Sperm-activating proteins obtained from the herring eggs

Spermatozoa of the Pacific herring, Clupea pallasii, were immotile in a solution of isotonic to seminal plasma, but swimming was initiated in a hypertonic solution such as 60% water. The motility of herring spermatozoa became active in the presence of freshly ovulated eggs, suggesting that ovulated eggs release the sperm-activating proteins, which are prerequisite to successful fertilization. Five species of herring sperm-activating proteins (HSAP) with different pI values (4.8, 4.9, 5.0, 5.1 and 5.4) were purified from the egg-conditioned medium by gel filtration and isoelectric focusing. Molecular mass of the HSAP (pI=5.1), the major species of the five HSAPs, was determined to be 8.1 KDa by mass spectrometry. Complementary DNA clones encoding herring sperm-activating proteins were isolated from a herring ovarian complementary DNA library and amino acid sequences were deduced. The herring sperm-activating protein(s) is a secretory product(s) with strong homology to Kazal-type trypsin inhibitors, such as mammalian acrosin inhibitors. The sperm-activating proteins were globally distributed in the outermost layer of the egg chorion and its gene was expressed in the follicle cells that surround developing oocytes. Both HSAPs and HSAP fragment (HSAP-E) could induce the membrane depolarization and increase in the intracellular Ca²⁺ concentration of herring spermatozoa. These results suggest that, in the Pacific herring, trypsin inhibitor like proteins are synthesized in the follicle cells, secreted, accumulated in the egg chorion during oocyte development and released into the milieu at spawning to activate the motility of spermatozoa at the time of gamete interaction.