Pre-fertilization egg coat dynamics in Phallusia philippinensis (Tunicata, Ascidiacea)

In externally fertilizing species such as ascidians, gametes are released into the surrounding seawater, which makes the fertilization process highly sensitive to environmental conditions. In the ascidian Phallusia philippinensis, the oviductal fluid has an acidic pH of 5.65 (Sensui et al., 2023), markedly lower than that of seawater (approximately 8.2). Consequently, eggs experience a rapid and substantial pH shift when released into the external environment. The vitelline coat (VC), an extracellular matrix surrounding the oocyte, serves both as a structural barrier and as a mediator of essential sperm-egg interactions during fertilization. In this study, we report for the first time a pH-dependent, pre-fertilization elevation of the VC in the ascidian P. philippinensis. The eggs of P. philippinensis exhibit rapid VC elevation that occurs independently of fertilization after release into seawater. This process appears to be driven by osmotic pressure and enzymatic structural loosening of the VC, rather than cortical granule exocytosis, which is well known to initiate the modification and hardening of the fertilization envelope in organisms such as sea urchins and mammals. Consistent with this, VC elevation occurs independently of fertilization and does not function as a polyspermy block in this species. These findings reveal a previously unrecognized pH-responsive property of the ascidian egg coat and provide new insights into a seawater pH-triggered mechanism regulating egg coat structure.