Critical Role of the Cortical Alveolus Protease Alveolin in Chorion Hardening In Vivo at Medaka Fertilization

Alveolin is a cortical alveolus proteinase that is secreted in the perivitelline space (PVS) at fertilization to act on the chorion. Purified alveolin is known to induce chorion hardening in vitro by processing zona pellucida B (ZPB), a major chorion component. However, in vivo function of alveolin remains unclear; thus, in this study, the effects of alveolin efficiency (Alv(−/−)) at the organism level were investigated using the medaka, Oryzias latipes. The Alv(−/−) fertilized eggs were mechanically fragile; however, they developed normally and left offspring as long as they were carefully handled before hatching. A mechanical press test showed that the Alv(−/−) fertilized eggs were six times more fragile than the wild-type eggs. They were 35% larger owing to the enlarged PVS, 34% thinner, and permeable to even 10 kDa FITC-dextran. These results are consistent with the transmission electron microscopy observation that the periphery of the inner layers was highly porous in the Alv(−/−) chorion. In chorion hardening, the alveolin-mediated processing of ZPB and the transglutaminase (TGase)-mediated crosslinking of chorion components are the key steps. This study was the first to show that alveolin also processed TGase concomitantly with ZPB, which greatly facilitated the crosslinking. Thus, alveolin was concluded to be the primary trigger for chorion hardening in vivo. Furthermore, fertilization in a balanced salt solution could partially improve the impaired chorion hardening of the Alv(−/−) eggs fertilized in water, probably through an alveolin-independent mechanism.