Figure 1.
C. elegans gonad, F-series prostaglandin structures, and PUFA metabolism pathways.
(A) The C. elegans hermaphrodite reproductive tract. A differential interference contrast (DIC) image of an adult hermaphrodite is shown above with a diagram of half the uterus below. Males inject sperm through the vulva (V) into the uterus. Sperm become motile by extending a pseudopod and crawl around fertilized embryos (E) toward the spermatheca (S, yellow outline). Oocytes (O) enter the spermatheca during oocyte maturation and ovulation. The uterus is divided into 3 zones for quantification of sperm distribution. Scale bar, 0.1 mm. (B) Human F-series prostaglandin structures showing carbon numbering and important energy-induced cleavage sites characteristic of F-series prostaglandins. z1 cleavage causes loss of 44 Da from the parent ion and z2 cleavage, together with methyl terminal loss, generates the product ion m/z 193. Prostaglandin precursors for each class are shown in parentheses. (C) Diagram of C. elegans PUFA synthesis pathways. Δ9 and Δ12 desaturase activities generate substrates for the n3 desaturase FAT-1, the Δ6 desaturase FAT-3, and the Δ5 desaturase FAT-4 [31]. Fatty acids are abbreviated as in 20:4n6, which has 20 carbons with four double bonds, the first occurring at the n6 position. LA, linoleic acid; ALA, linolenic acid; GLA, γ-linolenic acid; STA, stearidonic acid; DGLA, dihomo-γ-linolenic acid; O3AA, omega-3 arachidonic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid.
Figure 2.
Sperm guidance in wild-type and fat mutant hermaphrodites.
Uterine sperm distribution 1 hr after mating wild-type or mutant hermaphrodites to wild-type males. A representative image is shown to the left and average zone distribution ± standard deviation is to the right. The spermatheca (S) is outlined in yellow. Asterisks indicate nonspecific gut autoflourescence. fat-2 and fat-3 mutants have reduced distance from vulva to spermatheca relative to the wild type, a feature that may influence distribution values. MT, MitoTracker channel; N, number of gonads scored; V, vulva. Scale bar, 20 µm. See Materials and Methods for zone definitions.
Table 1.
Sperm motility values in wild-type and mutant hermaphrodite uteri.
Figure 3.
Prostaglandins in wild-type and fat mutant extracts.
(A) MRM chromatograms of wild-type and fat mutant extracts. Liquid chromatography retention time (min) is shown on the X-axis and for selected prostaglandin isomers. Bolded retention times indicate prostaglandin isomers shown in Figure 4. Prostaglandin standard retention times are shown at the top. The dot diagram to the left indicates relative PUFA levels in the corresponding strains [33]. DGLA, dihomo-γ-linolenic acid; O3AA, omega-3 arachidonic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; cps, counts per second. (B) CePGF2 quantification in wild-type and fat mutant extracts. Error bars are SD. (C) Summary of MRM mass transitions and retention times for chemically synthesized standards. RT, retention time.
Figure 4.
Collision induced decomposition patterns of selected C. elegans and human prostaglandins.
LC-MS/MS of authentic standards (top) compared to selected C. elegans prostaglandins highlighted in Figure 3A. Red color indicates ions shared by the standard and an unknown prostaglandin. Blue color indicates ions that are not shared. m/z is on the X-axis. C. elegans MS/MS data are from fat-1(wa9) fat-4(wa14) mutant extracts (F1 class), fat-1(wa9) extracts (F2 class), and wild-type extracts (F3 class). RT, retention time.
Table 2.
LC-MS/MS data summary for chemically synthesized F-series prostaglandin standards.
Table 3.
LC-MS/MS data for selected C. elegans F-series prostaglandins.
Figure 5.
Effect of AA supplementation on sperm guidance in wild-type and fat mutant hermaphrodites.
Sperm distribution 1 hr after mating AA supplemented wild-type or mutant hermaphrodites to non-supplemented wild-type males. A representative image is shown to the left and average zone distribution ± standard deviation is to the right. See Figure 2 for sperm distribution in non-supplemented fat mutants. The spermatheca (S) is outlined in yellow. V, vulva; MT, MitoTracker channel; N, number of gonads scored. Scale bar, 20 µm.
Figure 6.
Effect of EPA supplementation on sperm guidance in wild-type and fat mutant hermaphrodites.
Sperm distribution 1 hr after mating EPA supplemented wild-type or mutant hermaphrodites to non-supplemented wild-type males. A representative image is shown to the left and average zone distribution ± standard deviation is to the right. See Figure 2 for sperm distribution in non-supplemented fat mutants. The spermatheca (S) is outlined in yellow. V, vulva; MT, MitoTracker channel; N, number of gonads scored. Scale bar, 20 µm.
Figure 7.
Prostaglandins in adult wild type and germline-deficient glp-4 mutants.
(A) MRM chromatograms of synchronized 1–2 day adult wild-type and glp-4(bn2) mutants. The F2 class was detected with mass transition m/z 353/193 and the F3 class was detected with mass transition m/z 351/193. Liquid chromatography retention time (min) is shown on the X-axis and for selected prostaglandin isomers. Cps, counts per second. (B) CePGF2 quantification in wild-type and glp-4 mutant extracts. Error bars are SD.
Figure 8.
Effect of cytochrome P450 enzymes on sperm guidance and prostaglandin metabolism.
(A) Sperm distribution 1 hr after mating wild-type or mutant hermaphrodites to wild-type males. A representative image is shown to the left and average zone distribution ± standard deviation is to the right. The spermatheca (S) is outlined in yellow. V, vulva; MT, MitoTracker channel; N, number of gonads scored. Scale bar, 20 µm. (B) MRM chromatograms of wild-type and cyp-32A2(tm2711) mutants. The F1 class was detected with mass transition m/z 355/311, the F2 class was detected with mass transition m/z 353/193, and the F3 class was detected with mass transition m/z 351/191. Liquid chromatography retention time (min) is shown on the X-axis and for selected prostaglandin isomers. Cps, counts per second.
Figure 9.
F-series prostaglandins in C. elegans and mouse tissues.
MRM chromatograms comparing F-series prostaglandins in C. elegans and mouse tissue extracts. The F1 class was detected with mass transition m/z 355/311, the F2 class was detected with mass transition m/z 353/193, and the F3 class was detected with mass transition m/z 351/193. Liquid chromatography retention time (min) is shown on the X-axis and for selected prostaglandin isomers. Cps, counts per second. The C. elegans MRM chromatograms are from Figure 3A. The mouse lung is extremely rich in PGE2, which was detectable with mass transition m/z 351/193.