32 ALLOCATION OF INNER CELL MASS AND TROPHECTODERM CELLS OF NUCLEAR TRANSFER EMBRYOS CULTURED IN MEDIUM SUPPLEMENTED WITH EPIDERMAL GROWTH FACTOR AND INSULIN-LIKE GROWTH FACTOR-I

Abstract

This study was conducted to determine the additive effects of exogenous growth factors and different macromolecules during in vitro oocyte maturation (IVM) and sequential embryo culture of nuclear transfer (NT) embryos. Oocytes were matured in TCM-199 supplemented with 10% fetal calf serum (FCS), 50 mg mL^–1 sodium pyruvate, 1% penicillin/streptomycin (10 000 U mL^–1 penicillin G, 10 000 mg mL^–1 streptomycin), 5 mg mL^–1 LH, and 0.5 mg mL^–1 FSH without growth factors (Treatment 1) or with 50 ng mL^–1 epidermal growth factor (EGF; Treatment 2) or with 50 ng mL^–1 EGF and 100 ng mL^–1 insulin-like growth factor-I (IGF-1; Treatment 3). Cloned bovine embryos were produced by transferring granulosa cells into enucleated meiosis II oocytes. Following activation, reconstructed embryos were cultured in Quinn's Advantage Cleavage Medium (QACM) supplemented with 8 mg mL^–1 essentially fatty-acid free (FAF) BSA for 72 h. Then, developing embryos from granulosa cells were cultured in Sequential Quinn's Advantage Blastocyst Medium (QABM) supplemented with 4 mg mL^–1 essentially FAF-BSA (Sigma-Aldrich, St. Louis, MO, USA) + 5% FCS (Group 1), 4 mg mL^–1 BSA + 5% FCS+100 ng mL^–1 IGF 1 (Group 2), and 4 mg mL^–1 BSA + 5% FCS+100 ng mL^–1 IGF-1+50 ng mL^–1 EGF (Group 3) for an additional 5 days under low oxygen tension (5% CO₂, 5% O₂, 90% N₂) at 38.5°C in high humidity conditions. Maturation rates of oocytes matured in the presence of EGF (75.5%) and EGF+IGF-I combination (75.0%) were significantly higher than those of oocytes matured (63.8%) in the presence of only FCS (P < 0.05). The developing NT embryos derived from granulosa cells of the Anatolian Grey Cattle showed no significant differences in fusion (53.62%, 53.25%, 57.36%), cleavage (67.98%, 74.20%, 66.80%), or blastocyst rates (32.65%, 29.47%, 41.77%) among culture groups (P > 0.05). When 13 to 23 embryos per group were examined by using differential staining, the results showed that the IGF-I alone and combination with EGF in the sequential embryo culture medium (Group 2: 46.61%. and Group 3: 41.37%) significantly increased the number of inner cell mass (ICM)/total blastocyst cell ratio in comparison with Group 1 (29.32%, no IGF-I and EGF; P < 0.05). Our results showed that the addition of growth factors to IVM medium and sequential culture medium changed the cell ration of cloned bovine embryos to the advance of ICM without changing total cell number. Supplementation of media with growth factors can alter the allocation of ICM and trophectoderm cells in NT embryos.