title: Characteristics of preimplantational development of porcine parthenogenetic diploids relative to the existence of amino acids in vitro.
abstract: The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of hyaluronic acid (mWM), and a two-step culture system in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01-5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05-5.0 mg/ml of PVA (49%-53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%-20% vs. 39%, P < 0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3-5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of nonpolar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage rates of the diploids after compaction and the total number of cells in the blastocysts (53.7 +/- 2.7) and inner cell mass (12 +/- 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.
authors.affiliation: Department of Life Science, Graduate School of Science and Technology, Kobe University, Kobe City, Hyogo 657-8501, Japan.
authors.forename: Nguyen
authors.initials: N
authors.lastname: Van Thuan
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Harayama
authors.forename: Masashi
authors.initials: M
authors.lastname: Miyake
mesh_terms: D000596:Amino Acids / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002478:Cells, Cultured; D003470:Culture Media; D003571:Cytochalasin B / Q000494:pharmacology; D004171:Diploidy; D004558:Electric Stimulation; D005260:Female; D006820:Hyaluronic Acid / Q000008:administration & dosage; D009865:Oocytes / Q000502:physiology*; D009994:Osmolar Concentration; D010312:Parthenogenesis; D011142:Polyvinyl Alcohol / Q000008:administration & dosage; D012710:Serum Albumin, Bovine / Q000008:administration & dosage; D013552:Swine
chemical_list: D000596:Amino Acids; D003470:Culture Media; D012710:Serum Albumin, Bovine; D003571:Cytochalasin B; D011142:Polyvinyl Alcohol; D006820:Hyaluronic Acid