Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1

Abstract

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 ubiquitindependent 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.

Role of Ubiquitin C-Terminal Hydrolase-L1 in Antipolyspermy Defense of Mammalian Oocytes1

“…The deubiquitinating enzymes (DUBs) have been implicated, for example, in cell growth, differentiation, oncogenesis, and development and in regulation of chromosome structure [6]. One such DUB, ubiquitin C-terminal hydrolase-L1 (UCHL1), is highly abundant in mammalian oocytes [7]. It is also expressed in neurons and in the testis [8,9]; however, abnormal expression of UCHL1 is also found in many primary lung tumors [10,11] and in colorectal cancer [12].…”

Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

“…UCHL1 is essential for maintaining the ubiquitin-proteasome system and is abundantly expressed in mouse ova (Sekiguchi et al 2006). Similarly, previous studies have identified UCHL1 as a maternal protein necessary for oocyte maturation (Ellederova et al 2004, Massicotte et al 2006, Susor et al 2007). Thus, a logical potential conclusion would be that the NLRP5 protein level is potentially regulated by the ubiquitin-proteasome system for oocyte maturation.…”

“…In fact, ubiquitin mRNA levels are upregulated when the NLRP5 protein decreases during oogenesis and embryogenesis (Robert et al 2002, Pennetier et al 2006, Ohsugi et al 2008. Furthermore, a UCHL1 inhibitor prevents germinal vesicle (GV) breakdown and the metaphase I-anaphase transition in porcine oocytes by downregulating the ubiquitin-proteasome system (Susor et al 2007). Thus, excess NLRP5 protein resulting from downregulating the ubiquitin-proteasome system prevents complete oocyte maturation.…”

Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals

“…Ubiquitin C-terminal hydrolase L1 (UCHL1), a UPP enzyme with hydrolase and ligase activity, is one of the most abundant proteins in porcine oocytes (Susor et al 2007). The modulation of UCHL1 activity leads to polyspermy during porcine and mouse IVF, suggesting that UCHL1 is involved in the regulation of sperm incorporation and/or events leading to the establishment of the ZP or oolemma block to polyspermy (Sekiguchi et al 2006;Yi et al 2007).…”