spe-12 Encodes a Sperm Cell Surface Protein That Promotes Spermiogenesis in Caenorhabditis elegans

Corresponding author: Samuel Ward, MCB Department, The University of Arizona, Life Sciences South, Rm. 452, Tucson, AZ 85721., samward{at}u.arizona.edu (E-mail)

Communicating editor: R. K. HERMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.

COMMON sets of signal transduction molecules direct cell differentiation events in diverse tissues and organisms. Mitogen-activated protein (MAP) kinase signaling pathways in the nematode Caenorhabditis elegans, for example, are known to play a role in vulval cell induction (HAN and STERNBERG 1990 ), male spicule development (CHAMBERLIN and STERNBERG 1994 ), meiotic cell cycle progression (CHURCH et al. 1995 ), and embryogenesis (reviewed in KAYNE and STERNBERG 1995 ). In Drosophila melanogaster, similar signaling pathways control photoreceptor differentiation (SIMON et al. 1991 ; reviewed in WASSARMAN et al. 1995 ) and other aspects of development (reviewed in PERRIMON 1994 ). Induction of these pathways typically results in the transcriptional regulation of sets of genes that direct the new developmental fate.

Signaling systems that induce the differentiation of cells that are incapable of synthesizing new gene products may be expected to utilize some novel mechanisms of relaying information and rearranging cellular architecture. One such well-studied example is the signaling cascade that activates anucleate platelets, leading to their morphogenesis and aggregation (reviewed in BODY 1996 ). The surface of these cells is rich in receptors, including not only the commonly used integrins and members of the immunoglobulin superfamily, but also several specific and novel glycoproteins. Upon binding of receptors to exposed subendothelial ligands, signal transducers (including G-proteins, phospholipase C, and protein kinase C) collaborate to activate the cells. Thus, platelet activation is accomplished using a combination of general and more specific signaling proteins.

The initiation of C. elegans spermiogenesis (the final differentiation event of spermatids) and subsequent cellular rearrangements also take place in the absence of new gene product synthesis, since ribosomes and most mRNA are not segregated to the spermatid during the final meiotic division (WARD et al. 1981 ; PAVALKO and ROBERTS 1989 ). Analysis of the components required to transduce the signal initiating spermiogenesis may illustrate how such a specialized signaling pathway differs from the many well-characterized pathways that result in transcriptional regulation.

During spermiogenesis, spermatids activate by converting from immotile, symmetrical cells to asymmetrical crawling spermatozoa. Activation is induced only when crawling is required and thus, not surprisingly, is temporally and spatially distinct in hermaphrodites and males. Hermaphrodites produce spermatids transiently in both arms of the bilobed gonad before irreversibly switching to oogenesis. Soon after the completion of meiosis, spermatids activate to spermatozoa (reviewed in WARD 1986 ; KIMBLE and WARD 1988 ; L'HERNAULT 1997 ). During this striking conversion, membranous organelles fuse with the plasma membrane and a pseudopod extends from the cell body. A novel cytoskeleton composed of major sperm protein (MSP) filaments assembles in the pseudopod; the organized treadmilling of MSP filaments is thought to provide the motive force that enables spermatozoa to crawl (reviewed in ROBERTS and STEWART 1997 ). Spermatozoa crawl to the spermathecae where they position themselves for fertilization. Males synthesize spermatids and store them until mating occurs. Once ejaculated into the hermaphrodite uterus, spermatids activate in response to an unidentified signal that likely originates from male seminal fluid. These freshly activated spermatozoa crawl to the spermathecae where they join hermaphrodite-derived spermatozoa in competition for fertilization of her oocytes (WARD and CARREL 1979 ). Male-derived spermatozoa take nearly complete precedence over hermaphrodite-derived spermatozoa, fertilizing the overwhelming majority of oocytes (WARD and CARREL 1979 ; LAMUNYON and WARD 1995 ) before they are used up.

How is the dramatic morphogenesis from spermatid to spermatozoon initiated? Among the many spermatogenesis-defective mutants is a class that compromises the spermatid's ability to initiate spermiogenesis. These mutants, spe-8, spe-12 (L'HERNAULT et al. 1988 ), spe-27 (MINNITI et al. 1996 ), and spe-29 (J. NANCE and S. WARD, unpublished results) are unlike other mutants in that their Spe phenotype is affected by mating. Virgin hermaphrodites are self-sterile; their spermatids arrest and never form spermatozoa. Males, however, are fertile. Their spermatids are able to initiate and complete spermiogenesis after ejaculation. Surprisingly, mating by any male "transactivates" mutant hermaphrodite-derived spermatids, initiating spermiogenesis and resulting in newly acquired self-fertility. These observations led to a model in which spermatids initiate spermiogenesis in response to either a male-supplied or endogenous hermaphrodite "activator" (SHAKES and WARD 1989A ; MINNITI et al. 1996 ; L'HERNAULT 1997 ). Mutations in spe-8, spe-12, spe-27, or spe-29 would prevent spermatids from properly responding to hermaphrodite activator; male activator would rescue these mutant spermatids by bypassing the function of SPE-8, SPE-12, SPE-27, and SPE-29. Though these mutations affect spermatids from both sexes, as judged by defects after exposure of both hermaphrodite- and male-derived spermatids to the in vitro activator pronase (SHAKES and WARD 1989A ; MINNITI et al. 1996 ; J. NANCE and S. WARD, unpublished results), the role of the wild-type gene products in male-derived spermatids, if any, was unknown.

Here we analyze the role of SPE-12 in hermaphrodite spermiogenesis initiation and present evidence that the gene product functions in male-derived spermatids as well. In a continuing molecular characterization of genes that affect this signaling pathway (MINNITI et al. 1996 ), we identify the spe-12 gene and show that it encodes a sperm plasma membrane protein.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Worm strains and handling:
Worms were maintained on nematode growth (NG) agar plates spotted with Escherichia coli strain OP50 and were manipulated physically and genetically as described by BRENNER 1974 . All strains were stored at 15°; experiments were conducted at 24.5°, the restrictive temperature for each temperature-sensitive mutant used in this study. The following recessive mutants, derivatives of the wild-type N2 (var. Bristol) strain, were used:

• LGI: spe-8(hc50, hc53, hc134ts) (L'HERNAULT et al. 1988 ); dpy-5(e61) and unc-13(e51) (BRENNER 1974 ); spe-12(hc76, hc149, hc152) (L'HERNAULT et al. 1988 and this study); fer-1(hc13ts) (WARD and MIWA 1978 ); and spe-9(hc88ts) (L'HERNAULT et al. 1988 ; SINGSON et al. 1998 ).

• LGIV: fem-1(hc17ts) (NELSON et al. 1978 ), unc-24(e138) (BRENNER 1974 ), fem-3(q23ts) (HODGKIN 1986 ), and dpy-20(e1282ts) (HOSONO et al. 1982 ).

• LGV: him-5(e1490) (HODGKIN et al. 1979 ). hcEx052 is a nonintegrated transgenic array and is described in RESULTS.

Isolation of new spe-12 alleles:
The canonical spe-12(hc76) allele was used to isolate new spe-12 alleles in an F₁ noncomplementation screen. N2 males were mutagenized with ethyl methanesulfonate (BRENNER 1974 ) and mated to spe-12(hc76) dpy-5(e61) hermaphrodites. A total of 2060 non-Dpy hermaphrodite F₁ larvae were individually isolated, allowed to mature, and scored for self-fertility at 24.5° by examining plates for eggs or oocytes. Sterile F₁ were rescued by mating to N2 males. Non-Dpy F₂ sterile hermaphrodites from this P₀ cross were isolated and backcrossed to N2 multiple times to remove unwanted deleterious mutations. Candidate spe-12 alleles were tested for their failure to complement spe-12(hc76), each other, and the underlying deficiency nDf25. Linkage to the LGI marker unc-13 (0.5 cM from spe-12) was established by trans-linkage analysis (BRENNER 1974 ). Two new spe-12 alleles, hc149 and hc152, were isolated.

Worm synchronization and brood-size determination:
Synchronized worms were obtained from gravid hermaphrodites by allowing them to lay eggs for a 2-hr window. Synchronized eggs were grown at 24.5°. To determine brood sizes, hermaphrodites were transferred to fresh plates daily until no new eggs were laid in a 24-hr period; the resulting progeny were counted.

Sperm counts:
Worms were synchronized by the alkaline hypochlorite method (L'HERNAULT and ROBERTS 1995 ), grown for 52 hr at 24.5°, picked as late larval hermaphrodites to individual plates, and allowed to mature for 18 additional hours. The number of eggs or oocytes that each worm laid was recorded. To stain nuclei, worms were fixed in Carnoy's solution and stained with 4',6-diamidino-2-phenylindole (DAPI). Sperm nuclei were identified by their compactness and position in either the spermatheca or the uterus. The total sperm population was determined by scanning through focal planes and summing individual nuclei.

DNA transformation:
Cosmid or plasmid DNA purified over a QIAGEN (Santa Clarita, CA) column and resuspended in TE (pH 7.4; AUSUBEL et al. 1995 ) was coinjected with the transformation marker pRF4, which contains the dominant rol-6(su1006) allele (KRAMER et al. 1990 ). DNA mixtures were injected into the gonadal syncytium of young adult spe-12 hermaphrodites at a concentration of 100 ng/µl of tester DNA and 100 ng/µl of pRF4 (MELLO and FIRE 1995 ). After recovery, worms were placed with four spe-12(hc76) males and allowed to mate overnight. Transformed hermaphrodite progeny of injected worms were recognized by their Rol phenotype, picked to individual plates as virgins, and scored for self-fertility.

Molecular biology:
Standard techniques were used to isolate and manipulate DNA and RNA (AUSUBEL et al. 1995 ). Enrichment for poly(A)⁺ mRNA was performed using the Invitrogen (Carlsbad, CA) Fast Track 2.0 kit following the manufacturer's protocol.

spe-12 cDNAs were isolated by probing a custom Stratagene (La Jolla, CA) UNIZAP library derived from him-5(e1490) adult-male-enriched mRNA (VARKEY et al. 1995 ; MINNITI et al. 1996 ; ACHANZAR 1997 ) with the rescuing 3.6-kb BstXI fragment of genomic clone pCS102 using the manufacturer's protocol. Differential Northern blots (containing 2 µg of separated poly(A)-enriched RNA per lane) were hybridized (AUSUBEL et al. 1995 ) with the 503-bp XhoI fragment of spe-12 cDNA cJN1201, which represents the last two-thirds of the spe-12 transcript. Stripped blots were reprobed with the complete spe-29 cDNA. The spe-29 3' untranslated region (UTR) overlaps the 3' UTR of another hermaphrodite-specific gene (J. NANCE and S. WARD, unpublished results). Detection of this message in the fem-1(hc17) lane provided a loading control.

DNA was sequenced as detailed by the manufacturer using the USB (Cleveland, OH) Sequenase reagent kit. Original cDNA sequence was initially generated by sequencing into clone cJN1201 from both ends of the insert using T3 and T7 vector primers; the remainder of the cDNA and the genomic sequence (using pCS102 as template) was generated by the primer walking technique (AUSUBEL et al. 1995 ). Both the cJN1201 cDNA and corresponding exons and intervening sequences from the genomic clone pCS102 were sequenced completely on both strands to ensure accuracy (GenBank accession no. U57624). The genomic sequence from pCS102 is identical to a region of cosmid T02E1 (which overlaps rescuing cosmid ZK260) representing the predicted gene T02E1.1, which was sequenced and analyzed by the C. elegans Genome Project (WILSON et al. 1994 ).

Identification of the spe-12 molecular lesions:
Overlapping genomic fragments, amplified by PCR from single wild-type or spe-12 worms (WILLIAMS 1995 ), were sequenced using the USB (Cleveland, OH) Sequenase PCR product sequencing kit. To confirm a lesion, new genomic DNA spanning the mutated base was amplified and sequenced. Mutations from all three spe-12 alleles were predicted to create restriction fragment length polymorphisms (RFLPs; hc76 and hc149 each introduce an ApoI site; hc152 introduces an XbaI site). The presence of these RFLPs was verified by amplifying genomic DNA flanking the predicted site from single mutant worms, digesting with the appropriate restriction enzyme, and analyzing the digested products in direct comparison to similarly prepared N2 DNA.

Antibody production and Western analysis:
The peptide acetyl-CEVKEDFERTVEDLD, representing the last 14 amino acids of the predicted spe-12 translation product (serine 241 was changed to cysteine), was synthesized (QCB, Hopkinton, MA) and coupled to maleimide-activated KLH (Pierce, Rockford, IL) via the cysteine sulfhydryl group (as detailed by the manufacturer). Conjugated carrier protein was purified and injected into rabbits (Pocono Rabbit Farm and Laboratory, Canadensis, PA) using the company's standard protocol. Polyclonal serum 12756 was produced in this manner and will be referred to as anti-SPE-12 serum.

For Western analysis, spermatid proteins were separated by SDS-PAGE (12.5% acrylamide) and electroblotted as described by the electroblotting chamber manufacturer (Hoefer Scientific Instruments, San Francisco). Integrity of samples was monitored by staining blots with Ponceau S (0.1% in 5% acetic acid) and examining protein bands. After rinsing off Ponceau S with water, blots were blocked in 5% powdered milk, 1% bovine serum albumin in Tween tris-buffered saline (TTBS) (AUSUBEL et al. 1995 ) and incubated first with a 1:1000 dilution (in blocking buffer) of anti-SPE-12 serum, and then with a 1:20,000 dilution of HRP-conjugated donkey-anti-rabbit antibody (Amersham International, Buckinghamshire, UK). Blots were washed in TTBS three times (15 min, 10 min, 5 min) after incubation with each antibody. Supersignal substrate (Pierce, Rockford, IL) was used to generate HRP-catalyzed chemiluminescence as described by the manufacturer; film was exposed to the blots for varying times and developed. MSP was detected (in addition to Ponceau S visualization) by incubating and developing insufficiently blocked blots where all common sperm proteins were visible. The protein was recognized by its distinctive low molecular weight and abundance (BURKE and WARD 1983 ).

Spermatid isolation and surface proteolysis:
Spermatids isolated from him-5(e1490) males were purified as described by L'HERNAULT and ROBERTS 1995 using the alternative method for large-scale sperm isolation. To increase yields, worms were grown on peptone-enriched plates (LEWIS and FLEMING 1995 ) and purified males were allowed to feed overnight on plates before harvesting spermatids. Spermatids were kept on ice until treated (see below).

Spermatid surface proteins were digested as described by PAVALKO and ROBERTS 1989 . Briefly, 10⁶ freshly isolated spermatids in sperm medium (SM) containing 10 mM sodium azide (sodium azide prevents the protease-initiated conversion of spermatids into spermatozoa) were treated with either 0.66 mg/ml or 6.6 µg/ml of Streptomyces griseus protease (product P-6911; Sigma, St. Louis) or buffer alone, for 5 min at room temperature. Spermatids were pelleted in a microcentrifuge, treated with 10 µl of 2x sample buffer (AUSUBEL et al. 1995 ), boiled for 5 min, respun briefly, and then loaded onto a 12.5% polyacrylamide gel. Western analysis to detect SPE-12 and MSP was performed as described above.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mating induces a partial functional bypass of SPE-12:
Hermaphrodite-derived spermatids from spe-8, spe-12, spe-27, or spe-29 mutants can become spermatozoa that yield self-progeny following mating with any male. The extent of this male-mediated activation of hermaphrodite-derived spermatids (referred to as transactivation) differs for various mutants. We compared the effectiveness of transactivation among various spe-12 and spe-8 mutants to determine if this difference is a consequence of varying levels of loss of function among the mutants (Figure 1). While all mutants exhibited an increase in self-fertility after mating, each spe-8 mutant displayed a different level of transactivation. The spe-8 mutants hc53 and hc134 produced self-broods of over 30 after mating, while the least self-fertile spe-8 mutant, hc50, averaged <5 self-progeny after transactivation. A brood of 5 after spe-8(hc50) transactivation is similar to that observed for spe-12(hc76) and spe-12(hc152) mutants. Since these spe-12 mutants are predicted to behave as nulls (see molecular characterization below), this suggests that the null phenotype of spe-8 and spe-12 mutants is similar and the more efficiently transactivated spe-8 mutants (hc53 and hc134) are hypomorphic.

View larger version (14K): In this window In a new window Download PPT slide   Figure 1. Self-fertility of virgin and mated spe-8 and spe-12 hermaphrodites. Late larval hermaphrodites (24.5°) were kept as virgins or were mated to four late larval fer-1(hc13); him-5(e1490) males. Self-progeny broods were counted. Virgin wild-type hermaphrodites produced 202 ± 12 (n = 10) progeny; when mated to fer-1(hc13); him-5(e1490) males, wild-type hermaphrodites produced 206 ± 8.3 self-progeny (n = 13). Error bars represent the SEM. Sample size is 18–20.

Differing levels of transactivation between mutants could be due to sperm defects that occur either during spermiogenesis or fertilization. Alternatively, since oocytes are produced in excess of sperm in C. elegans (WARD and CARREL 1979 ), the number of spermatids present within the mutant hermaphrodite at the time of mating could account for this level of self-fertility. We compared spermatid populations in spe-12(hc76), spe-8(hc134), and wild-type virgin hermaphrodites to determine if fewer spermatids were present in spe-12 mutants at the time of mating. Fewer sperm remained in spe-12(hc76) and spe-8(hc134) than in wild type since oocytes squeegee noncrawling spermatids into the uterus and out the vulva (Figure 2; L'HERNAULT et al. 1988 ; MINNITI et al. 1996 ). spe-8 and spe-12 mutants, however, retained equivalent numbers of spermatids. Moreover, there were roughly 25 times more spermatids present in spe-12 hermaphrodites than the average number of self-progeny observed in the transactivation assays (shown in Figure 1). Thus, poor transactivation likely reflects a property of the spermatids themselves: only about 4% of spe-12(hc76) hermaphrodite-derived spermatids and 30% of spe-8(hc134) hermaphrodite-derived spermatids proceed to fertilize oocytes after mating, whereas nearly every hermaphrodite-derived spermatozoon in wild type successfully fertilizes an oocyte (WARD and CARREL 1979 ).

View larger version (14K): In this window In a new window Download PPT slide   Figure 2. Sperm retention of wild-type, spe-8(hc134), and spe-12(hc76) hermaphrodites. Synchronized young adult virgin hermaphrodites were fixed and stained as described in MATERIALS AND METHODS. The number of eggs or oocytes that each individual laid before fixation was determined: N2 (n = 7) laid an average of 31 ± 5 eggs; spe-8(hc134) (n = 13) laid 0.1 ± 0.1 eggs, 24 ± 3 oocytes; spe-12(hc76) (n = 15) laid no eggs, 24 ± 2 oocytes. Sperm nuclei present in the spermathecae and uterus were counted. Error bars represent the SEM.

Comparison of the transactivation phenotype of spe-8 and spe-12 mutants, in addition to molecular evidence presented below, suggests that spe-12 mutants are genetically null and have no functional spe-12 gene product. Yet mutant self-spermatids can still be transactivated, indicating that mating results in a functional bypass of the spe-12 gene product. However, spe-12 transactivation is invariably poor, and this inefficiency appears to be a property of the mutant sperm themselves. Thus, after mating, only some spe-12 self-spermatids successfully activate and fertilize oocytes, implying that the functional bypass of SPE-12 is only partial and is not an adequate substitute for the wild-type gene product.

spe-12 male-derived spermatids are partially defective:
Since only a fraction of spe-12 hermaphrodite-derived spermatids can be transactivated to fertilize oocytes after mating occurs, we suspected that spe-12 male-derived spermatids might also be compromised. If mutant male-derived spermatids were impaired in their ability to activate, only a fraction of inseminated spermatids might activate, resulting in fewer outcross sperm reaching their necessary destination, the spermatheca. Such an activation impairment would result in males that are fertile yet incapable of siring the number of progeny that wild-type males can sire. Alternatively, reduced mutant male fertility could result from spermatids that activate normally after insemination, but form impaired spermatozoa that are less likely than wild type to fertilize oocytes.

We examined the competency of spe-12(hc76) male-derived sperm by comparing the fertility of wild-type, spe-8(hc134), and spe-12 males in single-male mating assays. To avoid overlooking subtle defects in spermatozoon function, we tested the ability of sperm to fertilize oocytes in increasingly competitive spermathecal environments. Wild-type and spe-12 male fertility was examined in fem-1(hc17) hermaphrodites, which make no interfering self-sperm (NELSON et al. 1978 ); fer-1(hc13) hermaphrodites, which produce defective self-sperm that cannot crawl and are swept out of the spermatheca (WARD and MIWA 1978 ; ARGON and WARD 1980 ); and spe-9(hc88) hermaphrodites, which make crawling spermatozoa that localize to the spermatheca but cannot fertilize oocytes (L'HERNAULT et al. 1988 ; SINGSON et al. 1998 ). As shown in Figure 3, spe-12 males were as fertile as wild-type or spe-8(hc134) males when mated to fem-1 (hc17) or fer-1(hc13) hermaphrodites, but spe-12 male fertility was greatly reduced in comparison to wild-type or spe-8(hc134) males when mated to spe-9(hc88) hermaphrodites.

View larger version (18K): In this window In a new window Download PPT slide   Figure 3. Fertility of wild-type, spe-8(hc134), and spe-12(hc76) males when mated to fem-1(hc17), fer-1(hc13), or spe-9(hc88) sterile hermaphrodites. Both males and hermaphrodites were synchronized and maintained as virgins until 64 ± 1 hr after being laid at 24.5°. Each hermaphrodite was mated with one male for 24 hr before hermaphrodites were removed to fresh plates. Progeny from each cross were counted; individuals that laid no progeny were judged to be unmated and were not factored into determining male fertility. Error bars represent the SEM. Sample size is 18–50.

spe-12 male-derived spermatozoa outcompete wild-type hermaphrodite-derived spermatozoa:
spe-12 male fertility is clearly compromised, but why is this effect influenced by the hermaphrodite genotype? spe-9 hermaphrodites were the only mutants used in the single-male mating assays that produce crawling spermatozoa. Could spe-9 hermaphrodite-derived spermatozoa interfere directly with the success of spe-12 male-derived spermatozoa, resulting in lower fertility? Normally, wild-type male-derived spermatozoa take nearly complete precedence over hermaphrodite-derived spermatozoa and sire a disproportionate number of progeny (WARD and CARREL 1979 ). If spe-12 male-derived spermatozoa were abnormal after activation so that they were unable to compete with hermaphrodite-derived spermatozoa and were lost, male fertility would be lower. To test the competitiveness of spe-12(hc76) male-derived sperm, we placed them in competition with wild-type hermaphrodite-derived sperm (Figure 4). Control wild-type male-derived spermatozoa clearly outcompeted hermaphrodite-derived spermatozoa, siring nearly 100% of the progeny. spe-12 male-derived spermatozoa also took precedence over hermaphrodite-derived spermatozoa since their outcross progeny were produced in a burst following mating. spe-12 male-derived spermatozoa failed to take complete precedence presumably because so few total outcross progeny were sired (mean = 19; SEM = 2.7) when compared to wild-type males (mean = 175; SEM = 7.3). Similar incomplete precedence has been observed in wild type when males transfer few sperm (S. WARD, unpublished observation). It is likely that a minimum number of male-derived spermatozoa must reach each spermatheca before taking complete precedence, as is the case in the closely related hermaphroditic nematode C. briggsae, where about 50 male-derived spermatozoa are required per spermatheca for this to occur (LAMUNYON and WARD 1997 ). These results again demonstrate the lack of fertility of spe-12 males but show that once spermatozoa form, they are normal and outcompete hermaphrodite-derived spermatozoa like wild type.

View larger version (20K): In this window In a new window Download PPT slide   Figure 4. Competitiveness of wild-type or spe-12 male-derived spermatozoa vs. wild-type hermaphrodite-derived spermatozoa. Three males and one unc-24 dpy-20 hermaphrodite, all synchronized to the adult molt ± 2 hr (24.5°), were allowed to mate for 12 hr. Hermaphrodites were transferred twice daily until their death or until no more progeny were laid. The number of self (Unc Dpy) and outcross (wild-type) progeny laid in each interval was determined; the percentage of progeny that were outcross is shown. Error bars represent the SEM; where not visible, error was smaller than the symbol. Sample size is 10 (wild type) or 20 (spe-12).

Since spe-12 male-derived spermatozoa compete effectively, the sterilizing effect of hermaphrodite-derived sperm on spe-12 male-derived sperm must take place prior to activation and is likely indirect. One indirect effect of sperm in the spermathecae is a stimulation of oocyte maturation (WARD and CARREL 1979 ; J. MCCARTER, B. BARTLETT, T. DANG and T. SCHEDL, personal communication). Thus spe-9 hermaphrodites might be expected to lay more oocytes in the mating interval than either fer-1 or fem-1 hermaphrodites since they retain sperm. This is indeed the case. During the 64- to 88-hr (24.5°) developmental window, which corresponds to the age of hermaphrodites when mated in the single-male mating assays, spe-9 hermaphrodites laid more oocytes (125 ± 8; n = 22) than fer-1 hermaphrodites (86 ± 9; n = 22) and many more than fem-1 hermaphrodites (15 ± 2; n = 29). Male-derived sperm are deposited into the hermaphrodite as immotile spermatids that can be expelled out of the vulva by eggs or oocytes as they are laid (BARKER 1994 ). If spe-12 male-derived spermatids activated too slowly after deposition in the hermaphrodite, they would be swept out before they could crawl, especially within a hermaphrodite that laid oocytes at a high rate. Thus, an activation defect in spe-12 male-derived spermatids could contribute to their poor success following mating.

spe-12 encodes a predicted novel transmembrane protein:
To determine the nature of the spe-12 gene product, we identified and sequenced the spe-12 gene. Prior genetic mapping placed spe-12 very near lin-10 on LGI (L'HERNAULT et al. 1988 ). Genetic transformation of spe-12 hermaphrodites with genomic DNA from this region identified ZK260 as a cosmid that restored self-fertility to mutant hermaphrodites. Both a subcloned 7.1-kb SacI fragment (pCS102) and a 3.6-kb BstXI fragment contained within this subclone rescued spe-12 hermaphrodites. One line of transgenic spe-12(hc76) hermaphrodites (hcEx052), transformed with pCS102 and the dominant roller transformation marker pRF4, produced 177 ± 11 (n = 11) self-progeny at 20°; untransformed virgin animals rarely produced any self-progeny, while wild type produced 257 ± 14 (n = 10) self-progeny. The presence of the transgenic array in the germ line was essential for rescue since we always observed rollers in broods from transgenic spe-12; hcEx052 hermaphrodites, even if the transgenic parent was not obviously rolling (n = 36 rollers, n = 9 nonrollers). Consistent with observed inheritance patterns of transgenic arrays (MELLO et al. 1991 ), some roller hermaphrodites were half-sterile, producing eggs in one-half of the bilobed reproductive tract and oocytes in the other (2 of 38), while other rollers were completely sterile (2 of 38); these animals were presumed to be mosaics that were missing the array in half or all of the germ line, respectively. The hcEx052 transgenic array also restores self-fertility to hc149 and hc152 hermaphrodites, confirming that members of the spe-12 complementation group are allelic (data not shown).

To identify the gene responsible for the rescuing activity, we probed a phage cDNA library synthesized from him-5(e1490) adult male-enriched RNA with the rescuing 3.6-kb BstXI fragment. Screening 5 x 10⁵ plaques, we identified eight isogenic clones; the longest, cJN1201 (839 bp), encodes a full-length cDNA as judged by the presence of an initiator methionine preceded by an inframe stop codon near the insert 5' end and a poly(A) tail at the insert 3' end. Alignment of the cJN1201 cDNA and pCS102 genomic sequences revealed a single gene containing five introns (Figure 5A and Figure B). The full-length cDNA encodes a predicted 255-amino-acid translation product that is novel. While no regions of the protein are similar to known proteins or functional domains, the presence of a putative N-terminal signal sequence and a potential membrane-spanning domain near the C terminus indicate that SPE-12 is likely an integral membrane protein (Figure 5A).

View larger version (44K): In this window In a new window Download PPT slide   Figure 5. The spe-12 locus and mutations. (A) Genomic sequence of the spe-12 gene from the first base of the longest cDNA isolated, cJN1201, to the site of polyadenylation. 5' and 3' untranslated regions and introns are in lowercase, predicted translated regions are in caps. Amino acids of the putative signal sequence and proposed membrane-spanning region are underlined. These regions were strongly predicted using the SignalP (NIELSEN et al. 1997 ) and TMpred (HOFMANN and STOFFEL 1993 ) database comparison programs. Bases mutated in the various spe-12 alleles are in boldface. (B) Graphical representation of spe-12. Exons are represented by boxes, introns and untranslated regions by thin lines. The translational start site is marked as ATG. Molecular lesions associated with individual spe-12 alleles are indicated. The regions encoding the predicted signal sequence and putative transmembrane domain are hatched.

We compared the sequences of mutant and wild-type cDNA and genomic PCR fragments to identify the molecular lesions associated with spe-12 (Figure 5B). The hc76 and hc152 alleles each contain nonsense codons that are predicted to terminate translation upstream of the transmembrane domain and C terminus. hc149 harbors a change in a conserved 5' intronic splice site. To confirm that this lesion alters the normal splicing pattern, we examined hc149 transcript lengths by RT-PCR. spe-12(hc149) cDNAs were smaller than those from the other alleles and wild type and lacked exon III; this changes the downstream reading frame, resulting in a premature stop codon that prevents translation of the transmembrane domain and C terminus (data not shown). Since all three alleles are predicted to make truncated proteins that lack the transmembrane domain, mutant SPE-12, if synthesized, would likely be mislocalized or topologically altered. In addition, mutant hermaphrodites are equally sterile as homozygotes and trans-heterozygotes in all allelic combinations and show no additional abnormalities when in trans to the underlying deficiencies nDf24 or nDf25 (L'HERNAULT et al. 1988 and data not shown). These results suggest that all spe-12 mutants are genetically null.

spe-12 is expressed in the sperm-producing germ line:
We and others have failed to detect any differences between spe-12 and wild-type worms except in the sperm (L'HERNAULT et al. 1988 ; SHAKES and WARD 1989B ), suggesting that the spe-12 gene product is essential only in the sperm-producing germ line. To determine if spe-12 is expressed in the sperm and their germ-line precursors, we looked for spe-12 mRNA by differential Northern analysis. mRNA from worms with a male soma and sperm-producing germ line [him5(e1490) males], a hermaphrodite soma and oocyte-producing germ line [fem-1(hc17)], or a hermaphrodite soma and sperm-producing germ line [fem-3(q23gf)] was probed with the spe-12 cDNA cJN1201. A 1.0-kb transcript was evident only in worms containing a sperm-producing germ line and conspicuously absent in worms containing an oocyte-producing germ line (Figure 6A; compare him-5 and fem-3gf lanes to fem-1); fem-1 mRNA was loaded properly and remained intact since a control hermaphrodite-specific message was readily detected (Figure 6B, fem-1 and fem-3gf lanes). Thus, spe-12 is expressed in the sperm-producing germ line in adult animals, as expected.

View larger version (20K): In this window In a new window Download PPT slide   Figure 6. Differential Northern analysis of the spe-12 transcript. Poly-A+ RNA from purified him-5(e1490) males (male soma, sperm-producing germ line), fem-1(hc17) hermaphrodites (hermaphrodite soma, oocyte-producing germ line), and fem-3(q23gf) hermaphrodites (hermaphrodite soma, sperm-producing germ line) was probed with (A) spe-12 cDNA. (B) Blots were stripped and hybridized with a hermaphrodite-specific probe as a loading control for lanes containing hermaphrodites (see MATERIALS AND METHODS).

SPE-12 is localized to the spermatid plasma membrane:
As a tool to determine the localization of the SPE-12 protein, we raised polyclonal antibodies against a C-terminal spe-12 peptide representing the last 14 amino acids of the protein. By Western analysis, we were able to detect a protein of the predicted size of SPE-12 (~35 kD) present in purified him-5 spermatids only when probing with anti-SPE-12 immune serum; preimmune serum failed to recognize this protein (data not shown). To determine if this protein was SPE-12, we compared protein from similarly prepared spe-12(hc76) spermatids and him-5 spermatids by Western analysis, probing with anti-SPE-12 immune serum (Figure 7A). Although Ponceau S staining of the blot prior to incubation with antibody revealed that the spe-12 sperm protein samples were intact and levels MSP were similar between samples (Figure 7A, bottom), we detected an antigenic band only in the him-5 sperm sample. Since spe-12(hc76) mutants have an early nonsense codon predicted to prevent translation of the antigenic C-terminal region, the truncated mutant protein, even if stable, should not be recognized by anti-SPE-12 serum.

View larger version (53K): In this window In a new window Download PPT slide   Figure 7. Localization of SPE-12. (A) Protein from 3.6 x 106 spermatids purified from him-5(e1490) or spe-12(hc76) males was separated and immunoblotted with anti-SPE-12 antibodies. As a loading control, blots were stripped and MSP was detected (shown at bottom). (B) 1.0 x 106 intact spermatids from purified males were treated as indicated. Spermatids in lane 3 were incubated in a 1:100 dilution of the protease used for samples in lane 2. Sodium azide was added to prevent cells from activating. Western analysis was performed using anti-SPE-12 antibodies. As a control to demonstrate the inability of protease to penetrate the spermatid plasma membrane, blots were stripped and cytoplasmic MSP was detected (shown at bottom). See MATERIALS AND METHODS for details.

Since SPE-12 contains a predicted internal transmembrane domain, we wondered if it was indeed inserted into a spermatid membrane. To determine if SPE-12 is present in the spermatid plasma membrane, we purified intact him-5 spermatids, digested away surface proteins with proteases, and analyzed remaining proteins by Western analysis (Figure 7B). Though most proteins including the cytosolic major sperm protein appeared similar in intensity in both treated and untreated spermatid samples (Figure 7B, bottom), full-size SPE-12 almost completely disappeared in surface-digested spermatids (Figure 7B, compare lane 2 to lane 1). SPE-12 was not degraded when spermatids were treated with diluted (1:100) protease, indicating that the protein is not simply unstable (Figure 7B, lane 3). A smaller fragment (~25 kD) of widely varying intensity is often visible only in digested spermatid samples (faintly visible in lane 2); since this band is never as robust as the full-length SPE-12 band, we believe this represents an unstable degradation intermediate. Interestingly, this band is significantly larger than would be expected if SPE-12 were cleaved at or near the transmembrane domain. Perhaps all degradation products except this higher molecular weight intermediate are destroyed before samples are processed entirely.

These experiments strongly suggest that within spermatids, SPE-12 is predominantly localized to the plasma membrane. Residual full-size SPE-12 seen in the digested samples is not likely due to incomplete digestion, since longer protease incubation times did not diminish its strength (data not shown). Although there could be a small protected pool of protein within spermatids, we feel it is more likely that this faint band is due to SPE-12 within spermatocytes (which comprise <5% of purified spermatid samples), where the protein is probably synthesized.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Spe-12 phenotype and spermiogenesis initiation:
The Spe-12 phenotype is common to a set of genes that, when mutated, compromise the ability of spermatids to initiate spermiogenesis, or activate. Rather than complete this final differentiation, spe-8, spe-12, spe-27, or spe-29 hermaphrodite-derived spermatids arrest their development and remain impotent. Previous studies established that once a hermaphrodite is mated, male- and hermaphrodite-derived mutant spermatids can activate and form functional spermatozoa (L'HERNAULT et al. 1988 ; SHAKES and WARD 1989A ). Therefore, these mutant spermatids have the components necessary for the morphological rearrangements that occur during spermiogenesis, but lack some constituent(s) required for activation in the absence of mating.

Our experiments demonstrate that transactivation of spe-12 hermaphrodite-derived spermatids by male seminal fluid is never efficient, since the number of spermatids present within a mutant virgin hermaphrodite (~100) greatly exceeds the number of self-progeny that are produced after mating (~4). Genetically, all spe-12 mutants appear to be null so it is likely that inefficient transactivation, rather than no transactivation, is the null phenotype. These observations suggest that spe-12 hermaphrodite-derived spermatids are partly compromised in their ability to activate after mating takes place. This partial incapacitation is also observed in spe-12 males, which exhibit defects in fertility that are best explained by a similar defective-activation phenotype.

We propose a model for spermatid activation in which SPE-12, perhaps in conjunction with SPE-8, SPE-27, and SPE-29, functions in reception or transduction of a spermiogenesis initiation signal, or "activator," that originates from either the hermaphrodite or male somatic gonad. spe-12 mutations would prevent effective signaling if spermatids were exposed only to the hermaphrodite activator (explaining virgin hermaphrodite sterility) and would compromise signaling if spermatids were exposed to the male activator (explaining inefficient transactivation and partial male sterility). SPE-12 localization to the sperm cell surface suggests that it is not an extrinsic activator, but more likely a receptor or a member of the reception pathway.

Though neither the hermaphrodite nor male activators have been identified, there is evidence for their existence. When MINNITI et al. 1996 successfully artificially inseminated hermaphrodites with washed wild-type male-derived spermatids, they demonstrated that hermaphrodites contain an activation signal; since outcross progeny were produced, the artificially inseminated spermatids must have received a signal once inside the hermaphrodite to initiate and complete spermiogenesis. Inseminations with spe-27 male-derived spermatids repeatedly failed to yield progeny, consistent with these mutant sperm being incapable of responding to hermaphrodite activator. Male activator, likely a component of the seminal fluid as is the case in the parasitic nematode Ascaris suum (SEPSENWOL and TAFT 1990 ), represents the activity responsible for transactivating spe-12 hermaphrodite-derived spermatids.

Several observations described in this study conflict with a prior model of spermiogenesis initiation proposed by SHAKES and WARD 1989A . In this initial model, spermatids from either sex could utilize one of two pathways to begin activation. In one pathway, hermaphrodite activator would signal spermatids to begin activation. SPE-8, SPE-12, SPE-27, and SPE-29 would be necessary components of this pathway, so mutant spermatids could not activate when exposed only to hermaphrodite activator. A distinct male-supplied activator would signal spermatids to begin activation via an independent pathway. SPE-8, SPE-12, SPE-27, and SPE-29 would not be required for response to male activator, explaining why mutant spermatids could be transactivated and why males were fertile. These two functionally redundant pathways would differ only in the signals that initiated them and in at least some of the components (SPE-8, SPE-12, SPE-27, and SPE-29) required for their execution. Our observations that spe-12 hermaphrodite- and male-derived spermatids activate inefficiently after mating occurs demonstrate that SPE-12 functions irrespective of the source of the activation signal and is therefore not restricted to the hermaphrodite pathway. Moreover, these observations eliminate the requirement for two separate activators and two distinct pathways. The Spe-12 phenotype could be explained if male and hermaphrodite activators were chemically identical yet male activator were more potent due to higher concentration or longer duration of exposure to spermatids. In this revised model, hermaphrodite activator would be too weak to activate spe-12 spermatids, but male activator would be effective enough to accomplish this at least some of the time.

Biologically, a stronger male-supplied activation signal might be expected. Male-derived spermatids must activate quickly since eggs can expel immotile freshly inseminated spermatids out of the vulva (BARKER 1994 ). On the contrary, most hermaphrodite-derived sperm are synthesized before there are oocytes present in the reproductive tract that might displace them. The urgency to activate hermaphrodite-derived spermatids is likely not as pressing as it is with inseminated male-derived spermatids. The peculiar effects we observed that hermaphrodite genotype plays on mutant male fertility could be explained by the time constraint placed on male-derived spermatid activation. spe-12 males were significantly less fertile than wild-type males when mated to spe-9 hermaphrodites, which laid the most oocytes during the mating interval. These oocytes could easily expel most freshly inseminated spermatids that have not yet formed the pseudopod that allows them to crawl upstream to the spermathecae. Perhaps spe-12 spermatids activate more slowly than wild type, spending more time in this vulnerable state and increasing their chances of expulsion. spe-12 males were nearly as fertile as wild-type males when mated to hermaphrodites that laid very few oocytes (fem-1) in the mating interval, supporting this hypothesis, but were not significantly less fertile when mated to hermaphrodites that laid intermediate numbers of oocytes (fer-1), contrary to what might be expected. This could be explained if the average time between oocyte expulsions (out of the vulva) was shorter than the amount of time required by male-derived spermatids to initiate and complete spermiogenesis only in spe-9 hermaphrodites and not in fem-1 and fer-1 hermaphrodites.

Alternatively, direct interaction between hermaphrodite-derived spermatozoa and spe-12 male-derived spermatozoa could lower fertility. Our observation that spe-12 male-derived spermatozoa outcompete wild-type hermaphrodite-derived spermatozoa makes this possibility unlikely. Their competitiveness also suggests that, once activated, spe-12 spermatozoa function normally.

Possible roles of SPE-12 in a spermatid signaling pathway:
In vitro, spermatids can be activated by increasing the intracellular pH or by digesting the surface of the cells with proteases. In the parasitic nematode A. suum, pH is tightly controlled during spermatogenesis and has been demonstrated to directly affect the assembly state of MSP (KING et al. 1994 ). Activation of Ascaris spermatids results in a rise in intracellular pH. This increase is followed by the assembly of the MSP cytoskeleton. It is likely that pH regulation is a key step in activation of C. elegans spermatids as well, since increasing the intracellular pH of spermatids with triethanolamine (TEA) results in their activation and conversion to functional spermatozoa (WARD et al. 1983 ; LAMUNYON and WARD 1994 ). Functional SPE-12 is not required for activation of spermatids in TEA (SHAKES and WARD 1989B ), so a pH increase, if a normal feature of spermiogenesis, occurs after initiation has begun. Perhaps a rise in pH, as simulated by TEA treatment, is an endpoint of the spermiogenesis initiation signaling pathway. When treated with proteases, however, spe-12 (and spe-8, spe-27, spe-29) spermatids arrest after extending immotile spiky projections while wild-type spermatids form crawling spermatozoa (SHAKES and WARD 1989A ). In vitro protease activation could have little relevance to normal mechanisms of spermatid activation, but it is clearly abnormal when spe-12 is mutated. Since SPE-12 is positioned on the cell surface, perhaps it is a direct target of an activating protease in vivo. Such a protease-dependent activation of cell-surface receptors has been documented; the human G-protein-coupled receptors PAR1, PAR-2, and PAR-3 are activated only after specific extracellular proteases cleave away the inhibitory amino terminus, allowing a region of the receptor itself to act as a tethered ligand in cis (reviewed by MACEY 1998 ; OLIVIER et al. 1998 ). A simple test of this hypothesis would be to examine spermatozoa that were activated in vivo for any changes in SPE-12 molecular weight that might indicate a proteolytic cleavage. Unfortunately, anti-SPE-12 serum is not sufficiently sensitive to detect the protein on Western blots unless purified male-derived spermatid proteins are probed, so we currently cannot examine the state of the protein in in vivo-activated spermatozoa, which cannot be purified in bulk.

Though it shares no sequence similarity with known receptors, SPE-12 resembles many receptor types, such as the receptor tyrosine kinases (reviewed in WHITE 1991 ) or the cytokine receptors (reviewed in IHLE 1995 ), both in its topology and its cell-surface localization. However, SPE-12 could not function as the sole receptor of all activation signals since null mutant spermatids can still activate, though inefficiently, after mating occurs. SPE-12 could also play less glamorous roles in this signaling pathway, potentially functioning as a signal-amplifying coreceptor, much like the CD4 or CD8 proteins in T lymphocytes (reviewed in WEISS and LITTMAN 1994 ), transporting or anchoring other signaling components, or transducing the activation signal downstream from its reception. As more components of this distinctive signaling pathway are identified genetically and molecularly, we will be more likely to assign roles to the novel members, such as SPE-12, and perhaps identify the special features of a pathway that operates solely by rearranging preexisting components.

	ACKNOWLEDGMENTS

We thank E. Davis, C. LaMunyon, S. McKnight, P. Muhlrad, H. Smith, and W. Van Voorhies for reviewing the manuscript. C. LaMunyon provided insightful discussion on sperm competition. We owe additional thanks to an anonymous reviewer, whose suggested revisions improved the manuscript. Many strains used in this study were kindly provided by the Caenorhabditis elegans Genetics Center that is funded by the National Institutes of Health (NIH) Center for Research Resources. Cosmid clones were graciously provided by A. Coulson. This work was funded in part by a Howard Hughes Medical Institute grant (71109-52130) to the University of Arizona (C.S.), a predoctoral training grant to J.N. from the NIH (T32-CA09213), and an NIH grant (GM-25243) to S.W.

Manuscript received December 2, 1998; Accepted for publication January 28, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ACHANZAR, W. E., 1997  A nematode gene required for sperm vesicle fusion. J. Cell Sci. 110:1073-1081[Abstract].

ARGON, Y. and S. WARD, 1980  Caenorhabditis elegans fertilization-defective mutants with abnormal sperm. Genetics 96:413-433[Abstract/Free Full Text].

AUSUBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. G. SEIDMAN et al. (Editors), 1995 Current Protocols in Molecular Biology. John Wiley & Sons, New York.

BARKER, D. M., 1994  Copulatory plugs and paternity assurance in the nematode Caenorhabditis elegans.. Anim. Behav. 48:147-156.

BODY, S. C., 1996  Platelet activation and interactions with the microvasculature. J. Cardiovasc. Pharmacol. 27:S13-S25.

BRENNER, S., 1974  The genetics of Caenorhabditis elegans.. Genetics 77:71-94[Abstract/Free Full Text].

BURKE, D. J. and S. WARD, 1983  Identification of a large multigene family encoding the major sperm protein of Caenorhabditis elegans.. J. Mol. Biol. 171:1-29[Medline].

CHAMBERLIN, H. M. and P. W. STERNBERG, 1994  The lin-3/let-23 pathway mediates inductive signalling during male spicule development in Caenorhabditis elegans.. Development 118:297-324[Abstract].

CHURCH, D. L., K.-L. GUAN, and E. J. LAMBIE, 1995  Three genes of the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans.. Development 121:2525-2535[Abstract].

HAN, M. and P. W. STERNBERG, 1990  let-60, a gene that specifies cell fates during C. elegans vulval induction, encodes a ras protein. Cell 63:921-931[Medline].

HODGKIN, J. A., 1986  Sex determination in the nematode Caenorhabditis elegans: analysis of tra-3 suppressors and characterization of the fem genes. Genetics 114:15-52[Abstract/Free Full Text].

HODGKIN, J. A., H. R. HORVITZ, and S. BRENNER, 1979  Nondisjunction mutants of the nematode Caenorhabditis elegans.. Genetics 91:67-94[Abstract/Free Full Text].

HOFMANN, K. and W. STOFFEL, 1993  TMbase—a database of membrane spanning protein segments. Biol. Chem. Hoppe-Seyler 347:166.

HOSONO, R., K. HIRAHARA, S. KUNO, and T. KURIHARA, 1982  Mutants of Caenorhabditis elegans with dumpy and rounded head phenotype. J. Exp. Zool. 224:135-144.

IHLE, J. N., 1995  Cytokine receptor signalling. Nature 377:591-594[Medline].

KAYNE, P. S. and P. W. STERNBERG, 1995  Ras pathways in Caenorhabditis elegans.. Curr. Opin. Genet. Dev. 5:38-43[Medline].

KIMBLE, J., and S. WARD, 1988 Germ-line development and fertilization, pp. 191–214 in The Nematode Caenorhabditis elegans, edited by W. B. WOOD. Cold Spring Harbor Laboratory Press, Plainview, NY.

KING, K. L., J. ESSIG, T. M. ROBERTS, and T. S. MOERLAND, 1994  Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH. Cell Motil. Cytoskeleton 27:193-205[Medline].

KRAMER, J. M., R. P. FRENCH, E. C. PARK, and J. J. JOHNSON, 1990  The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen. Mol. Cell. Biol. 10:2081-2090[Abstract/Free Full Text].

L'HERNAULT, S. W., 1997 Spermatogenesis, pp. 271–294 in C. elegans II, edited by D. L. RIDDLE, T. BLUMENTHAL, B. J. MEYER and J. R. PRIESS. Cold Spring Harbor Laboratory Press, Plainview, NY.

L'HERNAULT, S. W., and T. M. ROBERTS, 1995 Cell biology of nematode sperm, pp. 273–302 in Caenorhabditis elegans: Modern Biological Analysis of an Organism, edited by H. F. EPSTEIN and D. C. SHAKES. Academic Press, San Diego.

L'HERNAULT, S. W., D. C. SHAKES, and S. WARD, 1988  Developmental genetics of chromosome I spermatogenesis-defective mutants in the nematode Caenorhabditis elegans.. Genetics 120:435-452[Abstract/Free Full Text].

LAMUNYON, C. W. and S. WARD, 1994  Assessing the viability of mutant and manipulated sperm by artificial insemination of Caenorhabditis elegans.. Genetics 138:689-692[Abstract].

LAMUNYON, C. W. and S. WARD, 1995  Sperm precedence in a hermaphroditic nematode (Caenorhabditis elegans) is due to competitive superiority of male sperm. Experientia 51:817-823[Medline].

LAMUNYON, C. W. and S. WARD, 1997  Increased competitiveness of nematode sperm bearing the male X chromosome. Proc. Natl. Acad. Sci. USA 94:185-189[Abstract/Free Full Text].

LEWIS, J. A., and J. T. FLEMING, 1995 Basic culture methods, pp. 3–30 in Caenorhabditis elegans: Modern Biological Analysis of an Organism, edited by H. F. EPSTEIN and D. C. SHAKES. Academic Press, San Diego.

MACEY, M. G., 1998  The protease-activated receptors and their cellular expression and function in blood-related cells. Br. J. Haematol. 101:1-9.

MELLO, C., and A. FIRE, 1995 DNA transformation, pp. 452–482 in Caenorhabditis elegans: Modern Biological Analysis of an Organism, edited by H. F. EPSTEIN and D. C. SHAKES. Academic Press, San Diego.

MELLO, C. C., J. M. KRAMER, D. STINCHCOMB, and V. AMBROS, 1991  Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10:3959-3970[Medline].

MINNITI, A. N., C. SADLER, and S. WARD, 1996  Genetic and molecular analysis of spe-27, a gene required for spermatogenesis in Caenorhabditis elegans hermaphrodites. Genetics 143:213-223[Abstract].

NELSON, G. A., K. K. LEW, and S. WARD, 1978  Intersex, a temperature-sensitive mutant of the nematode C. elegans.. Dev. Biol. 66:386-409[Medline].

NIELSEN, H., J. ENGELBRECHT, S. BRUNAK, and G. VON HEIJNE, 1997  Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1-6[Abstract/Free Full Text].

OLIVIER, D., C. U. CORVERA, M. STEINHOFF, and N. W. BUNNETT, 1998  Proteinase-activated receptors: novel mechanisms of signaling by serine proteases. Am. J. Physiol. 274:C1429-C1452.

PAVALKO, F. M. and T. M. ROBERTS, 1989  Posttranslational insertion of a membrane protein on Caenorhabditis elegans sperm occurs without de novo protein synthesis. J. Cell. Biochem. 41:57-70[Medline].

PERRIMON, N., 1994  Signalling pathways initiated by receptor protein tyrosine kinases in Drosophila. Curr. Opin. Cell Biol. 6:260-266[Medline].

ROBERTS, T. M. and M. STEWART, 1997  Nematode sperm: amoeboid movement without actin. Trends Cell Biol. 7:368-373[Medline].

SEPSENWOL, S. and S. J. TAFT, 1990  In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum.. Cell Motil. Cytoskeleton 15:99-110[Medline].

SHAKES, D. C. and S. WARD, 1989a  Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis. Dev. Biol. 134:189-200[Medline].

SHAKES, D. C. and S. WARD, 1989b  Mutations that disrupt the morphogenesis and localization of a sperm-specific organelle in Caenorhabditis elegans.. Dev. Biol. 134:307-316[Medline].

SIMON, M. A., D. D. L. BOWTELL, G. S. DODSON, T. R. LAVERTY, and G. M. RUBIN, 1991  Ras1 and a putative guanine nucleotide exchange factor performs crucial steps in signaling by the Sevenless tyrosine kinase. Cell 67:701-716[Medline].

SINGSON, A., K. B. MERCER, and S. W. L'HERNAULT, 1998  The C. elegans spe-9 gene encodes a sperm transmembrane protein that contains EGF-like repeats and is required for fertilization. Cell 93:71-79[Medline].

VARKEY, J. P., P. J. MUHLRAD, A. N. MINNITI, B. DO, and S. WARD, 1995  The Caenorhabditis elegans spe-26 gene is necessary to form spermatids and encodes a protein similar to the actin-associated proteins kelch and scruin. Genes Dev. 9:1074-1086[Abstract/Free Full Text].

WARD, S., 1986 Asymmetric localization of gene products during the development of Caenorhabditis elegans spermatozoa, pp. 55–75 in Gametogenesis and the Early Embryo. Alan R. Liss, New York.

WARD, S. and J. S. CARREL, 1979  Fertilization and sperm competition in the nematode Caenorhabditis elegans.. Dev. Biol. 73:304-321[Medline].

WARD, S. and J. MIWA, 1978  Characterization of temperature-sensitive, fertilization-defective mutants of the nematode Caenorhabditis elegans.. Genetics 88:285-303[Abstract/Free Full Text].

WARD, S., Y. ARGON, and G. A. NELSON, 1981  Sperm morphogenesis in wild-type and fertilization-defective mutants of Caenorhabditis elegans.. J. Cell Biol. 91:26-44[Abstract/Free Full Text].

WARD, S., E. HOGAN, and G. A. NELSON, 1983  The initiation of spermiogenesis in Caenorhabditis elegans.. Dev. Biol. 98:70-79[Medline].

WASSARMAN, D. A., M. THERRIEN, and G. M. RUBIN, 1995  The Ras signaling pathway in Drosophila. Curr. Opin. Genet. Dev. 5:44-50[Medline].

WEISS, A. and D. R. LITTMAN, 1994  Signal transduction by lymphocyte antigen receptors. Cell 76:263-274[Medline].

WHITE, M. F., 1991  Structure and function of tyrosine kinase receptors. J. Bioenerg. Biomembr. 23:63-82[Medline].

WILLIAMS, B. D., 1995 Genetic mapping with polymorphic sequence-tagged sites, pp. 81–96 in Caenorhabditis elegans: Modern Biological Analysis of an Organism, edited by H. F. EPSTEIN and D. C. SHAKES. Academic Press, San Diego.

WILSON, R., R. AINSCOUGH, K. ANDERSON, C. BAYNES, and M. BERKS et al., 1994  2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans.. Nature 368:32-38[Medline].

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