蕨类植物精细胞运动器和细胞骨架的研究

介绍了近年来蕨类植物游动精子运动器和细胞骨架的研究进展。游动精子由配子体精子器中的非运动细胞发育形成,其分化过程包括了运动器官和细胞骨架的合成和组装。精子发生过程中形成的运动器的各部分结构包括鞭毛、基体、多层结构及附属结构;基体是细胞中新形成的结构,在不同类群的蕨类植物中分别由双中心粒、分支生毛体和生毛体产生。鞭毛、基体和多层结构中的微管带形成了游动精子三个独特的微管列阵,由于微管蛋白的后修饰作用这些微管列阵十分稳定;centrin是运动器中的重要成分, 但功能尚不清楚,可能和细胞骨架及运动器的构建有关。     

傅氏凤尾蕨精子发生超微结构的研究

超微结构研究显示傅氏凤尾蕨（Pteris fauriei Hieron）精子发生过程包括生毛体、多层结构和鞭毛等运动细胞器重新发生,环状线粒体形成,核塑形等过程,最后形成一个螺旋形的游动精子,这与其他真蕨类精子发生过程相似。本研究观察到的一些新现象包括：精细胞在分化早期呈极性,细胞核位于精细胞的近极端,生毛体、线粒体和质体等细胞器主要分布远极端;在生毛体分化早期,可见大量微管从其发出,其周围线粒体丰富;基体分化经历了前中心粒、中心粒和基体3个阶段,它们的内部结构不同;研究表明生毛体内的不定形物质是微管组织者,多层结构、附属微管带及鞭毛等细胞器均由不定形物质分化形成;精细胞在分化过程中产生了丰富的膜结构,它们可能为精核塑形提供原料。本研究报道了傅氏凤尾蕨精细胞分化的一些细节,这有助于进一步揭示蕨类植物精子发生的细胞学机制。     

水蕨精子发生过程中中心体蛋白和微管蛋白的免疫荧光定位

采用透射电镜技术和免疫荧光标记技术对水蕨精子发生的超微结构以及中心体蛋白和微管蛋白在精子发生过程中的动态表达进行了观察。研究发现：（1）生毛体分化早期周围有放射状微管分布,这与线粒体向生毛体的聚集有关。（2）免疫荧光观察表明,中心体蛋白仅定位于生毛体、基体和鞭毛带上,自生毛体至基体阶段呈现明亮的荧光标记,在核塑形、鞭毛形成至精子成熟阶段,中心体蛋白荧光标记随着鞭毛的发生而逐渐减弱,至游动精子阶段中心体蛋白荧光标记信号几乎消失。（3）微管蛋白早期荧光标记与中心体蛋白标记形相同,在生毛体、鞭毛带、基体等运动细胞器上呈现明亮荧光标记,但微管蛋白随着鞭毛的发生其荧光标记越来越强。从二者的时空表达特征可以推断,中心体蛋白主要是运动细胞器的组织者,而非这些运动细胞器的结构成分,其功能是参与或负责中心粒、基体和鞭毛的发生。     

蕨类植物分株紫萁精子发生过程中生毛体和多层结构的超微结构

应用电镜技术对蕨类植物分株紫萁(Osmunda cinnamomea L. var.asiatica Fernald)精子发育过程中的生毛体和多层结构的超微结构进行了研究.生毛体在幼精子细胞中出现,正在分化的生毛体略呈球状,球状体的中央由一团染色深的颗粒状物质构成,外围分化出若干柱状体.已分化的生毛体由柱状体分散或辐射状排列构成,呈球状,球体中心不含染色深的物质.多层结构位于精子细胞内的基体和巨大线粒体之间,刚形成时仅由片层构成,片层相互平行排列形成片层带.多层结构在分化中期由微管带、片层带和蚀斑三层构成.多层结构在分化末期又形成附属微管带、嗜锇冠和嗜锇层.微管带从多层结构长出,沿细胞核的表面伸展,并与核膜之间形成复合结构.基体由柱状体转变而成,它向两端生长,在远端产生鞭毛的轴丝,在近轴端形成楔状结构.本文首次详细阐明了原始薄囊蕨分株紫萁生毛体和多层结构发育的超微结构特点,并与其他蕨类进行了比较,发现其片层带出现在微管带形成之前.     

蕨类植物分株紫萁精子发生过程中生毛体和多层结构的超微结构

应用电镜技术对蕨类植物分株紫萁(Osmunda cinnamomea L. var．asiatica Fernald)精子发育过程中的生毛体和多层结构的超微结构进行了研究。牛毛体在幼精了细胞中出现,正在分化的生毛体略呈球状,球状体的中央由一团染色深的颗粒状物质构成,外围分化出若干柱状体。已分化的生毛体由柱状体分散或辐射状排列构成,呈球状,球体中心不含染色深的物质。多层结构位于精子细胞内的基体和巨大线粒体之间,刚形成时仅由片层构成,片层相互平行排列形成片层带。多层结构在分化中期由微管带、片层带和蚀斑三层构成。多层结构在分化末期又形成附属微管带、嗜锇冠和嗜锇层。微管带从多层结构长出,沿细胞核的表面伸展,并与核膜之间形成复合结构。基体由柱状体转变而成,它向两端生长,在远端产生鞭毛的轴丝,在近轴端形成楔状结构。本文首次详细阐明了原始薄囊蕨分株紫其生毛体和多层结构发育的超微结构特点,并与其他蕨类进行了比较,发现其片层带出现在微管带形成之前。     

海金沙精细胸中生毛体发育及多层细胞起源的超微结构研究

蕨类植物海金沙（Ｌｙｇｏｄｉｕｍ ｊｏｐｏｎｉｃｕｍ （Ｔｈｕｎｂ．）Ｓｗ．）的游动精子发育过程中,生毛体在精母细胞质中出现,它是直径为０．５～０．６μｍ的椭球,其结构紧密,由辐射排列的具轮辐结构的管状亚单位和无定形基质组成。大量微管从生毛体伸向细胞质。随着精细胞的发育,生毛体细胞变得松散,亚单位分化形成的中心粒彼此分开矿散到外围,中心为无定形物质。伴随着中心粒的分化,多层结构出现,一端与无定形基     

海金沙精细胞中生毛体发育及多层结构起源的超微结构研究

蕨类植物海金沙（LygodiumJaponicum（Thunb．）Sw．）的游动精子发育过程中,生毛体在精母细胞的细胞质中出现,它是直径为0．5－0．6μm的椭球体,其结构紧密,由辐射排列的具轮辐结构的管状亚单位和无定形基质组成。大量微管从生毛体伸向细胞质。随着精细胞的发育,生毛体结构变得松散,亚单位分化形成的中心粒彼此分开扩散到外围,中心为无定形物质。伴随着中心粒的分化,多层结构出现,一端与无定形基质相连。多层结构由外侧的微管带及内侧的片层组成,形成后与一线粒体相连,移向靠近核的位置,并正对着核上出现凹点。研究发现在精原细胞后期出现一团絮状结构,为无定形基质,其中有深染色的小管状结构分布,同时可见微管从絮状结构边缘伸出,这一絮状结构可能与生毛体的产生有一定的关系。     

绵马鳞毛蕨精母细胞和游动精子超微结构特征的研究

应用电镜技术对蕨类植物绵马鳞毛蕨(RYOPTERIS CRASSIRHIZOMA Nakai)精母细胞和游动精子的超微结构特征进行了研究。精母细胞为多边形,细胞质内含有丰富的线粒体、质体、内质网、高尔基体等常见的细胞器．在细胞质中还可见到一些同心圆膜状结构,位于质膜的附近或精母细胞的角偶。同心圆膜状结构由双层膜环绕构成,外被l层单位膜。精母细胞与精子器的璧细胞之间形成了分离腔。在精母细胞质膜外形成了嗜锇层,这些结构的形成说明精母细胞已经开始与雄配子体逐渐分离,进入独立发育的阶段。尽管精母细胞之间也有嗜锇层的形成,但嗜锇层是不连续的,其上有一些空隙,精母细胞之间可通过空隙进行物质和信息的交流。成熟的精子细胞外被l层透明的薄膜,里面为游动精子。螺旋状。由环状细胞器环绕3～4圈构成．这些环状细胞器包括多层结卡构、微管带、巨大线粒体、鞭毛带和1个长形浓缩的细胞核。游动精子的后端为一些泡囊化的细胞质．其中包括一些残存的线粒体、造粉质体及大的囊泡等。当成熟的精子细胞排出精子器后。其内的游动精子挣脱透明质膜的束缚,摆脱后端的囊泡,成为1条游动精子。本文还对绵马鳞毛蕨和其它蕨类植物精子的超微结构特征进行了比较。     

褐菖■精细胞晚期的变化及精子结构研究

本文研究卵胎生硬骨鱼褐菖（Ｓｅｂａｓｔｉｓｃｕｓｍａｒｍｏｒａｔｕｓ）精细胞的成熟变化和精子结构。褐菖精细胞发育晚期已具有硬骨鱼类精子的结构雏形：细胞核的背面较平坦,腹面稍外鼓,呈弧面;染色质浓缩成团块状,核的腹侧和后端的染色质较致密;中心粒复合体由近端中心粒和基体组成,近端中心粒和基体排成“Ｌ”形;近端中心粒向细胞核的背侧伸出中心粒附属物,中心粒附属物由９条微管组成,９条微管围成一筒状结构,类似轴丝。在晚期精细胞形成精子的过程中,中心粒附属物和近端中心粒相继退缩以至消失不见,同时细胞核后端的形状也随着发生变化。中心粒附属物和近端中心粒的相继消失可以看作是成熟的最后标志。精子的中心粒复合体由基体及其上方的基体帽组成,袖套接于核的后端,其中约有３０～４０个线粒体;鞭毛从袖套腔中伸出,鞭毛的中心结构是轴丝;轴丝外方为细胞质形成的侧鳍,在鞭毛的近核段,轴丝两侧的侧鳍较宽且不对称。     

蕨类植物桂皮紫萁颈卵器和精子器形态和发育的研究

利用扫描电镜技术和树脂切片技术对蕨类植物桂皮紫萁（Osmunda cinnamomeaL.var.asiatica Fernald)的颈卵器和精子器的形态和发育进行了细致的研究。颈卵器发生于雌配子体的腹面,颈部由4列壁细胞构成,6-7个细胞高,内部含有颈沟细胞,腹沟细胞和卵细胞,卵细胞在整个发育过程中,造粉体和囊泡最为显著,颈卵器内的卵细胞成熟时产生卵膜和分离腔。精子器发生于雄配子体的边缘及腹面,由7-8个壁细胞螺旋状围绕而成,壁细胞内为产精组织,精子成熟时精子器盖细胞开裂释放出游动精子。     

Ultrastructure of the spermatozoid of Lycopodium obscurum (Lycopodiaceae)

Ultrastructural observations reveal that the spermatozoid of Lycopodium obscurum is crescent shaped and contains two posteriorly directed flagella that are inserted at the front of the cell. The nucleus is broad and elongated with a narrow posterior projection or nuclear diverticulum. Spline microtubules (MTs) number 180 at their maximum and provide the framework for the cell. These MTs extend from the anterior of the locomotory apparatus and along the outermost surface of the nucleus, with a central shank of 14–17 MTs encircling the cell for at least one-third gyre beyond the nucleus. The two basal bodies are slightly staggered and positioned at the front of the cell over a highly elongated multilayered structure (MLS). The MLS extends laterally around the cell anterior and curves posteriorly over the nucleus. One large anterior mitochondrion is situated subjacent to the MLS, while numerous small mitochondria are scattered near or among the lobes of the single plastid. The plastid rests on the inner nuclear surface and contains numerous large starch grains. This cell differs from that of L. cernuum, the only other species of Lycopodium examined to date, in that it is more elongated and has an anterior-posterior orientation of the nucleus, basal bodies, MLS, and spline. Comparisons with coiled gametes of bryophytes and Selaginella suggest that some degree of coiling and cell streamlining may be ancestral in archegoniate spermatozoids.     

FINE STRUCTURE OF THE SPERMATOZOID OF ZAMIA WITH SPECIAL REFERENCE TO THE FLAGELLAR APPARATUS

The locomotor apparatus of the spermatozoid of Zamia integrifolia consists of numerous flagella having the typical 9 + 2 substructure connected through basal bodies to a spiral band of complex structure. Basal bodies have a fine structure somewhat resembling that found in algae, mosses, and ferns, but they are much longer. They are composed of a circle of 9 double fibers just beneath the plasma membrane, changing to 9 doublets interconnected by fibrils in a star-pattern, giving over to a centriolar type of 9 triplet fibers embedded in an electron-dense layer of the spiral band, and ending in a “cartwheel” configuration. A system of microtubules arranged in a spiral, secondary to the flagellated spiral, is thought to underlie the plasma membrane in flagellated regions. It is suggested that this system accounts for “euglenoid” movements of the sperm. Other details of cellular fine structure are described.     

Motile Gametes of Land Plants: Diversity,Development, and Evolution

Referee: Professor Jeffrey Duckett, School of Biological Sciences, Queen Mary and Westfield College, University of London, Mile End Road, London, E1 4NS, UK Spermatogenesis is a morphogenetic system in plants that is unparalled in its potential to yield diverse and informative structural and developmental data. The unquestionable homology of terrestrial plant spermatozoids to each other and to gametes of related lineages allows an examination of cellular evolution and provides sound data for phylogenetic analyses. In this review we examine the architecture and ontogeny of motile male gametes among major groups of land plants. We begin with a historical perspective that emphasizes the utility of spermatogenesis in understanding cellular evolution and in determining phylogenetic relationships. A cladistic analysis of data based solely on spermatogenesis and a conceptual phylogeny based on combined morphological and molecular data serve as the basis for the comprehensive discussion of architectural and developmental features of plant spermatozoids. Spermatozoids of green plants have two fundamental architectural designs: biflagellated or multiflagellated. Biflagellated gametes vary among basal archegoniates and charophytes in degree of coiling, position, and substructure of the basal bodies and number of organelles. Hornwort spermatozoids are simple, bilaterally symmetrical, and uniquely exhibit a right-handed coil. An autapomorphy among setaphytes (a clade containing mosses and liverworts) is the production of coiled biflagellated sperm cells with dimorphic staggered basal bodies. Like bryophytes, gametes of most lycophytes are biflagellated; two exceptions are Isoëtes and Phylloglossum, taxa that independently evolved multiflagellated sperm cells with approximately 20 flagella. Developmental information, especially related to the origin and development of the locomotory apparatus, are essential to determine structural homology among these taxa. Evaluation of the more complicated multiflagellated gametes of other vascular plants reveals similarities that support a monophyletic fern, Equisetum and Psilotum assemblage. Autapomorphies of this clade include the arrangement of the microtubular cytoskeleton, origin of the locomotory apparatus, and structural details of the basal bodies and multilayered structure. Sperm cell development in archegoniates involves the complete transformation of virtually every cellular component. Crucial to this process are proteinaceous elements of the cytoskeleton. Complex microtubule arrays unique to these cells include the spline, basal bodies, and flagella. The discrete microtubule-organizing centers (MTOCs) that generate these cytoskeletal arrays are equally complex and enable the examination of molecular constituents and ontogenetic modifications. The protein centrin is found in a variety of structures, including the diverse MTOCs and the locomotory apparatus. Actin plays a role in organellar shaping and positioning as well as in cytoplasmic deletion and the maintenance of spatial integrity in the mature cell. We conclude with an overview of the current and potential utility of male gametogenesis as an informative system in approaching fundamental questions relating to cellular differentiation and motility. Characterization of motility mutants will elucidate genetic control of structure-function relationships among cellular components, while biochemical and molecular investigations provide crucial data on the mechanism of development. The examination of spermatogenesis in additional taxa is essential to characterize further developmental variations. Moreover, such studies provide a more comprehensive understanding of plant biodiversity at the cellular level and lead to even greater phylogenetic resolution from this elegant morphogenetic system.     

DEVELOPMENT OF THE FLAGELLAR APPARATUS AND FLAGELLAR POSITION IN THE COLONIAL GREEN ALGA PLATYDORINA CAUDATA (CHLOROPHYCEAE)1

The ultrastructure of the flagellar apparatus in pre-inversion and inversion stages of Platydorina resembles that of Chlamydomonas in having 180° rotational symmetry and clockwise absolute orientation. Basal bodies are in a “V” configuration and connected by one distal and two proximal fibers. Alternating two- and four-membered microtubular rootlets are cruciately arranged. During maturation, the basal bodies rotate and separate, and 180° rotational symmetry is lost. Simultaneously, each proximal fiber detaches from one of the functional basal bodies, and the distal fiber detaches from both. The mature apparatus has widely separated and nearly parallel basal bodies. Flagellar orientation in Platydorina is completed just after inversion and a flattening of the colony called intercalation, resulting in the pairs of flagella of neighboring cells extending from the colony in opposite directions in an alternating fashion. Flagellar orientation and separated basal bodies minimize the interference between the flagella of neighboring cells. Basal bodies and rootlets of the two intercalated halves of a colony rotate, resulting in the effective strokes of the flagella of every cell being towards the colonial posterior. The flagella of each cell beat with an effective stroke in the direction of the two inner rootlets. The flagella have an asymmetrical ciliary type beat. The rotated, separated, and parallel basal bodies, together with the nearly parallel rootlets probably are adaptations for movement of this colonial volvocalean alga. The flagellar apparatus in immature stages of Platydorina lends support to the suggestion that the alga has evolved from a Chlamydomonas-like ancestor.     

Ultrastructural characterization of the locomotory cytoskeletal system of the spermatozoid inGinkgo biloba

Summary The development of the locomotory cytoskeletal system of sperm is carefully coordinated with the development of the sperm inGinkgo biloba. Here we report further ultrastructural characterization of the locomotory cytoskeletal system in the developing spermatid and mature spermatozoid, particularly with respect to the initiation and early development of the flagellar apparatus. A multilayered structure (MLS) assembles from an electron-dense matrix that self-organizes after blepharoplast breakup and then further elongates. At the tail of the assembling MLS, the spline microtubules connect to an anterior beak of the nuclear envelope. Nuclear-pore complexes are found on the nuclear envelope close to this beak. The mitochondria which elongate and line up one behind the other are tightly associated with the MLS. The MLS ofG. biloba is composed of an upper layer of parallel spline microtubules and a lower layer consisting of a fibrous lamellar strip composed of paralled fibers about 9 nm in diameter. Higher-magnification images show that the fully assembled fibers of the lamellar strip consist of subunits which suggest that protofilaments are involved in the assembly processes. A unique cytoskeletal system of the spermatozoid inG. biloba is given by the anterior bundle of microtubules. This bundle, in which microtubules are arranged parallel to each other, forms between the plasmalemma and the MLS and is about 214–392 nm in cross section. These microtubules expand spirally along the MLS band. Other details of cellular fine structure of the mature spermatozoid are described.     

Comparative development of the spermatozoids of cycads andGinkgo biloba

Cycads andGinkgo biloba are the only extant seed plants that produce flagellated male gametes. Superficially, the cells of both are similar in structure and function. In both the motile organelles arise from multicentriolar bodies, the blepharoplasts, and, in both forms, these give rise to a complex fibrous band, the multilayered structure (MLS), which bears numerous flagella. Generally speaking, these structures are much alike in cycads andGinkgo. However, there are marked differences in details of their development, particularly in the presence of a “nucellar beak” inGinkgo.     

Immunofluorescent localization of cytoskeletal tubulin and actin during spermatogenesis inPteridium aquilinum (L.) Kuhn

Summary Details concerning the appearance and behaviour of blepharoplasts during spermatogenesis, and the assembly of the cytoskeletal motile apparatus of spermatids were elucidated by immunofluorescence microscopy using antibodies to tubulin and actin, applied to material prepared from antheridia of the fernPteridium aquilinum (L.) Kuhn. Blepharoplast immunofluorescence with antitubulin first appears as spheres at the future spindle poles prior to the last spermatogenous division. Developing spermatids each have one blepharoplast, which gives rise to a triangular layer corresponding to the incipient microtubule ribbon. Compared to the ribbon, immunoreactivity of the multilayered structure is relatively weak. Intensely fluorescing basal bodies appear, increase in number, and become arranged in rows along two edges of the microtubule ribbon as it widens and elongates. Along the dorsal edge is a dense file of basal bodies spaced at about 0.3 m intervals, parallel to each other and oriented at 145° to the multilayered structure. This spacing and orientation is maintained throughout spermatid development. Basal bodies at the opposite edge are initially oriented at 115° to the multilayered structure but become rearranged into small groups that rotate so that the angle is reduced to 55–70° by the time the assembly of flagella commences on both sets of basal bodies. By this stage the microtubule ribbon has encircled about 2/3 of the nuclear circumference and the nucleus is assuming a crescent shape. In fully developed spermatozoids the groups of basal bodies are oriented at 25° to the multilayered structure, parallel to the long body of the now helical nucleus. Immunofluorescence using antiactin showed that towards the completion of nuclear shaping, actin forms a strip along the helical multilayered structure. Detergent-extraction of mature spermatozoids revealed that actin is associated also with the flagellar band, particularly with basal bodies.Abbreviations MLS multilayered structure - MT microtubule     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.