290 EFFECTS OF HYALURONAN ON IN VITRO DEVELOPMENT OF PORCINE EMBRYOS CULTURED IN A CHEMICALLY DEFINED MEDIUM

Abstract

Hyaluronan (HA), a glycosaminoglycan present in follicular and oviductal fluids, has been related to sperm capacitation, fertilization and embryo development. We have previously developed an in vitro-production (IVP) system of porcine embryos, where porcine blastocysts can be produced by IVF and IVC in chemically defined media and can develop to full-term by transfer to recipients. The application of a chemically defined medium to IVP in pigs allows the analysis of the physical action of substances on the development of pre-implantation embryos. In the present study, the effects of HA on the development of porcine embryos in a chemically defined medium were investigated. Porcine presumptive zygotes were produced by IVM and IVF of COC from pre-pubertal gilts and frozen-thawed ejaculated boar semen. The zygotes were cultured in Porcine Zygote Medium (PZM)-5 containing different concentrations of HA (0 [control], 1, 2, 5, 10, 20 and 50 μg mL⁻¹) until 6 days after IVF, and representative specimens were fixed for cell counting and transmission electron microscopy. Data of percentages and cell numbers were statistically analyzed by one-way ANOVA and Fisher’s PLSD test. The percentage of embryos that developed to the blastocyst stage (15.8% [23/144] to 19.5% [27/139]) did not differ among treatments. However, addition of 5 or 10 μg mL⁻¹ HA increased (P < 0.05) the total number of cells in blastocysts (56.1 and 58.3 cells [n = 22 and 23], respectively) compared to control (no HA, 42.0 cells [n = 23]). To evaluate proliferation rates of inner cell mass (ICM) and trophectoderm (TE), embryos were cultured in PZM-5 for various periods of exposure to 10 μg mL⁻¹ HA. The numbers of ICM and TE cells in Day-6 blastocysts cultured in the presence of exogenous HA from Day 0 to Day 3 (18.3 and 34.4 cells, respectively [n = 38]) or Day 6 (17.9 and 35.9 cells, respectively [n = 36]) were significantly (P < 0.05) higher than those cultured without HA through the culture period (13.5 and 24.2 cells, respectively [n = 26]). In the presence of HA from Day 3 to 6, only the number of TE cells (37.1 cells [n = 33]) increased (P < 0.05), compared to PZM-5 alone. Differences in ultrastructure were noticed among blastocysts cultured with or without 10 mg mL⁻¹ HA. Blastocysts cultured with HA had mainly mature mitochondria while many mitochondria appeared morphologically immature in the blastocysts cultured without HA. Lipid droplets in the blastocysts cultured with HA seemed to be more homogeneous in comparison with those in the blastocysts cultured in PZM-5 alone. Further differences were seen in the numbers of lysosome-like structures, which were greater in blastocysts cultured with HA. This study demonstrates that exogenous HA improves cell proliferation and normality of ICM and TE in porcine embryos cultured in a chemically defined medium, depending on the exposure periods to HA. (Supported by MAFF, Japan and STINT, Sweden.)