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First published online 29 August 2007
doi: 10.1242/dev.004770

Development 134, 3517-3525 (2007)
Published by The Company of Biologists 2007
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The testis-specific proteasome subunit Pros{alpha}6T of D. melanogaster is required for individualization and nuclear maturation during spermatogenesis

Lei Zhong and John M. Belote*

Department of Biology, Syracuse University, 130 College Place, Syracuse, NY 13244, USA.


Figure 1
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  		Fig. 1. The expression patterns of Pros{alpha}6-GFP and Pros{alpha}6T-GFP during spermatogenesis. (A-H) Pros{alpha}6-GFP; (A'-H')Pros{alpha}6T-GFP. (A,A') Apical ends of testes showing early spermatogonial stages. (B,B') 16-cell primary spermatocyte cysts (dotted lines, also in C-E). (B) Arrow, nucleolus; arrowhead, nucleus. (C,C') Late primary spermatocyte cysts. (D,D') Cysts undergoing meiosis I. (E,E') 64-cell onion-stage spermatid cysts. Nuclei are brightly fluorescing and nebenkerns are dark. (F,F') Elongated spermatid bundles. Nuclear bundles are brightly fluorescing in F' (arrows). (G,G') Spermatid bundles undergoing individualization. In G' the arrowhead indicates elongated and condensed nuclei and the arrow indicates spermatid bundles at the onset of individualization. (H,H') Seminal vesicles containing stored mature sperm (arrows). Scale bars: 20 µm in A-G,A'-G'; 150 µm in H,H'.

 

Figure 2
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  		Fig. 2. Proteasomal `speckles' associated with the individualization complex. (A) Elongated spermatid bundles showing Pros{alpha}6T-GFP speckles (arrows, green). (B-E) Speckles (green) are located close to the actin cones (red): (B) Pros{alpha}6T-GFP, (C) Pros{alpha}6-GFP, (D) Pros{alpha}3T-GFP, (E) Pros{alpha}3-GFP. (F) Wild-type IC stained with anti-Pros{alpha}7 antibody (green) and Alexa Fluor 635 phalloidin (red). (G-I) Pros{alpha}6T-GFP (G, green) in an IC stained with anti-Pros{alpha}7 antibody (H, red). (I) The merged image of G and H with the yellow signal indicating colocalization. Scale bars: 20 µm.

 

Figure 3
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  		Fig. 3. Generation of the Pros{alpha}6T1 mutant. (A) Pros{alpha}6T knockout construct. The bent arrow to the left of the Pros{alpha}6T coding region (hatched area) indicates the transcription direction. The two arrows beneath the Pros{alpha}6T coding region indicate the positions of PCR primers used to verify the targeted Pros{alpha}6T mutation. (B) Results of male fertility tests showing mean number of progeny per male±s.e.m.

 

Figure 4
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  		Fig. 4. Phase-contrast microscopy of whole testes and pre-individualization stages of spermatogenesis of wild-type and Pros{alpha}6T1 mutant males. (A,C) Wild-type and Pros{alpha}6T1 testes. (B,D) Higher magnification of the seminal vesicle in A and C (boxed), respectively. (E-L) Comparison of different spermatogenic stages of wild-type (E-H) and Pros{alpha}6T1 (I-L) testes. (E,I) Primary spermatocyte cysts at the 16-cell stage. (F,J) Onion (64-cell) stage spermatid cysts. (G,K) Early stage of elongating spermatid cysts. (H,L) Fully elongated spermatid cyst bundles. Scale bars: 150 µm in A-D,H,L; 10 µm in E-G,I-K.

 

Figure 5
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  		Fig. 5. The individualization complexes are disrupted in Pros{alpha}6T1 mutant testes. (A-D)Testes stained with Alexa-Fluor-phalloidin (green) showing actin cones of the ICs. (A,B) Actin cones (green) in wild-type ICs move synchronously, although occasionally a few actin cones lag behind (arrow). (C,D) In Pros{alpha}6T1 testes, actin cone synchronization is disrupted. The further the ICs move along the tail bundles, the more scattered the actin cones become (compare D with C). (E,F) Wild-type (E) and Pros{alpha}6T1 (F) testes stained with anti-Pros{alpha}7 antibody (green) and Alexa-Fluor-phalloidin (red). Speckles (arrow) can be observed in the wild-type testis, whereas they are absent in the Pros{alpha}6T1 testis. Two cystic bulges are shown in F. The actin cones are disorganized as a result of the mutation. Scale bars: 20 µm.

 

Figure 6
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  		Fig. 6. Caspase activation is affected in Pros{alpha}6T1. (A,B) Anti-active Dronc staining (red) is observed in cystic bulges in wild-type testes (A), whereas it is absent in Pros{alpha}6T1 testes (B). (C,D) Visualization of active Ice with anti-cleaved-caspase-3 antibody (CM1; red). CM1 staining is observed in the cystic bulge (arrow) and elongated spermatid bundles (arrowheads) in wild-type testes (C). However, CM1 staining is not detected in the cystic bulge (arrow) in Pros{alpha}6T1 testes (D), and is weaker in elongated spermatid bundles (arrowheads) compared with wild type. Actin cones were visualized by Alexa-Fluor-phalloidin staining (green). Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. Pros{alpha}6T1 mutant testes show abnormalities in nuclear bundles. Wild-type or Pros{alpha}6T1 spermatid nuclei stained with TO-PRO3. (A) Wild-type nuclei are long, fully condensed and remain in register. (B) Some of the Pros{alpha}6T1 nuclei are not fully condensed and they are often scattered. (C) Spermatid nuclei of the mutant are often contorted and frequently form circles (arrow). (D) Proportion of normal, scattered and curled spermatid nuclear bundles in young (black bars) versus old (hatched bars) flies. Scale bars: 20 µm.

 

Figure 8
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  		Fig. 8. The Pros{alpha}6T1 mutant delays the disappearance of histone H2AvD but protamine incorporation is not affected. Images are of increasing age, left to right. Wild-type (A-F,M-R) or Pros{alpha}6T1 (G-L,S-X) testes expressing either H2AvD-GFP (D-F,J-L; green) or ProtA-GFP (P-R,V-X; green) were stained with TO-PRO3 (A-C,G-I,M-O,S-U; red) to visualize nuclei. In wild-type testes, H2AvD-GFP signal is barely detectable in elongating and condensing nuclei (D), and completely disappears in mature nuclear bundles (E) and nuclei in individualized sperm (F). However, elongating and condensing nuclei in Pros{alpha}6T1 testes still contain significant amounts of H2AvD-GFP (J), although the GFP signal disappears in mature nuclear bundles (K) and curled nuclei (L). ProtA-GFP is highly expressed in elongating and condensing nuclei in wild-type testes (P), and persists in the mature nuclear bundles (Q) and nuclei in individualized sperm (R). In Pros{alpha}6T1 testes, ProA-GFP is observed in elongating and condensing nuclei (V), mature nuclear bundles (W) and curled nuclei (X). Scale bar: 20 µm.

 




© The Company of Biologists Ltd 2007