Regulation of ubiquitin-proteasome pathway on pig oocyte meiotic maturation and fertilization.
Degradation of proteins mediated by the ubiquitin-proteasome pathway (UPP) plays essential roles in the eukaryotic cell cycle. The main aim of the present study was to analyze the functional roles and regulatory mechanisms of the UPP in pig oocyte meiotic maturation, activation, and early embryo mitosis by drug treatment, Western blot analysis, and confocal microscopy. By using the hypoxanthine-maintained meiotic arrest model, we showed that the meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated in a dose- and time-dependent manner by two potent and cell-permeable proteasome inhibitors. Both the mitogen-activated protein kinase (MAPK) kinase inhibitor U0126 and the maturation-promoting factor inhibitor roscovitine overcame the stimulation of germinal vesicle breakdown induced by proteasome inhibitors. The phosphorylation of MAPK and p90rsk and the expression of cyclin B1 increased in a dose- and time-dependent manner when treated with proteasome inhibitors during oocyte in vitro-maturation culture. Both U0126 and roscovitine inhibited the phosphorylation of MAPK and p90rsk, and the synthesis of cyclin B1 stimulated by proteasome inhibitors. When matured oocytes were pretreated with proteasome inhibitors and then fertilized or artificially activated, the second polar body emission and the pronuclear formation were inhibited, and the dephosphorylation of MAPK and p90rsk as well as the degradation of cyclin B1 that should occur after oocyte activation were also inhibited. We also investigated, to our knowledge for the first time, the subcellular localization of 20S proteasome alpha subunits at different stages of oocyte and early embryo development. The 20S proteasome alpha subunits were accumulated in the germinal vesicle, around the condensed chromosomes at prometaphase, with spindle at metaphase I and II, the region between the separating chromosomes, and especially the midbody at anaphase I and telophase I, the pronucleus, and the nucleus in early embryonic cells. In conclusion, our results suggest that the UPP is important at multiple steps of pig oocyte meiosis, fertilization, and early embryonic mitosis and that it may play its roles by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.
Huo
Li-Jun
LJ
State Key Laboratory of Reproductive Biology, Institute of Zoology, Graduate School of the Chinese Accademy of Sciences, Chinese Academy of Sciences, Beijing 100080, China.
Fan
Heng-Yu
HY
Liang
Cheng-Guang
CG
Yu
Ling-Zhu
LZ
Zhong
Zhi-Sheng
ZS
Chen
Da-Yuan
DY
Sun
Qing-Yuan
QY
D000818:Animals; D002970:Cleavage Stage, Ovum / Q000502:physiology; D003470:Culture Media / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004558:Electric Stimulation; D005260:Female; D005307:Fertilization in Vitro; D007976:Leupeptins / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000502:physiology; D008540:Meiosis / Q000187:drug effects / Q000502:physiology; D009865:Oocytes / Q000166:cytology* / Q000201:enzymology*; D010312:Parthenogenesis / Q000502:physiology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D013552:Swine; D025801:Ubiquitin / Q000378:metabolism*
D003470:Culture Media; D019926:Cyclin B; D056744:Cyclin B1; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde