IGFs mediate the action of LH on oocyte maturation in zebrafish

Abstract

LH signaling is required for oocyte maturation in fish and other vertebrates. However, the downstream factors mediating LH signaling are largely unexplored in fish. In this study, we investigated whether IGFs could mediate LH action on oocyte maturation in zebrafish. Our results show that all igfs, including igf1, igf2a, igf2b, and igf3, are dynamically expressed during folliculogenesis, with the expression of igf3 reaching its maximal level in full grown stage follicles. The expression of igfs is regulated by LH through a cAMP pathway in intact follicles as well as in primary cultured follicular cells, with igf3 expression being the most sensitive to human chorionic gonadotropin (hCG) treatment. Moreover, recombinant zebrafish IGF-2a, IGF-2b, and IGF-3 proteins significantly enhanced oocyte maturation via IGF-1 receptors (IGF-1rs), with IGF-3 exhibiting the most potent stimulatory action on oocyte maturation. Furthermore, we have demonstrated that IGF-3 or hCG treatment could stimulate IGF-1rs phosphorylation, and hCG-induced oocyte maturation could be attenuated by IGF-1r inhibitors as well as by an anti-IGF-3 antiserum in vitro and in vivo, indicating that the IGF system especially IGF-3 plays a crucial role in mediating LH action on oocyte maturation. In addition, igf3 expression is significantly attenuated in LH β-subunit (lhb) mutant zebrafish and treatment with recombinant IGF-3 could partially rescue the oocyte maturation defects of the lhb mutants in vitro and in vivo. Collectively, our results clearly demonstrated that IGFs, particularly the gonad-specific IGF-3, act as important mediators of LH action on oocyte maturation in zebrafish.

Figure 1.

Expression of igf1, igf2a, igf2b, and igf3 during folliculogenesis. A, RT-PCR detection of igf1, igf2a, igf2b, and igf3 expression in the ovary of adult fish. RT(+), RNA with reverse transcription; RT(−), RNA without reverse transcription; M, marker. B, Real-time PCR detection of igf1, igf2a, igf2b, and igf3 expression in the ovary of adult fish. C–F, Temporal expression of igf1 (C), igf2a (D), igf2b (E), and igf3 (F) in the follicles of different stages isolated from the ovaries of adult fish; each value representing the mean value ± SEM of quadruplicate assays of 3 independent experiments (*, P < .05; **, P < .01; ***, P < .001 vs control).

Figure 2.

Regulation of igf expression by hCG. A, Dose dependence of hCG action (2-h treatment) on the expression of igf1, igf2a, igf2b, and igf3 in FG stage follicles. B, Time dependence of hCG action (at 10 IU/mL of hCG) on the expression of igf1, igf2a, igf2b, and igf3 in FG stage follicles. C, Morphology of primary cultured zebrafish ovarian follicular cells after 6 days of incubation. The 2 types of epithelial cells present in the culture with distinct morphologies. G, granulosa cells; T, theca cells. D, Dose response of hCG action (2-h treatment) on the expression of igf1, igf2a, igf2b, and igf3 in primary cultured follicular cells. E, Dose response of IBMX action (2-h treatment) on the expression of igf1, igf2a, igf2b, and igf3 in primary cultured follicular cells. F, Dose response of forskolin action (2-h treatment) on the expression of igf2a, igf2b, and igf3 in primary cultured follicular cells. The expression levels of igfs were normalized to gapdh and expressed as a percentage of the control. Each value represents the mean value ± SEM of quintuplicate assays from 3 independent experiments (*, P < .05; **, P < .01; ***, P < .001 vs control).

Figure 3.

Recombinant zebrafish IGFs stimulate oocyte maturation in vitro and in vivo. A, Incubation of intact FG follicles with 2μM recombinant zebrafish IGF-2a, IGF-2b, or IGF-3 protein for 17 hours significantly induced GVBD. B, PI staining of intact follicles (left panel) and denuded oocytes (right panel). C, Incubation of denuded oocytes with 2μM recombinant zebrafish IGF-2a, IGF-2b, or IGF-3 protein for 4 hours significantly induced GVBD. D, Gross morphology of ovaries dissected from adult zebrafish after injection of 10-IU hCG or 1ug recombinant zebrafish IGF-2a, IGF-2b, or IGF-3 protein into zebrafish after 4 hours. Each picture is the representative result of triplicate assays from 3 independent experiments. E, Quantitative assessment of oocyte maturation induction by injection of 10-IU hCG or 1ug recombinant zebrafish IGF-2a, IGF-2b, or IGF-3 protein after 4 hours. Each value represents the mean value ± SEM of quintuplicate assays from 3 independent experiments (*, P < .05; **, P < .01; ***, P < .001 vs control).

Figure 4.

IGFs stimulate oocyte maturation through activating IGF-1rs in zebrafish. A, Inhibition of IGF-induced (at 2μM) GVBD by IGF-1r inhibitors (10μM NVP-AEW541 or NVP-ADW742). B, DHP-induced (at 10 ng/mL) (a) and EGF-induced (at 100 ng/mL) (b) oocyte maturation was not significantly blocked by IGF-1r inhibitors (10μM). C, Western blotting results demonstrated the stimulation of IGF-3 (at 2μM) on IGF-1r phosphorylation and expression of cyclin B. Numbers on top stand for the treatment time. Mature, mature oocytes. D, Western blotting results demonstrated the stimulation of cyclin B by IGF-3 depends on the activation of IGF-1r. Each value represents the mean value ± SEM of triplicate assays from 3 independent experiments (***, P < .001 vs control).

Figure 5.

IGF-1r phosphorylation could be activated by hCG and hCG-induced oocyte maturation could be blocked by IGF-1r inhibitors in vitro and in vivo. A, Phosphorylation of IGF-1rs activated by treatment with hCG (at 50 IU/mL) in FG stage follicles. B, Inhibition of hCG-induced (50 IU/mL) GVBD by in vitro treatment of FG stage follicles in vitro with IGF-1r inhibitors (10μM NVP-AEW541 or NVP-ADW742). C, Significant inhibition of hCG-induced oocyte maturation after in vivo administration of hCG (10 IU/fish) with or without IGF-1r inhibitor (10mM NVP-ADW742, 4 μL/fish). D, Inhibition of hCG-induced (50 IU/mL) GVBD by treatment with an anti-IGF-3 antiserum (AS) (1:25 dilution) but not by the preimmune serum (PIS) (1:25 dilution) of FG stage follicles in vitro for 18 hours (a). Inhibition of hCG-induced oocyte maturation in vivo after injection of hCG (10 IU/fish) by AS or PIS (1.5 μL/fish) (b). Each value represents the mean value ± SEM of quintuplicate assays from 3 independent experiments (*, P < .05; **, P < .01 vs control).

Figure 6.

Expression of igfs in FG stage follicles of lhb mutant fish and partial rescue of defects in oocyte maturation by IGF-3. A, Relative expression of igf1, igf2a, igf2b, and igf3 in FG stage follicles of the wild-type (lhb^+/+) and lhb mutant (lhb^m/m) zebrafish. B, Recombinant zebrafish IGF-3 protein (2μM) induced maturation of FG follicles isolated from the ovary of lhb mutant fish. Each value represents the mean value ± SEM of triplicate assays from 3 independent experiments (*, P < .05; **, P < .01; ***, P < .001 vs control). C, Gross morphology of ovaries dissected from lhb mutant (lhb^m/m) zebrafish after administration of hCG (10 IU/fish) or IGF-3 (1ug/fish). Each picture is the representative result of triplicate assays from 3 independent experiments. D, Quantitative assessment of oocyte maturation induction in lhb^m/m zebrafish by injection of 10-IU hCG or 1ug recombinant zebrafish IGF-3 protein after 4 hours. Each value represents the mean value ± SEM of triplicate assays from 3 independent experiments. (*, P < .05 vs control).

Figure 7.

IGFs mediate LH action on oocyte maturation: a proposed model. IGF-2a, IGF-2b, and IGF-3 are all expressed in the follicular cells and subject to regulation by LH signaling, with IGF-3 being the most important IGF subtype in mediating LH action on oocyte maturation.