In large-scale genetic screens for egg-laying defective (Egl) mutants, a single temperature-sensitive allele of the gene egl-32, n155, was isolated 12. Pharmacological tests designed to characterize gene activity relative to the HSNs did not identify the cellular or anatomical basis for the egl-32 egg-laying defect. Here we present evidence that the cellular basis of the egl-32 egg-laying phenotype is a defect in sperm. We used two assays to quantify egg laying, an egg retention and an egg laying assay (Methods). In the egg retention assay, mutant egl-32 animals retain about twice as many eggs as wild-type animals (Figure 1a). In the egg-laying assay, mutant egl-32 animals lay about half as many eggs per time period as wild-type animals (Figure 1b).

Because food cues play an important role in egg laying, we were interested in determining whether egl-32 mutant animals have a normal response to food. In the absence of food wild-type animals lay fewer eggs. This is also true of egl-32 animals (Figure 1b) suggesting that the defect in egl-32 is not at the level of chemosensation or sensory processing of food cues. The one existing allele of egl-32, n155, displays a temperature-sensitive egg-laying defect at the nonpermissive temperature of 25°C. We performed a temperature shift assay to determine the critical period for egl-32 activity. The temperature sensitive period is between 24 and 48 hours after egg collection at 25°C, which corresponds to the L4 stage of development (Figure 2). This result was interesting because egg laying does not occur at this stage. However, this is the time during which vulval morphogenesis occurs and the HSNs innervate their targets. Moreover, it is the one and only time during which hermaphrodites produce sperm 7.

In transformation experiments, we identified a cosmid, C26G6, which could rescue the egl-32 phenotype. Subclones of this cosmid containing the predicted open reading frame (ORF) T08G11.2 can also rescue egl-32 (Figures 3a &3b). Several lines of evidence, however, suggest that egl-32 does not encode T08G11.2, but rather that they are interacting loci. First, when we sequenced T08G11.2 from egl-32(n155) mutants we could find no change to the genome. We extended our sequencing of this region from the previous ORF to the next ORF and still could not find a change to the genome. Second, SNP mapping was performed (data not shown), that confirmed that egl-32 is separable from T08G11.2. Third, we performed a complementation test between egl-32 and a T08G11.2 deletion allele, tm336. The trans-heterozygotes tm336/n155 retained an average of 13 eggs, significantly fewer than n155 homozygotes (p = 1.74 × 10^-14). Thus, tm336 complements egl-32, supporting the conclusion that they are separate but genetically interacting genes.

A BLAST search revealed that T08G11.2 has three homologues, B0207.11, F44F4.10 and F42G4.6 in C. elegans (Figure 3c). T08G11.2 and its three homologues have all been identified as sperm enriched transcripts 13. T08G11.2 and its closest homologue, B0207.11, have about a 10,000-fold enrichment in sperm. A similar fold enrichment is found in the sperm specific proteins (ssp-11, ssp-16, ssp-19, ssq-1, ssq-2, sss-1 and sss-2). T08G11.2 encodes a small novel protein of 282 amino acids. It contains an SH2-like domain. However, it lacks the critical arginine present in functional SH2 domains. To characterize further the biological roles of these sperm enriched proteins, we obtained deletion alleles from the C. elegans knockout consortium. tm336 contains a deletion in T08G11.2, tm322 in the ORF B0207.11, and tm332 in the ORF F44F4.10. These animals all retained far fewer eggs than wild-type animals (Figure 1a), suggesting the absence of an egg-laying defect. However, older embryos were often seen in mutant hermaphrodites, which would be consistent with an egg-laying defect. We therefore determined the egg-laying rate for tm336, the T08G11.2 mutant, to resolve this issue (Figure 1c). The data demonstrate that tm336 is, in fact, egg-laying defective based on rates of egg laying. The decreased egg retention is therefore not due to increased egg laying, but may reflect a reduced ovulation rate instead (see below).

We became interested in the possibility that sperm play an active role in egg laying when we discovered that the egl-32 interacting gene T08G11.2 and its three homologues are all highly expressed in sperm. This hypothesis is supported by the temperature shift assay, which revealed that the L4 stage of development, the stage at which hermaphrodites produce sperm, is the critical period for egl-32 activity (Figure 2). We investigated the possibility that sperm are playing an active role in egg laying by performing mating experiments. In all of these experiments, the mutation him-5 was included in the genetic background to facilitate recovery of males. Males were stained with a vital dye for a minimum of 2 hours and allowed to mate with stage-matched young adult hermaphrodites. Hermaphrodites were then examined for the presence or absence of fluorescent sperm as an indicator of the success or failure of mating. If the egl-32 egg-laying defect is due to a defect in the sperm, then we might expect that the introduction of wild-type sperm could correct this defect. Consistent with this hypothesis, the experiments revealed that the introduction of wild-type sperm, via mating, into egl-32 mutant animals rescued the egg-laying defect (Table 1). More strikingly, the introduction of mutant egl-32 sperm into wild-type animals was sufficient to induce an egg-laying defective phenotype (Table 1). The presence of males in itself did not lead to this change in egg-laying behavior, since control animals on the same plates without detectable stained sperm were not affected significantly. In addition, in control experiments with egl-32;him-5 hermaphrodites mated with egl-32;him-5 males, the hermaphrodites retained an average of 31 eggs. This increase in egg retention was not statistically significant, and demonstrates that mating per se does not rescue the egg-laying defect.

To determine the effect of egl-32 mutant sperm in the absence of endogenous hermaphroditic sperm we performed mating experiments using female fog-2 animals. These mutants make oocytes but not sperm, and so are not self-fertile 14. Consistent with our previous mating experiments, fog-2 animals mated with egl-32 males retained significantly more eggs than fog-2 females mated with wild-type males (Table 1). To confirm that the egg-laying defect is due to the inactivation of egl-32, we repeated these experiments at 20°C, a permissive temperature. At this temperature, there is no difference between females mated with egl-32 and those mated with wild-type males, consistent with the conclusion that egl-32 provides the relevant activity. Finally, we determined that the introduction of sperm cannot rescue every egg-laying defect. We tested egl-33 mutant animals by introducing wild-type sperm into egl-33 hermaphrodites (Table 1). The animals containing stained sperm did not retain fewer eggs than those not mated with wild-type males.

These mating experiments established the existence of a sperm-derived signal, dependent on egl-32 activity, which promotes egg laying. The best characterized sperm signaling molecules are the Major Sperm Proteins (MSPs), which are necessary for oocyte maturation and ovulation 11. In mutant female animals producing only oocytes, the oocytes will not mature and ovulation occurs at a very low rate. But, when MSPs are introduced, the oocytes are induced to mature and the sheath cells surrounding the ovary are stimulated to contract resulting in more frequent ovulations. Since a sperm-derived signal regulates ovulation rates, we tested whether our mutants have altered ovulation rates. We have found that the ovulation rate in egl-32(n155) mutant animals is slightly, but significantly reduced (Figure 4). The ovulation rate in tm336 mutants, in which the egl-32 interacting gene, T08G11.2 is deleted, is even more significantly reduced (Figure 4).

In our mating experiments, we found that egl-32 mutant male sperm can suppress egg laying even in the presence of wild-type hermaphrodite sperm. The question arises as to why the hermaphrodite sperm are not sufficient to promote normal egg-laying rates. One possible explanation is that egg laying is coupled to fertilization. Since male sperm out-compete hermaphrodite sperm for fertilization of oocytes, in matings with egl-32 males the defective male sperm will be preferentially used for fertilization even in the presence of wild-type hermaphrodite sperm. We were therefore interested in determining if fertilization is required for the sperm-derived signal regulating egg laying. To determine if fertilization is required we mated egl-32 hermaphrodites with fertilization-defective males. Sperm from spe-9 and spe-38 mutants are structurally normal, can crawl into the spermatheca, but are incapable of binding to and fertilizing an oocyte 1516. In these mating experiments, we found that neither spe-9 nor spe-38 mutant sperm could rescue the egl-32 defect, unlike wild-type sperm (Table 1). This result eliminates the possibility that the rescuing activity is provided either by seminal fluid or by the physical act of mating rather than by some factor in the sperm. We propose instead that fertilization is coupled to the regulation of egg-laying rates.

Interestingly, spe-9 sperm are capable of signaling to promote meiotic maturation 151617, but we find them insufficient to promote egg laying. Thus, these two sperm functions are separable genetically. Since MSPs are the active factor in promoting meiotic maturation, we asked whether a defect in MSP production or localization could also underlie the egg-laying defects of egl-32(n155) and tm336 mutants. MSP has previously been found to be present in the uterus, where it could theoretically be involved in regulating egg laying. MSP localization was visualized by immunohistochemistry (Figure 5). We found that in both mutant backgrounds, detectable levels of MSP are released and are present in the uterus, arguing against a role for MSP in regulating egg laying downstream of egl-32 and T08G11.2. Although we cannot exclude the existence of subtle differences in MSP localization or accessibility, or the existence of isoform-specific differences in these mutants, our results suggest that the egg-laying defects are not due to a loss of MSP signaling. This conclusion is consistent with the observation that regulation of meiotic maturation and of egg laying are genetically separable.

The following worm stocks used in this study were obtained from the Caenorhabditis Genetics Center (CGC): egl-32(n155) I, spe-9(hc88) I, him-5(e1490) V, fog-2(q71) V. The following deletion alleles were obtained from Dr. Shohei Mitani, National Bioresource Project for the nematode, Tokyo Women's Medical University School of Medicine, Japan: tm336 I, tm322 I, tm332 II. In addition to phenotypes reported here, tm322, the deletion allele of B0207.11, has an Unc phenotype that cosegregates with the deletion. The spe-38 I; him-5 V animals were obtained from Andrew Singson. We generated egl-32;him-5 and spe-9;him-5 by standard techniques. Worms were raised at 25°C, unless otherwise noted, and grown on EZ worm plates (E. Lambie, personal communication; Per liter: 550 mg Tris-Cl, 240 mg Tris-OH, 3.1 g Bacto Peptone, 8 mg cholesterol, 2.0 g NaCl, 200 mg streptomycin sulfate, 20 g agar).

L4 larvae were placed on plates with food and allowed to mature at 25°C overnight to become young adults. The young adults were move to individual plates that either contained or lacked food. The worms were allowed to lay eggs at 25°C for 2 hours. The number of eggs (or progeny, as sometimes was the case with the egg-laying defective animals) present on the plate after 2 hours was then counted.

L4 larvae were placed on plates with food and allowed to mature at 25°C overnight into young adults. The young adult worms were then placed on an agar pad on a glass slide in a drop of 10 mM sodium azide. The worms were then viewed under a 40 × objective lens (Zeiss Axioskop). The number of eggs retained in the uterus was counted.

L4 larvae were placed on plates with food and allowed to mature at 25°C overnight into young adults. Worms were then placed in a small drop of OP50 on an agar pad. The number of eggs/oocytes contained in the uterus and spermathecae were counted. The worms were then transferred to individual plates containing food and allowed to lay eggs at 25°C for 5 hours. The worms were then again placed on a slide with an agar pad in a small drop of OP50 and the eggs retained were again counted. The number of eggs present on the plate was also counted. The ovulation rate was determined by taking the number of eggs retained originally and subtracting it from the number of eggs retained after 5 hours plus the number of eggs laid during the 5 hours. The average number of eggs/oocytes ovulated in 1 hour and per gonad arm was then calculated.

Eggs were placed on a plate and allowed to hatch at either 15°C or 25°C. At various times after hatching the worms where shifted to a higher or lower temperature 22; the worms that hatched at 25°C were shifted down to 15°C; the worms hatched at 15°C were shifted up to 25°C. The number of eggs retained in the young adults was then counted as above. 10 – 30 animals were scored at each time point. Two hours of development at 15°C was considered equivalent to one hour of development at 25°C.

The following clones and subclones were injected, along with rol-6 as a marker gene, in to the gonad of egl-32(n155) animals: C45D10, C26G6, F53B6, H14D01, F32H2, W06D4, F36F2, W10D5, ZK858, H07C08, H32K16, F25H5, C45G3, H07C08 and subclones of C26G6. We screened for rolling animals that were no longer egg-laying defective.

All animals used in mating experiments included the him-5 mutation in the genetic background. The hermaphrodites, or female fog-2 animals, used in the mating experiments were selected as L4 larvae were allowed to mature overnight at 25°C. The males used in the mating experiments were stained with SYTO-17 23 as follows:

Worms were washed off plates with M9 solution and transferred to a 1.5 ml microcentrifuge tube. The worms were pelleted by microcentrifugation. The supernatant was removed. The dye was prepared by taking 3 μl of 5 mM stock solution into 1497 μl M9 to make a 10 μM solution. (occasionally a solution as concentrated as 100 μM was used. This was done only when the dye was old and appeared to be losing its ability to fluoresce strongly). 500 μl of dye was added to the worm pellet. 250 μl of dyed worms were transferred to two clean dry plates. The plates were wrapped in aluminum foil and places at 25°C for a minimum of 2 hours. To remove the dye solution the worms were pelleted in a fresh 1.5 ml microcentrifuge tube. The supernatant was removed. The worms were resuspended in a few drops of M9 and transferred to a fresh plate for recovery. Once the plates were dry stained males were picked to plates containing the young adult hermaphrodites and allowed to mate for a minimum of 2 hours. The hermaphrodites were then transferred to an agar pad on a microscope slide and placed in a drop of 10 mM sodium azide. The number of eggs retain in the uterus was then counted under a 40 × objective lens.

Immunohistochemistry using anti-MSP monoclonal antibodies was performed as described previously 17.