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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

Actin caps and NBs are disrupted in hemizygous shibire mutant testes after heat pulse. (A), (B) Wild type (A) and hemizygous shi ts1 mutant (B) testes labeled with FITC:phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (blue) after 30 minutes at 29°C. Regions populated by the coiled-up sperm bundles are marked by the dotted lines. Note that the base (arrows) is slightly enlarged in shi ts1 hemizygous testis. Figures (b), (c) of (A) as well as (B) are enlarged views of the regions marked in (a) of (A), and (a) of (B), respectively. The figures in the (c) of A and (B) indicate the DAPI labeled NBs. The white arrowheads indicate actin caps and arrows indicate the NBs in respective figures. The red star indicates a highly disrupted NB and red arrowhead indicates abnormal actin caps in figures (b) and (c) of (B). (C) Isolated cysts stained with RITC: phalloidin (red) and DAPI (blue) from (a, b) wild type control and (c, d) the shi ts1 hemizygous mutant testes before and after 30 minutes at 29°C. Arrows indicate actin caps in figures (c) and (d). (D) Histograms indicate average NBs in a testis in wild type and shi ts1 adults after 30 minutes of incubation at 18°C (grey filled bars) and at 29°C (open bars), respectively. The error bars indicate ± S. E. M. The number of specimen (n) for each bar and the pair wise test of significance (p values) are indicated on each figure. The non-significant (ns), significant (*) and very significant (**) differences are indicated on each set of bars linked by the horizontal lines.
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Figure 7: Actin caps and NBs are disrupted in hemizygous shibire mutant testes after heat pulse. (A), (B) Wild type (A) and hemizygous shi ts1 mutant (B) testes labeled with FITC:phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (blue) after 30 minutes at 29°C. Regions populated by the coiled-up sperm bundles are marked by the dotted lines. Note that the base (arrows) is slightly enlarged in shi ts1 hemizygous testis. Figures (b), (c) of (A) as well as (B) are enlarged views of the regions marked in (a) of (A), and (a) of (B), respectively. The figures in the (c) of A and (B) indicate the DAPI labeled NBs. The white arrowheads indicate actin caps and arrows indicate the NBs in respective figures. The red star indicates a highly disrupted NB and red arrowhead indicates abnormal actin caps in figures (b) and (c) of (B). (C) Isolated cysts stained with RITC: phalloidin (red) and DAPI (blue) from (a, b) wild type control and (c, d) the shi ts1 hemizygous mutant testes before and after 30 minutes at 29°C. Arrows indicate actin caps in figures (c) and (d). (D) Histograms indicate average NBs in a testis in wild type and shi ts1 adults after 30 minutes of incubation at 18°C (grey filled bars) and at 29°C (open bars), respectively. The error bars indicate ± S. E. M. The number of specimen (n) for each bar and the pair wise test of significance (p values) are indicated on each figure. The non-significant (ns), significant (*) and very significant (**) differences are indicated on each set of bars linked by the horizontal lines.

Mentions: Testes from the shi ts1 hemizygous males grown at 18°C had no apparent defect in NB organization and actin cap morphology (Figure 7A). However, some of the actin caps (white arrowheads, Figure 7B, b) and a few NBs (red *, Figure 7B, c) appeared disrupted after 30 minutes at 29°C. In addition, a few actin caps were found without the associated nuclei (red arrowheads, Figure 7B, b). Interestingly, F-actin levels in the disrupted actin caps were not reduced (Figure 7C), indicating that shibire/dynamin functions were required to maintain the attachments to sperm heads. In addition, there was a small but significant increase in the numbers of mature NBs at the testis base after the heat treatment (Figure 7D). Since the gonial precursors form at an hourly rate, cysts are expected to mature at the same rate. Therefore, the small increase in the NBs after a 30 minute pulse is quite significant. This indicated that shibire could regulate premature sperm release. This would also suggest that the sperm maturation process is very dynamic and the shibire/dynamin function is constitutively required to maintain the association between the sperm heads and the head cyst cell. However, this failed to resolve whether the shibire/dynamin acts at the actin caps.


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 and NBs are disrupted in hemizygous shibire mutant testes after heat pulse. (A), (B) Wild type (A) and hemizygous shi ts1 mutant (B) testes labeled with FITC:phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (blue) after 30 minutes at 29°C. Regions populated by the coiled-up sperm bundles are marked by the dotted lines. Note that the base (arrows) is slightly enlarged in shi ts1 hemizygous testis. Figures (b), (c) of (A) as well as (B) are enlarged views of the regions marked in (a) of (A), and (a) of (B), respectively. The figures in the (c) of A and (B) indicate the DAPI labeled NBs. The white arrowheads indicate actin caps and arrows indicate the NBs in respective figures. The red star indicates a highly disrupted NB and red arrowhead indicates abnormal actin caps in figures (b) and (c) of (B). (C) Isolated cysts stained with RITC: phalloidin (red) and DAPI (blue) from (a, b) wild type control and (c, d) the shi ts1 hemizygous mutant testes before and after 30 minutes at 29°C. Arrows indicate actin caps in figures (c) and (d). (D) Histograms indicate average NBs in a testis in wild type and shi ts1 adults after 30 minutes of incubation at 18°C (grey filled bars) and at 29°C (open bars), respectively. The error bars indicate ± S. E. M. The number of specimen (n) for each bar and the pair wise test of significance (p values) are indicated on each figure. The non-significant (ns), significant (*) and very significant (**) differences are indicated on each set of bars linked by the horizontal lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Actin caps and NBs are disrupted in hemizygous shibire mutant testes after heat pulse. (A), (B) Wild type (A) and hemizygous shi ts1 mutant (B) testes labeled with FITC:phalloidin (green) and 4',6-diamidino-2-phenylindole (DAPI) (blue) after 30 minutes at 29°C. Regions populated by the coiled-up sperm bundles are marked by the dotted lines. Note that the base (arrows) is slightly enlarged in shi ts1 hemizygous testis. Figures (b), (c) of (A) as well as (B) are enlarged views of the regions marked in (a) of (A), and (a) of (B), respectively. The figures in the (c) of A and (B) indicate the DAPI labeled NBs. The white arrowheads indicate actin caps and arrows indicate the NBs in respective figures. The red star indicates a highly disrupted NB and red arrowhead indicates abnormal actin caps in figures (b) and (c) of (B). (C) Isolated cysts stained with RITC: phalloidin (red) and DAPI (blue) from (a, b) wild type control and (c, d) the shi ts1 hemizygous mutant testes before and after 30 minutes at 29°C. Arrows indicate actin caps in figures (c) and (d). (D) Histograms indicate average NBs in a testis in wild type and shi ts1 adults after 30 minutes of incubation at 18°C (grey filled bars) and at 29°C (open bars), respectively. The error bars indicate ± S. E. M. The number of specimen (n) for each bar and the pair wise test of significance (p values) are indicated on each figure. The non-significant (ns), significant (*) and very significant (**) differences are indicated on each set of bars linked by the horizontal lines.
Mentions: Testes from the shi ts1 hemizygous males grown at 18°C had no apparent defect in NB organization and actin cap morphology (Figure 7A). However, some of the actin caps (white arrowheads, Figure 7B, b) and a few NBs (red *, Figure 7B, c) appeared disrupted after 30 minutes at 29°C. In addition, a few actin caps were found without the associated nuclei (red arrowheads, Figure 7B, b). Interestingly, F-actin levels in the disrupted actin caps were not reduced (Figure 7C), indicating that shibire/dynamin functions were required to maintain the attachments to sperm heads. In addition, there was a small but significant increase in the numbers of mature NBs at the testis base after the heat treatment (Figure 7D). Since the gonial precursors form at an hourly rate, cysts are expected to mature at the same rate. Therefore, the small increase in the NBs after a 30 minute pulse is quite significant. This indicated that shibire could regulate premature sperm release. This would also suggest that the sperm maturation process is very dynamic and the shibire/dynamin function is constitutively required to maintain the association between the sperm heads and the head cyst cell. However, this failed to resolve whether the shibire/dynamin acts at the actin caps.

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