Proteomic Analysis of Porcine Oocytes During in Vitro Maturation Reveals Essential Role for the Ubiquitin C-Terminal Hydrolase-L1

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 identified 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. For the highest used concentration of C30 (i.e., To investigate overall changes in protein biosynthesis 100 mM), about 25% oocytes were still blocked in the involved in the in vitro porcine oocyte maturation, 2-DE GV stage, while 75% underwent GVBD and progressed

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Figure 1 Two-dimensional gel electrophoresis of proteins synthesized during in vitro maturation of porcine oocytes. Following labeling by [35S]- methionine, samples of 100 oocytes were lysed and cell lysates were subjected to 2-DE followed by autoradiography. The representative images for gels from oocytes in each stage of in vitro maturation are shown: (A) GV, germinal vesicle; (B) M I, the first meiosis; (C) M II, the second meiosis. To compare and analyze the images of the gels in experiments, four independently prepared samples for GV (initial), M I, and M II (final) stages of oocyte maturation were evaluated using PDQuest software, version 7.1. (D) The Master, a synthetic image containing the data from all the gels included in this MatchSet was created. Significant protein spots with differences at the level of P!0.05 between initial and final stages are indicated by their SSP numbers and marked by a circle. The protein spots designated by a square correspond to identified proteins described in previous paper (Ellederova et al. 2004).

to M I. On the contrary, the group of control oocytes Regulation of CDK1 activity and monoubiquitin level in cultured in the inhibitor-free medium comprised more the course of UCH-L1 inhibition than 80% of M II oocytes (Table 2). To assess whether UCH-L1 inhibition inﬂuences activity When the oocytes that had been cultured with of CDK1 which is controlled by the availability of cyclin 100 mM C30 for 4, 8, 12, and 28 h were transferred B1 and level of monoubiquitin, we examined the effect to inhibitor-free medium and cultured until the total of 100 mM C30 using a kinase assay and Western blot time period was 44 h, nearly half of the oocytes treated for 8 h in the presence of C30 were affected and respectively. The double kinase assay measured the remained in M I stage, while the second half was ability of CDK1 kinase and myelin-binding protein capable to reach M II. In the presence of inhibitor for the (MBP) kinase (corresponding to ERK1/2 MAP kinase) to ﬁrst 12 h of culture, about 20% of oocytes were still kept phosphorylate histone H1 and MBP respectively. Both in GV stage and about 50% reached M I, while only histone H1 kinase and MBP kinase possess minimum about 30% of oocytes completed maturation to M II activity levels at GV stage (time 0) but they are highly stage. The presence of C30 for 28 h followed by 16-h active after 20–24 h of culture, e.g., at the time when culture in MM results to the approximately same GVBD is observed. The activity of H1 kinase declines distribution of oocyte stages comparable to 12-h temporarily in untreated oocytes after 28 h of culture inhibition (Table 3). corresponding to metaphase I–anaphase transition. On www.reproduction-online.org Reproduction (2007) 134 559–568

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Figure 2 Histograms of the protein spots with regulated protein synthesis during in vitro maturation of porcine oocytes. Relative spot volume intensities corresponding to the rate of protein synthesis with significant differences between initial and final stages of meiosis. The intensities and significance were calculated and graphically presented by PDQuest software (using Student’s t-test implemented in this software) for protein spots selected by the criterion of difference at the level of P!0.05 for samples corresponding to germinal vesicle stage (GV, initial stage, left columns), and the second meiosis (M II, final stage, right columns) of oocyte maturation. Mass spectrometric identification revealed UCH-L1 in spot number SSP1108. the contrary, H1 kinase activity in C30-treated oocytes proteomic analyses with respect to relatively good increases continuously, peaking at 28 h of culture and availability of oocytes and its utilization in biomedical then declining slowly until 44 h of culture. On the other research (Vodicka et al. 2005). Pig oocytes at a GV stage hand, MBP kinase activity increases gradually and can be obtained from ovaries of slaughtered noncycling remains at a high level for up to 36 h of culture in both gilts by the aspiration of antral follicles of about 3–5 mm. control and inhibitor-treated oocytes, although the onset Their in vitro meiotic maturation resulted in the of activity appears to be delayed in the presence of metaphase II stage in w85% oocytes. inhibitor. Furthermore, as revealed by specific In a previous study, we profiled constituent proteins of immunodetection, a temporary decrease in the level of pig oocytes using 2-DE and mass spectrometry. Remark- monoubiquitin occurs in C30-treated oocytes after 28 h ably, among identified proteins we found several of culture compared with untreated oocytes (Fig. 5). proteins, including peroxiredoxins, ubiquitin carboxyl- terminal hydrolase isozyme L1, and spermine synthase, which are even more abundant than actin, usually the Discussion most abundant protein in somatic cells (Ellederova et al. A major challenge for the improvement of poor 2004). Surprisingly, UCH-L1 alone constituted w6% of developmental potential of in vitro-produced embryos, total identified oocyte proteins. their successful implantation, and maintenance of In this study, we have focused on overall changes in pregnancy is to elucidate molecular networks underlying protein biosynthesis involved in in vitro porcine oocyte fine mechanisms of reproductive processes. Here, we maturation. We considered only biosynthetically labeled utilized a proteomics approach and model of porcine proteins that were significantly different at the 95% oocytes maturing in vitro after their release from follicles. probability level in comparison between initial (GV) and We considered this animal model suitable to initiate final (M II) stages of oocyte maturation. Using these

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Figure 3 Tryptic peptide mass map of pig UCH-L1 protein. Peak labels cor- C respond to [MCH] ions of the individual peptides and amino acid positions of these fragments in UCH-L1 sequence. Underlined amino acid stretches of UCH-L1 sequence were verified by peptide mass mapping. Signals of trypsin autoproteolytic fragments are labeled with the letter T . criteria, we found 16 protein spots that displayed sequence identity (Wilkinson et al. 1989). While reproducible quantitative differences in relative inten- UCH-L3 is ubiquitously distributed, UCH-L1 is nor- sities. Mass spectrometry identified satisfactorily only mally selectively expressed in the neuronal cells and in one up-regulated protein, UCH-L1. In addition to the the testis/ovary (Wilkinson et al. 1992, Kon et al. 1999). results showing the increase in biosynthesis, further In addition to its hydrolase activity, it can act as an immunoblot analysis showed that overall level of ubiquitin-protein ligase and catalyze ubiquityl transfer UCH-L1 increases during maturation. These findings to a lysine residue of another ubiquitin molecule. demonstrated a possibility that this protein plays a major role in pig oocyte function and its characterization indicated that ubiquitin-dependent processes would play an essential role during maturation of oocytes. Ubiquitination and ubiquitin-dependent proteolysis are the major routes of intracellular protein degradation. Ubiquitin tagging of target proteins is a four-step process, including the activation of protein-ligating enzymes, and it operates in all eukaryotic cells (Doherty et al. 2002). The monoubiquitin-conjugated target protein can serve as an ubiquitylation substrate for repeated ubiquitination leading to polyubiquitination of the protein target. These polyubiquitylated proteins are then degraded by a multicomponent protease system, the 26S proteasome. The small peptides resulting from degradation are further processed for antigen presen- tation or hydrolyzed by other proteases. The role of deubiquitination in the regulation of this process is not clear, however, it may restrict the length of the ubiquitin chain, thus preventing selection for proteasomal Figure 4 Immunoblot analysis of UCH-L1 during in vitro maturation of degradation. Deubiquitinating enzymes are subdivided porcine oocytes. (A) Protein lysates prepared from oocytes in GV into ubiquitin-specific proteases and ubiquitin carboxy (initial), M I, and M II (final) stages of oocyte in vitro maturation were terminal hydrolases (UCHs). Mammalian UCHs, the examined on immunoblot using a specific antibody-recognizing UCH- isoenzymes UCH-L1 and UCH-L3, are small proteins L1. (B) Additional 2-DE protein separation followed by immunoblot consisting of about 220 amino acids with O40% revealed the presence of three protein spots corresponding to UCH-L1. www.reproduction-online.org Reproduction (2007) 134 559–568

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Table 1 Morphology of pig oocytes treated with C30 inhibitor for 28 h.

Treatment of oocytes Total no. of oocytes GV oocytes (%) M I oocytes (%) M II oocytes (%) Control 28 h 129 11 (8.6)a,b 103 (79.8)a,b 15 (11.6)a,b 10 mM C30 57 7 (12.3) 44 (77.2) 6 (10.5) 50 mM C30 82 31 (37.8)a 44 (53.7)a 7 (8.5)a 100 mM C30 124 83 (66.9)b 38 (30.7)b 3 (2.4)b

Within groups (columns), values with the same letter are signiﬁcantly different (P!0.001). GV, germinal vesicle stage of oocytes; M I, metaphase I stage of oocytes; M II, metaphase II stage of oocytes.

However, this ligase activity requires dimerization of and rat oocytes (Josefsberg et al. 2000). While the the enzyme (Liu et al. 2002). The enzyme has a variety maturation of pig and rodent oocytes is differentially of functions. UCH-L1, which constitutes about 5% of regulated in many aspects, the present results the brain-soluble proteins, appears to be associated supported the hypothesis that metaphase I–anaphase with the stabilization of monoubiquitin in neural cells transition may be regulated by ubiquitin-dependent and it is a component of inclusions that are indicative of proteasome mechanisms in these species. The block neurodegenerative diseases including Parkinson’s of metaphase I–anaphase transition was not disease and Alzheimer’s disease (Lowe et al. 1990, completely reversible. Only about 30% of oocytes Osaka et al. 2003). Furthermore, it has been suggested arrested at GV stage by 100 mM C30 present for 12 or that UCH-L1 also functions as a regulator of apoptosis 28 h of the culture could resume meiosis to M II stage in spermatogonial cell and sperm maturation. The testes following culture in inhibitor-free medium until 44 h. of the gracile axonal dystrophy mouse, which carries an Furthermore, inhibition of UCH-L1 resulted in intragenic deletion of the Uchl1 gene and thus lacks elevated histone H1 kinase (MPF) activity after 28 h UCH-L1 expression, have a reduced ubiquitin level thus of culture. This is another possible explanation and are resistant to cryptorchid injury-mediated germ of meiotic arrest at M I stage since metaphase cell apoptosis (Kwon et al. 2005). I–anaphase transition needs decrease in MPF activity. To study regulatory role of UCH-L1 during pig Since it is widely accepted that inactivation of MPF is oocyte meiotic maturation, we used specific inhibition associated with cyclin B1 degradation by ubiquitin– of this enzyme. By utilizing high-throughput screening proteasome pathway (Tokumoto et al. 1997), we to find inhibitors and traditional medical chemistry, expect that high MPF activity observed in oocytes Liu et al. (2003) identified a small-molecule inhibitor cultured in the presence UCH-L1 inhibitor reflects a belonging to the class of isatin O-acyl oximes that restrictionincyclinB1ubiquitinationandasa selectively inhibit UCH-L1 when compared with its consequence failure in its degradation by the isoform UCH-L3. We used the representative of this proteasome system. This result is further corroborated group, marked C30, which has IC50 value of 0.88 mM by a low level of monoubiquitin in oocytes treated by for UCH-L1. Then GV stage cumulus-enclosed oocytes C30 inhibitor for 28 h in comparison with those were treated with C30, GVBD was inhibited after 28 h treated in inhibitor-free medium. of culture, and most of the oocytes were arrested at M In summary, a proteomic approach followed by I stage after 44 h. This is in contrast to the study of verification functional study revealed a major regulatory Sun & Nagai (2003) showing that specific inhibition of role for UCH-L1 in pig oocyte maturation and its proteasome by MG-132 or lactacystin blocked the first essential requirement in completion of the first meiosis meiosis resumption when observed at 44 h of culture and its transition to anaphase. The mechanisms by which (Sun et al. 2004). On the contrary, our results are hydrolase as well as ligase activities of this enzyme affect consistent with the reports showing that specific the balance of ubiquitination and contribute to time and proteasome inhibition by MG-132 blocked the spatial control of ubiquitin-dependent processes during metaphase I–anaphase transition of meiosis in mouse oocyte maturation require further study.

Table 2 Morphology of pig oocytes treated with C30 inhibitor for 44 h.

Treatment of oocytes Total no. of oocytes GV oocytes (%) M I oocytes (%) M II oocytes (%) Control 44 h 122 5 (4.1)a,b,c 16 (13.1)a,b,c 101 (82.8)a,b,c 10 mM C30 148 7 (4.7)a 66 (44.6)a 75 (50.7)a 50 mM C30 175 11 (6.3)b 91 (52.0)b 73 (41.7)b 100 mM C30 216 54 (25.0)c 138 (63.9)c 24 (11.1)c

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Table 3 Morphology of pig oocytes treated with 100 mM C30 inhibitor for different culture time; followed by culture in inhibitor-free medium to the 44 h of total time.

Treatment of oocytes Total no. of oocytes GV oocytes (%) M I oocytes (%) M II oocytes (%) Control 44 h 46 2 (4.4) 6 (13.0)a,b,c 38 (82.6)a,b,c 4 h C30C40 h 48 1 (2.1) 7 (14.6) 40 (83.3) 8 h C30C36 h 68 0 (0.0) 31 (45.6)a 37 (54.4)a 12 h C30C32 h 77 15 (19.5) 37 (48.0)b 25 (32.5)b 28 h C30C16 h 69 10 (14.5) 38 (55.1)c 21 (30.4)c

Materials and Methods Oocyte collection and culture Materials Ovaries, collected from slaughtered pigs, were transported in physiological saline at 20 8C to the laboratory. The ovaries Parker’s medium H-199 was from Sevapharma (Prague, Czech were briefly washed for 20 s in 70% ethanol and then Republic) and estrous cow serum (OCS), including inactivation twice in physiological saline. The oocytes were obtained for 30 min at 56 8C, was prepared in our laboratory and stored by aspiration of antral follicles about 5 mm in diameter. K at 80 8C. Immobiline dry strips (immobilized pH gradient, Only oocytes surrounded by compact cumuli (COC; pH 3–10 non-linear, 7 cm) and ampholytes (pH 3–10) were cumulus–oocyte complex) were used for culture. COCs purchased from Amersham Pharmacia Biotech; 3-((3-cholami- were cultured in droplets of maturation medium (MM) dopropyl) dimethylammonio)-2-hydroxyl-1-propanesulfonate supplemented with 15% OCS and 20 ml Suigonan PG-600 (CHAPS), urea, and dithiothreitol (DTT) were from Amersham Intervet (International BV, Boxmeer, Holland) at 38.5 8Cin Pharmacia Biotech; acrylamide/bis-acrylamide 30% solution, an atmosphere of 5% CO2 (Pavlok et al. 2005). The samples Tris–HCl, agarose, iodoacetamide, thiourea, glycine, silver were collected at 0 (GV; germinal vesicle stage), 28 (M I; nitrate, protease inhibitor complete tablets (Complete, Mini, the first meiosis), and 44 h (M II; the second meiosis) during cat. No. 04693124001), and trifluoroacetic acid (TFA) were spontaneous in vitro maturation. At the end of culture, the from Sigma; ammonium persulfate, SDS, and N0,N0,N00,N00- cumulus cells of the oocytes were removed by vortexing. tetramethylenediamine were from Bio-Rad; and tributyl Denuded oocytes were then washed thrice in PBS and were phosphine was purchased from Fluka (Buchs, Switzerland). stored at K80 8C until use in the experiment. Morphological evaluation of oocytes was used to verify GV (intact GV), M I (the first meiotic spindle), or M II (extrusion of the first polar body) stage of in vitro maturation and quality of the oocytes collected for 2-DE. The oocytes were mounted on microscope slides with Vaseline, covered with a cover glass, and fixed in ethanol:acetic acid (3:1) for 24 h. Staining was performed with 2% orcein in 50% aqueous acetic acid and 1% sodium citrate. The slides were then placed in 40% acetic acid and observed with a phase contrast NU Zeiss microscope (Jena, Germany). The collection of the oocytes used for the proteomic study was based on the criteria that at least 85% of oocytes reached an appropriate maturation stage.

Sample preparation and two-dimensional gel electrophoresis For analytical 2-DE, samples of 100 oocytes were labeled with 50 mCi [35S]-methionine for 4 h at time 0, 24, and 40 h Figure 5 Regulation of CDK1 activity and monoubiquitin level in the during in vitro culture. Following labeling, oocytes were course of UCH-L1 inhibition. To assess the effect of UCH-L1 inhibition lysed in 30 ml of lysis buffer containing urea (9 M), CHAPS on the activity of CDK1 and the level of monoubiquitin, we examined the effect of 100 mM C30 using a kinase assay and Western blot (4% w/v), Tris (40 mM), DTT (70 mM), 2% (v/v) ampholytes respectively. (A) Using Histone H1 and MBP double kinase assay, (pH 3–10), protease inhibitors (Complete, Mini, Sigma, cat. CDK1 kinase and MAP kinase activities were measured in oocytes in No. 04693124001, used according to the manufacturer’s absence or presence of C30 via their capacity to phosphorylate external recommendation), and 0.003% (v/v) bromophenol blue. The substrates histone H1 and myelin basic protein respectively. (B) Protein samples were loaded by gel rehydration on 7 cm immobi- lysates prepared from oocytes cultured in maturation medium (MM) for lized, pH 3–10, nonlinear gradient strips for 2-DE. The 0, 28, and 44 h in absence or presence of C30 were examined on separations were performed as described by Hochstrasser immunoblot using a speciﬁc antibody-recognizing monoubiquitin. et al. (1992). The isoelectric focusing was carried out in a www.reproduction-online.org Reproduction (2007) 134 559–568

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Multiphore II apparatus (Amersham Pharmacia Biotech) with Inhibition of meiotic maturation by inhibitor specific a 3500 V power supply. The second dimension was done on toward UCH-L1 12% SDS-polyacrylamide gels using a MiniProtean II cell To investigate the effect of UCH-L1 inhibition on meiosis (Bio-Rad). An autoradiography was performed to visualize resumption, specific inhibitor of UCH-L1 marked C30 (Liu et al. protein spots. Autoradiographed gel films were scanned 2003) and kindly provided by Peter T Lansbury,Jr (Harvard Center using a laser densitometer (Duo Scan, AGFA, 2088!1872 for Neurodegeneration and Repair) was added to the MM at the pixels, 16 bits/pixel) generating 7.5 Mb images. The images beginning of culture of cumulus-enclosed GV oocytes. Meiosis were evaluated by PD Quest analysis software version 7.1 progression was evaluated at 28 or 44 h after culture. To evaluate cell (Bio-Rad). For each gel, the spots were detected and further impact and reversibility of the inhibitory effect, the GV quantified automatically using default spot detection. A oocytes were cultured in inhibitor-containing medium for the manual spot editing was performed and the results were in first 2, 8, 12, or 28 h, then washed thrice and placed into fresh agreement with those of the visual inspection. Quantifi- inhibitor-free MM with supplements of OCS and Suigonan cation of spots was done in terms of parts per million. To PG-600 until the total time of cultivation reached 44 h. In control compare and analyze the images of the gels in experiments, groups, oocytes were cultured for either 28 or 44 h in the the MatchSet Tool was used and the Master, a synthetic inhibitor-free medium supplemented by DMSO in the amount image containing the data from all the gels in the MatchSet, equal to working C30 solution. Stock solution of C30 was was created. Four independently prepared samples for initial prepared in DMSO at the concentration of 5 mM and kept frozen (GV) and final (M II) stages of oocyte maturation were at K20 8C. Final working solution was diluted immediately evaluated. The Analysis Set Manager, including Student’s before usage. t-test, was used for determination of significant protein spot differences at the level of P!0.05. Histone H1 and myelin basic protein (MBP) double kinase double assay Identification of UCH-L1 by mass spectrometry CDK1 kinase and MAP kinase activities were measured in oocytes via their capacity to phosphorylate external substrates A CBB-stained protein spot corresponding to the same spot on histone H1 and MBP respectively according to Motlı´k et al. radiolabeled and silver stained gels was excised from the gel, (1996). At each time interval during the culture, ten oocytes cut into small pieces, and washed several times with 10 mM per sample were lysed in 5 ml homogenization buffer DDT, 0.1 M 4-ethylmorpholine acetate (pH 8.1) in 50% containing 40 mM MOPS (pH 7.2), and inhibitors of acetonitrile (MeCN). After complete destaining, the gel was phosphatases (20 mM NaF, 20 mM para-nitrophenyl phos- washed with water, shrunk by dehydration in MeCN, and phate, 40 mM b-glycerophosphate, 0.2 mM Na3VO4), and reswelled again in water. The supernatant was removed and the proteases (10 mM EGTA, 0.2 mM EDTA, 2 mM benzamidine, gel was partly dried in a SpeedVac concentrator. The gel pieces 20 mg/ml leupeptin and 20 mg/ml aprotinin, 1 mM phenyl- were then reconstituted in a cleavage buffer containing 0.01% methylsulphonyl fluoride) by three cycles of freezing/thawing 2-mercaptoethanol, 0.1 M 4-ethylmorpholine acetate, 10% on dry ice. The kinase reaction was initiated by an addition MeCN, and sequencing grade trypsin (20 ng/ml; Promega). of 5 ml kinase buffer containing 10 mg/ml histone H1, and After overnight digestion, the resulting peptides were extracted 5 mg/ml MBP, together with 10 mCi/ml [g-32P] ATP to 40% MeCN/0.1% TFA. (Amersham Pharmacia Biotech). The reaction was stopped A solution of a-cyano-4-hydroxycinnamic acid (Sigma) in after 30 min by the addition of SDS-PAGE sample buffer and aqueous 30% MeCN/30% MeOH/0.2% TFA (10 mg/ml) was boiling for 3 min. After electrophoresis on 15% SDS-PAGE used as a MALDI matrix. A 1 ml sample was deposited on the gel, the gels were stained with Coomassie Blue R250, MALDI target and allowed to air-dry at room temperature. After destained overnight, dried, and autoradiographed. complete evaporation, 1 ml of the matrix solution was added. Positive ion MALDI mass spectrum was measured on a Bruker BIFLEX II reflectron time-of-flight mass spectrometer (Bruker- Immunoblotting Franzen, Bremen, Germany) equipped with SCOUT 26 sample Oocyte extracts used for 1-D immunoblotting were prepared inlet and a nitrogen laser (337 nm; Laser Science, Cambridge, by lysis of ten oocytes in reduced SDS buffer and separated MA, USA). The spectrum was acquired in the mass range of 700– by 15% SDS-PAGE. 2-D immunoblotting was performed 4500 Da and calibrated internally using the monoisotopic [MC C using samples of 200 oocytes which were lysed and proteins H] ions of autoproteolytic fragments of trypsin (842.5 and were separated by 2-DE as described above. The proteins 2211.1 Da). were then transferred to Immobilon P (Millipore, Bedford, The peak list created using flexAnalysis 2.2 program with MA, USA) membrane using a semidry blotting system SNAP peak detection algorithm was searched for the protein (Biometra, Schoeller Instruments, Prague, Czech Republic). identification using MASCOT search engine against SwissProt Blots were incubated with low-fat milk dissolved in 0.5% 51.2 database subset of mammalian proteins with the following Tween-20 in Tris-buffered saline, pH 7.4. The antibody search settings: peptide tolerance of 100 ppm, missed cleavage specific toward UCH-L1 (Chemicon, Temecula, CA, USA) site value set to one, fixed carbamidomethylation of cysteine, and monoclonal Anti-Ubiquitin (Clone 6C1, Sigma) was and variable oxidation of methionine. diluted in the ratio of 1:1000 in 5% low-fat milk and

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Downloaded from Bioscientifica.com at 10/03/2021 04:10:57AM via free access Porcine oocytes and UCH-L1 during maturation 567 incubated with the membrane overnight at 4 8C. Following Goto T, Jones GM, Lolatgis N, Pera MF, Trounson AO & Monk M 2002 washing and addition of secondary antibody (Jackson Identification and characterisation of known and novel transcripts ImmunoResearch Laboratories, West Grove, PA, USA), expressed during the final stages of human oocyte maturation. Molecular Reproduction and Development 62 13–28. immunodetected proteins were visualized by ECL-plus Hochstrasser DF, Frutiger S, Paquet N, Bairoch A, Ravier F, Pasquali C, chemiluminiscent kit according to manufacturer’s instructions Sanchez JC, Tissot JD, Bjellquist B, Vargas R et al. 1992 Human liver (Amersham Pharmacia Biotech). protein map: a reference database established by microsequencing and gel comparison. Electrophoresis 13 992–1001. 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Robert C, Gagne D, Bousquet D, Barnes FL & Sirard MA 2001 Vodicka P, Smetana K Jr, Dvorankova B, Emerick T, Xu YZ, Ourednik J, Differential display and suppressive subtractive hybridization used to Ourednik V & Motlik J 2005 The miniature pig as an animal model for identify granulosa cell messenger RNA associated with bovine oocyte Biomedical research. Annals of the New York Academy of Sciences 1049 developmental competence. Biology of Reproduction 64 1812–1820. 161–171. Sasaki R, Nakayama T & Kato T 1999 Microelectrophoretic analysis of Wilkinson KD, Lee KM, Deshpande S, Duerksen-Hughes P, Boss JM & Pohl J changes in protein expression patterns in mouse oocytes and pre- 1989 The neuron specific protein PGP 9.5 is a ubiquitin carboxyl- implantation embryos. Biology of Reproduction 60 1410–1418. terminal hydrolase. Science 288 874–877. Sun QY & Nagai T 2003 Molecular mechanisms underlying pig oocyte Wilkinson KD, Deshpande S & Larssen CN 1992 Comparison of neuronal maturation and fertilization. Journal of Reproduction and Development (PGP9.5) and non-neuronal ubiquitin C-terminal hydrolases. 49 347–359. Biochemical Society Transactions 20 631–637. Sun QY, Fuchimoto D & Nagai T 2004 Regulatory roles of ubiquitin- Zeng F & Schultz RM 2003 Gene expression in mouse oocytes and proteasome pathway in pig oocyte meiotic maturation and fertilization. preimplantation embryos: use of suppression subtractive hybridization Theriogenology 62 245–255. to identify oocyte- and embryo-specific genes. Biology of Reproduction Tokumoto T, Yamashita M, Tokumoto M, Katsu Y, Horiguchi R, 68 31–39. Kajiura H & Nagahama Y 1997 Initiation of cyclin B degradation by the 26S proteasome upon egg activation. Journal of Cell Biology Received 13 February 2007 138 1313–1322. First decision 15 March 2007 Vitale AM, Calvert ME, Mallavarapu M, Yurttas P, Perlin J, Herr J & Coonrod S 2007 Proteomic profiling of murine oocyte maturation. Revised manuscript received 27 June 2007 Molecular Reproduction and Development 74 608–616. Accepted 10 July 2007

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