Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by a H-ATPase inhibitor: Effects on the hydration mechanism

1 2 In teleost oocytes, yolk proteins (YP)-derived from the yolk precursors vitellogenins are 3 partially cleaved into free amino acids and small peptides during meiotic maturation prior to 4 ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration 5 which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). 6 However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), 7 such as the killifish (Fundulus heteroclitus), in which oocyte hydration is driven by K ions. 8 Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation9 associated proteolysis of YPs is poorly understood. Here, lysosomal cysteine proteinases 10 potentially involved in YP processing, cathepsins L, B and F (CatL, CatB and CatF), were 11 immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During 12 oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF 13 disappeared from yolk organelles and CatL became inactivated, while CatB proenzyme was 14 processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major 15 YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of 16 bafilomycin A1 (BA1), a specific inhibitor of vacuolar-type H-ATPase (V-ATPase), underwent 17 maturation in vitro, but acidification of yolk globules, activation of CatB and proteolysis of YPs 18 were prevented. In addition, MIS plus BA1-treated oocytes accumulated less K than those 19 stimulated with MIS alone; hence oocyte hydration was reduced. These results suggest that 20 CatB is the major protease involved in yolk processing during the maturation of killifish 21 oocytes, whose activation requires acidic conditions maintained by a V-ATPase. Also, the data 22 indicate a link between ion translocation and YP proteolysis, suggesting that both events may be 23 equally important physiological mechanisms for oocyte hydration in benthophil teleosts. 24 25 Fundulus heteroclitus; oocyte maturation; hydration; V-ATPase; cathepsin 26 27