140 EXPRESSION OF LEPTIN LIGAND AND RECEPTOR AND EFFECT OF EXOGENOUS LEPTIN SUPPLEMENTATION ON IN VITRO DEVELOPMENT OF PORCINE IN VITRO FERTILIZED AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Abstract

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro-fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. In Exp. 1, in vitro-matured porcine oocytes and embryos in 2-, 4-, and 8-cell as well as morula and blastocyst stages derived from IVF or SCNT were immunostained for leptin ligand and receptor with their specific antibodies. The expression of leptin ligand and receptor proteins was detected in oocytes and all stages of IVF and SCNT embryos. The IVF (Exp. 2; n = 635, 630, 633, 635, respectively) or SCNT oocytes (Exp. 3; n = 256, 258, 251, 258, respectively) were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (1, 10, 100, or 1000 ng/mL) of leptin. For the control group, IVF (n = 635) or SCNT embryos (n = 249) were cultured without leptin supplementation (0 ng/mL leptin). Embryo development and cell number in blatoscysts after differential staining according to a modified staining procedure (Thouas et al. 2000 Reprod. Biomed. Online 3, 25–29) were evaluated. The IVF or SCNT embryos were randomly distributed, and experiments were replicated at least 11 times. The differences in embryo development among experimental groups were analyzed using one-way ANOVA after arcsine transformation (without arcsine transformation for cell number of blastocysts) to maintain homogeneity of variance. Post hoc analyses to identify between-group differences were performed using the LSD test. In SCNT embryos, the cleavage rate was not different in leptin-treated groups (73.8, 77.5, 75.7, or 78.7%, respectively) compared to the control (76.3%). The rate of blastocyst formation (at 166 h after the day of injection of donor cells) in SCNT embryos was significantly increased (P < 0.05) in 1000 ng/mL leptin-supplemented group (20.2%) compared with the control (12.9%) and 1 ng/mL leptin-supplemented (12.5%) groups. Supplementing mNCSU-23 with 1000 ng/mL leptin also significantly increased (P < 0.05) the number of total cells (54.6) and trophectoderm (TE) cells (39.1) in SCNT blastocysts (n = >25) compared with the control [45.1 (total cells) and 31.6 (TE cells)] and 10 ng/mL leptin-supplemented group [44.4 (total cells) and 31.7 (TE cells)]. In IVF embryos, leptin supplementation did not affect pre-implantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor proteins in porcine in vitro matured oocytes, IVF and SCNT embryos, and the embryotropic role of leptin in the SCNT embryo development. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1) and the Korean Ministry of Science and Technology (M10310060000-03B4606-00000).