Partial characterisation of a spawning pheromone in the herring Clupea harengus pallasi

The production of a pheromone-like spawning substance by male herring is confirmed and biochemical properties of the substance are characterised. A behavioural bioassay is described in which 0.25–0.5 mL of test solution was added to 46 L of water containing a single male herring. The effect of the solution was judged by the degree of extension of the herring gonadal papilla and release of milt. Treatment with fresh milt, extracts of milt, or extracts of mature testes resulted in papilla extension, often with milt release; extracts of ovaries or immature testes were not effective. The milt and testicular extracts retained their bioactivity during purification, including removal of substances soluble in petroleum ether, elution from C-18 Sep-Pak cartridges, and elution from C-18 high-performance liquid chromatography (HPLC) columns. The bioactive substances do not appear to be proteins or peptides, because (i) bioactivity was retained in milt and testicular extracts after the large proteins were precipitated by acetone−HCl solution and discarded, and (ii) bioactivity was present after the remaining soluble peptides were degraded by incubation with pronase E, proteinase K, or protease type VI enzymes. The pheromone is also not a neutral lipid or nonpolar fatty acid, as bioactivity remained in the aqueous phase after extraction with petroleum ether. Rather, the proposed pheromone(s) eluted from Sep-Pak C-18 cartridges with 30% acetonitrile and subsequently from the reverse-phase HPLC column in fractions 33–48 with a gradient of acetonitrile. Several polar steroids, conjugated steroids, and prostaglandin F2α were also shown to elute in this region, whereas a variety of other less polar compounds eluted later. Finally, after the post-Sep-Pak material was extracted with dichloromethane, bioactivity was shown to be present to a lesser extent in the dichloromethane fraction (containing free steroids) and to a greater extent in the water phase (containing conjugated steroids). The bioactivity of the water phase was reduced by incubation with glucuronidase but was eliminated by a combination of glucuronidase and sulfatase treatment. Various synthetic free steroids, conjugated steroids, prostaglandins, and amino acids were tested in the bioassay, but none mimicked the effect of the crude or semipurified testicular and milt extracts. We conclude that at least one substance is active as a pheromone-like spawning substance in males. These substances show hydrophobic properties similar to those of polar steroids, prostaglandins, or their conjugated forms, and at least one form likely contains a sulfate or glucuronide group. The role of a male pheromone may be synchronisation of spawning in schools of herring.