L-Kynurenine, an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon

Abstract

Many animals employ sex pheromones to find mating partners during their reproductive seasons. However, most sex pheromones of vertebrates remain to be identified. Over the past 20 years, steroids and prostaglandins have been identified as sex pheromones in several fishes. These pheromones are broadly termed "hormonal pheromones" because they or their precursors act as hormones in these fishes. Hitherto, no other type of sex pheromone has been unambiguously identified in teleost fish. Here we report the identification of a "nonhormonal pheromone" in teleost fish. The urine of the reproductively mature female masu salmon (Oncorhynchus masou) contains a male-attracting pheromone. Bioassay-guided fractionation yielded an active compound that was identical to L-kynurenine in spectral and chromatographic properties. L-Kynurenine is a major metabolite of L-tryptophan in vertebrates. This pheromone elicits a male-specific behavior at even picomolar concentrations; its electrophysiological threshold is 10(-14) M. L-Kynurenine is a reasonable substance for female masu salmon to advertise their readiness for mating.

Fig. 1.

Pheromonal activity of OFU and the first purification. MMU, mature male urine; IFU, immature female urine. (A) Y-maze tests of spermiating males to the urine of adult salmon. BW, blank water. Each graph represents mean values ± SEM (n = 16). ∗, P = 0.0100, compared by using the Wilcoxon signed rank test. (B) Activities of OFU and eluates from the polystyrene column in the new assay. CS, control solution (50% ethanol); WS, wash solution; 50, 50% ethanol eluate; 80, 80% ethanol eluate; 100, 100% ethanol eluate. Scores are represented as mean ± SEM (n = 14). ∗, P = 0.0011; ∗∗, P = 0.0003, compared by using the Mann–Whitney U test.

Fig. 2.

Schematic representation of the concise bioassay system devised for the isolation of a sex pheromone from the urine of ovulated female masu salmon. This figure will help in understanding the protocol of the bioassay and the behavioral response shown in Movie 1.

Fig. 3.

Second and third purifications of a pheromone. CS, control solution (70% ethanol); Frc, fraction. (A) Elution profile of gel filtration using a Sephadex LH-20 column and the activity of the fractions. Inset shows UV spectrum of the highest peak. Scores are represented as mean ± SEM (n = 12–23). (B) Elution profile of HPLC (Inset) and the activity of the fractions. Scores are represented as mean ± SEM (n = 12–29). ∗∗, P < 0.0001; ∗, P = 0.0263, compared with CS by using the Mann–Whitney U test.

Fig. 4.

LC-MS and stereochemical analyses of the pheromone and its distribution in masu salmon urine. (A) Liquid chromatography–mass spectrometry analysis of the major peak obtained by HPLC. The mass spectra of the pheromone and authentic l-kynurenine are shown in the insets on the right. The inset on the left indicates the precursor, N-formylkynurenine. (B) Marfey's analysis (16) of the natural pheromone (upper part of B) and authentic d- and l-kynurenine. Inset shows the structure of the pheromone. (C) HPLC analysis of original urine samples. MMU, mature male urine; IFU, immature female urine.

Fig. 5.

Pheromonal activities of l-kynurenine and related compounds. Behavioral scores are represented as mean ± SEM. Each compound (100 μl) was introduced into the flume. DW, distilled water. ∗, P < 0.005; ∗∗, P < 0.0005; ∗∗∗, P < 0.0001, compared with DW by using the Mann–Whitney U test. (A) Activity in response to 10⁻⁴ M tryptophan and kynurenines (final concentrations of 10⁻⁹ M). Trp, tryptophan; Dk, d-kynurenine; Lk, l-kynurenine; Lks, l-kynurenine sulfate (n = 11). (B) Dose-response relationship of l-kynurenine sulfate (n = 9–18). The dilution rate of the samples was 10⁵. (C) Representative EOG responses recorded from a spermiating male to log molar concentrations of l-kynurenine (10⁻¹⁵ to 10⁻⁵ M). BW, blank water; −5 Ser, 10⁻⁵ M l-serine. (D) The EOG of the masu salmon in response to l- and d-kynurenine (n = 4–6). Response (%) is the value relative to that elicited by 10⁻⁵ M l-serine. The values are represented as mean ± SEM. IM, immature male; SM, spermiating male; OF, ovulated female (rarely the river resident form); RM, sexually regressed male. (E) Specific response of spermiating males to 10⁻⁴ M l-kynurenine that was introduced into the assay system (final concentrations of 10⁻⁹ M; n = 11–15).