title: Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs.
abstract: This study was conducted to evaluate the effects of treatment with the cAMP modulators cilostamide and/or forskolin during pre-IVM culture on meiotic progression, gap junction communication, intraoocyte cAMP level and glutathione content, embryonic development after parthenogenesis, and somatic cell nuclear transfer in pigs. Cumulus-oocyte complexes were cultured for 24 hours in unsupplemented medium or media containing 20 μM cilostamide and/or 50 μM forskolin. After pre-IVM, oocytes were cultured for 41 to 44 hours in a standard IVM medium to induce oocyte maturation. When the nuclear status of oocytes was examined after pre-IVM for 24 hours, a higher (P < 0.01) proportion of oocytes treated with forskolin (85.5%) and cilostamide + forskolin (92.6%) remained at the germinal vesicle stage compared with untreated (20.6%) and cilostamide-treated oocytes (54.7%). cAMP level in pre-IVM oocytes was significantly increased by combined treatment with cilostamide + forskolin (21.38 fmol/oocyte) relative to the no pre-IVM control, no treatment, cilostamide, and forskolin groups (2.85, 1.88, 1.74, and 8.95 fmol/oocyte, respectively). Forskolin with or without cilostamide significantly maintained open-gap junction communication relative to no treatment. Blastocyst formation in parthenogenesis was significantly (P < 0.01) improved by forskolin (65.3%) relative to other treatments (28.3% to 48.1%). Supplementation of pre-IVM with dibutyryl cAMP showed similar blastocyst formation as forskolin treatment (61.1% and 61.0%, respectively). In somatic cell nuclear transfer, simultaneous treatment with cilostamide + forskolin significantly (P < 0.05) increased embryonic development to the blastocyst stage (42.9%) relative to the no pre-IVM, control, and cilostamide groups (32.3, 28.6, and 32.8%, respectively). The glutathione contents in pre-IVM oocytes were increased by no treatment, forskolin, and cilostamide + forskolin (1.38, 1.39, and 1.27 pixels/oocyte, respectively) compared with no pre-IVM and cilostamide (1.00 and 0.99 pixels/oocyte, respectively; P < 0.05). Our results reported that the meiotic progression of immature pig oocytes could be reversibly attenuated by cAMP, whereas treatment with cilostamide and forskolin during pre-IVM had positive effects on developmental competence of oocytes in pigs, probably by improving cytoplasmic maturation.
authors.affiliation: Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
authors.forename: Bola
authors.initials: B
authors.lastname: Park
authors.affiliation: Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
authors.forename: Hanna
authors.initials: H
authors.lastname: Lee
authors.affiliation: Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
authors.forename: Yongjin
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
authors.forename: Fazle
authors.initials: F
authors.lastname: Elahi
authors.affiliation: Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
authors.forename: Joohyeong
authors.initials: J
authors.lastname: Lee
authors.affiliation: Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Korea.
authors.forename: Seung Tae
authors.initials: ST
authors.lastname: Lee
authors.affiliation: Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Korea.
authors.forename: Choon-Keun
authors.initials: CK
authors.lastname: Park
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea.
authors.forename: Sang-Hwan
authors.initials: SH
authors.lastname: Hyun
authors.affiliation: Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea; Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea. Electronic address: eslee@kangwon.ac.kr.
authors.forename: Eunsong
authors.initials: E
authors.lastname: Lee
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D003994:Bucladesine / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D046149:Embryo Culture Techniques / Q000662:veterinary; D004624:Embryo Transfer; D005260:Female; D017629:Gap Junctions / Q000502:physiology*; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008540:Meiosis; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D015363:Quinolones / Q000494:pharmacology*; D013552:Swine / Q000196:embryology / Q000502:physiology*
chemical_list: D000276:Adjuvants, Immunologic; D010726:Phosphodiesterase Inhibitors; D015363:Quinolones; D005576:Colforsin; C021294:cilostamide; D003994:Bucladesine
keywords: Cyclic AMP; Forskolin; Nuclear transfer; Oocyte maturation