Structural and immunocytochemical characterization of microtubule organizing centers in pteridophyte spermatogenous cells

Summary During the development of the spermatogenous cells, the pteridophyteCeratopteris richardii produces three structurally well-defined microtubule organizing centers (MTOCs). The blepharoplast, a spherical body that occurs during the last two spermatogenous divisions, organizes two microtubule (MT) arrays, one associated with a nuclear indentation and the other that organizes the spindle apparatus for the final divisions. After the last spermatogenous division, the blepharoplast reorganizes to produce two new putative MTOCs: the lamellar strip (LS) of the multilayered structure (MLS), which apparently organizes the spline microtubule array, and an amorphous zone (AM), that connects the basal bodies. Thin and semi-thin sections of this tissue were probed with antisera which recognize MTOCs in lower eukaryotes and animals to determine if any of these structures contain MTOC-associated proteins or epitopes recognized by monoclonal antisera. Gamma tubulin antibodies, which recognizeonly the minus ends of MTs in mammalian cells, label along the MT in all arrays found in the pteridophyte spermatogenous cells. Kinetochore MTs are unlabelled near the kinetochore, however. The monoclonal antibodies MPM-2 and C-9, that recognize centrosomal and nuclear epitopes in mammalian cells, label the interphase nucleus, the cytoplasm of mitotic cells, and the blepharoplast during both nuclear indentation and spindle formation. Double labelling of the blepharoplast-containing cells with anti-tubulin and either MPM-2 or C-9 reveals that the blepharoplast-associated fluorescence is the focus of the tubulin arrays. Centrin labels the reorganizing blepharoplast, the MLS, the AM, and a stellate pattern in the transition region of the flagella. These data indicate the usefulness of the structurally well-recognized MTOCs in pteridophyte spermatogenous cells in investigation of land plant MTOCs.     

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.     

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。     

银杏精子细胞生毛体及其它细胞器的超微结构

生毛体与嗜锇颗粒是银杏 (Ginkgobiloba)精子细胞中最具有标志性的结构。生毛体是细胞质内一直径为 3～ 4μm的圆球形结构 ,它由一个电子致密的核心和由此向周边发散出的辐射状中心粒组成 ,致密核心上具有微管结构的弱电子染色区域 ,并有微管从生毛体延伸到细胞质。嗜锇颗粒直径为 1 0～ 2 0μm,呈圆球状 ,位于生毛体和细胞核之间 ,其相对的另一侧存在纤维颗粒体。在精子细胞质内 ,特别是嗜锇颗粒和生毛体周围线粒体、质体、内质网、高尔基体等细胞器丰富。银杏精子细胞核较大 ,在细胞核内 ,核仁结构呈球形 ,电子染色致密的颗粒区在周围 ,而纤维组分则在圆球的中间。在核膜表面布满了分布不均匀的核孔复合体。     

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     

银杏精细胞结构及受精作用的细胞学研究

采用常规石蜡制片技术和环氧树脂半薄切片技术,对银杏的精细胞结构及受精过程进行了研究。结果表明,临近受精前,精原细胞分裂形成两个半球形的精细胞,每个精细胞内含液泡状结构、生毛体和纤维性颗粒体各一个。两精细胞的液泡状结构其位置可同时或分别位于近极面和远极面,这种位置的变动可能是鞭毛摆动导致精细胞的旋转所引起,这说明银杏的精细胞在花粉管内也许即可以旋转运动。在银杏受精前后,珠孔端颈卵器室附近的珠心组织表现出向上隆起、出现受精滴、皱褶等规律性变化,这些现象是判断银杏受精时期的良好形态特征。在银杏受精时带有鞭毛的完整精细胞进入颈卵器,随后鞭毛带及精细胞质遗留于颈卵器口下方、卵细胞上面,仅精核进入卵细胞。进入卵细胞的精核直径约30μm,小于成熟精细胞的精核直径（约40μm）。这些对探讨银杏的系统地位及裸子植物的生殖演化具有一定意义。     

Ultrastructure of Blepharoplast and Other Organelles of the Spermatid in Ginkgo biloba

Both blepbaroplast and osmiophilic globule were characteristic structures to the spermatid of Ginkgo biloba. The blepharoplast of Ginkgo biloba ranged from 3 ～ 4 μm in diameter and consisted of a number of basal centrioles radiating from an electron dense core that contained electron-lucent areas with microtubule structure. Microtubules extended radially from the blepharoplast into the cytoplasm. A large round osmiopbilie globule with a diameter of about 10～20/μm, was located between the blepharoplast and the nucleus, while a filbrillogranular body in the cytoplasm was opposite to the osmiophilic globule. There were numerous mitochondria, plastids, endoplasmic reticulia and dictiosomes in the cytoplasm, particularly around the blepharoplast and the osmiophilic globule of sperm cells. The nucleus of spermatid in Ginkgo biloba was large and roundly elliptical in shape. The large spheroidal nucleolus was the most obvious structure in the nucleus, There were two regions in the nucleolus distinguished by TEM: A ring-shaped granular component, which contained maturing ribosomal precursor particles; and a centrally placed fibrillar component. The nuclear pore complexes in the nuclear envelope were plentiful but not evenly distributed.     

An Ultrastructural Study of the Development of Blepharoplast and the Origin of Multilayered Structure in the Sperm of Lygodium japonicum

The blepharoplast, an elliptical, compact spherical body ranging 0.5～0.6 /μm in diameter, appears in the cytoplasm of the sperm mother cell during the sperm development of Lygodium japonicum (Thunb.) Sw. It consists of cartwheel tubular subunits arranged in radiant pattern and amorphous material. Numerous microtubules extended from the blepharoplast into cytoplasm. In the process of sperm cell development, the blepharoplast became loose, and the centrioles originated from the subunits separated from each other and dispersed towards the surface, with the amorphous material in the center. Accompanying the differentiation of the centrioles, muhilayered structure with the spline ( i. e. microtubular band) and lamella strata came into existence, with one end attaching to the amorphous material in the center. Then the muhilayered structure (MIS) associated with a mitochondrion and moved towards the nucleus, and a dent facing the MIS is formed in the nucleus. A cotton-like structure consisting of amorphous material and dardyed small tubular structure appeard in late stage of spermatogenesis. Microtubules were seen protruding from the surface of the cotton-like structure. It is suggested that this structure might be relevant to the origin of the blepharoplast.