglia, K. (2008). Sperm cell architecture, insemination, and fertilization in the model fern, Ceratopteris richardii. Sex Plant Reprod 21:153-167.
  4. Renzaglia, K. S., Dengate, S. B., Schmitt, S. J., Duckett, J. G. (2002). Novel features of Equisetum arvense spermatozoids: insights into pteridophyte evolution. New Phytol 154:159-174.
  5. Renzaglia, K. S., Duff, R. J. T., Nickrent, D. L. and Garbary, D. J. (2000). Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny. Philos Trans R Soc Lond B Biol Sci 355(1398): 769-793.
  6. Renzaglia, K. S. and Garbary, D. J. (2001). Motile male gametes of land plants: Diversity, development, and evolution. Crit Rev Sci 20:107-213.
  7. Renzaglia, K. S., Johnson, T. H., Gates, H. D. and Whittier, D. P. (2001). Architecture of the sperm cell of Psilotum. Am J Bot 88(7): 1151-1163.
  8. Renzaglia, K. S., Rasch, E. M. and Pike, L. M. (1995). Estimates of nuclear DNA content in bryophyte sperm cells: phylogentic considerations. Am J Bot 82: 18-25.
  9. Southworth, D. and Cresti, M. (1997). Comparison of flagellated and nonflagellated sperm in plants. Am J Bot 84:1301-1311.

简介

在陆地植物中生产的唯一的活细胞是雄性配子(精子),它们在开花植物和大多数裸子植物中被还原成非鞭毛细胞。 虽然盘绕的建筑是普遍的,但是种植植物鞭毛细胞的复杂性在种子植物银杏和苏铁中每个配子的苔藓植物双歧杆菌数量增加到成千上万的鞭毛。 鞭毛数量的多样性与细胞大小和形状的巨大差异有关。 扫描电子显微镜(SEM)在表征陆地植物各种动态配子的外形形态,包括鞭毛的细胞形态和排列方面发挥了重要作用。 由于释放游泳精子的大小和稀缺性,难以集中精力并准备在SEM中观察。 在这里,我们详细介绍了SEM制备技术,可以在植物组中产生精子细胞的良好保存。
【背景】土地植物的运动配子是非常多样化的,通过涉及重新定位细胞组分和组装复杂运动器官的转化来发展(Renzaglia和Garbary,2001)。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。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多变化,从苔藓植物(苔藓,>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)。男性配子提供了丰富的生物学信息,包括生物多样性和细胞分化和进化(Garbary et al。,1993; Renzaglia et al。,1995; Renzaglia and Garbary,2001 ; Renzaglia等人,2000; Lopez-Smith和Renzaglia,2008; Lopez和Renzaglia,2014)。在用于表征植物精子的微观技术的范围内,SEM提供了阐明细胞形状和鞭毛数量,长度和排列的最直接手段。与TEM观察一起,SEM研究导致配子结构和细胞器内容与植物谱系(Renzaglia et al。,2001和2002; Lopez-Smith和Renzaglia,2008)的比较描述。

关键字:鞭毛, 雄配子, 扫描电子显微镜观察, 游动精子

材料和试剂

扫描电子显微镜(SEM)和运动精子细胞结构

  1. 移液器提示1-200μl(Carolina Biological Supply,目录号:215055)
  2. 巴斯德移液器(Fisher Scientific,目录号:13-678-4)
    制造商:Corning,目录号:C7095B5X。
  3. 1.5 ml离心管(USA Scientific,目录号:1615-5500)
  4. 玻璃盖玻片22 x 22毫米(费雪科学,目录号:12-542B)
  5. 具有成熟精子细胞的雄性植物
    1. > la>>>>。。。。。。。。
    2. Conocephalum conicum

    3. Ceratopteris richardii
  6. Sorensens磷酸盐缓冲液,0.2M,pH7.2(Electron Microscopy Sciences,目录号:11600-10)
  7. 戊二醛(Electron Microscopy Sciences,目录号:16120)
  8. 二甲胂酸钠缓冲液,0.2M,pH 7.4(电子显微镜科学,目录号:11652)
  9. 四氧化锇(Electron Microscopy Sciences,目录号:19150)
  10. 乙醇(Decan Laboratories,目录号:2705HC)
  11. 六甲基二硅氮烷(HMDS)(Electron Microscopy Sciences,目录号:16700)
  12. 0.01 M磷酸盐缓冲液(pH 7.2)(见配方)
  13. 0.05 M磷酸盐缓冲液(pH 7.2)(见配方)
  14. 0.02 M磷酸缓冲液(pH 7.2)(见配方)
  15. 2.5%戊二醛(见食谱)
  16. 0.05 M二甲胂酸钠缓冲液(pH 7.2)(见配方)
  17. 2%四氧化锇水溶液(见配方)

设备

  1. 扫描电子显微镜(SEM)(FEI,型号:Quanta TM 450 FEG)
  2. 振动筛(Thermolyne,型号:M-16715)
  3. 离心机(Thermo Fisher Scientific,Thermo Scientific TM ,型号:mySPIN TM 6,目录号:75004061)
  4. 烤箱(一般信号,型号:重力对流)
  5. SEM样品安装支柱,铝开槽头(电子显微镜科学,目录号:75220)
  6. Samdri 790临界点烘干机(Tousumis)
  7. 溅射涂布机(Denton Vacuum,型号:Desk V)

程序

扫描电子显微镜(SEM)和运动精子细胞结构:
成熟精子细胞的SEM检查使得快速可视化和比较整个配子结构与费力的TEM观察和细胞重建相比。成熟细胞中配子结构和鞭毛组织的多样性在SEM中很容易看出(图1)。


图1.植物精子细胞的扫描电子显微照片。 :一种。 Pellia epiphylla ,一种简单的thalloid liverwort。这是苔藓植物产生的最长的精子细胞之一。两个鞭毛(f)在插入细胞前方交错交错(a),细胞体由长的圆柱形核(n)组成,后部具有质体和线粒体(箭头)。 B. conocephalum conicum ,一种复杂的thalloid hepwort。这个精子细胞比Pellia 小得多。两个鞭毛(f)前面(a)插入,并且比细胞体长得多,细胞体由圆柱形核(n)和后细胞质组成,在这种情况下,这不是完全延伸的。 Equ se ar。。。。。。。。。。。。。。。。。。。。。。大约36个基底体(bb)沿着细胞的前线圈附着鞭毛(f)。核(n)延伸到细胞后(不可见)。镰刀菌(Ceratopteris richardii),一种叶片孢子蕨。该细胞四次缠绕,并且具有约80个鞭毛(f)前方插入(a)。圆柱形核(n)延伸三个线圈,并在细胞后方呈锥形。棒=5.0μm(A),1.0μm(B,C,D)。


  1. 使用切除的雄性组织(雄性配子体的吸虫),在0.01M磷酸盐缓冲液(pH7.2)(见食谱)的每个液滴(100μl)中在牙蜡板上盖几个花药以延缓蒸发,并在室温下放置一夜,以促进精子的释放。或者,将切除的烟酰胺置于1.5ml Eppendorf离心管中,加入1ml 0.01M磷酸盐缓冲液(pH7.2)以完全覆盖组织并放置过夜。第二天可以去除和丢弃炎症组织。游泳精子将在缓冲区。
  2. 取出组织,并将液滴放入含有1毫升2.5%戊二醛的1.5毫升Eppendorf离心管中(参见食谱)在0.05M二甲胂酸钠缓冲液(pH 7.2)中(见食谱)。
  3. 轻轻摇动管,并在室温下将精子悬浮液固定1 h 注意:从这一点上,通过在每个溶液变化之间以100×g离心5分钟浓缩精子。因为精子是无色的,离心后很可能看不到颗粒。
  4. 将戊二醛上清液除去至0.1ml,将颗粒置于管底部。在1ml 0.05M二甲胂酸钠缓冲液中漂洗固定的精子细胞三次(每次10分钟)
  5. 在1ml 2%四氧化锇水溶液中修复组织20分钟(参见食谱)
  6. 在1ml双蒸高压灭菌水(10分钟)中冲洗标本两次,每次25%,50%和75%乙醇(10分钟),100%乙醇(10分钟)中两次,然后在100%六甲基二disilizane(HMDS; 10分钟)
  7. 将1毫升HMDS中的精液细胞吸入固定在标准铝针式安装支架上的玻璃盖玻片上,并在60℃烘箱中干燥20分钟。
  8. 安装支柱上的样品被溅射涂覆有导电金属(钯 - 金)的超薄层(大约390nm),其阻止非导电生物样品的充电,增加了来自样品表面的二次电子改善了地形对比度,并最大限度地减少了对样品的损伤 基本步骤:
    1. 真空下溅射涂布机
    2. 氩气气体
    3. 施加电压
    4. 开放气体到特定排放物
    5. 大衣与大约 390nm钯金
  9. 查看高分辨率SEM。

注意:细胞通常聚集在盖玻片的边缘周围,因此最好将一小滴精子细胞悬浮液放在盖玻片中间的HMDS中干燥。

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. 0.02 M磷酸盐缓冲液(pH 7.2)
    将1份0.20 M Sorenson磷酸盐缓冲液(pH 7.2)加入到9份双蒸压高压灭菌水中
  4. 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)
  5. 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5份双蒸煮蒸压水
  6. 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

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. Garbary,D.J.,Renzaglia,K.S。和Duckett,J.G。(1993)。 陆地植物的系统发育:基于雄性配子发生的分层分析。植物系统Evol 188:237-269。
  2. Lopez,R.A。和Renzaglia,K.S。(2014)。在开发过程中,多鞭毛虫精子细胞沐浴在阿拉伯半乳聚糖蛋白质中。 Am J Bot 101(12):2052-2061。
  3. Lopez-Smith,R。和Renzaglia,K。(2008)。 蕨类植物中的精子细胞结构,授精和受精,Cer藜richardii 。 Sex Plant Reprod 21:153-167。
  4. Renzaglia,K.S.,Dengate,S.B.,Schmitt,S.J.,Duckett,J.G。(2002)。 新的Phytol 154:159-174。
  5. Renzaglia,K.S.Duff,R.J.T.,Nickrent,D.L。和Garbary,D.J。(2000)。 早期土地植物的植物和生殖创新:对统一的系统发育的影响。 > Philos Trans R Soc Lond B Biol Sci 355(1398):769-793。
  6. Renzaglia,K.S。和Garbary,D.J。(2001)。 土地植物的动态男性配子:多样性,发展和进化。 Crit Rev Sci 20:107-213。
  7. Renzaglia,K.S.,Johnson,T.H.,Gates,H.D。和Whittier,D.P。(2001)。 Psilotum 精子细胞的体系结构 Am J Bot 88(7):1151-1163。
  8. Renzaglia,K.S.,Rasch,E.M。和Pike,L.M。(1995)。 苔藓植物精子细胞中核DNA含量的估计:系统学考虑 Am J Bot 82:18-25。
  9. 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。
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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. and Schmitt, S. J. (2017). Scanning Electron Microscopy of Motile Male Gametes of Land Plants. Bio-protocol 7(19): e2570. DOI: 10.21769/BioProtoc.2570.
  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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