Sol Sepsenwol (2013) CIL:44001, Ascaris sp., sperm. CIL. Dataset

The sequences in this videotape illustrate the unusual crawling motility of the amoeboid sperm of the nematode parasite, Ascaris suum, an intestinal parasite of pigs. Ascaris sperm contain no myosins or tubulins, and only a trace of actin -- all of which were discarded in a residual body early in spermatogenesis. There are 5 stages of activation of the ovoid spermatids to crawling spermatozoa. Activation in vivo or in vitro is initiated by a protein from the vas deferens of the male worm, which is thought to be a specific protease. During activation, refringent granules in the cell coalesce into a large refringent body and the pseudopod forms, makes contact with the substrate and pulls the cell along. In the sequence of in vitro activation, spermatids from the seminal vesicle of a male worm are exposed to activator protein, put into a glass coverslip chamber under anaerobic conditions and observed with phase- contrast time-lapse microscopy on a heated stage. Next shown is an inactive spermatid in freeze-frame - among the numerous intracellular vesicles are mitochondria, sperm-specific membraneous organelles, refringent granules and a condensed nucleus. About 3-4 min after activation, the pseudopod rapidly forms and extends, and the cell body attaches to the glass. Villipodia are already forming and moving over the pseudopod at this early stage, a phenomenon that parallels the development of the pseudopod cytoskeleton. Next is seen the cells at about 8-10 min after activation when the fully-developed pseudopod gyrates above the cell body, which is still cemented to the glass. The refringent granules in the cell have fused into a large doughnut-shaped refringent body of unknown function. At stage 4, the pseudopod begins to make contact with the glass, seen as dark areas with phase-contrast microscopy; the prominent rapid membrane flow is due to the movement of villipodia. Once contact between villipodia and glass takes place at stage 5, the pseudopod flattens out and the cell begins to crawl. In the upper right-hand corner of this sequence, the cell body is still attached in some cases, and is stretched out as the pseudopod gains traction. The rapid movement seen over the surface of the pseudopod, especially clear on tethered cells, is due to the formation of villipodia at the leading edge of the pseudopod and their migration toward the pseudopod-cell body junction, where they disappear. Branched structures within the pseudopod move in unison with the villipodia. These are the fiber complexes, the dynamic cytoskeletal superstructures largely responsible for ascaris sperm locomotion. When well-attached, the cells move forward at the same speed that the villipodia move rearward. There seems to be chemotactic behavior among sperm cells - in these short sequences, crawling spermatozoa change direction to follow passing cells. Unlike the sperm of most other nematodes, ascaris sperm are anaerobic and are sensitive to oxygen diffusing into the chamber. The next sequences were taken with a video-disc system at the Madison integrated microscopy resource. The branched fiber complexes move rearward as the cell moves forward. As the cell turns to the left, new complexes form at the left edge, but the old complexes do not change their position relative to each other. Next is seen the aging phenomenon again: it enables separation of the two, normally synchronized, but independent processes which allow the sperm to move forward: (1) the assembly of the fiber-complex cytoskeleton and formation of villipodia at the leading edge of the pseudopod, and (2), the disassembly of cytoskeleton at the pseudopod-cell body junction. Seen here is the cessation of forward extension -- or cytoskeletal assembly -- of the pseudopod, but the cell body is still pulled forward as the cytoskeleton continues to be disassembled at the base of the pseudopod. Next is shown another sequence which suggests chemotactic behavior among sperm. The cell entering from the right and moving to the top of the circle of cells was originally some distance from the original group of cells. The last sequence is in real time and focuses on the fiber complexes. Most of the cells are not crawling yet because their pseudopods have not attached to the glass, but the rapid movement of villipodia, and the fiber complexes which form the villipodia, continues.
See also: Sepsenwol S, Ris H, Roberts TM. (1989). A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode Ascaris suum. J Cell Biol 108:55-66.

本录像带中的序列展示了猪肠道寄生虫线虫Ascaris suum的变形虫精子不寻常的爬行运动能力。Ascaris精子中不含肌球蛋白或微管蛋白，仅含有微量的肌动蛋白——这些成分均在精子发生早期被丢弃在残留体内。卵圆形精子团至游动精子发生激活的过程分为五个阶段。无论是体内还是体外激活，均由雄虫输精管中的一种蛋白质引发，该蛋白质被认为是一种特定的蛋白酶。在激活过程中，细胞中的折光颗粒会聚集成一个大型的折光体，伪足形成并与底物接触，从而拉动细胞前进。在体外激活的序列中，雄虫精囊中的精子团被暴露于激活蛋白中，置于无氧条件下的玻璃载玻片室中，并使用相位差显微镜在加热平台上进行时间推移观察。随后展示的是冻结帧中的非活性精子团——其中包含众多细胞内囊泡，如线粒体、特异性精子膜质细胞器、折光颗粒和浓缩的细胞核。激活后约3-4分钟，伪足迅速形成并延伸，细胞体附着在玻璃上。此时，绒毛状伪足已经形成并在伪足上移动，这一现象与伪足细胞骨架的发展相平行。大约在激活后8-10分钟时，可见细胞，此时成熟的伪足在细胞体上方盘旋，而细胞体仍然附着在玻璃上。细胞中的折光颗粒已融合成一个未知功能的巨大环状折光体。在第四阶段，伪足开始与玻璃接触，这在相位差显微镜下表现为暗区；明显的快速膜流动是由于绒毛状伪足的运动。一旦绒毛状伪足与玻璃接触在第五阶段，伪足变平，细胞开始爬行。在这一序列的右上角，细胞体在某些情况下仍然附着，随着伪足获得牵引力而伸展。在伪足表面观察到的快速运动，尤其是在固定细胞中，是由于伪足前端的绒毛状伪足形成并向伪足-细胞体连接处迁移，在那里它们消失。伪足内的分支结构与绒毛状伪足同步移动。这些是纤维复合体，是负责Ascaris精子运动的主要动态细胞骨架超结构。当细胞牢固附着时，其移动速度与绒毛状伪足向后移动的速度相同。似乎精子细胞之间存在趋化行为——在这些短序列中，游动精子团会改变方向以跟随通过的细胞。与其他大多数线虫精子不同，Ascaris精子为厌氧性，对进入室内的氧气扩散敏感。接下来的序列使用Madison集成显微镜资源中的视频光盘系统拍摄。分支纤维复合体在细胞向前移动时向后移动。当细胞向左转时，新的复合体在左侧边缘形成，但旧复合体相对于彼此的位置没有改变。接下来观察到的是衰老现象的再次出现：它使得两个通常同步但独立的过程分离，这两个过程允许精子向前移动：（1）纤维复合体细胞骨架的组装和在伪足前端的绒毛状伪足形成；（2）伪足-细胞体连接处细胞骨架的解聚。在此观察到的是伪足向前延伸——或细胞骨架组装——的停止，但细胞体仍然被向前拉动，因为细胞骨架在伪足底部继续解聚。接下来展示的是另一个表明精子之间存在趋化行为的序列。从右侧进入并移动到细胞圆圈顶部的细胞最初与原始细胞群有一定距离。最后一个序列是实时拍摄的，聚焦于纤维复合体。大多数细胞尚未开始爬行，因为它们的伪足尚未附着在玻璃上，但绒毛状伪足及其形成的纤维复合体的快速运动仍在继续。