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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01868

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The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization

Indrani Chatterjee¹, Alissa Richmond², Emily Putiri^1,*, Diane C. Shakes² and Andrew Singson^1,

¹ Waksman Institute and Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA
² Department of Biology, College of William and Mary, Williamsburg, VA 23187, USA

View larger version (16K): [in a new window]   Fig. 1. spe-38 mutant worms are sterile. (A) Quantification of hermaphrodite self-fertility with (asEx67 [A.1-A.2], asEx72 [A.1+]) or without rescuing transgenes. (B) Quantification of male fertility. Four males were crossed to single dpy-5 hermaphrodites that were then allowed to produce non-Dpy outcross progeny for one day. (A,B) Progeny counts of the indicated genotypes (x-axis) were performed on a minimum of eight individuals or crosses. Error bars indicate s.d.

View larger version (117K): [in a new window]   Fig. 2. The morphology of spe-38 mutant sperm is indistinguishable from that of wild-type sperm. (A-C) White arrows indicate examples of spermatozoa in Nomarski DIC images. In vitro-activated sperm from wild-type (A) and spe-38(eb44) (B) male worms appear identical. (C) In vivo-activated hermaphrodite sperm within the spermatheca of an unmated spe-38(eb44) hermaphrodite are indistinguishable from wild-type sperm (A). Some of these sperm are in direct contact with the oocyte. (D-F) Transmission electron micrographs of spe-38(eb44) sperm within spe-38(eb44) hermaphrodites. The ultrastructural details of spe-38 sperm are indistinguishable from those of wild type, including the sperm chromatin mass (N), the pseudopod (P) and the fused membranous organelles (MOs). (F) Close-up view of MOs in spe-38(eb44) sperm highlights the fusion pore (black arrow) surrounded by an electron-dense collar. Scale bars: in A, 5 µm for A-C; 1 µm in D,E; 0.5 µm in F.

View larger version (70K): [in a new window]   Fig. 3. Meiotic maturation and ovulation. (A-C) Three sequential Nomarski DIC micrographs showing normal meiotic maturation, ovulation and sperm-oocyte contact in a spe-38(eb44) hermaphrodite. The black arrows indicate the position of the same oocyte as it moves from the oviduct (A) to the spermatheca (site of sperm storage; B) and finally to the uterus (C). The oocyte spent 7 minutes in contact with sperm in the spermatheca without being fertilized. The uterus is filled with previously unfertilized oocytes. (D) A wild-type hermaphrodite has a uterus that is filled with developing embryos. Unlike unfertilized oocytes, developing embryos are oval and are enclosed within a clearly defined eggshell. In all panels, the white arrows indicate examples of sperm and their position.

View larger version (72K): [in a new window]   Fig. 4. spe-38 sperm exhibit normal transfer and migratory behavior, but fail to penetrate oocytes. (A-E) Partial images of whole-mount DAPI-stained hermaphrodites or genetic females, in which the oviduct (left), spermatheca (large arrowheads) and uterus (right) are shown. In such DAPI-stained preparations, the small dense chromatin mass of mature spermatozoa distinguishes them from other somatic- and germ-cell types. (A) In an unmated wild-type hermaphrodite, numerous sperm (small bright spots) are present within the spermatheca. (B) An unmated fem-1 female lacks sperm within her spermatheca, and her uterus is filled with unfertilized, endomitotic (emo) oocytes (arrows). (C-E) Upon mating, sperm from either wild-type (C) or spe-38(eb44) males (D,E) populate the spermatheca of fem-1 females. (F) In a newly fertilized wild-type oocyte, the sperm chromatin remains as a single, highly condensed DNA mass, while the oocyte chromosomes are undergoing their meiotic divisions. (G,H) Sperm chromatin masses were never observed in either young (G) or older (H) emo oocytes from the uteri of unmated spe-38 hermaphrodites. (I,J) Young (I) and older (J) emo oocytes from the uteri of fog-2 females crossed with spe-38; him-5 males. (K) A series of developing embryos within the uterus of a spe-38 hermaphrodite mated to wild-type males. A condensed sperm chromatin mass is visible in the meiotic-stage embryo (far left). The broken lines outline the oocytes and embryos.

View larger version (18K): [in a new window]   Fig. 5. spe-38 sperm accelerate ovulation rates and participate in sperm competition. (A) The level of ovulation in unmated spe-38 and wild-type (N2) hermaphrodites is significantly higher than in unmated fem-1 controls (*P<0.001, when compared fem-1 at 25°C, Student's t-test). (B) Quantitative analysis of fertilized and unfertilized oocytes laid by unmated fem-1 females, as well as fem-1 females crossed to either wild-type him-5 males or mutant spe-38; him-5 males. Sperm from wild-type and spe-38 males induce comparable ovulation rates, which are significantly higher than those of the unmated fem-1 controls (*P<0.001, Student's t-test). (C) Hermaphrodite sperm from morphologically marked (dpy-5), but otherwise wild-type, hermaphrodites are out-competed by male sperm from either wild-type him-5 or mutant spe-38(eb44); him-5 males. In both cases, the self-fertility of mated dpy-5 hermaphrodites was significantly depressed when compared with unmated controls (*P<0.001, Student's t-test). All counts for the indicated genotypes were performed on a minimum of eight individuals or crosses. Error bars indicate s.d.

View larger version (19K): [in a new window]   Fig. 6. Molecular cloning of the spe-38 gene. (A) Genetic and physical maps of the spe-38 region of chromosome I. Expanded views below the genetic map show the relative position of informative single nucleotide polymorphisms (SNPs), and the structure/orientation of the Y52B11A.1 (spe-38) and Y52B11A.2 genes. Exons are indicated by the blue and green boxes. The transgenic rescue profile of DNA fragments containing these genes is shown on the right. (B) Expanded view showing the intron and exon structure of the spe-38 gene. Exons are indicated by the green boxes and are numbered from right to left to keep orientation consistent with the genetic map. The 270 bp deleted in the spe-38(eb44) mutant strain are indicated by the bracket under the structural representation of the gene. (C) PCR characterization of the spe-38(eb44) mutation. Primers designed to amplify an 1.5 kb DNA fragment from wild-type worms amplify a smaller DNA fragment from spe-38(eb44) homozygous animals. Marker sizes are indicated to the right of the gel.

View larger version (41K): [in a new window]   Fig. 7. The amino acid sequence of SPE-38 and a visual comparison of the predicted structure of SPE-38 with that of several tetraspan integral membrane proteins that are thought to function in cell-cell interactions. (A) The predicted and aligned amino acid sequences (single letter code) of C. elegans (Ce-SPE-38) and C. briggsae (Cb-SPE-38) SPE-38 proteins. Yellow shading indicates identical amino acids; green shading indicates similar amino acids; predicted transmembrane domains are shown in bold blue letters. The sequence used to generate the anti-SPE-38 sera is underlined, and the first amino acid missing in the spe-38(eb44) mutation (aspartic acid) is indicated by red shading. (B) A schematic representation of Ce-SPE-38 and Cb-SPE-38, and comparisons with other tetraspan integral membrane proteins. Transmembrane domains are indicated with blue boxes; the black arrow indicates the loop domain used for peptide synthesis and the red arrow indicates the aspartic acid residue position in eb44 noted in A. The mutant gene is not predicted to code for any wild-type protein sequence beyond this residue. The schematics for the various proteins are derived from previous reports (Boucheix and Rubinstein, 2001; Heiman and Walter, 2000; Hemler, 2003; Tsukita and Furuse, 1999), and/or from our hydropathy plot and domain structure analysis (see Materials and methods).

View larger version (76K): [in a new window]   Fig. 8. Localization of SPE-38 in spermatids and spermatozoa. SPE-38 was detected using a peptide polyclonal antibody against a putative extracellular loop domain of SPE-38 (SPE-38101–114). (A) DIC (top) and epifluorescence (bottom) images of in vivo-activated wild-type spermatids and spermatozoa. Permeabilized cells were cryomethanol fixed and permeabilized with Triton X-100. Live cells were incubated with antibody prior to fixation and were not permeabilized. Insets are enlargements of cells from the same or equivalent fields. (B) Sperm prepared as described in A, but incubated with anti-SPE-38 antibody that was pre-incubated with competing peptide. (C) DIC (top) and epifluorescence (bottom) images of in vitro-activated spe-38(eb44) and wild-type sperm. Cells were paraformaldehyde fixed and premeabilized with Triton X-100. (D) Western blot of wild-type and spe-38(eb44) adult males. Each lane contains exactly 400 male worms. Equivalent loading was confirmed by Coomassie Blue staining. The sequence coding the antigenic peptide (SPE-38101–114) is deleted in eb44 mutants.

View larger version (90K): [in a new window]   Fig. 9. The dynamic localization of SPE-38 requires MO fusion, but SPE-38 is not required for the dynamic localization of other sperm membrane proteins. (A) Confocal/multiphoton images of wild-type spermatids stained with DAPI (blue), anti-SPE-38 antibody (red) and the monoclonal antibody 1CB4 (Okamoto et al., 1985), which binds to an MO-associated antigen (green). Partial co-localization (yellow) of SPE-38 and 1CB4 staining is shown in the merged image. (B) Anti-SPE-38 immunolocalization in fer-1(hc17ts) mutant sperm, which fail to undergo MO fusion during spermiogenesis. Staining is detectable in fixed, permeabilized spermatids and spermatozoa, but not in live, non-permeabilized cells. (C) Localization of the 1CB4 antigen (green), SPE-9 (red) and DNA (blue) in wild-type and spe-38 sperm.