In this chapter, I hope to convince the reader that Drosophila spermatogenesis is an ideal system for the cytogeneticist. Spermatogenesis is relatively simple, dispensable for adult viability, and amenable to genetic, cell biological, and biochemical approaches. The stages of spermatogenesis are well defined, the cells are large, easily accessible, and easily identified. Because spermatogenesis initiates with a stem cell division, there is continuous production through the life of the fly. Therefore, a normal adult testis presents all of the stages of spermatogenesis as a spatio-temporal array (see Fig. 1A). Excellent and comprehensive reviews of the genetics of spermatogenesis and ultrastructure of wild-type spermatogenesis are available (1,2). In this introduction, I will review only the essential features needed to interpret the results of experiments performed according to the protocols given here.
Spermatogenesis follows a multistep differentiation program involving dramatic changes in cell cycle dynamics, gene expression, and morphogenesis. The transformation of a 15-^m-diameter round spermatid into a 1.8-mm-long mature motile sperm is a truly remarkable act of cellular remodeling. This includes changes in mitochondrial morphology that occur nowhere else in the fly; mitochondria aggregate, fuse, and wrap to give a characteristic Nebenkern mitochondrial derivative at the onion stage. The centriole is transformed into a flagellar basal body embedded in the nuclear envelope. Flagellal elongation is accompanied by elongation of the two mitochondrial derivatives. Nuclear shaping and chromatin condensation transforms a round nucleus into a bear-claw-shaped
From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ
Fig. 1. Phase-contrast microscopy of wild-type testes. The stages of spermatogenesis are easily visible with phase-contrast optics in gently squashed preparations. (A) A whole wild-type testis, cut near the distal end, with most of the elongating spermatids spilled out through the cut. There is a temporal progression of cell types from very
Fig. 1. Phase-contrast microscopy of wild-type testes. The stages of spermatogenesis are easily visible with phase-contrast optics in gently squashed preparations. (A) A whole wild-type testis, cut near the distal end, with most of the elongating spermatids spilled out through the cut. There is a temporal progression of cell types from very highly compact structure. Some processes and gene products are shared with other tissues and developmental stages; others are spermatogenesis-specific. Male meiosis, for example, is much more similar to mitosis than to female meiosis. Many mitotic gene products are used for male meiosis and, therefore the application of a few simple techniques for studying testes can reveal much about the mechanics of cell division. For example, we have learned about the maintenance of sister chromatid cohesion through analysis of mei-S332 and ord. Analysis of weak ord mutants suggests Ord is required for proper centromeric cohesion after arm cohesion is released at the metaphase I-anaphase I transition. Ord activity appears to promote centromeric cohesion during meiosis II. Mei-S332 protein is localized to the centromeric region in meiosis; its destruction at the metaphase II-anaphase II transition allows sister chromatid separation. A balance between the activity of Mei-S332 and Ord is required for proper regulation of meiotic cohesion (3-7). Analysis of asp alleles indicates a role for Asp protein in the normal meiotic and mitotic spindle structure. Immunolocalization of Asp in spermatocytes revealed that it is required for the bundling of microtubules at spindle poles, but it is not an integral centrosome component implicated in microtubule nucleation (8,9). There has been a longstanding debate over the role of asters in determining the position of the cleavage furrow of cytokinesis. Recent evidence from Drosophila spermatogenesis has shown that asters are not required for cytokinesis, because asterless mutants undergo cytokinesis (10). Instead, it appears that the cleavage furrow depends on the central spindle (9). Analysis of cell cycle regulatory genes has revealed that differentiation can continue in the absence of cell cycle progression. Mutation of the meiosis-specific Cdc2-activator twine blocks progression through the meiotic divisions; however, the cells continue with aspects of spermatid differentiation, including axoneme elongation and nuclear shaping (11-14).
A notable feature of the recent completion of the genome sequencing and continuation of the expressed sequence tag (EST) sequencing projects has been early stages at the apical end (top) to nearly mature sperm at the distal end (bottom), leading into the seminal vesicle (*). (B-H) Higher magnification of specific stages of spermatogenesis; (B) polar spermatocytes. (C) mature primary spermatocytes; (D) part of a cyst in metaphase-anaphase I; (E) part of a secondary spermatocyte cyst (meiotic interphase); (F) a telophase II cyst; (G) onion-stage spermatids (right) and comet stage of early spermatid elongation (left); (H) part of a cyst late in elongation before indi-vidualization. (I-L) Hoechst 33342 labeling of DNA in wild-type live squashes. (I) Primary spermatocytes with decondensed chromosomes visible as three discrete regions in each nucleus; (J) prophase of meiosis I, partially condensed chromosomes are visible; (K) leaf blade stage, DNA is compact within the nucleus, faint staining of the mitochondrial DNA is seen in the Nebenkern; (L) tightly clustered and fully shaped nuclei in nearly mature bundles of elongated spermatids, before individualization.
the identification of a large set of testis-specific transcripts. Andrews et al. (15) sequenced 3141 testis ESTs, representing 1560 contigs, of which 47% were not represented in the 80,000 ESTs sequenced by the Berkley Drosophila Genome Project (BDGP) from other tissues. Sixteen percent had not even been predicted as genes on the first annotation of the genome sequence, and only 11% corresponded to known named genes. This study highlights how little we know and how much more there is to learn about the genes required to carry out this most remarkable cellular process of spermatogenesis. Fortunately, a large set of new male sterile mutants, the tool we need to take a genetic approach to understanding spermatogenesis, is now available. A large-scale mutagenesis screen was undertaken in the Charles Zuker lab and yielded over 12,000 new viable mutagenized chromosomes. These were tested for male and female sterility by Barbara Wakimoto and Dan Lindsley, yielding approx 2000 male sterile lines (cited in ref. 16). This collection supplements our existing battery of male sterile mutants (e.g., those generated by Castrillon et al. ). They are available to the whole community and have formed the basis of a new wave of excitement and analysis of cytogenetics in Drosophila spermatogenesis (18-20).
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