Attribution of Cortical Granules to Formation of Fertilization Envelopes and Polyspermy Block in Urechis unicinctus

Cortical reaction and polyspermy block are well defined in most marine invertebrates. In Urechis species, the function of cortical granules (CGs) is not yet known, and there is controversy on whether the cortical reaction occurs, or the fertilization envelope (FE) is attributed to CG releases or functions to prevent polyspermy. This study was carried out to determine the cortical reactions and functions of the FE in Urechis unicinctus. Artificial insemination of the eggs revealed that CG release occurred to give rise to perivitelline space (PS) into the final FE. Both PS and final FE effectively blocked polyspermy. The final FE was accomplished within 10 min after sperm-egg initial binding. No massive release of CGs occurred within the early phase of 5 min after the initial binding, initially and the PS seemed to playa role to prevent polyspermy. The CG massively released its content into the PS in late phase of FE formation, and differentiated PS into five intermediate layers. The layers opened into each other by anastomosis, so that the final FE consisted of two layers, the inner layer ($15{\mu}m$ in thickness) and the outer layer ($1{\mu}m$ in thickness). The outer layer derived from vitelline layer and the inner layer consisted of PS and CG secretions. Immunofluorescence and confocal laser microscopy revealed that the spermatozoon took up residence in the egg cortex during FE formation and successive meioses of the fertilized egg. These results suggest that both PS and final FE of U. unicinctus were equivalent to the early and late block, respectively, of other marine animals.