Characterization of a Novel Bile Alcohol Sulfate Released by Sexually Mature Male Sea Lamprey (Petromyzon marinus)

Li W (2013) Characterization of a Novel Bile Alcohol Sulfate Released by Sexually Mature Male Sea Lamprey (Petromyzon marinus). PLoS ONE 8(7): e68157. doi:10.1371/journal.pone.0068157 Characterization of a Novel Bile Alcohol Sulfate Released by Sexually Mature Male Sea Lamprey (Petromyzon marinus ) Ke Li 0 Cory O. Brant 0 Michael J. Siefkes 0 Hanna G. Kruckman 0 Weiming Li 0 Michel Renou, INRA-UPMC, France 0 1 Department of Fisheries and Wildlife, Michigan State University , East Lansing, Michigan , United States of America, 2 Great Lakes Fishery Commission , Ann Arbor, Michigan , United States of America A sulphate-conjugated bile alcohol, 3,12-diketo-4,6-petromyzonene-24-sulfate (DKPES), was identified using bioassayguided fractionation from water conditioned with sexually mature male sea lamprey (Petromyzon marinus). The structure and relative stereochemistry of DKPES was established using spectroscopic data. The electro-olfactogram (EOG) response threshold of DKPES was 1027 Molar (M) and that of 3-keto petromyzonol sulfate (3 KPZS; a known component of the male sea lamprey sex pheromone) was 10210 M. Behavioural studies indicated that DKPES can be detected at low concentrations by attracting sexually mature females to nests when combined with 3 KPZS. Nests baited with a mixture of DKPES and 3 KPZS (ratio 1:29.8) attracted equal numbers of sexually mature females compared to an adjacent nest baited with 3 KPZS alone. When DKPES and 3 KPZS mixtures were applied at ratios of 2:29.8 and 10:29.8, the proportion of sexually mature females that entered baited nests increased to 73% and 70%, respectively. None of the sexually mature females released were attracted to nests baited with DKPES alone. These results indicated that DKPES is a component of the sex pheromone released by sexually mature male sea lamprey, and is the second biologically active compound identified from this pheromone. DKPES represents the first example that a minor component of a vertebrate pheromone can be combined with a major component to elicit critical sexual behaviors. DKPES holds considerable promise for increasing the effectiveness of pheromone-baited trapping as a means of sea lamprey control in the Laurentian Great Lakes. - Bile alcohols and bile acids are amphipathic multifunctional end products of cholesterol metabolism in vertebrates [1], [2]. In vertebrate animals, bile alcohols are synthesized in hepatocytes, conjugated by esterification with sulfate to form bile salts, converted to strong acids at the terminal carbon of the side chain, and shown to have substantial structural diversity across species [13]. Recent studies have shown that the olfactory epithelia of teleost fishes and jawless vertebrates are acutely sensitive to bile acids [46]. Bile salt molecules are ideal for chemical communication because of their stability and water solubility [7]. The sea lamprey (Petromyzon marinus) provides a useful model for studying intraspecific chemical communication mediated by bile salts [6]. These semelparous, jawless vertebrates rely on bile salts released from conspecifics to coordinate reproduction [811]. Previous research has demonstrated that 3-keto petromyzonol sulfate (3 KPZS), a bile salt derivative, is a potent sex pheromone released by sexually mature male sea lamprey, and elicits upstream movement in sexually mature females [1214]. Behavioral studies have indicated that there are additional components of the male sex pheromone that play roles in inducing additional behaviors, such as attraction and nest retention, in sexually mature females [15]. During in-stream behavioral bioassays, water conditioned with sexually mature males, which presumably contained all the components of the male sea lamprey sex pheromone, attracted more sexually mature females to artificial nests, retained females at nests longer, and induced a greater number of nesting and spawning behaviors than when 3 KPZS alone was applied, indicating that other constituents may be critical for these behaviors [15]. The male sea lamprey sex pheromone is hypothesized to contain additional components, some of which may function more locally around nests to attract female mates [16]. This study reports the structure and biological activity of a novel bile salt, 3,12-diketo-4,6-petromyzonene-24sulfate (DKPES), recently isolated from the male sea lamprey sex pheromone, which represents the first example of the importance of minor components in vertebrate pheromone communication. Structure Elucidation of DKPES DKPES was isolated as a colorless oil. The molecular formula, C24H33NaO6S, was deduced from its HRESIMS (m/z 495.1782 [M+Na]+), (See Supplementary Data), which corresponds to C24H33Na2O6S (calcd, 495.1793). The MS/MS fragmentation showed a 96.9603 fragment, which corresponds to HSO42. The 13C chemical shift for C-24 (dC 69.4 ppm) was similar to a sulfate dH (mult., J in Hz) half-ester OSO3Na [17]. The 1H NMR spectrum, (See Figure S1, S2, S3, S4, S5, S5, S7), showed two deshielded methyl singlets (dH 1.17, s, H-18; dH 1.24, s, H-19) and one methyl doublet (dH 0.87, d) with chemical shifts corresponding to methyl groups H3-21. The results of the 1H NMR spectrum revealed the sulfated steroidal nature of DKPES. The 13C NMR spectrum of DKPES displayed 24 carbon signals. Two carbonyl carbon signals appeared at dC 201.9 (qC) and 215.5 (qC), and four olefin carbon resonances were located at dC 124.5 (CH), 165.7 (qC), 129.4 (CH), and 141.4 (CH). Using the gHSQC spectrum (See Supplementary Data), the remaining carbon resonances were three methyl carbons, eight methylene carbons, five methine carbons, and two quaternary carbons, indicating that DKPES was a tetracyclic structure with eight degrees of unsaturation. The major part of the tetracyclic steroidal backbone could be assembled through the interpretation of COSY and HMBC correlations. The gCOSY experiment showed two spinning systems (indicated in bold in Figure 1), while a series of gHMBC correlations displayed the connection of these fragments (Figure 1). Characteristic gHMBC correlations, exhibited by the two methyl singlets Me-18 and Me-19, confirmed the cyclization of four rings and the location of a ketone at position of C-12 (Figure 1).The correlations from H2-2 to C-4, from H-4 to C-2, C-4, and C-6, from H-6 to C-4 and C-8, and from H-7 to C-5 and C-9 indicated that the conjugated ketene was placed at C-4, D4(5), and D6(7) (Figure 1). The carbon ring skeleton of DKPES was originally known to be one of the minor unsaturated acidic metabolites degraded by cholic acid under anaerobic conditions by Pseudomonas sp [18]. An overall comparison of 1H and 13C NMR data (Table 1) revealed that the main tetracyclic skeleton of DKPES was similar to 21-hydroxypregna-4, 6-diene-3, 12, 20-trione and 20R-hydroxypregna-4, 6-diene-3,12-dione, which had been isolated from the bark and twigs of Nerium oleander L. from the family of Apocyaceae [19]. The Compound DKPES possess (...truncated)