REPRODUCTIONRESEARCH Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1 Andrej Susor, Zdenka Ellederova, Lucie Jelinkova, Petr Halada1, Daniel Kavan1, Michal Kubelka and Hana Kovarova Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska str. 89, 277 21 Libechov, Czech Republic and 1Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic Correspondence should be addressed to H Kovarova; Email: [email protected] 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 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. Reproduction (2007) 134 559–568 Introduction is expected that the important information necessary for full meiotic and developmental competence of oocytes The mammalian oocyte is the cornerstone of reproductive could be retained at the protein level. Recent findings biology. Recent advantages in technologies such as suggest that the coordination of a large number of events assisted reproduction, nuclear transfer, and embryonic is controlled by a network of protein interactions and/or or adult stem cell derivation have been significant, and development in this field is enormous. In spite of this posttranslational modifications, such as phosphorylation progress, our knowledge of molecular networks under- (Dai et al. 2003, Kraft et al. 2003). The M-phase lying fine mechanisms of the reproductive processes promoting factor (MPF) is one of the main regulators of remains elusive. Deeper understanding of these meiosis. The CDK1 kinase, the catalytic subunit of MPF, is mechanisms should help to improve poor developmental stored in oocytes as an inactive protein and its activation potential of in vitro-produced embryos, their successful needs, at the first level, the association with cyclin B1 implantation, and maintenance of pregnancy. which is known as the regulatory subunit of MPF and Fully grown mammalian oocytes are arrested at the whose synthesis and degradation oscillates during the cell dictyate stage of meiosis I (M I) and can be induced to cycle (Pines & Hunter 1990, Jackman et al. 2003). Other resume the meiotic cell cycle by a surge of luteinizing reports demonstrated, however, that mitogen-activated hormone or after the release from their follicles in vitro. protein (MAP) kinases have an important role during Although several in vitro culture systems for mammalian M-phase. They can be involved not only in spindle oocyte maturation, fertilization, and embryo develop- assembly but also in regulation of cap-dependent ment have been established, the final outcome is still not translation initiation and its contribution to the changes satisfactory (Prather & Day 1998, Nagai 2001, Pavlok in overall protein synthesis during in vitro meiotic et al. 2005). Transcriptional activity of the mammalian maturation of mammalian oocytes (Sun & Nagai 2003, oocyte rapidly decreases during maturation; therefore, it Ellederova et al. 2006, 2007). The precise timing of q 2007 Society for Reproduction and Fertility DOI: 10.1530/REP-07-0079 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/03/2021 04:10:57AM via free access 560 A Susor and others protein synthesis and degradation plays an important role of biosynthetically labeled oocyte proteins was per- in controlling oocyte maturation (Moor & Dai 2001, formed. Four gels for each type of cell population were Coenen et al. 2004). In this context, the involvement of used for comparative analysis of protein profiles of GV, the ubiquitin–proteasome complex, an essential M I, and M II stages of oocyte maturation. Using component of the proteolytic pathway in eukaryotic PDQuest analysis software version 7.1 (Bio-Rad), an cells, has been demonstrated in the regulation of pig average of 220G44 (nZ4) and 201G38 (nZ4) protein oocyte meiotic maturation and fertilization; however, spots was mapped for GV and M I oocytes respectively, the major regulatory molecular events are unknown (Sun while 120G23 (nZ4) were detected from M II oocytes et al. 2004, Pines & Lindon 2005). (Fig. 1). A statistical comparison between the initial GV Until recently, there have been relatively few studies and final M II groups of gels using Student’s t-test examining genomes and proteomes of germ cells as well as implemented in PDQuest software identified 16 protein embryos at a global level (Colonna et al. 1989, Latham spots that were significantly (P!0.05) attenuated or et al. 1991, Sasaki et al. 1999). Molecular investigations of amplified during in vitro maturation. Most of the 16 gene expression or protein composition of germ cells or significantly altered protein spots were down-regulated embryos have been sparse due to the paucity of sample during oocyte meiotic division, but four were up-regu- cells and sufficiently sensitive procedures to analyze and lated. Among these four spots, the protein in one spot identify them. With the progress of technologies, including (SSP 1108) from the Coomassie-stained gel was ident- linear amplification of cDNA populations, it has been ified by mass spectrometry. Attempts to identify other possible to consider gene profiling in this biological model selected proteins failed due to their low abundance or of extremely limited availability (Robert et al. 2001, Goto the incomplete knowledge of the pig genome. Repre- et al. 2002, Dalbies-Tran & Mermillod 2003, Zeng & sentative protein features are presented in Fig. 2. Schultz 2003). In addition, the number of functional Among the spots whose biosynthesis was apparently proteomic analyses identifying biologically relevant up-regulated in M II stage of oocyte matured in vitro, candidate proteins which may be involved in the SSP 1108 corresponded to UCH-L1. The protein spot regulation of oocyte maturation, embryo development, was unambiguously identified by peptide mass or oviductal proteome is gradually increasing in spite of the mapping approach (for details see Materials and limited availability of human germ cells and lack of Methods). Briefly, the protein was digested directly in complete genome sequences of other mammalian species the gel by trypsin and the generated peptide mixture (Georgiou et al. 2005, Horiguchi et al. 2006, Massicotte was analyzed by MALDI mass spectrometry. Seven et al. 2006, Vitale et al. 2007). peptide fragments covering 35% of UCH-L1 sequence In this study, we used a proteomics approach to were found in the obtained MALDI spectrum (Fig. 3). analyze changes in protein synthesis of porcine oocytes In addition, steady-state level of UCH-L1 revealed by during in vitro maturation. Comparative analysis of the specific immunodetection was high and it increased oocytes at the initial and final stages of meiotic division slightly in the course of meiotic division. More detailed characterized candidate proteins that are differentially 2-DE followed by immunoblot revealed the presence synthesized during in vitro maturation. While the of three spots corresponding to UCH-L1 protein with biosynthesis of many of these proteins was significantly the most acidic form increased at the final stage of decreased, we found four proteins with increase in oocyte maturation when compared with the initial biosynthetic rate, which are supposed to play a critical stage (Fig. 4). role during oocyte meiotic maturation. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was ident- ified by mass spectrometry. In addition, two-dimensional Inhibition of meiotic progression by UCH-L1 inhibition gel electrophoresis (2-DE) followed by immunoblot When COCs were cultured in maturation medium revealed the presence of three spots corresponding to (MM) containing 10, 50, or 100 mM C30 for 28 h, UCH-L1 protein with the most acidic form increased at meiosis resumption and GV breakdown (GVBD) were the final stage of oocyte maturation compared with the inhibited in oocytes treated with 50 and 100 mM C30, initial stage. An essential requirement of this oocyte while 10 mM C30 did not affected these steps. In protein for the process of meiosis in pig model was comparison, most oocytes underwent GVBD and verified using a specific inhibitor of UCH-L1. progressed to M I in control group treated in inhibitor-free medium (Table 1). The oocytes treated with 10, 50, or 100 mM C30 for Results 44 h were not capable to reach successfully M II stage in the presence of either 10 or 50 mM concentrations of C30 Protein patterns of biosynthesis during in vitro maturation and approximately half of the oocytes remained in M I of porcine oocytes and identification of UCH-L1 stage.