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Pamula Harrison
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In Drosophila, all the 64 clonally derived spermatocytes differentiate in syncytium inside two somatic-origin cyst cells. They elongate to form slender spermatids, which are individualized and then released into the seminal vesicle. During individualization, differentiating spermatids are organized in a tight bundle inside the cyst, which is expected to play an important role in sperm selection. However, actual significance of this process and its underlying mechanism are unclear.
We show that dynamic F-actin-based processes extend from the head cyst cell at the start of individualization, filling the interstitial space at the rostral ends of the maturing spermatid bundle. In addition to actin, these structures contained lamin, beta-catenin, dynamin, myosin VI and several other filopodial components. Further, pharmacological and genetic analyses showed that cytoskeletal stability and dynamin function are essential for their maintenance. Disruption of these F-actin based processes was associated with spermatid bundle disassembly and premature sperm release inside the testis.
Altogether, our data suggests that the head cyst cell adheres to the maturing spermatid heads through F-actin-based extensions, thus maintaining them in a tight bundle. This is likely to regulate mature sperm release into the seminal vesicle. Overall, this process bears resemblance to mammalian spermiation.
Spermiogenesis offers a good model for investigating the molecular basis of large-scale cellular morphogenesis and movement. In Drosophila , 64 haploid sperm develop from a single gonial precursor through several well-defined morphogenetic steps [1, 2]. This entire process happens in three distinct developmental stages: (1) the formation of 64 spermatids from a gonial precursor; (2) the elongation of spermatids from nearly spherical to around 1.8 mm long cells; and (3) the individualization of the elongated spermatids into mature sperm, which then enter the seminal vesicle (SV) [2]. Altogether, it involves large-scale changes in cell shape and internal reorganization [3, 4]. Every spermatogonial cell is encapsulated by two somatic-origin cyst cells within the testicular lumen as they form at the testis apex. Subsequent developments occur within this cyst capsule. At the end of the process, the individualized sperm coil up inside the cyst capsule at the base of the testis before entering the SV [4]. Studies in Drosophila indicated that defective sperm fail to enter the SV as they fail to remain attached to the head cyst cell during the coiling process [1]. Thus, the dynamics of sperm head attachment to the cyst cell are likely to play a major role in this quality control exercise.
In this paper we report the results of a systematic analysis of the final stages of sperm maturation before their release in Drosophila testis. We show that the somatic-origin head cyst cell grows F-actin based membranous projections into the interstitial spaces between the mature spermatid heads at the start of individualization. Immunohistochemical analysis showed that these F-actin-rich processes contained markers of filopodia and also proteins found in the AJ. Pharmacological manipulations of the F-actin and microtubule dynamics further revealed that these structures are dynamic and are involved in maintaining mature spermatids in a tight parallel bundle. Finally, a genetic screen identified that shibire (dynamin) function is essential to maintain the integrity of these F-actin-based structures and the sperm bundle at the final stage of maturation. Altogether our data provide an initial set of descriptions for further cellular and molecular analysis of spermiation in Drosophila .
Additional F-actin accumulations were observed at the rostral tips of the NBs (arrows, Figure 1B) at the start of individualization. Subsequently, the F-actin grew as cap-like structures around the spermatid nuclei during individualization (Figure 1C). The F-actin densities were found around rostral tips of individual nuclei and acrosomes of an NB (Figure 1D), marked by the sneaky-GFP [20]. At a later stage when the individualized and mature sperm coiled up inside the cyst, the F-actin densities were mostly found around the lateral sides of the nuclei (arrowhead, Figure 1E). This is likely to correspond to a stage when sperm were about to be released as both the acrosomes and the NB appeared unpacked (arrow, Figure 1E). These F-actin based structures will be referred as 'actin caps' in the subsequent discussion. The actin caps were also observed to form inside the head cyst cell covering the rostral ends of maturing spermatid bundles (Figure 1F) and occasionally 'empty' actin caps not associated with the NB were also observed inside the testis. These observations raised an obvious question about the cellular origin of the actin caps.
Transmission electron microscopy (TEM) studies further showed that the nuclei of the 'individualized' spermatids were tightly invested with plasma membrane (arrows, Figure 2A) and contained very little cytoplasm. They were embedded into the head cyst cell (HC, Figure 2A) with electron dense material around (fine arrows, Figure 2A; see also the inset). An earlier study reported that membrane bound projections containing microfilaments are extended from the head cyst cell and interspersed between the sperm heads after individualization [4]. We also found some tightly packed NBs (arrows, Figure 2B) with dense material around them near the head cyst cell perimeter (fine arrows, Figure 2B; see also the inset). Together with the previous results this suggested that the actin caps are likely to form inside the head cyst cells and therefore, unlikely to be a part of the spermatids.
Transmission electron microscopy images of transverse sections of mature sperm heads inside a cyst at the base of a wild-type testis (magnification 28,000). (A) Cross-section view of mature and individualized sperm nuclei (arrows) inside the head cyst cell (HC, arrowheads). The stage is determined according to an earlier description [4]. Inset shows enlarged view of the nuclei (N) tightly invested with plasma membrane (white arrowhead). The nuclei are embedded in an electron dense material (fine arrows) and membranous projections filled the interstitial space (marked with *). (B) Nuclei (arrows) of post-individualized spermatids inside the head cyst cell are tightly packed with electron dense material around them (fine arrows). They were also placed at one side of the HC. Inset shows 10 enlarged view of part of the bundle. The interstitial space (*) is packed with electron dense material.
A large number of vesicles were reported to accumulate inside the head cyst cell around the rostral tips of the embedded sperm heads [4]. This was also revealed in the DIC images of the isolated head cyst cells. The punctate distributions of myosins in this region further suggested that these motors might be involved in vesicle transport into the actin cap projections. Myosin VI was known to associate with the clathrin-coated pits inside the cell during endocytosis as well as with dynamic membrane ruffles in the migrating epithelial cells [33]. We found that the clathrin light chain-GFP (clc-GFP) was enriched in punctate spots around the actin caps (arrows, Figure 4E, d) in the w; UAS-clc-GFP Actin5cGal4 testis. Clathrin is involved in coated vesicle assembly [38] and plays an important role in dynamin-mediated vesiculation inside the cell and the clc is an integral part of the clathrin complex. Therefore, this observation further supported the hypothesis that vesicular traffic from the head cyst cells could supply membrane to the actin cap projections. Altogether the immunolocalization results indicated that actin caps are filopodia-like extensions and likely to attach to the sperm heads during individualization.
Mature spermatid heads remain in a tight bundle attached to the testis wall during the coiling stage. (A), (B) Sets of time lapse images of the testis base, expressing the sneaky-GFP transgene [20], were projected together to show the relative movement of the acrosome bundles of a cyst inside the testis. Each frame is labeled with a specific false color as per the list shown in (B). The blue arrows and arrowheads point to the positions of acrosome bundles in the first frame while the white arrows and arrowheads indicate the final position. The arrows indicate the compacted set, which is likely to belong to the post-individualization stages, while the arrowheads indicate the acrosomes of the elongating/pre-individualization stage spermatids. (A) The compacted acrosome bundle (arrows) found near the base of the testis remain confined in the region as indicated by the positions of the blue and white arrow. Some acrosomes (green arrow) are occasionally found to move away from the bundle. This is considered to belong to the defective sperm that are lost during coiling. In comparison the acrosomes of the elongating spermatids (arrowheads) are loosely organized and move rapidly towards the testis base as evident from the positions of the blue and white arrowheads. (B) The mobility of acrosome bundles (blue and white arrows) near the base of the testis increased after 30 minutes of 5 μM vinblastine (vinb ) treatment. (See Additional files 2 and 4 for details.)
The association between head cyst cells and spermatids was predicted to play an important role in spermiation [4]. The above results indicated that the head cyst cells are likely to adhere to the maturing sperm heads through F-actin-rich projections containing several filopodial markers. Dynamin plays a key role in membrane reorganization process and our immunolocalization data suggested that it could play an important role in actin cap assembly or maintenance as well. The shibire gene codes for a dynamin homologue in Drosophila , which is shown to play an important role in endocytosis [45] and various other cellular functions [46]. Previous studies have also shown that shi ts1(a conditional mutant allele of shibire ) hemizygous flies rapidly paralyze within a few minutes at non-permissive temperatures [47]. Thus, the conditional shi ts1alleles provided a good tool to further test the role of dynamin in actin caps and the latter's role in sperm maturation/release.
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