Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1.
In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.
Susor
Andrej
A
Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska str. 89, 277 21 Libechov, Czech Republic.
Ellederova
Zdenka
Z
Jelinkova
Lucie
L
Halada
Petr
P
Kavan
Daniel
D
Kubelka
Michal
M
Kovarova
Hana
H
D000818:Animals; D016203:CDC2 Protein Kinase / Q000032:analysis / Q000378:metabolism; D004527:Egg Proteins / Q000032:analysis* / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005307:Fertilization in Vitro; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D008540:Meiosis / Q000187:drug effects / Q000502:physiology*; D004676:Myelin Basic Protein / Q000032:analysis / Q000378:metabolism; D009865:Oocytes / Q000737:chemistry* / Q000378:metabolism; D010091:Oximes / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism; D013057:Spectrum Analysis; D013552:Swine; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004527:Egg Proteins; D007211:Indoles; C525086:LDN 57444; D004676:Myelin Basic Protein; D010091:Oximes; D025801:Ubiquitin; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase; D043222:Ubiquitin Thiolesterase; D046988:Proteasome Endopeptidase Complex