Fabrizio J J, Hime G, Lemmon S K, Bazinet C
Department of Biological Sciences, St. John's University, Jamaica, NY 11439, USA.
Development. 1998 May;125(10):1833-43. doi: 10.1242/dev.125.10.1833.
The morphogenesis of spermatids generally takes place within a syncytium, in which all spermatid nuclei descended from a primary spermatocyte remain connected via an extensive network of cytoplasmic bridges. A late step in sperm maturation therefore requires the physical resolution of the syncytium, or cyst, into individual cells, a process sometimes referred to as sperm individualization. Despite the identification of specialized machinery involved in the individualization of Drosophila spermatids (Tokuyasu, K. T., Peacock, W. J. and Hardy, R. W. (1972) Z. Zellforsch 124, 479-506), and of many Drosophila genes mutable to male-sterile phenotypes, little is known of the mechanisms by which this extensive remodeling of the cyst is accomplished. Here, the identification of a major cytoskeletal component of the individualization complex as actin is confirmed with a simple fluorescence assay. Using rhodamine-phalloidin as a probe, the individualization complex is readily visualized forming around bundles of spermatid nuclei at one end of highly elongated cysts, then translocating along the length of the cysts. The structure of the individualization complex in a male-sterile clathrin heavy chain (Chc) mutant is observed to be reduced or disrupted relative to wild-type, consistent with the individualization-deficient phenotype of this mutant. Using the fluorescence assay, a sampling of male-sterile mutant phenotypes in which spermatogenesis proceeds to the assembly of highly elongated cysts distinguishes at least four different phenotypic classes: (1) mutations (nanking class) that block or significantly retard the assembly of the actin-based individualization complex around the nuclear bundle, (2) mutations (dud class) in which the individualization complex assembles in/around the nuclear bundle, but fails to translocate down the cyst, (3) mutations (mulet class) that allow the assembly of a morphologically normal individualization complex around the nuclear bundle, but result in a breakdown in the complex after it begins to translocate down the cyst, and (4) mutations (purity of essence class) that allow the assembly of a motile but morphologically altered or reduced individualization complex. Individualization also fails in a number of mutants with altered nuclear shape, consistent with the hypothesis that spermatid nuclei provide a physical scaffolding for the assembly of the individualization complex. Genetic analysis suggests that a substantial number of additional loci with phenotypes distinguishable with this assay remain to be identified. The large proportion of male-sterile mutations resulting in a late block to spermatogenesis, in which highly elongated cysts fail to be individualized, suggest a substantial susceptibility of this process to a broad range of cellular perturbations. The massive reorganization of cyst cytoplasm required at individualization is expected to be a correspondingly complex function requiring exquisite coordination of multiple cytoplasmic functions, and may account for the previously noted high frequency with which Drosophila genes are mutable to male-sterile phenotypes.
精子细胞的形态发生通常在一个合胞体中进行，其中所有源自初级精母细胞的精子细胞核通过广泛的细胞质桥网络保持连接。因此，精子成熟的后期步骤需要将合胞体或细胞囊物理分解为单个细胞，这个过程有时被称为精子个体化。尽管已经鉴定出参与果蝇精子细胞个体化的特殊机制（德久安夫、皮科克、哈迪，1972年，《细胞研究杂志》第124卷，第479 - 506页），并且发现了许多导致雄性不育表型的果蝇基因，但对于细胞囊这种广泛重塑是如何完成的机制却知之甚少。在这里，通过一种简单的荧光检测方法证实了肌动蛋白是个体化复合体的一种主要细胞骨架成分。使用罗丹明 - 鬼笔环肽作为探针，可以很容易地观察到个体化复合体在高度细长的细胞囊一端围绕精子细胞核束形成，然后沿着细胞囊的长度移动。相对于野生型，观察到雄性不育的网格蛋白重链（Chc）突变体中个体化复合体的结构减少或被破坏，这与该突变体的个体化缺陷表型一致。利用荧光检测方法，对精子发生过程进行到高度细长细胞囊组装阶段的雄性不育突变体表型进行抽样分析，至少区分出四种不同的表型类别：（1）阻止或显著延迟基于肌动蛋白的个体化复合体围绕核束组装的突变（南京类），（2）个体化复合体在核束内/周围组装，但未能沿细胞囊向下移动的突变（哑炮类），（3）允许在核束周围组装形态正常的个体化复合体，但在其开始沿细胞囊向下移动后导致复合体解体的突变（骡类），以及（4）允许组装可移动但形态改变或缩小的个体化复合体的突变（本质纯度类）。在一些核形状改变的突变体中，个体化也会失败，这与精子细胞核为个体化复合体的组装提供物理支架的假设一致。遗传分析表明，还有大量具有可通过该检测方法区分表型的其他基因座有待鉴定。大量导致精子发生后期受阻的雄性不育突变，即高度细长的细胞囊未能个体化，表明这个过程对广泛的细胞扰动具有高度敏感性。个体化过程中细胞囊细胞质的大规模重组预计是一个相应复杂的功能，需要多种细胞质功能的精确协调，这可能解释了之前提到的果蝇基因易发生雄性不育表型突变的高频率现象。
