27 THE HISTONE DEACETYLASE INHIBITOR 4-IODO-SUBEROYLANILIDE HYDROXAMIC ACID IMPROVES TOTAL CELL NUMBER IN PIG NUCLEAR TRANSFER BLASTOCYSTS

Abstract

Treatment of reconstructed pig clones with the histone deacetylase inhibitor (HDACi) Scriptaid immediately after nuclear transfer (NT) and activation results in increased cloning efficiency. Aberrant gene expression examined in NT blastocyst stage embryos is only partially corrected by Scriptaid use; therefore, 2 other HDACi were examined in this study including the class I and II HDACi, suberoylanilide hydroxamic acid (SAHA) and its hydrophobic derivative 4-iodo-SAHA (I-SAHA). Blastocyst rates and total cell numbers were examined across 6 treatment groups (1 μM SAHA, 10 μM SAHA, 1 μM I-SAHA, 10 μM I-SAHA, 0.5 μM Scriptaid and no HDACi treatment). Nuclear transfer was performed on enucleated MII oocytes using 3 different cell lines. Clones were electrically fused and activated, treated with HDACi for 14 to 16 h and cultured to the blastocyst stage in PZM3 under low oxygen tension for 7 days. Blastocyst number was calculated from the total number of fused oocytes. Blastocysts were then fixed in 4% paraformaldehyde and total cell number was determined by Hoechst staining of nuclei. The results from all 3 cell lines were pooled and 782 embryos were examined for blastocyst development from 7 replicates. All statistical analysis was performed by SAS 9.1 and means were separated by least significant difference (P < 0.05). The treatment group 10 μM SAHA had the highest blastocyst rate of 41.9% (n = 124) and was significantly different than no HDACi treatment (29.2%, n = 161; P < 0.003). There was no significant difference in blastocyst rates between 1 μM SAHA, 10 μM SAHA, 1 μM I-SAHA and 0.5 μM Scriptaid with blastocyst rates of 31.6% (n = 168), 41.9% (n = 124) 34.2% (n = 76) and 40.2% (n = 179), respectively (P < 0.05). Treatment with 10 μM I-SAHA significantly decreased development when compared with the other HDACi treatments (17.6%, n = 74, P < 0.05). There was no interaction between treatment and cell line for blastocyst rates (P > 0.45). Total cell number was significantly higher in blastocysts from the 1 μM I-SAHA (37.9, n = 20) treatment group when compared with Scriptaid (29.9, n = 50) and no HDACi treatment (29.4, n = 42; P < 0.04). There were no significant improvements in total cell number between the other concentrations (P > 0.05). Additionally, there was also a significant interaction between cell line used for nuclear transfer and the total cell number (P < 0.002). Two treatments were selected to determine if 10 μM SAHA and 1 μM I-SAHA treatment postnuclear transfer was compatible with term development. Six embryo transfers were performed and 5 recipient pigs became pregnant and developed to term. The results of this study show that treatment with the HDACi, SAHA and I-SAHA postnuclear transfer has the same blastocyst rates as the commonly used HDACi, Scriptaid. Additionally, treatment with 1 μM I-SAHA improves total cell number when compared with Scriptaid or no HDACi treatment.

Funding was provided by Food for the 21st Century.