Effects of the Isoquinolinesulfonamide H-8 on Fundulus heteroclitus Ovarian Follicles: Role of Cyclic Nucleotide-Dependent Protein Kinases on Steroidogenesis and Oocyte Maturation In Vitro

The possible role of cyclic nucleotide-dependent protein kinases in mediating the stimulatory actions of Fundulus heteroclitus pituitary extract (FPE) during ovarian steroidogenesis and oocyte maturation in vitro was investigated. Follicle-enclosed oocytes were cultured in the presence of FPE and/or N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a compound that inhibits protein kinase A (PKA) and cGMP-dependent protein kinase. H-8 alone (0.1–1 mM) promoted oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. However, the process of GVBD initiated by H-8 was much slower than that triggered by 17 α,20 β-dihydroxy-4-pregnen-3-one (17,20 βP), the natural inducer of oocyte maturation in F. heteroclitus. Treatment with H-8 also increased 17,20 βP production by the follicles and the accumulation of this steroid in the media was much slower than that initiated by FPE. However, in contrast to the FPE action on the oocyte, which is mediated by 17,20 βP, the stimulatory action of H-8 on GVBD appears to be independent of follicular steroid production, since aminoglutethimide (AGI), an inhibitor of steroidogenesis, did not-block H-8-induced GVBD while inhibiting H-8-induced 17,20 βP production. Moreover, addition of H-8 to FPE-treated follicles significantly reduced 17,20 βP secretion and the percentage of GVBD. These results provide further support for the involvement of PKA in the mechanism by which FPE stimulates ovarian steroidogenesis in F. heteroclitus. Furthermore, the fact that H-8 alone increased 17,20 βP levels may imply that basal follicular production of this steroid could be induced by inactivation of cyclic nucleotide-dependent protein kinases. Data also indicate that inhibition of PKA and/or c-GMP-dependent protein kinase in the oocyte may be involved in the mechanism leading to resumption of meiosis in this species.