Dibutyryl cyclic amp

Neural stem cells (NSCs) were generated and differentiated into neural cultures as described previously^{72 (link)}. For patterning, NSCs were plated on polyornithin-/laminin-coated culture flasks at 1E4 cells/cm² in DMEM/F-12 with GlutaMax (#31331093) and neurobasal medium (#21103049) supplemented with 1X B27 (ThermoFisher #12587010), 1X N2 (ThermoFisher #17502048), 50 µM 2-mercaptoethanol (ThermoFisher #31350010) and 100 ng/ml FGF-8 (Peprotech), 200 ng/ml sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma) and cultured for one week. For differentiation, the resultant progenitors were plated at 5E4 cells/cm² in basal medium supplemented with 20 ng/ml BDNF, 10 ng/ml glial cell-derived neurotrophic factor (GDNF; Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), and 100 μM ascorbic acid 2-phosphate.

Immature GV (germinal vesicle) eggs were collected from the ovaries of wild-type B6D2F1 or Juno KO female mice (9–16 weeks old), 46 h after the injection of pregnant mare serum gonadotropin or anti-inhibin antibody (CARD HyperOva, Kyudo). Antral follicles, suspended in FHM medium supplemented with 250 μM dibutyryl-cyclic AMP, were punctured with 26G needles (Sigma). After dissociation of the cumulus cells by a glass pipette, the eggs were transferred to the medium supplemented with 5 μg ml⁻¹ cytochalasin B (Sigma), and mRNAs (50 ng ml⁻¹) were then injected into the eggs, using a piezo-micromanipulator with a glass capillary needle. The eggs were washed three times, and were cultured in TYH medium containing 10% fetal bovine serum (Biowest) for in vitro maturation. After 14 h, the zona were removed from the MII eggs, using a micromanipulator27 (link). Pre-loading of Hoechst 33342 into the denuded eggs was performed, as previously described2 (link), and after three washes with fresh TYH medium, the eggs were subjected to mJUNO immunostaining or sperm–egg fusion assays. In one experiment, considering a failure of superovulation, 2 to 4 Juno KO female mice were assigned to an experimental group and 2 WT mice were assigned to a control group.

Nicotine was purchased from Nacalai Tesque (Kyoto, Japan). Thapsigargin and ionomycin were purchased from FUJIFILM Wako Pure Chemical Industries, Ltd. Fluo-4-AM and PowerLoad^™ concentrate were purchased from Molecular Probe (Eugene, OR, USA). Acetylcholine, d-tubocurarine, nifedipine and dibutyryl cyclic AMP were purchased from Sigma-Aldrich (St. Louis, MO, USA). Omega-conotoxin GVIA and α-conotoxin AuIB were from the Peptide Institute, Inc. (Osaka, Japan). Atropine was obtained from Katayma Chemical Industries Co., Ltd. (Osaka, Japan). All the other chemicals used were of analytical grade.

Prior to survival assay and RT-PCR, human retinoblastoma cell lines Y79 (# HTB-18, ATCC, Manassas, VA) and WERI Rb-1 (# HTB-169, ATCC) were maintained in flasks with DMEM Glutamax medium (#21885, Invitrogen) containing 1000 U/ml penicilin and streptavidin (#15140-122, Invitrogen) and 10% fetal calf serum (#16000-044, Invitrogen) at 37°C, 85% humidity and 5% CO2. For immunocytochemistry, Y79 and WERI-Rb-1 cells were cultured on BD Falcon 8 wells glass chambers slides (#354108, Becton-Dickinson, Franklin Lakes, NJ) previously coated with 25 µg/ml of laminin-1 (L2020, Sigma, St. Louis, MO). Cells were seeded at 60.000 cells/cm2 and cultured for a week in DMEM Glutamax medium (#21885, Invitrogen) supplemented with 250 µM dibutyryl cyclic AMP (Sigma) 1000 U/ml penicilin and streptavidin (#15140-122, Invitrogen) and 10% Lipumin (#F11-014, PAA Laboratories, Pasching). As described previously [23] (link), [24] , those conditions allowed the adherence of the retinoblastoma cell lines on the glass surface, therefore easing the immunocytochemistry staining process and analysis.

The murine mesodermal endothelial progenitor cell line T17b was cultured and differentiated according to established protocols [24 (link),30 (link)]. Briefly, T17b EPCs were seeded onto cell culture flasks coated with bovine skin gelatin type B (Sigma-Aldrich, Schnelldorf, Germany). For cell cultivation, high glucose DMEM GlutaMAX^® (Gibco/Life Technologies, Carlsbad, CA, USA) was used containing 20% fetal calf serum (Biochrom), 100 U/mL penicillin (Biochrom), 100 µg/mL streptomycin (Biochrom), 1 mM nonessential amino acids (Gibco), 2 mM HEPES buffer pH 7.5 (Gibco) and 0.1 mM 2-mercaptoethanol (Gibco). By supplementing 0.5 mM dibutyryl cyclic AMP and 1 µM all-trans retinoic acid (Sigma-Aldrich), endothelial differentiation was induced.