Inhibition of the ubiquitin-proteasome system leads to delay of the onset of ZGA gene expression.
In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation. However, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins. Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryos. First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used these oocytes throughout this study. Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the G1 phase of the first cell cycle. Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i.e., the hsp70.1, MuERV-L, eif-1a and zscan4d genes. As a result, we found that onset of expression of the four examined ZGA genes was delayed in both normally developed 2-cell embryos and arrested 1-cell embryos. Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos.
Shin
Seung-Wook
SW
Division of Biological Science, Graduate School of Biology-Oriented Science and Technology, Wakayama, Japan.
Tokoro
Mikiko
M
Nishikawa
Satoshi
S
Lee
Hyang-Heun
HH
Hatanaka
Yuki
Y
Nishihara
Takuji
T
Amano
Tomoko
T
Anzai
Masayuki
M
Kato
Hiromi
H
Mitani
Tasuku
T
Kishigami
Satoshi
S
Saeki
Kazuhiro
K
Hosoi
Yoshihiko
Y
Iritani
Akira
A
Matsumoto
Kazuya
K
D000818:Animals; D056744:Cyclin B1 / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015977:Eukaryotic Initiation Factor-1 / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D061988:Proteasome Inhibitors; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D025801:Ubiquitin / Q000037:antagonists & inhibitors*; D015053:Zygote / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure
C534700:Ccnb1 protein, mouse; D056744:Cyclin B1; D004791:Enzyme Inhibitors; D015977:Eukaryotic Initiation Factor-1; D018840:HSP70 Heat-Shock Proteins; D007976:Leupeptins; C499132:MuERV-L protein, mouse; D061988:Proteasome Inhibitors; D011506:Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D025801:Ubiquitin; C534888:Zscan4d protein, mouse; C026022:eukaryotic peptide initiation factor-1A; C081884:heat-shock protein 70.1; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde