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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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F-actin-rich structures cap the mature nuclei at the beginning of sperm individualization. (A) Wild-type testis stained with fluorescein isothiocyanate (FITC):phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (red), respectively. Spermatid NBs associated with investment cones (*), at the early (arrows) and intermediate (arrowheads) stages of individualization, and of the coiled-up stages (fine arrows) are marked. Rostral ends of the NBs moved very slowly towards the SV; see Additional files 1 and 2. (B), (C) DAPI (blue) and rhodamine isothiocyanate (RITC):phalloidin (red) stained isolated cysts show F-actin organization at the rostral ends of spermatids during individualization. (B) Arrow indicates F-actin (red) accumulation at the rostral ends of spermatids at the beginning of the investment cone (arrowhead) assembly. (C) Overlay of the differential interference contrast (DIC) picture (gray scale) of the isolated NB containing the needle shaped nuclei (blue) and the F-actin (arrows) cap at the rostral ends. (D), (E) Isolated cysts from sneaky-GFP (green) testis strained as above. (D) NB of a post individualized spermatids. Actin caps appeared around the acrosomes marked by sneaky-GFP (arrows). (E) NB of a relatively later stage bundle (post coiling) found at the testis base. F-actin staining disappeared from the rostral ends but increased laterally (arrowhead). This is presumed to be the penultimate stage before the sperm is released. (F) Combined DIC (Grey) and epifluorescence image of the head cyst cell and associated spermatids stained with anti-Lamin (Dm0) (green), DAPI (blue) and RITC: phalloidin (red). The cell perimeter (broken line) and Lamin-rich membrane folds inside the cell (arrows) are marked. Lamin also localized along the F-actin extensions (arrowheads).
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Figure 1: F-actin-rich structures cap the mature nuclei at the beginning of sperm individualization. (A) Wild-type testis stained with fluorescein isothiocyanate (FITC):phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (red), respectively. Spermatid NBs associated with investment cones (*), at the early (arrows) and intermediate (arrowheads) stages of individualization, and of the coiled-up stages (fine arrows) are marked. Rostral ends of the NBs moved very slowly towards the SV; see Additional files 1 and 2. (B), (C) DAPI (blue) and rhodamine isothiocyanate (RITC):phalloidin (red) stained isolated cysts show F-actin organization at the rostral ends of spermatids during individualization. (B) Arrow indicates F-actin (red) accumulation at the rostral ends of spermatids at the beginning of the investment cone (arrowhead) assembly. (C) Overlay of the differential interference contrast (DIC) picture (gray scale) of the isolated NB containing the needle shaped nuclei (blue) and the F-actin (arrows) cap at the rostral ends. (D), (E) Isolated cysts from sneaky-GFP (green) testis strained as above. (D) NB of a post individualized spermatids. Actin caps appeared around the acrosomes marked by sneaky-GFP (arrows). (E) NB of a relatively later stage bundle (post coiling) found at the testis base. F-actin staining disappeared from the rostral ends but increased laterally (arrowhead). This is presumed to be the penultimate stage before the sperm is released. (F) Combined DIC (Grey) and epifluorescence image of the head cyst cell and associated spermatids stained with anti-Lamin (Dm0) (green), DAPI (blue) and RITC: phalloidin (red). The cell perimeter (broken line) and Lamin-rich membrane folds inside the cell (arrows) are marked. Lamin also localized along the F-actin extensions (arrowheads).

Mentions: An adult testis contains multiple cysts at different stages of differentiation. They are recognized by the nuclear morphology of the spermatids and the relative positions in the testis. For instance, the needle-shaped nuclei of the elongated spermatids are loosely organized in the cyst (*, Figure 1A, Additional files 1 and 2) at the start of individualization and these are generally found at about 250 μm from the base. The F-actin-rich investment cones (also known as F-actin cones) form around each nucleus during the individualization stage and move towards the caudal ends in synchrony [1,3,5,6]. The NBs of the individualizing spermatids (arrowheads, Figure 1A) are found in a region 200–255 μm from the testis base (Figure 1A), and they move further towards the SV in subsequent developmental stages (fine arrows, Figure 1A) until the mature sperm enter the SV. The entire process was estimated to take nearly 20 hours [1,3]. Therefore, the rostral ends of the maturing spermatid bundles are likely to traverse around 250 μm to the SV in as much time or longer.


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)

F-actin-rich structures cap the mature nuclei at the beginning of sperm individualization. (A) Wild-type testis stained with fluorescein isothiocyanate (FITC):phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (red), respectively. Spermatid NBs associated with investment cones (*), at the early (arrows) and intermediate (arrowheads) stages of individualization, and of the coiled-up stages (fine arrows) are marked. Rostral ends of the NBs moved very slowly towards the SV; see Additional files 1 and 2. (B), (C) DAPI (blue) and rhodamine isothiocyanate (RITC):phalloidin (red) stained isolated cysts show F-actin organization at the rostral ends of spermatids during individualization. (B) Arrow indicates F-actin (red) accumulation at the rostral ends of spermatids at the beginning of the investment cone (arrowhead) assembly. (C) Overlay of the differential interference contrast (DIC) picture (gray scale) of the isolated NB containing the needle shaped nuclei (blue) and the F-actin (arrows) cap at the rostral ends. (D), (E) Isolated cysts from sneaky-GFP (green) testis strained as above. (D) NB of a post individualized spermatids. Actin caps appeared around the acrosomes marked by sneaky-GFP (arrows). (E) NB of a relatively later stage bundle (post coiling) found at the testis base. F-actin staining disappeared from the rostral ends but increased laterally (arrowhead). This is presumed to be the penultimate stage before the sperm is released. (F) Combined DIC (Grey) and epifluorescence image of the head cyst cell and associated spermatids stained with anti-Lamin (Dm0) (green), DAPI (blue) and RITC: phalloidin (red). The cell perimeter (broken line) and Lamin-rich membrane folds inside the cell (arrows) are marked. Lamin also localized along the F-actin extensions (arrowheads).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: F-actin-rich structures cap the mature nuclei at the beginning of sperm individualization. (A) Wild-type testis stained with fluorescein isothiocyanate (FITC):phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (red), respectively. Spermatid NBs associated with investment cones (*), at the early (arrows) and intermediate (arrowheads) stages of individualization, and of the coiled-up stages (fine arrows) are marked. Rostral ends of the NBs moved very slowly towards the SV; see Additional files 1 and 2. (B), (C) DAPI (blue) and rhodamine isothiocyanate (RITC):phalloidin (red) stained isolated cysts show F-actin organization at the rostral ends of spermatids during individualization. (B) Arrow indicates F-actin (red) accumulation at the rostral ends of spermatids at the beginning of the investment cone (arrowhead) assembly. (C) Overlay of the differential interference contrast (DIC) picture (gray scale) of the isolated NB containing the needle shaped nuclei (blue) and the F-actin (arrows) cap at the rostral ends. (D), (E) Isolated cysts from sneaky-GFP (green) testis strained as above. (D) NB of a post individualized spermatids. Actin caps appeared around the acrosomes marked by sneaky-GFP (arrows). (E) NB of a relatively later stage bundle (post coiling) found at the testis base. F-actin staining disappeared from the rostral ends but increased laterally (arrowhead). This is presumed to be the penultimate stage before the sperm is released. (F) Combined DIC (Grey) and epifluorescence image of the head cyst cell and associated spermatids stained with anti-Lamin (Dm0) (green), DAPI (blue) and RITC: phalloidin (red). The cell perimeter (broken line) and Lamin-rich membrane folds inside the cell (arrows) are marked. Lamin also localized along the F-actin extensions (arrowheads).
Mentions: An adult testis contains multiple cysts at different stages of differentiation. They are recognized by the nuclear morphology of the spermatids and the relative positions in the testis. For instance, the needle-shaped nuclei of the elongated spermatids are loosely organized in the cyst (*, Figure 1A, Additional files 1 and 2) at the start of individualization and these are generally found at about 250 μm from the base. The F-actin-rich investment cones (also known as F-actin cones) form around each nucleus during the individualization stage and move towards the caudal ends in synchrony [1,3,5,6]. The NBs of the individualizing spermatids (arrowheads, Figure 1A) are found in a region 200–255 μm from the testis base (Figure 1A), and they move further towards the SV in subsequent developmental stages (fine arrows, Figure 1A) until the mature sperm enter the SV. The entire process was estimated to take nearly 20 hours [1,3]. Therefore, the rostral ends of the maturing spermatid bundles are likely to traverse around 250 μm to the SV in as much time or longer.

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