COMP-1 promotes competitive advantage of nematode sperm
- University of Utah, United States
Figures
Figure 1 with 2 supplements
Isolation of the male precedence mutant me69 in a genetic screen.
(A) Screening assay for mutants with reduced male precedence, showing outcomes for mating failure, mating by wild-type males, and mating by males with less-competitive sperm. (B) me69 males have …
Figure 1—figure supplement 1
COMP-1 is highly conserved within the Caenorhabditis genus and present in related parasitic species.
Alignment of C. elegans COMP-1 with its orthologs from other nematode species. Yellow highlighting represents amino acids conserved with the C. elegans protein. Bars above the sequence indicate the …
Figure 1—figure supplement 2
COMP-1 transgenes rescue the male precedence defects of comp-1 mutants.
(A, B) The jnSi109[Pcomp-1::COMP-1] transgene, which contains a 3.9 kb region surrounding F37E3.3, rescues the precedence defect of (A) comp-1(me69) and (B) comp-1(gk1149) males in crosses to dpy-4 …
Figure 2 with 1 supplement
The comp-1 mutant has defects in male–male sperm competition.
(A) comp-1 male sperm are outcompeted by wild-type male sperm. Wild-type and/or comp-1(gk1149) males were mated sequentially to fog-2 hermaphrodites; second-mated males harbored the transgene mIs11(G…
Figure 2—figure supplement 1
The comp-1 mutant has defects in male–male sperm competition.
comp-1 male sperm are outcompeted by wild-type male sperm. Wild-type and/or comp-1(gk1149) males were mated sequentially to fog-2 hermaphrodites; first-mated males harbored the transgene …
Figure 3
comp-1 male sperm have long-term precedence defects.
(A) Crosses with comp-1 males result in a low percentage of cross progeny. (B) The number of cross progeny sired by comp-1 increases at late time points. (C) Crosses with comp-1 males do not …
Figure 4 with 1 supplement
COMP-1 is expressed and functions in sperm of both males and hermaphrodites.
(A–D) Images of jnSi118[Pcomp-1::GFP::H2B] adult males (A, B) and hermaphrodites (C, D), which express the comp-1 reporter in developing sperm. int, intestinal autofluorescence. Scale bar (A–D), 30 …
Figure 4—figure supplement 1
A COMP-1::GFP transgene rescues the male precedence defects of comp-1 mutants.
Expression of COMP-1::GFP rescues the precedence defect. comp-1(gk1149); jnSi171[Pcomp-1::COMP-1::GFP] males have a wild-type precedence pattern in crosses to dpy-4 hermaphrodites. Precedence assays …
Figure 5
comp-1 sperm have normal organization and size.
(A–D) Wild-type (A, B) and comp-1(gk1149) (C, D) spermatozoa stained for mitochondria using Mitotracker. (E–H) Wild-type (E, F) and comp-1 (G, H) spermatozoa expressing PEEL-1::GFP (membranous …
Figure 6
comp-1 sperm have defects in migration and spermathecal accumulation.
(A) Schematic of hermaphrodite gonad arm showing zones used to quantify sperm position. (B, C) Localization of wild-type (B) and comp-1(gk1149) (C) Mitotracker-labeled male sperm 1–1.5 hr after …
Figure 7 with 1 supplement
comp-1 male sperm can migrate normally but have context-dependent defects in cell morphology.
(A) comp-1(gk1149) sperm can migrate in vivo at speeds equivalent to wild-type sperm. Mitotracker-labeled males were crossed to N2 hermaphrodites and time-lapse images of sperm migrating through …
Figure 7—figure supplement 1
comp-1 sperm can crawl and be activated in vitro.
(A) comp-1 sperm can crawl at wild-type velocities in vitro. Wild-type and comp-1(gk1149) spermatids were treated with TEA for 30 min and velocity was obtained from time-lapse images collected every …
Figure 8
Model: comp-1 sperm have localization defects that result in failure to compete with wild-type sperm.
(A) Wild-type male sperm (blue) migrate to the region of the spermathecae, where they displace hermaphrodite self sperm (pink) and preferentially fertilize oocytes. Oocytes fertilized by male sperm …
Tables
Table 1
me69 is linked to chromosome I
| Marker* | Genetic position* | Genomic position† | Haw/+ frequency‡ |
|---|---|---|---|
| WBVar00240399 | I:0.91 | I:6350803 | 1/16 |
| WBVar00172772 | II:0.12 | II:6789208 | 8/16 |
| WBVar00067953 | III:−0.31 | III:8318640 | 10/16 |
| WBVar00188750 | IV:1 | IV:4625317 | 3/16 |
| WBVar00240687 | V:0.88 | V:8177520 | 9/16 |
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*
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†
WormBase WS243 (accessed 30 August 2014).
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‡
me69; him-5 males were crossed to CB4856 Hawaiian hermaphrodites, F1 males were crossed back to me69; him-5 hermaphrodites, and F2 males were assayed for precedence defects in crosses to spe-8; dpy-4 hermaphrodites. Animals scoring as mutant (me69 homozygotes) were scored by PCR and restriction digest for centrally-located SNPs on each chromosome. Animals lacking Hawaiian alleles at all loci tested were considered self progeny and excluded from analysis.
Table 2
Mapping of me69 on chromosome I
| No. F2s* | WBVar 00240 394† | WBVar 00240 397 | WBVar 00240 399 | WBVar 00155 231 | WBVar 00240 416 | WBVar 00240 407 | WBVar 00159 097 | WBVar 00240 414 | WBVar 00161 629 |
|---|---|---|---|---|---|---|---|---|---|
| 825026 | 5482531 | 6351803 | 8646304 | 10614690 | 11472093 | 12433167 | 13066381 | 14154889 | |
| 16 | B/B | B/B | B/B | ||||||
| 6 | H/B | B/B | B/B | ||||||
| 2 | H/B | B/B | B/B | B/B | B/B | B/B | B/B | H/B | H/B |
| 3 | B/B | B/B | B/B | B/B | B/B | B/B | B/B | H/B | H/B |
| 1 | H/B | H/B | H/B | B/B | B/B | B/B | B/B | B/B | B/B |
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F2 males from the cross described in Table 1 were scored for SNPs across chromosome I. Animals were either homozygous Bristol (B/B) or heterozygous for the Hawaiian allele (H/B) at each SNP.
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*
Number of F2 males showing each pattern.
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†
SNP designation and genomic position on chromosome I. Wicks et al. (2001); WormBase.
Table 3
Construction of entry plasmids used to generate targeting constructs
| Fragment description | Fragment length | Forward primer | Reverse primer | Vector | Plasmid name |
|---|---|---|---|---|---|
| comp-1 promoter | 712 | GGGACAACTTTGTATAGAAAAGTTGCCAGTTCCTCGCCTAGCTTTC | GGGACTGCTTTTTTGTACAAACTTGATGCTTTTGATTCGATAGATGATCC | pDONR P4-P1r | pJMH1 |
| comp-1 coding region | 1921 | GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGACGTTGGTCGAATCGAAAC | GGGACCACTTTGTACAAGAAAGCTGGGTCTTATTTGCGCTGGAATTGATC | pDONR 221 | pJMH2 |
| comp-1 coding region without stop codon | 1918 | GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGACGTTGTCGAATCGAAAC | GGGACCACTTTGTACAAGAAAGCTGGGTATTTGCGCTGGAATTGATC | pDONR 221 | pJMH3 |
| comp-1 3’ region | 561 | GGGGACAGCTTTCTTGTACAAAGTGGAAGAACTTACGGAAGAATATG | GGGGACAACTTTGTATAATAAAGTTGATGCGTTCTCATCAGGCTTC | pDONR P2r-P3 | pJMH4 |
| peel-1 coding region* without stop codon | 3279 | GGGGACAAGTTTGTACAAAAAAGCAGGCTGCTTAATGCGCTTTGGTAAG | GGGGACCACTTTGTACAAGAAAGCTGGGTCTGGATTTTCAACACTTGGATC | pDONR 221 | pJMH20 |
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*
Table 4
Description of targeting constructs used to generate transgenic worm strains
| Construct | Position 1 pDONR P4-P1r | Position 2 pDONR 221 | Position 3 pDONR P2r-P3 | Destination vector | Locus |
|---|---|---|---|---|---|
| Pcomp-1::comp-1::comp-1 3’ region | pJMH1 | pJMH2 | pJMH4 | pCFJ150* | ttTi5605 |
| Pcomp-1::H2B::GFP::comp-1 3’ region | pJMH1 | pCM1.35† | pJMH4 | pCFJ150 | ttTi5605 |
| Ppeel-1::comp-1::tbb-2 3’ region | Ppeel-1 [4-1]‡ | pJMH2 | pCM1.36† | pCFJ150 | ttTi5605 |
| Ppeel-1::comp-1::GFP::unc-54 3’ region | Ppeel-1 [4-1] | pJMH3 | pGH50§ | pCFJ150 | ttTi5605 |
| Ppeel-1::comp-1::mCherry::unc-54 3’ region | Ppeel-1 [4-1] | pJMH3 | mCherry::unc-54 3’ region§ | pCFJ150 | ttTi5605 |
| Ppeel-1::peel-1::GFP::unc-54 3’ region | Ppeel-1 [4-1] | pJMH20 | pGH50 | pCFJ212* | cxTi10816 |
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