55 NUCLEUS CHANGES AND DEVELOPMENT OF PORCINE RECONSTRUCTED (NT) AND PARTHOGENETICALLY ACTIVATED (PA) EMBRYOS

Abstract

Nuclear reprogramming is characterized by functional modification(s) of the transferred nucleus that allows it to direct normal embryo development with the potential to grow to term. The aim of our study was to investigate the process of nuclear changes in reconstructed and activated embryos as well as their developmental competence. All chemicals used were from Sigma Chemicals (St. Louis, MO, USA). Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries of prepurbetal gilts and matured for 42 h in vitro. The cumulus cells were removed by adding in 1 mg mL ⁻¹ hyaluronidase in TLP-HEPES. For the NT experiment, oocytes with first polar body were cultured in 0.4 μg mL⁻¹ demecolcine for 1 h. A protruding membrane was removed by micromanipulator and a single donor nucleus from fetal fibroblast was injected subzonally. Fusion was conducted immediately after transfer in 0.3 M mannitol, 0.5 mM HEPES, 0.1% PVA, and 0.1 mM MgCl₂ in a fusion chamber with parallel electrodes set 1 mm apart using a singe DC pulse of 125 V mm⁻¹ for 80 s. Activation was done 2–4 h after fusion in the same medium as fusion but with 0.1 mM CaCl₂ added; embryos were cultured in 5 μg mL⁻¹ cytochalasin B and 10 μg mL⁻¹ cyclohexamide for 6 h. The embryos were cultured in glucose-free NCSU-37 containing 4 mg mL⁻¹ BSA as basic medium supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2, and then in basic medium with 5.55 mM D-glucose from Days 2–6 (Kikuchi K et al. 2002 Biol. Reprod. 66, 1033–1041). Non-manipulated oocytes (PA) were electrically activated as stated above. For observing the changes of donor cells, some reconstructed oocytes were fixed 2 h after fusion, prior to activation, and some 12 h after activation in acetic acid:ethanol (1:3) and stained in 1% orcein. The activated oocytes were fixed at 12 h and stained as stated above. There were 47.5% (38/80) of reconstructed oocytes with premature chromosome condensation (PCC), and 23.7% (19/80) with nuclear swelling two hours after fusion. Pronuclear like formation 12 h after activation was 45% (27/60) and 83.3% (50/60) in NT and PA, respectively. The blastocyst rate was 8.3% (5/60) and 46% (69/150) for NT and PA, respectively. The results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of NT embryos to the blastocyst stage.