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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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Actin caps are extended from the head cyst cell. (A) Optical section from the SG18.1Gal4 UAS-GFP/UAS-GFP:actin testes stained with rhodamine isothiocyanate (RITC):phallodin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue) show GFP and GFP-actin localizations in the head cyst cell nucleus (arrowhead) and in the actin cap (arrows), respectively. (B), (C) A similar staining of the SG18.1Gal4 UAS-GFP:actin/+ testis shows prominent GFP:actin localization in the actin cap (arrows, (B)) and also in the investment cones (C). (D) The actin cap (arrows) is also labeled by GFP:actin in pCOGGal4/Y; UAS-GFP:actin/+ testis. (E)-(G) Confocal sections from the DAPI (blue) stained w pCOGGal4 UAS-myosin VII:GFP/Y testis show myosin-VII:GFP (green) localization around the nuclei bundles (NBs) (arrows, (E)) at the testis base. (F) Higher magnification image of a single NB show prominent myosin VII:GFP localization along the NB (arrowheads) and also in the head cyst cell (arrows). (G) RITC:phalloidin (red) and DAPI (blue) staining further revealed that the myosin VII:GFP is enriched in the actin cap (arrows). The arrowheads indicate an empty actin cap. (See Additional file 3 for a detailed analysis of expression pattern in testes.)
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Figure 3: Actin caps are extended from the head cyst cell. (A) Optical section from the SG18.1Gal4 UAS-GFP/UAS-GFP:actin testes stained with rhodamine isothiocyanate (RITC):phallodin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue) show GFP and GFP-actin localizations in the head cyst cell nucleus (arrowhead) and in the actin cap (arrows), respectively. (B), (C) A similar staining of the SG18.1Gal4 UAS-GFP:actin/+ testis shows prominent GFP:actin localization in the actin cap (arrows, (B)) and also in the investment cones (C). (D) The actin cap (arrows) is also labeled by GFP:actin in pCOGGal4/Y; UAS-GFP:actin/+ testis. (E)-(G) Confocal sections from the DAPI (blue) stained w pCOGGal4 UAS-myosin VII:GFP/Y testis show myosin-VII:GFP (green) localization around the nuclei bundles (NBs) (arrows, (E)) at the testis base. (F) Higher magnification image of a single NB show prominent myosin VII:GFP localization along the NB (arrowheads) and also in the head cyst cell (arrows). (G) RITC:phalloidin (red) and DAPI (blue) staining further revealed that the myosin VII:GFP is enriched in the actin cap (arrows). The arrowheads indicate an empty actin cap. (See Additional file 3 for a detailed analysis of expression pattern in testes.)

Mentions: To further establish this point we used two Gal4 stocks, (a) SG18.1Gal4 and (b) pCOGGal4 , which were found to express in the head cyst cells during the final stages of sperm maturation (Additional file 3). Although the SG18.1Gal4 expression was also found in the spermatocytes at an earlier stage, the pCOGGal4 expression was limited to the cyst cells from the very beginning (Additional file 3). The expression of UAS-actin:GFP in SG18.1Gal4 background marked the actin caps (arrows, Figures 3A and 3B) and the F-actin cones (Figure 3C). However, only the actin caps were marked in the pCOGGal4/Y; UAS-actin:GFP/+ testis (arrows, Figure 3D). Furthermore, the recombinant myosin-VII:GFP expression in the w pCOGGal4 UAS-ck:GFP stock clearly marked the actin caps at the rostral ends of mature NBs (arrows, Figure 3E–G). These observations confirmed that the actin caps are indeed formed inside the head cystcells. In addition, the actin caps were always found attached with the spermatid heads in squash preparations. This suggested that these F-actin-based extensions of the head cyst cell are likely to adhere to the maturing sperm heads. However, TEM studies did not reveal obvious cellular junctions between the sperm heads and the cyst cells.


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)

Actin caps are extended from the head cyst cell. (A) Optical section from the SG18.1Gal4 UAS-GFP/UAS-GFP:actin testes stained with rhodamine isothiocyanate (RITC):phallodin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue) show GFP and GFP-actin localizations in the head cyst cell nucleus (arrowhead) and in the actin cap (arrows), respectively. (B), (C) A similar staining of the SG18.1Gal4 UAS-GFP:actin/+ testis shows prominent GFP:actin localization in the actin cap (arrows, (B)) and also in the investment cones (C). (D) The actin cap (arrows) is also labeled by GFP:actin in pCOGGal4/Y; UAS-GFP:actin/+ testis. (E)-(G) Confocal sections from the DAPI (blue) stained w pCOGGal4 UAS-myosin VII:GFP/Y testis show myosin-VII:GFP (green) localization around the nuclei bundles (NBs) (arrows, (E)) at the testis base. (F) Higher magnification image of a single NB show prominent myosin VII:GFP localization along the NB (arrowheads) and also in the head cyst cell (arrows). (G) RITC:phalloidin (red) and DAPI (blue) staining further revealed that the myosin VII:GFP is enriched in the actin cap (arrows). The arrowheads indicate an empty actin cap. (See Additional file 3 for a detailed analysis of expression pattern in testes.)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Actin caps are extended from the head cyst cell. (A) Optical section from the SG18.1Gal4 UAS-GFP/UAS-GFP:actin testes stained with rhodamine isothiocyanate (RITC):phallodin (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue) show GFP and GFP-actin localizations in the head cyst cell nucleus (arrowhead) and in the actin cap (arrows), respectively. (B), (C) A similar staining of the SG18.1Gal4 UAS-GFP:actin/+ testis shows prominent GFP:actin localization in the actin cap (arrows, (B)) and also in the investment cones (C). (D) The actin cap (arrows) is also labeled by GFP:actin in pCOGGal4/Y; UAS-GFP:actin/+ testis. (E)-(G) Confocal sections from the DAPI (blue) stained w pCOGGal4 UAS-myosin VII:GFP/Y testis show myosin-VII:GFP (green) localization around the nuclei bundles (NBs) (arrows, (E)) at the testis base. (F) Higher magnification image of a single NB show prominent myosin VII:GFP localization along the NB (arrowheads) and also in the head cyst cell (arrows). (G) RITC:phalloidin (red) and DAPI (blue) staining further revealed that the myosin VII:GFP is enriched in the actin cap (arrows). The arrowheads indicate an empty actin cap. (See Additional file 3 for a detailed analysis of expression pattern in testes.)
Mentions: To further establish this point we used two Gal4 stocks, (a) SG18.1Gal4 and (b) pCOGGal4 , which were found to express in the head cyst cells during the final stages of sperm maturation (Additional file 3). Although the SG18.1Gal4 expression was also found in the spermatocytes at an earlier stage, the pCOGGal4 expression was limited to the cyst cells from the very beginning (Additional file 3). The expression of UAS-actin:GFP in SG18.1Gal4 background marked the actin caps (arrows, Figures 3A and 3B) and the F-actin cones (Figure 3C). However, only the actin caps were marked in the pCOGGal4/Y; UAS-actin:GFP/+ testis (arrows, Figure 3D). Furthermore, the recombinant myosin-VII:GFP expression in the w pCOGGal4 UAS-ck:GFP stock clearly marked the actin caps at the rostral ends of mature NBs (arrows, Figure 3E–G). These observations confirmed that the actin caps are indeed formed inside the head cystcells. In addition, the actin caps were always found attached with the spermatid heads in squash preparations. This suggested that these F-actin-based extensions of the head cyst cell are likely to adhere to the maturing sperm heads. However, TEM studies did not reveal obvious cellular junctions between the sperm heads and the cyst cells.

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