Title

The protein phosphatase inhibitor, okadaic acid, elicits several components of zebrafish (Danio rerio) oocyte maturation in vitro

Abstract

In vitro treatment of fully grown immature zebrafish oocytes with 17-α, 20-β dihydroxyprogesterone (DHP) resulted in germinal vesicle migration (GVM) and breakdown or dissolution (GVD), ooplasmic clearing (OC, as assessed by CAMMA, Lessman et al., 2007 Mol. Reprod. Dev. 74:97-107), yolk protein changes (YP), and blastodisc formation (BF). Experiments were conducted to study the effect of okadaic acid (OA), a specific inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), on zebrafish oocyte maturation. Immature oocytes treated with OA (1 μg/ml) alone, showed GVM and GVD, OC, YP, and BF. The GVD ED50 for OA was 29.9 nM, for cantharidin the ED50 was 213.6 nM, and for calyculin the ED50 was 23.9 nM. These ED50s are consistent with the higher doses required for PP1 inhibition compared with 10-100 fold lower doses for PP2A inhibition. The time course for maturation with OA was delayed relative to DHP-induced maturation. In addition, OA-matured oocytes exhibited an intermediate level of YP compared to DHP. OA and DHP were synergistic in promoting oocyte maturation. Microarray data indicated the presence of PP1 and PP2A mRNA in follicle-enclosed oocytes. Column chromatography, dot and western blots indicated the presence of PP2A subunit B contained within large complexes in homogenates of ovary, large ovarian follicles and eggs. Together, these data support the hypothesis of PP1 and/or PP2A involvement in oocyte maturation.

Publication Title

Zebrafish: Topics in Reproduction, Toxicology and Development

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