278 EFFECT OF GLYCOSAMINOGLYCAN SUPPLEMENT ON PORCINE PREIMPLANTATION EMBRYO DEVELOPMENT AND EXPRESSION OF RECEPTORS FOR GLYCOSAMINOGLYCANS IN PORCINE EMBRYOS

Abstract

The present study investigated the effect of glycosaminoglycan (GAG) supplement on developmental competence of porcine in vitro fertilized (IVF) embryos and GAG receptor expression in the porcine embryos. In vitro-matured oocytes were inseminated with frozen-thawed boar semen and cultured in North Carolina State University (NCSU)-23 medium in the absence or presence of various GAGs (hayaluronic acid, heparin or both). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos and blastocysts at Days 2 and 7, respectively. Differential staining was performed in blastocysts at Day 7. All data were analyzed by ANOVA using a Generalized Linear Model (SAS). Inseminated oocytes were cultured in NCSU-23 supplemented with different concentrations (0, 0.1, 0.5 or 1.0 mg mL⁻¹) of hyaluronic acid (in Experiment 1) or heparin (in Experiment 2). Supplementing NCSU-23 with 0.5 mg mL⁻¹ hyaluronic acid significantly increased (P < 0.05) the total number of cells (55.9) and the number of trophectoderm (TE) cells (41.7) compared with the other culture groups (44.7, 45.0 and 31.3, 31.8, respectively). The rate of blastocyst formation was significantly increased (P < 0.05) in the 1.0 mg mL⁻¹ heparin-supplemented group (21.8%) compared with that in the control culture group (16.4%). In experiment 3, inseminated oocytes were cultured in NCSU-23 supplemented with 0, 0.1 mg mL⁻¹ heparin, 0.5 mg mL⁻¹ hyaluronic acid, or 0.1 mg mL⁻¹ heparin + 0.5 mg mL⁻¹ hyaluronic acid. The rate of blastocyst formation was significantly increased (P < 0.05) in the 1.0 mg mL⁻¹ heparin group (21.5%) and the 1.0 mg mL⁻¹ heparin + 0.5 mg mL⁻¹ hyaluronic acid group (22.8%) compared with that of control culture group (16.6%). In experiment 4, total RNA was prepared from oocytes, 2-, 4-, and 8-cell stages, morulae, and blastocysts and reverse-transcribed using the First-strand cDNA Synthesis kit (Amersham Biosciences, Piscataway, NJ, USA). The cDNA was subjected to polymerase chain reaction with primers specific for hyaluronic acid receptor (CD44) and heparin binding protein (HIP). The expression of CD44 gene was detected in oocytes, and in 2- and 4-cell stages;; HIP gene was detected in all preimplanatation-stage embryos. In conclusion, the present study demonstrated the embryotrophic role of GAG and expression of GAGs receptors in porcine IVF embryos. This study was supported by the Advanced Backbone IT Technology Development (IMT 2000-C1-1).