BACKGROUND
egg laying in c. elegans is a carefully monitored process.
genetic studies of egg-laying defective  mutants have revealed significant insight into the neuronal, developmental, and environmental control of egg laying behavior  <cit> .
egg laying is biphasic, alternating between an active phase when eggs are laid, and an inactive phase  <cit> .
the hormone serotonin is necessary to initiate the active phase of egg laying.
serotonin is produced by the hermaphrodite specific neurons   <cit> .
hermaphrodites integrate many internal and external cues in deciding whether or not to enter the active phase.
wild-type hermaphrodites under laboratory conditions release embryos from the uterus at a similar stage in their development.
retaining fertilized eggs for too long endangers the mother's life, as larvae that hatch internally will consume and destroy the mother.
there are circumstances, however, such as in the absence of food, where egg retention may be desirable, as this increases the odds that the progeny will be laid in a favorable environment.
in addition, when eggs are retained, they eventually hatch internally and devour the hermaphrodite's body before escaping the cuticle, allowing maternal resources to be sacrificed to the progeny.
accordingly, in the absence of food, worms enter the active phase less frequently, and animals begin to bloat with eggs  <cit> .
neurotransmitters that inhibit egg laying are also known.
for example, the endocrine uv <dig> cells produce tyramine which acts to inhibit egg laying  <cit> .
this inhibitory pathway may allow egg-laying behavior to respond to the internal cue of the presence of fertilized eggs in the uterus  <cit> .
in studies of the egg-laying defective mutant egl- <dig>  we have now uncovered another internal cue, the presence of sperm, which stimulates egg laying.
c. elegans hermaphrodites produce sperm during the fourth larval  stage  <cit> .
the sperm are amoeboid not flagellated, and they are stored in the spermathecae.
when the worms undergo their final molt to become adults they switch to producing exclusively oocytes and will do so for the remainder of their lives.
as the oocytes mature they are ovulated and pass though the spermathecae where they come in contact with sperm.
the oocytes are fertilized, pass into the uterus, and are eventually laid.
unlike in humans, almost all of the sperm in the hermaphrodite are used to fertilize oocytes  <cit> .
the number of sperm limits the number of potential offspring a self-fertilizing hermaphrodite is capable of producing, while matings with males will increase the number of progeny  <cit> .
males produce exclusively sperm beginning in the l <dig> stage and throughout their adult lives.
if a male is allowed to mate with a hermaphrodite his sperm will crawl into the spermatheca where they will be preferentially used to fertilize the oocytes <cit> .
previous work has revealed that c. elegans sperm release factors that stimulate oocyte maturation and ovulation  <cit> .
here we describe evidence that sperm also promote egg laying, the release of embryos from the uterus.
RESULTS
characterization of the egg-laying defects of egl- <dig> mutants
in large-scale genetic screens for egg-laying defective  mutants, a single temperature-sensitive allele of the gene egl- <dig>  n <dig>  was isolated  <cit> .
pharmacological tests designed to characterize gene activity relative to the hsns did not identify the cellular or anatomical basis for the egl- <dig> egg-laying defect.
here we present evidence that the cellular basis of the egl- <dig> egg-laying phenotype is a defect in sperm.
we used two assays to quantify egg laying, an egg retention and an egg laying assay .
in the egg retention assay, mutant egl- <dig> animals retain about twice as many eggs as wild-type animals .
in the egg-laying assay, mutant egl- <dig> animals lay about half as many eggs per time period as wild-type animals .
because food cues play an important role in egg laying, we were interested in determining whether egl- <dig> 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- <dig> animals  suggesting that the defect in egl- <dig> is not at the level of chemosensation or sensory processing of food cues.
the one existing allele of egl- <dig>  n <dig>  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- <dig> activity.
the temperature sensitive period is between  <dig> and  <dig> hours after egg collection at 25°c, which corresponds to the l <dig> stage of development .
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  <cit> .
egl- <dig> interacts with t08g <dig>
in transformation experiments, we identified a cosmid, c26g <dig>  which could rescue the egl- <dig> phenotype.
subclones of this cosmid containing the predicted open reading frame  t08g <dig>  can also rescue egl- <dig> .
several lines of evidence, however, suggest that egl- <dig> does not encode t08g <dig> , but rather that they are interacting loci.
first, when we sequenced t08g <dig>  from egl- <dig> 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 , that confirmed that egl- <dig> is separable from t08g <dig> .
third, we performed a complementation test between egl- <dig> and a t08g <dig>  deletion allele, tm <dig>  the trans-heterozygotes tm336/n <dig> retained an average of  <dig> eggs, significantly fewer than n <dig> homozygotes .
thus, tm <dig> complements egl- <dig>  supporting the conclusion that they are separate but genetically interacting genes.
a blast search revealed that t08g <dig>  has three homologues, b <dig> , f44f <dig>  and f42g <dig>  in c. elegans .
t08g <dig>  and its three homologues have all been identified as sperm enriched transcripts  <cit> .
t08g <dig>  and its closest homologue, b <dig> , have about a  <dig> -fold enrichment in sperm.
a similar fold enrichment is found in the sperm specific proteins .
t08g <dig>  encodes a small novel protein of  <dig> amino acids.
it contains an sh2-like domain.
however, it lacks the critical arginine present in functional sh <dig> domains.
to characterize further the biological roles of these sperm enriched proteins, we obtained deletion alleles from the c. elegans knockout consortium.
tm <dig> contains a deletion in t08g <dig> , tm <dig> in the orf b <dig> , and tm <dig> in the orf f44f <dig> .
these animals all retained far fewer eggs than wild-type animals , 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 tm <dig>  the t08g <dig>  mutant, to resolve this issue .
the data demonstrate that tm <dig> 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 .
mating experiments
we became interested in the possibility that sperm play an active role in egg laying when we discovered that the egl- <dig> interacting gene t08g <dig>  and its three homologues are all highly expressed in sperm.
this hypothesis is supported by the temperature shift assay, which revealed that the l <dig> stage of development, the stage at which hermaphrodites produce sperm, is the critical period for egl- <dig> activity .
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- <dig> was included in the genetic background to facilitate recovery of males.
males were stained with a vital dye for a minimum of  <dig> 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- <dig> 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- <dig> mutant animals rescued the egg-laying defect .
more strikingly, the introduction of mutant egl- <dig> sperm into wild-type animals was sufficient to induce an egg-laying defective phenotype .
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- <dig> hermaphrodites mated with egl-32;him- <dig> males, the hermaphrodites retained an average of  <dig> eggs.
this increase in egg retention was not statistically significant, and demonstrates that mating per se does not rescue the egg-laying defect.
*statistically significant difference between mated and control animals, p <  <dig> .
all experiments are performed at 25°c, unless otherwise indicated.
data ranges give standard deviation.
to determine the effect of egl- <dig> mutant sperm in the absence of endogenous hermaphroditic sperm we performed mating experiments using female fog- <dig> animals.
these mutants make oocytes but not sperm, and so are not self-fertile  <cit> .
consistent with our previous mating experiments, fog- <dig> animals mated with egl- <dig> males retained significantly more eggs than fog- <dig> females mated with wild-type males .
to confirm that the egg-laying defect is due to the inactivation of egl- <dig>  we repeated these experiments at 20°c, a permissive temperature.
at this temperature, there is no difference between females mated with egl- <dig> and those mated with wild-type males, consistent with the conclusion that egl- <dig> provides the relevant activity.
finally, we determined that the introduction of sperm cannot rescue every egg-laying defect.
we tested egl- <dig> mutant animals by introducing wild-type sperm into egl- <dig> hermaphrodites .
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- <dig> activity, which promotes egg laying.
the best characterized sperm signaling molecules are the major sperm proteins , which are necessary for oocyte maturation and ovulation  <cit> .
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- <dig> mutant animals is slightly, but significantly reduced .
the ovulation rate in tm <dig> mutants, in which the egl- <dig> interacting gene, t08g <dig>  is deleted, is even more significantly reduced .
fertilization is required for egl- <dig> activity
in our mating experiments, we found that egl- <dig> 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- <dig> 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- <dig> hermaphrodites with fertilization-defective males.
sperm from spe- <dig> and spe- <dig> mutants are structurally normal, can crawl into the spermatheca, but are incapable of binding to and fertilizing an oocyte  <cit> .
in these mating experiments, we found that neither spe- <dig> nor spe- <dig> mutant sperm could rescue the egl- <dig> defect, unlike wild-type sperm .
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.
msp localization in egg-laying defective mutants
interestingly, spe- <dig> sperm are capable of signaling to promote meiotic maturation  <cit> , 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- <dig> and tm <dig> 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 .
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- <dig> and t08g <dig> .
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.
discussion
mutant egl- <dig> hermaphrodites retain about twice as many eggs, lay about half as many eggs and ovulate at a slightly, but significantly slower rate than wild-type animals.
the reduction in egg laying rate is not due to a defect in chemosensation or sensory processing of food cues as egl- <dig> animals respond normally to food cues.
several lines of evidence suggest that the defect in egl- <dig> is due to an abnormality of the sperm.
first, the egl- <dig> gene interacts with t08g <dig> , a gene that is highly expressed in sperm, to promote proper egg laying in c. elegans.
injection of t08g <dig>  into egl- <dig> mutant animals rescues their egg-laying defective phenotypes.
second, egl- <dig> is a temperature-sensitive mutation.
temperature shift assays reveal that the l <dig> stage of development is the critical period for egl- <dig> activity.
this was an interesting finding because this is not when eggs are being laid.
however, it is the one and only time that a hermaphrodite produces sperm.
third, mating experiments reveal that wild-type sperm introduced, via mating, into egl- <dig> animals rescues their egg-laying defective phenotype.
this is not true of all egg-laying defective mutants, as the egg-laying defect of egl- <dig> could not be rescued via mating.
fourth, egl- <dig> mutant sperm are sufficient to confer the egg retention phenotype in both wild-type hermaphrodites and females.
we have also determined that fertilization of oocytes is required to mediate egl- <dig> activity.
mutant spe- <dig> and spe- <dig> sperm, which are incapable of fertilizing oocytes, were unable to reduce the number of eggs retained by mutant egl- <dig> hermaphrodites when introduced via mating.
thus, we conclude that egl- <dig> activity is required for a sperm-derived signal that promotes egg laying and acts at or after fertilization.
this signal is unlikely to be msp, since msp localization is not detectably altered in egl- <dig>  furthermore, the regulation of meiotic maturation/ovulation and egg laying are at least partially separable genetically as seen in egl- <dig> mutants, which have significant defects in egg laying but only a slightly reduced ovulation rate , and in spe- <dig> mutants, which are defective in promoting egg laying but not in promoting meiotic maturation  <cit> .
a role for seminal fluid in regulating female oocyte maturation and ovulation, among other behaviors, has been well established in drosophila  <cit> .
in particular, the seminal fluid hormone acp70a  stimulates oogenesis and acp26aa  stimulates ovulation.
similarly, factors in mammalian seminal fluid, such as tgfβ and prostaglandin, promote changes in the female to facilitate embryo survival and implantation  <cit> .
the rapid evolution of male seminal fluid proteins suggests a role in male-female sexual conflict, in which the male benefits most by increasing the quantity of offspring, while the female benefits more by selecting high quality mates  <cit> .
in c. elegans, although competition between hermaphrodite and male sperm occurs  <cit>  it might be expected that the hermaphrodite's sperm would play a limited role in regulating its own reproductive behaviors due to the lack of sexual conflict.
this, apparently, is not the case.
instead, hermaphrodite sperm actively signal to promote oocyte maturation and ovulation via the msps  <cit>  and, as shown here, to promote egg laying via an egl-32-dependent activity.
sperm signaling in the hermaphrodite may exist to ensure prudent investment of resources.
in the absence of sperm, ovulation would be an energetically wasteful behavior.
similarly, a sperm-derived signal may exist to coordinate the rate of fertilization to the rate of egg laying.
as long as sperm are present the rate of egg laying is close to the rate of ovulation resulting in a fairly constant number of eggs present in the uterus.
older, wild-type worms normally bloat with eggs.
these older hermaphrodites may become defective in egg laying because they no longer contain the internal cue present in sperm to promote egg laying.
conversely, egg laying in young adults is inhibited until the uterus becomes sufficiently filled with eggs, possibly by an inhibitory effect of the uv <dig> cells that is blocked when mechanical deformation by eggs occurs  <cit> .
it is possible that the sperm signal modifies or coordinates with the uterine-occupancy signal to effect the appropriate rate of egg laying throughout the nematode's life history.
CONCLUSIONS
our data demonstrate the existence of a novel sperm-derived signal that regulates rates of egg laying in the nematode c. elegans.
this signal is unlikely to be dependent on the known sperm signaling molecule msp.
the novel signal requires sperm fertilization of oocytes to function.

 egg laying in c. elegans is a carefully monitored process. genetic studies of egg-laying defective mutants have revealed significant insight into the neuronal, developmental, and environmental control of egg laying behavior. egg laying is biphasic, alternating between an active phase when eggs are laid, and an inactive phase. the hormone serotonin is necessary to initiate the active phase of egg laying. serotonin is produced by the hermaphrodite specific neurons. hermaphrodites integrate many internal and external cues in deciding whether or not to enter the active phase. wild-type hermaphrodites under laboratory conditions release embryos from the uterus at a similar stage in their development. retaining fertilized eggs for too long endangers the mother's life, as larvae that hatch internally will consume and destroy the mother. there are circumstances, however, such as in the absence of food, where egg retention may be desirable, as this increases the odds that the progeny will be laid in a favorable environment. in addition, when eggs are retained, they eventually hatch internally and devour the hermaphrodite's body before escaping the cuticle, allowing maternal resources to be sacrificed to the progeny. accordingly, in the absence of food, worms enter the active phase less frequently, and animals begin to bloat with eggs. neurotransmitters that inhibit egg laying are also known. for example, the endocrine uv cells produce tyramine which acts to inhibit egg laying. this inhibitory pathway may allow egg-laying behavior to respond to the internal cue of the presence of fertilized eggs in the uterus. in studies of the egg-laying defective mutant egl- we have now uncovered another internal cue, the presence of sperm, which stimulates egg laying. c. elegans hermaphrodites produce sperm during the fourth larval stage. the sperm are amoeboid not flagellated, and they are stored in the spermathecae. when the worms undergo their final molt to become adults they switch to producing exclusively oocytes and will do so for the remainder of their lives. as the oocytes mature they are ovulated and pass though the spermathecae where they come in contact with sperm. the oocytes are fertilized, pass into the uterus, and are eventually laid. unlike in humans, almost all of the sperm in the hermaphrodite are used to fertilize oocytes. the number of sperm limits the number of potential offspring a self-fertilizing hermaphrodite is capable of producing, while matings with males will increase the number of progeny. males produce exclusively sperm beginning in the l stage and throughout their adult lives. if a male is allowed to mate with a hermaphrodite his sperm will crawl into the spermatheca where they will be preferentially used to fertilize the oocytes. previous work has revealed that c. elegans sperm release factors that stimulate oocyte maturation and ovulation. here we describe evidence that sperm also promote egg laying, the release of embryos from the uterus.  characterization of the egg-laying defects of egl- mutants in large-scale genetic screens for egg-laying defective mutants, a single temperature-sensitive allele of the gene egl- n was isolated. pharmacological tests designed to characterize gene activity relative to the hsns did not identify the cellular or anatomical basis for the egl- egg-laying defect. here we present evidence that the cellular basis of the egl- egg-laying phenotype is a defect in sperm. we used two assays to quantify egg laying, an egg retention and an egg laying assay. in the egg retention assay, mutant egl- animals retain about twice as many eggs as wild-type animals. in the egg-laying assay, mutant egl- animals lay about half as many eggs per time period as wild-type animals. because food cues play an important role in egg laying, we were interested in determining whether egl- 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- animals suggesting that the defect in egl- is not at the level of chemosensation or sensory processing of food cues. the one existing allele of egl- n 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- activity. the temperature sensitive period is between and hours after egg collection at 25°c, which corresponds to the l stage of development. 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. egl- interacts with t08g in transformation experiments, we identified a cosmid, c26g which could rescue the egl- phenotype. subclones of this cosmid containing the predicted open reading frame t08g can also rescue egl-. several lines of evidence, however, suggest that egl- does not encode t08g, but rather that they are interacting loci. first, when we sequenced t08g from egl- 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, that confirmed that egl- is separable from t08g. third, we performed a complementation test between egl- and a t08g deletion allele, tm the trans-heterozygotes tm336/n retained an average of eggs, significantly fewer than n homozygotes. thus, tm complements egl- supporting the conclusion that they are separate but genetically interacting genes. a blast search revealed that t08g has three homologues, b, f44f and f42g in c. elegans. t08g and its three homologues have all been identified as sperm enriched transcripts. t08g and its closest homologue, b, have about a -fold enrichment in sperm. a similar fold enrichment is found in the sperm specific proteins. t08g encodes a small novel protein of amino acids. it contains an sh2-like domain. however, it lacks the critical arginine present in functional sh domains. to characterize further the biological roles of these sperm enriched proteins, we obtained deletion alleles from the c. elegans knockout consortium. tm contains a deletion in t08g, tm in the orf b, and tm in the orf f44f. these animals all retained far fewer eggs than wild-type animals, 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 tm the t08g mutant, to resolve this issue. the data demonstrate that tm 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. mating experiments we became interested in the possibility that sperm play an active role in egg laying when we discovered that the egl- interacting gene t08g and its three homologues are all highly expressed in sperm. this hypothesis is supported by the temperature shift assay, which revealed that the l stage of development, the stage at which hermaphrodites produce sperm, is the critical period for egl- activity. 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- was included in the genetic background to facilitate recovery of males. males were stained with a vital dye for a minimum of 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- 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- mutant animals rescued the egg-laying defect. more strikingly, the introduction of mutant egl- sperm into wild-type animals was sufficient to induce an egg-laying defective phenotype. 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- hermaphrodites mated with egl-32;him- males, the hermaphrodites retained an average of eggs. this increase in egg retention was not statistically significant, and demonstrates that mating per se does not rescue the egg-laying defect. *statistically significant difference between mated and control animals, p <. all experiments are performed at 25°c, unless otherwise indicated. data ranges give standard deviation. to determine the effect of egl- mutant sperm in the absence of endogenous hermaphroditic sperm we performed mating experiments using female fog- animals. these mutants make oocytes but not sperm, and so are not self-fertile. consistent with our previous mating experiments, fog- animals mated with egl- males retained significantly more eggs than fog- females mated with wild-type males. to confirm that the egg-laying defect is due to the inactivation of egl- we repeated these experiments at 20°c, a permissive temperature. at this temperature, there is no difference between females mated with egl- and those mated with wild-type males, consistent with the conclusion that egl- provides the relevant activity. finally, we determined that the introduction of sperm cannot rescue every egg-laying defect. we tested egl- mutant animals by introducing wild-type sperm into egl- hermaphrodites. 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- activity, which promotes egg laying. the best characterized sperm signaling molecules are the major sperm proteins, which are necessary for oocyte maturation and ovulation. 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- mutant animals is slightly, but significantly reduced. the ovulation rate in tm mutants, in which the egl- interacting gene, t08g is deleted, is even more significantly reduced. fertilization is required for egl- activity in our mating experiments, we found that egl- 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- 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- hermaphrodites with fertilization-defective males. sperm from spe- and spe- mutants are structurally normal, can crawl into the spermatheca, but are incapable of binding to and fertilizing an oocyte. in these mating experiments, we found that neither spe- nor spe- mutant sperm could rescue the egl- defect, unlike wild-type sperm. 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. msp localization in egg-laying defective mutants interestingly, spe- sperm are capable of signaling to promote meiotic maturation, 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- and tm 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. 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- and t08g. 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. discussion mutant egl- hermaphrodites retain about twice as many eggs, lay about half as many eggs and ovulate at a slightly, but significantly slower rate than wild-type animals. the reduction in egg laying rate is not due to a defect in chemosensation or sensory processing of food cues as egl- animals respond normally to food cues. several lines of evidence suggest that the defect in egl- is due to an abnormality of the sperm. first, the egl- gene interacts with t08g, a gene that is highly expressed in sperm, to promote proper egg laying in c. elegans. injection of t08g into egl- mutant animals rescues their egg-laying defective phenotypes. second, egl- is a temperature-sensitive mutation. temperature shift assays reveal that the l stage of development is the critical period for egl- activity. this was an interesting finding because this is not when eggs are being laid. however, it is the one and only time that a hermaphrodite produces sperm. third, mating experiments reveal that wild-type sperm introduced, via mating, into egl- animals rescues their egg-laying defective phenotype. this is not true of all egg-laying defective mutants, as the egg-laying defect of egl- could not be rescued via mating. fourth, egl- mutant sperm are sufficient to confer the egg retention phenotype in both wild-type hermaphrodites and females. we have also determined that fertilization of oocytes is required to mediate egl- activity. mutant spe- and spe- sperm, which are incapable of fertilizing oocytes, were unable to reduce the number of eggs retained by mutant egl- hermaphrodites when introduced via mating. thus, we conclude that egl- activity is required for a sperm-derived signal that promotes egg laying and acts at or after fertilization. this signal is unlikely to be msp, since msp localization is not detectably altered in egl- furthermore, the regulation of meiotic maturation/ovulation and egg laying are at least partially separable genetically as seen in egl- mutants, which have significant defects in egg laying but only a slightly reduced ovulation rate, and in spe- mutants, which are defective in promoting egg laying but not in promoting meiotic maturation. a role for seminal fluid in regulating female oocyte maturation and ovulation, among other behaviors, has been well established in drosophila. in particular, the seminal fluid hormone acp70a stimulates oogenesis and acp26aa stimulates ovulation. similarly, factors in mammalian seminal fluid, such as tgfβ and prostaglandin, promote changes in the female to facilitate embryo survival and implantation. the rapid evolution of male seminal fluid proteins suggests a role in male-female sexual conflict, in which the male benefits most by increasing the quantity of offspring, while the female benefits more by selecting high quality mates. in c. elegans, although competition between hermaphrodite and male sperm occurs it might be expected that the hermaphrodite's sperm would play a limited role in regulating its own reproductive behaviors due to the lack of sexual conflict. this, apparently, is not the case. instead, hermaphrodite sperm actively signal to promote oocyte maturation and ovulation via the msps and, as shown here, to promote egg laying via an egl-32-dependent activity. sperm signaling in the hermaphrodite may exist to ensure prudent investment of resources. in the absence of sperm, ovulation would be an energetically wasteful behavior. similarly, a sperm-derived signal may exist to coordinate the rate of fertilization to the rate of egg laying. as long as sperm are present the rate of egg laying is close to the rate of ovulation resulting in a fairly constant number of eggs present in the uterus. older, wild-type worms normally bloat with eggs. these older hermaphrodites may become defective in egg laying because they no longer contain the internal cue present in sperm to promote egg laying. conversely, egg laying in young adults is inhibited until the uterus becomes sufficiently filled with eggs, possibly by an inhibitory effect of the uv cells that is blocked when mechanical deformation by eggs occurs. it is possible that the sperm signal modifies or coordinates with the uterine-occupancy signal to effect the appropriate rate of egg laying throughout the nematode's life history.  our data demonstrate the existence of a novel sperm-derived signal that regulates rates of egg laying in the nematode c. elegans. this signal is unlikely to be dependent on the known sperm signaling molecule msp. the novel signal requires sperm fertilization of oocytes to function. 
previous work has revealed that c. elegans sperm release factors that stimulate oocyte maturation and ovulation. here we describe evidence that sperm also promote egg laying, the release of embryos from the uterus. we have studied the egg-laying defective mutant, egl- and found that the cellular basis of the egl- egg-laying phenotype is likely a defect in sperm. in insects and in mammals, male sperm and seminal fluid provide signaling factors that influence various aspects of female physiology and behavior to promote reproductive success and to compete with other males. most importantly, introduction of mutant egl- sperm into wild-type hermaphrodites or females is sufficient to induce an egg-laying defective phenotype. third, mating experiments have revealed that wild-type sperm can rescue the egg-laying defect of egl- mutant animals.
 in insects and in mammals, male sperm and seminal fluid provide signaling factors that influence various aspects of female physiology and behavior to promote reproductive success and to compete with other males. it is less apparent how important such signaling is in the context of a self-fertile hermaphrodite species. we have addressed this question in the nematode caenorhabditis elegans, which can reproduce either by hermaphrodite self-fertilization or by male-hermaphrodite mating.  we have studied the egg-laying defective mutant, egl- and found that the cellular basis of the egl- egg-laying phenotype is likely a defect in sperm. first, the time of egl- action coincides with the timing of spermatogenesis in the hermaphrodite. second, egl- interacts with genes expressed in sperm. third, mating experiments have revealed that wild-type sperm can rescue the egg-laying defect of egl- mutant animals. most importantly, introduction of mutant egl- sperm into wild-type hermaphrodites or females is sufficient to induce an egg-laying defective phenotype.  previous work has revealed that c. elegans sperm release factors that stimulate oocyte maturation and ovulation. here we describe evidence that sperm also promote egg laying, the release of embryos from the uterus. 