The <i>hc130</i> mutation alters <i>zipt-7</i>.<i>1</i>, which encodes a ZIP family transporter.

<p>(A) Genetic map of linkage group IV (upper) and a corresponding portion of the physical map (lower). Blue line indicates the frequency of CB4856 SNP alleles in homozygous <i>hc130</i> mutant animals, and red shows the inferred position of the <i>hc130</i> mutation. (B) Diagram of the physical map showing <i>zipt-7</i>.<i>1</i> gene structure and portions of flanking genes. Predicted exons are boxes, coding regions are black, and untranslated regions are gray. The extent of the <i>ok971</i> deletion mutation and the positions of <i>hc130</i> and <i>as42</i> are marked. (C) A maximum likelihood tree illustrating evolutionary relationships between predicted ZIP proteins from <i>Caenorhabditis elegans</i> (red), <i>Drosophila melanogaster</i> (green), <i>Homo sapiens</i> (blue), and <i>Saccharomyces cerevisia</i> (yellow). The ZIP7 family is circled. (D) An alignment of predicted ZIP7 proteins from <i>C</i>. <i>elegans</i> (ZIPT-7.1 and ZIPT-7.2), <i>D</i>. <i>melanogaster</i> (Catsup), and <i>H</i>. <i>sapiens</i> (ZIP7). Identical residues are marked “*” and similar ones “:”; chemical properties are indicated by color according to ClustalX conventions. The individual numerical values for panel A can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.s008" target="_blank">S1 Data</a>.</p

Model for ZIPT-7.1 function.

<p>(A) Model of the genetic function of <i>zipt-7</i>.<i>1</i>. The black line shows the plasma membrane, and arrows and bars indicate positive and negative interactions, respectively. Genes that mutate to sperm activation defective or constitutive are green or red, respectively. (B) Model of the biochemical function of ZIPT-7.1 at three times during sperm activation: (1) Primed spermatid: prior to activation, spermatids are primed to respond—ZIPT-7.1 (dark blue) is inactive and localized to the membrane of a vesicle that has a high internal concentration of stored zinc (orange circles) and is presumably the membranous organelle (light blue). The cytoplasm has a low concentration of zinc, and the equilibrium of zinc-regulated proteins (purple) is shifted towards unbound. (2) Release: a sperm-activating signal results in activation of ZIPT-7.1 (orange arrow), zinc begins to flow into the cytoplasm, and zinc-regulated proteins begin binding zinc. (3) Activated spermatozoon: the cytoplasmic concentration of zinc is now high, and zinc-regulated proteins are bound to zinc. The pseudopod extension begins (lower), and the membranous organelles fuse with the plasma membrane. Fusion of membranous organelles is based on previously published observations of this event [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.ref028" target="_blank">28</a>], and our results do not directly address whether ZIPT-7.1 is relocalized during this process.</p

Genetic and physical interactions of ZIPT-7.1 with the SPE-8 activation pathway.

<p>(A) Self progeny of <i>spe-6(hc163)</i> III<i>; zipt-7</i>.<i>1(ok971)/+</i> IV<i>; him-5(e1490)</i> V hermaphrodites were analyzed for active sperm in the male gonad (the <i>spe-6(</i>−<i>)</i> phenotype) or hermaphrodite sterility (the <i>zipt-7</i>.<i>1(</i>−<i>)</i> phenotype). About 25% of the animals were predicted to be homozygous <i>zipt-7</i>.<i>1(</i>−<i>)</i> mutants (red values). The number and fraction of progeny of each sex displaying these phenotypes are shown. Photomicrographs show DIC images of representative male gonads (large panel) and dissected sperm (small panel). Scale bars are 10 μm. About 25% of hermaphrodites were sterile, indicating that <i>spe-6(</i>−<i>)</i> did not suppress the <i>zipt-7</i>.<i>1(</i>−<i>)</i> phenotype. About 25% of males did not have prematurely active sperm, indicating that <i>zipt-7</i>.<i>1(</i>−<i>)</i> suppressed the <i>spe-6(</i>−<i>)</i> phenotype. (B) Photomicrograph showing the density of yeast colonies at three plating dilutions. The split-ubiquitin yeast two-hybrid study was conducted with ZIPT-7.1 expressed from the bait plasmid (green) and other proteins expressed from the prey plasmid (blue). The box marks robust colony growth with SPE-4, indicating a protein–protein interaction. (C) Males were classified as having inactive spermatids (red), characteristic of both <i>zipt-7</i>.<i>1(</i>−<i>)</i> and the wild type, active sperm (dark blue), characteristic of <i>swm-1(</i>−<i>)</i>, or a mixture of spermatids and active sperm (light blue). From left to right, <i>N</i> = 39, 56, 50, and 54; dotted lines show the percentage of animals that displayed each phenotype, and boxes show 95% confidence intervals. All strains included <i>him-5(e1490)</i> and either <i>zipt-7</i>.<i>1(ok971)</i> or <i>swm-1(ok1193)</i>, as indicated. The individual numerical values for panels A and C can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.s008" target="_blank">S1 Data</a>. DIC, differential interference contrast.</p

<i>zipt-7</i>.<i>1</i> is required for sperm activity in both sexes.

<p>(A,B) Values are total number of eggs (A) and unfertilized oocytes (B) laid by hermaphrodites in the same 5-day span. Box and whisker plots show the mean (dotted line), 25th to 75th percentiles (box), and 10th to 90th percentiles (whiskers). Points falling outside of this range are marked individually. <i>N</i> indicates number of broods scored. The deletion allele was <i>zipt-7</i>.<i>1(ok971)</i>, and all strains contained <i>him-5(e1490)</i>. (C) Photomicrograph of the uterus and spermatheca of a wild-type hermaphrodite that carried the <i>him-5(e1490)</i> mutation. A yellow “s” marks a group of sperm, and a red “e” marks each embryo. The inset shows a 4-fold expansion of the boxed region, which contains the spermatheca (green arrow in the main image; green outline in the inset). (D) Photomicrograph of a <i>zipt-7</i>.<i>1(ok971)</i> hermaphrodite that also carried the <i>him-5(e1490)</i> mutation. A yellow “s” marks each sperm in the uterus, and a red “o” marks each unfertilized oocyte. The area in the orange box is expanded 4-fold in the left inset, and the area in the yellow box is expanded 4-fold in the right inset to show the empty spermatheca. (C,D) In the insets, some sperm are indicated by dotted yellow circles. Anterior is left and ventral is down. Scale bars are 50 μm. (E) Total broods from crosses between either wild-type males and <i>spe-8 dpy-5</i> sterile hermaphrodites (<i>N</i> = 23) or <i>zipt-7</i>.<i>1(ok971)</i> males and <i>spe-8 dpy-5</i> sterile hermaphrodites (<i>N</i> = 39). All males carried the <i>him-5</i> mutation. (F) Total broods from crosses between either wild-type males and <i>fog-2</i> females (<i>N</i> = 13) or <i>zipt-7</i>.<i>1(ok971)</i> males and <i>fog-2</i> females (<i>N</i> = 14). All males carried the <i>him-5</i> mutation. In A, B, and F, statistical significance was calculated using the Mann-Whitney <i>U</i> test, whereas in E, it was determined with a 2 × 2 contingency table. The individual numerical values for panels A, B, E, and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.s008" target="_blank">S1 Data</a>.</p

ZIPT-7.1 is expressed and functions in the germ line to control sperm activation.

<p><b>(A)</b> Bands represent transcript levels determined by semiquantitative RT-PCR. Genotypes are labeled above, target transcripts at the left, and germ line phenotypes below; <i>act-1</i> is a loading control. For each lane, RNA was isolated from batches of five worms (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#sec017" target="_blank">Materials and methods</a>). The alleles were <i>him-5(e1490)</i>, <i>fem-3(q96)</i>, <i>fem-1(hc17</i>ts<i>)</i>, <i>fog-1(q253</i>ts<i>)</i>, and <i>glp-4(bn2</i>ts<i>)</i>. (B) Diagram illustrating the insertion site of the GFP coding sequence into the endogenous <i>zipt-7</i>.<i>1</i> locus to generate the <i>zipt-7</i>.<i>1(ibp18)</i> strain. (C) Sets of photomicrographs from two extruded male gonads. The location of the images in the gonad arm is indicated with a red box on the cartoon; distal is up and proximal to the right. The bright-field images show cell morphology (left), the antibody stain shows the expression of GFP::ZIPT-7.1 (middle), and the DAPI stain shows cell nuclei (right). Genotypes were <i>him-5(e1490)</i> and <i>him-5(e1490) zipt-7</i>.<i>1(ibp18)</i>. Scale bar is 10 μm. (D) Reproductive defects caused by <i>zipt-7</i>.<i>1</i> RNAi treatment. The F₁ progeny of mothers injected with dsRNA were each scored for whether they laid eggs, oocytes, or both. Dotted lines show the average percentage for each phenotype and sum to 100%, the size of the boxes indicates 95% confidence limits, and the color indicates the phenotype according to the key. The <i>rrf-1</i> genotype and RNAi treatment are indicated at the bottom (<i>N</i> = 200, 248, and 315, left to right). Wild-type animals are susceptible to RNAi in germ cells and most somatic cells, whereas the <i>rrf-1</i> mutant animals are susceptible to RNAi in germ cells but resistant in most of the somatic cells [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.ref017" target="_blank">17</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.ref019" target="_blank">19</a>], as indicated in the diagram. The individual numerical values for panel D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.s008" target="_blank">S1 Data</a>. DIC, differential interference contrast; dsRNA, double-stranded RNA; GFP, green fluorescent protein; Oo, oocyte; Sp, sperm; RNAi, RNA interference; RT-PCR, Reverse transcription polymerase chain reaction.</p

Regulation of sperm activation by ZIPT-7.1 is conserved in nematodes.

<p>(A) Maximum likelihood phylogeny of all ZIP7 homologs identified in eight nematode species: <i>Caenorhabditis briggsae</i> (Cbr), <i>C</i>. <i>nigoni</i> (Cni), <i>C</i>. <i>remanei</i> (Cre), <i>C</i>. <i>tropicalis</i> (Ctr), <i>C</i>. <i>brenneri</i> (Cbn), <i>C</i>. <i>elegans</i> (Cel), <i>C</i>. <i>japonica</i> (Cja), and <i>Onchocerca volvulus</i> (Ovo). Sequences were obtained from <a href="http://wormbase.org" target="_blank">wormbase.org</a> and aligned using ClustalX, and calculations were done using PhyML with 100 bootstrap replications. Blue indicates the ZIPT-7.1 subfamily and green the ZIPT-7.2 subfamily. (B) <i>C</i>. <i>tropicalis zipt-7</i>.<i>1</i> mutations produced by gene editing. Gray numbers indicate positions in the coding sequence of the gene. Deleted nucleotides are shown as dashes, and inserted nucleotides are blue. We selected the frameshift alleles <i>v332</i> and <i>v334</i> as representative null alleles and the in-frame deletion <i>v335</i> as a nearly wild-type control. (C, D) Brood sizes of <i>ctr-zipt-7</i>.<i>1</i> hermaphrodites and males, presented as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.g002" target="_blank">Fig 2</a>. JU1373 is the wild-type strain of <i>C</i>. <i>tropicalis</i>. For D–F, wild-type males were <i>him-8(v287)</i> and mutant males were <i>him-8(v287); zipt-7</i>.<i>1(v332)</i>. (E) Photomicrographs of spermatids stained with Zinpyr-1 to reveal labile zinc levels, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.g005" target="_blank">Fig 5A</a>. Scale bars are 5 μm. (F) Quantitation of fluorescence intensity, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.g005" target="_blank">Fig 5B</a>. The individual numerical values for panels C, D, and F can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.s008" target="_blank">S1 Data</a>. Cbn, <i>C</i>. <i>brenneri</i>; Cbr, <i>C</i>. <i>briggsae</i>; Cel, <i>C</i>. <i>elegans</i>; Cja, <i>C</i>. <i>japonica</i>; Cni, <i>C</i>. <i>nigoni</i>; Cre, <i>C</i>. <i>remanei</i>; Ctr, <i>C</i>. <i>tropicalis</i>; Ovo, <i>Onchocerca volvulus</i>.</p

ZIPT-7.1 transports zinc in vitro and regulates zinc levels in vivo.

<p>(A) Photomicrographs of spermatids isolated from <i>him-5</i> males (upper) or <i>him-5 zipt-7</i>.<i>1(ok971)</i> males (lower). Spermatids were stained with Zinpyr-1 and visualized with fluorescence to reveal labile zinc (left) and DIC optics to show morphology (right). Scale bar is 5 μm. (B) Fluorescence intensity is (F-F₀)/F₀, where F is the fluorescence level of each spermatid (<i>N</i> = 50), and F₀ is background. Box and whiskers conventions are described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.g002" target="_blank">Fig 2</a>; comparisons used the Mann-Whitney <i>U</i> test. (C) Wild-type animals were cultured with 0 or 40 μM TPEN, a zinc chelator. RNA from a mixed-stage population was analyzed by qRT-PCR. Mean mRNA levels are shown relative to those for 0 μM TPEN, which was set to 1; error bars indicate SE (<i>N</i> = 4 trials). (D–I) Human HeLa cells expressing ZIPT-7.1 were double labeled with antibodies against ZIPT-7.1 (D, G, green) and the <i>Cis</i>-Golgi marker GM130 (E, red) or the lysosomal marker LMP2 (H, red). Confocal fluorescence micrographs of representative cells show ZIPT-7.1 localization, which appears to be at the nuclear envelope, Golgi, and lysosomes. Arrowheads indicate apparent colocalization of ZIPT-7.1 with GM130 or LMP2 (yellow), and the arrow indicates nuclear envelope localization. The boxed area highlights apparent colocalization and is enlarged 2.3-fold in the inset. Scale bar is 5 μm. (J–L) Human HEK293T cells expressing ZIPT-7.1 or an empty vector control were incubated with radioactive zinc solution, which contains a trace amount of radioactive ⁶⁵Zn, and the rate of zinc uptake was determined. Values are mean and SE (<i>N</i> = 4 trials). (J) Uptake as a function of total zinc concentration in the medium. ZIPT-7.1 expression significantly increased uptake (** <i>p</i> < 0.01; *** <i>p</i> < 0.001). (K, L) Competition experiments with nonradioactive ions were used to establish metal specificity. Assays contained 30 μM radioactive zinc solution and 300 μM nonradioactive zinc, magnesium, cadmium, iron, manganese, or calcium. For cells expressing ZIPT-7.1, comparison of the different metals reveals that zinc was significantly more effective as a competitor than other ions (Fe, Ca, <i>p</i> < 0.001; Mg, Cd, Mn, <i>p</i> < 0.01). For control cells, which reveal uptake mediated by endogenous proteins, comparison of the different metals reveals that zinc was also significantly more effective as a competitor than other ions (Mg, Fe, <i>p</i> < 0.001; Cd, Mn, Ca, <i>p</i> < 0.01). The individual numerical values for panels B, C, J, K, and L can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005069#pbio.2005069.s008" target="_blank">S1 Data</a>. DIC, differential interference contrast; qRT-PCR, quantitative RT-PCR; TPEN, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine.</p