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Theriogenology. 2016 Aug;86(3):757-65. doi: 10.1016/j.theriogenology.2016.02.029. Epub 2016 Mar 8.

Cilostamide and forskolin treatment during pre-IVM improves preimplantation development of cloned embryos by influencing meiotic progression and gap junction communication in pigs.

Author information

1
Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea.
2
Division of Applied Animal Science, College of Animal Life Science, Kangwon National University, Chuncheon, Korea.
3
Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea.
4
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.

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.

KEYWORDS:

Cyclic AMP; Forskolin; Nuclear transfer; Oocyte maturation

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