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A combination of recombinant human (rh) insulin-like growth factor-I (IGF-I) (25 nM) and the maturation-inducing hormone (MIH), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S; 72.5 nM), induced germinal vesicle breakdown (GVBD) in ovarian follicles of white bass incubated in vitro, whereas a four times greater concentration of each hormone was ineffective alone. These results indicate that IGF-I induces oocyte maturational competence (OMC) but not meiotic resumption in white bass. Culture medium concentrations of 20beta-S remained below detection limits for ovarian fragments incubated with rhIGF-I. Actinomycin D blocked GVBD in response to hCG but not to rhIGF-I plus 20beta-S, suggesting that IGF-I requires de novo translation but not transcription to induce OMC. Gap junction uncouplers, 1-octanol and 1-heptanol, and the phosphatidylinositiol 3-kinase (PI 3-K) inhibitors, wortmannin and LY 294002, attenuated hCG-, 20beta-S-, and rhIGF-I plus 20beta-S-induced GVBD. Although these inhibitors reduced hCG-induced progestin release, PI 3-K inhibitors did not alter MIH synthesis in some incubations and addition of 20beta-S to the incubations did not fully overcome the effects of either class of inhibitors, suggesting that decreasing MIH production is not their only inhibitory effect on gonadotropin (GtH) action. Our data suggest that gap junctions and PI 3-K activity are necessary for GtH and IGF-I to induce and maintain OMC in white bass. The induction of OMC but not meiotic resumption by IGF-I in white bass, compared with the induction of meiotic resumption but not OMC by IGF-I discovered in the congeneric striped bass suggests rapid evolution of the reproductive actions of IGF-I among temperate basses (genus Morone).

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.

Recombinant human (rh) insulin-like growth factor-I (IGF-I) was more potent than rhIGF-II at inducing in vitro germinal vesicle breakdown (GVBD), a marker for resumption of meiosis, in oocytes of striped bass. Treatment of ovarian fragments containing oocytes in intact follicles with rhIGF-I increased concentrations of estradiol-17beta and maturation-inducing steroid (MIS) 17,20beta, 21-trihydoxy-4-pregnen-3-one (20beta-S) in the culture medium and decreased testosterone levels. The follicles were too immature for oocytes to complete GVBD in response to 20beta-S (MIS incompetent) or hCG. Addition of 20beta-S to cultures did not increase the percentage of oocytes completing GVBD in response to rhIGF-I or rhIGF-II. Bovine insulin was without effect on GVBD or steroid production. Incubation of MIS-competent follicles with actinomycin D, cyanoketone, trilostane, 1-heptanol, or 1-octanol had no effect on rhIGF-I-induced GVBD, but attenuated hCG-induced GVBD and 20beta-S production. Cycloheximide inhibited rhIGF-I-induced GVBD. Collectively, these observations indicate that IGF-I can induce GVBD via MIS- and transcription-independent pathways without coupled gap junctions between oocytes and granulosa cells or among granulosa cells, but requires protein synthesis to do so. An rhIGF-I analogue that does not bind IGF-binding proteins, des(1,3)IGF-I, was more potent than rhIGF-I in inducing GVBD, suggesting ovarian IGF-binding proteins may inhibit IGF-I action.