Dibutyryl adenosine 3 5 cyclic monophosphate db camp

Bdph (mol. wt., 188.23) was synthesized in accordance with the previously described method (Mowry et al. 1949 ; Lin et al. 1984 ) and produced a light-yellow oily substance that is a mixture of 85% trans (Z)- and 15% cis (E)-isomers. Caffeine sodium benzoate (≥98%), dibutyryl adenosine 3′,5′-cyclic-monophosphate (db-cAMP, ≥97%), imidazole (≥99%) and tetrodotoxin (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO). Other drugs used in this study included aminophylline (Eizai, Tokyo, Japan), clonidine (Boehringer-Ingelheim, Ridgefield, CT) and NA (Breon, NY). The final concentrations of the aforementioned drugs in the medium were expressed in molarity.
Male Hartley guinea-pigs (500–600 g, each) were obtained from the Animal Center of the Ministry of Science and Technology, Taipei, Taiwan. The animals were housed in ordinary cages at 22 ± 1 °C with a humidity of 50–60% under a constant 12 h light/dark cycle and were provided with food and water ad libitum. Under a protocol (LAC-74-0032) approved by the Animal Care and Use Committee of Taipei Medical University on 14 October 1985, the GPVD were dissected under anaesthesia (pentobarbital 50 mg/kg, intraperitoneal injection).

GFP CETN2 CB6F1 ovaries in EmbryoMax M2 (EMD Millipore, Billerica, MA) were minced mechanically to collect follicles of various sizes (Suppl Fig. S6) and GV’s released using sterile needles. Cumulus cells were removed with a 75-µm tip (Stripper pipet; Origio Mid-Atlantic Diagnostics, Trumbull, CT). Oocyte diameters were determined with Elements software (Nikon USA, Melville, NY) after image capture on a Nikon digital sight camera (DS Fi1). Oocytes were maintained in KSOM until fixation. Mature oocytes were harvested after stimulation with an intraperitoneal injection of 7.5 IU PMSG (Sigma-Aldrich, St. Louis, MO) for 48 hrs and kept in M2 supplemented with 100 µg/ml dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP; Sigma-Aldrich) to prevent meiosis resumption. To initiate meiotic maturation, mature GVs were rinsed 3x in M2 and placed in KSOM in a 37 °C, 5% CO₂ humidified incubator.