Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells

Abstract

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95–100%), but distributed from GVI to GVIV (GVI 56.1 ± 9.1%, GVII 15.3 ± 1.4%, GVIII 21.5 ± 7.1%, GVIV 7.1 ± 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 ± 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1–25 μg mL^–1 resulted in 95–100% oocytes being arrested and synchronized at GVII. With >5 μg mL^–1 CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 μg mL^–1 CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6–12 h, metaphase I (MI) at 10–18 h and anaphase I/telophase I at 16–20 h. After 12 h preincubation with 5 μg mL^–1 CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.