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First published online 8 November 2006
doi: 10.1242/dev.02671

Development 133, 4871-4879 (2006)
Published by The Company of Biologists 2006
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Sperm plasma membrane breakdown during Drosophila fertilization requires Sneaky, an acrosomal membrane protein

Kathleen L. Wilson1, Karen R. Fitch2, Blaine T. Bafus1 and Barbara T. Wakimoto1,2,3,*

1 Department of Biology, University of Washington, Seattle, Washington 98195, USA.
2 Department of Genome Sciences and University of Washington, Seattle, Washington 98195, USA.
3 Center for Developmental Biology, University of Washington, Seattle, Washington 98195, USA.


Figure 1
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  		Fig. 1. The rCD2 protein marks the sperm plasma membrane. The panels show sperm produced by males homozygous for snky1 and a transgene expressing rat CD2 gene. The rCD2 protein (green) was detected by immunolocalization and nuclei (red) by PI or DAPI staining. In a cyst of elongating spermatids (A) and mature sperm (B), rCD2 is located on the plasma membrane. After entry into the egg, sperm retain a condensed nucleus (C) and rCD2 (D). The merged image (E) shows overlapping rCD2 and nuclear staining consistent with retention of the plasma membrane surrounding the head. Scale bar: 10 µm.

 

Figure 2
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  		Fig. 2 . Expression and structure of the snky transcript. (A,B) Northern blots probed with a genomic fragment that contained CG11281 detected a 2.7 kb transcript that was present in testis, absent in male carcasses (A) and reduced in size and abundance in snky1 testes compared to its parental strain (B). Bottom panel shows levels of rp49 mRNA in each lane as a loading control. (C) Transcript structure was determined by RT-PCR. Primers used for cDNA amplification are indicated by arrows. The transcript is contained within the AvaI genomic fragment, which was used as a probe for northern analysis. Fragment endpoints are labeled according to Drosophila Genome Project Release 4.0 coordinates. Transcription initiation site (+1) determined by 5' RACE is nucleotide 3L 13,131,973. Positions of the start and stop codons of the 2145 bp ORF (black box), the 72 bp intron and the predicted polyadenylation site are also indicated.

 

Figure 3
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  		Fig. 3. Predicted topology and deduced amino acid sequence of Snky. (A) Predicted features of Snky are shown with six transmembrane (TM) domains (blue cylinders) as predicted by the SOSUI program (Hirokawa et al., 1998Go), three highly conserved regions a-c (red bars), a predicted coiled-coil domain (orange) and conserved Cys residues (yellow circles). A C4-C4 RING finger is located near the carboxyl terminus. Asterisks (*) indicate location of residues altered in snkyZ mutants. (B) Deduced amino acid sequence showing features diagrammed above. TM domains (boxed blue), highly conserved regions a-c (red), coiled-coil domain (underlined) and conserved Cys residues (C, boxed yellow).

 

Figure 4
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  		Fig. 4. Multiple sequence alignment of Snky and related proteins. Three regions, denoted a, b and c, are the most highly conserved among Snky family members and are aligned here. Their locations in Snky are shown in Fig. 3. The sequence of Snky (D. melanogaster), the mouse (M. musculus) and human proteins are based on full-length cDNAs. The mosquito (A. aegypti) and zebrafish (D. rerio) sequences are based on conceptual translations of predicted genes.

 

Figure 5
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  		Fig. 5. Localization of Snky-GFP during spermatogenesis. The diagram depicts stages of spermatogenesis in which Snky-GFP expression was observed. Letters correspond to confocal images below (A-E). Primary spermatocytes (A) undergo two meiotic divisions to yield round spermatids (B). Differentiation follows (C-E), as spermatids undergo gradual condensation of nuclei (red), elongation of mitochondrial derivatives (blue) and sperm tails (black lines). Individualized, mature sperm are released from the base of the testis into the seminal vesicle (F). Confocal images show stages of spermatogenesis in males that were homozygous for snky1 and carried two copies of a transgene expressing Snky-GFP (green). Nuclei were visualized by DAPI-staining (red). In A-C, mitochondrial structures were detected with the dye MT-CMXRos (blue). (A) In primary spermatocytes, Snky-GFP and the mitochondrial dye give diffuse cytoplasmic signals. (B) In post-meiotic spermatids at the onion stage, clusters of Snky-GFP-containing spheres are adjacent to nuclei and mitochondrial derivatives. (C) Snky-GFP is visible in clusters of spheres along the length of elongating spermatids, but a prominent spot (arrow) is associated with each nucleus. (D) A single slightly oval Snky-GFP signal is associated with each spermatid nucleus as nuclear condensation begins. (E) In cysts of late spermatids, Snky-GFP appears as a thin oval signal distal to the needle-shaped sperm nucleus. (F) Individualized sperm present in the seminal vesicle contain Snky-GFP signal located at the apical tip and consistent with localization to the acrosome. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Localization of Snky-GFP and GFPsecr during fertilization. (A-D) GFP (green) was monitored in eggs fertilized by sperm from snky1 males that carried two copies of a snky-GFP transgene. Nuclei (red) were stained with DAPI. Insets to the right of each picture show higher magnification view of the GFP signal. (A) A single GFP signal is observed just apical to the condensed sperm nucleus and (B) more distantly as the sperm nucleus decondenses. (C) GFP is seen near the apposing male and female pronuclei and (D) by the chromosomes during prometaphase of the first embryonic cycle. (E-H) In eggs fertilized by sperm from males expressing GFPsecr, the GFP signals appear similar to those described for Snky-GFP. Scale bars: 3 µm in A,B,E,F; 6 µm in C,D,G,H; 2 µm in the higher magnification insets.

 




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