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Corresponding author: Samuel Ward, Department of Molecular and Cellular Biology, 452 Life Sciences South, 1007 Lowell St., Tucson, AZ 85721., samward{at}U.arizona.edu (E-mail)
Communicating editor: P. ANDERSON
 | ABSTRACT |
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Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa.
SPERM in the nematode Caenorhabditis elegans accumulate as immotile spherical spermatids, which are incompetent for fertilization. In response to extracellular signals, the cells undergo spermiogenesis, a rapid and dramatic morphological transformation to mature amoeboid spermatozoa, and immediately begin crawling. Because this maturation occurs without any new mRNA or protein synthesis, spermiogenesis initiation affords an opportunity to study a signaling pathway that acts post-translationally to regulate cellular morphogenesis in a genetically tractable organism. Moreover, nematode sperm provide a distinctive system for investigating the acquisition of cellular motility because they derive their crawling motility from an unconventional cytoskeleton containing neither actin filaments nor microtubules (reviewed in 
As they develop, C. elegans sperm become streamlined toward their sole task of encountering and fertilizing oocytes. Spermatocytes, the progenitors of spermatids, are biosynthetically active cells, producing all the components that will be needed in the mature spermatozoa. One key component, which will form the cytoskeleton responsible for motility, is a small protein called major sperm protein (MSP). Spermatocytes synthesize large amounts of MSP and package it into paracrystalline arrays called fibrous bodies (FBs), which form a complex with novel structures termed membranous organelles (MOs). Each spermatocyte divides twice in meiosis and segregates a subset of its contents to the resulting four haploid spermatids. Each spermatid receives a nucleus, mitochondria, FB-MOs, and other molecular machinery necessary for forming a pseudopod, for crawling, and for fertilization. Components not needed in the spermatids, including ribosomes, endoplasmic reticulum, Golgi, and the actin and tubulin cytoskeletons, are jettisoned to an anucleate residual body and subsequently degraded. After spermatids detach from the residual body, the FBs disassemble and release their MSP to the cytosol, where it remains largely depolymerized until spermiogenesis commences.
Within minutes after exposure to the (as yet unidentified) spermiogenesis initiation signal, MOs fuse with the plasma membrane at one side of the cell. A pseudopod extends from the opposite side. MSP polymerizes within the pseudopod, forming a dynamic cytoskeleton that pushes the tip of the pseudopod forward while simultaneously drawing the cell body forward (
Many mutants defective in sperm development have been isolated as self-sterile hermaphrodites that can be rescued by mating with wild-type males (
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Strains and general nematode methods:
Standard methods for culture and genetic analysis of C. elegans were performed as described (
Isolation of suppressor mutants:
To isolate suppressors of the temperature-sensitive hermaphrodite-self-sterile phenotype imposed by spe-27(it132), large populations of spe-27(it132) unc-22(e66) hermaphrodites were raised at the permissive temperature. The unc-22 mutation was included in the strain to preclude mating by males in the population because spe-27 mutant hermaphrodites are fertile when mated to males. unc-22(e66) males cannot copulate. The populations, which contained many L4 larvae and young adults, were mutagenized for 4 hr at 15° in 50 mM ethyl methanesulfonate (Sigma, St. Louis) as described by 
156,000 F1 mutagenized individuals were subjected to selection in this screen.
Dominance and complementation tests between suppressor mutants:
To test the suppressor mutants for dominance, young adult spe-27(it132) unc-22(e66); "sup-X" hermaphrodites were mated to spe-27(it132) males. F1 cross-progeny hermaphrodites (i.e., non-Unc) were scored for self-fertility at 25°. In almost every case, all the F1 cross-progeny were sterile, indicating that the suppressor mutation was recessive. For two mutants, all or most of the F1 cross-progeny were weakly self-fertile, indicating semidominance. Suppressor strains used in complementation tests were outcrossed at least twice to spe-27(it132) males to remove any secondary mutations. Outcrosses also indicated that the suppressor mutations are not true revertants of spe-27(it132) since the suppression phenotype always segregated independently from unc-22(e66), which is itself closely linked to the spe-27 mutation in the suppressor strains. For complementation tests spe-27(it132) unc-22(e66)/+ +; "sup-X"/+ males were mated to spe-27(it132) unc-22(e66); "sup-Y" hermaphrodites. One-half of the outcross (i.e., non-Unc) hermaphrodite progeny from this cross should be self-fertile if sup-X and sup-Y fail to complement; if the two mutants complement each other, no fertile cross-progeny are expected. All pairwise combinations of the five strongest suppressor mutants (hc163, hc164, hc165, hc166, and hc167) were tested for complementation. In addition, the strongest suppressing strain, hc163, was tested for complementation of the remaining nine strongest suppressing recessive mutants.
Testing suppression of null spe-27 alleles and other spermiogenesis mutants:
spe-27(it132) unc-22(e66); hc163 hermaphrodites were crossed to spe-27(it110) dpy-20(e1282) males. F1 male and hermaphrodite siblings were intercrossed, and their Dpy, non-Unc hermaphrodite progeny were scored for fertility at 25°. Similar crosses were performed to test hc163 suppression of spe-27(hc161) (
Genetic mapping of hc163:
We used trans-heterozygote linkage analysis with respect to dpy-1(e1) to assign hc163 to chromosome III. cis-heterozygote recombination mapping experiments with hc163 dpy-1(e1) and hc163 dpy-18(e364) double mutants were performed to map hc163 more accurately. Genetic mapping data are available on the world wide web at WormBase (http://www.wormbase.org).
Complementation crosses between hc163 and chromosome III deficiencies:
We tested chromosome III deficiencies tDf2, tDf7, ctDf3, and ctDf2 for their ability to complement hc163. hc163; spe-27(it132) unc-22(e66) hermaphrodites were crossed to tDf2/qC1 dpy-19(e1259) glp-1(q339); spe-27(it132) males. Individual F1 hermaphrodite progeny were scored for fertility at 25°. If hc163 complements the deficiency, then all F1 hermaphrodites should be sterile; if hc163 fails to complement the deficiency, then about one-half of the F1 hermaphrodites should be fertile. As a negative control, similar crosses were assayed using spe-27(it132) unc-22(e66) worms as the hermaphrodite parent. Similar complementation crosses and controls were done with the other three deficiencies.
Complementation tests between hc163 and spe-6(hc49):
We performed two different tests to determine if hc163 and spe-6(hc49) complement. First, we tested the ability of hc163 to complement the sterile phenotype of spe-6(hc49). Next, we tested whether spe-6(hc49) could complement the ability of hc163 to suppress the temperature-sensitive self-sterile phenotype imposed by spe-27(it132). For the first test, spe-6(hc49) vab-7(e1562) hermaphrodites were crossed to hc163 dpy-18 (e364); him-5(e1490) males. For the second test, spe-6(hc49) vab-7(e1562); spe-27(it132) hermaphrodites were mated to hc163 dpy-18(e364); spe-27(it132); him-5(e1490) males. F1 hermaphrodites were picked to individual plates and assayed for self-fertility at 25°. For both tests, individual F1 hermaphrodites were assayed for self-fertility at 25°.
Physical mapping of ctDf3 and tDf7 breakpoints:
The genome sequence around the spe-6 locus [yeast artificial chromosome (YAC) clone Y66D12, GenBank accession no.
AL161712] was determined and generously provided by John Sulston and Alan Coulson (Sanger Center, Cambridge, UK). To locate the left breakpoint of ctDf3 and the right breakpoint of tDf7 on the C. elegans physical map, we designed PCR primers targeting sequences in the region and assayed their ability to amplify targets from individual arrested embryos homozygous for either deficiency. ctDf3 or tDf7 mutant embryos were treated essentially as described by
Sequencing spe-6 mutants and cDNAs:
To sequence spe-6 wild-type and mutant alleles, fragments of the spe-6 genomic region of homozygotes were first amplified by single-worm PCR using similar conditions as used for mapping deficiency breakpoints. Four PCR reactions were run for each template, and the products were pooled and purified by filtration on a Microcon PCR purification filter (Millipore, Bedford, MA). cDNA clones for sequencing were amplified from a C. elegans spermatogenesis-enriched library constructed and generously provided by H. SMITH (unpublished results). Automated DNA sequencing was performed by the Laboratory of Molecular Systematics and Evolution (Arizona Research Labs, University of Arizona). Data were analyzed using the Wisconsin Package Version 10.0 for UNIX (Genetics Computer Group, Madison, WI) and FAKtory (
Microinjection transformation rescue:
Using TurboPfu polymerase (Stratagene, La Jolla, CA), we amplified a 3.8-kb fragment from wild-type worms containing the entire putative spe-6 gene (Y66D12A.20), including 180 bases 3' of the stop codon, and all of the upstream sequences, including the 5' portion of the immediately upstream gene (Y66D12A.21). For microinjection rescue, we injected a mixture containing the PCR fragment (2 ng/µl), linearized plasmid pRF4 (2 ng/µl;
Progeny counts:
To determine brood sizes, virgin L4 hermaphrodites were incubated on individual culture plates at 20° (unless otherwise noted) and transferred to fresh plates daily until they stopped laying eggs. To count dead and live progeny, plates were incubated for at least 24 hr after the parents were removed to allow all viable eggs to hatch. Live progeny and dead embryos were counted daily as they were aspirated from the plate.
Light microscopy:
Differential interference contrast (DIC) images of live worms or dissected testes in SM1 buffer (
Electron microscopy:
Four-day-old virgin spe-6(hc163); him-5(e1490) or him-5(e1490) males were prepared as described by
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolation and characterization of suppressors of a spermiogenesis initiation mutant:
To search for additional genes in the spermiogenesis initiation pathway and to reveal potential interactions among the four known genes, we selected for ethyl methanesulfonate-induced fertile suppressors of spe-27(it132), a temperature-sensitive missense allele. At 25°, homozygous it132 virgin hermaphrodites are self-sterile, but mated hermaphrodites are cross- and self-fertile (30 progeny, enabling us to grow a large population of homozygous mutants for secondary mutagenesis.
We isolated 32 recessive suppressors of it132 whose self-brood sizes (in a homozygous it132 genetic background) range from 5 to 82 progeny at 25°. We chose 14 suppressor strains with the highest self-fertility (14–82 progeny) for initial characterization. None of the suppressor mutations are alleles of spe-27. All 5 of the most fertile suppressors (hc163, hc164, hc165, hc166, and hc167) failed to complement each other, and hc163 failed to complement the remaining 9 recessive suppressor strains. Further genetic and phenotypic analysis was restricted to allele hc163, which suppressed spe-27(it132) to yield 82 ± 21 progeny at 25°, where spe-27(it132) produced no progeny and wild type produced 188 ± 47 progeny.
hc163 mutants suppress mutations in multiple genes required for spermiogenesis initiation:
To determine whether the Spe-27-suppression phenotype results from an allele-specific interaction with spe-27(it132), we tested whether hc163 could suppress two probable null alleles of spe-27, it110, and hc161 (
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hc163 is an allele of spe-6, a gene required for early events in sperm development:
We mapped hc163 and the other 13 suppressor mutations to chromosome III, 1 cM to the right of dpy-18. Complementation tests localized the mutation under deficiencies tDf7 and ctDf3 and between tDf2 and ctDf2 (Fig 1A). The combination of complementation and genetic mapping data suggested that all of these mutations are alleles of the same gene. We confirmed this by sequence analysis (described below) and identified four additional alleles of this gene in our collection of suppressors. Genetic analysis of the remaining nine suppressor strains, most of which have very low fertility, indicated that their mutations are not allelic to hc163. We will describe these mutants elsewhere.
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The only reported mutations in the genetic interval containing hc163 were in spe-6, a gene originally defined in our laboratory by four mutations—hc49, hc92, hc143, and hc146—which behave as loss-of-function or strong reduction-of-function alleles and affect an early stage of sperm development (
Identifying the spe-6 gene:
spe-6 is the only reported locus that genetically maps to a region of chromosome III deleted in deficiencies tDf7 and ctDf3, which suggested that this region might be physically small (Fig 1A). To locate this region on the physical map we performed PCR assays on individual dead embryos homozygous for tDf7 or ctDf3 using primer sets designed from the published DNA sequence in this region of the genome. We narrowed the tDf7/ctDf3 overlap to within a 12-kb region represented in YAC clone Y66D12 (GenBank accession no.
AL161712; Fig 1B).
We surveyed the genes in this region by performing a BLASTN search against the C. elegans expressed sequence tag (EST) nucleotide database (
spe-6 encodes a casein kinase 1:
BLASTP and TBLASTN searches of the public sequence databases revealed that the predicted SPE-6 protein (Wormpep ID WP:CE28799) is a member of the CK1 family of protein-serine/threonine kinases (Fig 2 and Fig 4). SPE-6 shares 30% identity and almost 50% similarity to yeast, mammalian, and plant CK1 proteins over their catalytic domains. C. elegans has a large CK1 subfamily, containing 90 members (286 amino acids (70-amino-acid C terminus whose sequences share no significant similarity with other proteins. On the basis of the longest 5' cDNA end detected, we tentatively assign the first in-frame methionine codon in exon 1 as the start codon, although the C. elegans Sequencing Consortium predicts an additional 9 amino acids at the N terminus of Y66D12A.20 (Fig 3).
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Identification of spe-6 mutations:
We identified mutations in all four of the spe-6 sterile alleles and 18 suppressor alleles (Fig 2 Fig 3 Fig 4). The canonical sterile allele, hc49, contains a premature stop codon predicted to result in a protein truncated at a position in the C-terminal domain. hc146 is identical to hc49 and may be a reisolate of the original mutation. Two independently isolated sterile alleles, hc92 and hc143, are both T185I substitutions. This is also likely to be a null mutation because this residue is universally conserved as a threonine or serine in all CK1 proteins (and most other protein kinases) examined, and it may participate in a hydrogen-bonding network that stabilizes the catalytic core of the enzyme (
As shown in Fig 4, SPE-6 mutations that lead to the spermiogenesis-defect suppression phenotype are distributed mainly throughout the catalytic domain of the protein. Some of the suppressor mutations are predicted to lie in regions of the protein recognized as important for its kinase activity, such as the ATP-binding region [e.g., hc167(S24P)] and the catalytic cleft [e.g., hc164, hc176, hc187 (all G133E), and hc168 (L135F)]. However, no suppressor mutations are in positions known to be essential for activity in related protein kinases. Several mutations are in regions of the protein where functions are not well characterized. Of the 18 suppressor alleles, 16 have single amino acid substitutions. hc190 has a mutation in the conserved 5' intronic G of the first intron, within the N terminus, and probably results in a misspliced message. hc171 has a disrupted 5' splice donor site in the final intron; the effects of this on its transcript and protein products are not known. Beside hc171, the only other mutation in the C terminus is hc172 (A322V).
Spe-6(hc163) phenotypes:
The suppression phenotype of spe-6 suppressor alleles indicates that, in mutant hermaphrodites, spermatids initiate spermiogenesis without requiring the activation signal transduced by the spe-8, -12, -27, -29 signaling pathway. Using transmission electron microscopy, we found that spermatids from hc163 mutant virgin males also activate precociously to form spermatozoa (Fig 5A). However, it is clear that only a small fraction of the spermatids develop into mature spermatozoa in males. Interspersed among the spermatozoa are what appear to be the cytoplasmic contents of degraded cells. The degraded cells were presumably spermatids because the zone of degradation is restricted to the proximal end of the testis, where spermatids normally accumulate. Additionally, nuclei in the degradation zone have the highly compact morphology of spermatid nuclei rather than the more typical structure of spermatocyte nuclei (Fig 5A). This cellular lysis appears similar to the necrosis-like cell death seen in several C. elegans mutants that affect ion transport in sensory neurons (
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In marked contrast to the sterile spe-6 mutants, whose membranous organelles lack fibrous bodies altogether, fully developed spermatocytes from hc163 mutants have normal looking FB-MO complexes. In early stage spermatocytes, however, the developing FB-MO complexes contain FBs with ragged edges, separated from the membrane of the MO (Fig 5F and Fig G).
hc163 appears to affect fertility more severely in males than in hermaphrodites. While mutant males copulate normally, their crosses are rarely successful, and those that are successful result in very few cross-progeny (data not shown). This is consistent with the severe ultrastructural defects and reduced number of spermatozoa we observed in mutant male testes. Hermaphrodites show minimal signs of sperm degradation by light microscopy (data not shown).
On the basis of their recessive behavior and their intermediate effects on fertility and testis ultrastructure, the spe-6 suppressors appear to be reduction-of-function alleles. We asked whether hc163 could be a gain-of-function allele, even though such mutations are usually dominant. If hc163 were a gain-of-function allele, then two doses of the mutation should cause a more severe phenotype (i.e., lower fertility) than a single dose, but this is not the case (Table 2). The recessive nature and partial sterile phenotypes of the other spe-6 suppressor mutants, together with the observation that their lesions are widely distributed throughout the predicted protein, support the notion that these suppressors are also reduction-of-function alleles.
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| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We show here that certain alleles of spe-6, a gene known from previous work to be required early in sperm development, can suppress mutations in all four of the known genes required for spermiogenesis initiation, one of the final events in sperm development. The proteins encoded by the three cloned spermiogenesis initiation genes, spe-12, spe-27, and spe-29, show no significant similarity to any other proteins in the sequence databases, and the nature of their activities is unknown. SPE-12 resides in the spermatid plasma membrane, and genetic interactions between spe-12, -27, and -29 suggest that their products may act together in a complex (
The role of spe-6 in spermiogenesis initiation:
Because an apparent reduction of SPE-6 activity suppresses spermiogenesis-defective mutations, we propose that the normal function of the SPE-6 protein kinase in spermatids is to restrain the spermiogenesis machinery, preventing it from engaging until the SPE-8, -12, -27, and -29 gene products have relayed the signal to commence spermiogenesis (Fig 6A). In this model, SPE-6 maintains phosphorylation of its target protein substrate(s), which in turn serves as a brake on spermiogenesis. When stimulated by an extracellular signal, the SPE-8, SPE-12, SPE-27, and SPE-29 gene products antagonize the SPE-6 kinase activity, releasing the brake so that spermiogenesis can proceed, forming the crawling spermatozoon (Fig 6B).
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Casein kinase 1 proteins are ubiquitous and promiscuous regulatory proteins, engaged in a wide range of cellular processes (
Because spermatids lack translational machinery, spermiogenesis must be controlled by post-translational regulation; therefore it is not surprising that nematode sperm employ protein phosphorylation. spe-6 is the first protein kinase gene demonstrated to regulate sperm development in C. elegans, but a protein-tyrosine kinase activity is key to sperm motility in the related nematode, Ascaris suum (
The role of spe-6 in early sperm development:
We were surprised to discover that the suppressor mutations are alleles of spe-6, a gene our laboratory had previously found to be involved in spermatocyte development (
Relating the functions of SPE-6:
How might SPE-6 perform apparently different functions in distinct stages of spermatogenesis? The need for SPE-6 for fibrous body formation in spermatocytes and its inhibitory role in spermiogenesis may reflect different phosphorylation targets in these two cells. Alternatively, SPE-6 may phosphorylate the same proteins in spermatocytes and spermatids, but these targets could lead to different effects based on their distinct cellular contexts. We cannot eliminate a third possibility—that the effect of spe-6 mutations on spermiogenesis may be only an indirect consequence of its earlier influence on spermatocytes. Defective spermatocytes may simply produce spermatids that are incapable of repressing spermiogenesis. However, the sheer number of spe-6 alleles that produce the suppression phenotype and the minimal ultrastructural defects observed in hc163 mutant spermatocytes argue against this possibility.
How is fibrous body formation in spermatocytes related to spermiogenesis initiation in spermatids? One common feature of these two processes is that each involves MSP polymerization. SPE-6 may regulate the state of MSP polymerization, although not by directly phosphorylating it, since MSP is not detectably phosphorylated in vivo (
Whatever the roles of SPE-6 in spermatocyte development and in spermiogenesis initiation, our analysis of suppressor allele hc163 illustrates that a single mutation can compromise the two processes to dramatically different extents. spe-6(hc163) homozygous hermaphrodites have virtually identical (moderate) fertility as hemizygotes, suggesting that the mutation causes only a slight reduction of function in the SPE-6 protein relative to wild type. Indeed, spe-6(hc163) exhibits very little effect on spermatocyte development. Yet, this allele strongly influences spermiogenesis initiation. The large number and widespread distribution of spe-6 suppressor mutations is consistent with the hypothesis that a small reduction in SPE-6 activity is sufficient to prevent SPE-6 from inhibiting spermiogenesis initiation.
This sensitivity of spermiogenesis initiation to small decreases in SPE-6 activity makes good biological sense. Sperm are the limiting gamete in C. elegans, so reproductive fitness depends directly on the number of functional sperm (
| ACKNOWLEDGMENTS |
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We extend thanks to our colleagues, Jeremy Nance, for stimulating discussions throughout this project, Craig LaMunyon, for help with statistics, and Harold Smith, for providing cDNA libraries. We thank Theresa Stiernagle, Michel Labouesse, and Barbara Robertson for providing nematode strains, and Brian James for helping with complementation tests. We are especially indebted to Alan Coulson, Lucy Matthews, and John Sulston for filling the "spe-6 chasm" of the C. elegans physical map. Finally, we thank C. LaMunyon, J. Nance, members of the Ward lab, a Genetics editor, and an anonymous reviewer for their insightful comments on the manuscript. Some nematode strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources. This work was supported by National Institutes of Health research grant GM25243 to S.W.
Manuscript received December 7, 2001; Accepted for publication February 18, 2002.
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isoform, PID g547766 (
isoform, PID g547763 (
