Mechanisms of TGFß in prostaglandin synthesis and sperm guidance in Caenorhabditis elegans

Abstract

Background:

The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance.

Results:

We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance.

Conclusion:

The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.

1 ∣. INTRODUCTION

Successful fertilization requires that sperm navigate the female reproductive tract and reach the oocyte.^1-4 Sperm motility has been extensively studied in aquatic animals where fertilization occurs externally.^5,6 However, less is known about the mechanisms that guide sperm to oocytes in internally fertilizing animals, especially in their native environments. Caenorhabditis elegans provide a powerful model to study sperm migration since their transparent epidermis allows visualization of sperm motility in live animals. Stained males can be mated with wild-type or mutant hermaphrodites and individual sperm can be tracked and recorded as it travels through the uterus toward the spermatheca and the ovulating oocyte.^7-11

In C elegans, sperm motility and guidance are modulated by a specific class of F-series PGs (PGFs).^12-14 PGs are lipid signaling molecules derived from C20 polyunsaturated fatty acids (PUFA). Classically, PUFAs, such as arachidonic acid, are converted into the PGH2 intermediate by Cox enzymes. Specific PG synthases then act on PGH2 to produce particular PGs, such as PGI2, PGE2, PGD2, and PGF2.^15,16 Interestingly, the C elegans genome does not encode cox-like enzymes. Unlike the wide array of PGs derived from the classical pathways, C elegans synthesize only F-series PGs, PGF1, PGF2, and PGF3, and the levels of these PGs are not affected by treatment with known Cox inhibitors.^12,13 Prior studies from our lab suggest that C elegans PG synthesis occurs mostly in the oocytes, and that PUFA precursors are transported from the intestine to the oocytes via yolk complexes. Animals that lack germline development, such as the glp-4 (bn2) mutants, or have faulty lipoprotein receptors, such as rme-2(b1008) mutants, have significantly lower levels of PGs.^13,14 Currently, the enzyme(s) responsible for C elegans PG synthesis has not been identified. However, the DAF-7 TGFß pathway has been implicated in regulating PGF metabolism.¹⁷

DAF-7 is a TGFß homolog that is secreted by ASI sensory neurons. Its expression is regulated by various environmental cues, such as food and pheromones. Similar to TGFß ligands in other organisms, DAF-7 functions as a homodimer and transmits its signals through the DAF-1 Type I and DAF-4 Type II heterotetrameric complex. Its downstream R-Smads DAF-8 and DAF-14 act to inhibit the Co-Smad DAF-3.^18-23 The involvement of the DAF-7 pathway in sperm guidance links environmental cues to sperm behavior and fertility. daf-1 mutant hermaphrodites exhibit low PG levels that correlate with the poor sperm guidance phenotype.¹⁷ It is unclear how this pathway modulates PG metabolism, but it is likely through both cell autonomous and nonautonomous mechanisms because daf-1 can act in the nervous system and germline to modulate sperm guidance.¹⁷

In this study, we first assess whether the low PG levels are responsible for the poor sperm guidance defect seen in daf-1 mutants. To this end, we injected PGF2α into the gonad of daf-1 animals and find that sperm guidance is significantly increased. We next investigate the mechanism by which daf-1 affects PG levels. in vitro assays using active worm lysates and AA suggest that daf-1 lysates can produce the same amount of PGF2α as wild-type animals, suggesting the PG synthesis machinery is being expressed in these animals. However, daf-1 fed with AA does not show changes in sperm guidance. Interestingly, injection of AA directly into the germline rescues sperm guidance in daf-1 animals. Together these data suggest that daf-1 mediates PG levels by regulating the accessibility of the AA precursor. Finally, we show that daf-3 suppresses the low PG levels seen in daf-1, and functions in the nervous system and germline to affect sperm guidance.

2 ∣. RESULTS

2.1 ∣. Reduced PG levels in daf-1 mutantscause poor sperm guidance

Our lab previously showed that the DAF-7 TGFß pathway can modulate sperm navigation in the hermaphrodite uterus.¹⁷ We recapitulated the previous findings using wildtype N2, daf-1, and daf-1;daf-3 mutant hermaphrodites. To this end, we mated fog-2(q71) males stained with a mitochondrial dye to the adult hermaphrodites. The distribution of fluorescent sperm was quantified 1 hour after mating. Sperm distribution was quantified by dividing the uterus into three zones and counting the fluorescent sperm in each zone. In wild-type animals, 80% of the sperm reached the spermatheca, where fertilization occurs. However, in daf-1 mutants, only 40% of the sperm reached the spermatheca. This sperm guidance defect was suppressed in the daf-1;daf-3 double mutants (Figure 1A, B). We previously linked the defect in sperm migration in daf-1 mutants to low levels of PGs, which induce sperm motility.^12,17 In this study, we used liquid chromatography tandem mass spectrometry operated in multiple reaction monitoring (MRM) mode to assess the level of PGs in N2, daf-1, and daf-1;daf-3 whole worm lipid extracts. F1, F2, and F3 series PGs were all decreased in daf-1 compared with wild-type (Figure 1C). Quantification of PGF2α suggested that PGF2α is reduced by 75% in daf-1 mutants, consistent with previous findings. daf-1;daf-3 double mutants showed a rescue of PG levels back to that of wild-type levels (Figure 1D), suggesting that PG metabolism, like that of the sperm guidance phenotype, was also modulated by daf-3.

FIGURE 1.

PGF2α rescues sperm guidance in daf-1 mutants. A, Sperm guidance assay of wild-type, daf-1, and daf-1;daf-3 adult hermaphrodites. Fog-2(q71) males were stained with MitoTracker CMXRos and mated to the hermaphrodites. B, Quantification of the percent of sperm accumulated in zone 3 in A. Statistics was assessed using a one-way ANOVA, P < .001. C. MRM spectrum of PGF1α (m/z 355/311, RT 11.78), PGF2α (m/z 353.2/193 RT 11.70), PGF3α (m/z 351/193, RT 11.24). N2: pink, daf-1: orange, daf-1;daf-3: blue. Asterisk denotes peak for the PG of interest based on retention times. Data are representative of three biological replicates. D. Quantification of PGF2α levels in whole worm lipid extracts. Statistics was assessed using a one-way ANOVA, P < .001. E. Percent of sperm accumulated in zone 3 of daf-1 mutant hermaphrodites injected with 100 μM PGF2α or PBS. A two sample t test was used for statistical analysis, P < .05

We next asked whether the reduced PG levels seen in daf-1 mutants were responsible for the corresponding sperm guidance defect. All three F series PGs act redundantly to affect sperm motility and migration.^12,13 To test whether the addition of PGF2α rescued the sperm guidance defect in daf-1 mutants, we injected PGF2α directly into the gonad of adult daf-1 hermaphrodites. The injected animals were allowed to rest for 2 to 3 hours before mating with stained males. Injection of PGF2α significantly increased the percent of sperm reaching the spermatheca (Figure 1E), suggesting that the PGs in C elegans were directly responsible for modulating sperm guidance.

2.2 ∣. Low PG in daf-1 is due in part to an inaccessibility of AA

daf-1 mutants consistently exhibit low PG levels that can be suppressed by daf-3, but the mechanism underlying this phenomenon is still unclear. AA is the precursor of PGF2α. fat-2(wa17) mutants, which lack Δ 12-fatty acid dehydrogenase and stearoyl-CoA 9-desaturase activity, and therefore lacking AA synthesis capabilities, have poor sperm guidance.¹⁴ fat-2(wa17) mutants fed with 100 μM AA rescued the sperm guidance defect (Figure 2A). To assess whether the addition of AA also rescued sperm guidance in daf-1 mutants, we fed these mutants with 100 μM AA. Feeding daf-1 with AA did not rescue sperm guidance, suggesting the defect in daf-1 mutants was downstream of AA synthesis (Figure 2B).

FIGURE 2.

daf-1 can synthesize PGF2α in vitro. A, Percent of sperm accumulated in zone 3 of fat-2(wa17) hermaphrodites fed with AA supplemented or control bacteria. B, Percent of sperm accumulated in zone 3 of daf-1(m40) hermaphrodites fed with AA supplemented or control bacteria. C, in vitro PG synthesis using active worm lysates. Equal amounts of lysate, as normalized by protein content, was used in each reaction across all three genotypes with varying AA concentration. Each condition was performed in triplicate. D, Percent of sperm accumulated in zone 3 of daf-1(m40) hermaphrodites injected with 100 μM AA or PBS. * indicate P < .05 in a two sample t test

The enzyme responsible for PG synthesis in C elegans is still unknown. However, we wanted to assess whether daf-1 mutants still produced the components needed to synthesize PGF. To this end, we generated worm lysates by lysing worm pellets in Tris-HCl buffer containing 1x protease inhibitors and incubated them with various concentrations of AA. daf-1 lysates generated the same level of PGF2α as wild-type and daf-1;daf-3 lysates, suggesting that the daf-1 mutants expressed the machinery needed to convert AA to PGF2α (Figure 2C). Our lab previously showed that the bulk of PGs are synthesized within the gonad, and our in vitro data suggested daf-1 mutants still retained the enzymatic machinery to convert AA to PGF2α. To assess whether the gonads of daf-1 mutants actively synthesized PGs in vivo, we injected AA directly into the gonad of daf-1 mutant animals. Sperm guidance of the AA injected animals showed significantly increased sperm accumulation in zone 3 compared with control (Figure 2D), suggesting that the PG synthesizing machinery was active in the oocyte. Together, these data suggested that the low PGF level and poor sperm guidance phenotype observed in daf-1 mutants was due, in part, to a lack of AA transport into the gonad.

2.3 ∣. Decreased AA availability in daf-1 isdue in part to decreased yolk transport

The specific gene(s) that is responsible for the direct conversion of PUFAs to PGs remains unidentified. However, our data suggest that one mechanism by which the TGFβ pathway modulates PG levels is by regulating the accessibility of PUFAs in the oocytes, where the bulk of PGs are synthesized.¹³ PUFA transport into oocytes is largely mediated through the RME-2 lipoprotein receptors on the surface of oocytes.¹⁴ PUFAs are packaged within lipoprotein particles and endocytosed by RME-2. We measured rme-2 expression in wild-type, daf-1, and daf-1;daf-3 animals using qPCR. We found that the expression of rme-2 was significantly decreased in daf-1 animals. This decrease in expression was suppressed in daf-1;daf-3 double mutants (Figure 3A). This data suggested that the decreased ability for PUFAs to enter the oocyte may contribute to the low PG levels found in daf-1 mutants.

FIGURE 3.

TGF β pathway regulates yolk endocytosis receptor RME-2. A, Expression fold change for rme-2 in wild-type (N2), daf-1, and daf-1;daf-3 adult hermaphrodites. cDNA was synthesized from 1 μg of total RNA. qPCR was performed using 2 μL of cDNA per reaction with cdc-42 as the endogenous control. Error bars represent SD of three biological replicates. B, Fluorescence microscopy of yp170 and daf-1;yp170 animals. Signal represents GFP-labeled yolk distribution

To qualitatively assess the effect of rme-2 expression on the distribution of yolk within the C elegans, daf-1 mutant animals were mated with yp170 mutant worms that had GFP labeled yolk lipoprotein. In yp170 animals, all the yolk was distributed between the intestine and oocytes. However, in daf-1;yp170 worms, a significant amount of yolk was found outside of these two tissues, as indicated by the puncta seen along the worm (Figure 3B). This qualitative assessment of yolk distribution in daf-1 animals suggested that the decreased rme-2 expression contributed to the dysregulation of yolk transport into the oocytes, thus contributing to the low PG levels. However, as see in Figure 3B, some yolk was still found within the oocytes, suggesting additional mechanisms existed to inhibit PG synthesis to the extent seen in Figure 1C. One hypothesis was that daf-1 also affected downstream pathways that regulate lipid content within the yolk complexes. We hypothesized that the amount of AA packaged into each unit of yolk was decreased in daf-1 mutants, such that fewer AA molecules were transported into the oocytes with each unit of yolk. Further studies are needed to quantify AA in pure yolk fractions.

2.4 ∣. daf-3 acts in the nervous system and possibly the germline to modulate sperm guidance

As seen in Figures 1 and 2, the PG and sperm guidance phenotypes observed in daf-1 mutants were suppressed by daf-3, suggesting these phenotypes were modulated by the canonical DAF-7 pathway, involving both daf-1 and daf-3. Previous studies showed that daf-1 functions in the nervous system and germline to affect sperm guidance, but little is known about where daf-3 functions.¹⁷ To understand whether daf-3 also functions in these tissues to suppress daf-1 activity, wild-type daf-3 was expressed under tissue specific promoters in daf-1;daf-3 double mutant hermaphrodites and sperm guidance was assessed in the transgenic animals. As seen in Figure 4, 80% of the sperm accumulated at the spermatheca of non-transgenic daf-1;daf-3 double mutant hermaphrodites. In the positive control animals, where wild-type daf-3 was expressed under the endogenous daf-3 promoter, only 65% of the sperm accumulate at the spermatheca. Sperm guidance in transgenic animals expressing wild-type daf-3 under the pan-neuronal promoter, rab-3p, resulted in significantly decreased sperm accumulation when compared with non-transgenic animals, suggesting DAF-3 function in the neurons was important for modulating sperm guidance. Wild-type daf-3 was also expressed under the muscle (myo-3p), gonadal sheath cells (lim-7p), intestine (ges-1p), and pharyngeal (myo-2p) promoters. Sperm guidance in these animals did not show any difference when compared with the non-transgenic animals, suggesting that the effect seen with rab-3p was specific to the neurons (Figure 4A).

FIGURE 4.

daf-3 functions in the nervous system and germline to modulate sperm guidance. A, Plasmids containing daf-3 cDNA under tissue specific promoters were injected into daf-1;daf-3 mutant hermaphrodites. Sperm guidance was assessed in transgenic animals. For each construct, more than one line was assessed. One-way ANOVA was used for statistical analysis, P < .05. B. RNAi was performed on daf-1 (white circles) or daf-1;rrf-1(black circles) hermaphrodites using the feeding method. Bacteria containing the daf-3 or control target constructs were used. One way ANOVA was used for statistical analysis. P < .001between daf-3 RNAi groups vs. control groups. No significance between groups within each RNAi condition

To assess the function of daf-3 in the germline, we used rrf-1 mutants. rrf-1 encodes an RNA dependent RNA polymerase that is involved in the production of siRNAs that function in RNA mediated interference (RNAi) in somatic tissues. All somatic tissues of rrf-1 mutants were initially thought to be resistant to RNAi while the germline remained sensitive. However, rrf-1 mutants exhibit RNAi sensitivities in the intestine in addition to the germline.²⁴ We show in Figure 4A that daf-3 did not function in the intestine to affect sperm guidance. To assess DAF-3 function in the germline, daf-1;rrf-1 double mutants were generated. Knockdown of daf-3 in daf-1;rrf-1 mutants led to a 20% increase in sperm accumulation in zone 3 compared with rrf-1 control (Figure 3B). Taken together, daf-3, similar to daf-1, functioned in the nervous system and germline to affect sperm guidance.

3 ∣. DISCUSSION

The mechanisms that guide sperm to the oocyte in internally fertilizing animals are not well studied largely due to the lack of visibility of the female reproductive tract. To overcome this limitation, C elegans was developed as a model to study sperm motility in the reproductive tract of live animals because of its transparent epidermis.^9-11 Labeled males can be tracked and recorded at single cell resolutions. Studies using the C elegans model have identified a specific class of F-series prostaglandins (PGFs) that are important for guiding the sperm to the spermatheca, or fertilization site. Mutants with low PGFs exhibit correspondingly poor sperm guidance, as defined by a failure of sperm to accumulate at the spermatheca.^12,13 While the enzyme(s) responsible for PGF synthesis remains unidentified, the DAF-7 TGFß pathway has been implicated in regulating both PGF levels and sperm guidance.¹⁷ However, the mechanisms are not well delineated. In this study, we show that the low PGF levels seen in daf-1 mutants are likely responsible for the sperm guidance defect. Furthermore, we provide evidence suggesting that the low PGFs detected in daf-1 mutants are due in part to the inaccessibility of the PUFA precursors, which is partly mediated through the RME-2 receptors. Finally, daf-3 suppresses the sperm guidance phenotype. In this study, we show that daf-3 also acts to regulate PG levels and can act in the nervous system and germline to regulate sperm guidance.

Lipid signaling molecules play important roles in the reproductive process of many animals.^25,26 In this study, we show that daf-1 mutants have low levels of PGFs that coincide with a sperm guidance defect. Both of these phenotypes can be suppressed by mutations in daf-3. PGFs can rescue sperm speed. In fat-2 mutants, which lack the synthesis of the PUFA precursors, the ability of sperm to reach the spermatheca is significantly reduced. Additionally, sperm in these animals travel very slowly and with little direction. Injection of any one of PGF1, PGF2, or PGF3 into the vulva of fat-2 worms can rescue sperm speed, but not directional velocity.^12-14 This suggests that these PGFs act redundantly in their ability to modulate sperm motility and that these PGs are not simply permissive cues. The current model for PG synthesis involves the oocyte. PUFA precursors are thought to be transported to the oocyte via yolk complexes and machinery in the oocytes then synthesize and secrete these PGs to provide a gradient to draw sperm toward the spermatheca and ovulating oocyte.^12,14 To test whether PGFs are indeed providing the directional cues that are guiding sperm toward the spermatheca, we injected PGF2α directly into the germline of daf-1 mutant animals. Injected animals showed increased sperm targeting to the spermatheca, suggesting that the exogenous PGFs injected into the oocyte are providing guidance cues to the sperm.

Since the enzyme(s) responsible for PG synthesis has yet to be identified in C elegans, little is known about how the DAF-7 pathway is affecting PG levels, and therefore sperm guidance. We show here that, unlike fat-2 mutants, feeding AA directly to daf-1 mutants does not rescue sperm guidance, suggesting that AA is not being converted to PGF2. However, in vitro assays using active daf-1 lysates and AA show robust PGF2α synthesis, similar to wild-type and daf-1;daf-3 lysates, suggesting that the daf-1 mutants are equipped with the machinery to synthesize PGF2α. These experiments suggest that the relatively low PGF2 production in daf-1, compared with wild-type may be associated with the inaccessibility of free PUFA precursors. To test this hypothesis, we injected AA directly into the daf-1 gonad and assessed sperm guidance, taking advantage of the fact that the adult C elegans germline exist as a syncytium, where all developing oogenic germ cells are exposed to the same intracellular milieu. We see that injected animals have significantly increased sperm accumulation at the spermatheca, suggesting that the AA is able to be converted to PGF2α and released into the uterus.

Further investigation is needed to delineate the specific mechanism that is preventing AA from reaching the active site in daf-1 mutants. Multiple mechanisms are likely involved. First defective PUFA transport seems to be the cause of poor AA accessibility. We find that daf-1 animals express significantly less of the lipoprotein receptor, rme-2, that is responsible for endocytosing yolk into the oocytes. In wild-type animals, almost all yolk complexes are either within the intestine or the oocytes and embryos. However, daf-1 animals exhibit ectopic yolk puncta outside of these tissues, suggesting a decreased efficiency of yolk transport. In addition, it is possible that PUFAs are not adequately released from the intestines. A previous study examining the PUFA content of whole worm lipid extracts suggest that daf-1 mutants contain increased free AA compared with wild-type animals.²⁷ Additionally, Greer et al, 2008, show that daf-1 mutants accumulate fat in the intestine, a phenotype that is suppressed by daf-3.²⁸ Further investigation is needed to assess the PUFA content of yolk lipoprotein particles that are being transported in TGFβ mutant animals. Interestingly, daf-1 and daf-3 do not seem to be needed in the intestine to alter sperm guidance, suggesting that they function outside of the intestine to modulate the packaging of AA into yolk lipoprotein particles. While our in vitro assay does not indicate degradation, we cannot rule out the possibility that PGF2α is being converted into other lipid species through cyp enzymes or degraded through another unidentified pathway.¹³

This study and a previous study from our lab have implicated daf-3 in regulating sperm guidance. daf-1;daf-3 animals exhibit sperm guidance phenotypes similar to that of wild-type animals. Here we show that daf-1;daf-3 mutants exhibit similar levels of PGFs as wild-type animals. We drove wild-type daf-3 under tissue specific promoters in daf-1;daf-3 double mutant animals and find that daf-3 exerts its effects on sperm guidance, and thus PG metabolism, by acting in the nervous system. To establish its function in the germline, we used rrf-1 mutants that exhibit RNAi sensitivity that is largely limited to the germline.²⁴ Our data using daf-3 RNAi in a rrf-1 background, in addition the tissue specific expression of wild-type daf-3, suggest that daf-3 may also function in the germline to affect sperm guidance. Future studies using CRISPR technology and ppw-1 mutants where the germline is refractory to RNAi should be used to confirm this conclusion.

Together, this data suggest the DAF-7 pathway functions cell autonomously and nonautonomously downstream of daf-3. RNA sequencing of wild-type, daf-1, and daf-1;daf-3 mutants showed many neuronal neurotransmitter transporters and ion channels genes that are differentially regulated between daf-1 and wild-type or daf-1;daf-3.²⁹ A similar cell nonautonomous function of the DAF-7 pathway was also shown with fat accumulation in the intestine.²⁸ Together with our data demonstrating that AA transport may be responsible for the low PGF levels in daf-1 mutants, we hypothesize that the DAF-7 pathway may exerts its nonautonomous effects by regulating important neuroendocrine factors that may affect the ability of AA to be released from its lipid precursors or packaged into lipoprotein particles. With respect to its autonomous functions, it is likely that the DAF-7 pathway regulates transcriptional regulators in the oocyte that may affect the transcription of genes such as the rme-2 endocytosis receptor or other enzymes that may participate in the metabolism of AA into metabolites other than PGF2α. Furthermore, we cannot exclude that enzymes that can catabolize PGF2α are also altered such that the PGF2α synthesized in the oocytes are quickly degraded or converted into other lipid species. Taken together, the data suggest that DAF-7 pathway likely modulates multiple signaling cascades to alter PG levels and sperm guidance. Future studies should aim to delineate these possible mechanisms.

4 ∣. EXPERIMENTAL PROCEDURES

4.1 ∣. C elegans strains and maintenance

C elegans were maintained at 16°C, unless otherwise indicated, and fed with NA22 E. coli on NGM plates. N2 Bristol (wild-type), daf-1(m40), daf-3(mgDf90), rrf-1 (pk1417), fat-2(wa17), and RT368 (YP170::tdimer2 + unc-119[+]) strains were purchased from the Caenorhabditis Genetics Center (CGC). The daf-1(m40);daf-3(mgDf90), daf-1(m40); rrf-1(pk1417), and daf-1(m40); yp170 strains were generated by genetic crosses. The fog-2(q71) strain was used for males.

4.2 ∣. Sperm guidance assays

The sperm guidance assays were performed as previously described.^9,13 Briefly, ~100 males were picked onto a NA22 Escherichia coli food dot containing 2 μL 1 mM MitoTracker CMXRos in DMSO and 10 μL M9 buffer. The males were allowed to stain overnight at 16°C in the dark. The stained males were picked onto a seeded NGM plate and allowed to rest before mating. To ensure the hermaphrodites are similar in age, L4 stage hermaphrodites were picked 1 day before the assay and transferred to 25°C. On the day of the assay, 10 to 15 adult hermaphrodites were anesthetized in 0.1% tricaine and 0.01% tetramisole hydrochloride in M9 buffer for 30 minutes. Approximately 50 stained males and the anesthetized hermaphrodites were transferred onto a mating plate containing a 5 mm drop of thick NA22 E coli. The worms were allowed to mate for 30 minutes in the dark. After 30 minutes, the mated hermaphrodites were transferred to a seeded plate and allowed to rest for 1 hour before mounting for microscopy. DIC and fluorescence images were acquired using an upright microscope with epifluorescence at 60x magnification.

To quantify sperm distribution, the uterus was divided into three zones, with zone 1 containing the vulva and zone 3 containing the spermatheca. Individual sperm was counted in each zone and value is reported as a percentage of the total sperm in the uterus. A one-way ANOVA or a two sample t test was used to test for significance.

4.2.1 ∣. Prostaglandin extraction

PGs from whole worms were extracted as previously described.^13,30 Briefly, synchronized worms were grown at 16°C until L4 and then shifted to 25°C for 24 hours. Worms were washed from the NGM plates using M9 buffer. Cleaned worm pellets were stored at −80°C in 0.5 g aliquots. To extract hydrophilic lipids, 0.5 g frozen worm pellet, 1 mL ice-cold 1:2 acetone/saline containing 0.005% butylated hydroxytoluene (BHT), 1 mL 0.5 mm zirconium oxide beads, and internal standard PGF2α-d9 (to 1 ng/mL final concentration) were added to each Bullet Blender tube. The Bullet Blender 5 homogenizer (Next Advance) was run for 3 minutes at speed 9. The homogenate was transferred to a 10 mL glass conical tube. The remaining beads were washed twice with 1 mL of 1:2 acetone/saline containing BHT and the supernatant from each wash was also transferred to the 10 mL glass conical tube. The glass conical tube was centrifuged at 4°C for 10 minutes at 1000 × g. The supernatant was transferred to a new glass conical tube and an equivalent volume of hexane was added to each tube. The tube was vortexed for 30 seconds and centrifuged again at 4°C for minutes at 1000 × g. The upper hexane phase and white debris in the interphase were discarded. Then, 2 M formic acid was added to the lower aqueous phase until the pH reached 3.5. An equal volume of chloroform was added to each tube and vortexed for 30 seconds. The tube was centrifuged at 4°C for 10 minutes at 1000 × g. The lower chloroform layer was transferred to a clean class conical tube and flushed with nitrogen gas. The tube was stored at −20°C overnight. The aqueous phase was removed and the remaining chloroform layer containing prostaglandins was dried in a Teflon-lined capped ½-Dram glass vial under nitrogen. For mass spectrometry analysis, the dried extracts were dissolved in 100 μL 80% methanol.

4.2.2 ∣. Chromatography and mass spectrometry

LC-MS/MS analyses of commercial standards and tissue extracts were performed as previously described using a system consisting of a Shimadzu Prominence HPLC with a refrigerated auto sampler (Shimadzu Scientific Instruments, Inc., Columbia, MD) and an API 4000 (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada) triple quadrupole mass spectrometer. Briefly, the chromatographic separation was performed on a Synergy hydro RP-C18 column pre-equilibrated with 0.1% formic acid. The mobile phase consists of 0.1% formic acid [A] and acetonitrile containing 0.1% formic acid [B] and was pumped at a flow rate of 0.2 mL/min. The gradient started with 10% B and went up to 80% B from 0 to 11 minutes, 80% to 100% B from 11 to 14 minutes and returned back to 10% B at 16 minutes. The column effluent was introduced into the mass spectrometer using an ESI interface operating in negative ion mode. Nitrogen was used as a nebulizer and curtain gas (CUR = 10). The col-lision gas, collision energy and temperature were set at 10, −35 eV and 600°C, respectively. The LC-MS/MS system was controlled by BioAnalyst 1.4.2 software. For comparative analysis of different strains, the extracts and standards were run consecutively.

To determine PGF2α concentration, a stock solution of PGF2α (1 μg/mL in 80% MeOH) was serially diluted with the same solvent to obtain 20, 10, 5, 1, 0.5, and 0.1 ng/mL concentrations. The samples were analyzed by LC-MS/MS operated in the multiple reaction monitoring (MRM) mode with mass transition m/z 353/193 in negative ion mode. The standard curve exhibited excellent linearity in the range of concentration 0.1 to 20 ng/mL with a correlation coefficient > 0.99. Average PGF2α concentration was calculated using the MultiQuant software 2.1.1 from Sciex from three MRM analyses using three independent sample extractions per genotype.

4.2.3 ∣. Lipid supplementation and injection

For supplementation assays, arachidonic acid (Cayman Chemicals) was diluted in thick NA22 E coli to a final concentration of 100 μM. Then, 250 μL of the thick bacteria containing 100 μM AA was added and spread onto an unseeded 6 cm plate. The bacteria was allowed to dry and two gravid hermaphrodites were transferred onto the plate. The worms were maintained at 16°C. F1 generation worms were picked at the L4 stage onto a freshly made plate containing 100 μM AA. The worms were transferred to 25° C for 24 hours before using for the sperm guidance assay.

For lipid injections, L4 stage worms were picked to a new plate and transferred to 25°C for 24 hours before injection; 100 μM AA or PGF2α in PBS was microinjected directly into the gonad of the worms. Successful injections were transferred onto a seeded plate and allowed to rest at 25°C for 2 to 3 hours before assessing sperm guidance. PBS injections were used as control.

4.3 ∣. In vitro PG synthesis assay

The biochemical PG synthesis assay was performed as previously described.³¹ Briefly, synchronized worms were grown at 16°C until the L4 stage and shifted to 25°C for 24 hours. The worms were washed with M9 and stored as 2 g pellets. Two-gram worm pellets were homogenized using the Bullet Blender in 1 mL 10 mM Tris-HCl buffer with 1x protease inhibitors and 1 mL 0.5 mm zirconium oxide beads. The Bullet Blender was run twice at speed 9 for 4 minutes each. The homogenate was centrifuged for 10 minutes at 13.2 × 1000 × g. The supernatant was transferred to a new tube. The protein concentration in the supernatant was determined using the Pierce BCA protein assay kit (Fisher) following the kit's protocol. For each biochemical reaction, equal protein was used per reaction and PGF2α-d9 was used as the internal standard. The reaction was incubated at room temperature for 30 minutes, and the reaction was stopped with the addition of ice-cold 0.1% (vol/vol) formic acid in methanol and PGF2α-d9 (1 ng/mL) was added as the internal standard. The reaction was centrifuged for 15 minutes at 13.2 × 1000 × g. The supernatant was transferred to a clean glass vial and dried under a nitrogen stream. The dried samples were resuspended in 100 μL 80% methanol and analyzed by mass spectrometry.

4.4 μ. rme-2 qPCR

RNA was extracted from synchronized, 1 day old adult hermaphrodites. Then, 250 mg of frozen worms were homogenized with a Bullet Blender 5 (Next Advance) and 0.5 mm zirconium beads. Total RNA was extracted with Trizol (Invitrogen) following the kit's protocol. cDNAs were synthesized from 1 μg of RNA using the Superscript IV Reverse Transcriptase (Invitrogen) kit and random hexamers following the provided protocol. Real-time PCR was performed in the Bio-Rad CFX Connect Real-Time System with Syber Green master mix (Bio-Rad) following the provided protocol. Each sample was run in triplicate. Three biological samples were run for N2 (wild-type), daf-1(m40), and daf-1(m40);daf-3 (mgDf90) double mutants. The PCR protocol used was: 95°C for 2 minutes; 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The primers used are fwd: 5′-TCGACTGGATTGGTGGAAACG-3′ and rev: 5′-AAGGATTCTGACCTGAGACCCAT-3′. The relative expression fold change was calculated using the 2^-ddCT method. cdc-42 was used as a control.

4.4.1 ∣. RNA mediated interference

The feeding method was employed for RNAi experiments.^17,32 RNAi bacterial strains for the empty vector L4440 and daf-3 were obtained from the feeding library and sequenced for verification. Single colonies were expanded in overnight cultures containing tetracycline and ampicillin. One milliliter of the overnight bacteria was transferred to 25 mL of LB containing tetracycline and ampicillin. Then, 100 mM IPTG was added 1 hour after inoculation and the culture was allowed to shake for an additional 4 hours. Bacterial pellets were resuspended in M9 buffer and spread on unseeded NGM plates containing tetracycline, ampicillin, and IPTG. L4 stage hermaphrodites were transferred to the RNAi plates and allowed to grow for 24 to 48 hours at 25°C before assessing sperm guidance.

4.4.2 ∣. Transgenic lines

The plasmid daf-3p::daf-3CDs::SL2::GFP::UNC-54 3′UTR was generated in the pGEM5Z backbone using the Gibson Assembly Master Mix following the kit's protocol. An XbaI and NotI site was incorporated around the 5′ and 3′ end of the promoter sequence respectively. To generate the other constructs used in this study, the daf-3p sequence was removed via restriction digest and other promoter sequences were incorporated using the Gibson Assembly Master Mix. Then, 30 ng/μL of each construct was injected into the gonad of gravid daf-1;daf-3 hermaphrodites. Injected worms were maintained at 16°C and transgenic lines were selected based on GFP expression. Multiple independent lines were generated for all strains.