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F-actin-based extensions of the head cyst cell adhere to the maturing spermatids to maintain them in a tight bundle and prevent their premature release in Drosophila testis.

Desai BS, Shirolikar S, Ray K - BMC Biol. (2009)

Bottom Line: Disruption of these F-actin based processes was associated with spermatid bundle disassembly and premature sperm release inside the testis.This is likely to regulate mature sperm release into the seminal vesicle.Overall, this process bears resemblance to mammalian spermiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, India. bela@tifr.res.in

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

Immunohistochemical characterization of the actincap. (A) Confocal images of head cyst-cells in intact testis containing mature and individualized sperm nuclei from the basal regions of testes, stained with: ras, DE-cadherin, crumbs and lamin antisera (all in green), as well as rhodamine isothiocyanate (RITC): phalloidin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate staining in the membranous folds inside the head cyst cell and arrowheads indicate the head cyst cell nuclei. (B) Isolated mature spermatids without the head cyst cells as found in the testis squash preparations stained with lamin (Dm0), armadillo, tubulin and ERp72 antisera (indicated at the left side panel of each set). (C), (D) Similar preparations stained with the dynamin (a), syndapin (b) and WASP (c) as well as (D) Syntaxin (green). Arrows indicate positions of the actin caps. (E) Head cyst cells inside testis immunostained with the (a) ck/myosin VII and (b) jar/myosin VI, respectively, or expressing (c) the sqh-GFP and (d) the clathrin light chain:GFP (clc-GFP ), respectively. (a) ck (green) staining (arrows) coincided with the membranous fold and (b) anti-jar stained the actin cap (arrows) as well as a few punctate spots in the cytoplasm (arrowheads). (c) The sqh-GFP localized along the actin caps (arrowheads) and in punctate spots (arrows) in the cytoplasm. (d) clc-GFP mostly localized in the punctate spots (arrows) in the cytoplasm. The sqh-GFP transgene is expressed through its own promoter in sqh homozygous mutant background and the UAS-clc-GFP is expressed by using the actin5CGal4 (see Additional file 6 for details).
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Figure 4: Immunohistochemical characterization of the actincap. (A) Confocal images of head cyst-cells in intact testis containing mature and individualized sperm nuclei from the basal regions of testes, stained with: ras, DE-cadherin, crumbs and lamin antisera (all in green), as well as rhodamine isothiocyanate (RITC): phalloidin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate staining in the membranous folds inside the head cyst cell and arrowheads indicate the head cyst cell nuclei. (B) Isolated mature spermatids without the head cyst cells as found in the testis squash preparations stained with lamin (Dm0), armadillo, tubulin and ERp72 antisera (indicated at the left side panel of each set). (C), (D) Similar preparations stained with the dynamin (a), syndapin (b) and WASP (c) as well as (D) Syntaxin (green). Arrows indicate positions of the actin caps. (E) Head cyst cells inside testis immunostained with the (a) ck/myosin VII and (b) jar/myosin VI, respectively, or expressing (c) the sqh-GFP and (d) the clathrin light chain:GFP (clc-GFP ), respectively. (a) ck (green) staining (arrows) coincided with the membranous fold and (b) anti-jar stained the actin cap (arrows) as well as a few punctate spots in the cytoplasm (arrowheads). (c) The sqh-GFP localized along the actin caps (arrowheads) and in punctate spots (arrows) in the cytoplasm. (d) clc-GFP mostly localized in the punctate spots (arrows) in the cytoplasm. The sqh-GFP transgene is expressed through its own promoter in sqh homozygous mutant background and the UAS-clc-GFP is expressed by using the actin5CGal4 (see Additional file 6 for details).

Mentions: Therefore, to further understand the role of the actin caps, we decided to characterize its molecular composition. Regulation of actin dynamics and AJs are known to play crucial roles in epithelial cell morphogenesis and movement during development. In addition, the basolateral protrusive activity of these cells that penetrate into the neighboring cells to promote the formation of adhesive contacts is suggested to involve actin [21]. Immunostaining revealed that prominent AJ markers such as DE-cadherin and crumbs were enriched in the head cyst cells (arrows, Figure 4A). The Drosophila lamin (Dm0), armadillo (beta-catenin), tubulin and ERp72 were also enriched at the actin caps (arrows, Figure 4B). ERp72, which seemed to be enriched more caudally than the others, is an ER resident protein found in testes specific AJs [22,23]. This suggested that actin caps could adhere to the sperm heads through AJs. Actin-based membrane extensions such as filopodia and invadopodia are also known to require dynamin, syndapin and WASP [19,24-27]. These were also associated with the actin caps (arrows, Figure 4C, a, b and 4C, c). In addition, syntaxin [28], a t-SNARE involved in membrane fusion events, was present along the actin cap extensions (arrows, Figure 4D). Thus, actin caps contained proteins characteristic of both filopodia and AJs. They are likely to adhere to sperm heads through beta-catenin, which is an integral component of AJ in the epithelial cells [29] and testes [19].


F-actin-based extensions of the head cyst cell adhere to the maturing spermatids to maintain them in a tight bundle and prevent their premature release in Drosophila testis.

Desai BS, Shirolikar S, Ray K - BMC Biol. (2009)

Immunohistochemical characterization of the actincap. (A) Confocal images of head cyst-cells in intact testis containing mature and individualized sperm nuclei from the basal regions of testes, stained with: ras, DE-cadherin, crumbs and lamin antisera (all in green), as well as rhodamine isothiocyanate (RITC): phalloidin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate staining in the membranous folds inside the head cyst cell and arrowheads indicate the head cyst cell nuclei. (B) Isolated mature spermatids without the head cyst cells as found in the testis squash preparations stained with lamin (Dm0), armadillo, tubulin and ERp72 antisera (indicated at the left side panel of each set). (C), (D) Similar preparations stained with the dynamin (a), syndapin (b) and WASP (c) as well as (D) Syntaxin (green). Arrows indicate positions of the actin caps. (E) Head cyst cells inside testis immunostained with the (a) ck/myosin VII and (b) jar/myosin VI, respectively, or expressing (c) the sqh-GFP and (d) the clathrin light chain:GFP (clc-GFP ), respectively. (a) ck (green) staining (arrows) coincided with the membranous fold and (b) anti-jar stained the actin cap (arrows) as well as a few punctate spots in the cytoplasm (arrowheads). (c) The sqh-GFP localized along the actin caps (arrowheads) and in punctate spots (arrows) in the cytoplasm. (d) clc-GFP mostly localized in the punctate spots (arrows) in the cytoplasm. The sqh-GFP transgene is expressed through its own promoter in sqh homozygous mutant background and the UAS-clc-GFP is expressed by using the actin5CGal4 (see Additional file 6 for details).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683793&req=5

Figure 4: Immunohistochemical characterization of the actincap. (A) Confocal images of head cyst-cells in intact testis containing mature and individualized sperm nuclei from the basal regions of testes, stained with: ras, DE-cadherin, crumbs and lamin antisera (all in green), as well as rhodamine isothiocyanate (RITC): phalloidin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue). Arrows indicate staining in the membranous folds inside the head cyst cell and arrowheads indicate the head cyst cell nuclei. (B) Isolated mature spermatids without the head cyst cells as found in the testis squash preparations stained with lamin (Dm0), armadillo, tubulin and ERp72 antisera (indicated at the left side panel of each set). (C), (D) Similar preparations stained with the dynamin (a), syndapin (b) and WASP (c) as well as (D) Syntaxin (green). Arrows indicate positions of the actin caps. (E) Head cyst cells inside testis immunostained with the (a) ck/myosin VII and (b) jar/myosin VI, respectively, or expressing (c) the sqh-GFP and (d) the clathrin light chain:GFP (clc-GFP ), respectively. (a) ck (green) staining (arrows) coincided with the membranous fold and (b) anti-jar stained the actin cap (arrows) as well as a few punctate spots in the cytoplasm (arrowheads). (c) The sqh-GFP localized along the actin caps (arrowheads) and in punctate spots (arrows) in the cytoplasm. (d) clc-GFP mostly localized in the punctate spots (arrows) in the cytoplasm. The sqh-GFP transgene is expressed through its own promoter in sqh homozygous mutant background and the UAS-clc-GFP is expressed by using the actin5CGal4 (see Additional file 6 for details).
Mentions: Therefore, to further understand the role of the actin caps, we decided to characterize its molecular composition. Regulation of actin dynamics and AJs are known to play crucial roles in epithelial cell morphogenesis and movement during development. In addition, the basolateral protrusive activity of these cells that penetrate into the neighboring cells to promote the formation of adhesive contacts is suggested to involve actin [21]. Immunostaining revealed that prominent AJ markers such as DE-cadherin and crumbs were enriched in the head cyst cells (arrows, Figure 4A). The Drosophila lamin (Dm0), armadillo (beta-catenin), tubulin and ERp72 were also enriched at the actin caps (arrows, Figure 4B). ERp72, which seemed to be enriched more caudally than the others, is an ER resident protein found in testes specific AJs [22,23]. This suggested that actin caps could adhere to the sperm heads through AJs. Actin-based membrane extensions such as filopodia and invadopodia are also known to require dynamin, syndapin and WASP [19,24-27]. These were also associated with the actin caps (arrows, Figure 4C, a, b and 4C, c). In addition, syntaxin [28], a t-SNARE involved in membrane fusion events, was present along the actin cap extensions (arrows, Figure 4D). Thus, actin caps contained proteins characteristic of both filopodia and AJs. They are likely to adhere to sperm heads through beta-catenin, which is an integral component of AJ in the epithelial cells [29] and testes [19].

Bottom Line: Disruption of these F-actin based processes was associated with spermatid bundle disassembly and premature sperm release inside the testis.This is likely to regulate mature sperm release into the seminal vesicle.Overall, this process bears resemblance to mammalian spermiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, India. bela@tifr.res.in

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus