Effect of Growth Factors and Hormones during In Vitro Growth Culture of Cumulus-Oocyte-Complexes Derived from Small Antral Follicles in Pigs

Abstract

This study evaluated the effect of various growth factors and hormones in an in vitro growth (IVG) medium on the in vitro maturation (IVM) and developmental competence of oocytes derived from small antral follicles (SAFs) in pigs. Cumulus-oocyte complexes (COCs) derived from SAFs were either untreated or treated with epidermal growth factor (EGF), insulin-like factor-1 (IGF-1), insulin, or growth hormone (GH) for 2 days of IVG. Following IVG, COCs were cultured for maturation, and IVM oocytes were induced for parthenogenesis (PA). During IVG, the nuclear maturation of oocytes was significantly increased by the insulin treatment compared to other treatments. Moreover, the insulin treatment significantly increased blastocyst formation after PA relative to the No-IVG, control, EGF, and GH treatments. The cumulus expansion score after IVG-IVM was significantly higher in the insulin group than in the other groups. The glutathione (GSH) contents in IVM oocytes were increased through treatment with IGF, insulin, and GH compared to those of No-IVG oocytes. The level of reactive oxygen species (ROS) in IVM oocytes in all treatment groups was significantly lower after IVG culture than in the No-IVG group. The maturation-promoting factor (MPF) activity after IVM in the insulin-treated oocytes was significantly higher than that of the oocytes treated with EGF, IGF-1, and GH. In conclusion, this study demonstrates that insulin treatment during IVG culture improves the maturational and developmental competence of oocytes derived from SAFs in pigs through its effect on cumulus cell expansion and cytoplasmic microenvironments, such as GSH, ROS, and MPF activity.

Keywords: growth factor; in vitro growth culture; insulin; pig; small antral follicle.

Figure 1

Morphology of cumulus–oocyte complexes (COCs) after in vitro growth (IVG) and in vitro maturation (IVM) culture. COCs without IVG culture (A) and COCs that had been cultured for 2 days in an IVG medium supplemented with None (D), epidermal growth factor (EGF) (G), insulin-like growth factor-1 (IGF-1) (J), insulin (M), and growth hormone (GH) (P). No-IVG COCs and IVG COCs were cultured for 22 h in an IVM medium with FSH and human chorionic gonadotrophin (B,E,H,K,N,Q) and then cultured for an additional 22 h in an FSH and human chorionic gonadotrophin-free medium (C,F,I,L,O,R). Scale bar = 200 μm.

Figure 2

Maturation-promoting factor (MPF) activity (mean ± SEM) after in vitro maturation of small antral follicle-derived oocytes that were treated with various growth factors or hormones during in vitro growth (IVG). MPF activity was analyzed in four replicates by sampling 50 mature oocytes for each treatment. Different letters (a–c) in the bar indicate significant differences among treatment groups (p < 0.05).