Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states.
This study demonstrated the effects of phosphate on the 2-cell block of AKR/N mouse embryos at the molecular level and focused on changes in the kinase activity and the phosphorylation state of cdc2, which is shown to regulate the cell division cycle. Removal of phosphate from the culture medium dramatically increased developmental rates to the 4-cell (91.8%) and blastocyst (42.6%) stages compared with those of embryos cultured in 1.17 mM phosphate (3.3% and 0%, respectively). The rate of development to the 4-cell stage was significantly inhibited by 0.001 mM phosphate (p < 0.05), and no morula formation was observed at 1.0 mM. The patterns of cdc2 kinase activity during the first cell cycle in AKR/N embryos were similar to those of control MCH embryos, showing the highest activity at M phase and low activity during the interphase. The phosphorylated form of cdc2 increased during the interphase, indicating that the synthesis of cyclin B and accumulation of inactive pre-maturation-promoting factor (pre-MPF) as well as abrupt dephosphorylation of cdc2 at the first cleavage correlated with the activation of cdc2 kinase. When phosphate was absent, the activation pattern of cdc2 kinase during the second cell cycle in AKR/N embryos was similar to that in the first cell cycle. On the other hand, no dephosphorylation of cdc2 was observed and the kinase activity remained at a low level until 56 h after insemination in the presence of phosphate, although an increase in phosphorylated cdc2 was observed as in the phosphate-free group. Treatment of AKR/N embryos arrested at the 2-cell stage with okadaic acid resulted in the dephosphorylation and activation of cdc2, confirming the presence of a sufficient amount of pre-MPF. These results show that phosphate has a deteriorative effect on the in vitro development of AKR/N embryos and suggest that this effect was not on the synthesis of cyclin B but on the dephosphorylation of phosphorylated cdc2.
Haraguchi
S
S
Department of Reproductive and Developmental Biology, University of Tokyo, Japan.
Naito
K
K
Azuma
S
S
Sato
E
E
Nagahama
Y
Y
Yamashita
M
M
Toyoda
Y
Y
D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D004622:Embryo, Mammalian / Q000166:cytology* / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro; D005947:Glucose / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006824:Hybridization, Genetic; D015151:Immunoblotting; D016200:Maturation-Promoting Factor / Q000502:physiology; D000090204:Mesothelin; D051379:Mice; D008806:Mice, Inbred AKR; D019319:Okadaic Acid / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology*; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism
D004791:Enzyme Inhibitors; D006657:Histones; C000715528:Msln protein, mouse; D010710:Phosphates; D019319:Okadaic Acid; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase; D016200:Maturation-Promoting Factor; D005947:Glucose; D000090204:Mesothelin