Induction of sex pheromone secretion and observations of the secretory pathway in the Closterium peracerosum-strigosum-littorale complex

Several sex pheromones function in the process of sexual reproduction in the Closterium peracerosum-strigosum-littorale complex. The secretory pathways of two sex pheromones, protoplast release-inducing protein (PR-IP) and PR-IP Inducer, were studied by means of Western blot analysis and immunoelectron microscopy. PR-IP Inducer is secreted from mating-type minus (mt−) cells and induces the release of PR-IP from mating-type plus (mt+) cells. PR-IP causes release of the protoplast from mt− cells. Our studies were carried out using an experimental system that induces the secretion of sex pheromones in mt+ or mt− cells cultured separately, without mixing. Active secretion of PR-IP Inducer occurs 6-12 h after inoculation of mt− cells in a nitrogen-depleted medium, and the secretion of PR-IP is substantial by 8 h after inoculation of mt+ cells into medium containing PR-IP Inducer. In mt+ and mt− cells that actively secrete the pheromones, immunogold particles that label PR-IP Inducer and PR-IP were detected inside and along the surfaces of cell walls, vesicles 80-100 nm in diameter, Golgi cisternae, and in the region of the trans-Golgi networks. As gold particles were distributed entire the cell wall, exocytosis of pheromones may occur throughout the cell surface. This study is the first report to detect algal sex pheromones by means of electron microscopy.