Fig. 4: Identification, germline dependence, and biological activity of dipeptide medip#1.
a Chemical structures of medip#1–3 (26–28) and MS2 fragmentation of medip#1 (26) in ESI- mode. b Synthesis of medip#1 and related molecules. c Ion chromatograms for m/z 346.2125 and 349.2294, corresponding to medip#1 and D3-medip#1, from exo-metabolome extracts of him-5 animals supplemented with D3-Met and a synthetic sample containing medip#1 (26) and its Leu-derived isomer (25). d, f Abundance of medip#1 in exo-metabolome extracts of (d) him-5 relative to WT, e 1:1 male:hermaphrodite mixtures relative to WT hermaphrodites, and f indicated germline mutants relative to WT. Data represent four (d), two (e), and four or six (f) biologically independent experiments, and error bars represent mean ± s.e.m. P values were calculated by two-sided Welch t tests; ND, not detected. g Developmental stage-dependent production of medip#1 in WT (purple) and him-5 (light blue) animals. Data are from three independent biological replicates, except for two independent biological replicates for day 7. h Abundance of medip#1 in the exo-metabolome of male-enriched C. briggsae him-8 mutants relative to WT C. briggsae. Data represent five biologically independent experiments. i Faster acquisition of oocytes in hermaphrodites exposed to 2 nM of medip#1 (red) compared to paired controls (blue). Boxes represent the two inner quartiles, horizontal lines represent medians, and whiskers extend to 1.5× of the box data. Data represent one experiment with n = 25 animals for each condition; P values were calculated by Kolmogorov–Smirnov test. j Hermaphrodites exposed to 2 nM of medip#1 (red) ovulate earlier than controls (blue). Data represent one experiment with n = 25 animals for each condition. The differences at 54 h and 56 h are significant (P = 1.7 × 10−3 and P = 7 × 10−3, respectively, as calculated using binomial test). k Faster pharyngeal pumping in hermaphrodites treated with 2 nM of medip#1 (red) than in untreated controls (blue). Boxes represent the two inner quartiles, horizontal lines represent medians, and whiskers extend to 1.5× of the box data. Data represent one experiment with n = 30 animals for each condition; P value was calculated by Kolmogorov–Smirnov test. l Timepoint of first egg-laying of isolated worms on different concentrations of medip#1 and nacq#1 compared to untreated isolated worms (ISO, control) and grouped worms (high density, HD). Data represent four biologically independent experiments, except for 10 µM medip#1 and 1 pM nacq#1 (one experiment), and 10 pM nacq#1 (three experiments), with the total number of animals used for each condition indicated above the x axis. P values were calculated by two-sided Welch t test, comparing indicated conditions with ISO control. m Mean lifespan of medip#1-treated WT animals compared to untreated control. Data represent three to four biologically independent experiments, each using 15–20 animals per plate; the total number of plates used for each condition is indicated above the x axis. P values were calculated by two-sided t test, comparing indicated conditions with untreated control. Source data are provided as a Source Data file.
